Synthesis, Comparative Photosensitizing Efficacy, Human Serum Albumin (Site II) Binding Ability, and Intracellular Localization Characteristics of Novel Benzobacteriochlorins Derived from vic-Dihydroxybacteriochlorins

Article abstract of DOI:10.1021/jm030341y

In a sequence of reactions, methyl mesopyropheophorbide a, mesochlorin e₆ trimethyl ester, mesochlorin p₆ trimethyl ester, mesopurpurin-18-N-hexylimide methyl ester, and mesopurpurin-18-N-3,5-bis(trifluoromethyl)benzylimide methyl es

Full text of DOI:10.1021/jm030341y

J . Med. Chem. 2003, 46, 5349-5359
5349
Syn th esis, Com p a r a tive P h otosen sitizin g Effica cy, Hu m a n Ser u m Albu m in (Site
II) Bin d in g Ability, a n d In tr a cellu la r Loca liza tion Ch a r a cter istics of Novel
Ben zoba cter ioch lor in s Der ived fr om vic-Dih yd r oxyba cter ioch lor in s
Guolin Li,† Andrew Graham,‡ Yihui Chen,† Mahabeer P. Dobhal,†,| J anet Morgan,‡ Gang Zheng,†
Andrei Kozyrev,† Allan Oseroff,‡ Thomas J . Dougherty,† and Ravindra K. Pandey†,§,
*
Photodynamic Therapy Center, Department of Dermatology, Department of Nuclear Medicine and Radiology,
Roswell Park Cancer Institute, Buffalo, New York 14263
Received J uly 14, 2003
In a sequence of reactions, methyl mesopyropheophorbide a, mesochlorin e6 trimethyl ester,
mesochlorin p6 trimethyl ester, mesopurpurin-18-N-hexylimide methyl ester, and mesopurpurin-
18-N-3,5-bis(trifluoromethyl)benzylimide methyl ester were synthesized from chlorophyll-a.
These chlorins on reacting with osmium tetraoxide produced the corresponding vic-dihydroxy-
bacteriochlorins. The 8-vinylchlorins obtained by refluxing the related vic-dihydroxybacterio-
chlorins in o-dichlorobenzene were individually treated with dimethylacetylenedicarboxylate
(DMAD) under Diels-Alder reaction conditions. The intermediate adducts on 1,8-diazabicyclo-
[5.4.0]undec-7-ene (DBU) treatment rearranged to the corresponding stable benzobacterio-
chlorins, exhibiting the longest wavelength absorption in the range of 737 to 805 nm. In
preliminary in vitro (RIF tumor cells) and in vivo screening (C3H/HeJ mice bearing RIF tumors),
some of these compounds were found to be quite effective. Under similar treatment conditions
(drug dose: 5.0 µmol/kg; light dose: 135 J /cm2, tumors were exposed to light for 30 min at 24
h postinjection), the benzobacteriochlorins containing N-substituted-imide ring system produced
enhanced photosensitizing efficacy with limited skin phototoxicity. These compounds were also
found to bind to site II of human serum albumin (HSA). However, no correlation between the
binding constant values and photosensitizing efficacy was observed. A competitive intracellular
localization study of these novel structures with Rhodamine-123 (a mitochondrial probe)
indicated their preferential localization in mitochondria, without producing any specific
3
3
displacement of H-PK11195 (PBR probe, H-labeled 1-(2-chlorophenyl)-N-methyl-N-(1-meth-
ylpropyl)-3-isoquinoline carboxamide). These results suggest that the mitochondrial peripheral
benzodiazepine receptor (PBR) is not the cellular binding site for this class of compounds.
In tr od u ction
sensitizers that show the ability to localize at the tumor
site in high concentrations and may be able to treat
large tumors.
Among tetrapyrrolic systems, chlorins and bacterio-
chlorins have been proposed as potentially useful can-
didates for the use in photodynamic therapy (PDT)
where strong absorption in the visible or near-IR region
of the spectrum can be used to photoactivate dyes
previously located in targeted (neoplastic) tissues.1
Photoactivations of compounds at long-wavelength ab-
sorption (700-800 nm) may be able to treat larger
tumors. Some naturally occurring bacteriochlorins have
previously been reported as effective photosensitizers
both in vitro and in vivo.2 However, most of them are
extremely sensitive to oxidation, which results in rapid
transformation to the chlorin state (λmax 640 nm).2
Furthermore, if a laser is used to excite the bacterio-
chlorin in vivo, oxidation may result in the formation
of a new chromophore absorbing outside the laser
window, thus reducing the photodynamic efficacy. To
render PDT more applicable to tumor therapy, there is
need for long wavelength absorbing and stable photo-
In recent years, several approaches have been quite
successful for preparing stable bacteriochlorins exhibit-
ing long wavelength absorption near-IR region. The first
approach describes the in situ conversion of bacterio-
chlorophyll-a into bacteriopurpurin-18 methyl ester,3
which under appropriate reaction conditions can be
converted into a highly stable (both in vitro and in vivo)
bacteriopurpurinimide system.4 The second approach is
to prepare a series of metallobacteriochlorophylls in
which the central magnesium metal is replaced with
other diamagnetic metals.5 Some of the compounds in
these two series are reported to be effective photosen-
sitizers.4,5 The third approach deals with the utility of
Diels-Alder reaction for the preparation of stable
bacteriochlorins. For example, it has been shown that
pyrrole units containing vinyl groups at the diagonal
positions of the porphyrin molecule on subjecting to
double Diels-Alder reaction could be converted into
novel bacteriochlorins.6,7 These compounds exhibit long-
wavelength absorption near 800 nm but produced
limited PDT efficacy in mice implanted with RIF
tumors. For developing a general synthesis of stable
bacteriochlorins, Morgan et al.8 converted the octaeth-
* To whom correspondence should be addressed. Phone: 716-845-
3203. Fax: 716-845-8920. E-mail: ravindra.pandey@roswellpark.org.
† Photodynamic Therapy Center.
‡ Department of Dermatology.
§ Department of Nuclear Medicine and Radiology.
| On leave from the Department of Chemistry, University of Raj-
asthan, J aipur 302004, India.
10.1021/jm030341y CCC: $25.00 © 2003 American Chemical Society
Published on Web 11/01/2003

5350 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
Li et al.
ylporphyrins to the corresponding ketochlorins by fol-
lowing the pinacol-pinacolone approach, which in a
sequence of reaction was converted into the correspond-
ing vinylchlorin as a mixture of two positional isomers.
These isomers were individually reacted with DMAD,
and the corresponding bacteriochlorins were isolated in
good yields with long wavelength absorption near 700
nm. The spectroscopic properties of these compounds
resemble those of porphyrinones rather than bacterio-
chlorins. In preliminary in vivo screening these com-
pounds produced limited in vivo efficacy. Recently
Cavaleiro et al.9 have shown that tetraphenylporphyrin
(TPP) can be converted into a mixture of pyrrolidine-
fused chlorin, isobacteriochlorin, and bacteriochlorin on
refluxing with N-methylglycine and paraformaldehyde.
This procedure, however, produced the desired bacte-
riochlorin (λmax 732 nm) as a minor product. The same
authors10 also reported the synthesis of glycoconjugated
isoxazolidine-fused bacteriochlorins by heating the mix-
ture of porphyrin with an excess of sugar nitrone, and
the resulting bacteriochlorin was isolated in low yield
(about 9%). Bonnet et al.11 extended diimide approach
that had been used for the preparation of m-THPC (m-
tetrahydroxyphenylporphyrin), for converting the meso-
substituted porphyrins into the corresponding bacterio-
chlorins.
Chang et al.12 have previously shown that octaethyl-
chlorin on reacting with osmium tetraoxide produce the
corresponding vic-dihydroxybacteriochlorin system
in excellent yield. Roswell Park and University of
CaliforniasDavis groups together extended this meth-
odology to the pheophorbide a and chlorin e6 series,13
and the resulting bacteriochlorins exhibited strong
absorption in the red region of the electronic spectra
(730-750 nm). Unfortunately, these compounds did not
produce any significant in vivo photosensitizing activ-
ity.14 In our attempt to prepare 8-vinyl chlorins, we
investigated the stability of the vic-dihydroxybacterio-
chlorins under various conditions and the formation of
the corresponding desired product was found to depend
on the reaction conditions used.15-17 We extended the
thermolysis approach using 1,2-dichlorobenzene as a
solvent for the preparation of 8-vinylpurpurin-18 methyl
ester which on reacting with dimethyl acetylenedicar-
boxylate (DMAD) under Diels-Alder reaction conditions
produced benzobacteriopurpurin with long wavelength
absorption at 795 nm.18 Unfortunately, the six-mem-
bered fused anhydride ring system present in this
molecule was found to be unstable in vivo and produced
the corresponding chlorin p6 analogue with a significant
blue shift in its in vivo absorption.19
bis(trifluoromethyl)benzylimide 5b were used as sub-
strates and were prepared by following the literature
procedures.20,21 These compounds were converted into
the corresponding bacteriochlorins 16-20 by following
the reaction sequences depicted in Scheme 1. For
example, methyl mesopyropheophorbide a 1b obtained
from methylpyropheophorbide a 1a was reacted with
OsO4, and the corresponding vic-dihydroxybacteriochlo-
rin 6 was obtained in 65% yield as a mixture of two
isomers (cis-hydroxy groups up or down relative to ring
D). Bacteriochlorin 6 on refluxing in o-dichlorobenzene
for 1.5 h produced 8-vinyl pyropheophorbide a 11 in 54%
yield. The structure of this 8-vinyl product 11 was
confirmed by 2D NMR studies (H-H COSY and ROE-
SY). Reacting 11 with DMAD in refluxing toluene under
inert N2 atmosphere produced the intermediate Diels-
Alder adduct. The intermediate adduct22 was isolated
but not characterized and immediately reacted with 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU) at room temper-
ature to give the desired bacteriochlorin 16 in 36%
converted yield in two steps. Following a similar ap-
proach, mesochlorin e6 trimethyl ester 2b, mesochlorin
p6 trimethyl ester 3b, mesopurpurin-18-N-hexylimide
methyl ester 4b, and mesopurpurin-18-N-3,5-bis(trif-
luoromethyl)benzylimide methyl ester 5b were con-
verted in to the related 8-vinyl analogues 12-15,
respectively. Reaction of these vinyl derivatives with
DMAD as a dienophile produced the corresponding
benzobacteriochlorins 17-20 (Scheme 1). The interme-
diate Diels-Alder adduct 21 (Scheme 2) (before convert-
ing it into benzobacteriochlorin 17) was characterized
1
by H NMR, H-H COSY NMR, and HRMS analyses.
Other Diels-Alder adducts (intermediate products)
after purification were not characterized and immedi-
ately converted into the corresponding benzobacterio-
chlorins 16 and 18-20.
At the thermolysis step, the vic-dihydroxybacterio-
chlorins 6-10 in refluxing o-dichlorobenzene though
mainly produced the desired 8-vinylchlorins 11-15, it
also generated other byproducts in minor quantities.
These products were identified as the related 8-ke-
tochlorins as well as the corresponding symmetrical and/
or unsymmetrical dimers.23 The nature of the carbon-
carbon linkages joining these chromophores in a dimeric
form was found to be dependent on the substituents
present at the peripheral position of the macrocycle.23
Benzobacteriochlorins 16-20 reported here were
obtained as a mixture of diastereomers due to the
formation of two new chiral centers at positions C-7 and
C-84 generated after Diels-Alder reaction/DBU rear-
rangement. The structures of these benzobacteriochlo-
rins were confirmed by 1H NMR and HRMS spectra.
The 1H NMR signal assignment for benzobacteriochlorin
16 was achieved by 2D NMR studies (H-H COSY and
ROESY), and the resonances for the substituents in
other benzobacteriochlorins were assigned by analyzing
The present work describes the synthesis and biologi-
cal evaluation of a series of benzobacteriochlorins
derived from the related 8-vinylchlorins which are stable
in vivo. Under Diels-Alder reaction conditions these
chlorins were converted into the corresponding stable
bacteriochlorins exhibiting long wavelength absorption
in the range of 737-805 nm.
1
their H-H COSY NMR spectra and comparing their H
NMR data with that of bacteriochlorin 16.
In the electronic absorption spectra, the long wave-
length bands were observed in the range of 737-805
nm, and the shifts were significantly found to depend
on the nature of the substituents present at the periph-
eral positions of the chromophore. The benzobacterio-
chlorins 19 and 20, containing a fused-imide ring
Resu lts a n d Discu ssion
Ch em istr y. In this study, methyl mesopyropheophor-
bide a 1b , mesochlorin e6 trimethyl ester 2b , meso-
chlorin p6 trimethyl ester 3b, methyl mesopurpurin-18-
N-hexylimide 4b, and methyl mesopurpurin-18-N-3,5-

Novel Benzobacteriochlorins
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5351
Sch em e 1a
a
Reagents: a. (i) Zn(OAc)2, (ii) H2, Pd/C, (iii) TFA; b. OsO4, H2S; c. 1,2-dichlorobenzene; d. (i) DMAD, (ii) DBU.
Sch em e 2
showed the maximum red shift and exhibited a strong
absorption near 800 nm (Figure 1).
Biologica l Stu d ies. Benzobacteriochlorins 16-20
were insoluble in water, and for biological studies, these
compounds were formulated in 1% Tween 80/5% dex-
trose solution and filtered through a 0.22 µm syringe
filter. The concentration of the photosensitizers in the
filtrate was calculated on the basis of their extinction
coefficient values, using the Beer-Lambert equation.24
Compounds 16-20 were evaluated for both in vitro and

5352 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
Li et al.
F igu r e 1. The electronic absorption spectra of benzobacte-
riochlorins 16-20 at a concentration of 10 µM in dichloro-
methane.
F igu r e 3. In vivo photosensitizing efficacy of benzobacterio-
chlorin 19 in mice (5 mice/group, bearing RIF tumors) at
variable concentrations (1.0, 2.0, 3.5, and 5.0 µmol/kg). The
tumors were exposed to a laser light (805 nm, 135 J /cm2, 75
mWcm2) at 24 h postinjection.
F igu r e 2. In vitro PDT efficacy of benzobacteriochlorin in RIF
tumor cells at a dose of 1.0 µM at 48 h MTT. The cells were
treated with light (737-805 nm, 0-6 J /cm2, 18 mW/cm2) after
4 h incubation (see the text). Control: Cells exposed to light
without any photosensitizer. None of the photosensitizers
produced any dark toxicity at 1.0 µM concentration (data not
shown).
in vivo photosensitizing efficacy and a comparative
study was also conducted to determine a possible
correlation between the intracellular localization and
human serum albumin Site II (benzodiazepine binding
site) affinity to their photosensitizing efficacy.
F igu r e 4. In vivo photosensitizing efficacy of benzobacterio-
chlorins 16-20 in C3H mice (5 mice/group) bearing RIF
tumors at a dose of 5.0 µmol/kg. The mice were treated with
laser light at their respective longest wavelength absorption
(determined by in vivo reflectance spectroscopy) of the photo-
sensitizer (737-805 nm, 135 J /cm2, 75mWcm2) at 24 h postin-
jection. Control: 12 mice were exposed to light without
incubating the cells with photosensitizer.
In Vitr o P h otosen sitizin g Effica cy. Following the
experimental details described previously,4 benzobac-
teriochlorins 16-20 were tested for in vitro efficacy on
radiation-induced fibrosarcoma (RIF) tumor cells at
variable drug/light doses. A standard MTT assay was
performed at a fixed drug concentration (1.0 µM), and
the cells were exposed to variable light doses (1.0 to 6.0
J /cm2) after 4 h incubation. [The optimal drug concen-
tration for compound 19 was initially determined by
evaluating the photosensitizing efficacy at variable
concentration and the in vitro activity of other com-
pounds was then compared under similar experimental
conditions]. From the results summarized in Figure 2
it can be seen that among all the bacteriochlorins,
compound 19 produced the best efficacy.
In Vivo P h otosen sitizin g Activity. The in vivo
efficacy of benzobacteriochlorins 16-20 was determined
in C3H mice transplanted with RIF tumors (see Ex-
perimental Section). To determine the drug dose, the
benzobacteriochlorin 19 that was found to be most
effective in vitro was first evaluated at four different
doses (1.0, 2.0, 3.5, and 5.0 µmol/kg) and the tumors (5
mice/group) were exposed to light (135 J /cm2) for 30 min
at 24 h postinjection. The tumor growth (to reach > 400
mm3) was monitored daily for 30 days. As can be seen
from the results summarized in Figure 3, the best tumor
response was observed at a dose of 5.0 µmol/kg (2/5 mice
were tumor-free on day 30) and no apparent toxicity was
observed. Therefore, for a comparative study, all bac-
teriochlorins 16-20 were then evaluated under similar
treatment conditions and the results are depicted in
Figure 4. As can be seen, compared to mice (5 mice/
group) used for a control experiment, all photosensitiz-
ers showed significant delay in tumor regrowth. How-
ever, among all bacteriochlorins, compounds containing
N-substituted-imide ring system 19 and 20 were most
effective. For example, compound 19 produced 40%
tumor response (2/5 mice were tumor free on day 30),
and under similar treatment conditions bacteriochlorin
20 was slightly less effective, producing 20% tumor
response (1/5 mice was tumor-free on day 25) but
complete tumor regrowth was observed by day 30. Other
compounds 16-18 showed some delay in tumor re-
growth, but were certainly less effective than bacterio-
benzochlorin 19.
Sk in P h ototoxicity. The major problem associated
with porphyrin-based compounds is long lasting skin
phototoxicity.25 Therefore, the phototoxicity of benzo-
bacteriochlorinimide 19 at a dose of 5.0 µmol/kg was

Novel Benzobacteriochlorins
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5353
mitochondria and/or the lysosomes.26,27 The newly syn-
thesized benzobacteriochlorins 16-20 with partition
coefficient values in the range of 5.66-8.74 (16: 5.66,
17: 6.25; 18: 6.35; 19, 7.13; and 20, 8.74) were
investigated for sites of localization by following the
experimental procedure described previously.4 All bac-
teriochlorins were found to localize to the same subcel-
lular regions as Rhodamine-123, suggesting selectivity
toward mitochondria (a representative example is shown
in Figure 6).
P er ip h er a l Ben zod ia zep in e R ecep t or Bin d in g
Stu d ies. In previous studies it has been implied by us
and others that certain photosensitizers that show
mitochondrial localization exhibit peripheral benzodi-
azepine receptor (PBR) binding which may be an
important target for PDT.28,29 Therefore, the ability of
the newly synthesized benzochlorins to displace 3H
PK11195 (known PBR binding probe) from its specific
cellular binding site [the intelligent quotient (IQ) site
on PBR] was determined. Our preliminary experiments
with increasing concentrations of mitochondrially local-
ized benzobacteriochlorins 16-20 did not indicate any
specific displacement of 3H-PK11195. These results are
in contrast to those obtained from the hexyl ether
derivative of pyropheophorbide-a (HPPH)28 and suggest
that the PBR is not the target-site for the effective
benzobacteriochlorin analogues (Figure 7).
Hu m a n Ser u m Albu m in (Site II) Bin d in g Stu d -
ies. Human serum albumin (HSA) is the most abundant
protein in human blood plasma. Many compounds,
especially amphiphilic drugs and some endogenous
substances, bind reversibly and with high affinity to
HSA.30 The formation of this complex decreases the
concentration of unbound molecule in the plasma and
thereby affects the ligand’s distribution, pharmaco-
kinetics, toxicity, and ultimately its rate of excretion.
Previous studies on various porphyrin- and chlorin-
based photosensitizers revealed there is a well-cor-
related relationship between photodynamic activity and
HSA Site-II binding affinity to drugs, i.e. the photo-
dynamically active compounds were generally found to
bind to Site II of HSA.31,32 Therefore, benzobacterio-
chlorins 16-20 were also subjected to HSA Site II
binding ability following the literature procedure.31,32
As can be seen from Table 1 and Figure 8 (only a
representative example is shown), benzobacteriochlorins
16-20 competitively displaced DP (dansyl-L-proline),
the Site II probe of HSA, with the binding constant
values ranging from 1.06 × 107 to 1.75 × 107 M-1. The
binding constant values of benzobacteriochlorins 16-
20 to HSA (Site II) were determined by fluorescence
titration method. The theoretical basis and its applica-
tion on our experiment had been described in detail
F igu r e 5. Skin phototoxicity vs days: Bacteriochlorin 19 at
a dose of 5.0 µmol/kg was injected (iv) in12 mice (swiss mice,
4 mice/group). The hind foot of each mouse was exposed to
light (similar to PDT conditions); the first group was exposed
after 24 h postinjections and the other three groups at 48, 72,
and 96 h postinjection, respectively. For details, see Results
and Discussion).
investigated. For this study, bacteriochlorin 19 at a dose
of 5.0 µmol/kg was injected (iv) to four groups of mice
(Swiss mice, 3 mice/group). One of the hind feet of each
mouse in the first group was exposed to light (at the
therapeutic dose) at 24 h postinjection. The subsequent
groups (3 mice/group) were exposed at 48, 72, and 96
h, respectively. In each group, the unexposed hind feet
were used as control. Foot response was judged using a
0-3 scale: 0-0.1 ) No apparent difference from nor-
mal, 0.3 ) slight edema, 0.5 ) moderate edema, 0.75 )
large edema, 1.0 ) large erythema with exudate, 1.2 )
moderate edema with slightly crusty appearance, 1.5
) definite erythema, 1.65 ) slightly damaged and or
slight fusion of toes; 2.0 ) most toes are fused but no
change in general shape; 2.5 ) foot shapeless with no
toes, 3.0 ) only stub of foot remaining. Response >2.0
indicates unacceptably severe normal tissue reaction.25
Compared to Photofrin at approximately an equi-
effective in vivo dose (8.3 µmol/kg) that shows slight
damage and slight fusion of toes (score: 1.65-1.80), the
benzobacteriochlorin 19 produced limited phototoxicity.
Only a moderate edema (score: 0.5) was observed on
day 1 at 24 h postinjection of the drug. At 48-72 h
postinjection, no skin phototoxicity was observed, sug-
gesting considerable tumor selectivity. The results are
summarized in Figure 5.
In tr a cellu la r Loca liza tion . It has been shown that
depending on the nature of the chromophore, effective
photodynamic agents show very diverse patterns of
localization, which also is based on lipophilicity, charge,
and amphiphilicity. The predominant localization sites
for most effective photosensitizers are reported to be
F igu r e 6. Comparative intracellular localization of benzobacteriochlorins 19 (1.0 µM) and Rhodamine123 (0.5 µM) in RIF tumor
cells incubated for 24 and 0.5 h respectively. The images (false colors) were obtained by fluorescence light microscopy.

5354 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
Li et al.
treriochlorins 16-20 with long wavelength absorption
near 737-805 nm were synthesized in moderate overall
yield. In preliminary in vitro and in vivo studies, among
the bacteriochlorins investigated, benzobacteriochlorin
19 was found to be most effective and showed reduced
skin phototoxicity compared to Photofrin at their re-
spective therapeutic doses. Similarly to other known
porphyrin-based effective photosensitizers, benzochlo-
rins 16-20 also were found to localize in mitochondria
and exhibited competitive binding to site II of HSA (a
peripheral diazepine-binding site). No direct correlation
between the HSA (Site II) binding constant values of
various photosensitizers with photosensitizing efficacy
was observed. However, this technique could be used
as a simple in vitro screening tool in selecting the
compounds for in vivo studies. The detailed biological
evaluation (pharmacokinetic and pharmacodynamic
characteristics) of benzobacteriochlorin 19 and a series
of the related N-substituted analogues with variable
lipophilicity are currently in progress.
F igu r e 7. Displacement of 3H-PK11195 by PK11195 and
benzobacteriochlorins 16-20 atvariable concentrations. Com-
3
pounds 16-20 produce limited displacement of H-PK11195
indicating their limited peripheral benzodiazepine receptor
binding ability.
Ta ble 1. HSA Site II Binding Abilities of Various
Benzobacteriochlorins
benzobacteriochlorin
16
17
18
19
20
a
K (×107 M-1
)
1.06
1.75
1.44
1.61
1.60
a
The binding constant values of benzobacteriochlorins to HSA
(Site II).
Exp er im en ta l Section
1H and 19F NMR spectra were recorded in CDCl3 solutions
at 400 MHz Brucker instrument. Chemical shifts are reported
in ppm with CDCl3 as internal standard (for 1H, 7.26 ppm)
and TFA as external standard (for 19F, 0.00 ppm). Proton peak
assignments were based on 2D NMR (H-H COSY and/or
ROESY) analysis. UV-vis spectra were recorded on a Varian
(Cary-50 Bio) spectrophotometer. Column chromatographic
separations were performed over silica gel 60 (70-230 mesh)
or neutral alumina (Brockmann grade III, ∼150 mesh).
Preparative TLC was performed on silica 20 × 20 cm TLC
plates (Analtech). Methylpheophorbide a, the starting material
used for the preparation of a series of desired benzobacterio-
chlorin analogues was isolated from Spirulina pacifica by
following the literature procedure.
Meth yl Mesop yr op h eop h or bid e a (1b). A solution of Zn-
(OAc)2‚2H2O (1.11 g) in methanol (45 mL) was added to a
solution of methyl pyropheophorbide a 1a (1.11 g) in dichlo-
romethane (75 mL). The mixture was stirred at room temper-
ature for 2 h. The reaction mixture was then washed with
water (4 × 100 mL), and the organic layer was collected and
dried over Na2SO4. Solvent was removed with a rotavapor at
reduced pressure, and the residue was dissolved in THF (100
mL). Et3N (0.2 mL) and Pd/C (10%, 100 mg) were then added
to above solution. The resultant mixture was hydrogenated
(with a hydrogen balloon) at room temperature for 15 h and
then filtered through a pad of Celite. The solvent was removed
with a rotavapor at reduced pressure, and the residue was
treated with TFA (30 mL) for 1 h at room temperature. The
reaction mixture was poured into ice and extracted with CH2-
Cl2 until the water layer was clear. The CH2Cl2 layers were
combined and washed with water (2 × 200 mL) and 5%
NaHCO3 (1 × 200 mL). The CH2Cl2 layer was collected and
dried over Na2SO4. The solvent was removed after filtration,
and the residue was purified by column chromatography over
alumina eluted with CH2Cl2/EtOAc (v/v 10/1). The title com-
pound (1.09 g) was obtained with an overall yield of 98%.
Mesoch lor in e6 Tr im eth yl Ester (2b). Following the
procedure described for the preparation of 1b, the title
compound was obtained in 96% yield from chlorin 2a .
F igu r e 8. Langmuir plot of binding between DP and HSA
Site II with benzobacteriochlorin 19. Concentration of HSA
was kept constant (1.0 µM). Bacteriochlorin 19 was evaluated
at 1.0 and 2.0 µM concentrations. Concentrations of DP was
varied from 0 to 2.0 µM. Data were simulated with a competi-
tive binding model. Nonlinear least-squares curve fittings were
performed with Origin 5.0 (Microcal Software Inc.) on IBM-
PC computer.
previously.32 The following formula was derived from a
competitive binding model that was used for this study:
rA ) 2KAAu/{1 + KAAu + KBBt - KBPt (
[(1 + KAAu + KBPt - KBBt)2 + 4KBBt +
4KAKBAuBt]1/2
}
where rA is the number of probes binding with each HSA
molecule. KA is the binding constant of the probe (DP).
KB is the binding constant of the drug (benzobacterio-
chlorins 16-20). Au is the concentration of unbound
probe. Bt is the total concentration of the drug. Pt is the
total concentration of the HSA. The data analysis was
performed on an IBM-PC with Origin (version 5.0 for
Windows, Microcal Software Inc.). Nonlinear least-
squares curve fittings method was used for the calcula-
tion of KB.
Mesoch lor in p6 Tr im eth yl Ester (3b). Starting from 3a
and following the procedure described for the preparation of
1b, the title compound was obtained in 92% yield from
compound 3a . UV-vis in CH2Cl2 [λmax (ꢀ)]: 398 (150305), 496
1
(11402), 527 (4164), 605 (5453), 656 (40749). H NMR δ 9.68
(1H, s, meso H), 9.31 (1H, s, meso H), 8.57 (1H, s, meso H),
5.15 (1H, dd, J ) 8.8, 2.4 Hz, H-17), 4.37 (1H, q, J ) 7.3 Hz,
H-18), 4.22, 4.16, 3.63, 3.52, 3.29, 3.25 (each 3H, s, CH3-2, CH3-
7, CH3-12 and 3 × methyl esters), 3.82 (2H, q, J ) 7.6 Hz,
Con clu sion
In summary, starting from chlorophyll-a, an easily
available natural product, a series of stable benzobac-

Novel Benzobacteriochlorins
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5355
CH3CH2-3 or CH3CH2-8), 3.74 (2H, q, J ) 7.6 Hz, CH3CH2-3
or CH3CH2-8), 2.36, 2.20, 2.04, 1.87 (each 1H, m, CH3-
OOCCH2CH2-17), 1.85 (3H, d, J ) 7.4 Hz, CH3-18), 1.73 (3H,
t, J ) 7.5 Hz, CH3CH2-3 or CH3CH2-8), 1.70 (3H, t, J ) 7.5
Hz, CH3CH2-3 or CH3CH2-8), -0.84 (2H, br, 2 × NH). MS (ESI)
m/z 649.3 ([M + Na]+, 100). Anal. (C36H42N4O6) C, H, N.
18), 1.63 (3H, t, J ) 7.5 Hz, CH3CH2-3), 1.18, 1.12 (3H, t, J )
7.6 Hz, CH3CH2-8), 0.17, 0.07, -0.09, -0.19 (2H, br, 2 × NH).
MS (ESI) m/z 683.4 ([M + Na]+, 100). Anal. (C36H44N4O8‚1.5H2O)
C, H, N.
vic-7,8-Dih ydr oxym esopu r pu r in -18-N-h exylim ide Meth -
yl Ester (9). Mesopurpurin-18-N-hexylimide methyl ester 4b
(578 mg) was reacted OsO4 (500 mg) by following the procedure
described for the preparation of 6, the title compound (519 mg)
was obtained as a mixture of two isomers (1:1) in 85% yield.
The unreacted 4b (80 mg) was also recovered. A small amount
of the isomer mixture was separated with preparative silica
TLC plates using CH2Cl2/EtOAc (v/v 5/1) as developing solvent.
Th e fa ster m ovin g isom er : UV-vis in CH2Cl2 [λmax (ꢀ)]: 367
(112349), 411 (47574), 536 (42561), 757 (31650). 1H NMR δ
8.67 (1H, s, meso H), 8.49 (1H, s, meso H), 8.21 (1H, s, meso
H), 5.14 (1H, br d, J ) 8.5 Hz, H-17), 4.11 [3H, m, H-18 and
CH3(CH2)4CH2N], 3.89 (1H, s, OH), 3.60 (2H, q, J ) 7.7 Hz,
CH3CH2-3), 3.55, 3.54, 3.13 (each 3H, s, CH3-2, CH3-12 and
CH3OOCCH2CH2-17), 2.92 (1H, s, OH), 2.58 (3H, m, CH3-
OOCCH2CH2-17 and one proton of CH3OOCCH2CH2-17), 2.28
(2H, q, J ) 10.1 Hz, CH3CH2-8), 1.89 (4H, s, CH3-7 and one
proton of CH3OOCCH2CH2-17), 1.83 (2H, m, CH3CH2CH2-
CH2CH2CH2N), 1.68 (3H, d, J ) 7.3 Hz, CH3-18), 1.63 (3H, t,
J ) 7.7 Hz, CH3CH2-3), 1.50 (2H, m, CH3CH2CH2CH2CH2-
CH2N), 1.39 (4H, m, CH3CH2CH2CH2CH2CH2N), 1.28 (3H, t,
J ) 7.6 Hz, CH3CH2-8), 0.92 (3H, t, J ) 7.1 Hz, CH3CH2CH2-
CH2CH2CH2N), 0.47 (1H, s, NH), 0.11 (1H, s, NH). MS (ESI)
m/z 720.4 ([M + Na]+, 100). Th e slow er m ovin g isom er :
UV-vis in CH2Cl2 [λmax (ꢀ)]: 368 (99597), 412 (42859), 536
(38058), 762 (26798). 1H NMR δ 8.71 (1H, s, meso H), 8.47
(1H, s, meso H), 8.24 (1H, s, meso H), 5.21 (1H, dd, J ) 8.8,
2.3 Hz, H-17), 4.38 [2H, m, CH3(CH2)4CH2N], 4.17 (1H, q, J )
7.6 Hz, H-18), 3.60 (4H, q, J ) 7.6 Hz, CH3CH2-3), 3.58, 3.57,
3.13 (each 3H, s, CH3-2, CH3-12 and CH3OOCCH2CH2-17), 3.58
(1H, s, OH, overlapped with ring methyl or methyl of methyl
ester), 2.98 (1H, s, OH), 2.65 (1H, m, one proton of CH3-
OOCCH2CH2-17), 2.49 (2H, q, J ) 7.7 Hz, CH3CH2-8), 2.34
(2H, m, CH3OOCCH2CH2-17), 1.95 (2H, s, CH3-7), 1.94 (3H,
m, CH3CH2CH2CH2CH2CH2N and one proton of CH3OOCCH2-
CH2-17), 1.66 (3H, d, J ) 7.4 Hz, CH3-18), 1.62 (3H, t, J ) 7.7
Hz, CH3CH2-3), 1.56 (2H, m, CH3CH2CH2CH2CH2CH2N), 1.43
(4H, m, CH3CH2CH2CH2CH2CH2N), 1.17 (3H, t, J ) 7.2 Hz,
CH3CH2-8), 0.93 (3H, t, J ) 7.3 Hz, CH3CH2CH2CH2CH2-
CH2N), 0.44 (1H, s, NH), 0.02 (1H, s, NH). MS (ESI) m/z 720.4
([M + Na]+, 100). Anal. (C40H51N5O6) C, H, N.
vic-7,8-Dih yd r oxym esop u r p u r in -18-N-3,5-b is(t r iflu o-
r om eth yl)ben zylim id e Meth yl Ester (10). Mesopurpurin-
18-N-3,5-bis(trifluoromethyl)benzylimide methyl ester 5b (340
mg) was reacted with OsO4 (500 mg) by following the proce-
dure described for the preparation of 6, and the title compound
(263 mg) was obtained as a mixture of two isomers (1:1) in
74% yield. The unreacted 5b (20 mg) was also recovered. A
small amount of the isomer mixture was separated with
preparative silica TLC plates using CH2Cl2/EtOAc (v/v 5/1) as
developing solvent. Th e fa ster m ovin g isom er : UV-vis in
CH2Cl2 [λmax (ꢀ)]: 368 (107382), 413 (45711), 540 (44541), 756
(29415). 1H NMR δ 8.64 (1H, s, meso H), 8.49 (1H, s, meso H),
8.19 (1H, s, meso H), 7.91 (2H, s, 2 × CH at position 2 and 6
on benzene ring), 7.72 (1H, s, 1 × CH at position 4 on benzene
ring), 5.04 (1H, dd, J ) 8.8, 2.5 Hz, H-17), 4.95 (2H, m, CH2 of
benzyl), 4.10 (1H, q, J ) 7.4 Hz, H-18), 3.59 (2H, q, J ) 7.6
Hz, CH3CH2-3), 3.50, 3.48, 3.12 (each 3H, s, CH3-2, CH3-12
and CH3OOCCH2CH2-17), 2.54, 2.28, 2.14, 1.79 (3H, 1H, 1H,
1H, m, CH3OOCCH2CH2-17 and CH3CH2-8), 1.91 (1H, s, CH3-
7), 1.73 (3H, d, J ) 7.2 Hz, CH3-18), 1.63 (3H, t, J ) 7.5 Hz,
CH3CH2-3), 1.26 (3H, t, J ) 7.2 Hz, CH3CH2-8), 0.68 (1H, s,
NH), 0.36 (1H, s, NH). MS (ESI) m/z 862.4 ([M + Na]+, 100).
Anal. (C43H43F6N5O6) C, H, N. Th e slow er m ovin g isom er :
UV-vis in CH2Cl2 [λmax (ꢀ)]: 368 (107261), 414 (46658), 540
(44895), 764 (27995). 1H NMR δ 8.65 (1H, s, meso H), 8.44
(1H, s, meso H), 8.20 (1H, s, meso H), 8.14 (2H, s, 2 × CH at
position 2 and 6 on benzene ring), 7.78 (1H, s, 1 × CH at
position 4 on benzene ring), 5.62 (2H, m, CH2 of benzyl), 5.13
(1H, dd, J ) 8.9, 2.9 Hz, H-17), 4.16 (1H, q, J ) 7.2 Hz, H-18),
Mesop u r p u r in -18-N-h exylim id e Met h yl E st er (4b ).
Starting from 4a and following the procedure described for the
preparation of 1b, the title compound was obtained in 95%
yield. UV-vis in CH2Cl2 [λmax (ꢀ)]: 364 (35629), 417 (117252),
1
508 (6640), 554 (14738), 638 (6154), 694 (33848). H NMR δ
9.62 (1H, s, meso H), 9.22 (1H, s, meso H), 8.51 (1H, s, meso
H), 5.39 (1H, dd, J ) 8.3, 2.4 Hz, H-17), 4.46 [2H, m, CH3-
(CH2)4CH2N], 4.33 (1H, q, J ) 7.3 Hz, H-18), 3.84, 3.56, 3.25,
3.20 (each 3H, s, CH3-2, CH3-7, CH3-12 and methyl ester), 3.77
(2H, q, J ) 7.7 Hz, CH3CH2-3 or CH3CH2-8), 3.67 (2H, q, J )
7.7 Hz, CH3CH2-3 or CH3CH2-8), 2.67, 2.35, 1.99 [1H, 2H, 3H,
m, CH3OOCCH2CH2-17 and CH3(CH2)3CH2CH2N], 1.75 (3H,
d, J ) 7.3 Hz, CH3-18), 1.71 (3H, t, J ) 7.6 Hz, CH3CH2-3 or
CH3CH2-8), 1.68 (3H, t, J ) 7.6 Hz, CH3CH2-3 or CH3CH2-8),
1.62 (2H, m, CH3CH2CH2CH2CH2CH2N), 1.45 (4H, m, CH3-
CH2CH2CH2CH2CH2N), 0.95 [3H, t, J ) 7.2 Hz, CH3(CH2)3-
CH2CH2N], 0.02, -0.15 (each 1H, br, 2 × NH). MS (ESI) m/z
664.5 ([M + 1]+, 100). Anal. (C40H49N5O4‚0.5H2O) C, H, N.
Mesop u r p u r in -18-N-3,5-b is(t r iflu or om et h yl)b en zyl-
im id e Meth yl Ester (5b). Starting from 5a and following the
procedure described for the preparation of 1b, the title
compound was obtained in 92% yield. UV-vis in CH2Cl2 [λmax
(ꢀ)]: 362 (54373), 417 (174414), 508 (9263), 545 (26734), 641
1
(10167), 696 (53996). H NMR δ 9.54 (1H, s, H-10), 9.15 (1H,
s, H-5), 8.48 (1H, s, H-20), 8.24 (2H, s, 2 × CH at position 2
and 6 on the benzene ring), 7.81 (1H, s, CH at position 4 on
the benzene ring), 5.77 (2H, s, CH2 of benzyl), 5.33 (1H, m,
H-17), 4.33 (1H, q, J ) 7.5 Hz, H-18), 3.79, 3.56, 3.23, 3.15
(each 3H, s, CH3-2, CH3-7, CH3-12 and 1 × methyl ester), 3.74
(2H, q, J ) 7.7 Hz, CH3CH2-3 or CH3CH2-8), 3.61 (2H, q, J )
7.7 Hz, CH3CH2-3 or CH3CH2-8), 2.69, 2.38, 1.95 (1H, 2H, 1H,
m, CH3OOCCH2CH2-17), 1.77 (3H, d, J ) 7.2 Hz, CH3-18), 1.70
(3H, t, J ) 7.6 Hz, CH3CH2-3 or CH3CH2-8), 1.66 (3H, t, J )
7.6 Hz, CH3CH2-3 or CH3CH2-8), 0.25 (1H, br, NH), 0.04 (1H,
br, NH). MS (ESI) m/z 828.4 ([M + Na]+, 100). Anal.
(C43H41F6N5O4‚2H2O) C, H, N.
vi c-7,8-D ih y d r o x y m e t h y lm e s o p y r o p h e o p h o r b id e
a (6).13,15 Pyridine (1.0 mL) and a solution of OsO4 (1.0 g) in
Et2O (10 mL) were successively added to a solution of methyl
mesopyropheophorbide a (1b) (1.06 g) in dry CH2Cl2 (120 mL).
The mixture was stirred at room temperature for 24 h. H2S
gas was bubbled into the reaction mixture for 5 min to
decompose the unreacted OsO4. Nitrogen was then bubbled
into above mixture to remove H2S. The mixture was filtered
through a pad of Celite. The filtrate was evaporated, and the
residue was purified by column chromatography over silica gel
eluted with CH2Cl2/EtOAc (first v/v 10/1, then 5/1). The title
compound (735 mg) was obtained in 65% yield and the
unreacted starting material 1b (54 mg) was also recovered.
vic-7,8-Dih ydr oxym esoch lor in e6 Tr im eth yl Ester (7).13,16
Mesochlorin e6 trimethyl ester 2b (310 mg) was reacted with
OsO4 (350 mg) by following the procedure described for the
preparation of 6, and the title compound was obtained in 52%
yield (182 mg). The unreacted 2b (35 mg) was also recovered.
vic-7,8-Dih yd r oxym esoch lor in p6 Tr im eth yl Ester (8).
Mesochlorin p6 trimethyl ester 3b (367 mg) was reacted with
OsO4 (500 mg) by following the procedure described for the
preparation of 6 and the title compound (342 mg) was obtained
as a mixture of two isomers (2.4:1) in 83% yield. The unreacted
3b (21 mg) was also recovered. UV-vis in CH2Cl2 [λmax (ꢀ)]:
354 (97147), 382 (74713), 450 (3505), 480 (5558), 511 (21382),
723 (25789). 1H NMR δ 8.66, 8.65 (1H, s, meso H), 8.45 (1H, s,
meso H), 8.22, 8.19 (1H, s, meso H), 4.92, 4.86 (1H, m, H-17),
4.11 (1H, m, H-18), 4.14, 4.10, 3.54, 3.37, 3.14 (each 3H,
splitting s, CH3-2, CH3-12 and 3 × methyl esters), 3.61 (2H,
q, J ) 7.9 Hz, CH3CH2-3), 2.48 (2H, q, J ) 7.3 Hz, CH3CH2-
8), 2.32, 2.13, 2.04, 1.79 (total 4H, m, CH3OOCCH2CH2-17),
1.99, 1.91 (3H, s, CH3-7), 1.77, 1.74 (3H, d, J ) 7.5 Hz, CH3-

5356 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
Li et al.
3.54 (2H, q, CH3CH2-3, overlapped with ring methyls or methyl
of methyl ester), 3.56, 3.52, 3.07 (each 3H, s, CH3-2, CH3-12
and CH3OOCCH2CH2-17), 2.64, 2.37, 2.28, 1.89 (1H, 1H, 1H,
1H, m, CH3OOCCH2CH2-17), 2.48 (2H, m, CH3CH2-8), 1.93
(1H, s, CH3-7), 1.66 (3H, d, J ) 7.3 Hz, CH3-18), 1.61 (3H, t, J
) 7.6 Hz, CH3CH2-3), 1.16 (3H, t, J ) 7.3 Hz, CH3CH2-8), 0.73
(1H, s, NH), 0.29 (1H, s, NH). MS (ESI) m/z 862.4 ([M + Na]+,
100). Anal. (C43H43F6N5O6) C, H, N.
8-Vin ylm esopm esou r pu r in -18-N-h exylim ide Meth yl Es-
ter (14). vic-7,8-Dihydroxymesopurpurin-18-N-hexylimide meth-
yl ester 9 (519 mg) in o-dichlorobenzene (50 mL) was refluxed
for 1.5 h. After the standard workup by following the procedure
described for the preparation of 11, the resultant mixture was
purified by column chromatography over silica gel eluted with
CH2Cl2/EtOAc (v/v 20/1). The title compound was obtained in
41% (201 mg) yield. UV-vis in CH2Cl2 [λmax (ꢀ)]: 367 (35101),
422 (116543), 512 (8313), 547 (11085), 637 (5542), 690 (32946).
1H NMR δ 9.75 (1H, s, H-10), 9.25 (1H, s, H-5), 8.51 (1H, s,
H-20), 7.87 (1H, dd, J ) 18.0, 11.9 Hz, H-81), 6.08 (1H, d, J )
17.9 Hz, H-82 trans), 5.98 (1H, d, J ) 11.6 Hz, H-82 cis), 5.39
(1H, dd, J ) 8.8, 2.2 Hz, H-17), 4.45 (2H, m, NCH2CH2CH2-
CH2CH2CH3), 4.34 (1H, q, J ) 7.2 Hz, H-18), 3.77 (2H, q, J )
8.0 Hz, CH3CH2-3), 3.80, 3.56, 3.30, 3.25 (each 3H, s, CH3-2,
CH3-7, CH3-12, and 1 × methyl esters), 2.68, 2.38, 2.11 (1H,
2H, 1H, m, CH3OOCCH2CH2-17), 1.98 (2H, m, NCH2CH2CH2-
CH2CH2CH3), 1.76 (3H, d, J ) 7.2 Hz, CH3-18), 1.71 (3H, t, J
) 7.7 Hz, CH3CH2-3), 1.60 (2H, m, NCH2CH2CH2CH2CH2CH3),
1.44 (4H, m, NCH2CH2CH2CH2CH2CH3), 0.95 (3H, t, J ) 7.0
Hz, NCH2CH2CH2CH2CH2CH3), 0.02 (1H, br, NH), -0.15 (1H,
br, NH). MS (FAB) m/z 662.5 (MH+, 100). HRMS (FAB): Calcd
for C40H48N5O4, 662.3706 (M + H); Found 662.3685. Anal.
(C40H47N5O4) C, H, N.
8-Vin ylm eth ylm esop yr op h eop h or bid e a (11). vic-7,8-
Dihydroxymethylmesopyropheo phorbide a 6 (364 mg) was
dissolved in o-dichlorobenzene (30 mL). The solution was
refluxed for 1.5 h under an atmosphere of N2. After being
cooled to room temperature, the reaction mixture was loaded
on a short silica column and eluted with hexanes to remove
o-dichlorobenzene and then 10% MeOH/CH2Cl2 to give a
mixture. The mixture was further purified with silica gel
column eluted with 3% MeOH/CH2Cl2. The title compound (184
mg) was obtained in 54% yield. UV-vis in CH2Cl2 [λmax (ꢀ)]:
418 (143057), 509 (11678), 541 (7213), 602 (9703), 657 (53238).
1H NMR δ 9.22 (1H, s, H-10), 9.04 (1H, s, H-5), 8.47 (1H, s,
H-20), 7.66 (1H, dd, J ) 17.9, 11.8 Hz, H-81), 5.99 (1H, d, J )
17.4 Hz, H-82 trans), 5.88 (1H, d, J ) 9.7 Hz, H-82 cis), 5.13
(2H, dd, AB system, J ) 20.2 Hz, -COCH2-15), 4.46 (1H, dq,
J ) 7.7, 2.2 Hz, H-18), 4.25 (1H, m, H-17), 3.73 (2H, q, J ) 7.6
Hz, CH3CH2-3), 3.66 (3H, s, CH3OOCCH2CH2-17), 3.40 (3H,
s, CH3-12), 3.29 (3H, s, CH3-2), 3.20 (3H, s, CH3-7), 2.68 (1H,
m, one proton of CH3OOCCH2CH2-17), 2.58 (1H, m, one proton
of CH3OOCCH2CH2-17), 2.29 (2H, m, one proton of CH3-
OOCCH2CH2-17 and one proton of CH3OOCCH2CH2-17), 1.85
(3H, d, J ) 7.0 Hz, CH3-18), 1.71 (3H, t, J ) 7.6 Hz, CH3CH2-
3), 0.16 (1H, s, NH), -1.86 (1H, s, NH). MS (FAB) m/z 548.2
(M+, 100). HRMS (FAB): Calcd for C43H36N4O3, 548.2787;
Found 548.2763. Anal. (C34H36N4O3‚0.5H2O) C, H, N.
8-Vin ylm esop u r p u r in -18-N-3, 5-bis(t r iflu or om et h yl)-
ben zylim id e Meth yl Ester (15). vic-7,8-Dihydroxymesopur-
purin-18-N-3,5-bis(trifluoromethyl)benzylimide methyl ester
10 (243 mg) in o-dichlorobenzene (30 mL) was refluxed for 1.5
h. After workup by following the procedure for the preparation
of 11, the resultant mixture was purified by column chroma-
tography over silica gel eluted with CH2Cl2/acetone (v/v 99/1).
The title compound was obtained in 51% (120 mg) yield. UV-
vis in CH2Cl2 [λmax (ꢀ)]: 367 (45 775), 422 (152 778), 513
(10 230), 548 (17 650), 638 (8207), 693 (45 418). 1H NMR δ 9.71
(1H, s, H-10), 9.21 (1H, s, H-5), 8.48 (1H, s, H-20), 8.23 (2H, s,
2 × CH at position 2 and 6 on benzene ring), 7.84 (1H, dd, J
) 18.0, 10.7 Hz, H-81), 7.80 (1H, s, CH at position 4 on benzene
ring), 6.07 (1H, d, J ) 17.7 Hz, H-82 trans), 5.98 (1H, d, J )
11.8 Hz, H-82 cis), 5.76 (2H, s, CH2 of benzyl), 5.32 (1H, m,
H-17), 4.33 (1H, q, J ) 7.3 Hz, H-18), 3.78, 3.55, 3.27, 3.23
(each 3H, s, CH3-2, CH3-7, CH3-12, and 1 × methyl ester), 3.75
(2H, q, J ) 7.6 Hz, CH3CH2-3), 2.68, 2.38, 1.94 (1H, 2H, 1H,
m, CH3OOCCH2CH2-17), 1.76 (3H, d, J ) 7.1 Hz, CH3-18), 1.71
(3H, t, J ) 7.6 Hz, CH3CH2-3), 0.26 (1H, br, NH), 0.05 (1H,
br, NH). MS (ESI) m/z 804.5 (MH+, 100). HRMS (FAB): Calcd
for C43H40F6N5O4, 804.2985 (M + H); Found 804.2983. Anal.
(C43H39F6N5O4) C, H, N.
8-Vin ylm esoch lor in e6 Tr im eth yl Ester (12). A solution
of vic-7,8-Dihydroxymesochlorin e6 trimethyl ester 7 (491 mg)
in o-dichlorobenzene (40 mL) was refluxed for 1.5 h. After
workup (following the procedure described for the preparation
of 11), the resultant mixture was purified by column chroma-
tography over silica gel eluted with CH2Cl2/acetone (v/v 15/1).
The title compound was obtained in 41% yield (192 mg). UV-
vis in CH2Cl2 [λmax (ꢀ)]: 405 (145190), 501 (11460), 597 (4878),
1
651 (35620). H NMR δ 9.84 (1H, s, H-10), 9.41 (1H, s, H-5),
8.68 (1H, s, H-20), 8.03 (1H, dd, J ) 18.0, 11.0 Hz, H-81), 6.13
(1H, dd, J ) 17.0, 2.4 Hz, H-82 trans), 6.00 (1H, J ) 11.9, 1.9
Hz, H-82 cis), 5.31 (2H, dd, AB system, J ) 18.0 Hz, CH3-
OOCCH2-15), 4.48 (1H, q, J ) 7.4 Hz, H-18), 4.41 (1H, dd, J
) 9.8, 2.1 Hz, H-17), 3.86 (2H, q, J ) 7.6 Hz, CH3CH2-3), 4.28,
3.80, 3.66, 3.58, 3.42, 3.35 (each 3H, s, CH3-2, CH3-7, CH3-12,
and 3 × methyl esters), 2.58, 2.23, 1.79 (1H, 2H, 1H, m, CH3-
OOCCH2CH2-17), 1.77 (3H, d, J ) 6.9 Hz, CH3-18), 1.76 (3H,
t, J ) 8.3 Hz, CH3CH2-3), -1.27 (1H, br, NH), -1.30 (1H, br,
NH). MS (FAB) m/z 639.4 (MH+, 100). HRMS (FAB): Calcd
for C37H43N4O6 [M + H], 639.3182; Found 639.3168. Anal.
(C37H42N4O6) C, H, N.
Ben zoba cter ioch lor in s 16. DMAD (2.0 mL) was added to
a solution of 8-vinylmethylmesopyropheophorbide a 11 (95 mg)
in toluene (20 mL). The mixture was refluxed for 5 h under
nitrogen. After the mixture was cooled to room temperature,
another portion of DMAD (1.5 mL) was added, and the solution
was further refluxed for 2.5 h. The solvent and excess DMAD
were removed with rotavapor. The residue was purified with
preparative silica TLC using CH2Cl2/acetone (v/v 8/1) as
developing solvent. The intermediate benzobacteriochlorin (18
mg) and the unreacted 11 (55 mg) were isolated. The inter-
mediate compound (18 mg) was dissolved in CH2Cl2 (30 mL),
and 2 drops of DBU was added. The resulting solution was
stirred at room temperature for 5 min. The solvent was
removed, and the residue was purified with preparative silica
TLC using CH2Cl2/acetone (v/v 8/1) as developing solvent. The
title compound (18 mg) was obtained as a mixture of two
isomers (1:1) in 36% converted yield in two steps. UV-vis in
CH2Cl2 [λmax (ꢀ)]: 334 (38 860), 402 (36 471), 445 (28 623), 476
8-Vin ylm esoch lor in p6 Tr im eth yl Ester (13). A solution
of vic-7,8-Dihydroxymesochlorin p6 trimethyl ester 8 (342 mg)
in o-dichlorobenzene (30 mL) was refluxed for 1.5 h. After
standard workup (following the procedure described for the
preparation of 11), the resultant mixture was purified by
column chromatography over silica gel eluted with CH2Cl2/
EtOAc (v/v 15/1). The title compound (128 mg) was obtained
in 40% yield. UV-vis in CH2Cl2 [λmax (ꢀ)]: 405 (142 001), 502
(11 009), 601 (4725), 655 (35 171). 1H NMR δ 9.84 (1H, s, H-10),
9.35 (1H, s, H-5), 8.58 (1H, s, H-20), 7.97 (1H, dd, J ) 18.1,
10.9 Hz, H-81), 6.10 (1H, d, J ) 18.8 Hz, H-82 trans), 6.00 (1H,
J ) 12.0, H-82 cis), 5.14 (1H, dd, J ) 9.8, 2.2 Hz, H-17), 4.38
(1H, q, J ) 7.4 Hz, H-18), 3.83 (2H, q, J ) 7.6 Hz, CH3CH2-3),
4.22, 4.16, 3.62, 3.53, 3.37, 3.30 (each 3H, s, CH3-2, CH3-7,
CH3-12, and 3 × methyl esters), 2.39, 2.21, 2.08, 1.86 (each
1H, m, CH3OOCCH2CH2-17), 1.85 (3H, d, J ) 6.5 Hz, CH3-
18), 1.74 (3H, t, J ) 7.6 Hz, CH3CH2-3), -0.84 (2H, br, 2 ×
-NH). MS (FAB) m/z 624.4 (M+, 100). HRMS (FAB): Calcd for
C36H40N4O6, 624.2948; Found 624.2956. Anal. (C36H40N4O6‚
0.5H2O) C, H, N.
1
(40 414), 563 (9440), 666 (9705), 737 (21 686). H NMR δ 8.72
(1H, s, H-10), 8.11 (1H, s, H-5), 7.98, 7.97 (each 0.5H, s, H-20),
7.69 (1H, d, J ) 6.0 Hz, H-82), 7.09 (1H, d, J ) 6.3 Hz, H-81),
4.93, 4.92, 4.77 (0.5H, 0.5H, 1H, two sets of dd, AB system, J
) 20.0 Hz, -COCH2-15), 4.78, 4.77 (each 0.5H, s, H-84), 4.14
(1H, q, J ) 7.4 Hz, H-18), 3.97 (1H, m, H-17), 3.95 (3H, s, CH3-
OOC-83), 3.64, 3.63 (each 1.5H, s, CH3OOCCH2CH2-17), 3.57
(2H, q, J ) 7.7 Hz, CH3CH2-3), 3.37, 3.34 (each 1.5H, s, CH3-
12), 3.14, 3.13 (each 1.5H, s, CH3OOC-84), 3.08 (3H, s, CH3-2),

Novel Benzobacteriochlorins
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5357
2.51, 2.24 (each 2H, m, CH3OOCCH2CH2-17), 1.69 (3H, s, CH3-
7), 1.68 (3H, splitting d, CH3-18), 1.63 (3H, t, J ) 7.6 Hz, CH3-
CH2-3), 0.10, 0.08 (each 1H, br s, 2 × NH). MS (FAB) m/z 690.2
(M+, 100). HRMS (FAB): Calcd for C40H42N4O7, 690.3053;
Found 690.3062.
meso H), 7.70, 7.64 (1H, m, H-82), 7.14 (1H, m, H-81), 5.23 (1H,
m, H-17), 4.78, 4.62, 4.60, 4.48 (1H, d, d, d, s, H-84), 4.40 (2H,
m, NCH2CH2CH2CH2CH2CH3), 4.19 (1H, m, H-18), 3.61 (2H,
m, CH3CH2-3), 4.17, 4.95, 4.90, 4.67, 4.62, 4.58, 4.57, 4.16, 4.14,
4.13 (15H, each s, 3 × methyl esters, CH3-2 and CH3-12), 2.65,
2.35, 1.95 (1H, 2H, 3H, m, CH3OOCCH2CH2-17 and NCH2CH2-
CH2CH2CH2CH3), 1.68 (6H, m, CH3CH2-3 and CH3-18), 1.58
(2H, m, NCH2CH2CH2CH2CH2CH3), 1.54 (3H, splitting s, CH3-
7), 1.43 (4H, m, NCH2CH2CH2CH2CH2CH3), 0.95 (3H, t, J )
7.0 Hz, NCH2CH2CH2CH2CH2CH3), 0.60, 0.58, 0.27, 0.16, 0.14,
-0.14, -0.16 (2H, 2 × NH). MS (FAB) m/z 803.2 (M+, 100).
HRMS (FAB): Calcd for C46H53N5O8, 803.3894; Found 803.3895.
Ben zoba cter ioch lor in s 17. The intermediate benzobac-
teriochlorin 21 (15 mg) was dissolved in CH2Cl2 (30 mL), and
2 drops of DBU was added to above solution. The resultant
solution was stirred at room temperature for 5 min. The
solvent was removed, and the residue was purified with
preparative silica TLC using CH2Cl2/acetone (v/v 8/1) as
developing solvent. The title compound (15 mg) was obtained
as a mixture of four isomers (1:0.8:0.8:0.5) in quantitative
yield. UV-vis in CH2Cl2 [λmax (ꢀ)]: 330 (42 854), 399 (44 598),
Ben zoba cter ioch lor in s 20. DMAD (2.0 mL) was added to
a solution of 8-Vinylmesopurpurin-18-N-3,5-bis(trifluorometh-
yl)benzylimide methyl ester 15 (60 mg) in toluene (20 mL).
The mixture was refluxed for 20 h under N2. The solvent and
excess DMAD were removed with rotavapor. The residue was
purified with preparative silica TLC using CH2Cl2/EtOAc (v/v
30/1) as developing solvent. The intermediate benzobacterio-
chlorin (20 mg) was obtained, and the unreacted 15 was
recovered. The above intermediate compound was dissolved
in CH2Cl2 (30 mL) and 2 drops of DBU was added to above
solution. The resultant solution was stirred at room temper-
ature for 5 min. The solvent was removed and the residue was
purified with preparative silica TLC using CH2Cl2/EtOAc (v/v
40/1) as developing solvent. The title compound (10 mg) was
obtained as a mixture of isomers in 21% converted yield in
two steps. UV-vis in CH2Cl2 [λmax (ꢀ)]: 334 (41 591), 382
(42 244), 438 (68 667), 489 (42 896), 728 (11 417), 805 (34 089).
1H NMR δ 8.99 (1H, s, meso H), 8.30 (1H, splitting s, meso H),
8.19 (3H, s, 1 × meso H and 2 × phenyl H), 7.79 (1H, s, phenyl
H), 7.69 (1H, d, J ) 5.9 Hz, H-82), 7.12 (1H, d, J ) 5.6 Hz,
H-81), 5.70 [2H, s, CH2 of bis(trifluoromethyl)benzyl], 5.13 (1H,
m, H-17), 4.77 (1H, m, H-84), 4.16 (1H, m, H-18), 3.95, 3.60,
3.57, 3.16, 3.11 (each 3H, s, splitting s, s, s, s, CH3-2, CH3-12
and 3 × methyl esters), 3.59 (2H, m, CH3CH2-3, overlapped
with methyls), 2.65, 2.33, 1.89 (1H, 2H, 1H, m, CH3OOCCH2-
CH2-17), 1.75-1.60 (6H, m, CH3CH2-3 and CH3-18), 1.56 (3H,
s, CH3-7), 0.88, 0.86, 0.40, 0.38 (total 2H, br s, 2 × NH). 19F
NMR δ 13.04 (6F, s, 2 × -CF3). MS (FAB) m/z 945.5 (M+, 100).
HRMS (FAB): Calcd for C49H45F6N5O8, 945.3172; Found
945.3194.
In ter m ed ia te Ben zoba cter ioch lor in s 21. DMAD (2.0
mL) was added to a solution of 8-vinylmesochlorin e6 trimethyl
ester 12 (93 mg) in toluene (20 mL). The mixture was refluxed
for 5 h under nitrogen. The solvent and excess DMAD were
removed with rotavapor. The residue was purified with
preparative silica TLC using CH2Cl2/acetone (v/v 20/1) as
developing solvent. The title compound (27 mg) was obtained
as a mixture of two isomers (6:5) in 34% converted yield. The
unreacted 12 (29 mg) was also recovered. UV-vis in CH2Cl2
[λmax (ꢀ)]: 369 (96 055), 396 (86 594), 454 (4478), 482 (10 906),
511 (12 783), 664 (7439), 731 (49 978). 1H NMR δ 9.06 and
9.00 (each 1H, s, H-10), 8.58 and 8.54 (each 1H, s, H-5), 8.43
and 8.37 (each 1H, s, H-20), 7.16 (1H, dd, J ) 6.4, 2.8 Hz,
H-81), 7.10 (1H, dd, J ) 6.7, 2.2 Hz, H-81), 5.15 (2H, dd, AB
system, J ) 18.3 Hz, CH3OOCCH2-15), 5.10 (2H, dd, AB
system, J ) 18.5 Hz, CH3OOCCH2-15), 4.22, 4.19, 4.04, 3.96,
3.90, 3.88, 3.76, 3.75, 3.65, 3.62, 3.40, 3.37, 3.25, 3.22 (each
3H, s, 4 × ring methyl and 10 × methyl ester), 4.24 (2H, m, 2
× H-18), 4.18 (2H, m, 2 × H-17), 3.92 and 3.52 (each 2H, m,
2 × H-82), 3.70 (4H, m, 2 × CH3CH2-3), 2.51, 2.18, 1.80 (2H,
4H, 2H, 2 × CH3OOCCH2CH2-17), 2.00 and 1.94 (each 3H, s,
2 × CH3-7), 1.68 (6H, t, J ) 7.5 Hz, 2 × CH3CH2-3), 1.63 (6H,
d, J ) 7.2 Hz, 2 × CH3-18), -0.62, -0.75, -0.96, -1.13 (each
1H, s, 4 × NH). MS (FAB) m/z 780.2 (M+, 100). HRMS (FAB):
Calcd for C43H48N4O10, 780.3370; Found 780.3386.
1
470 (44 965), 570 (9911), 673 (8351), 744 (32 393). H NMR δ
9.07, 9.05, 9.00, 8.97, 8.70, 8.57, 8.38, 8.37, 8.35, 8.34, 8.27,
8.25 (3H, each s, 3 × meso H), 7.73, 7.66 (1H, m, H-82), 7.16
(1H, m, H-81), 5.08 (2H, m, CH3OOCCH2-15), 4.84, 4.81, 4.71,
4.61 (1H, s, s, d, d, H-84), 4.20, 4.19, 4.18, 3.95, 3.91, 3.89, 3.77,
3.75, 3.74, 3.73, 3.64, 3.63, 3.36, 3.36, 3.32, 3.22, 3.21, 3.18,
3.17, 3.09, 3.05 (21H, each s, 5 × methyl esters, CH3-2 and
CH3-12), 4.15 (2H, m, H-17 and H-18), 3.66 (2H, m, CH3CH2-
3), 2.51, 2.17, 1.72 (1H, 2H, 1H, m, CH3OOCCH2CH2-17),
1.80-1.50 (9H, m, CH3-7, CH3CH2-3 and CH3-18), -0.08,
-0.10, -0.37, -0.40, -0.46, -0.48, -0.74, -0.77 (2H, 2 × NH).
MS (FAB) m/z 780.2 (M+, 100). HRMS (FAB): Calcd for
C43H48N4O10, 780.3370; Found 780.3394.
Ben zoba cter ioch lor in s 18. DMAD (2.0 mL) was added to
a solution of 8-vinylmesochlorin p6 trimethyl ester 13 (54 mg)
in toluene (15 mL). The mixture was refluxed for 5 h under
N2. The solvent and excess DMAD were removed with ro-
tavapor. The residue was purified with preparative silica TLC
using CH2Cl2/acetone (v/v 15/1) as developing solvent. The
intermediate benzobacteriachlorin (crude) along with the
unreacted 13 (12 mg) was also recovered. The above interme-
diate compound was dissolved in CH2Cl2 (30 mL), and 2 drops
of DBU was added to above solution. The resultant solution
was stirred at room temperature for 5 min. The solvent was
removed, and the residue was purified with preparative silica
TLC using CH2Cl2/acetone (v/v 15/1) as developing solvent. The
title compound (13 mg) was obtained as a mixture of two
isomers (2:1) in 26% converted yield in two steps. UV-vis in
CH2Cl2 [λmax (ꢀ)]: 402 (38 000), 474 (30 000), 573 (8200), 687
1
(7400), 759 (20 000). H NMR δ 9.02, 8.98 (1H, each s, meso
H), 8.33 (1H, splitting s, meso H), 8.21, 8.20 (1H, each s, meso
H), 7.71, 7.70 (1H, each d, J ) 5.4 Hz, J ) 5.9 Hz, H-82), 7.15,
7.10 (1H, each d, J ) 5.4 Hz, J ) 6.1 Hz, H-81), 4.91, 4.89
(1H, m, H-17), 4.80, 4.78, 4.73, 4.53 (1H, each s, H-84), 4.15,
4.09, 3.95, 3.54, 3.40, 3.14, 3.10 (21H, each splitting s, 5 ×
methyl esters, CH3-2 and CH3-12), 4.13 (1H, m, H-18), 3.61
(2H, q, J ) 7.5 Hz, CH3CH2-3), 2.33, 2.08, 1.79 (1H, 2H, 1H,
m, CH3OOCCH2CH2-17), 1.75 (3H, splitting d, CH3-18), 1.66
(3H, s, CH3-7), 1.64 (3H, t, J ) 7.6 Hz, CH3CH2-3), 0.18, -
0.15 (each 1H, br, 2 × NH). MS (FAB) m/z 766.2 (M+, 100).
HRMS (FAB): Calcd for C42H46N4O10, 766.3214; Found
766.3228.
Ben zoba cter ioch lor in s 19. DMAD (2.0 mL) was added to
a solution of 8-Vinylmesopurpurin-18-N-hexylimide methyl
ester 14 (95 mg) in toluene (20 mL). The mixture was refluxed
for 5 h under N2. The solvent and excess DMAD were removed
with rotavapor. The residue was purified with preparative
silica TLC using CH2Cl2/acetone (v/v 50/1) as developing
solvent. The intermediate benzobacteriochlorin (55 mg) was
obtained and a small amount of the unreacted 14 was
recovered. The intermediate product was dissolved in CH2Cl2
(30 mL), 2 drops of DBU was added, and the resulting solution
was stirred at room temperature for 5 min. The solvent was
removed, and the residue was purified with preparative silica
TLC using CH2Cl2/acetone (v/v 50/1) as developing solvent. The
title compound (18 mg) was obtained as a mixture of four
isomers in 37% converted yield in two steps. UV-vis in CH2-
Cl2 [λmax (ꢀ)]: 341 (36740), 383 (39820), 436 (70 620), 483
(35 640), 668 (6820), 714 (9900), 801 (29 810). 1H NMR δ 9.11,
9.10, 9.03, 8.69, 8.64, 8.34, 8.33, 8.31, 8.23 (3H, each s, 3 ×
In Vivo P h otosen sitizin g Activity. C3H/HeJ mice were
injected intradermally with 2 × 105 RIF cells in 30 µL of
Hanks’s balanced salt solution without Ca2+ and Mg2+, into
the flank and allowed to grow until they were 4-5 mm in
diameter. The mice were injected (iv) with photosensitizers
(5.0 µmol/kg). At 24 h postinjection, the mice were restrained
in plastic holders and then treated with laser light from an

5358 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
Li et al.
(7) Yon-Hin, P.; Wijesekera, T. P.; Dolphin, D. A Convenient
Synthetic Route To The Bacteriochlorin Chromophore. Tetra-
hedron Lett. 1991, 32 (25), 2875-2878.
(8) Morgan, A. R.; Skalkos, D.; Garbo, G. M.; Keck, R. W.; Selman,
S. H. Synthesis and in vivo Photodynamic Activity of Some
Bacteriochlorin Derivatives Against Bladder-Tumors in Rodents.
J . Med. Chem. 1991, 34 (7), 2126-2133.
(9) Cavaleiro, J . A. S.; Neves, M. G. P. M.; Tome, A. C.; Silva, A. M.
S.; Faustino, M. A. F.; Lacerda, P. S.; Silva, A. M. G. Porphyrin
Derivatives: Synthesis And Potential Applications. J . Heterocycl.
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S. Cavaleiro, J . A. S.; Perrone, D.; Dondoni, A. Porphyrins In
1,3-Dipolar Cycloaddition Reactions With Sugar Nitrones. Syn-
thesis Of Glycoconjugated Isoxazolidine-Fused Chlorins And
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M. O.; Dougherty, T. J .; Smith, K. M. Pinacol-Pinacolone
Rearrangements In Vic-Dihydroxychlorins And Bacteriochlor-
ins: Effect of Substituents At the Peripheral Positions. J . Org.
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argon pumped dye laser in the range of 737-805 nm for a total
fluence of 135 J /cm2 at a fluence rate of 75 mW/cm2. The mice
(5 mice/group) were checked daily, the tumors were measured
using two orthogonal measurements L and W (perpendicular
to L), and the volumes were calculated using the formula V )
LW2/2 and recorded. Mice were considered cured if there was
no palpable tumor at 30 days post-PDT treatment.
P er ip h er a l Ben zod ia zep in e Recep tor Bin d in g Stu d -
ies. The experiment was performed as follows. A 50 µL amount
of drug solution with decreasing concentrations (3 × 10-4, 15
× 10-5, 3 × 10-5, 15 × 10-6, 3 × 10-6, 15 × 10-7, 3 × 10-7, 3
× 10-8, 3 × 10-9, and 3 × 10-10) were added into labeled tubes
(5 mL disposable glass borosilicate) containing 50 µL of cells
(1 × 106) and 50 µL of [3H]-PK11195 (final concentration 46
nM). The resultant samples were incubated at 4 °C for 1 h,
and then 3.0 mL of Tris buffer was added to each tube to stop
the displacement reactions. The solutions were then filtered
by vacuum on GF/C Whatman filters presoaked in Tris buffer
0.5% w/v polyethylenimine (to help prevent nonspecific binding
to the filter). The filters were washed with Tris buffer (3 × 4
mL) and transferred into scintillation vials. A 4 mL amount
of scintillation fluid (Universol, ICN) was added to each
scintillation vial, shaken to dissolve [3H]-PK11195, and then
kept dark to equilibrate for 1 h. The samples were counted in
a beta counter (Beckman LS 5801). Control samples replaced
cells and/or photosensitizer with Tris buffer. Data were
processed with Origin 5.0 (Microcal Software Inc., Northamp-
ton, MA).
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vs Non-Fluorinated Photosensitizers For Photodynamic Therapy.
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Ack n ow led gm en t. The authors are thankful to the
NIH (CA 55791) for the financial support. Partial
support by shared resources of the Roswell Park Cancer
Support Grant (P30CA16056) is also acknowledged. The
help rendered by J oseph Missert in extracting meth-
ylpheophorbide a from alga Spirulina pacifica and
Adam Sumlin for performing the skin phototoxicity
experiments is highly appreciated. The mass spectrom-
etry analyses of all the intermediates and the final
products were obtained by Beverly Chamberlin, Mass
Spectrometry Facility, MSU, East Lansing, MI, and at
Biopolymer Facility, RPCI, Buffalo, NY.
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