Structure-activity relationship among purpurinimides and bacteriopurpurinimides: Trifluoromethyl substituent enhanced the photosensitizing efficacy

Article abstract of DOI:10.1021/jm061036q

At similar lipophilicity, compared to the nonfluorinated purpurinimide 11, the corresponding fluorinated analog 8 with a trifluoromethyl substituent at the lower half (position-13²) of the molecule showed enhanced photosensitizing efficacy. The structural parameters established in purpurinimides (λ_max: 700 nm) were successfully translated to the bacteriopurpurin imide system 19 (λ_max: 792 nm) and within both series, a monotonic relationship between the lipophilicity and the in vivo PDT activity was observed. For preparing water-soluble compounds, the photosensitizers 8 and 19 were converted into the corresponding aminobenzyldiethylenetriamine pentaacetate conjugates 23 and 26. Acid treatment of purpurinimide 23 produced the corresponding water-soluble analog 24. Bacteriochlorin 26 under acidic or basic conditions mainly gave the decomposition products. At similar in vivo treatment conditions (C3H mice with RIF tumors and BALB-C mice with colon-26 tumors) the water-soluble purpurinimide 24 was found to be more effective than the methyl ester analog 8. These results suggest that besides overall lipophilicity the inherent charge of the photosensitizer also influences the PDT efficacy.

Full text of DOI:10.1021/jm061036q

1754
J. Med. Chem. 2007, 50, 1754-1767
Structure-Activity Relationship Among Purpurinimides and Bacteriopurpurinimides:
Trifluoromethyl Substituent Enhanced the Photosensitizing Efficacy
Amy Gryshuk,†,‡ Yihui Chen,† Lalit N. Goswami,† Suresh Pandey,† Joseph R. Missert,† Tymish Ohulchanskyy,§ William Potter,†
Paras N. Prasad,§ Allan Oseroff,‡ and Ravindra K. Pandey*,†,§
Chemistry DiVision, PDT Center, and Department of Dermatology, Roswell Park Cancer Institute, Buffalo, New York 14263, and
Institute of Lasers, Photonics and Biophotonics, State UniVersity of New York, Buffalo, New York 14221
ReceiVed August 29, 2006
At similar lipophilicity, compared to the nonfluorinated purpurinimide 11, the corresponding fluorinated
analog 8 with a trifluoromethyl substituent at the lower half (position-132) of the molecule showed enhanced
photosensitizing efficacy. The structural parameters established in purpurinimides (λmax: 700 nm) were
successfully translated to the bacteriopurpurin imide system 19 (λmax: 792 nm) and within both series, a
monotonic relationship between the lipophilicity and the in vivo PDT activity was observed. For preparing
water-soluble compounds, the photosensitizers 8 and 19 were converted into the corresponding aminobenzyl-
diethylenetriamine pentaacetate conjugates 23 and 26. Acid treatment of purpurinimide 23 produced the
corresponding water-soluble analog 24. Bacteriochlorin 26 under acidic or basic conditions mainly gave the
decomposition products. At similar in vivo treatment conditions (C3H mice with RIF tumors and BALB-C
mice with colon-26 tumors) the water-soluble purpurinimide 24 was found to be more effective than the
methyl ester analog 8. These results suggest that besides overall lipophilicity the inherent charge of the
photosensitizer also influences the PDT efficacy.
Introduction
Further, its accumulation in skin induces prolonged light
sensitivity for 5-10 weeks post-PDT treatment.
Over the past few decades, photodynamic therapy (PDTa)
has begun to gain worldwide attention either as a primary or as
an adjunctive treatment for solid cancers.1-3 PDT differs from
other forms of therapy in that the success of the treatment is
ineffective unless all the components (photosensitizer, light, and
oxygen status of the tumor) are utilized in combination. In the
presence of the drug-activating light, the photosensitizer un-
dergoes a photochemical reaction in which it transfers its long-
lived excited-state energy to ground state molecular oxygen
residing within the tumor. This interaction produces lethal
cytotoxic agents, primarily singlet oxygen (1O2) and other
reactive oxygen species (ROS), that aid in the destruction of
the malignant tissue.4 Therefore, the success of the treatment
depends upon the tumor-selectivity and photosensitizing proper-
ties of the photosensitizer used.
A hematoporphyrin derivative (purified HpD), the first-
generation photosensitizer, has most commonly been the choice
for PDT treatment of various forms of cancer due to its ability
to be easily synthesized and formulated.5 However, despite the
successes of the purified version of HpD, it suffers from certain
limitations due to a complex mixture of monomeric, dimeric,
and oligomeric porphyrins joined with ether, ester, and carbon-
carbon linkages.6 This complex chemical mixture makes it very
difficult to make any precise conclusion as to which portion of
the mixture is responsible for its photosensitizing ability. In
addition, the longest wavelength absorption of 630 nm limits
its penetrating ability to tumor depths of no more than 5 mm.7
To enhance the treatment of cancer and to overcome the
limitations associated with a purified version of HpD, efforts
in our laboratory were directed toward the synthesis of chemi-
cally pure new longer-wavelength absorbing photosensitizers8
related to purpurinimides and bacteriopurpurinimides. These are
porphyrin-based aromatic conjugated macrocycles in which the
presence of an additional imide ring fused at the meso-position
extends its long wavelength absorption to 700 and 800 nm.9
Such an inherent long-wavelength characteristic should provide
more efficient light penetration in tumor tissues as compared
to those photosensitizers that absorb light at 630 nm.10
Previously, our group reported the structure-activity relation-
ship (SAR) and quantitative SAR (QSAR) of a series of alkyl
ether analogs of pyropheophorbide-a11,12 and alkyl ether deriva-
tives of N-alkyl purpurinimides. The in vivo results demonstrated
that the overall lipophilicity of the molecules could be easily
altered by varying the length of the carbon chain, which resulted
in a significant difference in photosensitizing efficacy.13,14
In the pharmaceutical chemistry, fluorinated compounds in
general have shown a dramatic increase in biological efficacy
as compared to their nonfluorinated analogs.15 It has been
illustrated that fluorine, while maintaining a comparable size
to that of hydrogen (the van der Waals radii of F and H are
1.35 and 1.11, respectively) is sterically indistinguishable from
a host molecule, yet maintains different biological activity.16
Having fluorine in biologically active molecules brings desirable
benefits, but the trick is to introduce these fluorinated func-
tionalities at the right place. The Schlosser group at the Swiss
Federal Institute of Technology, Lausanne, have screened a
series of organofluorine compounds for medical applications,
and in certain structures, it was observed that introducing
fluorine into the lead structure enhanced the in vivo efficacy.17
Therefore, the present study was designed to compare the
photophysical and photosensitizing efficacy of a series of
fluorinated versus nonfluorinated purpurinimides and bacterio-
* To whom correspondence should be addressed. Ravindra K. Pandey,
Ph.D., Professor of Oncology, PDT Center, Roswell Park Cancer Institute,
Buffalo, New York 13263. Phone: 716-845-3203. Fax: 716-845-8920.
E-mail: ravindra.pandey@roswellpark.org.
† Chemistry Division.
‡ Department of Dermatology.
§ Institute of Lasers, Photonics and Biophotonics.
a Abbreviations: PDT, photodynamic therapy; SAR, structure-activity
relationship; QSAR, quantitative structure-activity relationship; ROS,
reactive oxygen species; DTPA, diethylenetriamine pentaacetate.
10.1021/jm061036q CCC: $37.00 © 2007 American Chemical Society
Published on Web 03/20/2007

Purpurinimides and Bacteriopurpurinimides
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1755
Scheme 1. Synthesis of Fluorinated and Nonfluorinated Purpurinimides
Scheme 2. Synthesis of Fluorinated Bacteriopurpurinimides
purpurinimides. Due to a close structural relationship between
the purpurinimide (ring D is reduced) and the bacteriopurpu-
rinimide systems (rings B and D are reduced), the synthetic
methodology developed for purpurinimides was successfully
extended for the preparation of related bacteriopurpurinimide
analogs.
Results and Discussion
(i) Chemistry of Purpurinimides and Bacteriopurpurin-
imides: Schemes 1 and 2 display the synthetic methodology
followed for the preparation of the fluorinated purpurinimides
and bacteriopurpurinimides with varying lipophilicity (see Table
1 for comparative Log P values). The starting materials for the
preparation of the desired products were extracted from Spir-
ulina Pacifica (contains chlorophyll-a) and Rb. sphaeroides
(contains bacteriochlorophyll-a). For the synthesis of purpurin-
imides, the methylpheophorbide-a derived from chlorophyll-a
was converted into purpurin-18 methyl ester by following the
literature procedure 1.18 It was then reacted with commercially
available 3,5-bis(trifluoromethyl) benzyl amine 3 and refluxed
in benzene. The reaction was monitored by UV-vis spectros-
copy, and the resulting product exhibited a red shift from 700
nm to ∼706 nm. Purpurinimide 5 was then reacted with HBr/
acetic acid, and the resulting bromo- derivative was not isolated,
but instead, the crude product was dried under vacuum and
immediately reacted with various alkyl alcohols, differing the
number of carbon units (methyl-, butyl-, heptyl-, or dodecyl-
alcohol) in the presence of anhydrous potassium carbonate and
dichloromethane to yield photosensitizers 7-10 of varying
lipophilicity. Purpurinimides 7-10 were purified on a silica prep
plate before subjecting them for in vitro/in vivo studies. To
investigate the effect of the fluorinated versus the nonfluorinated
analogs, purpurinimides 6 and 11 were also prepared by
following a similar approach, except the corresponding non-
fluorinated amine 2 was used as one of the starting materials
instead of the fluorinated amine.
This methodology was also extended for the preparation of
the fluorinated bacteriopurpurinimides, as shown in Scheme 2,
in which the starting material was extracted from Rb. sphaeroi-
des and converted into bacteriopurpurin methyl ester, 15. A

1756 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Gryshuk et al.
Table 1. Log P Values for the Fluorinated Purpurinimides (7-10, 24)
and Bacteriopurpurinimides (18-21)
reaction conditions, bacteriopurpurinimide 26 mainly produced
a complex mixture of the decomposition products. Various
attempts (both basic and acidic) to prepare water-soluble bac-
teriopurpurinimide 27 from 26 were unsuccessful (Scheme 3).
HPLC Analysis: The purity of the compounds was also
ascertained by Waters HPLC system containing Waters 600
controller, and Delta 600 pump and 996 photodiode array
detector. The purity was determined in both reverse and normal
phase HPLC conditions. For the reverse phase, symmetry C18,
5 µm, and 4.6 × 150 mm column (Waters) was used under an
isocratic setting of 100% MeOH for all the final compounds
(6-11, 13, 14, 18-21, and 26). For the normal phase,
Phenomenex Luna, 5 µm silica, and 4.6 × 250 mm column
was used under a gradient setting of 100% CH2Cl2 for 1 min
and then graded to 30% MeOH/CH2Cl2 over the next 30 min.
Purity of compound 24 was determined in reverse phase column
under isocratic 95% MeOH and 5% buffer (K2HPO4/KH2PO4
1:1; pH ) 7.0).
The photosensitizers were dissolved either in MeOH or in
CH2Cl2 for reverse and normal phase chromatography, respec-
tively. Solvent flow rate was kept constant at 1.00 mL/min and
detector was set at 415, 545, and 700 nm for purpurininimides
(6-11, 13, 14, and 24) and 367, 540, and 782 nm for
bacteriopurpurinimides (18-21 and 26). The final products were
obtained as a mixture of R- and S-isomers. As can be seen from
Table 2, most of the compounds showed a single peak, however,
in some cases, a nice separation of both the isomers was
observed. The final products were found to be >95% pure (for
the HPLC chromatograms of these compounds, see the Sup-
porting Information).
cmpd
7
8
9
10
18
19
20
21
24
calcd 9.43 10.91 12.13 14.67 8.82 10.30 11.83 14.38 -6.45
Log P
significant red shift in long-wavelength absorption of bacterio-
purpurin methyl ester 15 (815 nm) and the corresponding
fluorinated bacteriopurpurinimide 16 (820 nm) were observed
in organic and aqueous solutions. The product 17 (778 nm)
obtained after treatment with NaBH4 was purified via chroma-
tography and converted into a series of bacteriopurpurinimides
18-21 by following the synthetic strategy shown in Scheme
2 (Note: HBr/AcOH was replaced with HBr gas to avoid
any decomposition). As expected, similar to the purpurinimide
series, varying the length of the alkyl chain at position-3 of
bacteriopurpurinimide 17 also altered the overall lipophilicity
of the molecule. A linear relationship between the length of
O-alkyl chains and the overall lipophilicity was observed (see
Table 1).
(ii) Water-Soluble Photosensitizers: Similar to most of the
porphyrin-based compounds, the purpurinimides and bacterio-
purpurinimides exhibited limited water solubility and were
formulated in a 1% Tween 80/5% aqueous dextrose solution.
In our attempt to prepare water-soluble analogs, the most
effective fluorinated purpurinimide 8 and bacteriopurpurinimide
19 were converted into the corresponding monocarboxylic acid
derivatives 22 and 25, which on reacting with modified
aminophenyl-DTPA, containing five tert-butylester function-
alities, produced 23 and 26, respectively, in good yield. Reaction
of purpurinimide 23 with TFA at room temperature gave the
desired carboxylic acid 24 in quantitative yield and was highly
soluble in water (see Table 1 for Log P value). Under similar
Photophysical Characteristics of Purpurinimides and
Bacteriopurpurinimides: The electronic absorption spectra of
fluorinated purpurinimides and bacteriopurpurinimides were
Scheme 3. Synthetic Strategy for the Preparation of Water-Soluble Purpurinimides and Bacteriopurpurinimides

Purpurinimides and Bacteriopurpurinimides
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1757
Table 2. HPLC Retention Time of Purpurinimides and
Bacteriopurpurinimides
reverse phase (symmetry)
normal phase (phenomenex)
retention time
(min)
purity
(%)
retention time
(min)
purity
(%)
cmpd
6
7
8
24.96
13.48
21.83
35.93
67.01, 69.98
41.00
26.02
22.97
3.91
16.30, 17.60
26.28, 28.04
48.44, 55.82
4.79
96.7
98.9
98.2
98.1
99.4
95
97.2
96.6
95
95.9
99.9
95
95
96
14.00
13.93
14.32
6.90, 7.34
5.61, 6.03
14.50
14.07
14.09
14.05
10.05
10.95
10.43, 10.91
96.1
96.2
98.5
98.5
97.9
98.8
99.3
95.6
99.8
97.4
99.4
96.8
9
10
11
13
14
18
19
20
21
24
26
not suitable
18.77, 20.47
21.61
94.7
determined in anhydrous THF. The representative examples
from each series are shown in Figure 1. As can be seen, the
purpurinimides (i.e., 7-10) displayed a characteristic Soret band
at ∼417 nm, with various Q-bands and a characteristic long
wavelength absorption peak at ∼701 nm (ꢀ ) 58 000 in THF),
whereas the bacteriopurpurinimides (i.e., 18-21) displayed a
characteristic Soret band at ∼368 and 417 nm, with various
Q-bands and a characteristic long wavelength absorption peak
at ∼783 nm (ꢀ ) 40 530 in THF). The fluorescence spectra of
purpurinimide 8 and bacteriopurpurinimide 19 are shown in
Figure 2A,B, respectively, and as can be seen compared to
purpurinimide, the bacteriopurpurinimide exhibited weaker
absorptions. In both series, on exciting the longest wavelength
absorption, this produced a small difference (Stokes shift)
between the respective absorption and the emission peaks.
Steady-state measurements for the singlet oxygen (1O2)
generation of 8 and 19 were performed at room temperature
using the Fluorolog-3 Spectrofluorometer (Jobin Yvon). The
1O2 yields were determined by normalizing the results to Rose
Bengal, a known singlet oxygen producer (Q∆ ) 0.80 at 1270
nm).19 The graph below (Figure 3) displayed the differences in
1O2 generation between purpurinimide 8 and bacteriopurpurin-
imide 19. As a representative of the purpurinimide series, 8
Figure 2. (A) Fluorescence spectra [only the longest wavelength
emissions are shown] (a), (b), and (c) of purpurinimide 8 excited at
λ
max: 415, 700, and 544 nm, respectively, in THF (conc. 0.5 µM). (B)
Fluorescence spectra [only the longest wavelength emissions are shown]
(a), (b), (c), and (d) of bacteriopurpurinimide 19 excited at λmax: 366,
416, 535, and 784 nm, respectively, in THF (concd 0.5 µM). / Observed
on excitation at 416 nm.
1
1
produced a O2 yield Q∆ ≈ 0.60, whereas the O2 yield for 19
was two times lower (Q∆ ≈ 0.30). According to these data, 8
1
with a more efficient O2 yield may be predictive of a better
PDT response.
Figure 3. Singlet oxygen production efficiency of 8 and 19 referenced
to Rose Bengal in methanol. Samples absorbance was matched at the
wavelength of excitation (514 nm). Spectra were corrected with
background subtraction.
Effect of Substituents in Photosensitizing Efficacy: (a) In
Vitro Photosensitizing Efficacy. For determining the treat-
ment parameters, purpurinimide 8 was initially tested in
radiation-induced fibrosarcoma (RIF) cells at three different
concentrations (0.5, 1.0, and 2.5 µM) as a means to determine
the optimum drug dose. A drug and light dose-dependent
response was observed as determined by the MTT assay.20
A
drug concentration of 2.5 µM, together with a light dose of 4.0
J/cm2, produced the best efficacy with no significant dark
toxicity.
Among purpurinimides, the fluorinated photosensitizer 8
showed improved activity compared to the corresponding
nonfluorinated 6. Due to a significant difference in overall
lipophilicity (6, 9.47; 8, 10.91; Log P values), it was difficult
to draw any conclusions. Therefore, nonfluorinated purpurin-
imide 11, containing an O-heptyl side chain at position-3 with
a Log P value of 10.99 was prepared and evaluated for PDT
efficacy. As can be seen from Figure 4, 8 and 11, with similar
lipophilicity (8, 10.91; 11, 10.99) showed a significant difference
Figure 1. Electronic absorption spectra of the fluorinated photosen-
sitizers in anhydrous THF at equimolar concentrations (0.5 µM). (a)
Purpurinimide 8 (dotted line) and (b) bacteriopurpurinimide 19 (solid
line).

1758 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Gryshuk et al.
Figure 6. In vitro PDT photosensitivity of fluorinated bacteriopurpu-
rinimides 18-21 (2.5 µM) in RIF cells. The cells were exposed to
laser light at a dose of 3.2 mW/cm2. Dark control: cells were incubated
with photosensitizers, but not exposed to light. Light control: cells
were not incubated with photosensitizers, but were exposed to laser
light.
Figure 4. In vitro PDT photosensitivity of fluorinated purpurinimide
8 (2.5 µM) versus its related nonfluorinated analog 11 (2.5 µM) at
similar Log P values (10.91 and 10.99, respectively). The cells were
exposed to laser light at a dose of 3.2 mW/cm2. Dark control: cells
were incubated with photosensitizers, but not exposed to light. Light
control: cells were not incubated with photosensitizers, but were
exposed to laser light.
Figure 7. In vitro PDT photosensitizing efficacy of fluorinated
purpurinimide 8 (2.5 µM) and its water-soluble analog 24 (0.08 µM)
in colon-26 cells. The cells were exposed to laser light at a dose of 3.2
mW/cm2. Dark control: cells were incubated with photosensitizers, but
not exposed to light. Light control: cells were not incubated with
photosensitizers, but were exposed to laser light.
Figure 5. In vitro PDT photosensitivity of fluorinated purpurinimides
7-10 (2.5 µM) in RIF cells. The cells were exposed to laser light at a
dose of 3.2 mW/cm2. Dark control: cells were incubated with
photosensitizers, but not exposed to light. Light control: cells were
not incubated with photosensitizers, but were exposed to laser light.
in PDT photosensitivity. The fluorinated analog 8 was found
to be more photosensitive than the nonfluorinated 11.
carcinoma cell line (colon-26) at variable drug concentrations
and a significantly lower drug concentration of water-soluble
purpurinimide 24 (0.08 µM) was required to obtain a similar
PDT response from purpurinimide 8 (2.5 µM) when illuminated
at 4 J/cm2. As shown in Figure 7, both photosensitizers produced
a light-dependent PDT response with a 30-fold increase in PDT
efficacy for the water-soluble analog. Such a significant increase
in photosensitivity may be due to differences in intracellular
uptake or localization and these studies are currently in progress.
It could be hypothesized that both photosensitizers passively
diffuse into the cell but that their intracellular localization differs
such that 8 may be sequestered and only upon light treatment
redistribute to other sensitive organelles, such as the mitochon-
dria, which would help elicit an effective PDT-induced mech-
anism of cell death. On the other hand, the five carboxylic acid
groups on 24, which are anionic in nature, may allow 24 to
initially localize to a more active site, such as the mitochondria.
Once the advantage of fluorinated over the nonfluorinated
photosensitizers was established, our next objective was to
investigate the effect of overall lipophilicity on PDT photosen-
sitivity. Fluorinated purpurinimides 7-10 and bacteriopurpu-
rinimides 18-21 with variable lipophilicity were evaluated in
RIF under similar experimental conditions (drug concentration
of 2.5 µM, together with a light dose of 4.0 J/cm2 to yield a
fluence rate of 3.2 mW/cm2). As can be seen from the results
summarized in Figures 5 and 6, all photosensitizers were
effective in vitro with minimal to no dark phototoxicity.
However, increasing the overall hydrophobicity of the molecule
reduced its photosensitizing efficacy in vitro. A likely explana-
tion for those results may be due to differences in cellular
uptake. Experiments looking at intracellular uptake and localiza-
tion are currently in progress.
The in vitro results shown in Figures 5 and 6 indicate an
indirect relationship between the lipophilicity and the photo-
sensitizing efficacy. To explore things further, the biological
efficacy of purpurinimide 8 (Log P 10.91) and its water-soluble
analog 24 (Log P -6.45), with a drastic difference in their
overall lipophilicity, were also evaluated in vitro and in vivo.
The in vitro efficacy was initially determined in the murine colon
(b) Determination of Drug-Uptake By In Vivo Reflectance
Spectroscopy (IRS). The uptake of photosensitizers in tumor
versus skin is an important concern in developing effective
photosensitizers. The tumor and skin uptake was determined
by IRS21 at variable time points (0-48 h postinjection) but was
found to be most optimal at 24 h postinjection (Table 3). It had

Purpurinimides and Bacteriopurpurinimides
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1759
Table 3. Tumor to Skin Ratios of Photosensitizers 7-10, 18-21, and
24 at 24 h Postinjectiona
(c.1) Fluorinated versus Nonfluorinated Photosensitizers
as Methyl Esters: For in vivo studies, C3H mice were im-
planted subcutaneously with RIF tumors. A drug dose of 0.4
µmol/kg was used to evaluate the fluorinated and nonfluori-
nated photosensitizers. At 24 h postinjection, the mice (10
mice/group) were treated with the specific drug-activating
wavelength under the specified in vivo conditions for a total
light dose of 135 J/cm2 at a fluence rate of 75 mW/cm2. In
the purpurinimide series, initial experiments were performed
looking at the presence and position of the fluorinated sub-
stituent (Figure 8). Among the fluorinated photosensitizers (8
and 14; Log P ) 10.91), 8 displayed a better PDT response.
The corresponding nonfluorinated derivatives (6 and 13)
displayed only minimal PDT response with 10% of the mice
tumor-free by day 60. It was not as evident within the
nonfluorinated study, but overall, it was suggested that the
position of the substituent at the lower half of the macrocycle
(N-substitution) might play a significant role in enhancing the
PDT response.
In vivo
tumor to skin
ratio
std dev
(n ) 3)
PS
(λmax
)
Log P
7
8
9
10
24
18
19
20
21
704
708
709
705
705
788
791
788
791
9.43
10.91
12.13
14.67
-6.45
8.82
10.30
11.83
14.38
2.48
4.76
1.97
1.82
3.68
1.91
5.30
6.87
8.89
( 1.06
( 1.92
( 0.10
( 0.12
( 0.12
( 0.33
( 2.88
( 2.40
( 3.81
a In C3H mice bearing RIF tumors (determined by in vivo reflectance
spectroscopy.
been anticipated that as the Log P value increased, the photo-
sensitizer would be retained more selectively in the tumor.
(c) In Vivo Photosensitizing Efficacy. However, as can be
seen from Table 3, no direct correlations were observed between
the lipophilicity and photosensitizer uptake, except that the time
and wavelength at which to treat in vivo had been successfully
determined. Compared to the in vitro absorption characteristics
in THF, the in vivo absorption of each photosensitizer exhibited
a 4-5 nm red-shift (purpurinimides, ∼701 nm in THF shifted
to ∼704-709 nm in vivo; bacteriopurpurinimides, ∼783 nm
in THF shifted to ∼788-791 nm in vivo), which can reflect
the binding of the photosensitizers to various tissue components,
such as lipoproteins and human serum albumin site II.22
Under similar treatment conditions, fluorinated purpurinimide
8 was evaluated against its nonfluorinated analog, 11, with a
similar Log P value (8, 10.91; 11, 10.99) as a means to prove
the “proof of principle” that the N-aryl fluorinated substituent
enhanced the PDT response (Figure 9). At 60 days post-PDT,
there was a significant difference (*p < 0.0001) in the PDT
response of 8 in comparison to that of 11 (30% tumor-free for
8 versus 10% at day 60 for 11). This experiment illustrated the
Figure 8. Kaplan-Meier plot of the in vivo photosensitizing efficacy of nonfluorinated (6 and 13, Log P ) 9.43) and fluorinated (8 and 14, Log
P ) 10.91) photosensitizers evaluated in RIF/C3H. The mice were treated with laser light (705-708 nm, 135 J/cm2 at 75 mw/cm2) 24 h postinjection.
The control mice were measured for tumor regrowth (<400 mm3) and were not subjected to any photosensitizer or light.
Figure 9. Kaplan-Meier plot of the in vivo photosensitizing efficacy of 8 versus its nonfluorinated analog, 11 (similar Log P values), evaluated
in RIF/C3H. The mice were treated with laser light (705-708 nm, 135 J/cm2 at 75 mw/cm2) 24 h postinjection. The control mice were measured
for tumor regrowth (<400 mm3) and were not subjected to any photosensitizer or light.

1760 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Gryshuk et al.
Figure 10. Kaplan-Meier plot of the in vivo photosensitizing activity of fluorinated purpurinimides (7-10) and bacteriopurpurinimides (18-21)
varying in lipophilicity. The mice were treated with laser light (705-708 nm (7-10) and 788-791 nm (18-21), 135 J/cm2, 75 mw/cm2) at 24 h
postinjection. The control mice were measured for tumor regrowth (<400 mm3) and were not subjected to any photosensitizer or light.
Figure 11. Kaplan-Meier plot of the in vivo photosensitizing efficacy
of 8 and its corresponding water-soluble analog 24 at similar treatment
conditions (24 h postinjection). The control mice were measured for
tumor regrowth (<400 mm3) and were not subjected to any photosen-
sitizer or light.
Figure 12. Partial NMR spectra of (A) Bacteriopurpurinimide 19
(mixture of R- and S-isomers), (B) isomerically pure 19S-isomer, and
(C) isomerically pure 19R-isomer.
(c.2) Comparative Photosensitizing Efficacy of 8 and its
Water-Soluble Analog 24: The in vivo efficacy of photosen-
sitizers 8 and 24 was determined in both C3H mice bearing
RIF tumors and BALB/c mice inoculated with colon-26 tumors
at a dose of 0.7 µmol/Kg (treatment parameters: 135 J/cm2,
75mW/cm2 at 24 h postinjection). A similar response was
observed for both model systems with the BALB/c/colon-26
data presented. According to Figure 11, both photosensitizers
were effective and displayed long-term tumor cure.23 Compared
to the fluorinated purpurinimide 8, which yielded a 30% tumor-
free response at day 90, its corresponding water-soluble analog
24 displayed an enhanced tumor response with 70% of the mice
tumor-free.
There may be three possible explanations for the enhanced
photosensitizing efficacy of compound 24 over 8: (i) the five
carboxylic acids on 24 may be attributing to a difference in
intracellular localization, (ii) the PDT-induced mechanism of
action may differ between the two photosensitizers, or (iii) a
difference in tumor to skin uptake may play a role. The tumor
to skin ratio was evaluated in the Balb/c/colon-26 model, and
surprisingly, both 24 and 8 displayed optimal uptake at 24 h
postinjection. This new water-soluble fluorinated purpurinimide
exhibited a greater tumor to skin ratio (5.06), which may be a
importance of the N-aryl functionality containing the 3,5-bis-
(trifluoromethyl) benzyl groups.
Detailed biological studies with 8 and other fluorinated
photosensitizers varying the length of the alkyl ether chain
within both the purpurinimide (7-10) and the bacteriopurpu-
rinimide (18-21) series were then carried out under similar PDT
treatment conditions. We were interested to investigate if these
new longer-wavelength fluorinated photosensitizers mimic the
parabolic relationship between lipophilicity and PDT response
reported within the pyropheophorbide-a series.11,12 Instead, a
monotonic response was observed within both the fluorinated
purpurinimide and the bacteriopurpurinimide series (Figure 10).
As the length of the alkyl chain increased, the photosensitizing
activity enhanced and the O-dodecyl photosensitizers (10 and
21) displayed the best efficacy (50 or 60% tumor-free at day
60, respectively). The effect of lipophilicity was most evident
within the purpurinimide series (700 nm). Within the bacterio-
purpurinimide series (800 nm), the in vivo activity of photo-
sensitizers 20 and 21 was not significantly different (p )
0.8020). However, the tumor response results were comparable
with what had been observed within the 700 nm series of
compounds.

Purpurinimides and Bacteriopurpurinimides
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1761
Figure 13. Kaplan-Meier plot of the in vivo photosensitizing efficacy of 19 and its corresponding 19R- and 19S-isomers (0.4 µmol/kg; 135 J/cm2
at 75 mW/cm2, 24 h postinjection). The control mice were measured for tumor regrowth (<400 mm3) and were not subjected to any photosensitizer
or light.
factor for the better PDT outcome (compared to 8 with a ratio
of 3.38). Detailed biodistribution studies are required to confirm
this observation. These studies are currently in progress.
(c.3) Impact of Chirality on the Photosensitizing Efficacy
of the Bacteriopurpurinimide System: The alkyl ether analogs
of the purpurinimide and bacteriopurpurinimide systems contain
a chiral center at position-3 producing a mixture of diasteromeric
isomers (R- and S-configuration). For investigating the difference
(if any) of the individual isomers in photosensitizing efficacy,
one methyl ester photosensitizer, bacteriopurpurinimide 19, was
separated into the corresponding R- (19R) and S- (19S) isomers
by HPLC. The purity of the individual isomers was confirmed
with such a hydrophilic nature, the purpurinimide 24 showed a
significantly higher uptake in tumor than skin (tumor to skin
ratio: 5.06) at 24 h postinjection compared to that of 8 (tumor
to skin ratio of 3.38). The inherent charge as well as a significant
high tumor uptake could be the factors for an enhanced tumor
response (70% compared to 30% produced by purpurinimide
8). Attempts to convert bacteriopurpurinimide 26 bearing five
tert-butylester functionalities into the corresponding penta-
carboxylic acids under both acid and base conditions were
unsuccessful. In a modified synthetic approach, we plan to
replace the tert-butyl ester functionalities by the benzyl ester
substituents, which on hydrogenation under controlled conditions
should produce the desired water-soluble bacteriopurpurinimide.
The synthesis, the site(s) of localization of the foregoing
purpurinimides and bacteriopurpurinimides and the experiments
to understand its correlation in mechanism(s) of cell death are
currently in progress. We are also investigating the utility of
these fluorinated photosensitizers as “bifunctional agents” for
PDT and F-19 MR imaging.
1
by H NMR (Figure 12). The absolute stereochemistry of the
isomers was assigned by following the methodology reported
previously.24,25
Under similar treatment conditions, the PDT response of the
two isomers (19R and 19S) in the C3H mice bearing the RIF
tumors was compared to its parent mixture 19, and from the
results summarized in Figure 13, no significant difference in
photosensitizing efficacy between the two isomers (P values
for 19R, 0.9872, and 19S, 0.9744) was observed.
Experimental Section
Chemistry. Purpurin-18 methyl ester 1 and bacteriopurpurin-
18 methyl ester 15 were obtained from chlorophyll-a and bac-
teriochlorophyll-a, which in turn were extracted from Spirulina
pacifica and Rb. sphaeroides by following the methodology
previously reported from our laboratory.9,13,14 The 3,5-bis(trifluo-
romethyl) benzyl amine was purchased from Sigma-Aldrich and
used directly. The reactions were carried out under nitrogen in
degassed dried benzene and monitored by analytical thin layer
chromatography (TLC). Silica gel 60 (70-230 mesh, Merck) was
used for column chromatography. All the intermediates and the
Conclusion
The present study demonstrates the feasibility and success
of altering the lipophilicity of the purpurinimide system at
position-3 by varying the length of the alkyl ether chain. The
structural parameters established within the purpurinimide (700
nm) series 7-10 were then translated to the bacteriopurpurin-
imide (800 nm) 18-21. The in vivo photosensitizing results
within both series clearly indicated the superiority of the
O-dodecyl photosensitizers 10 and 21 in comparison to those
that were less lipophilic (O-methyl; 7 and 18). In contrast to
the alkyl ether analogs of pyropheophorbide-a, which produced
a parabolic relationship between the overall lipophilicity and
the photosensitizing efficacy, a monotonic relationship was
observed in both the purpurinimide and the bacteriopurpurin-
imide systems. Additionally, the chiral center at position-3,
which produces a mixture of diasteromeric isomers, did not
display significant differences (P ) 0.9744) in PDT response
when either separated (i.e., 19R or 19S) or left as a mixture 19.
Most of the porphyrin-based photosensitizers, except Npe6
or LS-11, that are under various phases of clinical trials are
insoluble in water, and formulating them in a suitable nontoxic
solvent is a major task. In our present study, we also report the
preparation of a water-soluble longer wavelength photosensitizer
24 (Log P ) -6.45 calculated by PALLAS program). Even
final compounds were characterized by UV-vis, 1H NMR, and 19
F
NMR spectroscopy (chemical shifts expressed in δ ppm are relative
to CDCl3 at 7.26 ppm) and high-resolution mass spectrometry.
3,5-Dimethylbenzylamine (2). The commercially available 3,5-
dimethylbenzyl bromide (0.6 g, 3 mmol) was reacted with 325 mg
NaN3, 350 mg (Bu)4NHSO4 (a phase transfer reagent), 6 mL
saturated NaHCO3, and 6 mL CHCl3 under N2 gas for ∼24 h. The
reaction was monitored periodically by analytical silica TLC in
hexane/CH2Cl2 (60:40) and at completion was washed with water/
NaHCO3 and CH2Cl2. The organic layer was collected, dried over
Na2SO4, and concentrated under vacuum, which yielded a clear
oily residue identified as the corresponding azide analog. This oily
residue was then hydrogenated under the presence of PtO2 (70 mg)
in MeOH (15 mL) overnight. The yellowish-colored residue 3,5-
dimethylbenzylamine (2) was used directly for the synthesis of 4.
Purpurin-18-N-3,5-bis(dimethyl)benzylimide (4). In brief, pur-
purin-18 methyl ester 1 (31 mg, 0.053 mmol) was reacted with 2

1762 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Gryshuk et al.
in 9 mL benzene and refluxed overnight to produce the desired
nonfluorinated purpurinimide, which exhibited a long wavelength
absorption at 707 nm characteristic of the formation of a fused six-
member imide ring system. The mixture was purified on a silica
analytical plate using 2% acetone in CH2Cl2 (yield: 19 mg, 51.4%).
UV-vis (ꢀ ) 53 600 at 705 nm in THF): 638.9 (1.22 × 103),
acetic acid was removed via high vacuum (∼1 h), yielding a dark
green/purple residue. To the dry residue was added 100 mg of
anhydrous K2CO3, 2 mL of methanol and 1.5 mL of dry CH2Cl2.
The reaction was stirred at RT under N2 gas for ∼1 h. At
completion, the reaction mixture was washed with water and
CH2Cl2, and the organic layer was collected, dried over Na2SO4,
and filtered. The filtrate was rotovapped to dryness, yielding a dark
purple crude material. The crude material was purified on silica
prep plates using a hexane/ethyl acetate (80:20) solvent system
(yield: 80 mg, 59.7%). Log P ) 9.43. UV-vis (ꢀ ) 58 000 at
700 nm in THF): 647.0 (1.14 × 104), 544.0 (2.63 × 104), 507.0
(1.06 × 104), 479.1 (7.71 × 103), 414.9 (1.55 × 105), 363.0 (5.53
× 104). 1H NMR (400 MHz, 3 mg/1 mL CDCl3, δ ppm): 9.75 and
9.63 (each s, 1H, for 10H and 5H), 8.52 (s, 1H, for 20H), 8.25 (s,
2H, 2 × ArH), 7.81 (s, 1H, ArH), 5.77 (m, 3H, N-CH2Ar and
31H), 5.34 (d, J ) 7.6 Hz, 1H, 17H), 4.35 (q, J ) 7.2 Hz, 1H,
18H), 3.68 (s, 3H, 12CH3), 3.50-3.60 (m, 8H, 172CO2CH3,
81CH2, 31OCH3), 3.31 (s, 3H, 7CH3), 3.19 (s, 3H, 2CH3), 2.64-
2.74 (m, 1H, 1 × 172H), 2.35-2.45 (m, 2H, 171H), 2.06 (m, 3H,
31CH3), 1.95-2.04 (m, 1H, 1 × 172H), 1.76 (d, J ) 6.8 Hz, 3H,
18CH3), 1.69 (t, J ) 7.4 Hz, 3H, 82CH3), 0.08 (br s, 1H, NH),
0.03 (br s, 1H, NH). 19F NMR (400 MHz, 3 mg/1 mL of CDCl3,
in reference to TFA, δ ppm): 13.28. Mass calculated for
C44H43N5O5F6, 835.32; found, 858.5 (M + Na). HRMS calculated
(M + H), 836.3246; found, 836.3224.
1
547.9 (1.62 × 104), 416.9 (1.43 × 105), 365.9 (4.14 × 104). H
NMR (400 MHz, 3 mg/1 mL CDCl3, δ ppm): 9.55 (s, 1H, 10H),
9.30 (s, 1H, 5H), 8.55 (s, 1H, 2OH), 7.86 (dd, J ) 18.0, 11.7, 1H,
31-CHdCH2), 6.9 (d, 2H, ArH), 6.37 (t, 1H, ArH), 6.25 (d, J )
18.0, 1H, 31-CHdCH2), 6.12 (d, J ) 11.7, 1H, 31-CHdCH2), 5.63
(m, 2H, NCH2Ar), 5.38 (m, 1H, 17-H), 4.35 (q, J ) 7.3, 1H, 18H),
3.80 (s, 3H, 12-CH3), 3.77 (s, 6H, Ar(CH3)2), 3.58 (q, J ) 7.8, 2H,
81CH2CH3), 3.54 (s, 3H, 172CO2CH3), 3.33 (s, 3H, 2-CH3), 3.12
(s, 3H, 7-CH3), 2.65-2.72 (m, 1H, 172CH2), 2.30-2.45 (m, 2H,
171CH2), 1.96-2.02 (m, 1H, 172CH2), 1.75 (D, J ) 7.0, 3H, 18-
CH3), 1.65 (t, J ) 7.0, 3H, 81CH2CH3), -0.05 (br s, 1H, NH),
-0.15 (br s, 1H, NH).18
Purpurin-18-N-3,5-bis(trifluoromethyl)benzylimide (5). The
purpurin-18 methyl ester 1 (1.0 g, 0.0017 mol) and commercially
available 3 (1.5 g, 0.0062 mol) in 15 mL of benzene was stirred
and refluxed for ∼24 h in the dark. The reaction was monitored
via analytical silica TLC in 2% acetone in CH2Cl2. At completion,
the mixture was concentrated under vacuum and purified on a silica
column (2% acetone in CH2Cl2). The appropriate eluates were
collected and evaporation of the solvent afforded 5 (yield: ∼650
mg, 50%). UV-vis (ꢀ ) 58 000 at 708 nm in THF): 650.0 (5.82
× 103), 549.0 (2.34 × 104), 511.1 (2.33 × 103), 416.9 (1.35 ×
105), 365.0 (4.60 × 104). 1H NMR (400 MHz, 3 mg/1 mL CDCl3,
δ ppm): 9.53 and 9.30 (each s, 1H, for 10-H and 5-H), 8.55 (s, 1H
for 20-H), 8.24 (s, 2H, 2 × ArH), 7.81 (s, 1H, 1 × ArH), 6.21 (dd,
J ) 17.44, 12.83 Hz, 1H), 5.77 (m, 2H, N-CH2Ar), 5.33 (d, J )
7.4 Hz, 1H, 17-H), 4.35 (m, J ) 6.12 Hz, 1H, 18-H), 3.80 (s, 4H),
3.55 (s, 6H), 3.33 (s, 4H), 3.12 (s, 4H), 2.171 (s, 1H), 1.964 (d,
2H), 1.77 (d, J ) 8.6 Hz, 4H), 1.64 (t, J ) 6.8, 5H), 1.54 (s, 4H),
0.114 (br s, 1H, 2 × NH). Mass calculated for C43H39N5O4F6,
803.29; found, 804.2 (M + H).18
3-Devinyl-3-(11-butoxyethyl)-purpurin-18-N-3,5-bis(trifluo-
romethyl)benzylimide (8). Compound 5 (200 mg, 0.25 mmol) was
reacted with ∼5 mL of 30% HBr in acetic acid for ∼2 h. The excess
acetic acid was removed via high vacuum (∼45 min), yielding a
dark green/purple residue. To the dry residue was added 100 mg
of anhydrous K2CO3, 1 mL of n-butanol, and 3 mL of dry CH2Cl2.
The reaction was stirred at RT under N2 gas for ∼1 h. At
completion, the reaction mixture was washed with water and
CH2Cl2, and the organic layer was collected, dried over Na2SO4,
and filtered. The filtrate was rotovapped to dryness to yield a dark
purple crude material. The crude material was purified on silica
prep plates using a hexane/ethyl acetate (80:20) solvent system.
The pure compound exhibited a characteristic absorption peak at
701 nm in CH2Cl2 (yield: 100 mg, 45.7%). Log P ) 10.91. UV-
vis (ꢀ ) 58 000 at 700 nm in THF): 644.1 (8.65 × 103), 544.0
(2.42 × 104), 507.9 (7.53 × 103), 478.0 (4.33 × 103), 415.1 (1.57
× 105), 364.0 (4.98 × 104). 1H NMR (400 MHz, 3 mg/1 mL CDCl3,
δ ppm): 9.75 and 9.63 (each s, 1H, for 10H and 5H), 8.52 (s, 1H,
for 20H), 8.25 (s, 2H, 2 × ArH), 7.81 (s, 1H, ArH), 5.77 (m, 3H,
N-CH2Ar and 31H), 5.34 (d, J ) 7.6 Hz, 1H, 17H), 4.35 (q, J )
7.2 Hz, 1H, 18H), 3.84 (s, 3H, 12CH3), 3.69 (m, 4H, 81CH2,
31OCH2-C3H7), 3.31 (s, 3H, 7CH3), 3.19 (s, 3H, 2CH3), 2.68 (m,
1H, 1 × 172H), 2.37 (m, 2H, 2 × 171H), 2.06 (m, 3H, 31CH3),
1.96 (m, 1H, 1 × 172H), 1.76 (d, J ) 6.8 Hz, 2H, 18CH3), 1.69 (t,
J ) 7.4 Hz, 3H, 82CH3), 1.35-1.55 (m, 4H, OCH2(CH2)2CH3),
0.89 (m, 3H, O-(CH2)3CH3), 0.17 (br s, 1H, NH), 0.05 (br s, 1H,
NH). 19F NMR (400 MHz, 3 mg/1 mL of CDCl3, in reference to
TFA, δ ppm): 13.05. Mass calculated for C47H49N5O5F6, 877.36;
found, 878.3 (M + 1). HRMS calculated (M + 1), 878.3716; found,
878.3723.
3-Devinyl-3-(11-heptoxyethyl)-purpurin-18-N-3,5-bis(trifluo-
romethyl)benzylimide (9). Compound 5 (85 mg, 0.11 mmol) was
reacted with 3.5 mL of 30% HBr in acetic acid for ∼2 h. The excess
acetic acid was removed via high vacuum (∼30 min), yielding a
dark green/purple residue. To the dry residue was added 19.6 mg
of dry K2CO3, 0.75 mL of n-heptanol, and 3 mL of dry CH2Cl2.
The reaction was stirred at RT under N2 gas for ∼1 h. At
completion, the reaction mixture was washed with water and
CH2Cl2, and the organic layer was collected, dried over Na2SO4,
and filtered. The filtrate was concentrated under vacuum to yield
a dark purple crude material. The crude material was purified on
silica prep plates using a hexane/ethyl acetate (80:20) solvent
system. The pure compound exhibited a characteristic absorption
peak at 701 nm in CH2Cl2 (yield: 44 mg, 43.6%). Log P ) 12.13.
UV-vis (ꢀ ) 58 000 at 700 nm in THF): 644.0 (9.14 × 103),
544.0 (2.53 × 104), 508.0 (8.70 × 103), 480.0 (5.45 × 103), 414.9
3-Devinyl-3-(11-butoxyethyl)-purpurin-18-N-3,5-bis(dimeth-
yl)benzylimide (6). Compound 4 (19 mg, 0.027 mmol) was reacted
with 1.5 mL 30% HBr in acetic acid as a means to activate the
vinyl group. After 1 h of stirring, the reaction was stopped and the
excess HBr/acetic acid was removed via high vacuum. To the
intermediate bromo analog was added 0.5 mL of butanol, 10 mg
of anhydrous K2CO3, and 2 mL of dry CH2Cl2. After ∼1 h of
stirring under N2 gas, the reaction was stopped and washed with
water and CH2Cl2, and the organic layer was collected, dried over
Na2SO4, and filtered. The filtrate was rotovapped to dryness and
aziotroped with water (if necessary) to remove any unreacted
alcohol. The crude compound was purified on analytical silica plates
using a hexane/ethyl acetate (70:30) solvent system, where it
exhibited a peak absorbance at 699 nm in CH2Cl2 (yield: 12 mg,
57.7%). Log P ) 9.47. UV-vis (ꢀ ) 53 600 at 698 nm in THF):
640.9 (7.68 × 103), 543.0 (2.10 × 104), 507.0 (7.97 × 103), 478.0
1
(4.80 × 103), 415.1 (1.44 × 105), 364.0 (4.72 × 104). H NMR
(400 MHz, 3 mg/1 mL CDCl3, δ ppm): 9.75 and 9.64 (each s, 1H,
for 10H and 5H), 8.53 (s, 1H, for 20H), 7.33 (s, 2H, 2 × ArH),
6.89 (s, 1H, ArH), 5.78 (q, J ) 6.8 Hz, 1H, 31CH), 5.62 (q, J )
12.8, 2H, NCH2Ar), 5.42 (d, J ) 8.2 Hz, 1H, 17H), 4.33 (q, J )
7.2 Hz, 1H, 18H), 3.83 (s, 3H, 12CH3), 3.60-3.72 (m, 4H, 81CH2
and 31-OCH2C3H7), 3.53 (s, 3H, 172CO2CH3), 3.30 (s, 3H, 7CH3),
3.18 (s, 3H, 2 CH3), 2.62-2.70 (m, 1H, 172H), 2.24-2.44 (m, 2H,
171H), 2.31 (s, 6H, 2 × Ar-CH3), 2.05 (m, 3H, 31CH3), 1.98-
2.04 (m, 1H, 172H), 1.75 (d, J ) 6.8 Hz, 3H, 18CH3), 1.64-1.78
(m, 2H, 31-OCH2CH2CH2CH3), 1.67 (t, J ) 7.8 Hz, 4H, 82CH3),
1.40-1.50 (m, 2H, 31O(CH2)2CH2CH3), 0.862 (m, 31O(CH2)3CH3),
-0.05 (br s, 1H, NH), -0.15 (br s, 1H, NH). Mass calculated for
C47H55N5O5, 769.42; found, 792.4 (M + Na). HRMS calculated,
769.4209; found, 769.4256.
3-Devinyl-3-(11-methoxyethyl)-purpurin-18-N-3,5-bis(trifluo-
romethyl)benzylimide (7). Compound 5 (125 mg, 0.16 mmol) was
reacted with 3 mL of 30% HBr in acetic acid for ∼2 h. The excess

Purpurinimides and Bacteriopurpurinimides
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1763
(1.53 × 105), 363.9 (5.20 × 104). 1H NMR (400 MHz, 3 mg/1 mL
CDCl3, δ ppm): 9.80 and 9.57 (each s, 1H, for 10H and 5H), 8.58
(s, 1H, for 20H), 8.29 (s, 2H, 2 × ArH), 7.86 (s, 1H, ArH), 5.75-
5.93 (m, 3H, N-CH2Ar and 31H), 5.40 (d, J ) 7.6 Hz, 1H, 17H),
4.40 (q, J ) 7.2 Hz, 1H, 18H), 3.78 (s, 3H, 12CH3), 3.62-3.75
(m, 4H, 81CH2, OCH2C6H13), 3.60 (s, 3H, 172CO2CH3), 3.36 (s,
3H, 7CH3), 3.23 (s, 3H, 2CH3), 2.66-2.80 (m, 1H, 1 × 172H),
2.35-2.52 (m, 2H, 2 × 171H), 2.10 (m, 3H, 31CH3), 1.96-2.07
(m, 1H, 1 × 172H), 1.84 (d, J ) 6.8 Hz, 3H, 18CH3), 1.75-1.81
(m, 2H, OCH2CH2(CH2)4CH3), 1.69 (t, J ) 7.4 Hz, 3H, 82CH3),
1.35-1.55 (m, 2H, O(CH2)2CH2(CH2)3CH3), 1.15-1.34 (m, 6H,
O(CH2)3(CH2)3CH3), 0.83 (m, 3H, O-(CH2)6CH3), 0.18 (br s, 1H,
NH), 0.09 (br s, 1H, NH). 19F NMR (400 MHz, 3 mg/1 mL of
CDCl3, in reference to TFA, δ ppm): 13.03. Mass calculated for
C50H55N5O5F6, 919.41; found, 942.4 (M + Na). HRMS calculated
(M + 1), 920.4185; found, 920.4167 (M + 1).
N2 gas. During this time, the long wavelength absorption shifted
from 699 to 666 nm. The solvent was removed and hexane was
added to the dried reaction mixture, and after 2 h in the freezer,
the recovered 1 was collected via filtration and the residue was
washed with excess hexane. The crystals were transferred back to
the reaction flask, dissolved in 10 mL of CH2Cl2/THF (1:1), and
treated with diazomethane. The intermediate were cyclized to the
corresponding cyclic imide on reacting with a catalytic amount of
KOH/MeOH solution for 4-5 min with vigorous stirring, and the
reaction was monitored by UV-vis spectroscopy (appearance of a
new peak at 706 nm). The reaction mixture was washed with 2%
acetic acid in water to neutralize the KOH and washed again with
water (3 × 250 mL). The solvent was removed and purified on a
silica column using a 3% MeOH in CH2Cl2 as an eluent (yield:
111 mg, 84.7%). UV-vis (ꢀ ) 58 000 at 706 nm in THF): 549.0
(2.51 × 104), 510.9 (6.33 × 103), 419.0 (1.75 × 105), 368.0 (6.05
× 104). 1H NMR (400 MHz, 3 mg/1 mL of CDCl3, δ ppm): 9.47
(s, 1H, 10H), 9.23 (s, 1H, 5H), 8.55 (s, 1H, 20H), 7.82 (dd, J )
18.0, 11.7, 1H 31CHdCH2), 6.24 (d, J ) 18.0, 1H, 31CHdCH2),
6.10 (d, J ) 11.7, 1H, 31CHdCH2), 5.40 (m, 1H, 171H), 4.46 (m
2H, NCH2CH2CH2CH3), 4.35 (q, J ) 7.3, 1H, 18H), 3.76 (s, 3H,
12CH3), 3.57 (s, 3H, 172CO2CH3), 3.54 (q, J ) 7.8, 2H, 81CH2-
CH3), 3.32 (s, 3H, 2CH3), 3.06 (s, 3H, 7CH3), 2.62-2.75 (m, 1H,
172CH2), 2.30-2.50 (m, 2H, 171CH2), 1.95-2.05 (m, 3H, 172CH2
and NCH2CH2CH2CH3), 1.78 (d, J ) 7.0, 3H, 18CH3), 1.58-1.70
(m, 5H, 81CH2CH3 and NCH2CH2CH2CH3), 1.12 (t, J ) 6.3, 3H,
NCH2CH2CH2CH3), -0.18 (br s, 1H, NH), -0.28 (br s, 1H, NH).18
3-Devinyl-3-[11-3,5-bis(dimethyl)benzyl]ethyl-purpurin-18-N-
butylimide (13). Further reaction of 12 with HBr/acetic acid
followed by addition of commercially available 3,5-dimethylbenzyl
alcohol with K2CO3 and CH2Cl2 gave the corresponding N-butyl
derivative 13. The crude product was purified on preparative silica
plate using 80:20 hexane/ethyl acetate. Each of these nonfluorinated
analogs maintained a Log P value of 9.43. UV-vis (ꢀ ) 53 600 at
698 nm in THF): 639.0 (8.08 × 103), 543.0 (2.10 × 104), 507.0
(8.37 × 103), 478.0 (5.20 × 103), 415.1 (1.46 × 105), 364.0 (4.97
× 104). 1H NMR (400 MHz, 3 mg/1 mL of CDCl3, δ ppm): 9.79
and 9.63 (each s, 1H, for 10H and 5H), 8.60 (s, 1H, for 20H), 7.79
(s, 1H, ArH), 6.96 (m, 2H, 2 × ArH), 5.91 (m, 1H, 31H), 5.44 (m,
1H, 17H), 4.57 (s, 2H, OCH2Ar), 4.49 (t, J ) 7.8 Hz, 2H,
N-CH2C3H7), 4.39 (m, 1H, 18H), 3.82 (s, 3H, 12CH3), 3.66 (q, J
) 7.6 Hz, 2H, 81CH2), 3.58 (s, 3H, 172CO2CH3), 3.34 (s, 3H,
7CH3), 3.12 (s, 3H, 2CH3), 2.70 (m, 1H, 1 × 172H), 2.46 (m, 1H,
1 × 172H), 2.20-2.40 (m, 1H, 1 × 171H), 2.25 (s, 6H, 2 ×
Ar-CH3), 2.12 (d, J ) 6.4 Hz, 3H, 32CH3), 2.00-2.05 (m, 3H, 1
× 172H and NCH2CH2CH2CH3), 1.80 (d, J ) 6.8, 3H, 8CH3),
1.62-1.70 (m, 5H, 82CH3 and N-CH2CH2CH2CH3), 1.12 (t, J )
7.4 Hz, 3H, N-CH2CH2CH2CH3), -0.09 (br s, 1H, NH), -0.14
(br s, 1H, NH). Mass calculated for C47H55N5O5, 769.42; found,
769/770 (M). HRMS calculated, 769.4209; found, 789.4234.
3-Devinyl-3-[11-3,5-bis(trifluoromethyl)benzyl]ethyl-purpurin-
18-N-butylimide (14). Compound 12 (111 mg, 0.18 mmol) was
reacted with ∼2.5 mL of 30% HBr in acetic acid for ∼1.5 h. The
excess acetic acid was removed via high vacuum (∼45 min). To
the dry residue was added the commercially available 3,5-bis-
(trifluoromethyl) benzyl alcohol (∼400 mg, 1.6 mmol), 28 mg of
anhydrous K2CO3, and ∼3 mL of CH2Cl2. The reaction was stirred
at RT under argon for ∼3 h. At completion (shift from 708 f 701
nm), the reaction mixture was washed with water and CH2Cl2, and
the organic layer was collected, dried over Na2SO4, and filtered.
The filtrate was rotovaporated to dryness, and the crude product
was purified on analytical silica prep plates using the 1.5% MeOH
in CH2Cl2 solvent system (yield: 13.8 mg, 10%). Log P ) 10.91.
UV-vis (ꢀ ) 58 000 at 700 in THF): 642.0 (7.66 × 103), 543.0
(2.16 × 104), 507.0 (8.19 × 103), 479.1 (4.75 × 103), 415.1 (1.51
3-Devinyl-3-(11-dodecyoxyethyl)-purpurin-18-N-3,5-bis(tri-
fluoromethyl)benzylimide (10). Compound 5 (204 mg, 0.25 mmol)
was reacted with 3 mL of 30% HBr in acetic acid for ∼2 h. The
excess acetic acid was removed via high vacuum (∼30 min),
yielding a dark green/purple residue. To the dry residue was added
100 mg of dry K2CO3, 1-1.5 mL of n-dodecanol, and 3 mL of dry
CH2Cl2. The reaction was stirred at RT under N2 gas for ∼1 h. At
completion, the reaction mixture was washed with water and
CH2Cl2, and the organic layer was collected, dried over Na2SO4,
and filtered. The filtrate was concentrated under vacuum to yield
a dark purple crude material. The excess dodecanol was aziotroped
with water. Initially, the compound was purified on a silica column
and then on an alumina column with CH2Cl2 as the solvent system
to help remove the remaining alcohol. The filtrate was collected,
rotavaporated to dryness, and additionally purified on silica prep
plates using a hexane/ethyl acetate (80:20) solvent system. Due to
the excessive purification demands, the percent yield of this
compound was lower compared to the other fluorinated purpurin-
imides (yield: 25.8 mg, 10.4%). Log P ) 14.67. UV-vis (ꢀ )
58 000 at 700 nm in THF): 643.0 (9.45 × 103), 544.0 (2.53 ×
104), 508.0 (8.66 × 103), 478.0 (5.77 × 103), 416.0 (1.60 × 105),
1
363.9 (5.42 × 104). H NMR (400 MHz, 3 mg/1 mL of CDCl3, δ
ppm): 9.81 and 9.53 (each s, 1H, for 10H and 5H), 8.6 (s, 1H, for
20H), 8.30 (s, 2H, 2 × ArH), 7.87 (s, 1H, ArH), 5.77 (m, 3H,
N-CH2Ar and 31H), 5.41 (d, J ) 6.9 Hz, 1H, 17H), 4.42 (m, 1H,
18H), 3.75 (s, 3H, 12CH3), 3.63-3.69 (m, 4H, OCH2C11C23,
81CH2), 3.61 (s, 3H, 172CO2CH3), 3.36 (s, 3H, 7CH3), 3.22 (s, 3H,
2CH3), 2.65-2.83 (m, 1H, 1 × 172H), 2.35-2.53 (m, 2H, 2 ×
171H), 2.12 (m, 3H, 31CH3), 1.96-2.07 (m, 1H, 1 × 172H), 1.85
(m, 3H, 18CH3), 1.74-1.82 (m, 2H, OCH2CH2(CH2)9CH3), 1.12-
1.54 (m, 18H, OCH2CH2(CH2)9CH3), 0.89 (m, 3H, O-(CH2)11CH3),
0.16 (br s, 1H, NH), 0.09 (br s, 1H, NH). 19F NMR (400 MHz, 3
mg/1 mL of CDCl3, in reference to TFA, δ ppm): 13.17. Mass
calculated for C55H66N5O5F6, 989.49; found, 1012.6 (M + Na).
HRMS calculated (M + 1), 990.4968; found, 990.4994 (M + 1).
3-Devinyl-3-(11-heptoxyethyl)-purpurin-18-N-3,5-dimethyl-
benzylimide (11). By following similar reaction conditions dis-
cussed for compound 6, compound 4 was reacted with HBr in acetic
acid, n-heptanol, and K2CO3 in CH2Cl2 to produce 11, which is
the nonfluorinated structural analog of 8 with similar lipophilicity.
Log P ) 11.00. UV-vis (ꢀ ) 53 600 at 698 nm in THF): 543.0
(1.63 × 104), 508.0 (2.18 × 103), 414.9 (1.52 × 105), 363.9 (4.17
× 104). 1H NMR (400 MHz, 3 mg/1 mL of CDCl3, δ ppm): 9.76
and 9.66 (each s, 1H for 5-H and 10-H), 8.54 (s, 1H, 20-H), 7.33
(s, 2H, 2 × ArH), 6.90 (s, 1H, 1 × ArH), 5.81-5.76 (m, J ) 6.8
Hz, 1H, 31CH), 5.68-5.58 (m, J ) 10.9, 2H, N-CH2Ar), 5.44 (d,
2H), 3.85 (s, 4H), 3.71-3.65 (m, 4H, 81CH2 and 31OCH2), 3.54
(s, 3H, 172CO2CH3), 3.50 (s, 2H), 3.31 (s, 3H, 7CH3), 3.20 (s, 4H),
2.32 (s, 11H), 2.17 (s, 9H), 2.06 (dd, 4H), 1.69 (t, J ) 7.3 Hz,
5H), 1.53 (s, 20H), 1.10 (t, J ) 6.8 Hz, 2H), 0.778 (m, 4H), -0.015
(br s, 1H, 2 × NH). Mass calculated for C50H61N5O5, 811.47; found,
835.5 (M + Na). HRMS calculated, 812.4751; found, 812.4746.
Purpurin-18-N-butylimide (12). Compound 1 (120 mg, 0.21
mmol) was reacted with 1 mL of n-butylamine (30.7 mg, 0.42
mmol) while stirring at RT in 10 mL of CH2Cl2 for ∼24 h under
1
× 105), 365.0 (5.21 × 104). H NMR (400 MHz, 3 mg/1 mL of
CDCl3, δ ppm): 9.73 and 9.68 (each s, 1H, for 10H and 5H), 8.60
(s, 1H, for 20H), 7.85 (s, 2H, 2 × ArH), 7.81 (s, 1H, ArH), 5.96
(q, J ) 6.8 Hz, 1H, 31H), 5.43 (d, J ) 8.0 Hz, 1H, 17H), 4.79 (s,
2H, O-CH2Ar), 4.48 (t, J ) 7.8 Hz, 2H, N-CH2C3H7), 4.38 (q, J
) 7.2 Hz, 1H, 18H), 3.86 (s, 3H, 12CH3), 3.69 (q, J ) 7.6 Hz, 2H,

1764 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Gryshuk et al.
81CH2), 3.57 (s, 3H, 172CO2CH3), 3.10 (s, 3H, 7CH3), 2.68 (m,
1H, 1 × 172H), 2.44 (m, 1H, 1 × 171H), 2.32 (m, 1 × 171H), 2.21
(d, J ) 6.4 Hz, 3H, 32CH3), 1.99 (m, J ) 5.4 Hz, 3H, N-CH2CH2-
CH2CH3 and 1 × 172H), 1.78 (d, J ) 6.8 Hz, 3H, 18CH3), 1.66
(m, 5H, N-CH2CH2CH2CH3 and 82CH3), 1.11 (t, J ) 7.4 Hz, 3H,
N-CH2CH2CH2CH3), -0.18 (br s, 1H, NH), -0.21 (br s, 1H, NH).
19F NMR (400 MHz, 3 mg/1 mL of CDCl3, in reference to TFA,
δ ppm): 12.60. Mass calculated for C47H49N5O5F6, 877.36; found,
879 (M + 2). HRMS calculated (M + 1), 878.3716; found,
878.3742 (M + 1).
(m, 2H, -CH2CH2COOCH3), 1.80 (d, J ) 7.17 Hz, 3H, 7-CH3),
1.70 (d, J ) 6.82 Hz, 3H, 18-CH3), 1.1 (t, J ) 6.46 Hz, 3H,
8-CH2CH3), -0.03 (s, 1H, NH), -0.65 (s, 1H, NH). Anal. Calcd
for C34H36N4O6‚H2O: C, 66.42; H, 6.24; N, 9.12. Found: C, 66.30;
H, 5.90; N, 8.99. Mass (m/e) calcd for C34H36N4O6, 596.3; found,
596.8 (M + 1). HRMS calcd, 596.2635; found, 596.2615.
Bacteriopurpurin-18-N-3,5-bis(trifluoromethyl)benzylimide
Methyl Ester (16). Compound 15 (250 mg, 0.42 mmol) and 3,5-
bis(trifluoromethyl)benzyl amine (3 g, 0.012 mol) were refluxed
in 25 mL of dry benzene under N2 gas for ∼24 h. According to
TLC (3% MeOH in CH2Cl2 on silica), there was a mixture of two
compounds, the desired 16 and a byproduct (3-acetyl-3[11-3,5-bis-
(trifluoromethyl)benzyl]ethyl-bacteriopurpurin-18-N-3,5-bis(trifluo-
romethyl) benzyl imide. The reaction was stopped and washed with
water and CH2Cl2. The organic layer was collected, dried over
Na2SO4, and concentrated under vacuum. As a means to convert
everything to the desired product, the crude material was dissolved
in CH2Cl2 and treated with dilute HCl (the reaction was monitored
by UV-vis.). The reaction yielded a single peak at 826 nm,
converting the majority of product to 16. Upon completion, the
reaction was immediately washed with water and CH2Cl2 (3×),
and the organic layer was collected, dried over Na2SO4, and
concentrated under vacuum. The crude material was purified on
silica prep plates in 1% acetone in CH2Cl2 (yield: 245 mg, 71%).
UV-vis (ꢀ ) 59 500 at 820 in THF): 544.9 (3.19 × 104), 414.9
(3.87 × 104), 363.9 (7.45 × 104). 1H NMR (400 MHz, 3 mg/1 mL
of CDCl3, δ ppm): 9.20 (s, 1H, 5-H), 8.80 (s, 1H, 10-H), 8.60 (s,
1H, 20-H), 8.19 (s, 2H, Ar-H), 7.80 (s, 1H, Ar-H), 5.74 (s, 2H,
NCH2Ar), 5.25 (m, 1H, 17-H), 4.22-4.32 (m, 2H, 7-H and 18-H),
4.05-4.15 (m, 1H, 8-H), 3.70 (s, 3H, 12-CH3), 3.55 (s, 3H, 172-
CO2CH3), 3.54 (s, 3H, 2-CH3), 3.17 (s, 3H, 3-COCH3), 2.60-2.70
(m, 1H, 1 × 171H), 2.43 (m, 3H, 81CH2 and 1 × 171H), 2.00-
2.10 (m, 1H, 1 × 172H), 1.85-1.95 (m, 1H, 1 × 172H), 1.81 (d,
J ) 5.8, 3H, 7-CH3), 1.72 (d, J ) 6.9, 3H, 18-CH3), 1.10 (t, J )
7.0, 3H, 82CH3), -0.35 (br s, 1H, NH), -0.61 (br s, 1H, NH).
Mass calculated for C43H41N5O5F6, 821.30; found, 844.4 (M + Na).
HRMS calculated, 821.3012; found, 821.3007.
3-Devinyl-3-(1′-butoxyethyl)-purpurin-18-N-3,5-bis(trifluo-
romethyl)benzyl-17-aminobenzyl-DTPA-imide (24). PS 8 (130
mg, 0.15 mmol) was dried under high vacuum, dissolved in dry
degassed THF (40 mL), and stirred under N2 gas. Upon addition
of LiOH solution (with minimal MeOH added to the reaction
mixture), the mixture changed from a dark/purple hue to green.
After ∼3 h, the reaction was stopped and washed with CH2Cl2.
The organic layer was collected, dried over Na2SO4, filtered, and
evaporated to dryness (yield: 130 mg, 100%). To this crude product
(130 mg, 0.15 mmol) was added amino-DTPA t-butyl protected
(170 mg), 20 mg of 4-(dimethylamino)pyridine (DMAP), and 49
mg of 1,3-dicyclohexylcarbodiimine (DCC) in ∼4 mL of dry
CH2Cl2. The reaction was stirred under N2 gas for ∼24 h, and excess
water (1-2 mL) was added to decompose DCC. The organic layer
was collected, dried over Na2SO4, filtered, and evaporated to
dryness. Excess CH2Cl2 was added to crystallize out DCC impurity
(urea) that was later removed by filtration. The product was purified
on silica prep plates using 8% MeOH in CH2Cl2 and then on an
Alumina III column, first eluting with CH2Cl2, followed by ethyl
acetate/CH2Cl2 (1:9). The final solvent system of ethyl acetate/
CH2Cl2 (1:3) yielded 105 mg of 23 (mass calculated for
C87H115N9O14F6, 1624.89; found, 1647.6 (M + Na + H)). For
deprotection, a small amount of concentrated trifluoroacetic acid
(TFA) was added to the DTPA-protected compound (∼9 mg) and
stirred at RT for ∼3 h. TFA was removed under high vacuum.
The residue was dissolved in 3 mL Na2HPO4 and then 3 mL
NaHPO4, which yielded 24 formulated in a water-based solution
at a pH of ∼7.4. Log P ) -6.45. UV-vis (ꢀ ) 41,181 at 711 in
H2O): 659.1 (1.19 × 104), 550.0 (2.24 × 104), 513.0 (7.22 × 103),
481.1 (5.78 × 103), 418.9 (1.06 × 105), 363.9 (7.08 × 104). HRMS
calculated, 1343.5337; found, 1343.5350.
3-Deacetyl-3(11-hydroethyl)-bacteriopurpurin-18-N-3,5-bis-
(trifluoro- methyl)benzylimide (17). Compound 16 (31 mg, 0.38
mmol) was initially dissolved in ∼20-25 mL of dry CH2Cl2 and
MeOH (∼5-10 mL). In small increments, a total of 60 mg of dry
NaBH4 was added, and the reaction mixture was stirred for 30 min.
After completion of the reaction (monitored by analytical TLC and
spectrophotometry), the contents were transferred to a beaker
containing ice + water. A 2% aqueous acetic acid solution was
slowly added to the reaction mixture until the solution became
neutral (pH ) ∼7). The mixture was then washed with water (100
mL), the organic layer was concentrated under vacuum, and the
residue was purified on silica prep plates in 2% acetone in CH2Cl2
(yield: 25 mg, 80.6%). UV-vis (ꢀ ) 40 530 at 778 in THF): 537.0
(3.94 × 104), 504.0 (3.94 × 103), 469.0 (3.01 × 103), 416.9 (4.47
Bacteriopurpurinimides. Synthesis of Bacteriopurpurin-18
Methyl Ester (15). Rb. sphaeroides (∼1.5 kg) and ∼3000 mL of
N-propanol were stirred overnight with a continuous flow of N2
gas bubbled through the mixture. The bacterial sludge was filtered
through a buchner funnel, and the blackish-blue/green filtrate was
collected (peak absorbance at 776 nm in N-propanol). A KOH
solution was added to the filtrate. With O2 bubbling through the
solution, the reaction was stirred for ∼1 h at RT. The reaction was
complete when a wavelength absorption shift occurred from 776
to 768 nm. At this point, the mixture was red; however, when
transferring to the ice + water solution, the mixture turned bluish-
green. While stirring, 5% H2SO4 was added dropwise until the
mixture reached a pH in the range of 2-3 (changed back to dark
red hue). The mixture was washed with water and CH2Cl2, and the
organic layer was collected, dried over Na2SO4, and filtered. The
filtrate was rotovapped to dryness and refluxed in THF for ∼30
min until a peak at ∼815 nm was noticeable. Hexane was added
to the residue, and the solid was collected via Buchner funnel
filtration. The filtrate mainly containing the carotene analogs was
discarded, and the solid, isolated as a carboxylic acid analog, was
treated with diazomethane and converted into the methyl ester. The
crude material was purified on a silica column using 2% acetone
in CH2Cl2 (yield: ∼2.2 g). UV-vis (in CH2Cl2): 364 nm (ꢀ 8.91
× 104), 412 nm (ꢀ 5.36 × 104), 545 nm (ꢀ 3.4 × 104), 815 nm (ꢀ
1
× 104), 367.0 (9.94 × 104), 347.0 (4.45 × 104). H NMR (400
1
1
MHz, 3 mg/1 mL of CDCl3, δ ppm): 8.80 (s, /2H, /25-H), 8.78
1
1
(s, /2H, /25-H), 8.56 (s, 1H, 10-H), 8.29 (s, 1H, 20-H), 8.19 (s,
2H, Ar-H), 7.80 (s, 1H, Ar-H), 6.18 (q, J ) 6.6, 1H, 31H), 5.72
(m, 2H, NCH2Ar), 5.19 (m, 1H, 17-H), 4.12-4.22 (m, 2H, 7-H
and 18-H), 3.95-4.02 (m, 1H, 8-H), 3.59 (s, 3H, 12-CH3), 3.56 (s,
1
3
1
3/2H, /2172CO2CH3), 3.55 (s, /2H, /2172CO2CH3), 3.24 (s, 3H,
2-CH3), 2.60-2.70 (m, 2H, 2 × 171H), 2.24-2.40 (m, 3H, 81CH2
and 1 × 172H), 2.05 (d, J ) 6.8, 3H, 31CH3), 1.85-1.95 (m, 1H,
1 × 172H), 1.78 (dd, J ) 3.0,3.5, 3H, 7-CH3), 1.68 (m, 3H, 18-
CH3), 1.09 (t, J ) 6.6, 3H, 82CH3), 0.28 (br s, 1H, NH); -0.11 (br
s, 1H, NH). HRMS calculated, 823.3168; found, 823.3173.
3-Deacetyl-3-(11-methoxyethyl)-bacteriopurpurin-18-N-3,5-
bis(trifluoro-methyl)benzylimide (18). A similar procedure as
described for 7-10 was followed for compounds 18-21, except
instead of using HBr/acetic acid, the HBr gas was used for the
preparation of the intermediate bromo derivative (under HBr/acetic
acid conditions, the bacteriopurpurinimides were found to be
unstable). Compound 17 (45 mg, 0.055 mmol) was dissolved in
∼30 mL of dry CH2Cl2. All air was removed from the reaction
1
5.53 × 104). H NMR (400 MHz, 3 mg/1 mL CDCl3, δ ppm):
9.21 (s, 1H, 5-H), 8.78 (s, 1H, 10-H), 8.62 (s, 1H, 20-H), 5.13 (m,
1H, 17-H), 4.30 (m, 2H, 1H for 7-H, 1H for 18-H), 4.08 (m, 1H,
8-H), 3.63 (s, 3H, 12-CH3), 3.57 (s, 3H, -COOCH3), 3.52 (s, 3H,
2-CH3), 3.15 (s, 3H, CH3C), 2.70 (m, 1H, -CHHCH2COOCH3),
2.42 (m, 2H, 8-CH2CH3), 2.35 (m, 1H, -CHHCH2COOCH3), 2.00

Purpurinimides and Bacteriopurpurinimides
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1765
flask and flushed with argon. Then under completely dry conditions,
HBr gas was passed through a needle and submerged into the
solution for ∼20 s. While stirring for ∼10 min, there appeared to
be a visible change from dark green to a purple hue of the solution.
The reaction mixture was then concentrated under high vacuum
for ∼1 h, which yielded a dark purple residue. To the residue was
added ∼30 mL of dry CH2Cl2, 90 mg of dry K2CO3, and 1.5 mL
of n-methanol, which was stirred under N2 gas for ∼30 min. Upon
completion, the contents were transferred to a beaker containing
ice + water, and the pH was adjusted to 2-2.5 by adding aqueous
acetic acid. After additional washes with water and CH2Cl2 (3×),
the organic layer was collected, dried over Na2SO4, and concentrated
under vacuum at room temperature to yield a dark purple crude
material. The crude material was purified on silica prep plates using
a hexane/ethyl acetate (80:20) solvent system. The pure compound
exhibited a characteristic absorption peak at 783 nm in CH2Cl2
(yield: 23.8 mg, 51.7%). Log P ) 8.82. UV-vis (ꢀ ) 40 530 at
784 in THF): 538.0 (3.91 × 104), 503.0 (5.63 × 103), 417.1 (5.49
NCH2Ar), 5.64 (m, 1H, 31H), 5.20 (m, 1H, 17-H), 4.19 (m, 2H,
7-H and 18-H), 4.00 (m, 1H, 8-H), 3.61 (s, 4H, 12-CH3 and 1 ×
31(OCH2C3H7)), 3.56 (s, 4H, 172CO2CH3 and 1 × 31(OCH2C3H7)),
3.22 (s, 3H, 2-CH3), 2.65 (m, 1H, 1 × 171H), 2.33 (m, 3H, 2H for
81CH2 and 1H for 171H), 2.00 (m, 1H, 1 × 172H), 1.99 (d, J )
6.5, 3H, 31CH3), 1.95 (m, 1H, 1 × 172H), 1.79 (d, J ) 6.6, 3H,
7-CH3), 1.70 (m, 5H, 18-CH3 and 31OCH2CH2C2H5), 1.28 (m, 2H,
31O(CH2)2CH2CH3), 1.12 (m, 3H, 82CH3), 0.88 (m, 3H, 3-CH3-
CH(O(CH2)3CH3)), 0.36 (br s, 1H, NH), -0.04 (br s, 1H, NH).
Mass calculated for C47H51O5F6, 879.38; found, 902.3 (M + Na).
HRMS calculated, 879.3799; found, 879.3798.
3-Deacetyl-3-(11-heptyloxyethyl)-bacteriopurpurin-18-N-3,5-
bis(trifluoro- methyl)benzylimide (20). A similar procedure as
described for 18 was followed. Compound 17 (45 mg, 0.055 mmol)
was dissolved in ∼30 mL of dry CH2Cl2. All air was removed from
the reaction flask and flushed with argon. Then under completely
dry conditions, HBr gas was passed through a needle and submerged
into the solution for ∼20 s. While stirring for ∼10 min, there
appeared to be a visible change from dark green to a purple hue of
the solution. The reaction mixture was then concentrated under high
vacuum for ∼1 h, which yielded a dark purple residue. To the
residue was added ∼30 mL dry CH2Cl2, 90 mg anhydrous K2CO3
and 1.5 mL n-heptanol which was stirred under N2 gas for ∼30
min. Upon completion, the contents were transferred to a beaker
containing ice water, and the pH was adjusted to 2-2.5. After
additional washes with water and CH2Cl2 (6×), the organic layer
was collected, dried over Na2SO4, and concentrated under vacuum
without heat to yield a dark purple crude material. The crude
material was purified on silica prep plates using a hexane/ethyl
acetate (80:20) solvent system. The pure compound exhibited a
characteristic absorption peak at 783 nm in CH2Cl2 (yield: 35.9
mg, 70.8%). Log P ) 11.83. UV-vis (ꢀ ) 40 530 at 784 in THF):
537 (3.90 × 104), 505.0 (5.49 × 103), 472.0 (5.23 × 103), 417.1
1
× 104), 367.0 (1.01 × 105), 346.0 (4.93 × 104). H NMR (400
1
1
MHz, 3 mg/1 mL of CDCl3, δ ppm): 8.76 (s, /2H, /25-H), 8.72
1
1
(s, /2H, /25-H), 8.56 (s, 1H, 10-H), 8.29 (s, 1H, 20-H), 8.19 (s,
2H, Ar-H), 7.78 (s, 1H, Ar-H), 5.72 (m, 2H, NCH2Ar), 5.54-
5.60 (m, 1H, 31H), 5.19 (m, 1H, 17-H), 4.12-4.22 (m, 2H, 7-H
and 18-H), 3.95-4.02 (m, 1H, 8-H), 3.60 (s, 3H, 31OCH3), 3.55
(s, 3H, 12-CH3), 3.50 (s, 3H, 172CO2CH3), 3.22 (s, 3H, 2-CH3),
2.58-2.68 (m, 1H, 1 × 171H), 2.24-2.40 (m, 3H, 81CH2 and 1 ×
171H), 2.00-2.10 (m, 1H, 1 × 172H), 1.98 (dd, J ) 2.5, 5.0, 3H,
31CH3), 1.85-1.95 (m, 1H, 1 × 172H), 1.78 (t, J ) 8.0, 3H, 7-CH3),
1.68 (d, J ) 7.6, 3H, 18-CH3), 1.12 (t, J ) 6.6, 3H, 82CH3), 0.30
(br s, 1H, NH), -0.09 (br s, 1H, NH). 19F NMR (400 MHz, 3
mg/1 mL of CDCl3, in reference to TFA, δ ppm): 13.14. Mass
calculated for C44H45N5O5F6, 837.33; found, 860.4 (M + Na).
HRMS calculated (M + 1), 838.3403; found, 838.3467.
1
(4.59 × 104), 367.0 (9.79 × 104), 346.9 (4.67 × 104). H NMR
3-Deacetyl-3-(11-butoxyethyl)-bacteriopurpurin-18-N-3,5-bis-
(trifluoro-methyl)benzylimide (19). A similar procedure as de-
scribed for 18 was followed. Compound 17 (24.6 mg, 0.030 mmol)
was dissolved in ∼15 mL of dry CH2Cl2. All air was removed from
the reaction flask and flushed with argon. Then under completely
dry conditions, HBr gas was passed through a needle and submerged
into the solution for ∼20 s. While stirring for ∼5 min, there
appeared to be a visible change from dark green to a purple hue of
the solution. The reaction mixture was then concentrated under high
vacuum for ∼1 h, which yielded a dark purple residue. To the
residue was added ∼15-20 mL of dry CH2Cl2, 50 mg of dry
K2CO3, and 1 mL of n-butanol, which was stirred under N2 gas for
∼35 min. Upon completion, the contents were transferred to a
beaker containing ice water, and the pH was adjusted in the range
of 2-2.5. After additional washes with water and CH2Cl2 (3×),
the organic layer was collected, dried over Na2SO4, and concentrated
under vacuum without heat to yield a dark purple crude material.
There were problems finding the best solvent system for purifica-
tion. Initially the crude material was purified on silica prep plates
using a 1-2% acetone in CH2Cl2 solvent system. After many
purification steps, the desired compound was finally purified using
1.5% MeOH in CH2Cl2 (yield: 13.1 mg, 49.6%). Log P ) 10.30.
UV-vis (ꢀ ) 40 530 at 785 in THF): 537.9 (3.86 × 104), 504.0
(5.25 × 103), 470.0 (4.56 × 103), 417.0 (4.44 × 104), 367.1 (9.66
1
1
(400 MHz, 3 mg/1 mL of CDCl3, δ ppm): 8.82 (s, /2H, /25-H),
8.78 (s, 1/2H, 1/25-H), 8.56 (s, 1H, 10-H), 8.27 (s, 1H, 20-H), 8.19
(s, 2H, Ar-H), 7.78 (s, 1H, Ar-H), 5.72 (m, 2H, N-CH2Ar), 5.63
(m, 1H, 31H), 5.17 (m, 1H, 17-H), 4.10-4.20 (m, 2H, 7-H and
18-H), 3.94-4.00 (m, 1H, 8-H), 3.60 (s, 4H, 12-CH3 and 1 ×
31OCH2C3H7), 3.54 (s, 4H, CO2CH3 and 1 × 31OCH2C6H13), 3.20
3
1
3
1
(s, /2H, /22-CH3), 3.21 (s, /2H, /22-CH3), 2.55-2.65 (m, 1H, 1
× 171H), 2.24-2.40 (m, 3H, 81CH2 and 1 × 171H), 2.00-2.10
(m, 1H, 1 × 172H), 1.98 (d, J ) 6.8, 3H, 31CH3), 1.85-1.95 (m,
1H, 1 × 172H), 1.78 (t, J ) 7.5, 3H, 7-CH3), 1.65-1.72 (m, 5H,
18-CH3 and 31OCH2CH2C5H11), 1.20-1.36 (m, 8H, 31O(CH2)2-
(CH2)4CH3), 1.05-1.15 (m, 3H, 82CH3), 0.78-0.82 (m, 3H, 31O-
(CH2)6CH3), 0.35 (br s, 1H, NH), -0.06 (br s, 1H, NH). 19F NMR
(400 MHz, 3 mg/1 mL of CDCl3, in reference to TFA, δ ppm):
13.14. Mass calculated for C50H57N5O5F6, 921.43; found, 944.5 (M
+ Na). HRMS calculated, 921.4264; found, 921.4259.
3-Deacetyl-3-(11-dodecyloxyethyl)-bacteriopurpurin-18-N-3,5-
bis(trifluoro-methyl)benzylimide (21). A similar procedure as
described for 18 was followed. Compound 17 (45 mg, 0.055 mmol)
was dissolved in ∼30 mL of dry CH2Cl2. All air was removed from
the reaction flask and flushed with argon. Then under completely
dry conditions, HBr gas was passed through a needle and submerged
into the solution for ∼20 s. While stirring for ∼10 min, there
appeared to be a visible change from dark green to a purple hue of
the solution. The reaction mixture was then concentrated under high
vacuum for ∼1 h, which yielded a dark purple residue. To the
residue was added ∼30 mL of dry CH2Cl2, 90 mg of dry K2CO3,
and 1.5 mL of n-dodecanol, which was stirred under N2 gas for
∼30 min. Upon completion, the contents were transferred to a
beaker containing ice + water and the pH was adjusted to 2-2.5,
and the contents were additionally washed with water, CH2Cl2 (6×),
and ethyl ether (3×). The residue remained oily in nature, so the
sample was run on a silica column first with CH2Cl2 so that the
excess dodecanol would be removed and then with hexane/ethyl
acetate (80:20) as a means to collect the sample. The eluent was
collected, dried over Na2SO4, and concentrated under vacuum
without heat to yield a dark purple crude material. Again the
1
× 104), 347.0 (4.51 × 104). H NMR (400 MHz, 3 mg/1 mL of
CDCl3, δ ppm, R-isomer): 8.78 (s, 5-H), 8.57 (s, 1H, 10-H), 8.28
(s, 1H, 20-H), 8.20 (s, 2H, Ar-H), 7.80 (s, 1H, Ar-H), 5.74 (m,
2H, NCH2Ar), 5.65 (m, 1H, 31H), 5.19 (m, 1H, 17-H), 4.18 (m,
2H, 7-H and 18-H), 4.00 (m, 1H, 8-H), 3.61 (s, 4H, 12-CH3 and 1
×
31(OCH2C3H7)), 3.56 (s, 4H, 172CO2CH3 and
1
×
31(OCH2C3H7)), 3.22 (s, 3H, 2-CH3), 2.64 (m, 1H, 1 × 171H), 2.33
(m, 3H, 2H for 81CH2 and 1H for 171H), 2.00 (m, 1H, 1 × 172H),
1.99 (d, J ) 6.5, 3H, 31CH3), 1.94 (m, 1H, 1 × 172H), 1.80 (d, J
) 6.6, 3H, 7-CH3), 1.70 (m, 5H, 18-CH3 and 31OCH2CH2C2H5),
1.27 (m, 2H, 31O(CH2)2CH2CH3), 1.11 (m, 3H, 82CH3), 0.89 (m,
3H, 3-CH3CH(O(CH2)3CH3)), 0.35 (br s, 1H, NH), -0.05 (br s,
1H, NH). S-isomer: 8.83 (s, 5-H), 8.58 (s, 1H, 10-H), 8.28 (s, 1H,
20-H), 8.20 (s, 2H, Ar-H), 7.80 (s, 1H, Ar-H), 5.76 (m, 2H,

1766 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Gryshuk et al.
1
material was purified on a silica prep plate using the same solvent
system as mentioned above to yield the desired compound (yield:
25.8 mg, 47.3%). Log P ) 14.38. UV-vis (ꢀ ) 40 530 at 785 in
THF): 537.1 (3.97 × 104), 506.0 (6.06 × 103), 470.0 (5.43 × 103),
utilized for acquisition of O2 emission spectra. A diode pumped
solid-state laser (Verdi, Coherent) at 532 nm was used to excite
the photosensitizers and reference samples at room temperature
(Rose Bengal purchased from Fisher Scientific, Hampton, NH). The
1O2 yield for Rose Bengal in MeOH solution is Q∆ ) 0.80.19 All
samples were dissolved in MeOH solution purchased from J.T.
Baker (Phillipsburg, NJ), which was used without purification. Once
dissolved in MeOH, the sample was transferred into a quartz cuvette
where it was placed directly in front of the entrance slit of the
spectrometer, and the exciting laser beam was directed at 90 degrees
relative to the collection of emission. Long-pass filters, 538AELP
and 950 LP (Omega Optical, U.S.A.), were used to attenuate the
excitation laser light and fluorescence from dyes as a means of
studying singlet oxygen phosphorescence peaked at around
1270 nm.
1
417.0 (4.91 × 104), 367.0 (9.97 × 104), 348.0 (4.83 × 104). H
1
NMR (400 MHz, 3 mg/1 mL of CDCl3, δ ppm): 8.82 (s, /2H,
1/25-H), 8.78 (s, 1/2H, 1/25-H), 8.56 (s, 1H, 10-H), 8.27 (s, 1H, 20-
H), 8.19 (s, 2H, Ar-H), 7.78 (s, 1H, Ar-H), 5.72 (m, 2H, NCH2-
Ar), 5.63 (m, 1H, 31H), 5.17 (m, 1H, 17-H), 4.10-4.20 (m, 2H,
7-H and 18-H), 3.94-4.00 (m, 1H, 8-H), 3.60 (s, 4H, 12-CH3 and
31OCH2C3H7), 3.54 (s, 4H, CO2CH3 and 31OCH2C6H13), 3.20 (s,
1
3
1
3/2H, /22-CH3), 3.21 (s, /2H, /22-CH3), 2.55-2.65 (m, 1H, 1 ×
171H), 2.24-2.40 (m, 3H, 81CH2 and 1 × 171H), 2.00-2.10 (m,
1H, 1 × 172H), 1.98 (d, J ) 6.8, 3H, 31CH3), 1.85-1.95 (m, 1H,
1 × 172H), 1.78 (t, J ) 7.5, 3H, 7-CH3), 1.65-1.72 (m, 5H, 18-
CH3 and 31OCH2CH2C5H11), 1.20-1.36 (m, 8H, 31O(CH2)2(CH2)4-
CH3), 1.05-1.15 (m, 3H, 82CH2CH3), 0.78-0.82 (m, 3H, 3-CH3-
CH(O(CH2)6CH3)), 0.35 (br s, 1H, NH), -0.06 (br s, 1H, NH).
Mass calculated for C55H67N5O5F6, 991.50; found, 1014.7 (M +
Na) and 1030.0 (M + K). HRMS calculated (M + 1), 992.5124;
found, 992.5168 (M + 1).
Cell Culture. The RIF tumor cells grown in alpha-minimum
essential medium (R-MEM; GIBCO Invitrogen Corporation), and
the murine colon carcinoma (Colon-26) tumor cells grown in RPMI-
1640 (GIBCO Invitrogen Corporation) with 10% FCS (BenchMark
FCS Triple 0.1 µm filtered - Gemini Bio-Products, Woodland CA),
L-glutamine, and P/S/N were maintained in 5% CO2, 95% air, and
100% humidity. The L-glutamine and P/S/N were purchased from
MediaTech Cellgro, and the Trypsin/EDTA solution 1× sterile was
purchased from Cascade Biologics. The 96-well and 6-well plates
were purchased from VWR. The MTT cell viability experiments20
were read using a Titertek Multiskan PLUS MKII plate reader with
Multiskan Interference Filter 560 nm (Flow Laboratories, Inc.), and
data were collected with the HyperTerminal Hilgraeve, Inc.,
program. All compounds were formulated in a 1% Tween 80/5%
aqueous dextrose solution and diluted in complete medium for all
in vitro experiments.
Determination of In Vitro Photosensitizing Activity. To assess
the photosensitizing ability of the purpurinimides and bacteriopur-
purinimides, the RIF cells were seeded in 96-well plates at a density
of 5 × 103 cells/well in R-MEM complete media. For determining
the photosensitizing ability of the water-soluble photosensitizer 24,
the colon-26 cells were seeded at 4 × 103 cells/well in RPMI-
1640 complete media. The next day, photosensitizers were added
at variable concentrations (1.25-20 µM). After the 24 h of
incubation in the dark at 37 °C, the cells were replaced with
complete media and exposed to 708 nm (7-11) or 791 nm (18-
21) at a dose rate of 3.2 mW/cm2 at various light doses (0.5-20
J/cm2). The dye laser (375; Spectra Physics, Mt. View, CA) was
excited by an argon-pumped laser tuned to emit drug-activating
wavelengths. Uniform illumination was accomplished using a 200-
µm diameter quartz optical fiber fitted with a graded index refraction
lens. Following illumination, the plates were incubated at 37 °C in
the dark for 48 h. Appropriate dark controls at variable drug doses
were also included. Following the 48 h incubation in the dark, the
plates were evaluated for cell viability using the MTT assay.20 At
this point, 10 µL of 4.0 mg/mL of solution of MTT dissolved in
PBS (Sigma Chemical Co., St. Louis, MO) was added to each well.
After the 4 h MTT incubation, the MTT + media were removed
and 100 µL of dimethyl sulfoxide was added to solubilize the
formazin crystals. The PDT efficacy was measured by reading the
96-well plate on a microtiter plate reader (Miles Inc., Titertek
Multiscan Plus MK II) at an absorbance of 560 nm. The results
were plotted as percent survival compared with the corresponding
dark (drug, no light) and light control (cells + light dose in J/cm2).
Each data point represents the mean from a typical experiment with
four replicate wells, and the error bars are the standard deviation
from three separate experiments.
DTPA Conjugate of Bacteriopurpurinimide (26). Compound
19 (60.0 mg, 0.068 mmol) was taken in a flask (100 mL) and
dissolved in an acetonitrile-methanol mixture (20:5 mL). The
resultant mixture was stirred, degassed three times, and filled with
N2 gas. To this mixture LiOH (500.0 mg) in H2O (20 mL) was
added, and the mixture was degassed again and kept for vigorous
stirring for 4 h at RT under a N2 atmosphere. The mixture was
partially concentrated, acidified with dilute acetic acid, and extracted
with dichloromethane (3 × 50 mL). Organic layers were separated,
combined, washed with water (3 × 50 mL), dried over sodium
sulfate, and concentrated. The crude acid 25 thus obtained was dried
under vacuum for several hours to remove the traces of water and
acetic acid and then used directly to couple with aminobenzyl-
DTPA. The above crude acid was dissolved in dry CH2Cl2 (30 mL)
and 4-aminobenzyl-(CO2t-Bu)-DTPA (108.0 mg, 0.136 mmol),
EDCI (26.2 mg, 0.136 mmol), and DMAP (16.7 mg, 0.136 mmol)
were added to it. The resultant mixture was stirred at room
temperature for 16 h under a N2 atmosphere, diluted with CH2Cl2
(100 mL), and washed with brine (50 mL). The organic layer was
separated, dried over sodium sulfate, and concentrated. The crude
product was purified on a silica gel column using MeOH/CH2Cl2
1
(5-10%) as eluent to give product 26 (yield: 50.0 mg; 45%). H
NMR (400 MHz, CDCl3): δ 8.81 (s, 1H, meso-H), 8.54 (s, 1H,
meso-H), 8.25 (s, 1H, meso-H), 8.14 (s, 2H, bis-CF3-C6H3), 7.95
(s, 1H, NH), 7.77 (m, 2H, bis-CF3-C6H3 and NH), 7.44 (d, 2H,
Ph-DTPA, J ) 7.6 Hz), 7.16 (d, 2H, Ph-DTPA, J ) 7.6 Hz), 5.83
(d, 1H, benzylic CH2, J ) 14.8 Hz), 5.73 (d, 1H, benzylic CH2, J
) 14.4 Hz), 5.61 (m, 1H, CH3CHObutyl), 5.17 (d, 1H, H-17, J )
8.4 Hz), 4.29 (m, 1H, H-8), 4.14 (m, 1H, H-18), 3.96 (m 1H, H-7),
3.59 (m, 4H, OCH2butyl and CH2-DTPA), 3.55 (m, 2H, CH2-
DTPA), 3.44 (ss, 3H, ring-CH3), 3.40 (m, 4H, 2CH2-DTPA), 3.32
(m, 2H, CH2-DTPA), 3.17 (s, 3H, ring-CH3), 3.06 (m, 1H, CH-
DTPA), 2.79-2.73 (m, 8H, 4CH2-DTPA), 2.56-2.47 (m, 2H, 172-
CH2), 2.34-2.29 (m, 2H, 8-CH2CH3), 2.05 (m, 1H, 171-CH2), 1.99
(d, 3H, CH3CHObutyl, J ) 6.4 Hz), 1.93 (m, 1H, 171-CH2), 1.79
(d, 3H, 18-CH3, J ) 6.4 Hz), 1.67 (d, 3H, 7-CH3, J ) 7.2 Hz),
1.46 (m, 4H, 2CH2butyl), 1.42 (s, 36H, 4CO2t-Bu), 1.39 (s, 9H,
CO2t-Bu), 1.14 (t, 3H, 8-CH2CH3, J ) 7.2 Hz), 0.86 (t, 3H, CH3-
butyl, J ) 5.2 Hz), 0.50 (br s, 1H, NH), 0.09 (br s, 1H, NH). HRMS
calculated for C87H117N9O14F6, 1625.8623; found, 1625.8646.
Methods for Determining the Photophysical Properties. The
final compounds were characterized by absorbance (CARY 50 Bio
UV-Visible Spectrophotometer with Varian v2.0 software), fluo-
rescence (Fluoromax II Fluorimeter with Thermo Galactic GRAMS/
32 v4.0 software), 1H NMR and 19F NMR (spectra obtained using
a Bruker 400 MHz spectrometer; chemical shifts relative to CDCl3
at 7.26 ppm and TFA 116.68 ppm), and mass spectrometry
(analyses performed at RPCI Biopolymer Facility).
Determination of In Vivo Uptake by In Vivo Reflectance
Spectroscopy (IRS). The female C3H mice 5-8 weeks of age were
obtained from the NCI Jackson Laboratory. At 7-14 weeks of age
the mice were inoculated s.c. with RIF (3 × 105 cells in 40 µL) on
the right posterior shoulder. The female BALB/c mice 6-8 weeks
of age obtained from the NCI Clarence Reeder were used for the
water soluble photosensitizer studies. At 8-14 weeks of age, the
mice were inoculated s.c. with colon-26 (1 × 106 cells in 50 µL)
on the right posterior shoulder. When tumors reached ∼7 mm3,
A SPEX 270M Spectrometer (Jobin Yvon) equipped with an
InGaAs photodetector (Electro-Optical Systems Inc., U.S.A.) was

Purpurinimides and Bacteriopurpurinimides
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1767
47, 4642-4745. (c) Pandey, R. K.; Dougherty, T. J. Synthesis and
photosensitizing activity of porphyrins joined with ester linkages.
Cancer Res. 1989, 49, 2042-2047. (d) Pandey, R. K.; Shiau, F. Y.;
Medforth, C. J.; Dougherty, T. J.; Smith, K. M. An efficient synthesis
of porphyrin dimers linked with carbon-carbon linkages. Tetrahedron
Lett. 1990, 31, 789-792.
the mice were anesthetized using ketamine xylazine intraperito-
neally. The optical power as a function of wavelength was recorded
before the i.v. injection of the photosensitizer. The measurements
were acquired through the use of bundle optical fibers that were
positioned on the surface of the tumor plus overlying skin and
normal skin. The drug at 2.5-5.0 µmol/kg was then injected (via
i.v. tail vein), and the absorbance spectrum of the drug postinjection
was recorded over time (up to 2 days p.i.). The in vivo drug
absorption spectrum was best displayed by determining the ratio
of the postinjection spectrum to the preinjection spectrum in the
tumor versus the skin. This experiment also determined the
wavelength and time at which to perform in vivo PDT treatments.
Determination of In Vivo Photosensitizing Activity. The day
before PDT light treatment, the mice with a tumor ∼4-5 mm3 in
diameter were injected intravenously with 0.2-1.0 µmol/kg of the
photosensitizer (photosensitizers were diluted in 1% Tween 80 in
HBSS; Sigma), and the hair over the treatment spot was removed
via depilation (with Nair). At 24 h p.i., the mice (10 per group)
were restrained in plastic plexiglass holders without anesthesia,
treated with a 1 cm2 drug-activating laser light spot from an argon-
pumped dye laser for a total fluence of 135 J/cm2 at a fluence rate
of 75 mW/cm2 (total power of 59-60 mW for a 30 min treatment).
The DCM Ext dye (Exciton, Dayton, OH) and the Styryl 8 dye
(Exciton, Dayton, OH - LDS 751) yielded tunable wavelengths
from 682 to 745 nm and 720-835 nm, respectively. The laser beam
was passed through an eight-way beam splitter. The power (in mW)
at each individual fiber could be individually set using the Brewster-
window-type attenuators and was constantly monitored during the
treatment. Post-PDT, the mice were observed daily, the tumors were
measured using two orthogonal measurements, L and W (perpen-
dicular to L), and the volumes were calculated, using the formula
V ) L‚W2/2, and recorded. Mice were considered cured if there
was no palpable tumor by day 60.
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M. Photodynamic therapy of cancer: Second and third generations
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Henderson, B. W.; Constantine, S.; Aoudia, M.; Rodgers, M. A. J.;
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Statistical Evaluation. All in vitro data were presented as the
mean of three replicate experiments with the standard deviation of
each. The GraphPad Prism program (v.3.0) was used for all in vivo
tumor response (Kaplan-Meier plots).23
Acknowledgment. Authors are thankful to Dr. Guolin Li
for his initial help in preparing the purpurinimide-aminoben-
zyl-DTPA conjugate and to Dr. Janet Morgan and Dr. David
Bellnier for valuable discussions. The financial support from
the NIH (CA55791) and the shared resources of the RPCI sup-
port grant (P30CA16056) is highly appreciated. A.L.G. thanks
the National Science Foundation under the Integrative Graduate
Education and Research Traineeship (Grant DGE0114330) for
funding the graduate fellowship. We thank the Mass Spectrom-
etry Facility, Roswell Park Cancer Institute, Buffalo, and the
Michigan State University for analyzing our samples.
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study of purpurinimide-based fluorinated versus nonfluorinated
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Supporting Information Available: 1H NMR spectra of
compounds 4-10, 13-20, 19S, and 26 and HPLC chromatograms
of compounds 6-11, 13, 14, 18-21, 24, and 26. This material is
available free of charge from http://pubs.acs.org.
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