Synthesis, spectroscopic, and in vitro photosensitizing efficacy of ketobacteriochlorins derived from ring-B and ring-D reduced chlorins via pinacol-pinacolone rearrangement

Article abstract of DOI:10.1021/jo201688c

In this report, we present a regioselective oxidation of a series bacteriochlorins, which on reacting with either ferric chloride (FeCl ₃) or 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) yielded the corresponding ring-B or ring-D reduced chlorins. The effect of the number of electron-withdrawing groups present at the peripheral position, with or without a fused isocyclic ring (ring-E), did not make any significant difference in regioselective oxidation of the pyrrole rings. However, depending on the nature of substituents, the intermediate bis-dihydroxy bacteriochlorins on subjecting to pinacol-pinacolone reaction conditions gave various ketochlorins. The introduction of the keto-group at a particular position in the molecule possibly depends on the stability of the intermediate carbocation species. The newly synthesized bacteriochlorins show strong long-wavelength absorption and produced significant in vitro (Colon26 cells) photosensitizing ability. Among the compounds tested, the bacteriochlorins containing a keto-group at position 7 of ring-B with cleaved five-member isocyclic ring showed the best efficacy.

Full text of DOI:10.1021/jo201688c

Article
pubs.acs.org/joc
Synthesis, Spectroscopic, and in Vitro Photosensitizing Efficacy of
Ketobacteriochlorins Derived from Ring-B and Ring-D Reduced
Chlorins via Pinacol−Pinacolone Rearrangement
Penny Joshi,† Manivannan Ethirajan,† Lalit N. Goswami,† Avinash Srivatsan,†,‡ Joseph R. Missert,†
and Ravindra K. Pandey*,†
‡
†Chemistry Division, PDT Center, Cell Stress Biology and Department of Pharmacology, Roswell Park Cancer Institute, Buffalo,
New York 14263, United States
S
* Supporting Information
ABSTRACT: In this report, we present a regioselective
oxidation of a series bacteriochlorins, which on reacting with
either ferric chloride (FeCl3) or 2,3-dichloro-5,6-dicyanoben-
zoquinone (DDQ) yielded the corresponding ring-B or ring-D
reduced chlorins. The effect of the number of electron-
withdrawing groups present at the peripheral position, with or
without a fused isocyclic ring (ring-E), did not make any
significant difference in regioselective oxidation of the pyrrole
rings. However, depending on the nature of substituents, the intermediate bis-dihydroxy bacteriochlorins on subjecting to
pinacol−pinacolone reaction conditions gave various ketochlorins. The introduction of the keto-group at a particular position in
the molecule possibly depends on the stability of the intermediate carbocation species. The newly synthesized bacteriochlorins
show strong long-wavelength absorption and produced significant in vitro (Colon26 cells) photosensitizing ability. Among the
compounds tested, the bacteriochlorins containing a keto-group at position 7 of ring-B with cleaved five-member isocyclic ring
showed the best efficacy.
of bacteriochlorins in the bacterial photosynthetic reaction
center20 and in the treatment of cancer by PDT. There are
several bacteriochlorophyll-a analogues, which are currently
under advanced human clinical trials (e.g., Tookad)21 or at the
advanced preclinical studies (bacteriopurpurinimides) for the
treatment of cancer.22 The only naturally occurring bacterio-
chlorin that is not involved in photosynthesis is the
tolyporphyrin isolated by Prinsep et al. from the blue-green
alga Tolypothrix nodosa.23 This compound, which enhances the
cytotoxicity of adriamycin or viablastine in SK-VLB cells at
doses as low as 1 mg/mL, is characterized as a multidrug
resistance (MDR) reversing agent. Kishi's group at Harvard
University synthesized the tolyporphyrin by the extension of
the Eschenmoser sulfide contraction/iminoester cyclization
method with long-wavelength absorption near 675 nm (ε =
22000).24 However, there are no reports regarding the in vivo
photosensitizing ability of this novel compound. Lindsey and
co-workers25 have also reported facile syntheses of certain
bacteriochlorins starting from pyrroles with variable lip-
ophilicity following multistep synthetic methodologies. Some
of these compounds exhibit interesting photophysical proper-
ties and could be potential candidates for PDT.
INTRODUCTION
■
In developing effective agents for photodynamic therapy
(PDT), the structure−activity relationship (SAR) and
quantitative structure−activity relationship (QSAR) studies
have been proven to be extremely useful.1−3 The Roswell Park
Cancer Institute (RPCI) group was the first to investigate the
SAR and QSAR studies on a series of the alkyl ether analogues
of pyropheophorbide-a (chlorophyll-a derivative) and observed
a parabolic relationship between overall lipophilicity and PDT
efficacy.4,5 Among the compounds investigated, 3-(1′-hexylox-
yethyl) derivative (HPPH) showed the best PDT efficacy
without any significant toxicity and is currently undergoing
phase I/II clinical trials for several indications.6−8 The SAR
approach has also been found useful in developing other
systems, for example, phthalocyanines,9 -tetra (m-hydroxyphenyl)
chlorins,10 extended porphyrins (texaphyrins),11 porphyceins,12
purpurinimides13−15 and bacteriopurpurinimides,16 and other
bacteriochlorin analogues.17,18 The biological studies have
indicated that besides overall lipophilicity, the position of the
substituents present in the photosensitizer(s) makes a tremen-
dous difference in long-term PDT efficacy.
Bacteriochlorins are a class of the tetrapyrrolic system in
which two pyrrole rings diagonal to each other are reduced.19
These chromophores exhibit long-wavelength absorption in the
range of 720−800 nm depending on the nature of substituents
present at the peripheral positions of the molecules. In recent
years, enormous interest has been generated due to the utility
One of the simplest methods that has been used extensively
for the conversion of porphyrins/chlorins to the corresponding
Received: March 1, 2011
Published: September 28, 2011
© 2011 American Chemical Society
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vic-dihydroxy chlorins and bacteriochlorins, respectively, is the
osmium tetroxide-mediated oxidation.26 These intermediate
“diols” on reacting under acidic conditions produce the
corresponding keto-chlorins and keto-bacteriochlorins, respec-
tively. The position of the keto-group in the resulting chlorins
and bacteriochlorins depends on the stability of the
intermediate carbocation, which is also influenced by the
nature of the substituents (electron withdrawing or electron
donating) present in the molecules (Scheme 1).
example, diol 8 gave 7-keto-bacteriochlorin 9 as a major and 9a
as a minor product, and it is exactly matched with the
previously reported procedure,26c whereas diol 10 under similar
reaction conditions gave mainly 18-keto-bacteriochlorin 11.
These results suggest that the formation of intermediate
carbocation species is certainly directing the position of the
keto-group, which is possibly also being influenced by
the electron-withdrawing acetyl group26 present at position 3
of the 7,8- and 17,18-dihydroxybacteriochlorins (Scheme 3).
For investigating the effect of the variable number of
electron-withdrawing groups in bacteriochlorin diols under
pinacol−pinacolone rearrangement conditions, the isocyclic
ring in methyl bacteriopheophorbide-a 12 was cleaved on
reacting with sodium methoxide, and the resulting bacterio-
chlorin 13 was obtained in excellent yield.33 Subsequent
treatment of 13 with collidine at refluxing temperature gave
rhodobacteriochlorin 14 as the minor (15%) and the
corresponding ring-B reduced chlorin 15 as the major product
(85%), which on reacting with osmium tetraoxide/pyridine
yielded 17,18-dihydroxybacteriochlorin diol 16 as an isomeric
mixture (cis-hydroxy groups up or down relative to ring-B).
Further treatment of 16 with concentrated sulfuric
acid gave 18-keto-bacteriochlorin 17 as a major product
(Scheme 4). As expected, the oxidation of bacteriochlorin 13
with DDQ and FeCl3 afforded the corresponding ring-D and
ring-B reduced chlorins 18 and 22, respectively, which on
reacting with osmium tetroxide produced the corresponding
diols 19 and 23 in quantitative yields. Interestingly, further
reaction of these diols with concentrated sulfuric acid gave a
mixture of 7-keto-bacteriochlorin 20 and 8-keto-bacteriochlorin
21 (from 19) as an isomeric mixture. Separation of these
mixtures (20/21) by the usual chromatographic technique was
not successful. Finally, the isomeric mixture was separated by
HPLC (column, Luna; eluting solvent, using ethyl acetate and
hexane as the eluting solvents) delivered the pure regioisomer
8-keto-isomer 20 and regioisomer 7-keto-isomer 21 were
isolated in 19 and 25%, respectively. Whereas diol 23 in which
the cis-hydroxyl groups were present in ring-D yielded only
18-keto-bacteriochlorin 17 (in which the β-ketoester function-
ality present at position 15 was cleaved) as a major product.
The elimination of β-keto-ester functionality was not observed
on treating 19 under similar reaction conditions, and these
results were surprising (Scheme 5). The results presented
herein confirm our previous findings,34 which suggests that the
position and presence of the number of electron-withdrawing
groups present at the peripheral position of chlorin diols makes
a significant impact in the stability of the intermediate
carbocation during the pinacol−pinacolone reaction, which
obviously determines the formation of the resulting keto-
chlorins under acid-catalyzed rearrangement. Therefore, in our
study to investigate the effect of more than two electron-
withdrawing groups under acid-catalyzed conditions, methyl
bacteriopheophorbide-a 12 containing a methoxycarbonyl
group at position 132 of the fused isocyclic ring was used as
a substrate, which on reacting with DDQ and FeCl3 produced
exclusively the ring-D reduced 24 and ring-B reduced chlorins
25, respectively (Scheme 6). Unfortunately, the corresponding
bacteriochlorin diols obtained by reacting 24 and 25 with
OsO4/pyridine as such or under acidic conditions at room
temperature were not stable and produced complex mixtures,
which were not characterized.
Chang and Sotiriou27 were the first to show that free-base
octaethylchlorin or its metalated analogue upon reaction with
osmium tetroxide can be converted into the corresponding
keto-bacteriochlorins in a reasonable yield. Bonnett et al.28
employed this approach to prepare bacteriochlorin diols by
reacting ketoethyl chlorin with osmium tetroxide. Some of
these analogues showed considerable PDT efficacy in vitro.
Pandey et al. in collaboration with Smith and co-workers
extended this approach to the pyropheophorbide-a, purpurin-
18, and purpurinimide systems, and a series of stable keto-
bacteriochlorin analogues were synthesized.29 Most of the
resulting bacteriochlorins showed long-wavelength absorption
in the range of 730−800 nm, and some of them were effective
both in vitro and in vivo.
So far, most of the keto-bacteriochlorins [the keto-group is
present at either position 7 or position 8 (ring-B)] investigated
for PDT efficacy are derived from ring-D reduced chlorins.30
The main objectives of the work presented herein were (i) to
develop an efficient synthetic approach for ring-B reduced
chlorins from certain selected bacteriochlorins derived from
naturally occurring bacteriochlorophyll-a and then convert
them into the corresponding ring-D ketobacteriochlorins
(17-keto- and 18 keto-) and (ii) to compare the photophysical
and photosensitizing abilities of these novel structures.
RESULTS AND DISCUSSION
■
For our present study, two types of bacteriochlorins 6 and 13
containing either a fused five-member isocyclic ring system or a
methoxycarbonyl group present at position 13 (ring-C pyrrole)
were used as a substrate, and these were obtained from
bacteriochlorophyll-a by following the literature procedure.31 We
have recently reported an efficient regioselective preparation of
ring-B and ring-D reduced chlorins from bacteriochlorins.32 We
extended this approach for bacteriopyropheophorbide-a, which on
treating with DDQ at room temperature produced mainly D-ring
reduced chlorin 5, whereas on treating 6 with ferric chloride
(FeCl3) produced ring-B reduced chlorin 7 in excellent yields
(Scheme 2). Analysis of the NMR data confirmed the structures of
both isomers. One of the distinct differences was the resonances of
the −CH2 protons of the five-member fused isocyclic ring, which
showed an ABX pattern at 5.22 ppm in compound 5 due to
reduced ring-D, whereas these protons appeared as a singlet at
5.44 ppm in compound 7 (ring-D oxidized).
For determining the photophysical and photosensitizing
abilities of various keto-bacteriochlorin isomers, the keto-group
was regioselectively introduced in either the ring-B or the ring-
D pyrrole ring of the bacteriochlorin system. For the synthesis
of the desired analogues, the ring-D and ring-B reduced
chlorins 5 and 7 were individually reacted with OsO4, and the
resulting diols 8 and 10 (both as isomeric mixtures, cis-diol up
and cis-diol down with respect to the diagonal reduced ring)
were isolated in 60% yield. Reaction of these diols
independently with concentrated sulfuric acid under pinacol−
pinacolone conditions produced some interesting results. For
The mechanism of the formation of keto-chlorins under
pinacol−pinacolone reaction is well established. However, the
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Scheme 1. Conversion of Chlorins to Keto-bacteriochlorins
Scheme 2. Regioselective Synthesis of Ring-D and Ring-B Chlorins (5 and 7, Respectively) from Bacteriochlorin 6
Scheme 3. Synthesis of Ring-B and Ring-D Reduced Ketobacteriochlorins from Ring-B and Ring-D Reduced Chlorins
compounds investigated in this study shows some interesting
results, and the mechanism for the formation of these analogues
is illustrated in Scheme 7. In brief, in the vic-dihydroxy analogue
containing methyl and ethyl groups present at adjacent
positions, the migration of the methyl group was preferred
over the ethyl group, and the ethyl group migrated product was
isolated in a minor quantity. Interestingly, the vic-dihydroxy
analogues containing methyl and propionic ester functionality
at adjacent positions on treating under similar acidic conditions
gave only the methyl migrated product (18-keto-), which of
course depends upon the stability of intermediate carbocation
species. The previous studies from others26c and our own
laboratory34 suggest that in the porphyrin system the number
of electron-withdrawing groups present at the peripheral
position of the porphyrin skeleton makes a remarkable
difference in the stability of intermediate cabocation(s),
which dictates the formation of the corresponding keto-
analogues.
The purity of ketobacteriochlorins 11, 17, 20, and 21 was
ascertained by HPLC, and the structures were assigned by
NMR and mass spectrometry analyses. The bacteriochlorins 20
and 21, which were initially isolated as isomeric mixture, were
separated into individual isomers by HPLC using Luna column,
eluted with 30% ethyl acetate−hexane. The retention times for
bacteriochlorins 11, 17, 20, and 21 were 12.20, 10.76, 21.13,
and 18.47 min, respectively (Figure 1). A slight shoulder in the
HPLC chromatogram of 21 could be due to repeated use of the
HPLC column for a long time. The purity of the product was
also confirmed by using a reverse phase HPLC column
(Symmetry C18 column, dimensions 4.6 mm × 150 mm),
eluted with 90% CH3OH and 10% H2O, and the flow rate was
adjusted to 1.0 mL/min. For details, see the Supporting
Information.
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Scheme 4. Synthesis of Rhodobacteriochlorin 14 and the Corresponding 18-Keto-bacteriochlorin 16
Scheme 5. Synthesis of 7-Keto- and 8-Keto-Ring-D Reduced and 18-Keto-Ring-B Reduced Bacteriochlorins
A detailed NMR study confirmed the structures of the
proposed bacteriochlorins. As can be seen from the results
summarized in Figure 2 (only partial NMR Spectra are shown)
the meso-protons at positions 5, 10, and 20 for bacteriochlorins
11, 17, 20, and 21 show a significant shift, which could be due
to variation in electron density at these positions. Furthermore,
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Scheme 6. Ring-B and Ring-D Reduced Chlorins Derived from Methyl Bacteriopheophorbide-a on Reacting with OSO4/H2SO4
Gave a Complex Mixture
Scheme 7. Proposed Mechanism for the Formation Ring-B and Ring-D Reduced Keto-bacteriochlorins Obtained from Their
Corresponding Diols under Pinacol−Pinacolone Reaction Conditions
Figure 1. HPLC chromatogram of bacteriochlorin 11, 17, 20, and 21 (for details, see the Supporting Information).
in 8-keto-bacteriochlorin 20, the 71-CH3 protons appeared as a
triplet around 0.5 ppm, whereas for the other isomer 21, these
protons were observed as a multiplet at 0.45−0.51 ppm, which
could be due to the presence of epimeric mixture. In compound 20,
the 7-CH3 was observed as a singlet at 1.82 ppm, while in
compound 21, it exhibited two singlets around 1.90/1.92 ppm
(epimeric mixture) for 8-CH3. The 7-CH2 and 8-CH2 in isomer 20
and 21 appeared as nice quartets at 2.56 and 2.62 ppm, respectively.
These assignments were further confirmed by 2D NMR studies.
To confirm the position of dialky groups (methyl/ethyl) or
methyl/propionic ester of the major regioisomers, NOESY
experiment was performed on isomers 11, 17, and 21. In the
case of compound 21, the resonances for the ethyl group at
position C-8 were chosen as a starting point for the
interpretation of the NOESY results. A peak at δ 2.62 for the
CH2CH3 protons showed NOE correlation with the adjacent
meso- proton at δ 8.97, which is C-10; the −CH3protons of the
ethyl group also showed a strong interaction with the methyl
proton, which is attached to the same carbon (C-8) as well as
adjacent meso-10-H proton. No correlation was observed
between the CH3 protons and the acetyl protons (C-3),
which further confirmed the proposed structural assignment for
21. Following a similar approach, the structures of compound
11 and 17 were also established (Figure 3).
Spectroscopic Properties of Keto-Bacteriochlorins. The
absorption and fluorescence characteristics of keto-bacteriochlorins
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Figure 2. Partial 1H NMR of keto-bacteriochlorins 11, 17, 20, and 21 (only meso-regions are shown). For details, see the Experimental Section and
Supporting Information.
Figure 3. NOE correlations of ketobacteriochlorins 11, 17, and 21.
Figure 4. Electronic absorption spectra and fluorescence emission spectra of keto-bacteriochlorins 9, 9a, 11, 17, 20, and 21 at equimolar
concentration (1.2 μM in CH2Cl2).
9, 9a, 11, 17, 20, and 21 as epimeric mixtures were measured in
dichloromethane. These bacteriochlorins exhibited the long-
wavelength absorptions at 725, 714, 715, 731, 737, and 732 nm
and fluorescence at 736, 727, 732, 735, 765, and 741, respectively.
Among all of the analogues, the 18-keto-bacteriochlorin 20
exhibited the largest Stokes shift (28 nm), whereas bacteriochlorin
17 lacked the five-member ring but, instead, containing a
methoxycarbonyl- functionality at position 13, showed a much
smaller shift of 4 nm. The observed shift was in the order of 20 >
11 > 9a > 9 > 21 > 17 (Figure 4).
In Vitro Photosensitizing Efficacy. The keto-bacterio-
chlorins 9, 9a, 11, and 17 and 20 and 21 (as an isomeric
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Figure 5. (A) In vitro photosensitizing efficacy of bacteriochlorins 9, 9a (containing a keto-group in ring-B), 11 (containing a keto-group in ring-D),
17, and 20/21 isomeric mixture (bearing keto-groups in ring-B) at variable concentrations and light doses. The Colon26 tumor cells were exposed to
light at 1 J/cm2 at 24 h postincubation, and the MTT assay was performed after 48 h. (B) In vitro dark toxicity of photosensitizers 9, 9a, 11, 17, and
20/21 incubated in Colon26 tumor cells for 24 h but not exposed to light. (C) The inset figure shows the PDT efficacy of the separated isomers 20
and 21 at similar concentrations as the parent mixture and exposed to light (dose, 1 J/cm2).
mixture and as individual isomers) were evaluated for in vitro
PDT efficacy in Colon26 tumor cells. The cells were incubated
with increasing concentrations of the photosensitizers (3−100
nM) for 24 h and were then exposed to variable light doses
(0−2 J/cm2) at an appropriate wavelength corresponding to
the long-wavelength absorption of each compound formulated
in 17% BCS in PBS. The MTT assay35 was performed after 48
h (for details, see the Experimental Section). Among the
photosensitizers evaluated, the bacteriochlorins 9 and 9a
containing a keto-group in ring-B were less effective than
bacteriochlorins 11 and 17 containing a keto-group in ring-D.
However, the photosensitizer containing a fused five-member
isocyclic ring showed lower activity than 17 bearing a −CO2Me
group at position 13. In contrast, bacteriochlorins 20 and 21 (as
a 43:57 mixture) containing a keto-group in ring-B showed
enhanced activity than 11 and 17. To investigate the efficacy of
the individual isomer, the isomeric mixture of 20 and 21 was
duly separated into individual isomers by HPLC (see Figure 1
and the Supporting Information), and the in vitro photo-
sensitizing efficacy of both the isomers was investigated under
similar experimental conditions except that the cells were
exposed to light at the appropriate long-wavelength absorptions
of the photosensitizers (bacteriochlorin 20: λmax, 737 nm; and
21: λmax, 732 nm). Under similar parameters, isomer 21
showed significantly higher efficacy than the isomer 20 (see the
inset in Figure 5C). As mentioned earlier, isomer 21 constitutes
a major part of the mixture 20/21 (Scheme 5), which could
explain the reason for similar PDT efficacy of the mixture as
compared to isomer 21.
which are otherwise difficult to synthesize. As compared to
naturally occurring bacteriochlorins, the keto-bacteriochlorins
obtained from the respective chlorins showed enhanced stability
with significant in vitro photosensitizing efficacy. The acetyl
group present at position 3 of the chlorin systems provides a
unique opportunity to alter the overall lipophilicity of the
molecules to investigate the effect of such modifications in in
vivo clearance and PDT efficacy. Easy access to these molecules
should also generate a great interest in developing new
supramolecular structures and synthetic models for under-
standing the bacterial photosynthetic reaction centers.
EXPERIMENTAL SECTION
■
All reactions were carried out in heat gun-dried glassware under an
atmosphere of nitrogen with magnetic stirring. Thin-layer chromatog-
raphy (TLC) was done on precoated silica gel GF PE sheets (layer
thickness, 0.25 mm) and aluminum oxide NF PE sheets. Column
chromatography was performed either over silica gel 60 (70−230
mesh) or neutral alumina. In some cases, preparative TLC plates were
also used for the purification. Solvents were purified as follows: trace
amounts of water and oxygen from THF were removed by refluxing
over sodium under an inert atmosphere. Dichloromethane was dried
over P2O5. Anhydrous DMF, triethylamine, pyridine, and other
common chromatographic solvents were obtained from commercial
suppliers and used without further purification. NMR spectra were
recorded on a 400 MHz spectrometer. All chemical shifts are reported
in parts per million (δ). 1H NMR (400 MHz) spectra were recorded at
room temperature in CDCl3 or CD3OD solutions and referenced to
residual CHCl3 (7.26 ppm) or TMS (0.00 ppm). EI-mass spectra were
carried out on a ion-trap mass spectrometer equipped with a
pneumatically assisted electrospray ionization source, operating in
positive mode. UV−visible spectra were recorded on FT UV−visible
spectrophotometer using dichloromethane/THF as solvent. All
photophysical experiments were carried out using spectroscopic
grade solvents.
The in vitro results depicted in Figure 5 suggest that besides
the position of the keto-group (ring-B vs ring-D), the nature of
the substituents present at the periphery of bacteriochlorin
system makes a significant impact in PDT efficacy. However,
further study with a series of analogues is required to establish a
“true” SAR, and these studies are currently in progress. Efforts
are also underway to investigate a correlation between the cell
uptake, intracellular localization, and STAT-3 dimerization36
with in vitro/in vivo PDT efficacy, and these results will be
published in an appropriate journal.
HPLC Method. HPLC analysis of final products was carried out
using a Waters Delta 600 System consisting of the 600 Controller, 600
Fluid Handling Unit, and 2998 Photodiode Array Detector equipped
with a Phenomenex Luna column, 5 μm particle size, with dimensions
4.6 mm × 250 mm. A gradient mobile phase program was used as
follows: starting at 30% ethyl acetate/70% hexane linear gradient to
70% ethyl acetate/30% hexane over 80 min; the flow rate was 1.0 mL/
min.
Methyl 3-Acetyl-17,18-dihydroxybacteriopyropheophor-
bide-a (10). Compound 7 (50.0 mg, 0.09 mmol) was taken in a
round-bottom flask (100 mL) and dissolved in 30 mL of dry
dichloromethane. To this were added OsO4 (100.0 mg) and pyridine
CONCLUSION
■
In summary, the work discussed in this manuscript describes an
efficient approach for the synthesis of ring-B and ring-D
reduced chlorins from naturally occurring bacteriochlorophyll-a,
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(1.0 mL), and the reaction mixture was stirred vigorously at room
temperature for 24 h. The reaction was monitored by UV−vis and
TLC. H2S gas was then bubbled into the reaction mixture for 5 min,
and then, the excess of H2S was removed by bubbling N2 gas for
another 30 min. Water was added to the reaction mixture and then
extracted with dichloromethane (100 mL), and the organic layer was
separated, washed with water, dried over sodium sulfate, and
concentrated. The crude thus obtained was chromatographed over
silica gel using 0.5−1% CH3OH/CH2Cl2 mixture as an eluent to give
UV−vis [ethyl ether, λmax nm (ε)]: 358 (11.8 × 104), 385 (6.76 ×
104), 525 (2.89 × 104), 680 (1.22 × 104), 749 (6.75 × 104); (in
CH2Cl2): 362 (10.8 × 104), 389 (5.81 × 104), 530 (2.84 × 104), 683
(1.11 × 104), 754 (6.27 × 104). 1H NMR δ (in CDCl3): 8.98 (s, 1H, 5-
H), 8.49 (s, 1H, 10-H), 8.41 (s, 1H, 20-H), 6.08 (s, 1H, 132-H), 4.27
(m, 2H, 1H for 7-H, 1H for 8-H), 4.02 (m, 2H, 1H for 17-H, 1H for
18-H), 3.85 (s, 3H, 12-CH3), 3.59 (s, 3H, 2-CH3), 3.49 (s, 3H, 132-
COOCH3), 3.45 (s, 3H, 17-CH2CH2 COOCH3), 3.16 (s, 3H, 3-
COCH3), 2.52 (m, 2H, 17-CH2CH2COOCH3), 2.34 (m, 2H, 17-CH2-
CH2COOCH3), 2.25 (m, 2H, 8-CH2CH3), 1.80 (d, J = 7.4 Hz, 3H, 7-
CH3), 1.73 (d, J = 7.9 Hz, 3H, 18-CH3), 1.12 (t, J = 7.2 Hz, 3H, 8-
CH2CH3), 0.47 (s, 1H, NH), −0.95 (s, 1H, NH). EIMS (m/z): 626
(M + 1). HRMS: calcd for C36H41N4O6 [MH]+, 625.3026; found,
625.3043.
3-Acetyl-bacteriochlorin 15-Glyoxilic Acid Trimethyl Ester
(13). Methyl bacteriopheophorbide-a 12 (100 mg, 016 mmol) was
taken in a round-bottom flask (100 mL), and dry THF (30 mL) was
added. A 0.3 mL amount of NaOMe (25% in CH3OH) was dissolved
in 10 mL of dry THF and added slowly via syringe to the reaction
mixture under vigorous stirring. The reaction mixture was stirred at
room temperature for 4 h and was quenched with 5% acetic acid−H2O
and extracted with dichloromethane (100 mL). The organic layer
separated, washed with brine, dried over sodium sulfate, and
concentrated to dryness. A trace of acetic acid was removed under
high vacuum. The crude product was redissolved in dichloromethane
and treated with diazomethane. The reaction mixture was stirred for
10 min, and then, an excess of diazomethane was removed by bubbling
N2 gas. The reaction mixture concentrated and chromatographed over
silica gel using 1−3% CH3OH/dichloromethane gradient as an eluent
to obtain the compound as a major product. Slow moving brown-red
band on silica. Yield, 40.0 mg (37.2%); mp > 260 °C (decomp.). UV−
vis λmax (in CH2Cl2): 782 nm (ε 5.19 × 104), 748 (10.4 × 104), 543
(3.12 × 104), 410 (5.27 × 104) and 363 (7.61 × 104).1H NMR (400
MHz, CDCl3): δ 9.14 (s, 1H, meso-H), 8.68 (s, 1H, meso-H), 8.54 (s,
1H, meso-H), 4.48 (m, 1H, 17-H), 4.26 (m, 1H, 8-H), 4.22 (m, 1H,
18-H), 4.10 (s, 3H, CO2Me), 4.07 (m, 1H, 7-H), 3.91 (s, 3H,
CO2Me), 3.53 (s, 3H, CO2Me), 3.51 (s, 3H, 12-CH3), 3.44 (s, 3H, 2-
CH3), 3.15 (s, 3H, COCH3), 2.31 (m, 2H, 172-CH2), 2.06−2.00 (m,
3H, 8-CH2CH3 and 171-CH2), 1.83 (d, 3H, 7-CH3, J = 7.2 Hz), 1.76
(d, 3H, 18-CH3, J = 7.6 Hz), 1.72 (m, 1H, 171-CH2), 1.06 (t, 3H, 8-
CH2CH3, J = 7.6 Hz), −0.45 (brs, 1H, NH), −0.53 (brs, 1H, NH).
EIMS (m/z): 671.3 (M + H). HRMS: calcd for C37H42N4O8,
670.3002; found, 670.3030.
Ring-B Reduced Chlorin (15). 3-Acetyl-bacteriochlorin 15-
glyoxilic acid trimethyl ester 13 (100.0 mg, 0.15 mmol) was refluxed
in collidine (15 mL) for 30 min. The progress of the reaction was
monitored by UV−vis and TLC. After completion, the reaction
mixture was concentrated to dryness using high vacuum and purified
on alumina (G-III) column using dichloromethane−hexane mixture as
the eluent. Yield, 50.0 mg (57.6%). UV−vis (CH2Cl2, λmax, nm, (ε):
410.0 (8.16 × 104), 509.0 (7.50 × 103), 683.1 (4.16 × 104). 1H NMR
(400 MHz, CDCl3): δ 10.50 (s, 1H, meso-H), 9.71 (s, 1H, meso-H),
9.60 (s, 1H, meso-H), 8.95 (s, 1H, meso-H), 4.60 (m, 1H, 8-H), 4.40 (s,
3H, CO2Me), 4.36 (m, 1H, 7-H), 4.12 (t, 2H, 171-CH2, J = 7.6 Hz),
3.77 (s, 3H, ring-CH3), 3.75 (s, 3H, ring-CH3), 3.70 (s, 3H, CO2Me),
3.35 (s, 3H, ring-CH3), 3.26 (s, 3H, COCH3), 3.18 (t, 2H, 172-CH2,
J = 7.6 Hz), 2.51−2.46 (m, 1H, 8-CHHCH3), 2.20−2.15 (m, 1H, 8-
CHHCH3), 1.92 (d, 3H, 7-CH3, J = 7.6 Hz), 1.13 (t, 3H, 8-CH2CH3,
J = 7.6 Hz), −1.79 (brs, 2H, NH). 13C NMR (100 MHz, CDCl3): δ
198.8, 173.5, 171.5, 168.9, 166.5, 152.7, 151.9, 140.8, 138.7, 138.1,
137.9, 137.3, 137.2, 133.5, 132.1, 130.1, 124.3, 101.9, 100.9, 96.7, 94.9,
57.4, 51.6, 47.8, 37.0, 33.3, 30.2, 29.6, 23.8, 21.9, 14., 13.2, 11.5, 10.8.
EIMS (m/z): 583.8 (M+ + 1). HRMS: calcd for C34H39N4O5 [MH]+,
583.2920; found, 583.2938.
1
10 as a mixture of cis-diols (49:51). Yield, 23.0 mg (44%). H NMR
(400 MHz, CDCl3): δ 9.08 and 9.02 (s, 1H, 5-H), 8.67 and 8.64 (s,
1H, 10-H), 8.30 and 7.96 (s, 1H, 20-H), 5.24 (dd, 1H, 13CHH, J = 20
Hz), 4.89 (dd, 1H, 13CHH), 4.30−4.25 (m, 1H, 8-H), 4.05 and 3.96
(m, 1H, 7-H), 3.45 and 3.43 (s, 3H, CO2Me), 3.38 and 3.34 (s, 3H,
12-CH3), 3.04 and 2.97 (s, 3H, 2-CH3), 2.89 and 2.24 (s, 3H,
COCH3), 2.96−2.88 (m, 2H, 171-CH2), 2.67−2.62 (m, 2H, 172-CH2),
2.41−2.36 (m, 2H, 8-CH2-CH3), 2.15 and 2.12 (s, 3H, 18-CH3), 1.95
and 1.72 (d, 3H, 7-CH3, J = 7.2 and 7.6 Hz), 1.11 and 1.02 (t, 3H, 8-
CH2CH3, J = 7.2 and 7.6 Hz), −0.09 and −0.03 (brs, 1H, NH), −1.26
and −1.23 (brs, 1H, NH). 13C NMR (100 MHz, CDCl3): δ 199.0,
198.9, 196.1, 196.0, 174.2, 174.1, 170.6, 170.7, 164.8, 164.6, 164.2,
164.0, 154.8, 154.6, 146.9, 146.7, 139.6, 139.1, 137.2, 136.9, 136.6,
136.5, 136.4, 136.1, 132.3, 132.2, 130.3, 129.6, 121.7, 120.9, 109.6,
109.4, 99.2, 99.0, 97.9, 97.8, 94.6, 94.4, 84.3, 83.8, 83.75, 83.74, 55.29,
55.28, 51.8, 51.7, 48.6, 48.5, 47.9, 47.8, 33.24, 33.2, 33.1, 33.0, 30.7,
30.1, 30.0, 28.9, 28.8, 23.1, 22.9, 20.9, 20.7, 11.0, 10.8, 10.7, 10.4. EIMS
(m/z): 621 (M+ + Na). HRMS: calcd for C34H39N4O6 [MH]+,
599.2870; found, 599.2880.
Methyl 3-Acetyl-18-keto-bacteriopyropheophorbide-a
(11). Compound 10 (15.0 mg, 0.02 mmol) was taken in a round-
bottom flask (100 mL) and dissolved in 15 mL of concentrated
H2SO4. The reaction mixture was stirred vigorously at room
temperature for 30 min and then poured into ice−water. The reaction
mixture was then extracted with dichloromethane (100 mL), and the
organic layer was separated, washed with water, dried over sodium
sulfate, and concentrated. The crude thus obtained was chromato-
graphed over silica gel using 1−3% CH3OH/CH2Cl2 mixture as an
eluent to get 11. Yield, 6.0 mg (42.8%). UV−vis (THF, λmax (nm) (ε):
715 (9.27 × 104), 650 (2.19 × 104), 518 (2.19 × 104), 432 (1.48 ×
1
105), and 385 (1.39 × 105). H NMR (400 MHz, CDCl3): δ 9.06 (s,
1H, 5-H), 8.55 (s, 1H, 10-H), 8.51 (s, 1H, 20-H), 5.32 (s, 2H, 13-
CH2), 4.38−4.33 (m, 1H, 7-H), 4.07−4.11 (m, 1H, 8-H), 3.56 (s, 3H,
CO2Me), 3.46 (s, 3H, 12-CH3), 3.38 (s, 3H, 2-CH3), 3.18 (s, 3H,
COCH3), 2.86−2.80 (m, 2H, 171-CH2), 2.41−2.35 (m, 1H, 8-CHH−
CH3), 2.17 - 2.06 (m, 3H, 172-CH2 and 1H of 8-CHH−CH3), 1.89/
1.88 (s, 3H, 17-CH3), 1.83 (d, 3H, 7-CH3 J = 6.4 Hz), 1.10−1.15
(distorted triplet, 3H, 8-CH2CH3), 0.2 (brs, 1H, NH), −0.88 (brs, 1H,
NH). 13C NMR (100 MHz, CDCl3): δ 207.7, 199.0, 196.5, 173.1,
165.9, 165.6, 144.9, 141.9, 138.7, 138.2, 138.1, 134.0, 133.8, 128.0,
122.2, 115.0, 112.9, 99.3, 97.6,94.0, 55.0, 54.7, 51.4, 49.2, 47.9, 33.3,
32.4, 30.1, 28.9, 22.8, 22.4, 13.6, 11.5, 10.7. EIMS (m/z): 603 (M+ +
Na). HRMS: calcd for C34H37N4O5[MH]+, 581.2764; found,
581.2770.
Methyl Bacteriopheophorbide-a (12). Rhodobacter sphaeroides
[containing bacteriochlorophyll a (6)] biomass (∼500 g) was
suspended in 1-propanol (2 L) and stirred at room temperature in
dark with constant nitrogen bubbling for 12 h. The blue-green extract
was filtered, and aqueous 0.5 N HCl (150−200 mL) was added to the
filtrate. After it was stirred for 25 min, the solution began to turn
reddish. The reaction mixture was then diluted with aqueous 5% NaCl
(1.5 L) and extracted with dichloromethane. The combined extracts
were washed with water, dried, and rotavaporated. The residue was
precipitated from hexanes to give crude bacteriopheophytin-a 6 (2 g)
with purity sufficient to proceed to the next step. Compound 6 was
dissolved in aqueous 80% TFA (300 mL) and stirred in the dark under
N2 at 0 °C for 2 h. The solution was then diluted with ice/water (600
mL) and extracted with dichloromethane. The combined organic
extracts were washed with water, treated with diazomethane, and
evaporated to dryness. The crude residue was precipitated
from hexanes to obtain the title compound (1.20 g); mp 222−224 °C.
Rhodobacteriochlorin (14). The second band from the column.
Yield, 9.0 mg (10.2%). UV−vis (CH2Cl2, λmax, nm, (ε): 355.0 (1.05 ×
1
105), 521.0 (2.22 × 104), 760 (8.43 × 104). H NMR (400 MHz,
CDCl3): δ 9.58 (s, 1H, meso-H), 9.32 (s, 1H, meso-H), 8.72 (s, 1H,
meso-H), 8.65 (s, 1H, meso-H), 4.43−4.33 (m, 3H, 8-H, 17-H, 18-H),
4.31 (s, 3H, CO2Me), 4.19−4.16 (m, 1H, 7-H), 3.63 (s, 3H, CO2Me),
8636
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3.62 (s, 3H, ring-CH3), 3.58 (s, 3H, ring-CH3), 3.20 (s, 3H, COCH3),
2.67−2.56 (m, 2H, 172-CH2), 2.42−2.35 (m, 3H, 8-CH2CH3 and 171-
CHH), 2.13−2.07 (m, 1H, 171-CHH), 1.83 (d, 3H, 7-CH3, J = 4.4
Hz), 1.81 (d, 3H, 18-CH3, J = 4.4 Hz), 1.11 (t, 3H, 8-CH2CH3, J = 7.6
Hz), −1.39 (brs, 1H, NH), −1.44 (brs, 1H, NH). EIMS (m/z): 584.5
(M+). 13C NMR (100 MHz, CDCl3): δ 198.8, 173.8, 168.5, 166.9,
166.0, 164.8, 164.6, 135.6, 135.0, 134.6, 133.9, 133.7, 132.2, 129.7,
119.7, 98.6, 98.5, 97.2, 96.3, 56.8, 54.8, 51.5, 47.6, 47.5, 33.2, 32.1,
30.9, 30.2, 29.6, 23.6, 23.5, 13.6, 13.2, 10.8. HRMS: calcd for
C34H41N4O5 [MH]+, 585.3077; found, 585.3059.
3-Acetyl-chlorin-15-glyoxilic Acid Trimethyl Ester (18). 3-
Acetyl-bacteriochlorin 15-glyoxilic acid trimethyl ester 13 (45 mg, 0.06
mmol) was dissolved in dichloromethane (15 mL). To this mixture
was added slowly a CH2Cl2 solution of DDQ (30 mg, 0.13 mmol).
The resulting mixture was stirred at room temperature for 10 min and
washed with water three times. The organic layer was separated and
dried over anhydrous Na2SO4, and solvent was removed under
vacuum. The residue obtained was purified with preparative plates
using 2% acetone/dichloromethane. Yield, 35.0 mg (78.6%). UV−vis
[CH2Cl2, λmax, nm, (ε)]: 410.9 (8.48 × 104), 504.9 (7.24 × 103), 546
1
(8.77 × 103), 692 (2.79 × 104). H NMR δ (in CDCl3): 9.94 (s, 1H,
3-Acetyl-17,18-bis Hydroxyl-Ring-B Reduced Chlorin (16).
Acetyl-chlorin-dimethyl ester 15 (50.0 mg, 0.08 mmol) was taken in a
round-bottom flask (100 mL) and dissolved in 30 mL of dry
dichloromethane. To this were added OsO4 (100.0 mg) and pyridine
(1.0 mL), and the reaction mixture was stirred vigorously at room
temperature for 24 h. The reaction was monitored UV−vis and TLC.
H2S gas was then bubbled into the reaction mixture for 5 min, and
then, the excess of H2S was removed by bubbling N2 gas for 30 min.
Water was added to the reaction mixture and extracted with
dichloromethane (100 mL), and the organic layer was separated,
washed with water, dried over sodium sulfate, and concentrated. The
crude reaction product thus obtained was chromatographed over silica
gel using 0.5−1% CH3OH/CH2Cl2 mixture as an eluent to give
product as a mixture of cis-diols (44:56). Yield, 30.0 mg (57%). UV−
vis λmax (in CH2Cl2) (ε): 358.1 nm (7.88 × 104), 388.1 nm (7.35 ×
5H), 9.68 (s, 1H, 10H), 8.70 (s, 1H, 20H), 4.66 (dd, 1H, 18 H, J = 6.8
Hz), 4.35 (m, 1H, 17H, J = 7.2 Hz), 4.15 (s, 3H, CO2Me), 3.90 (s, 3H,
CO2Me), 3.72 (m, 3H, 8 CH2 +171-CH2), 3.66 (s, 3H, CO2Me), 3.53
(s, 3H, 12 CH3), 3.73 (s, 3H, 2 CH3), 3.25 (s, 3H, 7-CH3), 3.20 (s,
3H, COCH3), 2.05- 2.17 (m, 3H, 172-CH2 +171-CH2), 1.68 (t, 3H,
8CH3, J = 7.6 Hz), 1.81 (d, 3H, 18 CH3, J = 8 Hz), 1.68 (t, 3H, 8CH3,
J = 7.6 Hz). 13C NMR (100 MHz, CDCl3): δ 198.5, 186.4, 173.2,
171.4, 167.4, 166.4, 163.5, 155.4, 150.1, 145.3, 140.6, 139.9, 137.4,
136.7, 135.6, 135.1, 135.0, 131.4, 121.1, 106.6, 105.4, 104.3, 94.7, 53.3,
52.8, 52.1, 51.5, 49.2, 33.2, 31.4, 30.9, 23.2, 19.4, 17.4, 13.4, 13.0, 11.1.
EIMS (m/z): 691.1 (M+ + Na). HRMS: calcd for C37H41N4O8
[MH]+, 669.2924; found, 669.2916.
3-Acetyl-7,8-dihydroxybacteriochlorin-15-glyoxilic Acid Tri-
methyl Ester (19). Compound 18 (30.0 mg, 0.04 mmol) was taken
in a round-bottom flask (100 mL) and dissolved in 30 mL of dry
dichloromethane (DCM). To this were added OsO4 (100 mg) and
pyridine (1.0 mL), and the reaction mixture was stirred vigorously at
room temperature for 24 h. The reaction was monitored UV−vis and
TLC. H2S gas was then bubbled into the reaction mixture for 5 min,
and then, an excess of H2S was removed by bubbling N2 gas for 30
min. Water was added to the reaction mixture and extracted with
DCM (100 mL), and the organic layer was separated, washed with
water, dried over sodium sulfate, and concentrated. The crude thus
obtained was purified with silica gel preparative plates using 5%
CH3OH/CH2Cl2 to give 19 as a mixture of cis-diols (38:62). Yield,
27.0 mg (64%). UV−vis [CH2Cl2, λmax, nm, (ε)]: 761 (7.1 × 104), 529
1
104), 524 nm (2.49 × 104), 751.9 nm (7.88 × 104). H NMR (400
MHz, CDCl3): δ 9.72 and 9.69 (s, 1H, meso-H), 9.34 and 9.33 (s, 1H,
meso-H), 8.83 and 8.79 (s, 1H, meso-H), 8.71 and 8.64 (s, 1H, meso-
H), 4.43−4.36 (m, 1H, 8-H), 4.27 and 4.20 (m, 1H, 7-H), 4.12 and
4.10 (s, 3H, CO2Me), 3.67 and 3.61 (s, 3H, CO2Me), 3.49 and 3.36 (s,
3H, ring-CH3), 3.31 (s, 3H, ring-CH3), 2.90 and 2.87 (s, 3H,
COCH3), 2.85 (m, 2H, 171-CH2), 2.76 and 2.66 (m, 2H, 172-CH2),
2.41 and 2.31 (m, 1H, 8-CHH−CH3), 2.11 and 2.00 (m, 1H, 8-CHH-
CH3), 2.06 and 1.87 (s, 3H, 18-CH3), 1.91 and 1.78 (d, 3H, 7-CH3, J =
7.6 Hz), 1.15 and 1.08 (t, 3H, 8-CH2CH3, J = 7.6 Hz), −1.30 and
−1.37 (brs, 1H, NH), −1.42 and −1.44 (brs, 1H, NH). 13C NMR
(100 MHz, CDCl3): δ 198.7, 198.6, 177.4, 177.3, 175.0, 170, 169.2,
169.1, 166.9, 166.3, 165.0, 163.9, 158.5, 158.4, 154.4, 154.2, 153.0,
143.0, 137.1, 137.0, 135.7,135.6, 135.5, 135.4, 134.9, 134.8, 133.3,
133.4, 120.0,119.9, 99.4, 99.2, 98.5, 98.3, 95.0, 96.1, 94.8, 94.2, 86.0,
86.4, 84.1, 84.0, 56.9, 57.1, 56.6, 56.5, 52.0, 51.9, 48.1, 47.8, 33.2, 33.1,
30.3, 30.2, 29.9, 30.0, 29.3, 29.2, 25.0, 25.1, 23.5, 23.3, 13.67, 13.64,
13.36, 13.14, 10.69, 10.64. EIMS (m/z): 617 (M++ 1). HRMS: calcd
for C34H40N4O7 [MH]+, 617.2975; found, 617.2980.
3-Acetyl-18-keto-bacteriochlorin (17). Bacteriochlorin diol 16
(25.0 mg, 0.04 mmol) was taken in a round-bottom flask (100 mL)
and dissolved in 15 mL of concentrated H2SO4. The reaction mixture
was stirred vigorously at room temperature for 30 min and then
poured into ice−water. The reaction mixture was then extracted with
dichloromethane (100 mL), and the organic layer was separated,
washed with water, dried over sodium sulfate, and concentrated. Crude
thus obtained was chromatographed over silica gel using 1−3%
CH3OH/CH2Cl2 mixture as an eluent to get the product; yield, 15.0
mg (61.9%). UV−vis [CH2Cl2, λmax, nm, (ε)]: 403.1 (7.68 × 104),
425.0 (9.10 × 104), 507.0 (1.17 × 104), 539.0 (2.72 × 103), 725.00
(8.70 × 104). 1H NMR (400 MHz, CDCl3): δ 10.30 (s, 1H, 5-H), 9.44
(s, 1H, 10-H), 8.88 (s, 1H, 20-H), 8.89 (s, 1H, 15-H), 4.48−4.51 (m,
1H, 7-H), 4.37 (s, 3H, 17-CO2Me), 4.24−4.28 (m, 1H, 8-H), 3.69 (s,
3H, 13-CO2Me), 3.68 (s, 3H, 2-CH3), 3.32 (s, 3H, 12-CH3), 3.24 (s,
3H, COCH3), 2.99−2.95 (m, 2H, 171-CH2), 2.49−2.39 (m, 2H, 8-
CHH−CH3 and 1H of 172-CH2), 2.20−2.10 (m, 2H, 8-CHH-CH3
and 172-CH2), 1.95/1.97 (singlets, 3H, 17-CH3), 1.86 (d, 3H, 7-CH3,
J = 7.2 Hz), 1.09−1.15 (m, 3H, 8-CH2CH3,), −1.50 (brs, 1H, NH),
−1.6 (brs, 1H, NH). 13C NMR (100 MHz, CDCl3): δ 208.6, 198.7,
173.1, 172.0, 166.5, 166.3, 162.8, 145.2, 136.8, 136.4, 135.1, 132.2,
131.5, 127.9, 123.4, 114.6, 98.6, 97.6, 94.6, 56.7, 53.4, 53.0, 52.2, 51.3,
48.3, 33.3, 30.4, 29.7, 28.8, 23.4, 23.0, 13.9, 12.9, 10.8; EIMS (m/z):
621.3 (M + Na). HRMS: calcd for C34H39N4O6 [MH]+, 599.2870;
obsd, 599.2882.
1
(3.47 × 104), 386 (9.9 × 104), 358 (1.3 × 105). H NMR δ (in
CDCl3): 9.31 and 9.28 (s, 1H, 5H), 8.95 and 8.93 (s, 1H, 10H), 8.55
and 8.50 (s, 1H, 20H), 4.37−4.39 (m, 1H, 18 H), 4.25−4.26 (m, 1H,
17H), 4.09 and 4.07 (s, 3H, CO2Me), 3.93 and 3.90 (s, 3H, CO2Me),
3.72−3.60 (m, 3H, 8 CH2 +171-CH2), 3.56 and 3.53 (s, 3H, CO2Me),
3.45 and 3.43 (s, 3H, 12 CH3), 3.38 and 3.34 (s, 3H, 2 CH3), 2.97 and
2.84 (s, 3H, 7 CH3), 2.26−2.41 (m, 3H, 172-CH2 + 171-CH2), 2.10
and 2.22 (s, 3H, COCH3), 1.67 and 1.80 (d, 3H, 18-CH3, J = 7.6 Hz),
0.68 and 0.86 (t, 3H, 8-CH3, J = 7.2 and 7.6 Hz), −0.41 and −0.44
(brs, 1H, NH), −0.49 and −0.53 (brs, 1H, NH). 13C NMR (100 MHz,
CDCl3): δ 198.2, 198.1, 186.4, 186.3, 173.3, 173.1, 169.7, 168.8, 166.6,
166.5, 166.1, 166.0, 165.8, 165.7, 164.8, 164.3, 163.2, 163.3, 161.5,
160.4, 136.0, 136.1, 135.9, 135.7, 134.3, 133.2, 132.7, 132.4, 132.0,
131.9, 131.5, 131.2, 120.7, 120.1, 108.8, 108.7, 100.3, 100.2, 98.6, 97.1,
96.5, 86.3, 85.8, 84.9, 82.7, 82.4, 53.3, 52.8, 52.1, 52.0, 51.9, 51.7, 51.6,
51.5, 49.1, 49.0, 32.9, 32.8, 31.3, 31.2, 31.0, 30.9, 22.7, 22.6, 20.7, 20.0,
13.8, 13.7, 13.3, 13.2, 12.8, 12.7, 8.3, 8.2. EIMS (m/z): 703 (M + H).
HRMS: calcd for C37H43N4O10 [MH]+, 703.2979; found, 703.3000.
3-Acetyl-8-keto- and 3-Acetyl-7-keto-bacteriochlorin-15-
glyoxilic Acid Trimethyl Ester (20 and 21). Compound 19
(20.0 mg, 0.04 mmol) was taken in a round-bottom flask (100 mL)
and dissolved in 15 mL of concentrated H2SO4. The reaction mixture
was stirred vigorously at room temperature for 30 min and then
poured into ice−water. The reaction mixture was then extracted with
dichloromethane (100 mL), and the organic layer was separated,
washed with water, dried over sodium sulfate, and concentrated. The
crude thus obtained was chromatographed using preparative plates
using 50% ethyl acetate/hexane to give an isomeric mixture of 20 and
21 (43:57). This isomeric mixture was then separated by HPLC using
the conditions described above in the Experimental Section to yield
pure isomer of 20 and 21.
3-Acetyl-8-keto-bacteriochlorin-15-glyoxilic Acid Trimethyl
Ester (20). Yield, 3.8 mg (19%). UV−vis [CH2Cl2, λmax, nm, (ε)]:
8637
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The Journal of Organic Chemistry
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392.1 (1.24 × 105), 508.0 (1.0 × 104), 546.0 (2.0 × 103), 737.0 (5.1 ×
4.38−4.40 (m, 1H, 8 H), 4.28−4.29 (m, 1H, 7H), 4.16 and 4.15 (s,
3H, CO2Me), 3.63 and 3.67 (s, 3H, CO2Me), 3.51 and 3.50 (s, 3H,
CO2Me), 3.43 and 3.42 (s, 3H, 12 CH3), 3.14 and 3.13 (s, 3H, 2
CH3), 3.10 and 3.09 (s, 3H, 18 CH3), 2.52 (s, 3H, 3COCH3), 2.31−
2.24 (m, 2H, 17-CH2), 2.19−2.24 (m, 2H, 8-CH2), 2.04−2.08 (m, 2H,
17-CH2), 1.73 and 1.89 (d, 3H, 7 CH3, J = 7.2 and 7.6 Hz), 1.08 (t,
3H, 8CH3, J = 5.6 Hz), −0.354 and −0.415 (brs, 1H, NH), −0.52 and
−0.60 (brs, 1H, NH). 13C NMR (100 MHz, CDCl3): δ 198.3, 198.4,
175.2 (2C ?), 174.4, 174.3, 171.0, 170.9, 170.1, 170.0, 167.2, 167.1,
166.2, 166.1, 160.2, 159.8, 159.5, 159.4, 158.2, 157.8, 153.0, 143.0,
137.4, 137.2, 135.6, 135.8, 134.1, 134.2, 133.6, 133.5, 131.6, 131.4,
131.3,129.8, 115.2, 115.0, 104.7, 104.3, 103.8, 103.9, 95.9, 96.0, 92.7,
92.8, 83.0, 83.2, 57.1, 57.0, 55.0, 52.6, 52.1, 52.0, 51.5, 51.5, 46.8, 47.4,
33.3, 33.5, 28.9, 29.5, 23.4, 23.3, 22.7,22.6, 19.2,19.3, 14.0, 14.1, 13.6,
13.5, 11.9, 11.8, 10.8, 10.7. EIMS (m/z): 725 (M+ + Na). HRMS:
calcd for C37H41N4O10 [M − H]+, 701.2823; found, 701.2855.
3-Acetyl-15-glyoxilic Acid-8-keto-bacteriochlorin Trimethyl
Ester (17). Compound 23 (15.0 mg, 0.02 mmol) was taken in a
round-bottom flask (100 mL) and dissolved in 15 mL of concentrated
H2SO4. The reaction mixture was stirred vigorously at room
temperature for 30 min and then poured into ice−water. The reaction
mixture was then extracted with dichloromethane (100 mL), and the
organic layer was separated, washed with water, dried over sodium
sulfate, and concentrated. The residue obtained was purified with silica
gel preparative plates using 2% acetone/dichloromethane to give 17 as
a mixture of cis-diols (47:53). Yield, 5.5 mg (43%). UV−vis [THF,
λmax, nm, (ε)]: 403.1(7.68 × 104), 425.0 (9.10 × 104), 507.0 (1.17 ×
1
104). H NMR (400 MHz, CDCl3): δ 9.45 (s, 1H, 5-H), 9.10 (s, 1H,
10-H), 8.51 (s, 1H, 20-H), 4.43−4.45 (m, 1H, 18-H), 4.21- 4.27 (m,
1H, 17-H), 4.11 (s, 3H, CO2Me), 3.91 (s, 3H, CO2Me), 3.56, 3.53,
and 3.51 (each s, 3H, 12-CH3, 2-CH3, and CO2Me), 3.18 (s, 3H,
COCH3), 2.56 (q, 2H, 7-CH2, J = 8 Hz), 2.36−2.37 (m, 1H, 171-
CH2), 2.04−2.09 (m, 3H, 2H of 172-CH2 and 1H of 171-CH2), 1.82
(s, 3H, 7-CH3), 1.74 (d, 3H, 18-CH3, J = 7.6 Hz), 0.50 (t, 3H, 7-
CH2CH3, J = 8 Hz), 0.19 (brs, 1H, NH), 0.13 (brs, 1H, NH). 13C
NMR (100 MHz, CDCl3): δ 209.2, 198.0, 185.8, 174.1, 173.2, 168.4,
166.4, 166.1, 163.25, 143.1, 140.39,137.8, 137.5, 137.4, 134.7, 133.8,
129.3, 127.8, 101.3, 98.4, 96.03, 55.1, 53.3, 52.0, 51.6, 49.9, 33.1,
31.5, 31.4, 31.2, 31.04, 30.9, 29.6, 22.6, 22.5, 13.4, 12.9, 12.8, 8.8. EIMS
(m/z): 707.3 (M+ + Na). HRMS: calcd for C37H41N4O9 [MH]+,
685.2874; found, 685.2890.
3-Acetyl-7-keto-bacteriochlorin-15-glyoxilic Acid Trimethyl
Ester (21). Yield, 4.5 mg (25%). UV−vis [CH2Cl2, λmax, nm, (ε)]:
385 (1.19 × 105), 428.0 (1.29 × 105), 511.0 (1.96 × 104), 544.0
(1.08 × 104), 732.0 (9.1 × 104). 1H NMR (400 MHz, CDCl3): δ 9.83
(s, 1H, 5-H), 8.97 (s, 1H, 10-H), 8.81 (s, 1H, 20-H), 4.49−4.52 (m,
1H, 18-H), 4.35−4.37 (m, 1H, 17-H), 4.15 (s, 3H, CO2Me), 3.97/3.96
(s, 3H, CO2Me), 3.58 and 3.54 (s, 9H, 12 CH3, 2-CH3 and CO2Me),
3.29 (s, 3H, COCH3), 2.62 (q, 2H, 8-CH2CH3, J = 7.8 Hz), 2.36−2.37
(m, 1H, 171 −CH2), 2.05−2.09 (m, 3H, 2H of 172-CH2 and 1H of
171-CH2), 1.90/1.92 (s, 3H, 8-CH3), 1.78−1.80 (m, 3H, 18-CH3),
0.45−0.51 (m, 3H, 8-CH2CH3), −1.07 (brs, 1H, NH), −1.01 (brs,
1H, NH). 13C NMR (100 MHz, CDCl3): δ 208.7, 198.1, 198.1, 186.4,
173.0, 169.2, 166.7, 166.3, 163.0, 162.95,162.93 147.7, 139.4, 136.7,
136.6, 135.6, 135.2, 134.6, 134.2, 133.3, 133.2, 98.9, 98.8, 97.3, 54.6,
53.2, 52.4, 51.6, 48.8, 33.3, 31.8, 31.7, 31.3, 30.5, 29.7, 23.2, 23.0, 22.9,
13.1, 13.0, 8.8, 8.7. EIMS (m/z): 707.3 (M+ + Na). HRMS: calcd for
C37H41N4O9[MH]+, 685.2874; found, 685.2852.
1
104), 539.0 (2.72 × 103), 725.00 (8.70 × 104). H NMR (400 MHz,
CDCl3 δ): 10.30 (s, 1H, meso-H), 9.44 (s, 1H, meso-H), 8.88 (s, 1H,
meso-H), 4.48−4.51 (m, 1H, 8-H), 4.37 (s, 3H, CO2Me), 4.24−4.28
(m, 1H, 7-H), 3.69 (s, 3H, CO2Me), 3.68 (s, 3H, 12CH3), 3.32 (s, 3H,
2-CH3), 3.24 (s, 3H, COCH3), 2.97−2.91 (m, 2H, 171-CH2), 2.49−
2.39 (m, 2H, 8-CHH−CH3 and 1H of 172-CH2), 2.09−2.15 (m, 2H,
8-CHH-CH3 and 172-CH2), 1.96 (d, 3H, 17-CH3, J = 7.2 Hz), 1.86 (d,
3H, 7-CH3, J = 7.2 Hz), 1.09−1.15 (m, 3H, 8-CH2CH3), 1.50 (brs,
1H, NH), −1.6 (brs, 1H, NH). EIMS (m/z): 621.3 (M + Na).
Ring-D Reduced Methyl Pheophorbide-a (24). Methyl
bacteriopheophorbide 12 (20 mg, 0.032 mmol) was dissolved in
dichloromethane (5 mL) under Ar atm. To this mixture, a solution of
DDQ (5.9 mg, 0.026 mmol) in dry CH2Cl2 was added slowly. The
resulting mixture was stirred at room temperature for 5 min, and the
entire reaction mixture was washed with water three times. The
organic layer was separated and dried over anhydrous Na2SO4, and
solvent was removed under vacuum. The residue obtained was purified
with preparative plates using 3% acetone/DCM affording 17 mg of the
3-Acetyl-15-glyoxilic Acid-Ring-B Reduced Chlorin Trimeth-
yl Ester (22). Acetyl-bacteriochlorin 15-glyoxilic acid trimethyl ester
13 (45 mg, 0.06 mmol) was dissolved in dichloromethane (20 mL).
To this mixture was added slowly a nitromethane solution of
FeCl3·6H2O (72 mg, 0.267 mmol). The resulting mixture was stirred
at room temperature for 30 min, quenched by the addition of 5 mL of
methanol, and washed with water three times. The organic layer was
separated and dried over anhydrous Na2SO4, and the solvent was
removed under vacuum. The residue obtained was purified with
preparative plates using 2% acetone/dichloromethane; yield, 39.0 mg
(87.2%). UV−vis [CH2Cl2, λmax, nm, (ε)]: 410.9 (8.09 × 104), 512.1
1
(1.00 × 104), 679 (3.28 × 104). H NMR δ (in CDCl3): 9.85 (s, 1H,
5H), 9.56 (s, 1H, 10H), 8.90 (s, 1H, 20H), 4.53 (q, 1H, 7-H, J = 24
Hz), 4.35 (m, 1H, 8H), 4.15 (s, 3H, CO2Me), 3.90 (s, 3H, CO2Me),
3.80 (s, 3H, CO2Me), 3.63 (s, 3H, 12 CH3), 3.52 (s, 3H, 2 CH3), 3.32
(s, 3H, 18 CH3), 3.26 (s, 3H, COCH3), 2.58−2.64 (m, 2H, 171-CH2),
2.43−2.45 (m, 1H, 8-CHH), 2.10−2.14 (m, 1H, 8-CHH), 1.89 (d,
3H, 7CH3, J = 6.8 Hz), 1.08 (t, 3H, 8- CH2CH3, J = 7.6 Hz), −1.2
(brs, 1H, NH), −1.60 (brs, 1H, NH). 13C NMR (100 MHz, CDCl3):
δ 198.4, 189.3, 173.0, 172.0, 169.9, 167.0, 161.8, 149.6, 149.4, 141.8,
138.5, 138.2, 137.8, 137.7, 137.4, 131.0, 130.8, 129.9, 127.5, 113.0,
102.8, 97.8, 96.0, 58.0, 54.0, 53.5, 51.6, 47.1, 35.0, 33.2, 30.0, 23.6,
23.4, 14.0, 12.6, 11.8, 10.8. EIMS (m/z): 691.1 (M+ + Na). HRMS:
calcd for C37H41N4O8[MH]+, 669.2924; found, 669.2912.
3-Acetyl-17,18-dihydroxy-15-glyoxilic Acid-Bacteriochlorin
Trimethyl Ester (23). Compound 22 (40.0 mg, 0.06 mmol) was
taken in a round-bottom flask (100 mL) and dissolved in 30 mL of dry
DCM. To this were added OsO4 (75.0 mg) and pyridine (1.0 mL),
and the reaction mixture was stirred vigorously at RT for 24 h. The
reaction was monitored by UV−vis and TLC. H2S gas was then
bubbled into the reaction mixture for 5 min, and then, an excess of
H2S was removed by bubbling N2 gas for another 30 min. Water was
added to reaction mixture and extracted with dichloromethane
(100 mL), and the organic layer was separated, washed with water,
dried over sodium sulfate, and concentrated. The crude thus obtained
was purified with silica gel preparative plates using 5% MeOH/
CH2Cl2. Yield, 23 mg (55%). 1H NMR δ (in CDCl3): 9.45 and 9.43 (s,
1H, 5H), 8.88 and 8.86 (s, 1H, 10H), 8.75 and 8.73 (s, 1H, 20H),
methyl pheophorbide-a. Yield, 17.0 mg (85%). UV−vis [CH2Cl2, λmax
,
nm, (ε)]: 691 (3.45 × 104). 1H NMR δ (in CDCl3): 9.95 (s, 1H, 5-H),
9.61 (s, 1H, 10-H), 8.77 (s, 1H, 20-H), 6.31 (s, 1H, 132-H), 4.53
(m, 1H for 18-H), 4.25 (m, 1H for 17-H), 3.89 (s, 3H, 12-CH3), 3.72
(s, 3H, 2-CH3), 3.63 (s, 3H, 132-COOCH3), 3.57 (s, 3H, 17-
CH2CH2COOCH3), 3.27 (s, 6H, 7-CH3 and 3-COCH3), 2.62 (m, 2H,
17-CH2CH2COOCH3), 2.28 (m, 4H, 2H of 17-CH2CH2COOCH3,
2H of 8-CH2CH3), 1.83 (d, J = 7.6 Hz, 3H, 18-CH3), 1.70 (t, J = 7.2
Hz, 3H, 8- CH2CH3). 13C NMR (100 MHz, CDCl3): δ 199.2, 189.5,
173.2, 169.4, 162.0, 153.2, 151.9, 148.8, 145.0, 139.1, 137.6, 135.9,
135.6, 134.4, 130.4, 129.8, 105.7, 104.0, 100.7, 94.2, 64.8, 52.9, 51.6,
51.4, 49.8, 33.4, 31.0, 29.8, 23.2, 19.4, 17.3, 13.8, 13.4, 12.2, 11.4, 11.2.
EIMS (m/z): 623 (M + 1). HRMS: calcd for C36H39N4O6 [MH]+,
623.2870; found, 623.2864.
Ring-B Reduced Methyl Pheophorbide-a (25). Methyl bacter-
iopheophorbide 12 (20 mg, 0.032 mmol) was dissolved in
dichloromethane (20 mL). To this mixture, a solution of FeCl3·6H2O
(24 mg, 0.089 mmol) in nitromethane was added slowly. The resulting
mixture was stirred at room temperature for 30 min, quenched by the
addition of 5 mL of methanol, and washed with water three times. The
organic layer was separated and dried over anhydrous Na2SO4, and
solvent was removed under vacuum. The residue obtained was purified
with silica gel preparative plates using 2% acetone/dichlorometha-
ne.Yield, 16.0 mg (80%). UV−vis [CH2Cl2, λmax, nm, (ε)]: 682
(3.65 × 104). 1H NMR δ (in CDCl3): 9.51 (s, 1H, 5-H), 9.35
8638
dx.doi.org/10.1021/jo201688c|J. Org. Chem. 2011, 76, 8629−8640

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Article
(s, 1H, 10-H), 8.77 (s, 1H, 20-H), 6.67 (s, 1H, 132-H), 4.53 (m, 1H
for 7-H), 4.30 (m, 1H for 8-H), 3.91 (m, 2H, 17-CH2CH2COOCH3),
3.81 (s, 3H, 12-CH3), 3.72 (m, 6H, 3H of 18-CH3 and 3H of 2-CH3),
3.60 (s, 3H, 132-COOCH3), 3.29 (s, 3H, 17-CH2CH2COOCH3),
3.23 (s, 3H, 3-COCH3), 2.90 (m, 2H of 8-CH2CH3), 2.08 (m, 2H, 17-
CH2CH2COOCH3), 1.88 (d, J = 7.2 Hz, 3H, 18-CH3), 1.15 (t, J = 7.2
Hz, 3H, 8-CH2CH3). 13C NMR (100 MHz, CDCl3): δ 203.3, 189,
172.9, 169.7, 166.4, 159.2, 155.4, 141.3, 140.2, 138.4, 133.3, 132.2,
127.9, 114.6, 113.3, 114.6, 100.1, 96.9, 96.2, 96.1, 66.2, 55.8, 53.1, 51.7,
48.3, 35.6, 33.3, 30.1, 29.2, 23.3, 22.1, 13.8, 11.9, 11.4, 10.8, 10.7. EIMS
(m/z): 623 (M + 1). HRMS: calcd for C36H39N4O6 [MH]+, 623.2870;
found, 623.2828.
In Vitro Photosensitizing Efficacy. The photosensitizing activity
of the compounds was determined as described previously.30 The
tumor cell lines used are Colon26 (mouse colon tumor). The Colon26
tumor cells were grown in RPMI-1640 medium supplemented with
10% fetal bovine serum, L-glutamine, penicillin, and streptomycin.
Tumor cells were maintained in an atmosphere of 5% CO2, 95% air,
and 100% humidity at 37 °C. For determining the PDT efficacy of the
compounds, the cells were plated in 96-well plates at a cell density of
3000 cell/well in complete media. After 16 h of incubation at 37 °C,
the photosensitizers were added at variable concentrations and
incubated at 37 °C for 24 h in the dark. Prior to light treatment,
the cells were replaced with drug-free complete media. Cells were then
illuminated with light from an argon-pumped dye laser set at 725−741
nm, respectively, for each of the drugs as per their absorption
wavelength in 17% bovine calf serum (BCS) measured before, at a
dose rate of 1.6 mW/cm2 for 0−2 J/cm2. After PDT, the cells were
incubated for a further 48 h at 37 °C in the dark. Following the 48 h
incubation, 10 μL of 5.0 mg/mL solution of 3-[4,5-dimethylthiazol-2-
yl]-2−5-diphenyltetrazoliumbromide (MTT) in PBS (Sigma, St.
Louis, MO) was added to each well. After 4 h of incubation at
37 °C, the MTT and the media were removed, and 100 μL of DMSO
was added to solubilize the formazan crystals. The 96-well plate was
read on a microtiter plate reader (BioTek Instruments, Inc., ELx800
Absorbance Microplate Reader) at an absorbance of 570 nm. The
results were plotted as a percent survival of the corresponding dark
(drug no light) control for each compound tested. Each data point
represents the mean from two separate experiments, with six replicate
wells, and the error bars are the standard deviation.
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ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra of compounds 10, 11, and 14−25
and 2D NMR spectra of compounds 11, 17, and 21. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: 716-845-3203. Fax: 716-845-8920. E-mail: ravindra.
pandey@roswellpark.org.
■
ACKNOWLEDGMENTS
■
The financial support from the NIH (CA127369, CA55791)
and the shared resources of the Roswell Park Cancer Center
support grant CA16056 are highly appreciated. The Mass
Spectrometry analyses were performed at the Biopolymer
Facility, Roswell Park Cancer Institute (Buffalo), and Mass
Spectrometry Facility at Michigan State University (East
Lansing, MI).
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