Synthesis of isoxazoline-linked chlorins and their in vitro cell viabilities

Article abstract of DOI:10.1142/S1088424610002690

A concise synthesis of isoxazoline-linked chlorins is described. This approach is carried out from methyl pyropheophorbide-a as the starting material via 1,3-dipolar cycloaddition of a vinyl group on the periphery with nitrile oxide to give regioselective

Full text of DOI:10.1142/S1088424610002690

Journal of Porphyrins and Phthalocyanines
J. Porphyrins Phthalocyanines 2010; 14: 859–865
DOI: 10.1142/S1088424610002690
Published at http://www.worldscinet.com/jpp/
Synthesis of isoxazoline-linked chlorins and their in vitro
cell viabilities
Jin-Jun Wang*a, Jia-Zhu Lia,b, Yun-Wei Lia,c, Judit Jakusd and Young Key Shimb
a Science and Engineering College of Chemistry and Biology, Yantai University, Yantai 264005, China
b PDT Research Institute, School of Nano Engineering, Inje University, Gimhae 621-749, Korea
c State Key Laboratory Breeding Base of Green Chemistry-synthesis Technology, Zhejiang University of Technology,
Hangzhou 310032, China
d Institute of Biomolecular Chemistry, Chemical Research Center, Hungarian Academy of Sciences,
Budapest 1025, Hungary
Received 11 May 2010
Accepted 30 July 2010
ABSTRACT: A concise synthesis of isoxazoline-linked chlorins is described. This approach is carried
out from methyl pyropheophorbide-a as the starting material via 1,3-dipolar cycloaddition of a vinyl
group on the periphery with nitrile oxide to give regioselective products with excellent yields. This method
represents an extensive and efficient entry into the functionalization of chlorins with a chlorophyll-α
skeleton. Moreover, we have examined a preliminary in vitro effect of these new derivatives on mouse
sarcoma S-180 cell line in photodynamic therapy.
KEYWoRDS: photodynamic therapy, photosensitizer, chlorine, methyl pyropheophorbide-a, isoxazole,
1,3-dipolar cycloaddition, mouse sarcoma S-180 cell.
In porphyrin chemistry, the 1,3-dipolar cycloaddition
INTRoDUCTIoN
reaction has been used to convert porphyrin into chlo-
Modification of the vinyl group at the 3-position in
rin and bacteriochlorin or to establish different chemi-
chlorins with a chlorophyll-α skeleton has been explored
as an important way to develop new photosensitizers for
cal moieties containing heteroatoms on the periphery
of an aromatic macrocycle [6]. Kozyrev et al. reported
photodynamic therapy (PDT) [1]. This double bond at the
the application of 1,3-dipolar cycloaddition with diaz-
chlorin periphery has chemical properties similar to those
omethane to the synthesis of pyrazolinyl- and cyclopro-
of an ethylene moiety linked with an aromatic ring and
pyl-substituted porphyrins and chlorins [7]. Recently, we
undergoes most of the chemical reactions of a vinyl group.
Several novel chlorins have been synthesized by modifying
demonstrated the reaction of methyl pheophorbide-α with
diazomethane and diazoethane [8]. Until now, however,
this vinyl group on the chromophore, such as its conversion
no study has reported on the same type of cycloaddition
to a formyl group by oxidization with osmium(III) tetroxide
to the vinyl group of these chlorins using nitrile oxides as
[2], to 1-bromoethyl by addition [3], to ethyl by reduction
a 1,3-dipolar reagent to give isoxazoline derivatives. We
[4], and to a substituted cyclobutyl or condensed ring moi-
postulated that a variety of functionalized chlorins might
ety by [2+2] or [4+2] cycloaddition [5]. An useful method-
be synthesized from the reaction of chlorins with other
ology for the functionalization of vinyl is the reaction with
1,3-dipolar compounds via a concerted cis-cycloaddition
1,3-dipoles. To test this hypothesis, we synthesized isox-
azoline-substituted chlorin by reacting methyl pyropheo-
mechanism. An olefinic moiety can react with 1,3-dipoles,
phorbide-a (MPPa) with nitrile oxides. In this study,
such as diazoalkanes, nitrile oxide, and azide to construct
novel isoxazoline-linked chlorins, a promising versatile
various five-membered heterocyclic structures.
starting material for further functionalization by chemi-
cal transformations, were synthesized via the 1,3-dipolar
cycloaddition reaction of chlorins with nitrile oxide.
SPP full member in good standing
We also carried out an in vitro assay of cell viability on
*Correspondence to: Jin-Jun Wang, email: wjj1955@163.com
Copyright © 2010 World Scientific Publishing Company

860
J.-J. Wang et al.
2'
2'
N
N
O 1'
1'
3'
4'
O
5'
3'
H
H
4'
5'
N
OH
NH
N
N
NH
N
N
+
NBS/N(Et)3
HN
HN
O
O
4
3
O
O
OCH3
OCH3
NH
N
N
NH
N
N
AcOH/reflux
Spirulina maxima
HN
HN
O
O
O
O
O
OCH3
1 MPa
2 MPPa
OCH3
OCH3
N
OH
O2N
O2N
O2N
O2N
NBS.N(Et)3
N
N
N
O
O
O
H
H
H
HO
NH
N
HN
NH
N
N
NH
N
+
+
N
HN
N
HN
O
O
NO2
O
N
O
O
O
OCH3
7
5
OCH3
6
OCH3
Scheme 1.
mouse sarcoma S-180 cell line with the aim of examining
preliminary PDT activities of these new derivatives.
thin layer chromatography (TLC). Under these condi-
tions, 3-[5′(R/S)-isoxazolinyl]-substituted chlorins were
obtained in excellent yields. The reaction results depended
on the choice of different nitrile oxide as 1,3-dipoles in
this cycloaddition. During the course of these investiga-
tions, we observed that treatment of MPPa 2 with phenyl
aldoxime under the same reaction conditions generated
chlorin 3 (68%) as the major adduct and chlorin 4 (3%)
as a trace amount of the constitutional isomer. The for-
mation and distribution of the product in these reactions
indicated that the 1,3-dipolar cycloaddition reaction of
chlorin derivative 2 with nitrile oxide at the 3-vinyl group
is particularly regioselective. We also attempted to use
p-nitrophenyl aldoxime as a precursor to nitrile oxide
in this 1,3-dipolar cycloaddition of MPPa 2 to afford a
mixture including standard adduct 5 (12%), the double
cycloaddition product 6 (9%), and the cycloaddition-ring
opening product 7 (58%). Compared to the 1,3-polar
cycloaddition of MPa 1 with p-nitrobenzonitrile oxide,
RESULTS AND DISCUSSIoN
Synthesis and characterization
Methyl pheophorbide-a (1, MPa) as dipolarphile
was extracted from the alga Spirulina maxima [9] and
converted into methyl pyropheophorbide-α (2, MPPa)
by thermal demethoxycarbonylation in refluxing ace-
tic acid. The corresponding hydroximoyl bromides
were first obtained by treating aromatic aldoximes with
N-bromosuccinimide (NBS) in an equimolar ratio in tet-
rahydrofuran at room temperature. Excess MPPa 2 was
added to these precursors before generating the nitrile
oxide by the slow addition of triethylamine over a period
of 15 min. Completion of the reaction was assessed using
Copyright © 2010 World Scientific Publishing Company
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SyntheSIS Of ISOxazOlIne-lInkeD ChlOrInS anD theIr in vitro Cell vIabIlItIeS
861
Slimy substance
N
N
OH
R
NBS/N(Et)3
N
N
N
O
O
H
H
R
NH
N
NH
N
NH
N
N
OH
N
OH
N
N
HN
N
HN
N
HN
NBS/N(Et)3
NBS/N(Et)3
O
O
O
8
9
2
O
O
O
a: R=4-CH3; b: R=4-Cl
c: R=4-OCH3; d: R=2-NO2
OCH3
OCH3
OCH3
Scheme 2.
this result showed that the 4-nitro group of benzonitrile
oxide has an important role in this reaction and prompted
us to study the 1,3-polar cycloaddition of chlorin deriva-
tives using another nitrile oxide linked with different
groups.
the high yield. However, when the reaction was carried
out using 4-pyridinyl aldoxime as a more active dipolar
precursor, an inseparable slimy mixture was obtained.
The higher yield and short reaction time demonstrated
that the electron withdrawal of the substituted group
had a positive effect on the cycloaddition. As expected,
the reactions involving the peripheral functional groups
of chlorin depended on the character of the substituted
group linked with the aromatic aldoximes: the stronger
the electron withdrawal by the aryl group linked to the
nitrile oxide, the greater the reactivity. The position of the
substituted groups on the aromatic aldoxime, in addition
to their character, also influenced the cycloaddition (e.g.
entry 7).
The possible formation process for 6 and 7 is out-
lined in Fig. 1. The 1,3-dipolar cycloaddition at 3-vinyl
in the congeneric reaction of chlorin 5a to the enolic
double bond in the exocyclic ring afforded chlorin 6 via
the dehydration of intermediate A under basic condi-
tions. As a reasonable explanation for the appearance of
chlorin 7, we postulated that the SN2 substitution attack
at the 4-position of the isoxazolinyl group on chlorin
5 by a hydroxide ion resulted in ring-opening to form
anionic intermediate B, which has an extensive delocal-
ized region. Lastly, hydroxyliminoyl-substituted chlorin
7 was generated from this anionic intermediate via the
seizure of a proton.
We explored the 1,3-dipolar cycloaddition of MPPa 2
with other nitrile oxide. The results of several attempted
cyclizations with various substituted benzonitrile oxides
to optimize the conditions are listed in Table 1. The
reactions with various aromatic aldoximes were moni-
tored until 2 had disappeared on TLC. With bases such
as K2CO3, NaOH, and NaOMe, the yield of adduct was
lower than that when Et3N was used. With 4- or 2-sub-
stituted phenyl aldoxime or 2-pyridinyl aldoxime, chlo-
rins 8 and 9a–d were formed with yields of 48% to 72%
at different times without any identifiable side products
[10]. The reaction results of chlorin with these 1,3-polar
cycloadditions were definitely related to the polarity of
the dipolar compounds. Surprisingly, the 1,3-dipolar
cycloaddition of MPPa 2 with active hydroximoyl bro-
mides, prepared from p-nitrophenyl aldoxime, can occur
in two regions. Chlorin 5 was formed by formal cycload-
dition with an ethylenic linkage at the 3-position in the
yields of 38%, and chlorin 6 was generated by cycload-
dition with an enolic double bond in the exocyclic ring
with relatively low yields. The cycloaddition-ring open-
ing product 7 derived from chlorin 5 was obtained in
Table 1. 1,3-cycloaddition between 1 and aromatic aldoxime as the precursor for aromatic nitrile oxide
Entry
Chlorin/aromatic aldoxime
Compounds
Time, h
Yield, %
1
2
3
4
5
6
7
MPPa 2/PhCH=NOH
MPPa 2/4-NO2PhCH=NOH
MPPa 2/2-PyrCH=NOH
3, 4
5, 6, 7
8
12
12
18
24
24
24
48
71
79
70
48
72
57
44
MPPa 2/4-CH3PhCH=NOH
MPPa 2/4-ClPhCH=NOH
MPPa 2/4-OCH3PhCH=NOH
MPPa 2/2-NO2PhCH=NOH
9a
9b
9c
9d
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J.-J. Wang et al.
O
N
O
O
O-
N
O
O
O
N
N
O
S-H
O
N
N
O
N
O
N
O
OH
N
-OH
OH
NH
N
NH
N
NH
N
N
NH
N
N
NO2
-H2O
7
6
NBS/N(Et)3
N
HN
N
HN
HN
HN
OH
H
O
OH
O
O
N
-OH
OCH3
O
O
O
O
NO2
5a
5
A
B
OCH3
OCH3
OCH3
Fig. 1. Possible mechanism of the production of compounds 6 and 7
All standard adducts comprised a pair of epimers and
with nitrile oxide. The C3a-H triplet appeared at δ = 7.00,
and mutual coupled two gem-protons of hydroxymethyl
group linked in 3a-position showed signals with large
coupling constant (J = 10.5 Hz) at δ = 4.59 and 4.35 ppm,
respectively.
their certain chemical shifts appeared in pairs in equal
1
intensities. The H NMR analyses of 3 and 4 clearly
indicate they were a pair of regioisomers. The chemical
shifts of the protons, linked in C3-position, were found at
δ = 7.00 and 6.85(6.86) ppm, while the two gem-carbon
protons of C3-heterocyclic ring appeared at δ = 3.95,
4.15 and 5.02, 5.34 ppm, respectively. These oppositely
shifted absorption peaks of isoxazsline rings showed
their attaching positions to the periphery of chlorins. In
In vitro photosensitizing efficacy
In the present study, viability of the cell using the meth-
odology reported by Ahn et al. [11] was determined by a
comparison with that of mTHPC and MPPa for new pho-
tosensitizers on mouse sarcoma S-180 cell line at 0.01,
0.1, 1 and 10 μM after PDT (Fig. 2). For PDT treatment,
compounds 7 and 8 among the tested photosensitizers
showed 20.3% and 21.7% cell viability at 0.1μM after
PDT, respectively. With an increase in the concentration of
1
the H NMR spectrum of chlorin 6 the chemical shifts
of the gem-protons at 132-position were not discovered
and the eight AB-type phenyl group signals appeared at
δ = 8.25, 8.13, 8.06, 7.13 ppm, each of which was ascrib-
able to two protons on symmetrical position of p-substi-
1
tuted phenyl group. These H NMR data demonstrated
that the 1,3-dipolar cycloaddition of
chlorin 6 with nitrile oxide formed
isoxazol ring fused with the exocy-
clic E-ring. Compared with the Qy
band (664nm) of normal adduct
5, the Qy absorption of 6 shifted
to 678 nm due to the expansion
of π-system by combining with
the conjugated heterocyclic ring.
1
The H NMR spectra of chlorin 7
as a pair of regioisomers showed
two C132-methylene signals with
large coupling constant (δ = 5.26
and 5.13 ppm, J = 19.9 Hz) to indi-
cate that the original exocyclic ring
was still intact, but newly linked
p-nitrobenzaldehyde oxime moiety
showed two sets of AB-type phenyl
peaks at δ = 8.06, 7.64 ppm and a
1H singlet [8.38(8.46) ppm] corre-
sponding to the proton attached to
C=N of the aldehyde oxime, which
reflected C3-vinyl group reacted
Fig. 2. Cell viability results of photosensitizers on mouse sarcoma S-180 cell line. Com-
pounds were tested at 0.01, 0.1, 1 and 10 μM concentrations in triplicate. mTHPC was
used as a reference compound. Mouse sarcoma S-180 cell line at 80% confluency in
96-well plates was used for in vitro PDT tests. Cells were illuminated with a lamp in a
wavelength range of 640–700 nm, with a peak at 660 nm, for 20 min. Total light dose
was 8.4 J. Statistical analyses were performed using unpaired Student’s t test
Copyright © 2010 World Scientific Publishing Company
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SyntheSIS Of ISOxazOlIne-lInkeD ChlOrInS anD theIr in vitro Cell vIabIlItIeS
863
Table 2. IC50 results of photosensitizers on mouse sarcoma
S-180 cell line
Chemical shifts are in δ values versus the internal stan-
dard tetramethylsilane and J values in Hz. The IR spectra
were measured with a Shimadzu FTIR 8300 instrument.
Elemental analysis were performed on a Perkin Elmer
240-C microanalyzer. All chemical reagents were com-
mercially available and purified with standard methods
before use. Methyl pyropheophorbide-a 1 was obtained
according to the method used by Smith [9]. All reactions
were monitored by thinlayer chromatography (TLC)
using Merck Silica gel 60 F254 precoated (0.2 mm
thickness) glass-backed sheets. Silica gel 60 (70–230 or
230–400 mesh, Merck) and neutral alumina (~150 mesh,
58 A0) were used for column chromatography. The alu-
mina was deactivated with 10% water (Brockmann Grade
IV) before use.
Compound mTHPC
3
4
5
6
7
IC50 (µM)
Compound
IC50 (µM)
0.064
8
0.098 0.091 0.495 0.601 0.067
9a 9b 9c 9d MPPa
0.096 0.099 0.345 0.085 6.812
0.056
the photosensitizer, the cell viability revealed decreasing
results, for example, the cell viability for compounds 7 and
8 was 20.3% and 21.7% at 0.1μM and 7.0% and 2.7% at
1μM after PDT, respectively. As for reference compound
mTHPC, it showed a promising effect after PDT. From
the experimental results, we observed that all compounds
showed an improved effect for cell death or cell viability as
the concentration of the photosensitizer increased. And all
compounds obtained showed better effect than that of MPPa.
Among all the compounds we obtained, compounds 7 and 8
showed high effects when compared with the others.
General procedure for the synthesis of isoxazoline-
linked chlorins
For each reaction, we prepared 3-[5′(R/S)-(3′-aryl-
isoxazolinyl]-substituted chlorin. The N-bromosuccin-
imide (NBS) (1.3 mmol) and the corresponding aromatic
aldoxime (1.5mmol) were dissolved in dry dichlo-
romethane (2 mL) in a 50-mL two-necked round-bottom
flask equipped with a magnetic stirrer and stirred for
20 min to finish the bromination. MPPa 2 (0.4 mmol) was
added to this mixture, and then triethylamine (1.5 mmol)
in 2 mL of dry dichloromethane was added slowly drop-
wise under nitrogen at room temperature over 15min.
The resulting mixture was stirred under nitrogen at the
same temperature and monitored using TLC. The reac-
tion mixture was partitioned with dichloromethane and
water. The obtained organic phase was washed with 1 N
HCl solution and brine, and dried over Na2SO4. The crude
products were purified by short column chromatography
over silica gel using n-hexane and ethyl acetate (6:1-2:1)
as the eluent.
Table 2 shows the IC50 values of these new photosensi-
tizers on mouse sarcoma S-180 cell line after PDT. The ref-
erence compound mTHPC showed a promising effect after
PDT (IC50 = 0.064μM), another reference compound MPPa
showed a relative low effect after PDT (IC50 = 6.812μM),
compounds 7 and 8 among the tested photosensitiz-
ers showed relatively high PDT effect (IC50 = 0.067 and
0.056 μM, respectively). compounds 8 was even better
than the reference compound mTHPC. While the double
cycloaddition product 6 showed relatively low PDT effect
(IC50 = 0.601μM) among the tested photosensitizers,
which probably due to its relatively higher lipophilic value
than other 3-[5′(R/S)-isoxazolinyl]-substituted products.
Furthermore, we are aiming to explore, in greater depth,
the other biological effects of these compounds for PDT.
In conclusion, we developed a new synthesis of isox-
azoline-linked chlorins from methyl pheophorbide-a via
cyclization with various aromatic nitrile oxides. The
synthetic methodology utilizing the 1,3-cycloaddition
of vinyl-substituted chlorins lays the groundwork for
important peripheral functionalization of the tetrapyrrolic
ring system. This reaction conveniently constructed five-
membered heterocycle moieties on the macrocycle, and
these modifications to the parent ring of chlorin deriva-
tives may be useful for generating novel new photosen-
sitizers for PDT. Among all the compounds we obtained,
compounds 7 and 8 showed relatively high PDT effects
(IC50 = 0.067 and 0.056 μM, respectively). Compound
8 (IC50 = 0.056 μM) was even better than the reference
compound mTHPC (IC50 = 0.064 μM).
3. Yield: 68%. UV-vis (CHCl3): λmax, nm (ε × 105) 410
(1.31), 505 (0.14), 536 (0.13), 608 (0.12), 666 (0.68). 1H
NMR (400 MHz, CDCl3): δ, ppm -1.95, 0.30 (each br s,
2H, NH), 1.57 (t, J = 7.4 Hz, 8b-CH3), 1.79 (1.78) (3H, d,
J = 7.0 Hz, 18-CH3), 2.14~2.29, 2.48~2.67 (each m, 4H,
17a + 17b-H), 3.44 (q, J = 7.4 Hz, 8a-CH2), 2.91 (2.90),
3.35, 3.46 (3.44), 3.59 (each s, 12H, CH3 + OCH3), 3.95
(dd, J = 9.8, 11.7 Hz, 1H, 4′-H), 4.15 (td, J = 4.6, 11.7
Hz, 1H, 4′-H), 4.22 (d, J = 8.7 Hz, 1H, 18-H), 4.43 (q,
J = 7.3 Hz, 1H, 17-H), 5.03 (d, J = 19.8 Hz, 1H, 132-H),
5.17 (dd, J = 2.1, 19.8 Hz, 1H, 132-H), 7.00 (t, J = 10.4
Hz, 1H, 5′-H), 7.42~7.48 (m, 3H, Ph-H), 7.81~7.87 (m,
2H, Ph-H), 8.54, 9.19 (9.17), 9.25 (9.21) (each s, each
1H, meso-H). IR (KBr): ν, cm-1 2958~2856 (C-H),
1735 (C=O), 1691 (C=N), 1620 (C=C). Anal. calcd. for
C41H41N5O4: C 73.74; H 6.19; N 10.49. Found: C 73.70;
H 6.25; N 10.55.
EXPERIMENTAL
4. Yield: 3%. UV-vis (CHCl3): λmax, nm (ε × 105) 411
(1.31), 506 (0.14), 537 (0.13), 608 (0.10), 665 (0.68).
1H NMR (400 MHz, CDCl3): δ, ppm -1.75, 0.40 (each
br s, 2H, NH), 1.66 (t, J = 7.7 Hz, 8b-CH3), 1.81 (1.80)
General
1
The H NMR spectra were recorded on a Varian-
400 MHz spectro-meter in solvent CDCl3 at 400 MHz.
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J.-J. Wang et al.
(d, J = 7.2 Hz, 18-CH3), 2.22~2.38, 2.51~2.74 (each m,
4H, 17a + 17b-H), 3.63 (q, J = 7.7 Hz, 2H, 8a-CH2), 2.96
(2.95), 3.39, 3.61 (3.60), 3.66 (each s, 12H, OCH3 +
CH3), 4.45-4.53 (m, 17-H + 18-H), 5.02 (td, J = 9.9, 3.1
Hz, 1H, 4′-H), 5.11 (d, J = 19.8 Hz, 1H, 132-H), 5.27 (d,
J = 19.8 Hz, 1H, 132-H), 5.34 (t, J = 9.9 Hz, 1H, 4′-H),
6.86 (6.85) (t, J = 10.7 Hz, 5′-H), 7.51~7.68 (m, 3H,
Ph-H), 8.32 (dd, J = 8.4, 1.3 Hz, 2H, Ph-H), 8.55, 9.37,
9.49 (9.48) (each s, each 1H, meso-H). IR (KBr): ν, cm-1
2962~2875 (C-H), 1730 (C=C), 1689 (C=N), 1627
(C=C). Anal. calcd. for C41H41N5O4: C 73.74; H 6.19; N
10.49. Found: C 73.53; H 6.31; N 10.66.
ν, cm-1 2968~2866 (C-H), 1728 (C=O), 1689 (C=N),
1624 (C=C). Anal. calcd. for C41H42N6O7: C, 67.38; H,
5.79; N, 11.50. Found: C, 67.45; H, 5.89; N, 11.54.
8. Yield: 70%. UV-vis (CHCl3): λmax, nm (ε × 105) 410
(1.31), 506 (0.13), 536 (0.12), 608 (0.09), 666 (0.65).
1H NMR (CDCl3): δ, ppm -1.80, 0.32, (each br s, 2H,
NH), 1.65(t, J = 7.7 Hz, 8b-CH3), 1.81 (d, J = 6.9 Hz,
3H, 18-CH3), 2.00~2.12, 2.24~2.38, 2.52~2.74 (each m,
4H, 17a + 17b-H), 3.04, 3.43, 3.61, 3.65 (each s, each
3H, OCH3 + CH3), 3.62 (q, 2H, J = 7.7 Hz, 8a-CH2),
4.14~4.26 (m, 1H, 18-H), 4.30 (d, J = 9.1 Hz, 1H, 17-H),
4.40, 4.51 (each d, 2H, J = 12.0 Hz, 4′-H), 5.12, 5.27
(each d, 2H, J = 19.9 Hz, 132-H), 7.20 (t, 1H, J = 12.0 Hz,
5′-H), 7.36~7.44 (m, 1H, Pyr-H), 7.90 (t, 1H, J = 7.9 Hz,
Pyr-H), 8.39 (d, 1H, J = 7.9 Hz, Pyr-H), 8.64~8.67 (m,
1H, Pyr-H), 8.59, 9.38, 9.47 (each s, each 1H, meso-H).
IR (KBr): ν, cm-1 2980~2893 (C-H), 1740 (C=O), 1699
(C=N), 1621 (C=C).Anal. calcd. for C40H40N6O4: C 71.84;
H 6.03; N 12.57. Found: C 71.90; H 6.08; N 12.71.
9a. Yield: 48%. UV-vis (CHCl3): λmax, nm (ε × 105)
411 (1.32), 506 (0.12), 536 (0.12), 607 (0.11), 665
(0.70). 1H NMR (CDCl3): δ, ppm -1.83, 0.28 (each br s,
2H, NH), 1.61 (t, J = 7.5 Hz, 8b-CH3), 1.81 (d, J = 7.2
Hz, 18-CH3), 1.98~2.12, 2.19~2.34, 2.47~2.72 (each
m, 4H, 17a + 17b-H), 2.43 (s, 3H, Ph-CH3), 3.58 (q,
J = 7.5 Hz, 2H, 8a-CH2), 3.01, 3.40, 3.59, 3.60 (each s,
each 3H, OCH3 + CH3), 4.02 (dd, J = 10.0, 7.0 Hz, 1H,
4′-H), 4.21~4.32 (m, 2H, 18-H + 4′-H), 5.09 (d, J = 19.8
Hz, 1H, 132-H), 5.25 (d, J = 19.8 Hz, 1H, 132-H), 7.08
(t, J = 11.8 Hz, 5′-H), 7.29 (d, J = 7.4 Hz, 2H, Ph-H),
7.78 (d, J = 7.4 Hz, 2H, Ph-H), 8.56, 9.35 (9.34), 9.39
(9.38) (meso-H). IR (KBr): ν, cm-1 2980~2890 (C-H),
1741 (C=O), 1699 (C=N), 1620 (C=C). Anal. calcd. for
C42H43N5O4: C 73.99; H 6.36; N 10.27. Found: C 74.05;
H 6.41; N 10.30.
5. Yield: 12%. UV-vis (CHCl3): λmax, nm (ε × 105) 410
(1.30), 506 (0.13), 536 (0.12), 608 (0.09), 664 (0.66). 1H
NMR (CDCl3): δ, ppm -1.86, 0.15 (each br s, 2H, NH),
1.65 (t, J = 7.5 Hz, 8b-CH3), 1.82 (d, J = 7.3 Hz, 3H,
18-CH3), 2.24~2.36, 2.54~2.64, 2.66~2.77 (each m, 4H,
17a + 17b-H), 3.03, 3.44, 3.61, 3.66 (each s, each 3H,
OCH3 + CH3), 3.63 (q, 2H, J = 7.7 Hz, 8a-CH2), 4.09
(dd, 1H, J = 17.1, 10.5 Hz, 4′-H), 4.32 (d, 17.1, 4′-H),
4.27~4.35 (m, 1H, 17-H), 4.51 (q, J = 7.3 Hz, 1H, 18-H),
5.13, 5.27 (each d, 2H, J = 19.9 Hz, 132-H), 7.24 (t, 1H,
J = 10.5 Hz, 5′-H), 8.05 (dd, 1H, J = 8.8, 2.9 2H, Ph-H),
8.35 (dd, 1H, J = 8.8, 3.2 Hz, 2H, Ph-H), 8.61, 9.31, 9.51
(each s, each 1H, meso-H). IR (KBr): ν, cm-1 2945~2880
(C-H), 1738 (C=C), 1702 (C=N), 1620 (C=C). Anal.
calcd. for C41H40N6O6: C 69.09; H 5.66; N 11.79. Found:
C 68.98; H 5.73; N 11.90.
6. Yield: 9%. UV-vis (CHCl3): λmax, nm (ε × 105) 414
(1.31), 518 (0.10), 550 (0.17), 618 (0.05), 678 (0.67). 1H
NMR (CDCl3): δ, ppm -1.96, -1.44 (each br s, 2H, NH),
1.73 (t, J = 7.8 Hz, 8b-CH3), 1.80 (d, J = 7.2 Hz, 18-CH3),
2.12~2.24, 2.46~2.62 (each m, 4H, 17a + 17b-H), 3.73
(q, J = 7.5 Hz, 2H, 8a-CH2), 3.35, 3.58 (3.57), 3.68, 3.74
(each s, each 3H, OCH3 + CH3), 4.22~4.38 (m, 2H, 18H +
4′-H), 4.58~4.67 (m, 4′-H), 4.87~4.93 (m, 1H, 17-H),
7.09 (t, J = 6.8 Hz, 5′-H), 7.70 (d, J = 8.5 Hz, 2H, Ph-H),
8.06 (d, J = 8.5 Hz, 2H, Ph-H), 8.13 (d, J = 8.5 Hz, 2H,
Ph-H), 8.25 (d, J = 8.5 Hz, 2H, Ph-H), 8.53, 9.62, 9.90
(9.89) (meso-H). IR (KBr): ν, cm-1 2985~2878 (C-H),
1740 (C=O), 1693 (C=N), 1615 (C=C). Anal. calcd. for
C48H42N8O8: C 67.12; H 4.93; N 13.05. Found: C 67.29;
H 4.98; N 13.10.
9b.Yield: 72%. UV-vis (CHCl3): λmax, nm (ε × 105) 411
(1.31), 507 (0.12), 536 (0.12), 607 (0.11), 665 (0.72). 1H
NMR (CDCl3): δ, ppm -1.85, 0.28 (each br s, 2H, NH),
1.64 (t, J = 7.5 Hz, 8b-CH3), 1.81 (d, J = 7.2 Hz, 18-CH3),
2.21~2.35, 2.50~2.74 (each m, 4H, 17a + 17b-H), 3.59
(q, J = 7.5 Hz, 2H, 8a-CH2), 3.02, 3.41, 3.61, 3.63 (each
s, each 3H, OCH3 + CH3), 4.02 (dd, J = 17.1, 10.0 Hz,
1H, 4′-H), 4.22 (dd, J = 17.1, 12.0 Hz, 1H, 4′-H), 4.30
(t, J = 6.6 Hz, 18-H), 4.49 (q, J = 7.2 Hz, 17-H), 5.11 (d,
J = 19.8 Hz, 1H, 132-H), 5.26 (d, J = 19.8 Hz, 1H, 132-H),
7.13 (t, J = 10.3 Hz, 5′-H), 7.47 (d, J = 8.4 Hz, 2H,
Ph-H), 7.82 (d, J = 8.4 Hz, 2H, Ph-H), 8.58, 9.33, 9.45
(9.46) (meso-H). IR (KBr): ν, cm-1 2981~2890 (C-H),
1744 (C=O), 1695 (C=N), 1621 (C=C). Anal. calcd. for
C41H40ClN5O4: C 70.12; H 5.74; N 9.97. Found: C 70.19;
H 5.78; N 10.06.
7. Yield: 58%. UV-vis (CHCl3): λmax, nm (ε × 105) 410
(1.32), 506 (0.13), 536 (0.12), 608 (0.09), 666 (0.63). 1H
NMR (CDCl3): δ, ppm -1.89, 0.18 (each br s, 2H, NH),
1.70 (t, J = 7.6 Hz, 8b-CH3), 1.81 (d, J = 7.0 Hz, 3H,
18-CH3), 2.21~2.34, 2.48~-2.59, 2.62~2.74 (each m, 4H,
17a + 17b-H), 3.31, 3.52, 3.60, 3.65 (each s, each 3H,
OCH3 + CH3), 3.70 (q, 2H, J = 7.6 Hz, 8a-CH2), 4.31 (d,
J = 10.4 Hz, 3b-H), 4.35 (td, J = 10.5 Hz, 5.4 Hz, 1H,
3b-H), 4.50 (q, J = 7.3 Hz, 1H, 18-H), 4.59 (dd, J = 10.5
Hz, 2.1 Hz, 3b-H), 5.13, 5.26 (each d, 2H, J = 19.9 Hz,
132-H), 7.00 (t, 1H, J = 6.8 Hz, 3a-H), 7.64 (d, 1H, J = 8.8
Hz, 2.1 Hz, 2H, Ph-H), 8.06 (d = d, 1H, J = 8.9 Hz, 1.6
Hz, 2H, Ph-H), 8.48 (8.46) (s, 1H, H-C=CN-OH), 8.63,
9.52 (9.51), 9.57 (each s, each 1H, meso-H). IR (KBr):
9c.Yield: 57%, UV-vis (CHCl3): λmax, nm (ε × 105) 411
(1.31), 506 (0.14), 537 (0.13), 607 (0.11), 665 (0.67). 1H
NMR (CDCl3): δ, ppm -1.82, 0.27 (each br s, 2H, NH),
1.61 (t, J = 7.6 Hz, 8b-CH3), 1.81 (3H, d, J = 6.9 Hz,
18- CH3), 2.22~2.36, 2.51~2.62, 2.63~2.72 (m, 4H, 17a +
17b-H), 3.59 (q, J = 7.6 Hz, 8a-CH2), 3.02, 3.41, 3.58,
Copyright © 2010 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2010; 14: 864–865

SyntheSIS Of ISOxazOlIne-lInkeD ChlOrInS anD theIr in vitro Cell vIabIlItIeS
865
3.62 (each 3H, CH3 + OCH3), 3.87 (s, 3H, PhOCH3),
after subtracting medium only control absorbance. Each
data point represents the mean from three separate exper-
iments with six replicates at each dose, and the standard
errors were less than 10%.
4.02 (1H, dd, J = 12.0, 17.0 Hz, 4′-H), 4.25 (1H, dd, J =
9.9, 17.0 Hz, 4′-H), 4.28 (d, 1H, J = 8.9 Hz, 18-H), 4.48
(q, 1H, J = 6.5 Hz, 17-H), 5.10, 5.26 (each d, each 1H,
J = 19.8 Hz, 132-H), 6.84~6.92 (m, 1H, 5′-H), 7.01 (d,
2H, J = 8.4 Hz, Ph-H), 7.83 (d, 2H, J = 8.4 Hz, Ph-H),
8.57, 9.36 (9.35), 9.43 (9.42) (meso-H). IR (KBr): ν,
cm-1 2977~2889 (C-H), 1740 (C=O), 1700 (C=N), 1616
(C=C). Anal. calcd. for C42H43N5O5: C 72.29; H 6.21; N
10.04. Found: C 72.35; H 6.28; N 10.11.
Acknowledgements
This work was supported by the open project of state
key laboratory breeding base of green chemistry-synthesis
technology (Zhejiang University of Technology) and
the natural science foundation of Shandong Province of
China (No. Y2008B49) and BK21.
9d. Yield: 44%. UV-vis (CHCl3): λmax, nm (ε × 105)
411 (1.31), 506 (0.12), 537 (0.11), 609 (0.09), 666 (0.69).
1H NMR (CDCl3): δ, ppm -1.85, 0.23 (br s, 2H, NH), 1.71
(t, J = 7.5 Hz, 8b-CH3), 1.81 (3H, d, J = 7.3 Hz, 18-CH3),
2.21~2.34, 2.51~2.61, 2.64~2.75 (each m, 4H, 17a +
17b-H), 3.74 (q, J = 7.5 Hz, 8a-CH2), 3.31, 3.51, 359, 3.68
(each s, each 3H, CH3 + OCH3), 4.22 (d, J = 7.5 Hz, 1H,
18-H), 4.34 (dt, J = 10.6, 3.2 Hz, 1H, 4′-H), 4.50 (q, J =
7.3 Hz, 1H, 17-H), 4.56 (dd, J = 10.6, 7.4 Hz, 4′-H), 5.13
(d, J = 19.8 Hz, 1H, 132-H), 5.28 (dd, J = 2.1, 19.8 Hz,
1H, 132-H), 7.12 (m, J = 10.4 Hz, 1H, 5′-H), 7.41~7.47
(m, 2H, Ph-H), 7.69 (d, 1H, J = 6.9 Hz, Ph-H), 7.98 (d,
1H, J = 7.4 Hz, Ph-H), 8.62, 9.08 (9.07), 9.56 (meso-H).
IR (KBr): ν, cm-1 2950~2882 (C-H), 1742 (C=O), 1696
(C=N), 1618 (C=C).Anal. calcd. for C41H40N6O6: C 69.09;
H 5.66; N 11.79. Found: C 69.14; H 5.73; N 11.82.
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Copyright © 2010 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2010; 14: 865–865