Functionalization of OEP-Based Benzochlorins to Develop Carbohydrate-Conjugated Photosensitizers. Attempt to Target β -Galactoside-Recognized Proteins

Article abstract of DOI:10.1021/jo030280b

meso-(2-Formylvinyl)octaethylporphyrin on reaction with cyanotrimethylsilane in the presence of various catalysts {copper triflate [Cu(OTf)₂], indium triflate [In(OTf)₃], or magnesium bromide diethyl etherate (MgBr₂·Et

Full text of DOI:10.1021/jo030280b

F u n ction a liza tion of OEP -Ba sed Ben zoch lor in s To Develop
Ca r boh yd r a te-Con ju ga ted P h otosen sitizer s. Attem p t To Ta r get
â-Ga la ctosid e-Recogn ized P r otein s(
Guolin Li,† Suresh K. Pandey,† Andrew Graham,†,‡ Mahabeer P. Dobhal,†,# Ricky Mehta,†,
Yihui Chen,† Amy Gryshuk,†,‡ Kate Rittenhouse-Olson,§ Allan Oseroff,‡ and
Ravindra K. Pandey*,†,|
Chemistry Division Photodynamic Therapy Center, Department of Dermatology, and Department of
Nuclear Medicine and Radiology, Roswell Park Cancer Institute, Buffalo, New York 14263, and
Department of Biotechnical and Clinical Laboratory Sciences and Department of Microbiology and
Immunology, SUNY at Buffalo, Buffalo, New York 14214
ravindra.pandey@roswellpark.org
Received September 2, 2003
meso-(2-Formylvinyl)octaethylporphyrin on reaction with cyanotrimethylsilane in the presence of
various catalysts {copper triflate [Cu(OTf)2], indium triflate [In(OTf)3], or magnesium bromide
diethyl etherate (MgBr2‚Et2O)} produced a mixture of the intermediate 3-hydroxy-3-cyanopropeno-
porphyrin, the corresponding trimethylsilyl ether derivative, and the unexpected propenochlorins.
The yields of the reaction products were found to depend on the reaction conditions and the catalysts
used. The intermediate porphyrins on treatment with concentrated sulfuric acid yielded the free-
base cyanobenzochlorins in major quantity along with several other novel benzochlorins as minor
products. Reduction of ethyl-3-hydroxy-1-pentenoate-porphyrin with DIBAL-H/NaBH4 and subse-
quent acid treatment provided the corresponding free-base 103-(2-hydroxyethyl)benzochlorin, which
upon a sequence of reactions gave a free-base benzochlorin bearing a carboxylic acid functionality
in good yield. It was then condensed with a variety of carbohydrates (glucosamine, galactosamine,
and lactosamine), and the related conjugates were screened using the galectin-binding-ability assay.
Among the carbohydrate conjugates investigated, the lactose and galactose analogues displayed
the galectin-binding ability with an enhancement of about 300-400-fold compared to lactose. In
preliminary studies, all photosensitizers (with or without carbohydrate moieties) were found to be
active in vitro [radiation-induced fibrosarcoma (RIF) tumor cells]. However, the cells incubated
with lactose (known to bind to â-galactoside-recognized proteins) prior to the addition of the
photosensitizers containing the â-galactose moiety (e.g., galactose and lactose) produced a 100%
decrease in their photosensitizing efficacy. Under similar experimental conditions, benzochlorin
without a â-galactoside moiety or the related glucose conjugate did not show any inhibition in its
photosensitizing efficacy. These results in combination with the galectin-binding data indicate a
possible â-galactoside-recognized protein specificity of the galactose- and lactose-benzochlorin
conjugates.
In tr od u ction
retained to some extent by the tumor and upon light
exposure to generate cytotoxic species (e.g., singlet
oxygen).2 So far, Photofrin (a porphyrin-based prepara-
tion) is the only photosensitizer that has been approved
all over the world for the treatment of various types of
cancer by PDT. One of the major problems associated
with Photofrin is its long-term skin phototoxicity. There-
fore, considerable efforts are underway in various labor-
atories to develop photosensitizers with improved tumor
selectivity.3
In recent years photodynamic therapy (PDT) has
received increasing attention as a new modality for the
selective treatment of solid tumors.1 The mechanism of
the tumor destruction is likely to require the interplay
of several biological responses. However, a key point
remains the ability of the photosensitizing agent to be
( A part of this work was previously published as a communication
in Chem. Commun. 2002, 1172-1173.
† Chemistry Division Photodynamic Therapy Center, Roswell Park
Cancer Institute.
(1) (a) Pandey, R. K. In Biomedical Photonics Handbook; Vo-Dinh,
T., Ed.; CRC Press: Boca Raton, FL, 2003; Chapter 37. (b) Dougherty,
T. J .; Gomer, C.; Henderson, B. W.; J ori, G.; Kessel, D.; Korbelik, M.;
Moan, J .; Peng, Q. J . Natl. Cancer Inst. 1998, 90, 889. (c) Dolmans, D.
E. J . G. J .; Fukumura, D.; J ain, R. K. Nat. Rev. Cancer 2003, 3 (5),
380.
(2) (a) Sharman, W. M.; Allen, C. M.; van Lier, J . E. Current Trends.
Drug Discovery Today; Elsevier Science: London, 1999; Vol. 4, p 507.
(b) Sharman, W. M.; Allen, C. M.; van Lier, J . E. Methods Enzymol.
2000, 319, 376. (c) Osterloh, J .; Vicente, M. G. H. J . Porphyrins
Phthalocyanines 2002, 6, 305.
‡ Department of Dermatology, Roswell Park Cancer Institute.
#
On leave from the Department of Chemistry, University of
Rajasthan, J aipur, India.
Undergraduate summer student (SUNY, Buffalo).
§ Department of Biotechnical and Clinical Laboratory Sciences and
Department of Microbiology and Immunology, SUNY at Buffalo.
| Department of Nuclear Medicine and Radiology, Roswell Park
Cancer Institute.
* Corresponding author.
10.1021/jo030280b CCC: $27.50 © 2004 American Chemical Society
Published on Web 12/12/2003
158
J . Org. Chem. 2004, 69, 158-172

Functionalization of OEP-Based Benzochlorins
For designing new photosensitizers, the balance be-
tween hydrophobicity and hydrophilicity is recognized as
an important factor.4 In this regard, various research
groups synthesized a variety of porphyrin analogues,5
including porphyrin-carbohydrate conjugates.6-10 It was
believed that the presence of the carbohydrate moiety
could enhance the membrane interaction, which may
increase their tumor selectivity. However, compared with
the parent molecules, none of the related carbohydrate
conjugates were evaluated for their affinity to target a
particular protein. Therefore, 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 the family of lectins defined
by a highly conserved carbohydrate recognition domain
and exhibit high affinity for â-galactoside.11 Because
galectins are involved in the modulation of cell adhe-
sion,12 cell growth,13 immune response,14 and angiogen-
esis,15 it is clear that changes in their expression play a
critical role in tumor progression. To confirm a “proof of
principle” concept, we recently reported an efficient
approach for the preparation of certain â-galactose-
conjugated purpurinimides (a type of chlorin).16 In pre-
liminary in vitro and in vivo studies, compared to the
nonconjugated analogues, the related carbohydrate con-
jugates produced an enhanced galectin-binding ability
with a remarkable increase in the photosensitizing
ability.17 Interestingly, compared to Photofrin, the pur-
purinimide-lactose conjugate also produced an enhanced
tumor selectivity (no skin phototoxicity was observed at
72 h postinjection).17 Currently, the synthesis and de-
tailed biological studies with a series of purpurinimides
and bacteriochlorin conjugates are in progress, and the
preliminary results are promising.
Encouraged with our previous findings, we were in-
terested in extending this approach to other compounds
known for their photosensitizing action and that could
serve as a prototype or lead molecule for the synthesis
of analogues for further biological testing. In general, for
designing an improved drug, besides increased potency,
greater selectivity, increased or decreased duration of
action, low toxicity, and increased stability, economics
is also considered a prime reason. Therefore, among the
second generation of photosensitizers, we were especially
interested in those benzochlorins, which could be derived
from the commercially available octaethylporphyrin
(OEP).18,19 One of the major problems associated with
OEP-based benzochlorin preparation is the difficulty in
the demetalation at the final step of the synthesis.18 We
have previously reported a simple and efficient approach
for the preparation of a series of fluorinated and nonflu-
orinated OEP-based benzochlorins (free-base and related
metal complexes) with variable lipophilicity.20 To inves-
tigate the effect of substituents in their photosensitizing
efficacy, these compounds were then evaluated for their
biological efficacy and, in general, compared to free-base
analogues. The related Zn(II) complexes were found to
be more effective.21 Our next objective was to extend this
methodology for introducing such functionalities that
could be easily modified to prepare a series of corre-
sponding carbohydrate conjugates for investigating their
galectin-binding ability.
(3) Pandey, R. K.; Zheng, G. In The Porphyrin Handbook; Kadish,
K. M., Smith, K. M., Guilard, R., Eds.; Academic Press: San Diego,
CA, 2000; Vol. 6 and references therein.
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2001, 5 (2), 105.
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R. K.; Vaughan, L.; Weishaupt, K. R.; Dougherty, T. J . Cancer Res.
1997, 57, 4000. (c) Pandey, R. K.; Sumlin, A.; Constantine, S.; Bellnier,
D. A.; Henderson, B. W.; Rodgers, M. A. J .; Smith, K. M.; Dougherty,
T. J . Photochem. Photobiol. 1993, 20, 44. (d) Pandey, R. K.; Constan-
tine, S.; Goff, D. A.; Kozyrev, A.; Dougherty, T. J .; Smith, K. M. Bioorg.
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R.; Potter, W. R.; Dougherty, T. J .; Pandey, R. K. Bioorg. Med. Chem.
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R.; Oseroff, A.; Dougherty, T. J .; Pandey, R. K. Photochem. Photobiol.
2002, 76, 555. (i) Chen, Y.; Graham, A.; Potter, W.; Morgan, J .;
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Resu lts a n d Discu ssion
Ch em istr y. Our initial approach was to introduce a
cyano group at the fused benzene ring of the benzochlorin
system, which could then be converted into the related
carboxylic acid or aminomethyl functionality under ap-
propriate reaction conditions. On a subsequent reaction
with various carbohydrates containing either an amino
acid or carboxylic acid functionality, the functionalized
benzochlorins should produce the desired conjugates.
Therefore, to achieve our goal, NiIIOEP (1) was converted
(9) Millard, P.; Hery, C.; Momemteau, M. Tetrahedron Lett. 1997,
38, 3731.
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R.; Dougherty, T. J .; Pandey, R. K. J . Org. Chem. 2001, 66, 8709.
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drasekharan, E. V.; Morgan, J .; Shibata, M.; Bellnier, D. A.; Henderson,
B. W.; Matta, K. L.; Oseroff, A. R.; Dougherty, T. J .; Pandey, R. K.
Unpublished results.
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Dougherty, T. J .; Pandey, R. K. J . Org. Chem. 2001, 66, 1316.
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T. J .; Pandey, R. K. Photochem. Photobiol. 2003, 77 (5), 561.
J . Org. Chem, Vol. 69, No. 1, 2004 159

Li et al.
SCHEME 1a
a
Reagents: (a) (CH3)3SiCN, Cu(OTf)2/CH2Cl2; (b) (CH3)3SiCN, Cu(OTf)2/THF; (c) (CH3)3SiCN, In(OTf)3/THF or CH2Cl2; (d) (CH3)3SiCN,
MgBr2‚Et2O/THF or CH2Cl2.
into the corresponding meso-(2-formylvinyl)octaethylpor-
phyrin 2 in excellent yield by following the Vicenta and
Smith approach.22 The reaction of 2 with cyanotrimeth-
ylsilane in combination with copper(II) triflate in CH2-
Cl2 mainly produced a mixture of novel-fused propeno-
chlorins 5 and 6 in a combined yield of 76%, and the
intermediate porphyrins 3 and 4 were obtained as minor
products. The formation of the reaction products was
found to depend on the reaction conditions used. For
example, the reaction was quite slow in dichloromethane,
and a relatively large amount of catalyst (50-75% mmol)
was required for the completion of the reaction. Replace-
ment of dichloromethane with tetrahydrofuran produced
mainly porphyrins 3 and 4, whereas chlorins 5 and 6
were isolated as minor products. During the workup
(washing the organic layer with water), porphyrin 3 was
found to be quite unstable and converted to porphyrin 4
as a major product. Attempts to purify porphyrin 4 by
silica or alumina (grade III) column chromatography
were unsuccessful, and the resulting product was identi-
fied as the starting porphyrin 2. The reaction mixture
containing mainly 4 was also found to be unstable on
alumina plates. However, we were able to isolate it in
pure form by silica gel G preparative TLC using hexanes/
CH2Cl2/EtOAc (v/v, 4:4:1) as the developing solvent.
However, during this process, a significant part (∼50%)
of it was converted into the starting porphyrin 2.
3 and 4 as major products, and porphyrins 5 and 6 were
isolated in minor quantities (∼20%) [Scheme 1]. In this
particular reaction, both indium(III) triflate and magne-
sium bromide diethyl etherate were found to be more
effective than copper(II) triflate. The literature survey
revealed23 that a number of Lewis acids [such as copper-
(II) triflate, magnesium bromide diethyl etherate, tita-
nium complexes, aluminum alkoxides, bismuth bromide,
indium fluoride, indium chloride, indium bromide, zinc
iodide, tin(II) triflate, and scandium(III) triflate] have
been reported as catalysts for the reaction of a carbonyl
compound with cyanotrimethylsilane to form cyanohy-
drin. However, to the best of our knowledge, this is the
first example that reports the utility of indium(III)
triflate as a catalyst for the reaction of a carbonyl
compound with cyanotrimethylsilane to form cyanohy-
drin under mild reaction conditions.
The formation of ketochlorin 7 and acetoxychlorin 8
by reacting chlorin 6 with TPAP/NMO24 (TPAP, tetra-
(23) (a) Bandini, M.; Cozzi, G. P.; Melchiorre, P.; Umani-Ronchi, A.
Tetrahedron Lett. 2001, 42, 3041 and references therein. (b) Chang,
C. W.; Yang, C. T.; Hwang, C. D.; Uang, B. J . Chem. Commun. 2002,
54. (c) Ward, D. E.; Hrapchak, M. J .; Sales, M. Org. Lett. 2000, 2 (1),
57. (d) Saravanan, P.; Anand, R. V.; Singh, V. K. Tetrahedron Lett.
1998, 39, 3823. (e) Loh, T. P.; Xu, K. C.; Ho, D. S. C.; Sim, K. Y. Synlett
1998, 369. (f) Komatsu, N.; Uda, M.; Suzuki, H. Tetrahedron Lett. 1997,
38 (41), 7215. (g) Zhou, X. G.; Huang, J . S.; Ko, P. H.; Cheung, K. K.;
Che, C. M. J . Chem. Soc., Dalton Trans. 1999, 3303. (h) Belokon, Y.;
Ikonnikov, N.; Moscalenko, M.; North, M.; Orlova, S.; Tararov, V.;
Yashkina, L. Tetrahedron: Asymmetry 1996, 7 (3), 851. (i) Belokon,
Y.; Moscalenko, M.; Ikonnikov, N.; Yashkina, L.; Antonov, D.; Voronts-
ov, E.; Rozenberg, V. Tetrahedron: Asymmetry 1997, 8 (19), 3245. (j)
Brunel, J . M.; Legrand, O.; Buono, G. Tetrahedron: Asymmetry 1999,
10, 1979. (k) Deng, H. B.; Isler, M. P.; Snapper, M. L.; Hoveyda, A. H.
Angew. Chem., Int. Ed. 2002, 41 (6), 1009. (l) Scholl, M.; Fu, G. C. J .
Org. Chem. 1994, 59, 7178.
With the replacement of the catalyst copper(II) triflate
with indium(III) triflate or with magnesium bromide
diethyl etherate in both THF and CH2Cl2, the same
reaction produced a mixture of porphyrin intermediates
(22) Vicenta, M. G. H.; Smith, K. M. J . Org. Chem. 1991, 56, 1407.
160 J . Org. Chem., Vol. 69, No. 1, 2004

Functionalization of OEP-Based Benzochlorins
SCHEME 2
SCHEME 3
propylammonium perruthenate; NMO, 4-methylmorpho-
line N-oxide) and acetic anhydride in pyridine, respec-
tively, confirmed the presence of the hydroxy group in
the molecule (Scheme 2). Our attempts to obtain suitable
crystals of 5 and 6 for X-ray crystallographic studies were
unsuccessful. However, a high quality of crystals of
compound 7 was obtained by using hexanes/CH2Cl2 as
the crystallizing solvent, and the X-ray crystallographic
studies confirmed the proposed structures.25 The X-ray
crystallographic studies indicated that porphyrin mac-
rocycle 7 exhibits a nonplanar distortion that was mainly
saddled with a mean deviation of the 24 macrocyclic
atoms from their least-squares plane of 0.283 Å. The
average Ni-N bond length was 1.935[6] Å (the number
in the square brackets indicates the deviation from the
mean) that is within the normal range for Ni porphy-
spectroscopy (COSY), rotating-frame Overhauser en-
hancement spectroscopy (ROESY), heteronuclear multiple-
quantum coherence (HMQC), and heteronuclear multiple-
bond correlation (HMBC) spectra (see the Supporting
Information).
The reaction of a crude mixture of porphyrins 3 and 4
with concentrated sulfuric acid produced the desired 3′-
cyanobenzochlorin 9 (71%) along with a mixture of three
other benzochlorins 10 (2%), 11 (5%), and 12 (1%) bearing
an amide group at various positions of the macrocycle
(Scheme 3). The formation of these benzochlorins derived
from porphyrins 3 and 4 was confirmed by treatment of
pure 4 with concentrated sulfuric acid. Interestingly, the
reaction of a mixture of propenochlorins 5 and 6 with
concentrated sulfuric acid under similar conditions pro-
duced a mixture of benzochlorins with amide substituents
11 (48%) and 12 (9%) and a 7-cyano-3′-ethylbenzochlorin
13 (14%). The mixture was separated into individual
compounds by column chromatography (alumina grade
III) and preparative silica TLC. The structures were
confirmed by extensive NMR studies (1H, 13C, H-H
COSY, and ROESY). The structure of 11 was also
confirmed with an X-ray crystallographic study.25
Several attempts to convert the cyanobenzochlorin 9
into the corresponding aminomethyl analogue by reacting
1
rinoids. The H and 13C NMR assignments for chlorins 6
and 7 were achieved by analyzing their H-H correlation
(24) (a) Pandey, R. K.; Smith, K. M.; Dougherty, T. J . J . Med. Chem.
1990, 33, 2032 and references therein. (b) Shiau, F.-Y.; Pandey, R. K.;
Ramaprasad, S.; Dougherty, T. J .; Smith, K. M. J . Org. Chem. 1990,
55, 2190. (c) Pandey, R. K.; Shiau, F.-Y.; Dougherty, T. J .; Smith, K.
M. Tetrahedron 1991, 47, 9571.
(25) Li, G.; Mehta, R.; Srikrishnan, T.; Nurco, D. J .; Tabaczynski,
W. A.; Alderfer, J . L.; Smith, K. M.; Dougherty, T. J .; Pandey, R. K.
Chem. Commun. 2002, 1172.
J . Org. Chem, Vol. 69, No. 1, 2004 161

Li et al.
SCHEME 4a
a
Reagents: (a) H2SO4; (b) (i) DIBAL-H, (ii) NaBH4; (c) 3 N HCl/EtOH; (d) (i) BrCH2CO2But, 25% NaOH, Bu4NHSO4; (e) TFA.
it with various reducing agents including BH3‚THF,
LiAlH4, and DIBAL-H/NaBH4 were unsuccessful. Reac-
tion of 9 under strong basic conditions did not produce
the desired benzochlorin with carboxylic acid functional-
ity; instead, benzochlorin 10 containing an amide group
at the 3′ position was isolated as a sole product. Attempts
to convert the cyano group in 9 into the corresponding
carboxylic acid with HCl or H2SO4 at variable reaction
conditions were unsuccessful and resulted in the recovery
of the starting material.
bearing a carboxylic acid functionality in 100% yield
(Scheme 4).
For investigating the galectin specificity and photo-
sensitizing efficacy of benzochlorin-carbohydrate conju-
gates, benzochlorin 21 was individually reacted with
acetylated galactosamine 22,26 glucosamine 25,27 and
lactosamine 28,26 and the corresponding benzochlorin-
carbohydrate conjugates 23, 26, and 29 were obtained
in 40%, 97%, and 43% yields, respectively (see the
Experimental Section). These compounds, when sub-
jected to the sodium methoxide treatment, produced the
corresponding hydroxy derivatives 24, 27, and 30 in 67-
95% yields depending on the substrates (Scheme 5).
Alternately, compound 27 was also prepared in 45% yield
by the reaction of 21 with glucosamine 32 using a mixed
anhydride method (isobutyl chloroformate and Et3N;28
Scheme 5). The basic idea for the preparation of the
benzochlorin-glucose conjugate was to investigate the
utility of â-galactoside (galactose and lactose) over other
carbohydrates (e.g., glucose).
From the NMR spectrum, an interesting phenomenon
was observed for the benzochlorin-glucosamine conju-
gate 27, which was isolated as a mixture of two isomers
(R-OH and â-OH at the 1′′ position of the glucosamine
part) in a ratio of 4:1 (R/â). The 2′′-H at the glucosamine
part of the conjugate was found to be easily exchanged
by deuterium to produce 27a in a mixture of CD3OD and
CDCl3 (NMR solvent: 6 drops of CD3OD and 0.6 mL of
CDCl3) during the evaporation of the solvent. The elec-
trospray ionization mass spectrometry (ESIMS) spectrum
of 27a produced a MH+ peak at 837.8 (100) cm-1 and [M
Because of our unsuccessful attempt to prepare a
benzochlorin with aminomethylene and carboxylic acid
functionalities at the 3′ position, we turned our attention
to our previous approach where porphyrin 14 in the
presence of concentrated sulfuric acid produced mainly
the corresponding dehydrated free-base porphyrin 16,
and the expected benzochlorin 15 was obtained in low
yield. To circumvent the formation of the dehydrated
product problem, 14 was first reduced with DIBAL-H/
NaBH4 to give the corresponding porphyrin 17 in 85%
yield, which on treatment with concentrated sulfuric acid
provided a mixture of the sulfonated benzochlorin 18b
and its Ni(II) complex 18a in good yield. The separation
and purification of 18a and 18b were quite difficult, and
the pure sample of 18b was obtained by using a Sephadex
LH-20 column eluted with MeOH/CH2Cl2 (v/v, 1:1). The
treatment of the crude mixture of 18a and 18b with 3 N
HCl in EtOH at a refluxing temperature gave a mixture
of 19a and 19b in a combined yield of 78% (from 17),
which was readily separated by silica column chroma-
tography. Reaction of 19a with sulfuric acid and then
with 3 N HCl/EtOH provided 19b in quantitative yield.
Benzochlorin 19b was then reacted with a large excess
of tert-butyl bromoacetate in the presence of NaOH and
Bu4HSO4, and benzochlorin 20 was obtained in quantita-
tive yield. Treatment of 20 with TFA produced 21,
(26) van Ameijde, J .; Albada, H. B.; Rob, M. J .; Liskamp, R. M. J .
J . Chem. Soc., Perkin Trans. 1 2002, 1042.
(27) Chauviere, G.; Bouteille, B.; Enanga, B.; de Albuquerque, C.;
Croft, S. L.; Dumas, M.; Perie, J . J . Med. Chem. 2003, 46 (3), 427.
(28) Furhop, J . H.; Demoulin, C.; Boettcher, C.; Koning, J .; Siggel,
U. J . Am. Chem. Soc. 1992, 114 (11), 4159-4165.
162 J . Org. Chem., Vol. 69, No. 1, 2004

Functionalization of OEP-Based Benzochlorins
SCHEME 5a
a
Reagents: (a) BOP/Et3N/CH2Cl2; (b) NaOMe/MeOH/CH2Cl2; (c) NaOH/H2O/EtOH; (d) 21, isobutyl chloroformate/Et3N/THF; (e) CD3OD/
CDCl3.
+ Na]+ at 859.7 cm-1. The FAB HRMS of 27a exhibited
an exact mass of 837.5039 (M + 1), indicating that the
molecular weight for 27a should be 836.5 g/mol and was
not consistent with the initially assigned structure.
However, the ESIMS spectrum of 27 (without treating
with 6 drops of CD3OD and 0.6 mL of CDCl3) showed a
molecular ion peak (M+) at 835.9 cm-1, which matched
with the molecular formula of C49H65N5O7. Upon further
analysis of the NMR results of 27 and 27a in pyridine-
d5 (assigned undoubtedly by H-H COSY and ROESY
NMR studies), it was observed that the resonance at δ
4.97 (1H, ddd, J ) 12.7, 9.0, 3.2 Hz, 2′′-H) for compound
27 was almost nonexistent for 27a . Besides, the reso-
nances of 27a at δ 8.08 (1H, s, 9′-H) and 5.96 (1 H, s,
1′′-H) appeared as singlets, while they were observed as
doublets with coupling constants (8.9 Hz for 9′-H and 3.2
Hz for 1′′-H) in the spectrum of 27. This is certainly due
to the exchange of hydrogen by deuterium at the 2′′
position of the glucose moiety (see Scheme 5). The above
findings were also confirmed by 13C NMR and DEPT-135
experiments (acquired in pyridine-d5), where the reso-
nance at δ 55.9 ppm in the 13C NMR spectrum of 27a
produced much less intensity than the corresponding
carbon resonance in that of 27. All of these experiments
were performed under similar conditions, using the same
relaxation delay (see the Supporting Information).
J . Org. Chem, Vol. 69, No. 1, 2004 163

Li et al.
SCHEME 6a
a
Reagents: (a) (i) 9-BBN, (ii) NaOH, H2O2; (b) Ph3P/phthalimide/DEAD; (c) NH2NH2/EtOH; (d) 21, DCC/DMAP.
SCHEME 7
It has been reported that the C-glycosyl derivatives
have a higher stability toward both chemical and enzy-
matic deglycosylation than the corresponding O- or
N-glycosyl compounds.29 To investigate its impact on PDT
efficacy, benzochlorin-C-galactose conjugates 42 and 43
were synthesized by following the reaction sequences as
depicted in Schemes 6 and 7. The benzylated galactose
derivative 34 was prepared by following the literature
procedure.30 Reaction of 34 with phthalimide in the
presence of Ph3P and diethyl azodicarboxylate gave 35
in 79% yield, which on subsequent treatment with
hydrazine monohydrate in EtOH afforded 36 in 91%
yield. Condensation of 21 with 36 by the dicyclohexyl-
carbodiimide (DCC) method gave 37 in excellent yield.
(29) (a) Bertozzi, C.; Bednarski, M. Carbohydr. Res. 1992, 223, 243.
(b) Dondoni, A.; Mariotti, G.; Marra, A. Tetrahedron Lett. 2000, 41,
3483. (c) Campbell, A. D.; Paterson, D. E.; Raynham, T. M.; Taylor, R.
J . K. Chem. Commun. 1999, 1599. (d) Lowary, T.; Meldal, M.;
Helmboldt, A.; Vasella, A.; Bock, K. J . Org. Chem. 1998, 63, 9657. (e)
Arya, P.; Ben, R. N.; Qin, H. P. Tetrahedron Lett. 1998, 39, 6131.
(30) Palomo, C.; Oiarbide, M.; Landa, A.; Gonzalez-Rego, M. C.;
Garcia, J . M.; Gonzalez, A.; Odriozola, J . M.; Martin-Pastor, M.; Linden,
A. J . Am. Chem. Soc. 2002, 124 (29), 8637.
164 J . Org. Chem., Vol. 69, No. 1, 2004

Functionalization of OEP-Based Benzochlorins
F IGURE 1. 1H NMR spectra of benzochlorins 10-12 (the signals labeled with + are the resonances of the residual CHCl3 in
CDCl3).
For the removal of the benzyl groups in the galactose
moiety by hydrogenation, compound 37 was first con-
verted into the corresponding Zn(II) complex 41, which
on hydrogenation provided 42 in 75% yield. Treatment
of 42 with TFA produced the free-base benzochlorin-
galactose conjugate 43 in 97% yield.
In our previous studies, compared to free-base ben-
zochlorins, the corresponding Zn(II) analogues were
found to be more effective for PDT efficacy.21 Therefore,
benzochlorins 21, 24, 27, and 30 were transformed into
the corresponding Zn(II) complexes 21a , 38, 39, and 40,
respectively (see the Experimental Section).
The structures of the newly synthesized benzochlorins
and the intermediates were confirmed by mass spectrom-
etry, elemental analyses, and extensive NMR studies (1H,
13C, COSY, and ROESY). Among all of the compounds,
the characterization of benzochlorins 10, 11, and 12 that
were formed after the sulfuric acid reaction was much
more challenging. Both compounds 10 and 11 showed the
molecular ion peak at 616.6 cm-1 (MH+), whereas ben-
zochlorin 12 produced the molecular ion peak 588.6 cm-1
(MH+) (-28), indicating a loss of an ethyl group. In the
electronic absorption spectra, all of these compounds
produced a characteristic absorption pattern of a typical
benzochlorin system, exhibiting the long wavelength
absorption near 660 nm. However, the NMR spectra of
these compounds were quite different (Figure 1). For
1
example, in the H NMR spectrum of benzochlorin 10,
the two protons of the formamide group were observed
at δ 6.12 and 5.98, and the resonances of the two ethyl
groups at the 7 position were observed at δ 3.25 (2H, m,
ABX system), 2.65 (2H, m, ABX system), and -0.04 (6H,
t, J ) 7.6 Hz). For compound 11, the two broad singlets
at δ 5.00 and 4.83 were assigned to two protons of the
formamide group and produced about a 1.1 ppm upfield
shift as compared to the corresponding benzochlorin 10.
This is possibly due to the tilting of the formamide group
toward the benzochlorin system, causing an anisotropic
J . Org. Chem, Vol. 69, No. 1, 2004 165

Li et al.
TABLE 1. Deter m in a tion of th e IC50 Va lu es a n d th e
Rela tive In h ibitor y Ca p a city (Rela tive P oten cy) of th e
Ben zoch lor in Der iva tives in a Solid -P h a se Assa y w ith
Su r fa ce-Im m obilized Asia lofetu in a n d Ga l-1 in Solu tion
compound
IC50 (µM)
relative potency
lactose
21a
38
39
40
1200
6.2
1
194
235
141
279
387
5.1
8.5
4.3
3.1
42
F IGURE 2. Titration of lactose by ELISA. The symbol on the
line with the error bars shows the standard error in the mean
based on three different measurements.
effect by the benzochlorin ring current. The ROESY
spectrum of 11 showed strong interactions among the
resonances at δ 8.00 (1H, d, J ) 8.5 Hz, 2′-H), 3.35 (2H,
q, J ) 7.7 Hz, 3′-CH2CH3), and 1.63 (3H, J ) 7.6 Hz,
3′-CH2CH3, partially overlapped with other signals),
indicating the presence of an ethyl group at the 3′
position, which possibly migrated during the intramo-
lecular cyclization from the 8 position of the starting
porphyrin 3 or 4. Compared to 10 and 11, in benzochlorin
12, the resonances for one of the ethyl groups were
missing. In the H-H COSY spectrum, the interactions
between the resonances at δ 9.62 (1H, d, J ) 8.2 Hz, 1′-
H) and 8.13 (1H, dd, J ) 7.3, 6.0 Hz, 2′-H) and the cross
peak between the resonances at δ 8.13 (1H, dd, J ) 7.3,
6.0 Hz, 2′-H) and 8.25 (1H, d, J ) 6.7 Hz, 3′-H) indicated
the absence of the ethyl group in 12 that was observed
at the 3′ position in benzochlorin 11. Further, the ROESY
analysis produced a strong interaction between 3′-H and
7-CH2CH3, which along with the 13C NMR data, mass
spectrometry, and elemental analyses confirmed the
proposed structure (Scheme 3).
A possible mechanism for the formation of 5 or 6 from
porphyrin 2 is possibly due to the cyanide displacement
(catalyzed by copper triflate or indium triflate) in inter-
mediate 3 or 4, followed by the Woodward-Hoffmann
[1,6] electrocyclization as suggested in our previous
paper.25 However, the mechanism of the migration of the
ethyl group to form 10 and 11 and its elimination to
produce 12 is not clear.
For drug development, a proper balance between
hydrophobicity and hydrophilicity is believed to be an
important factor, which influences the in vivo biodistri-
bution and clearance of the drug. The standard octanol/
water approach (“shake flask” method) that is generally
used to determine the overall lipophilicity of the molecule
(partition coefficient) was found to be unsuccessful for
the benzochlorin analogues. All photosensitizers with or
without a carbohydrate moiety stayed in the octanol
phase, suggesting an extremely high hydrophobic nature
for this class of compounds. The PALLAS program
(CompuDrug Chemistry Ltd., Budapest, Hungry), which
has been used successfully for calculating the overall
lipophilicity of other free-base photosensitizers,5g also has
some limitations and is not suitable for calculating the
log P values of the metalated analogues and the charged
molecules.
2 and 3). Among the above compounds, the benzochlorin-
galactose conjugates (38, IC50 ) 5.1 µM, and 42, IC50
)
3.1 µM) and the benzochlorin-lactose conjugate (40, IC50
) 4.3 µM) have a slight edge (∼2 times) over the
benzochlorin-glucose conjugate (39, IC50 ) 8.5 µM) and
the nonconjugate precursor (21a , IC50 ) 6.2 µM). This
further enforces the fact that the presence of the galac-
tose subunit is playing an important role. In addition to
â-galactoside, the benzochlorin unit has significant ga-
lectin-binding affinity, which is not surprising because
porphyrins are known to interact with almost all proteins
because of their lipophilic nature (presumably because
of the combination of the weak binding forces at the
microscopic level such as hydrogen bonding, π-π, and
other hydrophobic interactions). Therefore, relative to the
lactose moiety, the benzochlorin subpart in the lactose
and galactose conjugates is definitely a contributing
factor for the enhanced binding affinity of these com-
pounds to galectin.
In Vitr o P h otosen sitizin g Effica cy. Benzochlorins
with and without carbohydrate moieties were evaluated
for their in vitro efficacy in radiation-induced fibrosar-
coma (RIF) tumor cells known for their galectin-3 (Gal-
3) expression.31 A standard MTT assay was performed
at a fixed drug concentration (1.0 µM), and the cells were
exposed to light at variable light doses (1-5 J /cm2) after
a 4 h incubation.5i The in vitro results obtained from
benzochlorin 21a and the related carbohydrate analogues
38-40 and 42 are summarized in Figure 4. As can be
seen from Figure 4A, all compounds produced a similar
efficacy. To confirm the binding affinity of the galactose-
and lactose-benzochlorin conjugates (38, 40, and 42,
respectively) to the â-galectoside-recognized proteins, the
RIF tumor cells were preincubated with lactose alone to
block the â-galactoside binding sites before their treat-
ment with photosensitizers and light exposure. In a
typical experiment, lactose (100 µM) was added to RIF
tumor cells and incubated for 1 h. Photosensitizers with
or without the â-galactoside moiety (1.0 µM) were added
in an individual set of experiments. The cells were
incubated for an additional 3 h. After the treatment with
light (see Figure 4), the cells were resuspended in fresh
media. A total of 48 h later MTT was added, and the cells
were incubated for an additional 4 h. DMSO (100 µL) was
then added to each well to dissolve the formazine crystals.
The plates were read on a 96-well plate reader at an
absorbance of 560 nm. As can be seen from Figure 4B,C,
Ga lectin -Bin d in g Stu d ies. The enzyme-linked im-
munosorbent assay (ELISA) confirmed that the com-
pounds (21a , 38-40, and 42) bind to galectin with a
much higher affinity (141-387 times) than that of lactose
50% inhibitory concentration (IC50) ) 1.2 mM, a standard
â-galactoside used as a reference] (Table 1 and Figures
(31) Pandey, R. K.; et al. Unpublished results.
166 J . Org. Chem., Vol. 69, No. 1, 2004

Functionalization of OEP-Based Benzochlorins
F IGURE 3. Titration of benzochlorin 21a and its carbohydrate conjugates (38-40 and 42) by ELISA. The symbol on the line
with the error bars shows the standard error in the mean based on three different measurements.
the galactose- and lactose-benzochlorin conjugates (38,
40, and 42, respectively) in the presence of free lactose
produced 100% inhibition in their photosensitizing ef-
ficacy, whereas the related non-â-galactoside-benzochlo-
rin conjugate 21a , which was equally effective (Figure
4C), did not show any inhibition in the presence of free
lactose.
Incubation of the cells with free glucose (100 µM)
instead of lactose (Figure 4D) did not produce any
inhibition in the PDT efficacy of benzochlorin-carbohy-
drate conjugates 40 and 42. The related glucose conjugate
39 in the presence of either free glucose or lactose did
not diminish its photosensitizing efficacy. These results
suggest that benzochlorins conjugated with galactose and
lactose moieties bind to the â-galactose binding site of
the protein. No such specificity was observed with non-
carbohydrate benzochlorin 21a and the related glucose
conjugate 39.
chromophore with the aromatic residues of various
proteins, including galectins present in the cell surface.
Currently, the isolation and purification of other galectins
known for overexpression in various tumors (especially
Gal-3, which is highly expressed in brain tumors com-
pared to a normal brain34 and is a presurgical marker of
human thyroid carcinoma35) is in progress. Attempts are
also underway to conjugate those carbohydrates to ben-
zochlorins and other porphyrin-based photosensitizers
(containing flexible or rigid linkers with variable carbon
units) known for their enhanced binding to Gal-3 or other
tumor-specific galectins.
Exp er im en ta l Section
Melting points are uncorrected. Unless otherwise stated,
chemical shifts are reported in ppm and referenced to residual
1
solvent resonance peaks (CDCl3: for H, 7.26 ppm and for 13C,
1
77.2 ppm; and pyridine-d5: for H, 8.74 ppm and for 13C, 150.3
ppm, both referenced to the most downfield resonance; meth-
anol-d4: for H, 3.31 ppm). Hydrogen connectivity (C, CH, CH2,
1
Con clu sion
and CH3) information was obtained from DEPT-135 experi-
ments. Proton and carbon peak assignments were based on
2D NMR analysis (COSY, ROESY, HMQC, and HMBC).
Column chromatographic separations were performed over
silica gel 60 (70-230 mesh) or neutral alumina. Preparative
TLC was performed on silica 20 × 20 cm TLC plates.
P or p h yr in 4, Ch lor in s 5-8, a n d Ben zoch lor in s 9-13.
For the synthetic details and characterization of these com-
pounds including the detailed NMR data, please see the
Supporting Information.
P or p h yr in 17. To a solution of compound 14 (350 mg) in
dry toluene (30 mL) was dropwise added DIBAL-H (2.0 mL, 1
M in toluene) at -78 °C under N2. The mixture was main-
tained at this temperature for 45 min until the TLC showed
that all starting material was consumed. Water (1 mL) was
then added to quench the reaction. Solvent was removed with
rotavapor, and the residue was redissolved in a mixture of CH2-
Cl2 (80 mL) and MeOH (20 mL). NaBH4 (500 mg) was added
to the reaction mixture in three portions (about 1 min between
each portion). This mixture was stirred at room temperature
for 10 min before water (100 mL) was added. It was extracted
with CH2Cl2, washed with water, dried over Na2SO4, and
concentrated. The residue was purified over an alumina
column, eluting with CH2Cl2/EtOAc (v/v, 5:1) and MeOH/CH2-
We have developed a facile approach for functionalizing
OEP-based free-base benzochlorins. For determining the
effect of the nature of the carbohydrate moiety in
developing â-galectoside-recognized protein-specific pho-
tosensitizers for PDT, we extended our approach by
introducing a carboxylic acid functionality at the fused
exocyclic benzene ring system. It was then converted into
a series of carbohydrate conjugates and evaluated for the
galectin specificity. Among the conjugates subjected for
galectin-binding affinity (IC50), the galactose- and lactose-
benzochlorin conjugates produced the best binding affin-
ity, with a 235-387-fold enhancement compared to that
of lactose. These results suggest the specificity of the
galactose and lactose conjugates to proteins known for
their â-galectoside recognition sites and overexpression
in tumor cell surfaces (Gal-1 to Gal-12).11,32,33 Because of
the commercial availability of Gal-1, all binding studies
were performed with this particular protein. Interest-
ingly, the benzochlorin without a â-galactoside moiety
also produced a significant binding to Gal-1, and it could
be possibly due to a nonspecific interaction(s) of the
(32) Yu, F.; Finley, R. L.; Raz, A.; Kim, H. C. J . Biol. Chem. 2002,
277, 15819.
(33) Rudd, P. M.; Elliott, T.; Cresswell, P.; Wilson, I. A.; Dwek, R.
A. Science 2001, 291, 2370.
(34) Bresalier, R. S.; Yan, P.-S.; Byrd, J . C.; Lotan, R.; Raz, A. Cancer
1997, 80, 776.
(35) Orlandi, F.; Saggiorato, E.; Pivano, G.; Puligheddu, B.; Termine,
A.; Cappia, S.; Giuli, P. D.; Angeli, A. Cancer Res. 1998, 58, 3015.
J . Org. Chem, Vol. 69, No. 1, 2004 167

Li et al.
F IGURE 4. Comparative in vitro photosensitizing efficacy of various benzochlorins with and without carbohydrate moieties (1.0
µM) in RIF tumor cells. (A) Photosensitizers (38-40 and 42) incubated for 4 h (48 h MTT). Control: the cells incubated with
photosensitizers but not exposed to light. (B) Galactose conjugates 38 and 42 with and without free lactose (100 µM). (C)
Benzochlorin 21a and the related lactose conjugate 40 with and without free lactose. (D) Carbohydrate conjugate 39 with and
without free glucose (100 µM) and 40 and 42 with glucose (100 µM). (Note: The cells incubated with galactose and lactose conjugates
(38, 42, and 40) in the presence of free glucose did not produce any inhibition in their photosensitizing efficacy, whereas replacing
glucose with lactose at the same concentration showed 100% inhibition in their efficacy, suggesting the binding of the lactose and
galactose conjugates to those proteins known for recognizing â-galactosides. No inhibition in the photosensitizing efficacy of
benzochlorin 21a (without a galactose or a lactose moiety) in the presence of free lactose further suggests its nonspecificity to the
â-galactoside-recognized protein.)
Cl2 (5%) to provide 17 (280 mg, 85%) as red needles. Mp: 216-
218 °C. 1H NMR (CDCl3) δ: 9.47 (2H, s, 2 × meso-H), 9.45
(1H, s, 1 × meso-H), 9.05 (1H, d, J ) 15.1 Hz, 1′-H), 4.86 (1H,
dd, J ) 15.3, 4.5 Hz, 2′-H), 4.71 (1H, m, 3′-H), 4.04-3.68 (18H,
m, 8 × -CH2CH3 and 5′-H), 2.76 (1H, br s, -OH), 2.11 (1H,
br s, -OH), 1.90-1.68 (26H, m, 8 × -CH2CH3 and 4′-H). MS
(ESI) m/z: 690.5 (M+, 42), 713.6 (MNa+, 100). Anal. Calcd for
without further purification. An analytical sample of 18b was
purified with Sephadex LH-20 column using MeOH/CH2Cl2 (v/
v, 1:1) as the eluant. Data for 18b: dark blue solid. Mp: 220
°C (dec). UV-vis (MeOH) λmax, nm (ꢀ): 411 (88 027), 531
(5159), 564 (6695), 606 (8781), 660 (24 476). 1H NMR (CD3-
OD) δ: 9.51 (1H, d, J ) 8.7 Hz, 1′-H), 9.03 (1H, s, 15-H), 8.46
(1H, s, 20-H), 8.10 (1H, s, 5-H), 8.08 (1H, d, J ) 8.7 Hz, 2′-H),
4.62 (2H, t, J ) 7.7 Hz, 5′-H), 3.88 (2H, t, J ) 7.7 Hz, 4′-H),
3.77 (2H, q, J ) 7.5 Hz, 12-CH2CH3), 3.57 (4H, m, 3-CH2CH3
and 13-CH2CH3), 3.51 (2H, q, J ) 7.6 Hz, 2-CH2CH3 or 18-
CH2CH3), 3.40 (2H, q, J ) 7.6 Hz, 17-CH2CH3), 3.36 (2H, q, J
) 7.6 Hz, 2-CH2CH3 or 18-CH2CH3), 3.03 (2H, m, ABX system,
7-CH2CH3), 2.76 (2H, m, ABX system, 7-CH2CH3), 1.74 (3H,
t, J ) 7.6 Hz, 12-CH2CH3), 1.68 (3H, t, J ) 7.6 Hz, 3-CH2CH3),
1.64 (3H, t, J ) 7.6 Hz, 2-CH2CH3 or 18-CH2CH3), 1.57 (9H,
m, 3-CH2CH3, 17-CH2CH3, 2-CH2CH3, or 18-CH2CH3), 0.03
C
41H52N4NiO2: C, 71.21; H, 7.58; N, 8.10. Found: C, 71.32;
H, 7.59; N, 8.16.
Ben zoch lor in s 18a a n d 18b. Porphyrin 17 (300 mg) was
dissolved in sulfuric acid (40 mL, 95-98%), and the mixture
was stirred at room temperature for 2 h. It was then poured
into ice, extracted with CH2Cl2, washed with water and 5%
NaHCO3, dried over Na2SO4, and filtered. The solvent was
removed to provide a mixture of benzochlorins 18a and 18b
in a ratio of 1:4 approximately based on TLC (silica TLC, 10%
MeOH/CH2Cl2). The mixture was used for the next step
168 J . Org. Chem., Vol. 69, No. 1, 2004

Functionalization of OEP-Based Benzochlorins
(6H, t, J ) 7.1 Hz, 2 × 7-CH2CH3). MS (ESI, negative) m/z:
695.6 ([M - Na]-, 100).
) 8.8 Hz), 9.20 (1H, s), 8.55 (1H, s), 7.98 (1H, s), 7.91 (1H, d,
J ) 8.7 Hz), 4.30 (2H, s), 4.18 (2H, t, J ) 7.6 Hz), 3.86 (2H, q,
J ) 7.7 Hz), 3.79 (4H, m), 3.59 (6H, m), 3.51 (2H, q, J ) 7.7
Hz), 2.90 (2H, m, ABX system), 2.72 (2H, m, ABX system),
2.26 (1H, br s), 1.82 (3H, t, J ) 7.4 Hz), 1.75-1.60 (16H, m),
-0.01 (6H, t, J ) 7.5 Hz). MS (ESI) m/z: 675.5 (MH+, 100).
Anal. Calcd for C43H54N4O3‚1/2H2O: C, 75.52; H, 8.11; N, 8.19.
Found: C, 75.56; H, 7.88; N, 8.17.
Ben zoch lor in 21a . The title compound was obtained as a
dark green solid in quantitative yield by treating compound
21 with zinc acetate dihydrate by following the standard
procedure. UV-vis (MeOH) λmax, nm (ꢀ): 342 (23 438), 424
(92 815), 533 (4063), 579 (5000), 620 (10 078), 674 (46 876).
1H NMR (pyridine-d5) δ: 9.89 (1H, d, J ) 8.8 Hz), 9.61 (1H,
s), 9.13 (1H, s), 8.24 (1H, s), 8.17 (1H, d, J ) 8.8 Hz), 4.69
(2H, s), 4.53 (2H, t, J ) 7.5 Hz), 4.01 (2H, t, J ) 7.6 Hz), 3.91
(2H, q, J ) 7.5 Hz), 3.80 (2H, q, J ) 7.6 Hz), 3.70 (6H, m),
3.60 (2H, q, J ) 7.6 Hz), 3.13 (2H, m, ABX system), 2.82 (2H,
m, ABX system), 1.84 (3H, t, J ) 7.4 Hz), 1.80-1.64 (15H, m),
0.12 (6H, t, J ) 7.4 Hz). MS (ESI) m/z: 736.6 (M+, 100).
Com p ou n d 23. To a solution of 21 (35 mg, 0.05 mmol) and
22 (27 mg, 0.075 mmol) in dry CH2Cl2 (3 mL) were added Et3N
(6 drops) and the benzyl octyl phthalate (BOP) reagent (Chem.
Abstr. 56602-33-6, 28 mg, 0.06 mmol). The mixture was stirred
under N2 at room temperature for 18 h. It was then diluted
with CH2Cl2 (30 mL), washed with water, dried over Na2SO4,
filtered, and concentrated. The residue was purified with
preparative silica TLC using CH2Cl2/acetone (v/v, 8:1) as a
developing solvent to provide crude 23, which was further
purified by preparative silica TLC using hexanes/EtOAc (v/v,
1:2) as a developing solvent to give pure 23 (21 mg, 40%) as a
dark blue gummy solid. UV-vis (CH2Cl2) λmax, nm (ꢀ): 413
(117 733), 530 (7556), 563 (9564), 606 (11 381), 661 (32 135).
1H NMR (CDCl3) δ: 9.52 (1H, d, J ) 8.7 Hz), 9.20 (1H, s),
8.55 (1H, s), 8.00 (1H, s), 7.94 (1H, d, J ) 8.7 Hz), 7.54 (1H, d,
J ) 8.8 Hz), 5.48 (1H, d, J ) 3.3 Hz), 5.39-5.16 (3H, m), 4.18
(2H, dd, AB system, J ) 15.9 Hz), 4.11 (5H, m), 3.88 (2H, q, J
) 7.7 Hz), 3.80 (4H, m), 3.60 (6H, m), 3.52 (2H, q, J ) 7.6 Hz),
2.94 (2H, m, ABX system), 2.74 (2H, m, ABX system), 2.27
(1H, br s), 2.13 (3H, s), 2.07 (3H, s), 2.04 (3H, s), 2.03 (3H, s),
1.91 (1H, br s), 1.84 (3H, t, J ) 7.6 Hz), 1.76-1.60 (15H, m),
0.02 (6H, t, J ) 7.5 Hz). MS (FAB) m/z: 1004.3 (MH+, 100).
HRMS (FAB): calcd for C57H74N5O11 (M + H), 1004.5390;
found, 1004.5390.
Ben zoch lor in s 19a a n d 19b. To a solution of the foregoing
mixture of 18a and 18b in EtOH (30 mL) was added HCl (3
N, 45 mL). The mixture was refluxed for 4 h. After the
neutralization with saturated NaHCO3, the mixture was
extracted with CH2Cl2, washed with water, dried over Na2-
SO4, filtered, and concentrated. The residue was purified by
column chromatography on silica gel, eluting with CH2Cl2/
EtOAc (v/v, 40:1) to provide 19a (45 mg, 15% from 17) and
19b (167 mg, 63% from 17). Treatment of 19a with sulfuric
acid (following the procedure discussed at the preceding step)
and then with 3 N HCl/EtOH provided 19b in quantitative
yield. Data for 19a : dark blue solid. Mp: 288-290 °C. UV-
vis (CH2Cl2) λmax, nm (ꢀ): 420 (69 615), 622 (8933), 673
(33 883). 1H NMR (CDCl3) δ: 8.94 (1H, d, J ) 8.7 Hz), 8.86
(1H, s), 8.52 (1H, s), 7.78 (1H, s), 7.66 (1H, d, J ) 8.7 Hz),
4.21 (2H, t, J ) 7.0 Hz), 3.64 (2H, q, J ) 7.6 Hz), 3.59 (2H, t,
J ) 7.7 Hz), 3.54 (2H, q, J ) 7.7 Hz), 3.46 (6H, m), 3.38 (2H,
q, J ) 7.7 Hz), 2.76 (2H, m, ABX system), 2.53 (2H, m, ABX
system), 1.75-1.50 (18H, m), 0.08 (6H, t, J ) 7.2 Hz). MS (ESI)
m/z: 672.6 (M+, 100). Anal. Calcd for C41H50N4NiO‚3/2H2O: C,
70.29; H, 7.63; N, 8.00. Found: C, 70.07; H, 7.21; N, 7.93. Data
for 19b: dark blue solid. Mp: 275-277 °C. UV-vis (CH2Cl2)
λmax, nm (ꢀ): 413 (111 381), 528 (7235), 563 (9044), 607
(10 948), 661 (31 225). 1H NMR (CDCl3) δ: 9.59 (1H, d, J )
8.7 Hz, 1′-H), 9.32 (1H, s, 15-H), 8.66 (1H, s, 20-H), 8.08 (1H,
s, 5-H), 7.96 (1H, d, J ) 8.7 Hz, 2′-H), 4.22 (2H, t, J ) 7.4 Hz,
5′-H), 3.97 (2H, q, J ) 7.6 Hz, 12-CH2CH3), 3.88 (2H, q, J )
7.6 Hz, 13-CH2CH3), 3.69 (8H, m, 3, 17, 18-CH2CH3 and 4′-
H), 3.62 (2H, q, J ) 7.6 Hz, 2-CH2CH3), 2.97 (2H, m, ABX
system, 7-CH2CH3), 2.77 (2H, m, ABX system, 7-CH2CH3), 2.41
(1H, br s, -NH), 2.03 (1H, br s, -NH), 1.93 (3H, t, J ) 7.6 Hz,
12-CH2CH3), 1.86-1.71 (15H, m, 2, 3, 13, 17, 18-CH2CH3), 0.08
(6H, t, J ) 7.5 Hz, 2 × 7-CH2CH3). 13C NMR (CDCl3) δ: 175.6
(C), 157.2 (C), 153.6 (C), 146.8 (C), 145.4 (C), 142.7 (C), 140.5
(C), 140.0 (C), 139.9 (C), 139.4 (C), 139.2 (C), 138.2 (C), 134.8
(C), 130.3 (C), 129.0 (C), 127.2 (C), 125.7 (CH), 123.4 (CH),
114.0 (C), 107.1 (CH), 95.0 (CH), 87.1 (CH), 64.2 (CH2), 63.4
(C), 35.1 (CH2), 33.1 (CH2), 21.5 (CH2), 19.6 (CH2), 19.4 (CH2),
19.3 (CH2), 19.1 (CH2), 18.5 (CH3), 18.4 (CH3), 17.9 (CH3), 17.44
(CH3), 17.37 (CH3), 16.0 (CH3), 8.6 (CH3). MS (ESI) m/z: 617.5
(MH+, 100). Anal. Calcd for C41H52N4O‚3/2H2O: C, 76.48; H,
8.61; N, 8.70. Found: C, 76.35; H, 8.20; N, 8.62.
Ben zoch lor in 20. To a solution of 19a (270 mg) in CH2Cl2
(20 mL) were added tert-butyl bromoacetate (3 mL), 25%
NaOH (12 mL), and Bu4NHSO4 (500 mg). The mixture was
stirred at room temperature for 20 h, and TLC (alumina TLC,
CH2Cl2) confirmed the completion of the reaction. The mixture
was diluted with CH2Cl2 (50 mL), washed with water, dried
over Na2SO4, filtered, and concentrated. The residue was
purified by column chromatography on alumina, eluting with
hexanes/CH2Cl2 (v/v, 2:1) to provide 20 (313 mg, 98%) as a
dark blue solid. Mp: 124-126 °C. UV-vis (CH2Cl2) λmax, nm
(ꢀ): 413 (116 258), 530 (7698), 564 (9707), 607 (11 492), 661
(32 244). 1H NMR (CDCl3) δ: 9.50 (1H, d, J ) 8.7 Hz), 9.21
(1H, s), 8.56 (1H, s), 8.01 (1H, s), 7.96 (1H, d, J ) 8.8 Hz),
4.17 (2H, s), 4.14 (2H, t, J ) 7.7 Hz), 3.89 (2H, q, J ) 7.5 Hz),
3.81 (4H, m), 3.61 (6H, m), 3.53 (2H, q, J ) 7.6 Hz), 2.97 (2H,
m, ABX system), 2.72 (2H, m, ABX system), 2.30 (1H, br s),
1.92 (1H, br s), 1.85 (3H, t, J ) 7.5 Hz), 1.77-1.62 (15H, m),
1.55 (9H, s), 0.01 (6H, t, J ) 7.5 Hz). MS (ESI) m/z: 730.8 (M+,
100). Anal. Calcd for C47H62N4O3: C, 77.22; H, 8.55; N, 7.66.
Found: C, 77.09; H, 8.71; N, 7.71.
Com p ou n d 24. Compound 23 (17 mg) was dissolved in a
mixture of CH2Cl2 (9 mL) and MeOH (1 mL). Freshly prepared
sodium methoxide (0.3 mL, prepared from diluting 0.4 mL of
25% MeONa in MeOH with 5 mL of CH2Cl2) was added
dropwise. The mixture was stirred at room temperature for 5
min. It was then neutralized with AcOH, washed with water
and 5% NaHCO3, dried over Na2SO4, filtered, and concen-
trated. The residue was crystallized from hexanes/CH2Cl2
twice to provide 24. The filtrate was concentrated and passed
through a small short silica column, eluting with MeOH/CH2-
Cl2 (5%) to remove fast-moving impurities and then with
MeOH/CH2Cl2 (30%) to provide 24 (total 13 mg, 92%) as a dark
blue solid. Mp: 232-234 °C. UV-vis (MeOH/CH2Cl2, 30%)
λmax, nm (ꢀ): 412 (114 538), 530 (7547), 564 (9471), 606
(11 543), 661 (32 704). 1H NMR (a mixture of 0.6 mL of CDCl3
and 6 drops of CD3OD) δ: 9.45 (1H, d, J ) 8.7 Hz, 1′-H), 9.16
(1H, s, 15-H), 8.51 (1H, s, 20-H), 7.95 (1H, s, 5-H), 7.88 (1H,
d, J ) 8.9 Hz, 2′-H), 4.92 (1H, d, J ) 9.0 Hz, 1′′-H), 4.17 (2H,
s, 7′-H), 4.08 (2H, t, J ) 7.5 Hz, 5′-H), 3.89 (1H, d, J ) 2.6 Hz,
1 × sugar H), 3.81 (2H, q, J ) 7.7 Hz, 12-CH2CH3), 3.78-3.64
and 3.63-3.40 (17H, m, 2-, 3-, 13-, 17-, and 18-CH2CH3, 4′-H,
5 × sugar H), 2.84 (2H, m, ABX system, 7-CH2CH3), 2.67 (2H,
m, ABX system, 7-CH2CH3), 1.76 (3H, t, J ) 7.6 Hz, 12-
CH2CH3), 1.69-1.55 (15H, m, 2-, 3-, 13-, 17-, and 18-CH2CH3),
Ben zoch lor in 21. Compound 20 (262 mg) was dissolved
in TFA (15 mL). The resulting solution was stirred at room
temperature for 1 h. It was poured into cold water, extracted
with CH2Cl2, washed with water, dried with Na2SO4, and
filtered. After the solvent was removed, 21 (240 mg, 100%)
was obtained as a dark blue solid. Mp: 136-138 °C. UV-vis
(CH2Cl2) λmax, nm (ꢀ): 413 (112 659), 530 (7313), 564 (9181),
1
-0.05 (6H, t, J ) 7.2 Hz, 2 × 7-CH2CH3). H NMR (pyridine-
d5) δ: 9.70 (1H, d, J ) 8.7 Hz), 9.50 (1H, s), 9.24 (1H, d, J )
9.3 Hz), 8.88 (1H, s), 8.35 (1H, s), 8.14 (1H, d, J ) 8.8 Hz),
7.18 (1H, br s), 6.87 (1H, br s), 6.51 (1H, br s), 6.37 (1H, br s),
1
607 (10 970), 661 (30 810). H NMR (CDCl3) δ: 9.49 (1H, d, J
J . Org. Chem, Vol. 69, No. 1, 2004 169

Li et al.
6.06 (1H, t, J ) 9.3 Hz), 4.70 (1H, t, J ) 8.7 Hz), 4.66 (1H, br
s), 4.48 (4H, br s), 4.25 (4H, m), 3.87 (2H, q, J ) 7.4 Hz), 3.80
(4H, m), 3.64 (2H, q, J ) 7.6 Hz), 3.57 (6H, m), 2.99 (2H, m,
ABX system), 2.89 (3H, m), 2.55 (1H, br s), 1.79 (3H, t, J )
7.6 Hz), 1.76-1.62 (15H, m), 0.19 (6H, m). 13C NMR (a mixture
of 0.6 mL of CDCl3 and 6 drops of CD3OD) δ: 175.5, 171.8,
157.2, 153.4, 146.7, 145.4, 142.4, 140.5, 139.9, 139.6, 139.2,
138.1, 134.4, 129.6, 129.0, 126.9, 125.6, 123.4, 113.8, 106.9,
94.9, 86.9, 79.7, 76.6, 74.3, 73.0, 70.4, 70.3, 69.3, 63.3, 61.9,
32.9, 31.8, 21.3, 19.4, 19.2, 19.1, 18.9, 18.3, 17.7, 17.23, 17.17,
15.7, 8.4. MS (FAB) m/z: 836.3 (MH+, 100). HRMS (FAB):
calcd for C49H66N5O7 (M + H), 836.4963; found, 836.4960.
Com p ou n d 26. To a solution of 21 (91 mg, 0.13 mmol) and
25 (78 mg, 0.20 mmol) in dry CH2Cl2 (5 mL) were added Et3N
(15 drops) and the BOP reagent (71 mg, 0.16 mmol). The
mixture was stirred under N2 at room temperature for 2 h. It
was then diluted with CH2Cl2 (30 mL), washed with water,
dried over Na2SO4, filtered, and concentrated. The residue was
purified with preparative silica TLC using hexanes/EtOAc (v/
v, 1:2) as a developing solvent to provide 26 (130 mg, 97%) as
a dark blue solid. Mp: 133-135 °C. UV-vis (CH2Cl2) λmax, nm
(ꢀ): 413 (129 898), 530 (8554), 563 (10 693), 606 (12 646), 661
(35 520). 1H NMR (CDCl3) δ: 9.56 (1H, d, J ) 8.8 Hz), 9.23
(1H, s), 8.57 (1H, s), 8.01 (1H, s), 7.94 (1H, d, J ) 8.7 Hz),
6.68 (1H, d, J ) 9.4 Hz), 5.79 (1H, d, J ) 8.8 Hz), 5.30 (1H,
dd, J ) 10.0, 10.0 Hz), 5.17 (1H, dd, J ) 9.8, 9.8 Hz), 4.36-
4.21 (2H, m), 4.19-4.05 (5H, m), 3.91 (2H, q, J ) 7.4 Hz), 3.80
(5H, m), 3.62 (6H, m), 3.54 (2H, q, J ) 7.6 Hz), 2.93 (2H, m,
ABX system), 2.75 (2H, m, ABX system), 2.32 (1H, br s), 2.12
(3H, s), 2.09 (3H, s), 2.07 (3H, s), 2.04 (3H, s), 1.95 (1H, br s),
1.86 (3H, t, J ) 7.4 Hz), 1.77-1.63 (15H, m), 0.03 (6H, t, J )
7.2 Hz). MS (FAB) m/z: 1004.4 (MH+, 100). HRMS (FAB):
calcd for C57H74N5O11 (M + H), 1004.5390; found, 1004.5400.
Com p ou n d 27. Meth od A. When the same procedure as
that described for the foregoing compound is followed, 26 (70
mg) was treated with excess MeONa for 5 min. After workup
and purification with preparative silica TLC using MeOH/CH2-
Cl2 (10%) as a developing solvent, 27 (39 mg, 67%) was
obtained as a dark blue solid. Mp: 149-151 °C. UV-vis
(MeOH/CH2Cl2, 30%) λmax, nm (ꢀ): 413 (119 210), 530 (8015),
563 (10 023), 606 (11 790), 661 (32 544). 27 was a mixture of
two isomers with a ratio of 4:1 (R/â, referred to -OH at the 1′′
position). The NMR data was given for the R isomer. 1H NMR
(pyridine-d5) δ: 9.75 (1H, d, J ) 8.8 Hz, 1′-H), 9.49 (1H, s,
15-H), 8.89 (1H, s, 20-H), 8.34 (1H, s, 5-H), 8.25 (1H, d, J )
8.8 Hz, 2′-H), 8.09 (1H, d, J ) 8.9 Hz, 9′-H), 5.97 (1H, d, J )
3.2 Hz, 1′′-H), 5.86 (4H, very broad s, 4 × -OH), 4.97 (1H,
ddd, J ) 12.7, 9.0, 3.2 Hz, 2′′-H), 4.80 (2H, m, 3′′-H and 5′′-H),
4.58 and 4.45 (each 1H, each m, 6′′-H), 4.47 (2H, splitting s,
7′-H), 4.35 (1H, dd, J ) 9.2, 9.2 Hz, 4′′-H), 4.28 (2H, t, J ) 7.1
Hz, 5′-H), 3.87 (4H, m, 4′-H and 12-CH2CH3), 3.77 (2H, q, J )
7.7 Hz, 13-CH2CH3), 3.64 (2H, q, J ) 7.7 Hz, 17-CH2CH3), 3.58
(6H, m, 2-, 3-, and 18-CH2CH3), 3.03 (2H, m, ABX, 7-CH2CH3),
2.89 (2H, m, ABX, 7-CH2CH3), 2.84 (1H, br s, -NH), 2.54 (1H,
br s, -NH), 1.81 (3H, t, J ) 7.4 Hz, 12-CH2CH3), 1.77-1.62
(15H, m, 2-, 3-, 13-, 17-, and 18-CH2CH3), 0.21 (6H, m, 2 ×
7-CH2CH3).13C NMR (pyridine-d5) δ: 176.8 (C), 170.7 (C), 158.1
(C), 154.2 (C), 147.9 (C), 146.2 (C), 143.3 (C), 141.4 (C), 141.0
(C), 140.7 (C), 140.1 (C), 139.9 (C), 139.2 (C), 135.4 (C), 132.0
(C), 130.1 (C), 127.9 (C), 126.5 (CH), 124.9 (CH), 114.7 (C),
107.9 (CH), 96.2 (CH), 93.1 (CH), 87.9 (CH), 74.5 (CH), 73.9
(CH), 73.7 (CH), 73.2 (CH2), 71.9 (CH2), 64.2 (C), 63.7 (CH2),
55.9 (CH), 33.6 (CH2), 32.7 (CH2), 22.1 (CH2), 20.0 (CH2), 19.9
(CH2), 19.6 (CH2), 19.5 (CH2), 19.1 (CH3), 19.0 (CH3), 18.6
(CH3), 17.89 (CH3), 17.86 (CH3), 16.4 (CH3), 9.22 (CH3), 9.19
(CH3). MS (ESI) m/z: 835.9 (M+, 100). MS (FAB) m/z: 836.5
(MH+, 100). HRMS (FAB): calcd for C49H66N5O7 (M + H),
836.4963; found, 836.4958.
[1.0 mL, prepared by treating D-glucosamine hydrochloride 31
(750 mg, 3.48 mmol) to a solution of NaOH (139 mg, 3.48
mmol) in a mixture of water (2 mL) and EtOH (11 mL)] and
Et3N (0.2 mL) were added successively. The resulting reaction
mixture was stirred at room temperature for 2 h. The same
solution of 32 in H2O/EtOH (1.0 mL) was added. The final
mixture was stirred at room temperature for 17 h, poured into
water, and extracted with CH2Cl2 (2 × 100 mL). The organic
layer was washed with water (2 × 100 mL), 5% NaHCO3, and
again with water (100 mL). The dichloromethane layer was
separated, dried over Na2SO4, filtered, and concentrated. The
residue was purified with preparative silica TLC using acetone/
CH2Cl2/acetone (v/v, 1:9:10) as a developing solvent to provide
27 (14 mg, 45%) as a dark blue solid.
Com p ou n d 27a . For the preparation and characterization
of this compound including the detailed NMR and MS data,
please see the Supporting Information.
Com p ou n d 29. To a solution of 21 (96 mg, 0.14 mmol) and
28 (132 mg, 0.21 mmol) in dry CH2Cl2 (5 mL) were added Et3N
(10 drops) and the BOP reagent (75 mg, 0.17 mmol). The
mixture was stirred under N2 at room temperature for 17 h.
It was then diluted with CH2Cl2 (30 mL), washed with water,
dried over Na2SO4, filtered, and concentrated. The residue was
purified by preparative silica TLC using CH2Cl2/acetone (v/v,
7:1) as a developing solvent to provide 29 (79 mg, 43%) as a
dark blue solid. Mp: 150-153 °C. UV-vis (CH2Cl2) λmax, nm
(ꢀ): 413 (122 657), 530 (8078), 562 (10 097), 606 (12 011), 661
(33 800). 1H NMR (CDCl3) δ: 9.50 (1H, d, J ) 8.7 Hz), 9.19
(1H, s), 8.54 (1H, s), 7.98 (1H, s), 7.91 (1H, d, J ) 8.7 Hz),
7.36 (1H, d, J ) 9.3 Hz), 5.42-5.23 (3H, m), 5.11 (1H, dd, J )
10.9, 7.6 Hz), 5.04-4.90 (2H, m), 4.43 (1H, d, J ) 7.9 Hz), 4.39
(1H, d, J ) 12.3 Hz), 4.24-3.99 (7H, m), 3.87 (3H, m), 3.76
(6H, m), 3.59 (6H, m), 3.51 (2H, q, J ) 7.6 Hz), 2.90 (2H, m,
ABX system), 2.72 (2H, m, ABX system), 2.26 (1H, br s), 2.16
(3H, s), 2.10 (3H, s), 2.08 (6H, s), 2.01 (6H, s), 1.97 (3H, s),
1.89 (1H, br s), 1.83 (3H, t, J ) 7.5 Hz), 1.75-1.59 (15H, m),
-0.01 (6H, t, J ) 7.3 Hz). MS (FAB) m/z: 1292.5 (MH+, 100).
HRMS (FAB): calcd for C69H90N5O19 (M + H), 1292.6230;
found, 1292.6240.
Com p ou n d 30. Compound 29 (41 mg) was added to a
mixture of CH2Cl2 (9 mL) and MeOH (1 mL) and a MeONa
solution (0.5 mL, prepared from diluting 1.0 mL of 25%
MeONa in MeOH with 5 mL of CH2Cl2) dropwise. The mixture
was stirred at room temperature for 5 min. AcOH (0.1 mL)
was then added to the above mixture. Then 5% NaHCO3 was
added until the solution became slightly basic. The solvent was
removed, and the residue was extracted with MeOH/CH2Cl2
(25%) repeatedly (typically, 5 × 30 mL). The extracts were
combined, and then the solvent was removed. The residue was
redissolved in MeOH/CH2Cl2 (25%, 10 mL). Hexane was added
dropwise, and a dark blue solid was precipitated out. It was
filtered, and compound 30 (30 mg, 95%) was obtained as a dark
blue solid. Mp: 235-237 °C. UV-vis (MeOH/CH2Cl2, 30%)
λmax, nm (ꢀ): 412 (117 551), 530 (7401), 563 (9380), 606
(11 617), 661 (33 217). 1H NMR (pyridine-d5) δ: 9.71 (1H, d, J
) 8.9 Hz, 1′-H), 9.50 (1H, s, 15-H), 9.26 (1H, d, J ) 9.2 Hz,
9′-H), 8.88 (1H, s, 20-H), 8.35 (1H, s, 5-H), 7.88 (1H, d, J )
8.8 Hz, 2′-H), 6.41 (7H, very broad s, 7 × -OH), 6.05 (1H, t, J
) 9.2, 9.2 Hz, 1′′-H), 5.12 (1H, d, J ) 7.9 Hz, 1′′′-H), 4.65-
4.32 (10H, m, 7′-H and 8 × sugar H), 4.27 (3H, m, 5′-H and
2′′-H), 4.16 (2H, m, 2 × sugar H), 4.06 (1H, m, 1 × sugar H),
3.94-3.73 (6H, m, 4′-H and 12, 13-CH2CH3), 3.64 (2H, q, J )
7.5 Hz, 17-CH2CH3), 3.57 (6H, m, 2-, 3-, and 18-CH2CH3), 2.99
(2H, m, ABX, 7-CH2CH3), 2.90 (2H, m, ABX, 7-CH2CH3), 2.87
(1H, br s, -NH), 2.55 (1H, br s, -NH), 1.80 (3H, t, J ) 7.4 Hz,
12-CH2CH3), 1.77-1.62 (15H, m, 2-, 3-, 13-, 17-, and 18-
CH2CH3), 0.19 (6H, t, J ) 7.2 Hz, 2′- and 7-CH2CH3). 13C NMR
(pyridine-d5) δ: 176.8, 171.5, 158.2, 154.2, 147.9, 146.2, 143.3,
141.4, 141.0, 140.7, 140.0, 139.9, 139.2, 135.3, 131.8, 130.0,
127.9, 126.4, 124.8, 114.7, 107.9, 106.3, 96.2, 87.9, 82.5, 81.2,
78.7, 78.2, 77.7, 75.6, 74.4, 73.3, 72.9, 71.8, 70.5, 64.2, 62.52,
62.49, 33.5, 32.6, 22.0, 20.0, 19.8, 19.6, 19.4, 19.1, 19.0, 18.5,
Meth od B. To a solution of 21 (25 mg) in a mixture of dry
THF (10 mL) and Et3N (0.2 mL) was added the isobutyl
chloroformate (0.2 mL) at 0 °C. The mixture was stirred at
this temperature for 45 min. A solution of 32 in H2O/EtOH
170 J . Org. Chem., Vol. 69, No. 1, 2004

Functionalization of OEP-Based Benzochlorins
17.85, 17.82, 16.3, 9.2. MS (FAB) m/z: 998.3 (MH+, 100).
HRMS (FAB): calcd for C55H76N5O12 (M + H), 998.5491; found,
998.5494.
Hz), 3.26 (2H, m), 2.89 (2H, m, ABX system), 2.71 (2H, m, ABX
system), 2.26 (1H, s), 1.88 (1H, s), 1.82 (3H, t, J ) 7.6 Hz),
1.78-1.52 (17H, m), 1.46 (2H, m), 0.00 (6H, t, J ) 7.2 Hz).
MS (FAB) m/z: 1238.6 (MH+, 100). HRMS (FAB): calcd for
Com p ou n d 35. To a solution of compound 34 (14.04 g, 24.1
mmol) in dry THF (200 mL) were added Ph3P (6.96 g, 26.5
mmol) and phthalimide (4.30 g, 29.2 mmol). The resultant
solution was cooled to 0 °C, and diethyl azodicarboxylate (4.93
mL, 31.3 mmol) was added slowly (∼5 min) via a syringe. The
mixture was stirred at room temperature under N2 for 20 h.
It was diluted with CH2Cl2 (300 mL), washed with water, dried
over Na2SO4, filtered, and concentrated. The residue was
purified with column chromatography on silica gel, eluting
with hexanes/EtOAc (v/v, 2:1) first and then hexanes/EtOAc
(v/v, 3:2) to provide 35 (13.65 g, 79%) as a colorless oil.1H NMR
(CDCl3, TMS as ref, 0.00 ppm) δ: 7.82 (2H, m), 7.69 (2H, m),
7.36-7.19 (20H, m), 4.76-4.42 (8H, m), 3.97 (3H, m), 3.86-
3.60 (6H, m), 1.88-1.48 (4H, m). 13C NMR (CDCl3) δ: 168.4
(C), 138.76 (C), 138.68 (C), 138.54 (C), 138.39 (C), 133.9, 132.3,
128.45 (CH), 128.38 (CH), 128.08 (CH), 127.99 (CH), 127.88
(CH), 127.78 (CH), 127.68 (CH), 127.61(CH), 123.2, 76.9 (CH),
76.8 (CH), 74.5 (CH), 73.4 (CH2), 73.2 (CH2), 73.1 (CH2), 72.4
(CH), 70.9 (CH), 67.7 (CH2), 37.9 (CH2), 25.2 (CH2), 24.7 (CH2).
MS (ESI) m/z: 734.6 (MNa+, 100).
Com p ou n d 36. To a solution of 35 (13.30 g) in absolute
EtOH (300 mL) was added NH2NH2‚H2O (6 mL) dropwise. The
mixture was refluxed for 4 h (a white solid precipitated out
after the mixture was refluxed for a half hour). It was then
cooled to 0 °C with an ice bath. Concentrated HCl (37%, 60
mL) was slowly added to the reaction mixture (∼10 min). The
solvent was removed to dry, and then EtOH/H2O (v/v, 1:1, 200
mL) was added to the residue. The resulting mixture was
neutralized with NaOH (20%), and the pH was adjusted to 14
to give a clear pale yellow solution, which was then extracted
with Et2O (4 × 100 mL), washed with water, dried over Na2-
SO4, filtered, and concentrated. The residue was purified by
column chromatography on alumina (grade III), eluting with
MeOH/CH2Cl2 (2%) first and then with MeOH/CH2Cl2 (5%) to
provide 36 (9.89 g, 91%) as a pale yellow oil. 1H NMR (CDCl3,
TMS as reference, 0.00 ppm) δ: 7.42-7.20 (20H, m, 4 ×
-CH2C6H5), 4.78-4.42 (8H, m, 4 × -CH2C6H5), 3.98 (2H, s,
1′-H), 3.92 (1H, m), 3.86-3.75 (2H, m), 3.72 (1H, dd, J ) 7.1,
2.5 Hz), 3.63 (1H, dd, J ) 10.7, 4.3 Hz), 2.67 (2H, m, 1-H),
1.86 (2H, br s, 1-NH2), 1.66 (1H, m, one proton of 3-H), 1.54
(2H, m, one proton of 2-H and one proton of 3-H), 1.36 (1H,
m, one proton of 2-H). 13C NMR (CDCl3) δ: 138.8 (C), 138.7
(C), 138.5 (C), 128.5 (CH), 128.4 (CH), 128.1 (CH), 128.04 (CH),
127.95 (CH), 127.87 (CH), 127.71 (CH), 127.68 (CH), 127.63
(CH), 77.1 (CH), 77.0 (CH), 74.6 (CH), 73.4 (CH2), 73.3 (CH2),
73.2 (CH2), 72.3 (CH), 71.6 (CH), 67.9 (CH2), 42.1 (CH2), 30.4
(CH2), 24.6 (CH2). MS (APCI) m/z: 582.5 (MH+, 100).
C
80H96N5O7 (M + H), 1238.7310; found, 1238.7270.
Com p ou n d 38. The title compound was obtained as a deep
green solid in 85% yield by treating compound 24 with zinc
acetate dihydrate by following the standard procedure [the
compound was purified with a Sephadex LH-20 column,
eluting with MeOH/CH2Cl2 (v/v, 1:1)]. UV-vis (MeOH/CH2-
Cl2, 80%) λmax, nm (ꢀ): 343 (23 218), 425 (96 082), 535 (3964),
1
578 (5285), 621 (10 760), 675 (49 268). H NMR (pyridine-d5)
δ: 9.88 (1H, d, J ) 8.8 Hz), 9.61 (1H, s), 9.22 (1H, d, J ) 9.4
Hz), 9.14 (1H, s), 8.24 (1H, s), 8.02 (1H, d, J ) 8.8 Hz), 6.05
(1H, t, J ) 9.1 Hz), 4.70 (1H, t, J ) 9.1 Hz), 4.66 (1H, d, J )
2.9 Hz), 4.47 (4H, br s), 4.25 (4H, m), 3.92 (2H, q, J ) 7.4 Hz),
3.81 (4H, m), 3.70 (6H, m), 3.60 (2H, q, J ) 7.7 Hz), 2.97 (2H,
m, ABX system), 2.81 (2H, m), 1.85 (3H, t, J ) 7.4 Hz), 1.80-
1.65 (15H, m), 0.09 (6H, t, J ) 7.1 Hz). MS (FAB) m/z: 897.3
(M+, 100).
Com p ou n d 39. The title compound was obtained as a deep
green solid in 81% yield by treating compound 27 with zinc
acetate dihydrate by following the standard procedure [the
compound was purified with a Sephadex LH-20 column,
eluting with MeOH/CH2Cl2 (v/v, 1:1)]. UV-vis (MeOH/CH2-
Cl2, 80%) λmax, nm (ꢀ): 342 (24 369), 424 (94 966), 534 (4706),
1
578 (5962), 620 (10 668), 674 (47 588). H NMR (pyridine-d5)
δ: 9.92 and 9.89 (total 1H, d, J ) 8.7 Hz), 9.62 (1H, s), 9.15
(1H, s), 8.24 (1H, s), 8.62 and 8.13 (total 1H, d, J ) 8.9 Hz),
8.11 and 8.05 (1H, d, J ) 8.9 Hz), 7.27 (1H, br s), 7.05 (1H, br
s), 6.23 (1H, br s), 5.99 (1H, d, J ) 3.0 Hz), 5.54 (1H, br s),
4.92 (1H, m), 4.81 (2H, m), 4.60 (1H, m), 4.47 (2H, s), 4.43
(1H, m), 4.35 (1H, dd, J ) 9.1, 9.1 Hz), 4.28 (2H, t, J ) 7.4
Hz), 3.91 (4H, m), 3.80 (2H, m), 3.70 (6H, m), 3.61 (2H, m),
3.00 (2H, m, ABX), 2.82 (2H, m, ABX), 1.85 (3H, m), 1.80-
1.66 (15H, m), 0.09 (6H, m). MS (FAB) m/z: 898.3 (MH+, 100).
Com p ou n d 40. The title compound was obtained as a deep
green solid in 77% yield by treating compound 30 with zinc
acetate dihydrate by following the standard procedure [the
compound was purified with a Sephadex LH-20 column,
eluting with MeOH/CH2Cl2 (v/v, 1:1)]. UV-vis (MeOH/CH2-
Cl2, 80%) λmax, nm (ꢀ): 341 (23 683), 425 (97 439), 532 (4060),
1
579 (5413), 621 (10 962), 675 (49 667). H NMR (pyridine-d5)
δ: 9.88 (1H, d, J ) 8.7 Hz), 9.61 (1H, s), 9.25 (1H, m), 9.14
(1H, s), 8.24 (1H, s), 8.03 (1H, d, J ) 8.8 Hz), 6.04 (1H, dd, J
) 9.3, 9.3 Hz), 5.11 (1H, d, J ) 7.9 Hz), 4.60-4.31 (10H, m),
4.26 (3H, m), 4.15 (2H, m), 4.04 (1H, m), 3.92 (2H, q, J ) 7.6
Hz), 3.82 (4H, m), 3.70 (6H, m), 3.60 (2H, q, J ) 7.6 Hz), 2.97
(2H, m), 2.80 (2H, m), 1.85 (3H, t, J ) 7.3 Hz), 1.80-1.64 (15H,
m), 0.08 (6H, t, J ) 7.2 Hz). MS (ESI) m/z: 1082.7 (MNa+,
100).
Com p ou n d 37. A reaction flask containing a mixture of
21 (73 mg, 0.11 mmol), 36 (100 mg, 0.17 mmol), DCC (27 mg,
0.13 mmol), and DMAP (14 mg, 0.11 mmol) was dissolved in
CH2Cl2 (3 mL). The resulting solution was stirred at room
temperature under N2 for 15 h. Water (5 mL) was added, and
the mixture was stirred at room temperature for 15 min. It
was then diluted with CH2Cl2 (20 mL). The organic layer was
collected, dried over Na2SO4, filtered, and concentrated. The
residue was transferred into a 10 mL flask with CH2Cl2. The
solvent was removed, and CH2Cl2 (2 mL) was added to the
residue. The mixture was refrigerated for 1 h. It was filtered
and washed with CH2Cl2 (most of the urea was removed by
this process). The filtrate was concentrated, and the residue
was purified by column chromatography on silica gel, eluting
with CH2Cl2/acetone (v/v, 12:1) to provide 37 (112 mg, 83%)
as a dark blue gummy solid. UV-vis (CH2Cl2) λmax, nm (ꢀ):
413 (124 784), 530 (8035), 563 (9971), 607 (11 907), 662
(34 173). 1H NMR (CDCl3) δ: 9.50 (1H, d, J ) 8.7 Hz), 9.20
(1H, s), 8.54 (1H, s), 7.98 (1H, s), 7.90 (1H, d, J ) 8.7 Hz),
7.40-7.15 (20H, m), 6.59 (1H, t, J ) 5.8 Hz), 4.76-4.34 (8H,
m), 4.12 (2H, s), 4.09 (2H, t, J ) 7.5 Hz), 3.97-3.82 (5H, m),
3.82-3.68 (6H, m), 3.66-3.55 (8H, m), 3.51 (2H, q, J ) 7.6
Com p ou n d 41. The title compound was obtained as a deep
green solid in quantitative yield by treating compound 37 with
zinc acetate dihydrate by following the standard procedure.
UV-vis (MeOH) λmax, nm (ꢀ): 425 (91 154), 531 (4652), 573
(5909), 620 (10 373), 675 (48 783). 1H NMR (CDCl3) δ: 9.70
(1H, d, J ) 8.7 Hz), 9.26 (1H, s), 8.84 (1H, s), 7.95 (1H, s),
7.90 (1H, d, J ) 8.7 Hz), 7.45-7.17 (10H, m), 7.16-7.04 (5H,
m), 6.96-6.80 (5H, m), 5.84 (1H, br s), 5.77 (2H, br s), 4.73
(2H, s), 4.53 (1H, d, J ) 11.2 Hz), 4.35 (2H, dd, AB system, J
) 11.9 Hz), 4.23 (1H, m), 4.16 (1H, m), 4.09-3.89 (6H, m),
3.86 (1H, m), 3.75 (1H, m), 3.70-3.43 (11H, m), 3.36 (1H, m),
3.23 (2H, m), 3.11 (1H, m), 3.03 (1H, br s), 2.94 (1H, m), 2.84
(1H, m), 2.71 (2H, m), 2.57 (1H, m), 2.05 (1H, m), 1.91 (3H, t,
J ) 7.6 Hz), 1.77 (3H, t, J ) 7.6 Hz), 1.74 (3H, t, J ) 7.6 Hz),
1.70 (3H, t, J ) 7.5 Hz), 1.67 (3H, t, J ) 7.6 Hz), 1.56 (3H, t,
J ) 7.7 Hz), 1.06 (3H, m), 0.67 (1H, m), 0.24 (3H, t, J ) 7.2
Hz), -0.17 (3H, t, J ) 7.2 Hz). MS (FAB) m/z: 1300.7 (MH+,
100).
Com p ou n d 42. To a solution of 41 (42 mg) in EtOH (15
mL) was added Pd/C (10%, 150 mg). The mixture was stirred
at room temperature under a hydrogen atmosphere for 19 h.
J . Org. Chem, Vol. 69, No. 1, 2004 171

Li et al.
It was then filtered through a pad of Celite and washed with
MeOH/CH2Cl2 (10%). The filtrate was concentrated, and the
residue was purified by preparative silica TLC using MeOH/
CH2Cl2 (15%) as a developing solvent to afford pure 42 (23
mg, 75%) as a deep green solid. UV-vis (MeOH/CH2Cl2, 80%)
70.3, 69.8, 69.6, 68.1, 63.5, 62.4, 38.6, 33.2, 33.1, 31.9, 25.9,
21.5, 20.8, 19.5, 19.4, 19.2, 19.1, 18.5, 17.8, 17.4, 17.3, 16.0,
8.8, 8.5. MS (ESI) m/z: 900.6 (MNa+, 100). MS (FAB) m/z: 878.5
(MH+, 100). HRMS (FAB): calcd for C52H72N5O7 (M + H),
878.5432; found, 878.5430.
λmax, nm (ꢀ): 343 (23 613), 425 (94 829), 531 (4584), 578 (5652),
Deter m in a tion of Ga lectin Bin d in g by ELISA. A 96-
well microtiter plate were coated with asialofetuin (Sigma; 10
µg/mL; 100 µL/well) for 3 h at 37 °C and then washed three
times with phosphate-buffered saline (PBS) containing 0.05%
Tween 20 (PBS-T). The mixture containing equal volumes of
the Gal-1 concentration (Sigma; 1:50-fold dilution in 1% bovine
serum albumin (BSA) in PBS-T) and serially diluted glycoin-
hibitors (21a , 38-40, 42, and lactose), which had been
preincubated for 30 min at room temperature, were added in
duplicates to the wells (100 µL/well). The wells were incubated
for 1 h and then washed three times with PBS-T at 37 °C.
Anti-Gal-1 mouse monoclonal antibody (Novocastra Labora-
tories Ltd., U.K.; 1:100-fold dilution in 1% BSA-PBS-T; 100
µL/well) was added to each well and incubated for 1 h at 37
°C. After three washings with PBS-T, antimouse IgG (γ-chain
specific) alkaline phosphate conjugate (Sigma; 1:1000-fold
dilution in 1% BSA-PBS-T, 100 µL/well) was added to each
well and kept for 1 h at room temperature. Each well was
washed three times with PBS-T, and the substrate 4-nitro-
phenyl phosphate disodium salt hexahydrate (Sigma; 1 mg/
mL dissolved in 10% diethanolamine buffer containing 0.5 mM
magnesium chloride, pH 9.8, 100 µL/well) was added to each
well. After 1 h at room temperature, the plate was read on a
microplate autoreader (EL311s, Bio-Tek Instruments Inc.) at
405 nm. Experiments were repeated at three different time
points, and the data were plotted on the Microsoft Excel 2000
software. The IC50 values for compounds (21a , 38-40, and 42)-
and lactose were determined.
620 (10 676), 674 (48 859). 1H NMR (pyridine-d5) δ: 9.91 (1H,
d, J ) 8.8 Hz), 9.61 (1H, s), 9.14 (1H, s), 8.25 (1H, s), 8.21
(1H, t, J ) 5.7 Hz), 8.07 (1H, d, J ) 8.8 Hz), 6.65 (1H, d, J )
4.7 Hz), 6.43 (1H, d, J ) 5.4 Hz), 6.22 (1H, t, J ) 6.1 Hz), 6.19
(1H, d, J ) 4.5 Hz), 4.73 (1H, m), 4.66 (1H, br s), 4.52 (2H, m),
4.45 (1H, q, J ) 5.7 Hz), 4.42 (2H, s), 4.33 (1H, m), 4.27 (2H,
t, J ) 7.4 Hz), 4.25 (1H, partially overlapped with the signal
at δ 4.27), 3.94 (2H, q, J ) 7.4 Hz), 3.88 (2H, t, J ) 7.4 Hz),
3.81 (2H, q, J ) 7.5 Hz), 3.69 (8H, m), 3.61 (2H, q, J ) 7.6
Hz), 3.01 (2H, m, ABX system), 2.82 (2H, m, ABX system),
2.33-2.03 (3H, m), 1.97 (1H, m), 1.86 (3H, t, J ) 7.4 Hz), 1.80-
1.65 (15H, m), 0.09 (6H, t, J ) 7.2 Hz). MS (ESI) m/z: 962.9
(MNa+, 100).
Com p ou n d 43. 42 (32 mg) was treated with TFA (5 mL)
for 20 min. It was then poured into cold water (30 mL),
extracted with CH2Cl2, washed with water and NaHCO3 (5%),
dried over Na2SO4, filtered, and concentrated. The residue was
purified by preparative silica TLC using MeOH/CH2Cl2 (15%)
as a developing solvent to provide 43 (29 mg, 97%) as a dark
blue solid. Mp: 102-105 °C. UV-vis (MeOH/CH2Cl2, 30%)
λmax, nm (ꢀ): 412 (113 419), 529 (7528), 564 (9592), 606
(11 351), 659 (32 097). 1H NMR (CDCl3) δ: 9.51 (1H, d, J )
8.7 Hz, 1′-H), 9.20 (1H, s, 15-H), 8.56 (1H, s, 20-H), 7.99 (1H,
s, 5-H), 7.94 (1H, d, J ) 8.6 Hz, 2′-H), 6.20 (1H, br s, 9-H),
4.10 (2H, t, J ) 6.1 Hz, 5′-H), 4.02 (2H, splitting s, 7′-H), 3.85
(2H, q, J ) 7.6 Hz, 12-CH2CH3), 3.76 (3H, q, J ) 7.5 Hz, 13-
CH2CH3 and one proton of 4′-H), 3.65 (1H, t, J ) 6.0 Hz, one
proton of 4′-H), 3.63-3.45 (9H, m, 2-, 3-, 17-, and 18-CH2CH3
and 1′′-H), 3.36 (1H, m, 1 × sugar H), 3.21, 2.89, and 2.67 (4H,
4H, and 3H, all m, 5 × sugar H, 2 × 7-CH2CH3 and 10′-H),
2.20 (1H, br s, -NH), 2.01 (1H, br s, -NH), 1.84 (3H, t, J )
7.2 Hz, 12-CH2CH3), 1.74-1.59 (15H, m, 2-, 3-, 13-, 17-, and
18-CH2CH3), 1.07, 0.84, and 0.58 (2H, 1H, and 1H, all m, 11′-H
and 12′-H), 0.06 (3H, t, J ) 7.2 Hz, 7-CH2CH3), -0.09 (3H, t,
Ack n ow led gm en t. This work was supported by the
National Institutes of Health (CA 55791) and the shared
resources of the Roswell Park Cancer Center Support
Grant P30CA16056. The mass spectrometry analyses
were performed at the Biopolymer Facility of our
institute and the Mass Spectrometry facility, Michigan
State University, East Lansing, MI.
1
J ) 7.2 Hz, 7-CH2CH3). H NMR (pyridine-d5) δ: 9.73 (1H, d,
J ) 8.8 Hz), 9.49 (1H, s), 8.87 (1H, s), 8.36 (1H, s), 8.24 (1H,
t, J ) 5.7 Hz), 8.19 (1H, d, J ) 8.8 Hz), 6.65 (1H, br s), 6.42
(1H, br s), 6.22 (1H, br s), 6.19 (1H, br s), 4.73 (1H, m), 4.66
(1H, br s), 4.53 (2H, m), 4.45 (1H, m), 4.43 (2H, s), 4.32 (1H,
dd, J ) 8.8, 3.0 Hz), 4.27 (2H, t, J ) 7.4 Hz), 4.25 (1H, partially
overlapped with the signal at δ 4.27), 3.87 (4H, m), 3.78 (2H,
q, J ) 7.7 Hz), 3.69 (2H, q, J ) 7.4 Hz), 3.64 (2H, q, J ) 7.6
Hz), 3.57 (6H, m), 3.03 (2H, m, ABX system), 2.91 (3H, m),
2.55 (1H, br s), 2.34-2.04 (3H, m), 1.98 (1H, m), 1.80 (3H, t,
J ) 7.4 Hz), 1.76-1.61 (15H, m), 0.19 (6H, t, J ) 7.2 Hz). 13C
NMR (CDCl3) δ: 175.9, 170.3, 157.7, 153.6, 146.9, 145.7, 142.8,
140.8, 140.3, 140.2, 139.7, 139.4, 138.5, 134.5, 130.7, 129.1,
127.1, 125.6, 123.4, 113.9, 106.9, 95.1, 87.3, 74.7, 73.0, 70.4,
Su p p or tin g In for m a tion Ava ila ble: The experimental
details and the characterization (NMR, HRMS, and C, H, and
N analyses) of porphyrin 4, chlorins 5-8, benzochlorins 9-13,
and 27a ; the 1H NMR spectra of compounds 4-13, 17, 19a ,
19b, 20, 21, 23, 24, 26, 27, 27a , 29, 30, 35-37, and 43; 13C
NMR spectra of compounds 6, 7, 9-13, 19b, 24, 27, 27a , 30,
35, 36, and 43; and DEPT-135 NMR spectra of compounds 19b,
27, 27a , 35, and 36. The material is available free of charge
via the Internet at the http://pubs.asc.org.
J O030280B
172 J . Org. Chem., Vol. 69, No. 1, 2004