Core-modified porphyrins. Part 4: Steric effects on photophysical and biological properties in vitro

Bioorganic & Medicinal Chemistry 13 (2005) 2235–2251

Core-modiﬁed porphyrins. Part 4: Steric eﬀects on

photophysical and biological properties in vitro

Youngjae You,^a,*Scott L. Gibson,^bRussell Hilf,^b

Tymish Y. Ohulchanskyy^aand Michael R. Detty^a,*

^aInstitute for Lasers, Photonics, and Biophotonics, Department of Chemistry, University at Buﬀalo,

The State University of New York, Buﬀalo, NY 14260, USA

^bDepartment of Biochemistry and Biophysics, University of Rochester Medical Center, 601 Elmwood Avenue,

Box 607, Rochester, NY 14642, USA

Received 19 November 2004; revised 22 December 2004; accepted 22 December 2004

Abstract—21,23-Dithiaporphyrins (2–10) were designed and prepared as analogues of 5,20-diphenyl-10,15-bis(4-carboxylatometh-

oxy)phenyl-21,23-dithiaporphyrin (1) to examine the impact of steric bulk at the 5- and 20-meso positions as well as the impact of

symmetry. Changes at the meso positions had minimal impact on the UV–vis–near-IR absorption spectra, quantum yields for the

generation of singlet oxygen, and quantum yields for ﬂuorescence and some impact on values of the octanol/water partition coef-

ﬁcient. Of the compounds 1–10, 5-phenyl-20-(2-thienyl)-10,15-bis-(4-carboxylatomethoxy-phenyl)-21,23-dithiaporphyrin (3) showed

the greatest phototoxicity toward cultured R3230AC cells, with 68% cell kill at 1 · 10^À7M and irradiation with 5 J cm^À2of 350–

750 nm light. Results in this study suggest that smaller substituents on the meso ring and less symmetrical compounds are more eﬀec-

tive as photosensitizers than compounds with two bulky substituents at adjoining meso sites and a higher symmetry. The mitochon-

dria appear to be involved in the process of phototoxicity as determined by the inhibition of whole cell cytochrome c oxidase activity

in cells treated with 3 and light. No impact upon mitochondrial cytochrome c oxidase activity was observed in cells treated with 3

and no light. Fluorescence microscopy studies suggest that the mitochondria are not initial sites of accumulation of 3.

1. Introduction

photosensitizers and these are in late clinical trials for

various indications.^5,11,12Generally second-generation

photosensitizers are designed to be pure, single, identiﬁ-

able compounds with higher extinction coeﬃcients at

wavelengths above 600 nm.^8,13–19They are also designed

to achieve greater tumor speciﬁcity and targeted delivery

to malignant lesions.^20–31The 21,23-dithiaporphyrins

are unique entities as second-generation photosensitiz-

ers. The substitution of a chalcogen atom (S, Se) for

NH– at the 21- and 23-positions of the porphyrin

macrocycle red shifts the absorption maxima to

>690 nm.^19,32–34The core chalcogen atoms, S or Se,

exclude metal binding in the porphyrin core and 21,23-

dithiaporphyrins are more eﬃcient singlet oxygen gener-

ators than 21,23-diselenaporphyrins.^35–37

Photodynamic therapy (PDT) is a promising treatment

regimen for cancer that uses a dual-mode of selectiv-

ity:^1–9a photosensitizer, which absorbs light and gener-

ates cytotoxic singlet oxygen, localizes in or around a

tumor and is then irradiated in the lesion. The photosen-

sitizer, porﬁmer sodium (Photofrin), has gained regula-

tory approval for the treatment of various cancers

including lung, bladder, and oesophageal cancers in

numerous countries but suﬀers from several drawbacks

including persistent skin phototoxicity after treatment,

in some cases, for up to 3 months.¹⁰Several second-gen-

eration compounds have been developed to overcome

the deﬁciencies of Photofrin and other ﬁrst generation

The relationship between structural variations of the

21,23-core-modiﬁed porphyrins and the phototoxicity

of the core-modiﬁed porphyrins in vitro was established

previously.³²First, dithiaporphyrins are more photo-

toxic toward Colo-26 cells than diselenaporphyrins or

natural porphyrins containing four nitrogen atoms in

Keywords: Core-modiﬁed porphyrin; Dithiaporphyrin; Photodynamic

therapy; Mitochondria; Cytochrom c oxidase.

*

Corresponding authors. Tel.: +1 716 645 6800x2197; fax: +1 716 645

6963 (Y.Y.); tel.: +1 716 645 6800x2200; fax: +1 716 645 6963

(M.R.D.); e-mail addresses: yjyou@nsm.buﬀalo.edu; mdetty@

buﬀalo.edu

doi:10.1016/j.bmc.2004.12.048

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Y. You et al. / Bioorg. Med. Chem. 13 (2005) 2235–2251

S

6

2

S

7

3

S

N

S

HO

OH

O

8

1

S

9

4

S

1

5

0

Figure 1. Structures of 21,23-dithiaporphyrins with various group substitutions at the 10,15-meso positions.

their core.³²Second, by varying the number of sulfonate

or carboxylate groups from 1 to 4 on the outer porphy-

rin ring, we determined that compounds with two, cis–

meso, hydrophilic substituents are the most potent

in vitro photosensitizers toward Colo-26 cells.³⁴Third,

molecules with cis carboxylic acid groups at the meso

position are more potent than compounds with sulfo-

nate groups. Speciﬁcally, studies in vitro with the dicar-

boxylic acid compound, 5,20-diphenyl-10,15-bis(4-

carboxylatomethoxy)phenyl-21,23-dithiaporphyrin (1,

Fig. 1), required only 0.15 lM photosensitizer to

decrease the viability of R3230AC cells to 50% after

irradiation with 5 J cm^À2of 350–750 nm light, a concen-

tration that was 10-fold lower than that necessary for

the disulfonate compound to reach the same level of

cytotoxicity with the same light dose.³⁸

further alterations in the basic structure might lead to

more potent photosensitizers. Herein, we present the

synthesis of a series of new 21,23-dithiaporphyrins de-

signed to have diversity at the 5,10-meso aromatic rings

(Fig. 1). Results demonstrate that these structural alter-

ations did not greatly impact important photophysical

properties, that is, UV–vis–near-IR absorption spectra,

quantum yields for the generation of singlet oxygen, or

quantum yields for ﬂuorescence. However, biological

properties including dark and phototoxicity, cellular up-

take, subcellular localization, and dark and phototoxic-

ity toward mitochondrial cytochrome c oxidase in

isolated mitochondria or whole cells can be signiﬁcantly

aﬀected by structural modiﬁcations.

2. Results and discussion

The mitochondria are an important intracellular target

for PDT due to their critical role in energy production

and their involvement in apoptosis.³⁹For porphyrins

and porphyrin-related structures, accumulation of pho-

tosensitizer can occur in the membranes of organelles

such as the mitochondria or lysosomes.⁴⁰Earlier studies

with 1 indicated that the mitochondria were targets for 1

prior to irradiation of treated cells and that irradiation

lead to further mitochondrial damage.³⁸Costaining

studies of R3230AC cells with 1 and rhodamine 123 sug-

gested that the mitochondria were disrupted by the pres-

ence of 1 or that the presence of 1 excluded rhodamine

123 from the mitochondria.³⁸

Based on our earlier studies with 1, which showed

potent phototoxicity compared to derivatives with one,

three, or four carboxylic acids,³⁸we prepared the dithia-

porphyrins 2–10 of Figure 1 and examined the impact

of alterations to the head portion of the molecule,

namely the aromatic rings at meso positions 5 and 20,

on photophysical and biological properties. A small 2-

thienyl heteroaromatic ring or a larger 2,4,6-trimethyl-

phenyl were substituted for phenyl at the 5- and/or 20-

positions of the head portion of the molecule. A series

of alkyl substituents (methyl, ethyl, n-butyl, tert-butyl)

were also added to the 4-position of the meso-phenyl

substituents. Combined, these substitutions created mole-

cules with diﬀerent steric demands and with diﬀerent

symmetries.

Both the dark and phototoxicity studies and the mito-

chondrial studies with 1, prompted us to ask whether

Y. You et al. / Bioorg. Med. Chem. 13 (2005) 2235–2251

2237

hydrolyzed with NaOH in aqueous tetrahydrofuran

(THF) to give ﬁnal diacid compounds 2, 6, and 8–10.

c

b

Ph

OH

Ph

S

35

OTBS

34

2.1.2. Synthesis of unsymmetrical dicarboxylic acid

derivatives 3 and 7. The synthetic strategy used for the

synthesis of unsymmetrical porphyrins 3 and 7, was

elaborated in our previous report.³⁸2-(1-Hydroxy-1-

phenylmethyl)thiophene (34) was prepared in 68% yield

from thiophene using n-butyllithium (n-BuLi) as limiting

reagent (Scheme 1).³⁸Protection of the hydroxyl group

of 34 with tert-butyldimethylsilyl chloride (TBSCl) gave

TBS ether 35, in 86% yield.³⁸Compound 35 was then

lithiated with n-BuLi and treated with thiophene 2-car-

boxaldehyde or 2,4,6-trimethylbenzaldehyde. The

resulting alcohols were deprotected with tetrabutylam-

monium ﬂuoride (TBAF) to aﬀord unsymmetrical diols

36 and 37 in 43% and 68% isolated yields, respectively

(Scheme 1). The unsymmetrical diols were condensed

with 2,5-bis[1-(4-methoxyphenyl)-1-pyrrolomethyl]thio-

phene (18) to give the unsymmetrical 21,23-dithiapor-

phyrins, 38 and 39, in 11% and 14% isolated yields,

respectively (Scheme 3). Demethylation to phenols 40

and 41 with BBr₃, alkylation with ethyl bromoacetate

to diesters 42 and 43, and saponiﬁcation to give 21,23-

dithiaporphyrins 3 and 7 followed our standard proto-

cols (Scheme 3).³⁸

a

d

Ar

HO

Ar

Ph

S

OH

HO

OH

11, Ar = 2-thienyl

12, Ar = 4-MeC₆H₄

13, Ar = 4-EtC₆H₄

36, Ar = 2-thienyl

37, Ar = 4-t-BuC₆H₄

47, Ar = 2,4,6-Me₃C₆H₂

14, Ar = 4-t-BuC₆H₄

15, Ar = 4-BuC₆H₄

16, Ar = 2,4,6-Me₃C₆H₂

17, Ar = 4-MeOC₆H₄

Scheme 1. Reagents and conditions: (a) i. 2.5 equiv n-BuLi, ii. 2 equiv

aryl aldehyde; (b) i. 1 equiv n-BuLi, ii. 1 equiv benzaldehyde; (c)

TBSCl, DMAP, Et₃N; (d) i. 1 equiv n-BuLi, ii. 1 equiv aryl aldehyde,

iii. aqueous HCl.

2.1. Chemistry

2.1.1. Synthesis of symmetrical 21,23-dithiaporphyrins 2,

6, and 8–10. The synthesis of symmetric core-modiﬁed

porphyrins 2, 6, and 8–10 (Fig. 1) was based on our

previous synthetic methods to symmetrical derivatives.³⁴

As shown in Scheme 1, 2,5-bis[1-(4-aryl)-1-hydroxy-

methyl]thiophenes 11–17 were prepared from 2,5-dilith-

iothiophene and various aldehydes. 2,5-Bis[1-(4-

methoxyphenyl)-1-hydroxymethyl]-thiophene (17) was

converted to 2,5-bis[1-(4-methoxyphenyl)-1-pyrrolo-

methyl]-thiophene (18) using pyrrole and boron triﬂuoride

etherate (BF₃ÆOEt₂) (Scheme 2). Diols 11–15 were con-

densed with 18 in the presence of 2,3,5,6-tetrachlo-

robenzoquinone (TCBQ) and p-toluenesulfonic acid

monohydrate (TsOHÆH₂O) (or BF₃ÆOEt₂) in CH₂Cl₂to

give 21,23-dithiaporphyrins 19–23 in 9–20% isolated

yields (Scheme 2). Demethylation of 19–23 with BBr₃

in CH₂Cl₂gave phenolic core-modiﬁed porphyrins 24–

28, which were alkylated with ethyl bromoacetate and

K₂CO₃in acetone. The resulting esters 29–33 were

2.1.3. Synthesis of 2,4,6-trimethylphenyl-containing deriv-

atives 4 and 5. It was necessary to prepare 21,23-dithia-

porphyrins 4 and 5 bearing 2,4,6-trimethylphenyl

groups via an alternative synthetic route. Attempted

demethylation of the corresponding 5,20-bis(4-methoxy-

phenyl)-21,23-dithiaporphyrins with one or two 2,4,6-

trimethylphenyl substituents with BBr₃gave, in addition

to the desired bisphenols, monobrominated products as

determined by the appearance of M⁺+ Br peaks in the

1

mass spectra of these molecules. The H NMR spectra

of these molecules also showed multiple sets of 2,4,6-

trimethylphenyl peaks consistent with additional prod-

ucts. We were unable to purify/separate these mixtures

so further characterization of these materials was not

Ar

HO

Ar

OH

a

S

NH

HN

17, Ar = 4-MeOC₆H₄

18, Ar = 4-MeOC₆H₄

Ar

S

24, Ar = 2-thienyl, R = H

25, Ar = 4-MeC₆H₄, R= H

26, Ar = 4-EtC₆H₄, R = H

27, Ar = 4-t-BuC₆H₄, R = H

28, Ar = 4-BuC₆H₄, R = H

b

c

11-15 + 18

N

d

OR

RO

29, Ar = 2-thienyl, R = CH₂CO₂Et

30, Ar = 4-MeC₆H₄, R= CH₂CO₂Et

31, Ar = 4-EtC₆H₄, R = CH₂CO₂Et

32, Ar = 4-t-BuC₆H₄, R = CH₂CO₂Et

33, Ar = 4-BuC₆H₄, R = CH₂CO₂Et

19, Ar = 2-thienyl, R = Me

20, Ar = 4-MeC₆H₄, R=Me

21, Ar = 4-EtC₆H₄, R = Me

e

2, 6, 8-10

22, Ar = 4-t-BuC₆H₄, R = Me

23, Ar = 4-BuC₆H₄, R = Me

Scheme 2. Reagents: (a) pyrrole, BF₃ÆOEt₂; (b) TCBQ, TsOHÆH₂O, CH₂Cl₂; (c) BBr₃, CH₂Cl₂; (d) BrCH₂CO₂Et, K₂CO₃, acetone; (e) NaOH,

aqueous THF.

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Y. You et al. / Bioorg. Med. Chem. 13 (2005) 2235–2251

Ph

Ar

S

c

40, Ar = 2-thienyl, R = H

b

36 or 37 + 18

41, Ar = 4-t-BuC₆H₄, R = H

N

d

42, Ar = 2-thienyl, R = CH₂CO₂Et

43, Ar = 4-t-BuC₆H₄, R = CH₂CO₂Et

e

OR

RO

38, Ar = 2-thienyl, R = Me

39, Ar = 4-t-BuC₆H₄, R = Me

3 or 7

Scheme 3. Reagents: (a) pyrrole, BF₃ÆOEt₂; (b) TCBQ, TsOHÆH₂O, CH₂Cl₂; (c) BBr₃, CH₂Cl₂; (d) BrCH₂CO₂Et, K₂CO₃, acetone; (e) NaOH,

aqueous THF.

OH

Ar

HO

Ar'

OH

OTBS

a

S

OHC

44

b

16, Ar = 2,4,6-Me₃C₆H₂, Ar' = Ph

47, Ar = Ar' = 2,4,6-Me₃C₆H₂

a

c

Ar

46, Ar = 4-EtO₂CCH₂OC₆H₄

S

Ar

Ar'

HO

OH

S

45, Ar = 4-TBSOC₆H₄

NH

HN

Ar'

Ar

48, Ar = 2,4,6-Me₃C₆H₂, Ar' = Ph

49, Ar = Ar' = 2,4,6-Me₃C₆H₂

S

b

c

4 or 5

46 + 48 or 49

N

S

OCH₂CO₂Et

EtO₂CH₂CO

50, Ar = 2,4,6-Me₃C₆H₂, Ar' = Ph

51, Ar = Ar' = 2,4,6-Me₃C₆H₂

Scheme 4. Reagents and conditions: (a) TBSCl, Et₃N; (b) 0.5 equiv 2,5-Li₂SC₄H₂; (c) BrCH₂CO₂Et, K₂CO₃, acetone; (d) pyrrole, BF₃ÆOEt₂;

(e) TCBQ, TsOHÆH₂O, CH₂Cl₂; (f) NaOH, aqueous THF.

attempted. Alternative methods of demethylation

were examined including BCl₃with NaI or NaSEt in

DMF. However, reaction yields were poor with these

reagents.

in 90% and 87% isolated yields, respectively, after the

saponiﬁcation of 50 and 51.

2.2. Photophysical properties

An alternative approach to these products was devised

as shown in Scheme 4. 4-Hydroxybenzaldehyde was

protected with TBSCl to give 44, which was then added

to 2,5-dilithiophene to give 2,5-bis[1-(4-tert-butyldi-

methylsilyloxyphenyl)-1-hydroxymethyl]thiophene (45).

The TBS groups of 45 were deprotected with HCl and

the crude diol was alkylated with ethyl bromoacetate

in acetone to give 2,5-bis[1-(4-carboxylatometh-

oxy)phenyl-1-hydroxymethyl]thiophene (46).

2.2.1. Absorption maxima. Longer wavelength exciting

light is advantageous in PDT because it penetrates tissue

to a greater depth. The 21,23-dithiaporphyrinsꢀ band 1

absorbance maxima are all >690 nm. As shown in Table

1, changing the meso-aryl substituents in the dicarbox-

ylic acid derivatives 1–10 did not signiﬁcantly impact

the wavelength maxima of bands.^34,38

2.2.2. Quantum yields for the generation of singlet

oxygen. Singlet oxygen (¹O₂) is reported to be the toxic

agent responsible for the eﬀectiveness of the majority of

The addition of 2,4,6-trimethylbenzaldehyde to 2,5-

dilithiothiophene gave diol 16 in 53% isolated yield

and to lithiated 35 gave diol 47 in 47% isolated yield

(Scheme 1). Diols 16 and 47 were treated with pyrrole

and BF₃ÆOEt₂to give dipyrrolomethane compounds 48

and 49—each in 78% isolated yield (Scheme 3). The con-

densation of diol 46 with dipyrrolomethanes 48 and 49

in the presence of TCBQ and TsOHÆH₂O in CH₂Cl₂

gave diesters 50 and 51 in 13% and 22% isolated yields,

respectively. Dicarboxylic acids 4 and 5 were obtained

1

PDT photosensitizers.^1,3–5,41Thus, eﬃcient O₂genera-

tion during irradiation of the photosensitizer is impor-

tant for eﬀective treatment. Quantum yields of singlet

oxygen generation [/(¹O₂)] for dithiaporphyrins 1–7

1

were measured directly using the luminescence of O₂

compared to that of a rose bengal standard.⁴²For this

series of core-modiﬁed porphyrins, the average value

of /(¹O₂) was 0.80 with a range of 0.67 for 2 to 0.90

for 4 (Table 2).

Y. You et al. / Bioorg. Med. Chem. 13 (2005) 2235–2251

2239

Table 1. UV–vis–near-IR band maxima and molar absorptivities for dithiaporphyrins 1–10 in methanol^a

Compound

Soret

Band IV

Band III

Band II

Band I

1

435 (314)

442 (288)

438 (320)

434 (303)

434 (291)

438 (216)

438 (278)

436 (267)

437 (171)

437 (150)

513 (27.7)

520 (20.5)

516 (24.8)

512 (27.6)

513 (31.5)

517 (19.3)

516 (26.7)

515 (22.2)

516 (16.1)

516 (14.1)

549 (10.7)

558 (13.7)

553 (12.8)

546 (7.3)

547 (10.6)

552 (9.3)

551 (8.7)

550 (11.1)

551 (7.2)

552 (6.3)

632 (3.2)

640 (2.2)

636 (2.4)

633 (2.0)

633 (2.5)

637 (1.4)

635 (2.0)

635 (4.2)

637 (1.2)

636 (1.0)

698 (7.0)

707 (5.3)

702 (6.3)

697 (4.8)

697 (6.9)

702 (4.8)

700 (5.1)

700 (7.4)

701 (4.0)

700 (3.2)

2

3

4

5

6^b

7^b

8

9^b

10^b

^ak_max, nm (e · 10^À3M^À1cm^À1).

^bIn tetrahydrofuran.

Table 2. Quantum yields for the generation of singlet oxygen [/(¹O₂)],

quantum yields for ﬂuorescence (/_F), and octanol/water partition

coeﬃcients in pH 7.4 phosphate buﬀer (logD_7.4) for 1–10^a

9 were surprisingly hydrophilic with values of logD_7.4

of À0.14 to À0.55.

/(¹O₂)

/

F

logD_7.4

2.3. Biology

Compound

1

2

0.80 0.03

0.67 0.03

0.86 0.03

0.90 0.03

0.80 0.03

ND

0.007 0.001

0.009 0.002

0.007 0.002

0.006 0.001

0.005 0.001

ND

0.04 0.0191

À0.27 0.0057

À0.28 0.007

0.24 0.0061

0.28 0.0164

0.48 0.0079

À0.14 0.002

À0.21 0.110

À0.55 0.064

0.45 0.087

2.3.1. Intracellular accumulation of core-modiﬁed por-

phyrins into cultured R3230AC cells. Intracellular accu-

mulation of dithiaporphyrins was assessed in cultured

R3230AC cells. Cell monolayers were incubated with

dithiaporphyrins 1–10 for 24 h at concentrations of

5 · 10^À6and 1 · 10^À5M, cells were digested with 25%

Scintigest, and the ﬂuorescence in the cell digests was

determined. Intracellular dithiaporphyrin content was

calculated using standard ﬂuorescence curves obtained

from the individual dithiaporphyrins dissolved in 25%

Scintigest. The data are expressed in Figure 2 as fmol

dithiaporphyrin/cell. When 5 · 10^À6M dithiaporphyrin

was incubated with cultured R3230AC cells, dithiapor-

phyrins accumulated intracellularly at concentrations

ranging from 0.18 to 2.7 · 10^À15mol dithiaporphyrin/

cell. This intracellular dye content represents a range

of 2–27% of the dithiaporphyrin content in the culture

medium. The uptake of dithiaporphyrins 4–7 was

<10^À15mol/cell while the uptake of 1–3 and 8–10

was in the range of 1.2 · 10^À15–2.7 · 10^À15mol/cell.

3

4

5

6

7

8

9

10

ND

^aDetailed methods for measurements above are presented in Section 4.

ND (no data) represents measurements that were not performed.

2.2.3. Quantum yields for ﬂuorescence. The ﬂuorescence

signal emitted by photosensitizers has been used to

determine intracellular localization,⁴³to study pharma-

cokinetics in vivo,⁴⁴and to diagnosis malignancies.^45–47

Quantum yields for ﬂuorescence (/_F) for this series of

core-modiﬁed porphyrins were low (0.005–0.009, Table

2) compared to the standard rhodamine 6G ﬂuorescence

with a value of /_Fof 1.0. However, this weak ﬂuores-

cence could still be used to observe the intracellular

localization of the dithiaporphyrins (vide infra).

6.0

5.E-6 M

1.E-5 M

5.0

2.2.4. n-Octanol/water partition coeﬃcients. The pres-

ence of the carboxylic acid substituents necessitated

the use of buﬀer solutions for determining values of

the n-octanol/water partition coeﬃcient, P, for 1–10.

Values of the partition coeﬃcient were measured at

pH 7.4 in a phosphate buﬀer and are reported as values

of logD_7.4in Table 2. Values of logD_7.4were clearly im-

pacted by the aryl/heteroaryl substituents in the 5- and

20-positions with a range of À0.55 to 0.48. The substitu-

tion of a 2-thienyl ring for phenyl (comparison of 1 and

3) increased the hydrophilicity, logD_7.40.04 and À0.27

for 1 and 3, respectively. However, the addition of a sec-

ond 2-thienyl substituent in 2 had no further impact on

logD_7.4with logD_7.4of À0.28 for 2. Compounds 6 and

10 with two 4-tert-butylphenyl or two 4-n-butylphenyl

substituents were the most lipophilic compounds in the

series and were followed closely by compounds 4 and

5 with two and one 2,4,6-trimethylphenyl substituents,

respectively (Table 2). In contrast, dithiaporphyrins 7–

4.0

3.0

2.0

1.0

0.0

1

2

3

4

5

6

7

8

9

10

Dithiaporphyrin

Figure 2. Cellular uptake of 21,23-dithiaporphyrins in cultured

R3230AC rat mammary adenocarcinoma cells. Experimental condi-

tions are detailed in Section 4. Each bar represents the mean

intracellular uptake of each dithiaporphyrin incubated with

R3230AC cells for 24 h at 5 · 10^À6or 1 · 10^À5M. The intracellular

ﬂuorescence was obtained by comparing experimental values with

standard curves obtained from porphyrins dissolved in Scintigest.

Data are expressed as fmol porphyrin/cell, error bars are the SEM.

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Y. You et al. / Bioorg. Med. Chem. 13 (2005) 2235–2251

Compounds 4–7 bear bulky 2,4,6-trimethylphenyl (4

and 5) and 4-tert-butyl (6 and 7) substituents, which sug-

gests that steric factors in the meso substituents can im-

pact uptake.

750 nm light with dithiaporphyrins 1–3, 5, 7, and 8 while

dithiaporphyrins 4, 6, 9, and 10 displayed <35% cell kill

at this concentration. Among dithiaporphyrins 1–10,

dithiaporphyrin 3 exhibited the greatest phototoxicity

with 30% cell kill at 5 · 10^À8M, 68% cell kill at

1 · 10^À7M, and >90% cell kill at 5 · 10^À7M and the

same light dose (Fig. 3b).

At 1 · 10^À5M dithiaporphyrin (Fig. 2), the least uptake

was again observed with 4 and 6 bearing two bulky sub-

stituents, but the uptake of 5 with one 2,4,6-trimethyl-

phenyl substituent and 7 with one 4-tert-butylphenyl

were comparable to the uptake of 9 and 10 with

two 4-ethylphenyl and 4-n-butylphenyl substituents,

respectively. At 1 · 10^À5M dithiaporphyrin, the high-

est uptake was observed with 1–3 and 8, which

bear the smallest meso substituents. Again, uptake at

1 · 10^À5M dithiaporphyrin appears to be impacted by

steric eﬀects.

As was observed with respect to cellular uptake of

dithiaporphyrins 1–10, the eﬃcacy of the dithiaporphy-

rins as photosensitizers is sensitive to steric eﬀects. Dith-

iaporphyrin 1 with two phenyl substituents at the 5- and

20-meso positions and dithiaporphyrins 2 and 3, in

which one or both phenyl substituents were replaced

by the small 2-thienyl substituent, had comparable

phototoxicity. Similarly, the substitution of two 4-meth-

ylphenyl substituents at the 5- and 20-meso positions of

8 had little impact on phototoxicity. However, a slight

increase in the aryl steric bulk to 4-ethylphenyl as in

compound 9 was suﬃcient to give a statistically signiﬁ-

cant decrease in phototoxicity for 9 relative to 1 at

2.3.2. Dark and phototoxicity of core-modiﬁed porphyrins

toward cultured R3230AC cells. The dark toxicity and

phototoxicity of dithiaporphyrins 1–10 were determined

in cultures of R3230AC cells incubated with the dyes for

24 h prior to washing or irradiation. No signiﬁcant dark

toxicity was observed in R3230AC cells for any of the

core-modiﬁed porphyrins studied, even at the highest

porphyrin concentration used, 1 · 10^À5M (cell survival

P80%). At 1 · 10^À7M dithiaporphyrin with 5 J cm^À2

of 350–750 nm light, dithiaporphyrins 1–3, 5, 7, and 8

displayed 15–68% cell kill while dithiaporphyrins 4, 6,

9, and 10 showed no phototoxicity (Fig. 3a). Increasing

the dithiaporphyrin concentration to 5 · 10^À7M gave

phototoxicity of >80% cell kill with 5 J cm^À2of 350–

5 · 10^À8

,

1 · 10^À7

,

and 5 · 10^À7M photosensitizer

(P < 0.05). Similarly, compound 4 with two 2,4,6-tri-

methylphenyl substituents and compound 6 with two

4-tert-butylphenyl substituents showed no signiﬁcant

phototoxicity at 1 · 10^À7M (P > 0.05). In contrast,

compounds 5 and 7 with only one bulky substituent

were both eﬀective photosensitizers at 5 · 10^À7M with

>80% cell kill while compound 5 was also a photosensi-

tizer at 1 · 10^À7M with 56% cell kill. Compound 10

with two 4-n-butylphenyl substituents showed no signif-

icant phototoxicity (P > 0.05) over a concentration

range of 5 · 10^À8–1 · 10^À6M relative to dark controls.

Dark

Light

125

100

75

50

25

0

These data suggest that dithiaporphyrins bearing 4-

(carboxylatomethoxy)phenyl substituents in the 10-

and 15-positions can have one bulky aryl substituent in

the 5- or 20-position, but not two. In this study, a bulky

aryl substituent is deﬁned as 4-ethylphenyl, 4-n-butyl-

phenyl, 4-tert-butylphenyl, or 2,4,6-trimethylphenyl.

Phototoxicity does not appear to be solely dependent on

intracellular accumulation of the dithiaporphyrins. At

5 · 10^À6M, dithiaporphyrins 9 and 10 accumulated at

1.2 and 1.8 · 10^À15mol/cell but 9 and 10 showed rela-

tively little phototoxicity at 5 · 10^À7M relative to 5 or

7 whose intracellular concentrations were only 0.85

and 0.63 · 10^À15mol/cell, respectively. Clearly, other

factors such as subcellular localization of the dithiapor-

phyrins or relocalization of the photosensitizers during

light exposure may impact the phototoxicity of these

compounds.

none

1

2

3

4

5

6

7

8

9

10

(a)

µ

Dithiaporphyrin, 0.1

M

Dark

Light

125

100

75

50

25

0

0.00

0.25

0.50

0.75

1.00

2.3.3. Studies on the subcellular localization of 21,23-

dithiaporphyrins

(b)

Concentration 3,

µ

M

Figure 3. Cell viability of cultured R3230AC cells after photosensiti-

zation with 5 J cm^À2of 350–750 nm light in the presence of (a) 0.1 lM

21,23-dithiaporphyrins or (b) various concentrations of 3. Experimen-

tal conditions are detailed in Section 4. Each data point represents the

mean of at least three separate experiments performed in duplicate,

error bars are the SEM. Data are expressed as the surviving fraction of

viable cells relative to untreated controls.

2.3.3.1. Photosensitized inhibition of cytochrome c

oxidase in isolated mitochondrial suspensions. In order to

demonstrate that photodamage following irradiation of

21,23-dithiaporphyrins in the mitochondria could be

responsible for cellular phototoxicity, we initially exam-

ined the inhibition of cytochrome c oxidase in isolated

Y. You et al. / Bioorg. Med. Chem. 13 (2005) 2235–2251

2241

0.025

0.020

0.015

0.010

0.005

0.000

3, light

3, dark

4, light

4, dark

Dark

Light

125

100

75

50

25

0

0.1

0.2

Dithiaporphyrin 3, µM

Figure 5. Eﬀect of 3 on mitochondrial cytochrome c oxidase activity in

cultured whole R3230AC cells following irradiation. Cell culture and

light exposure conditions are detailed in Section 4. The activity values

represent the change in optical density (OD) units per minute for

1 · 10⁵cells that were exposed to 3 at indicated concentrations 24 h

after washing (dark) or 24 h after light exposure (light). Each data

point represents the mean of at least three separate experiments; the

error bars are the SEM.

0

90

180

270

360

Light, Jcm^-2

Figure 4. Eﬀect of compounds 3 and 4 (10 lM) on mitochondrial

cytochrome c oxidase activity in isolated mitochondrial suspensions in

the dark and with photosensitization. Details of experimental condi-

tions are described in Section 4. Data are expressed as the percent of

initial preirradiation cytochrome c oxidase activity in mitochondrial

suspensions. Each datum point represents the mean of three separate

experiments; the error bars are the SEM.

mitochondrial suspensions. Mitochondrial suspensions

were prepared from rat liver using a previously pub-

lished method.⁴⁸Mitochondrial suspensions were trea-

ted with 5 · 10^À6M solutions of 3 or 4 for 5 min. The

suspensions were centrifuged, the pellets resuspended

and irradiated with ﬁltered 530–750 nm light delivered

at 100 mW cm^À2. Aliquots were removed at various

times and cytochrome c oxidase activity was compared

to that of mitochondrial suspensions maintained in the

dark (Fig. 4). No signiﬁcant reduction in enzyme activ-

ity was observed for mitochondrial suspensions kept in

the dark while exposure of suspensions to 3 or 4 and

light resulted in a light-dose-dependent inhibition of

cytochrome c oxidase. At 360 J cm^À2, dithiaporphyrins

3 and 4 inhibited cytochrome c oxidase activity by

97% and 43%, respectively. These results suggest that

the dithiaporphyrin photosensitizers—even those bear-

ing a bulky substituent—can inhibit mitochondrial func-

tion if the dithiaporphyrins reach the mitochondria.

Figure 6. Fluorescence image of R3230AC cells treated with

5 · 10^À6M of 3 for 24 h. Cell culture conditions and experimental

details are detailed in Section 4.

2.3.3.2. Photosensitized inhibition of cytochrome c

oxidase in cultured whole R3230AC cells. We next exam-

ined the inhibition of mitochondrial cytochrome c oxi-

dase in cultured whole R3230AC cells using 3 and

5 J cm^À2of 350–750 nm light—the same light conditions

used in the phototoxicity studies. Thirty minutes after

the irradiation, at a time when there was no signiﬁcant

cytotoxicity, cells were harvested and cytochrome c oxi-

dase activity was assessed (Fig. 5). At concentrations of

61 · 10^À7M, no signiﬁcant inhibition of cytochrome c

oxidase was observed (P > 0.05). However, at

2 · 10^À7M 3, 60% of the enzyme activity was inhibited

after light exposure, which was signiﬁcantly diﬀerent

from dark and untreated controls (P < 0.05).

tion via ﬂuorescence microscopy. Cellular staining with

3 at 5 · 10^À6M resulted in globular cytosolic ﬂuores-

cence as shown in Figure 6. Under these conditions,

ﬂuorescence was not detected in the nucleus, the nuclear

membrane or the plasma membrane. Furthermore, the

ﬂuorescence pattern was not consistent with mitochon-

drial localization in R3230AC cells treated with the

mitochondrial stain rhodamine 123.³⁸Other studies

have demonstrated that staining of lipid droplets with

the fatty acid ﬂuorescence probe, C₁-BODIPY 500/510

C₁₂(Molecular Probes, Inc., Eugene, OR), showed sim-

ilar globular ﬂuorescence patterns in the cytosol.^49,50

3. Summary and conclusions

2.3.3.3. Intracellular localization of 3 via ﬂuorescence

microscopy. The ﬂuorescence of 21,23-dithiaporphyrin 3

was suﬃcient to make an image of subcellular localiza-

We have further developed synthetic routes to 21,

23-dithiaporphyrins with diﬀerent 5- and 20-meso

2242

Y. You et al. / Bioorg. Med. Chem. 13 (2005) 2235–2251

substituents with 4-caboxylatomethoxyphenyl substituents

as the 10- and 15-meso substituents. We prepared nine

new 21,23-dithiaporphyrins and compared their physical

and photophysical properties to those of 5,20-diphenyl-

10,15-bis(4-carboxylatomethoxyphenyl)-21,23-dithiapor-

phyrin (1).³⁸Changes in the meso substituents had little

eﬀect on the absorption spectra, quantum yields for the

generation of singlet oxygen, and quantum yields for

ﬂuorescence. The substituent changes did have some im-

pact on the n-octanol/water partition coeﬃcient, but

these changes showed no correlation with either cellular

uptake or cellular dark or phototoxicity.

for the sample chambers. Elemental analyses were con-

ducted by Atlantic Microlabs, Inc. Q-TOF 2 electro-

spray and ESI mass spectrometry and were conducted

by the Campus Chemical Instrumentation Center of

The Ohio State University (Columbus, OH) and the

Instrument Center of the Department of Chemistry at

the University at Buﬀalo. The purity of compounds 1–

10 was accessed by elemental analysis and the results

were within 0.4% of the theoretical values. 2,5-Bis[1-

(4-methoxyphenyl)-1-hydroxymethyl]thiophene (17),³⁴

2,5-bis[1-(4-methoxy-phenyl)-1-pyrrolomethyl]thiophene

(18),³²34,³⁴35,³⁸and 44⁵¹were prepared as previously

described. For biological studies, core-modiﬁed porphy-

rins 1–10 were dissolved in DMSO to make a stock

solution at 2 · 10^À3M. The stock solutions were used

after appropriate dilutions with sterilized doubly dis-

tilled water.

None of the 21,23-dithiaporphyrins 1–10 showed any

signiﬁcant dark toxicity following a 24 h incubation with

61 · 10^À5M 1–10 (>80% cell viability). However,

phototoxicity was structure dependent. The presence

of two bulky substituents, that is, substituents more

bulky than 4-methylphenyl at the 5- and 20-positions

(dithiaporphyrins 4, 6, 9, and 10), gave greatly decreased

phototoxicity relative to dithiaporphyrins 1–3 and 8

with two smaller substituents (phenyl, 2-thienyl, or 4-

methylphenyl) or dithiaporphyrins 5 and 7 with one

phenyl substituent and one bulky substituent (4-tert-

butyl or 2,4,6-trimethylphenyl) at the 5- and 20-positions.

Among the photosensitizers 1–3, 5, 7, and 8, dith-

iaporphyrin 3 with 5-phenyl-20-2-thienyl substituents

displayed the greatest phototoxicity with 68% cell kill

at a concentration of 1 · 10^À7M with 5 J cm^À2of 350–

750 nm light.

4.2. Chemistry

4.2.1. 2,5-Bis(2-thienyl-1-hydroxymethyl)thiophene (11).

Thiophene (4.4 g, 52 mmol) was added to a solution of

n-BuLi (80 mL of a 1.6 M solution in hexanes,

128 mmol) and TMEDA (20 mL, 133 mmol) in

200 mL of hexanes under an Ar atmosphere. The reac-

tion mixture was heated at reﬂux for 1 h, cooled to

ambient temperature, and transferred via a cannula to

a pressure-equalizing addition funnel. This dilithiothio-

phene suspension was then added dropwise to a solution

of 2-thiophenecarboxaldehyde (14.4 g, 128 mmol) in

200 mL of anhydrous THF cooled to 0 ꢁC, which had

been degassed with Ar for 15 min. After the addition

was complete, the mixture was warmed to ambient tem-

perature, 300 mL of NH₄Cl (aqueous 1 M solution) was

added, and the organic phase was separated. The aque-

ous phase was extracted with ether (3 · 300 mL). The

combined organic extracts were washed with water

(3 · 300 mL) and brine (300 mL), dried over MgSO₄,

and concentrated to give yellow oil. The crude product

was precipitated by the slow addition of hexanes to an

ether solution of 11 to give 13.0 g (80%) of predomi-

nantly one diastereomer of 11 as white amorphous pow-

Irradiation of cells in the presence of compound 3 at

2 · 10^À7M gives a decrease in cytochrome c oxidase

activity while dithiaporphyrin 3 at 2 · 10^À7M without

irradiation does not aﬀect cytochrome c oxidase activity

in whole cells. However, compound 3 does not appear to

localize in the mitochondria since images of 3-treated

cells by ﬂuorescence microscopy show globular ﬂuores-

cence inconsistent with mitochondrial localization. Pho-

tosensitizer 3 may relocalize following irradiation. We

are now trying to uncover the detailed biological re-

sponses after the irradiation with core-modiﬁed porphy-

rin 3 as well as in vivo antitumor eﬃcacy.

der. IR (KBr): 3390, 3104, 2869, 2247, 1604 cm^À1

.

¹H NMR (400 MHz, CDCl₃): d 2.69 (2H, br s), 6.31

(2H, s), 6.95 (2H, s), 7.05 (2H, s), 7.10 (2H, s), 7.36

(2H, s). ¹³C NMR (75 MHz, CDCl₃): d 68.9, 124.3,

125.0, 125.0, 125.6, 125.6, 127.1. High-resolution EI

MS: m/z 308.0000 (calcd for C₁₄H₁₂O₂S₃, 307.9999).

4. Experimental

4.1. General methods

Solvents and reagents were used as received from

Sigma–Aldrich Chemical Co. (St. Louis, MO) unless

otherwise noted. Cell culture medium was purchased

from GIBCO (Grand Island, NY). Fetal bovine serum

(FBS) was obtained from Atlanta Biologicals (Atlanta,

GA). Concentration in vacuo was performed on a Buchi

rotary evaporator. NMR spectra were recorded at 23 ꢁC

on a Varian Gemini-300, Inova 400, or Inova 500 instru-

ment with residual solvent signal as the internal stan-

dard: CDCl₃(d 7.26 for proton, d 77.16 for carbon).

Infrared spectra were recorded on a Perkin–Elmer FT-

IR instrument. UV–vis–near-IR spectra were recorded

on a Perkin–Elmer Lambda 12 spectrophotometer

equipped with a circulating constant temperature bath

4.2.2. 2,5-Bis[1-(4-methyl)phenyl-1-hydroxymethyl]thio-

phene (12). Diol 12 was prepared as described for the

preparation of 11 using thiophene (2.0 g, 25 mmol), n-

BuLi (34 mL of 1.6 M in hexanes, 55 mmol), TMEDA

(8.7 mL, 57 mmol), and 4-methylbenzaldehyde (5.7 g,

47 mmol). Product yield was 2.0 g (25%) of 12 as white

amorphous powder. IR (KBr): 3377, 2955, 2922,

2868, 1513 cm^À^{1 1}H NMR (400 MHz, 1:1 CDCl₃/

.

CD₃OD): d 2.19 (6H, s), 5.74 (2H, s), 6.52 (2H, s),

7.00 (4H, d, J = 7.6 Hz), 7.16 (4H, d, J = 7.6 Hz). ¹³C

NMR (75 MHz, 1:1 CDCl₃/CD₃OD): d 21.3, 81.8,

125.1, 127.2, 127.5, 127.8, 129.5, 138.1, 138.2, 146.6.

High-resolution ESI MS: m/z 347.1057 (calcd for

C₂₀H₂₀O₂S + Na, 347.1082).

Y. You et al. / Bioorg. Med. Chem. 13 (2005) 2235–2251

2243

4.2.3.

2,5-Bis[1-(4-ethyl)phenyl-1-hydroxymethyl]thio-

2,5-bis[1-(4-methoxyphenyl)-1-pyrrolomethyl]thiophene

(18, 4.6 g, 10 mmol), and TCBQ (12.6 g, 51 mmol) were

dissolved in 600 mL of CH₂Cl₂. BF₃ÆOEt₂(0.57 mL,

0.45 mmol) was added and the reaction mixture was stir-

red for 0.5 h in the dark. The reaction mixture was con-

centrated and the residue was redissolved in minimal

CH₂Cl₂. The crude product was puriﬁed via chromato-

graphy on basic alumina eluted with CH₂Cl₂. Porphyrin

19 was isolated as the ﬁrst red band. The crude product

was washed with acetone and was then recrystallized

from CH₂Cl₂/MeOH to give 0.96 g (13%) of 19 as a

purple solid; mp > 300 ꢁC. ¹H NMR (300 MHz,

CDCl₃): d 4.09 (6H, s), 7.35 (4H, d, J = 8.1 Hz), 7.57

(2H, d, J = 4.2 Hz), 7.93 (2H, d, J = 5.4 Hz), 7.96 (2H,

d, J = 3.3 Hz), 8.17 (4H, d, J = 8.1 Hz), 8.70 (2H, d,

J = 4.5 Hz), 8.87 (2H, d, J = 4.2 Hz), 9.69 (2H, s), 9.92

(2H, s). ¹³C NMR (75 MHz, CDCl₃): d 55.8, 77.2,

113.2, 125.6, 127.1, 128.9, 133.4, 133.7, 134.3, 134.8,

135.0, 135.6, 135.8, 142.0, 148.6, 148.9, 156.9, 157.4,

160.0. High-resolution Q-TOF MS: m/z 721.1144 (calcd

for C₄₂H₂₈N₂O₂S₄+ H, 721.1112).

phene (13). Diol 13 was prepared as described for the

preparation of 11 using thiophene (1.5 g, 19 mmol), n-

BuLi (26 mL of 1.6 M in hexanes, 41 mmol), TMEDA

(6.5 mL, 43 mmol), and 4-ethylbenzaldehyde (4.9 g,

37 mmol). Product yield was 5.0 g (76%) of 13 as white

amorphous powder. IR (KBr): 3410, 2962, 2928, 2872,

1514 cm^À^{1 1}H NMR (500 MHz, DMSO-d₆): d 1.14

.

(6H, t, J = 7.5 Hz), 2.55 (4H, q, J = 7.5 Hz), 5.79 (2H,

s), 6.03 (2H, s), 6.65 (2H, s), 7.15 (4H, d, J = 8.0 Hz),

7.28 (4H, d, J = 8.0 Hz). ¹³C NMR (75 MHz, 1:1

CDCl₃/CD₃OD): d 16.4, 28.5, 71.3, 123.4, 126.7, 128.1,

143.0, 143.2, 150.1. High-resolution ESI MS: m/z

352.1492 (calcd for C₂₂H₂₄O₂S, 352.1491).

4.2.4. 2,5-Bis[1-(4-tert-butyl)phenyl-1-hydroxymethyl]thio-

phene (14). Diol 14 was prepared as described for the

preparation of 11 using thiophene (1.4 g, 16 mmol), n-

BuLi (22 mL of 1.6 M in hexanes, 36 mmol), TMEDA

(5.6 mL, 37 mmol), and 4-tert-butylbenzaldehyde

(5.1 g, 32 mmol). Product yield was 4.0 g (60%) of 14

as white amorphous powder. IR (KBr): 3366, 2960,

2904, 2868, 1511 cm^À1. H NMR (500 MHz, DMSO-

4.2.8. 5,20-Bis(4-methylphenyl)-10,15-bis(4-methoxyph-

enyl)-21,23-dithiaporphyrin (20). Diol 12 (2.2 g,

6.8 mmol), thiophene 18 (3.1 g, 6.8 mmol), TsOHÆH₂O

(1.3 g, 6.8 mmol), and TCBQ (5.0 g, 20 mmol) were trea-

ted as described for the preparation of 19 to give 0.31 g

1

d₆): d 1.26 (18H, s), 5.79 (2H, m), 6.03 (2H, m), 6.66

(2H, m), 7.30 (4H, d, J = 6.5 Hz), 7.34 (4H, d,

J = 8 Hz). ¹³C NMR (126 MHz, DMSO-d₆): d 31.2,

34.2, 70.6, 122.7, 124.8, 125.7, 125.8, 142.0, 149.1,

149.4. High-resolution EI MS: m/z 408.2114 (calcd for

C₂₆H₃₂O₂S, 408.2118).

1

(6%) of 20 as a purple solid; mp > 300 ꢁC. H NMR

(400 MHz, CDCl₃): d 4.09 (6H, s), 7.35 (4H, d,

J = 8.1 Hz), 7.57 (2H, d, J = 4.2 Hz), 7.93 (2H, d, J =

5.4 Hz), 7.96 (2H, d, J = 3.3 Hz), 8.17 (4H, d,

J = 8.1 Hz), 8.70 (2H, d, J = 4.5 Hz), 8.87 (2H, d,

J = 4.2 Hz), 9.69 (2H, s), 9.92 (2H, s). ¹³C NMR

(75 MHz, CDCl₃): d 55.8, 77.2, 113.2, 125.6, 127.1,

128.9, 133.4, 133.7, 134.3, 134.8, 135.0, 135.6, 135.8,

142.0, 148.6, 148.9, 156.9, 157.4, 160.0. High-resolution

ESI MS: m/z 737.2308 (calcd for C₄₈H₃₆N₂O₂S₂+ H,

737.2296).

4.2.5.

2,5-Bis[1-(4-butyl)phenyl-1-hydroxymethyl]thio-

phene (15). Diol 15 was prepared as described for the

preparation of 11 using thiophene (1.3 g, 16 mmol), n-

BuLi (21 mL of 1.6 M in hexanes, 34 mmol), TMEDA

(5.4 mL, 36 mmol), and 4-butylbenzaldehyde (4.9 g,

30 mmol). Product yield was 4.0 g (63%) of 15 as color-

less oil as a mixture of meso- and d,l-diastereomers and

used for next reaction without further puriﬁcation. IR

(ﬁlm): 3364, 2955, 2928, 2857, 1512 cm^À^{1 1}H NMR

.

(500 MHz, 1:1 CDCl₃/CD₃OD): d 0.75–0.93 (6H, m),

1.30–1.40 (4H, m), 1.55–1.62 (4H, m), 2.54–2.60 (4H,

m), 5.36/5.87 (2H, s), 6.62–6.65 (2H, m), 7.10–7.17

(4H, m), 7.24–7.32 (4H, m). ¹³C NMR (75 MHz, 1:1

CDCl₃/CD₃OD): d 13.8, 22.4, 33.7, 35.4, 81.6, 125.0,

127.0, 128.6, 138.1, 142.9, 146.4. High-resolution ESI

MS: m/z 408.2118 (calcd for C₂₆H₃₂O₂S, 408.2111).

4.2.9. 5,20-Bis(4-ethylphenyl)-10,15-bis(4-methoxyphen-

yl)-21,23-dithiaporphyrin (21). Diol 13 (2.8 g, 8.0 mmol),

thiophene 18 (3.6 g, 7.9 mmol), TsOHÆH₂O (1.5 g,

7.9 mmol), and TCBQ (5.8 g, 24 mmol), were treated

as described for the preparation of 19 to give 1.2 g

1

(20%) of 21 as a purple solid; mp > 300 ꢁC. H NMR

(500 MHz, CDCl₃): d 1.54 (6H, t, J = 7.5 Hz), 3.02

(4H, q, J = 7.5 Hz), 4.12 (6H, s), 7.38 (4H, d,

J = 8.0 Hz), 7.67 (4H, d, J = 7.5 Hz), 8.19 (4H, d,

J = 7.0 Hz), 8.20 (4H, d, J = 6.5 Hz), 8.70–8.74 (4H,

m), 9.73 (4H, s). High-resolution ESI MS: m/z

765.2575 (calcd for C₅₀H₄₀N₂O₂S₂+ H, 765.2604).

4.2.6. 2,5-Bis[1-hydroxy-1-(2,4,6-trimethylphenyl)methyl]-

thiophene (16). Using thiophene (2.1 g, 25 mmol),

n-BuLi (34.3 mL of 1.6 M in hexanes, 55 mmol), TME-

DA (8.7 mL, 57 mmol), and mesitylaldehyde (7.0 g,

47 mmol), 16 was prepared as described in the method

for 11 to aﬀord 5.0 g (53%) of 16 as colorless oil. IR

4.2.10. 5,20-Bis(4-tert-butylphenyl)-10,15-bis(4-methoxy-

phenyl)-21,23-dithiaporphyrin (22). Thiophene 18 (3.0 g,

6.6 mmol), diol 14 (2.7 g, 6.6 mmol), and TCBQ (4.9 g,

20 mmol) were dissolved in 500 mL of CH₂Cl₂. TsOHÆ

H₂O (1.3 g, 6.6 mmol) was added and the reaction mix-

ture was stirred for 0.5 h at ambient temperature and

was then heated at reﬂux for 1 h in the dark. Porphyrin

22 was isolated as described for 19 to give 0.90 g (17%)

of 22 as a purple solid; mp > 300 ꢁC. ¹H NMR

(300 MHz, CDCl₃): d 1.82 (18H, s), 4.29 (6H, s), 7.56

(4H, s), 8.04 (4H, s), 8.39 (8H, br s), 8.94 (4H, s), 9.93

(KBr): 3408, 2921, 1611 cm^À^{1 1}H NMR (400 MHz,

.

DMSO-d₆): d 2.19 (6H, s), 2.21 (12H, s), 5.90 (2H, d,

J = 4.4 Hz), 6.13 (2H, d, J = 4.4 Hz), 6.27 (2H, d,

J = 7.6 Hz), 6.79 (4H, s). ¹³C NMR (75 MHz, DMSO-

d₆): d 20.1, 20.4, 67.2, 121.9, 129.4, 135.8, 136.1, 137.3,

147.5. High-resolution Q-TOF MS: m/z 403.1736 (calcd

for C₂₄H₂₈O₂S + Na, 403.1708).

4.2.7. 5,20-Bis(2-thienyl)-10,15-bis(4-methoxyphenyl)-

21,23-dithiaporphyrin (19). Diol 11 (3.1 g, 10 mmol),

2244

Y. You et al. / Bioorg. Med. Chem. 13 (2005) 2235–2251

(4H, s). ¹³C NMR (75 MHz, DMSO-d₆): d 31.8, 35.1,

55.7, 113.2, 124.5, 133.8, 134.0, 134.2, 134.3, 134.5,

134.7, 135.4, 135.6, 138.5, 148.1, 148.1, 151.0, 156.6,

156.7, 159.9. High-resolution ESI MS: m/z 821.3218

(calcd for C₅₄H₄₈N₂O₂S₂+ H, 821.3230).

8.0 Hz), 7.69 (4H, d, J = 7.5 Hz), 8.06 (4H, d,

J = 8.0 Hz), 8.08 (4H, d, J = 7.5 Hz), 8.59 (2H, d,

J = 4.5 Hz), 8.64 (2H, d, J = 4.5 Hz), 9.68 (2H, m),

9.72 (2H, s). High-resolution ESI MS: m/z 737.2285

(calcd for C₄₈H₃₆N₂O₂S₂+ H, 737.2291).

4.2.11. 5,20-Bis(4-butylphenyl)-10,15-bis(methoxyphen-

yl)-21,23-dithiaporphyrin (23). Diol 15 (3.4 g, 8.2 mmol),

thiophene 18 (3.5 g, 8.1 mmol), TsOHÆH₂O (1.6 g,

8.1 mmol), and TCBQ (6.0 g, 24 mmol), were treated

as described for the preparation of 19 to give 0.6 g

(9%) of 23 as a purple solid; mp 248–250 ꢁC. ¹H

NMR (500 MHz, CDCl₃): d 1.10 (6H, t, J = 7.0 Hz),

1.58–1.65 (4H, m), 1.90–1.97 (4H, m), 2.97 (6H, t, J =

8.0 Hz), 4.11 (6H, s), 7.39 (4H, d, J = 10.5 Hz), 7.65

(4H, d, J = 7.0 Hz), 8.18 (4H, d, J = 8.0 Hz), 8.21 (4H,

d, J = 8.0 Hz), 8.72 (4H, m), 9.73 (4H, s). High-resolu-

tion ESI MS: m/z 821.3235 (calcd for C₅₄H₄₈-

N₂O₂S₂+ H, 821.3230).

4.2.15. 5,20-Bis(4-tert-butylphenyl)-10,15-bis(4-hydroxy-

phenyl)-21,23-dithiaporphyrin (27). Dithiaporphyrin 22

(500 mg, 0.61 mmol) was treated with BBr₃(0.60 mL,

6.1 mmol) as described for the preparation of 24 to give

0.43 g (89%) of 27 as a dark blue solid; mp > 300 ꢁC. ¹H

NMR (400 MHz, 1:1 CDCl₃/CD₃OD): d 1.60 (18H, s),

7.29 (4H, d, J = 7.6 Hz), 7.83 (4H, d, J = 7.6 Hz), 8.08

(4H, d, J = 7.6 Hz), 8.15 (4H, d, J = 7.6 Hz), 8.69 (2H,

d, J = 3.6 Hz), 8.71 (2H, d, J = 4.4 Hz), 9.72 (2H, s),

9.76 (2H, s). ¹³C NMR (75 MHz, DMSO-d₆) d 35.7,

39.1, 119.0, 128.8, 136.8, 138.4, 138.5, 138.6, 138.7,

138.8, 139.9, 140.0, 142.5, 152.0, 152.4, 155.5, 160.8,

161.0, 161.9. High-resolution ESI MS: m/z 793.2932

(calcd for C₅₂H₄₄N₂O₂S₂+ H, 793.2917).

4.2.12. 5,20-Bis(2-thienyl)-10,15-bis(4-hydroxyphenyl)-

21,23-dithiaporphyrin (24). Dithiaporphyrin 19 (0.5 g,

0.69 mmol) was dissolved in 100 mL of CH₂Cl₂and

BBr₃(1.3 mL, 14 mmol) was added at 0 ꢁC. The result-

ing solution was stirred for 5 h at ambient temperature.

The reaction mixture was added to 200 mL of EtOAc

and 200 mL of saturated NaHCO₃. The organic layer

was separated and washed three times with brine, dried

over MgSO₄, and concentrated. The crude solid was

washed with 25% EtOAc/hexanes several times to give

0.42 g (87%) of 24 as a dark blue solid; mp > 300 ꢁC.

¹H NMR (400 MHz, DMSO-d₆): d 7.26 (4H, d,

J = 8.4 Hz), 7.62 (2H, d, J = 4.2 Hz), 8.01 (2H, br s),

8.01 (4H, d, J = 7.6 Hz), 8.18 (2H, d, J = 5.2 Hz), 8.62

(2H, d, J = 4.8 Hz), 8.75 (2H, d, J = 4.4 Hz), 9.72 (2H,

s), 9.88 (2H, s), 10.28 (2H, br s). ¹³C NMR (75 MHz,

DMSO-d₆): d 114.9, 125.1, 127.6, 130.3, 130.8, 133.7,

134.0, 135.1, 135.6, 135.8, 136.4, 140.5, 147.5, 147.7,

156.0, 156.2, 158.0. High-resolution Q-TOF MS: m/z

693.0754 (calcd for C₄₀H₂₄N₂O₂S₄+ H, 693.0799).

4.2.16. 5,20-Bis(4-butylphenyl)-10,15-bis(4-hydroxyphen-

yl)-21,23-dithiaporphyrin (28). Dithiaporphyrin 23

(430 mg, 0.52 mmol) was treated with BBr₃(0.50 mL,

5.2 mmol) as described for the preparation of 24 to give

1

0.38 g (92%) of 28 as dark blue solid; mp > 300 ꢁC. H

NMR (500 MHz, 1:1 CDCl₃/CD₃OD): d 1.06 (6H, t,

J = 7.0 Hz), 1.52–1.62 (4H, m), 1.86–1.93 (4H, m), 2.95

(6H, t, J = 8.0 Hz), 4.11 (6H, s), 7.35 (4H, d,

J = 6.5 Hz), 7.61–7.70 (4H, m), 8.12 (4H, d,

J = 8.0 Hz), 8.15 (4H, d, J = 7.5 Hz), 8.69 (2H, s), 8.73

(4H, d, J = 4.0 Hz), 9.70–9.80 (4H, m). High-resolution

ESI MS: m/z 793.2908 (calcd for C₅₂H₄₄N₂O₂S₂+ H,

793.2917).

4.2.17. Diethyl 5,20-bis(2-thienyl)-10,15-bis(4-carboxyl-

atomethoxyphenyl)-21,23-dithiaporphyrin (29). Dithia-

porphyrin 24 (0.29 g, 0.42 mmol), K₂CO₃(2.9 g,

21 mmol), ethyl bromoacetate (4.64 mL, 42 mmol) in

60 mL of acetone were heated at reﬂux for 15 h. The

reaction mixture was cooled to ambient temperature

and the K₂CO₃was removed by ﬁltration. The ﬁlter

cake was washed with acetone until the ﬁltrate was col-

orless. The combined ﬁltrates were concentrated. The

crude product was washed with MeOH to give 0.27 g

(75%) of 29 as a purple solid; mp 186–188 ꢁC. ¹H

NMR (400 MHz, CDCl₃): d 1.43 (6H, t, J = 7.2 Hz),

4.43 (4H, q, J = 6.8 Hz), 4.93 (4H, s), 7.36 (4H, d,

J = 8.4 Hz), 7.57 (2H, dd, J = 5.2, 3.2 Hz), 7.93 (2H, d,

J = 5.2 Hz), 7.96 (2H, d, J = 3.2 Hz), 8.17 (4H, d, J =

3.2 Hz), 8.67 (2H, d, J = 4.4 Hz), 8.87 (2H, d, J =

4.8 Hz), 9.66 (2H, s), 9.92 (2H, s). ¹³C NMR (75 MHz,

CDCl₃): d 14.4, 61.8, 65.9, 114.0, 125.7, 127.1, 129.0,

129.2, 133.5, 133.6, 134.4, 134.5, 134.7, 134.8, 134.9,

135.6, 135.7, 135.9, 141.9, 148.5, 149.0, 156.9, 157.4,

158.3, 169.1. High-resolution Q-TOF MS: m/z

865.1432 (calcd for C₄₈H₃₆N₂O₆S₄+ H, 865.1456).

4.2.13.

5,20-Bis(4-methylphenyl)-10,15-bis(4-hydroxy-

phenyl)-21,23-dithiaporphyrin (25). Dithiaporphyrin 20

(240 mg, 0.33 mmol) was treated with BBr₃(0.31 mL,

3.2 mmol) as described for the preparation of 24 to give

0.20 g (87%) of 25 as a dark blue solid; mp > 300 ꢁC. ¹H

NMR (400 MHz, 1:1 CDCl₃/CD₃OD): d 2.60 (6H, s),

7.17 (4H, d, J = 8.4 Hz), 7.51 (4H, d, J = 7.6 Hz), 7.97

(4H, d, J = 8.4 Hz), 8.00 (4H, d, J = 8.0 Hz), 8.56 (2H,

d, J = 4.4 Hz), 8.60 (2H, d, J = 4.4 Hz), 9.57 (2H, s),

9.63 (2H, s). ¹³C NMR (75 MHz, 1:1:1 CDCl₃/

CD₃OD/DMSO-d₆): d 26.6, 120.1, 133.6, 137.1, 139.1,

139.3, 139.5, 139.8, 140.8, 152.6, 153.1, 161.4, 161.6,

163.0. High-resolution ESI MS: m/z 709.1992 (calcd

for C₄₆H₃₂N₂O₂S₂+ H, 709.1983).

4.2.14. 5,20-Bis(4-ethylphenyl)-10,15-bis(4-hydroxyphen-

yl)-21,23-dithiaporphyrin (26). Dithiaporphyrin 21

(400 mg, 0.52 mmol) was treated with BBr₃(0.50 mL,

5.2 mmol) as described for the preparation of 24 to give

0.35 g (91%) of 26 as a dark blue solid; mp > 300 ꢁC. ¹H

NMR (500 MHz, 1:1 CDCl₃/CD₃OD): d 1.52 (6H, t,

J = 8.0 Hz), 3.01 (4H, q, J = 7.5 Hz), 7.33 (4H, d, J =

4.2.18. Diethyl 5,20-bis(4-methylphenyl)-10,15-bis(4-

carboxylatomethoxyphenyl)-21,23-dithiaporphyrin (30).

Dithiaporphyrin 25 (0.22 g, 0.31 mmol), K₂CO₃

(1.07 g, 7.8 mmol), and ethyl bromoacetate (0.86 mL,

7.8 mmol) in 60 mL of acetone were treated as described

Y. You et al. / Bioorg. Med. Chem. 13 (2005) 2235–2251

2245

for the preparation of 29 to give 0.24 g (88%) of 30 as a

purple solid; mp 134–136 ꢁC. ¹H NMR (500 MHz,

CDCl₃): d 1.42 (6H, t, J = 7.0 Hz), 2.71 (6H, s), 4.42

(4H, q, J = 7.0 Hz), 4.92 (4H, s), 7.36 (4H, d, J =

8.5 Hz), 7.62 (4H, d, J = 7.5 Hz), 8.13 (4H, d,

J = 8.0 Hz), 8.17 (4H, d, J = 8.5 Hz), 8.67 (2H, d,

J = 4.5 Hz), 8.69 (2H, d, J = 4.0 Hz), 9.68 (2H, s), 9.69

(2H, s). ¹³C NMR (126 MHz, CDCl₃): d 14.5, 21.8,

61.8, 66.0, 114.0, 128.4, 133.6, 134.4, 134.4, 134.6,

134.8, 135.0, 135.5, 135.6, 135.6, 138.0, 138.6, 148.1,

156.7, 156.8, 158.2, 169.2. High-resolution ESI MS:

m/z 881.2709 (calcd for C₅₄H₄₄N₂O₆S₂+ H, 881.2719).

4.2.22. 2-(1-Hydroxy-1-phenylmethyl)-5-[1-hydroxy-1-(2-

thienyl)methyl]thiophene (36). 2-[1-(tert-Butyldimethyl-

silyloxy)-1-phenyl methyl]thiophene⁴⁰(35, 5.0 g, 16

mmol) was added to a solution of n-BuLi (11 mL of

1.6 M in hexanes, 18 mmol) and TMEDA (2.5 mL,

16 mmol) in 150 mL of hexanes under an Ar atmo-

sphere. The reaction mixture was stirred at ambient tem-

perature for 30 min, and was transferred via cannula to

a pressure-equalizing addition funnel. The suspension of

2-lithio 35 was then added dropwise to a solution of

2-thiophenecarboxaldehyde (1.38 mL, 15 mmol) in

150 mL of anhydrous THF at 0 ꢁC, which was degassed

with Ar for 15 min. After addition was complete, the

mixture was warmed to ambient temperature and

300 mL of a 1 M solution of NH₄Cl was added and

the organic phase was separated. The aqueous phase

was extracted with ether (3 · 300 mL). The combined

organic extracts were washed with water (3 · 300 mL)

and brine (300 mL), dried over MgSO₄, and concen-

trated to give yellow oil. The oil was dissolved in a

1 M solution of Bu₄NF in THF (95 mL, 95 mmol) and

stirred at ambient temperature for 1 h at which point

100 mL of saturated aqueous NH₄Cl was added.

The resulting mixture was extracted with ether

(4 · 100 mL). The combined organic extracts were

washed with water (3 · 400 mL) and brine (400 mL),

dried over MgSO₄, and concentrated to brown oil.

The crude diol was puriﬁed by column chromatography

on SiO₂eluted with 25% EtOAc/hexanes to give 3.6 g

(73%) of predominantly one diastereomer of 36 as yel-

low amorphous powder. IR (KBr): 3468, 3085, 1455,

4.2.19. Diethyl 5,20-bis(4-ethylphenyl)-10,15-bis(4-carb-

oxylatomethoxyphenyl)-21,23-dithiaporphyrin (31). Dith-

iaporphyrin 26 (0.35 g, 0.47 mmol), K₂CO₃(0.66 g,

4.8 mmol), and ethyl bromoacetate (0.50 mL, 4.8 mmol)

in 50 mL of acetone were treated as described for the

preparation of 29 to aﬀord 0.36 g (83%) of 31 as a

purple solid; mp 238–240 ꢁC. ¹H NMR (500 MHz,

CDCl₃): d 1.42 (6H, t, J = 7.0 Hz), 1.54 (6H, t, J =

7.5 Hz), 3.02 (4H, q, J = 8.0 Hz), 4.42 (4H, q,

J = 7.0 Hz), 4.93 (4H, s), 7.39 (4H, d, J = 8.0 Hz), 7.67

(4H, d, J = 7.5 Hz), 8.19 (4H, d, J = 8.0 Hz), 8.21 (4H,

d, J = 8.0 Hz), 8.70 (2H, d, J = 4.5 Hz), 8.73 (2H, d,

J = 4.0 Hz), 9.71 (2H, m), 9.73 (2H, s). High-resolution

ESI MS: m/z 909.3015 (calcd for C₅₆H₄₈N₂O₆S₂+ H,

909.3027).

4.2.20. Diethyl 5,20-bis(4-tert-butylphenyl)-10,15-bis(4-

carboxylatomethoxyphenyl)-21,23-dithiaporphyrin (32).

Dithiaporphyrin 27 (0.40 g, 0.50 mmol), K₂CO₃

(0.70 g, 5.0 mmol), and ethyl bromoacetate (0.6 mL,

5.0 mmol) in 50 mL of acetone were treated as described

for the preparation of 29 to give 0.37 g (76%) of 32 as a

purple solid; mp 170–172 ꢁC. ¹H NMR (400 MHz,

CDCl₃) d 1.42 (6H, t, J = 7.6 Hz), 1.62 (18H, s), 4.42

(4H, q, J = 7.2 Hz), 4.93 (4H, s), 7.36 (4H, d,

J = 8.4 Hz), 7.83 (4H, d, J = 8.0 Hz), 8.18 (8H, d, J =

8.0 Hz), 8.68 (2H, d, J = 4.4 Hz), 8.73 (2H, d,

J = 4.4 Hz), 9.68 (2H, s), 9.72 (2H, s). ¹³C NMR

(75 MHz, CDCl₃): d 14.4, 31.8, 35.1, 61.7, 65.9, 113.9,

124.5, 133.4, 134.3, 134.4, 134.8, 135.0, 135.3, 135.5,

135.7, 138.4, 148.0, 148.1, 151.0, 156.6, 158.2, 169.1.

High-resolution Q-TOF MS: m/z 793.2932 (calcd for

C₅₂H₄₄N₂O₂S₂+ H, 793.2917).

1391 cm^À^{1 1}H NMR (300 MHz, CDCl₃): d 7.40 (d,

.

2H, J = 8.0 Hz), 7.30–7.36 (m, 2H), 7.25–7.30 (m, 1H),

7.21–7.30 (m, 1H), 6.97 (br s, 1H), 6.93 (dd, 1H,

J = 5.4, 2.3 Hz), 6.80 (d, 1H, J = 3.3 Hz), 6.69 (d, 1H,

J = 3.2 Hz), 6.17 (s, 1H), 5.95 (s, 1H), 2.54 (s, 1H),

2.41 (s, 1H). ¹³C NMR (75 MHz, CDCl₃): d 148.5,

147.3, 147.0, 143.0, 128.7, 128.2, 126.9, 126.4, 125.7,

125.2, 124.8, 124.6, 72.7, 68.8. High-resolution Q-TOF

MS: m/z 325.0314 (calcd for C₁₆H₁₄O₂S₂+ Na,

325.0333).

4.2.23. 2-[1-(4-tert-Butylphenyl)-1-hydroxymethyl]-5-(1-

hydroxy-1-phenylmethyl)-thiophene (37). Thiophene 35

(6.0 g, 19.7 mmol), n-BuLi (13.5 mL of 1 M in hexanes,

22 mmol), TMEDA (3.6 mL, 24 mmol), and 4-tert-

butylbenzaldehyde (4.0 mL, 24 mmol) were treated as

described for the preparation of 36 to give 3.0 g (43%)

of 37 as colorless oil. IR (ﬁlm): 3367, 2962, 2869,

4.2.21. Diethyl 5,20-bis(4-butylphenyl)-10,15-bis(4-carb-

(33).

oxylatomethoxyphenyl)-21,23-dithiaporphyrin

1

Using core-modiﬁed porphyrin 28 (0.35 g, 0.44 mmol),

K₂CO₃(0.61 g, 4.4 mmol), and ethyl bromoacetate

(0.49 mL, 4.4 mmol) in 50 mL of acetone, 33 was pre-

pared as described for the preparation of 29 to give

0.32 g (75%) of 33 as a purple solid; mp 120–122 ꢁC.

1711 cm^À1. H NMR (400 MHz, CDCl₃): d 1.31 (9H,

s), 5.94 (1H, s), 5.97 (1H, s), 6.70 (2H, s), 7.32–7.50

(9H, m). ¹³C NMR (126 MHz, CDCl₃): d 31.4, 34.6,

72.3, 72.4, 124.3, 124.3, 124.5, 124.5, 125.4, 126.1,

126.2, 126.4, 126.4, 127.9, 128.5, 140.0, 143.0, 147.9,

148.2, 150.9. High-resolution ESI MS: m/z 375.1387

(calcd for C₂₂H₂₄O₂S + Na, 375.1389).

¹H NMR (500 MHz, CDCl₃):

d

1.10 (6H, t,

J = 7.0 Hz), 1.42 (6H, t, J = 7.0 Hz), 1.58–1.66 (4H,

m), 1.90–2.00 (4H, m), 2.97 (6H, t, J = 7.5 Hz), 4.42

(4H, q, J = 7.0 Hz), 4.93 (4H, s), 7.29 (2H, d, J =

7.5 Hz), 7.39 (2H, d, J = 8.5 Hz), 7.65 (4H, d,

J = 7.5 Hz), 8.16–8.22 (8H, m), 8.69 (2H, d,

J = 4.5 Hz), 8.73 (2H, d, J = 4.5 Hz), 9.70–9.74 (4H,

m). High-resolution ESI MS: m/z 965.3658 (calcd for

C₆₀H₅₆N₂O₆S₂+ H, 965.3653).

4.2.24. 5-Phenyl-20-(2-thienyl)-10,15-bis(4-methoxyphen-

yl)-21,23-dithiaporphyrin (38). Diol 36 (3.1 g, 10 mmol)

and thiophene 18 (4.7 g, 10 mmol) in 600 mL of CH₂Cl₂

were treated with BF₃ÆOEt₂(0.57 mL, 4.5 mmol) and

TCBQ (13 g, 52 mmol) as described for the preparation

of 19 to give 0.84 g (11%) of 38 as a purple solid;

2246

Y. You et al. / Bioorg. Med. Chem. 13 (2005) 2235–2251

1

mp > 300 ꢁC. H NMR (300 MHz, CDCl₃): d 4.11 (6H,

s), 7.36 (4H, d, J = 8.1 Hz), 7.57 (1H, br s), 7.81 (3H, br

s), 7.92 (1H, d, J = 4.8 Hz), 7.96 (1H, br s), 8.17 (4H, d,

J = 8.4 Hz), 8.23 (2H, br s), 8.61 (1H, br s), 8.69 (2H, br

s), 8.88 (1H, d, J = 4.5 Hz), 9.67 (1H, d, J = 5.1 Hz),

9.70 (2H, br s), 9.91 (1H, d, J = 5.1 Hz). ¹³C NMR

(75 MHz, CDCl₃): d 55.8, 113.2, 127.0, 127.6, 128.2,

128.9, 133.3, 133.8, 134.3, 134.8, 135.4, 135.6, 135.7,

141.4, 160.0, 165.2. High-resolution Q-TOF MS: m/z

715.1615 (calcd for C₄₄H₃₀N₂O₂S₃+ H, 715.1547).

High-resolution ESI MS: m/z 737.2291 (calcd for

C₄₈H₃₆N₂O₂S₂+ H, 737.2297).

4.2.28. Diethyl 5-phenyl-20-(2-thienyl)-10,15-bis(4-carb-

oxylatomethoxyphenyl)-21,23-dithiaporphyrin (42). Dith-

iaporphyrin 40 (0.32 g, 0.47 mmol), K₂CO₃(0.64 g,

4.7 mmol), and 0.52 mL of ethyl bromoacetate in

50 mL of acetone were treated as described for the prep-

aration of 29 to aﬀord 0.36 g (90%) of 42 as a purple

1

solid; mp 198–200 ꢁC. H NMR (400 MHz, CDCl₃): d

1.43 (6H, t, J = 7.2 Hz), 4.43 (4H, q, J = 6.8 Hz), 4.93

(4H, s), 7.37 (4H, d, J = 8.4 Hz), 7.81 (3H, m), 7.92

(1H, d, J = 4.8 Hz), 7.96 (1H, d, J = 3.2 Hz), 8.17 (4H,

d, J = 8.4 Hz), 8.24 (2H, m), 8.66 (1H, d, J = 4.4 Hz),

8.68 (2H, m), 8.89 (1H, d, J = 4.4 Hz), 9.68 (1H, d,

J = 4.8 Hz), 9.69 (2H, s), 9.92 (1H, d, J = 4.8 Hz). ¹³C

NMR (75 MHz, CDCl₃): d 114.0, 125.4, 127.0, 127.6,

128.2, 128.9, 133.4, 134.0, 134.2, 134.3, 134.3, 134.4,

134.5, 134.6, 134.8, 135.4, 135.6, 135.7, 135.8, 141.3,

142.0, 147.8, 148.2, 148.5, 149.1, 156.7, 156.9, 157.3,

158.3, 169.1. High-resolution Q-TOF MS: m/z

859.1964 (calcd for C₅₀H₃₈N₂O₆S₃+ H, 859.1970).

4.2.25.

20-(4-tert-Butylphenyl)-5-phenyl-15,20-bis(4-

methoxyphenyl)-21,23-dithiaporphyrin (39). Diol 37

(3.9 g, 11 mmol) and thiophene 18 (5.0 g, 11 mmol) in

600 mL of CH₂Cl₂were treated with TsOHÆH₂O

(3.1 g, 11 mmol) and TCBQ (8.1 g, 33 mmol) as

described for the preparation of 19 to give 1.2 g (14%)

of 39 as a purple solid; mp 210–213 ꢁC. ¹H NMR

(300 MHz, CDCl₃): d 1.6 (9H, s), 4.0 (6H, s), 7.4

(3H, m), 7.62–7.98 (9H, m), 8.07–8.32 (5H, m),

8.60–8.80 (4H, m), 9.64–9.78 (4H, m). ¹³C NMR

(75 MHz, CDCl₃): d 31.8, 35.1, 55.7, 113.2, 114.0,

120.1, 124.6, 127.3, 127.6, 128.1, 128.2, 128.4, 133.7,

133.8, 133.9, 134.2, 134.3, 134.4, 134.5, 134.6, 134.7,

134.7, 134.9, 135.5, 135.5, 135.8, 138.4, 141.4, 142.7,

142.8, 147.8, 147.9, 148.0, 148.2, 151.1, 151.1, 156.4,

156.5, 156.7, 156.8, 158.8, 159.9. High-resolution ESI

MS: m/z 765.2604 (calcd for C₅₀H₄₀N₂O₂S₂+ H,

765.2631).

4.2.29. Diethyl 20-(4-tert-butylphenyl)-5-phenyl-10,15-

bis(4-carboxylatomethoxy-phenyl)-21,23-dithiaporphyrin

(43). Dithiaporphyrin 41 (0.55 g, 0.75 mmol), K₂CO₃

(1.0 g, 7.5 mmol), and ethyl bromoacetate (0.8 mL,

7.5 mmol) were treated as described for the preparation

of 29 to give 0.58 g (86%) of 43 as a purple solid; mp

1

136–138 ꢁC. H NMR (400 MHz, CDCl₃): d 1.27 (6H,

4.2.26. 5-Phenyl-20-(2-thienyl)-10,15-bis(4-hydroxyphen-

yl)-21,23-dithiaporphyrin (40). Dithiaporphyrin 38

(300 mg, 0.42 mmol) was treated with BBr₃(0.79 mL,

8.3 mmol) as described for the preparation of 24 to give

0.27 g (94%) of 40 as dark purple solid; mp > 300 ꢁC. ¹H

NMR (400 MHz, DMSO-d₆): d 7.26 (2H, d, J = 8.4 Hz),

7.26 (2H, d, J = 8.4 Hz), 7.61 (1H, dd, J = 5.0, 4.0 Hz),

7.83 (3H, m), 8.00 (1H, d, J = 3.6 Hz), 8.02 (2H, d,

J = 8.8 Hz), 8.02 (2H, d, J = 8.4 Hz), 8.17 (3H, m),

8.53 (1H, d, J = 4.4 Hz), 8.63 (2H, dd, J = 4.4, 2.4 Hz),

8.77 (1H, d, J = 4.8 Hz), 9.64 (1H, d, J = 5.2 Hz), 9.75

(2H, s), 9.88 (1H, d, J = 5.2 Hz), 10.26 (2H, br s). ¹³C

NMR (75 MHz, CDCl₃): d 115.3, 125.1, 128.2, 128.8,

130.7, 131.2, 134.0, 134.2, 135.3, 136.0, 136.3, 140.5,

140.8, 156.0, 156.2, 158.2. High-resolution Q-TOF

MS: m/z 687.1235 (calcd for C₄₂H₂₆N₂O₂S₃+ H,

687.1234).

t, J = 7.2 Hz), 1.62 (9H, s), 4.30 (4H, q, J = 7.2 Hz),

4.84 (4H, s), 7.38 (3H, br s), 7.69–7.93 (8H, m), 8.19

(3H, d, J = 8.0 Hz), 8.25 (2H, m), 8.67–8.79 (4H, m),

9.65–9.79 (4H, m). ¹³C NMR (75 MHz, CDCl₃): d

14.3, 31.8, 35.1, 61.6, 65.7, 113.9, 114.8, 120.6, 124.6,

127.6, 128.2, 128.6, 133.3, 133.4, 134.3, 134.3, 134.4,

134.4, 134.5, 134.8, 134.8, 135.0, 135.5, 135.9, 138.3,

141.3, 142.7, 147.7, 147.7, 148.0, 148.2, 151.1, 156.3,

156.4, 156.8, 157.0, 158.1, 169.0. High-resolution ESI

MS: m/z 909.3027 (calcd for C₅₆H₄₈N₂O₆S₂+ H,

909.3022).

4.2.30. 2,5-Bis[1-(4-tert-butyldimethylsilyloxyphenyl)-1-

hydroxymethyl]thiophene (45). Thiophene (3.3 g, 41

mmol) was treated with n-BuLi (51.5 mL of 1.6 M in

hexanes, 82 mmol), TMEDA (13.1 mL, 86 mmol), and

44 (19.0 g, 2.0 mmol) as described for the preparation

of 11 to give 6.5 g (28%) of 45 as colorless oil. IR (ﬁlm):

1

4.2.27. 20-(4-tert-Butylphenyl)-5-phenyl-10,15-bis(4-hydr-

oxyphenyl)-21,23-dithiaporphyrin (41). Dithiaporphyrin

39 (620 mg, 0.81 mmol) was treated with BBr₃

(0.80 mL, 8.1 mmol) as described for the preparation

of 24 to give 0.57 g (95%) of 41 as dark purple;

mp > 300 ꢁC. ¹H NMR (400 MHz, 1:1 CDCl₃/CD₃OD):

d 1.59 (9H, s), 7.28 (3H, t, J = 8.4 Hz), 7.62 (1H, t,

J = 8.4 Hz), 7.68–7.74 (3H, m), 7.77–7.87 (5H, m), 8.08

(1H, d, J = 8.0 Hz), 8.14 (2H, d, J = 7.2 Hz), 8.20–8.25

(2H, m), 8.63–8.75 (4H, m), 9.67 (4H, q, J = 5.2 Hz),

9.73 (1H, q, J = 4.8 Hz), 9.78 (2H, s). ¹³C NMR

(75 MHz, CDCl₃): d 31.6, 35.1, 114.9, 115.5, 121.8,

124.8, 126.5, 127.8, 128.4, 128.7, 132.7, 134.4, 134.6,

134.7, 134.8, 135.7, 136.0, 138.4, 141.4, 142.7, 148.0,

148.0, 148.1, 151.5, 156.4, 156.7, 156.8, 157.0, 157.9.

3410 cm^À1. H NMR (400 MHz, CDCl₃): d 0.21 (12H,

s), 1.00 (18H, s), 5.94 (2H, br s), 6.70 (2H, br s), 6.84

(4H, d, J = 8.4 Hz), 7.30 (4H, d, J = 8.4 Hz). ¹³C

NMR (75 MHz, CDCl₃): d À4.3, 18.3, 25.8, 72.4,

120.2, 124.4, 127.8, 135.9, 148.4, 155.6. High-resolution

ESI MS: m/z 579.2389 (calcd for C₃₀H₄₄O₄S₁Si₂+ Na,

579.2391).

4.2.31. Diethyl 2,5-bis[1-(4-carboxylatomethoxy)phenyl-

1-hydroxymethyl]thiophene (46). Diol 45 (6.5 g, 11.7

mmol) and TBAF (13 mL of 1 M in THF, 13 mmol)

were dissolved in 15 mL of THF, then the solution

was stirred for 1 h with several sonications. The solid

product was collected by ﬁltration and washed with ace-

tone to give 2.5 g (65%) of 2,5-bis-[1-(4-hydroxyphenyl)-

Y. You et al. / Bioorg. Med. Chem. 13 (2005) 2235–2251

2247

1-hydroxymethyl]thiophene as a white amorphous pow-

der, which was used for further reaction without further

puriﬁcation. Using 2,5-bis-[1-(4-hydroxyphenyl)-1-hydr-

oxymethyl]-thiophene(2.5 g, 7.6 mmol), K₂CO₃(4.2 g,

30 mmol), and ethyl bromoacetate (3.4 mL, 30 mmol)

in 50 mL of acetone were treated as described for the

preparation of 29 to give 0.37 g (76%) of 46 as colorless

oil. ¹H NMR (400 MHz, CDCl₃): d 1.29 (6H, t,

J = 7.6 Hz), 4.27 (4H, q, J = 7.2 Hz), 4.61 (4H, s), 5.93

(2H, s), 6.68 (2H, s), 6.88 (4H, d, J = 8.8 Hz), 7.34

(4H, d, J = 8.4 Hz). ¹³C NMR (75 MHz, CDCl₃): d

14.3, 61.5, 65.6, 72.2, 114.8, 124.4, 127.8, 136.5, 148.3,

157.8, 169.0. High-resolution ESI MS: m/z 523.1397

(calcd for C₂₆H₂₈O₈S₁+ Na, 523.1397).

yellow oil. ¹H NMR (400 MHz, DMSO-d₆): d 2.08

(12H, s), 2.19 (6H, s), 5.66 (2H, s), 5.85 (2H, s), 5.90

(2H, s), 6.47 (2H, s), 6.66 (2H, s), 6.80 (4H, s), 10.52

(2H, br s). ¹³C NMR (75 MHz, CDCl₃): d 20.9, 40.7,

107.0, 107.3, 108.5, 116.4, 125.0, 132.2, 135.8, 136.6,

137.4, 144.0. High-resolution ESI MS: m/z 479.2515

(calcd for C₃₂H₃₄N₂S + H, 479.2512).

4.2.35. Diethyl 5-phenyl-20-(2,4,6-trimethylphenyl)-

10,15-bis(4-carboxylatomethoxy-phenyl)-21,23-dithiapor-

phyrin (50). Diol 46 (4.7 g, 9.3 mmol) and thiophene 48

(4.1 g, 9.3 mmol) in 250 mL of CH₂Cl₂were treated with

TsOHÆH₂O (1.8 g, 9.3 mmol) and TCBQ (6.9 g,

28 mmol) as described for the preparation of 19 except

SiO₂was used as a stationary phase instead of basic

alumina to give 1.1 g (13%) of 50 as a purple solid; mp

4.2.32. 2-(1-Hydroxy-1-phenylmethyl)-5-[1-hydroxy-1-

(2,4,6-trimethylphenyl)methyl]-thiophene (47). Thiophene

35 (8.0 g, 26 mmol) was treated with n-BuLi (20 mL of

1 M in hexanes, 32 mmol), TMEDA (5.0 mL, 33 mmol),

and mesitaldehyde (4.6 mL, 32 mmol) as described for

the preparation of 36 to give 6.0 g (68%) of 47 as color-

less oil. IR (ﬁlm): 3402, 3029, 2920, 1708, 1610 cm^À1. ¹H

NMR (500 MHz, DMSO-d₆): d 2.19 (3H, s), 2.21 (6H,

s), 5.82 (1H, d, J = 3.5 Hz), 5.93 (1H, t, J = 4.0 Hz),

6.09 (1H, q, J = 4.5 Hz), 6.13 (1H, s), 6.32 (1H, d,

J = 2.5 Hz), 6.61 (1H, dd, J = 16.5, 3.0 Hz), 6.78 (2H,

s), 7.23 (1H, t, J = 7.0 Hz), 7.31 (2H, t, J = 7.5 Hz),

7.38 (2H, d, J = 5.6 Hz). ¹³C NMR (75 MHz, DMSO-

d₆): d 20.2, 20.4, 67.3, 70.7, 121.7, 121.8, 123.2, 126.1,

127.0, 128.0, 129.4, 135.9, 136.1, 137.1, 145.0, 148.2,

148.3, 148.4, 148.5. High-resolution EI MS: m/z

338.1332 (calcd for C₂₁H₂₂O₂S₁, 338.1335).

1

152–154 ꢁC. H NMR (400 MHz, CDCl₃): d 1.41 (6H,

t, J = 7.6 Hz), 1.87 (6H, s), 2.62 (3H, s), 4.41 (4H, q,

J = 6.8 Hz), 4.92 (4H, s), 7.29 (2H, s), 7.35 (4H, d,

J = 8.4 Hz), 7.75–7.81 (3H, m), 8.16 (2H, d,

J = 8.4 Hz), 8.18 (2H, d, J = 8.8 Hz), 8.22 (1H, s), 8.24

(1H, d, J = 2.8 Hz), 8.48 (1H, d, J = 4.4 Hz), 8.62 (1H,

d, J = 4.4 Hz), 8.66 (2H, q, J = 4.4 Hz), 9.41 (1H, d,

J = 5.2 Hz), 9.61 (1H, d, J = 5.2 Hz), 9.67 (2H, s). ¹³C

NMR (75 MHz, CDCl₃): d 14.4, 21.6, 61.7, 65.9,

113.9, 127.5, 128.1, 133.0, 133.4, 133.6, 133.7, 133.8,

134.2, 134.5, 134.6, 134.8, 135.0, 135.0, 135.5, 135.5,

135.7, 137.7, 138.1, 138.9, 141.4, 147.2, 148.0, 148.1,

148.1, 156.6, 156.7, 156.8, 158.2, 169.1. High-resolution

ESI MS: m/z 895.2903 (calcd for C₅₅H₄₆N₂O₆S₂+ H,

895.2887).

4.2.36. Diethyl 5,20-bis(2,4,6-trimethylphenyl)-10,15-

bis(4-carboxylatomethoxyphenyl)-21,23-dithiaporphyrin

(51). Compounds 46 (2.8 g, 5.6 mmol) and 49 (2.7 g,

5.6 mmol) in 500 mL of CH₂Cl₂were treated with TsO-

HÆH₂O (1.1 g, 5.6 mmol) and TCBQ (4.1 g, 17 mmol) as

described for the preparation of 19 to give 1.1 g (22%) of

51 as a purple solid; mp 160–162 ꢁC. ¹H NMR

(500 MHz, CDCl₃): d 1.42 (6H, t, J = 7.0 Hz), 1.90

(12H, s), 2.63 (6H, s), 4.42 (4H, q, J = 7.0 Hz), 4.93

(4H, s), 7.30 (4H, s), 7.36 (4H, d, J = 8.0 Hz), 8.16

(4H, d, J = 8.5 Hz), 8.48 (2H, d, J = 4.0 Hz), 8.62 (2H,

d, J = 4.0 Hz), 9.35 (2H, s), 9.66 (2H, s). ¹³C NMR

(75 MHz, CDCl₃): d 14.4, 21.6, 21.7, 61.7, 65.9, 113.9,

128.1, 132.8, 133.3, 133.5, 134.6, 134.9, 135.3, 135.4,

137.6, 138.1, 138.9, 147.3, 148.0, 156.6, 156.8, 158.2,

169.1. High-resolution Q-TOF MS: m/z 937.3315 (calcd

for C₅₈H₅₂N₂O₆S₂+ H, 937.3345).

4.2.33. 2-(1-Phenyl-1-pyrrolomethyl)-5-[1-pyrrolo-1-(2,4,

6-trimethylphenyl)methyl]-thiophene (48). Diol 47 (3.5 g,

10.2 mmol) was dissolved in excess pyrrole (28 mL),

and the resulting solution was degassed with Ar.

BF₃ÆOEt₂was added (0.3 mL, 2.0 mmol), and the result-

ing mixture was stirred for 1 h at ambient temperature.

The reaction was stopped by addition of CH₂Cl₂

(200 mL) followed by 40% NaOH (50 mL). The organic

layer was separated, washed with water (3 · 200 mL)

and brine (200 mL), dried over MgSO₄, and concen-

trated. The excess pyrrole was removed at reduced pres-

sure at ambient temperature. The residual oil was

puriﬁed via chromatography on SiO₂eluted with 25%

EtOAc/hexanes to give 3.5 g (78%) of 48 as yellow

1

oil. IR (ﬁlm): 3410, 2968, 2919, 1727 cm^À1. H NMR

(300 MHz, CDCl₃): d 2.36 (6H, s), 2.51 (3H, s), 5.80

(1H, s), 6.15 (1H, s), 6.23 (1H, s), 6.28 (1H, s), 6.39

(2H, br s), 6.85 (2H, M), 6.90 (2H, d, J = 3.0 Hz), 7.09

(2H, s), 7.44–7.64 (5H, m), 8.07 (1H, br s), 8.12 (1H,

br s). ¹³C NMR (75 MHz, CDCl₃): d 20.9, 20.9, 29.4,

40.7, 46.1, 103.8, 107.0, 107.6, 107.9, 108.4, 108.5,

116.4, 117.3, 125.1, 125.3, 127.1, 128.5, 128.7, 130.4,

132.1, 133.2, 135.7, 136.7, 137.5, 142.9, 144.8, 144.9,

145.1, 145.2. High-resolution ESI MS: m/z 437.2055

(calcd for C₂₉H₂₈N₂S + H, 437.2046).

4.2.37. 5,20-Bis(2-thienyl)-10,15-bis(4-carboxylatometh-

oxyphenyl)-21,23-dithiaporphyrin (2). Core-modiﬁed

porphyrin 29 (0.22 g, 0.25 mmol) was dissolved in

50 mL of THF and 20 mL of 1 M aqueous NaOH was

added. The resulting solution was stirred at ambient

temperature for 15 h. The solution was acidiﬁed by the

addition of 8.6 mL of acetic acid. The reaction mixture

was diluted with 100 mL of H₂O and the products were

extracted with EtOAc (3 · 100 mL). The combined or-

ganic extracts were dried over MgSO₄and concentrated.

The crude product was washed with several portion of

hexanes/MeOH to give 0.18 g (87%) of dithiaporphyrin

2 as a purple solid; mp 236–238 ꢁC. ¹H NMR

4.2.34. 2,5-Bis[1-pyrrolo-1-(2,4,6-trimethylphenyl)methyl]-

thiophene (49). Diol 16 (5.1 g, 13 mmol) in 35 mL of pyr-

role was treated with 0.3 mL of BF₃ÆOEt₂(2.7 mmol) as

for the preparation of 48 to give 5.0 g (78%) of 49 as

2248

Y. You et al. / Bioorg. Med. Chem. 13 (2005) 2235–2251

(400 MHz, CDCl₃): d 4.93 (4H, s), 7.43 (4H, d, J =

8.4 Hz), 7.61 (2H, dd, J = 4.8, 3.6 Hz), 8.00 (2H, d,

J = 2.8 Hz), 8.09 (2H, d, J = 5.6 Hz), 8.19 (4H, d, J =

8.4 Hz), 8.58 (2H, d, J = 4.4 Hz), 8.76 (2H, d,

J = 4.4 Hz), 9.68 (2H, s), 9.90 (2H, s). ¹³C NMR

(75 MHz, CDCl₃): d 65.5, 114.6, 126.1, 128.4, 131.1,

133.4, 134.5, 135.0, 135.3, 135.7, 136.0, 136.6, 137.0,

141.0, 148.2, 148.3, 156.6, 156.9, 158.8, 171.0. High-res-

olution Q-TOF MS: m/z 809.0901 (calcd for

C₄₄H₂₈N₂O₆S₄+ H, 809.0908). Anal. C, H, N.

135.5, 135.7, 135.8, 136.0, 138.4, 138.7, 147.2, 148.0,

148.1, 148.2, 156.8, 158.4, 171.5. High-resolution ESI

MS: m/z 839.2250 (calcd for C₅₁H₃₈N₂O₆S₂+ H,

839.2244). Anal. C, H, N.

4.2.41. 5,20-Bis(4-tert-butylphenyl)-15,20-bis(4-carboxy-

latomethoxyphenyl)-21,23-dithiaporphyrin (6). Dith-

iaporphyrin 32 (0.35 g, 0.42 mmol) in 40 mL of THF

was treated with 20 mL of 1 M aqueous NaOH as de-

scribed for the preparation of 2 to give 0.29 g (88%) of

1

dithiaporphyrin 6; mp > 300 ꢁC. H NMR (400 MHz,

4.2.38. 5-Phenyl-20-(2-thienyl)-10,15-bis(4-carboxylato-

methoxyphenyl)-21,23-dithiaporphyrin (3). Dithiaporph-

yrin 42 (0.18 g, 0.21 mmol) in 40 mL of THF was

treated with 20 mL of 1 M aqueous NaOH as described

for the preparation of 2 to give 0.15 g (89%) of dith-

iaporphyrin 3 as a purple solid; mp 226–228 ꢁC. ¹H

NMR (500 MHz, 1:1 CDCl₃/CD₃OD): d 4.90 (4H, s),

7.37 (4H, d, J = 8.5 Hz), 7.55 (2H, dd, J = 5.0, 4.0 Hz),

7.79 (3H, m), 7.93 (2H, d, J = 3.5 Hz), 8.14 (4H, d,

J = 8.5 Hz), 8.20 (2H, m), 8.63 (1H, d, J = 4.5 Hz),

8.66 (2H, d, J = 4.5 Hz), 8.85 (1H, d, J = 5.0 Hz), 9.67

(1H, d, J = 5.0 Hz), 9.69 (2H, s), 9.91 (1H, d,

J = 5.5 Hz). ¹³C NMR (75 MHz, 1:1 CDCl₃/CD₃OD):

d 65.6, 114.1, 125.7, 127.3, 127.8, 128.5, 129.3, 133.6,

134.3, 134.4, 134.5, 134.7, 134.9, 135.7, 135.9, 136.0,

141.3, 141.9, 147.9, 148.3, 148.6, 149.1, 156.9, 157.0,

157.5, 158.5, 171.5. High-resolution Q-TOF MS: m/z

803.1379 (calcd for C₄₆H₃₀N₂O₆S₃+ H, 803.1344).

Anal. C, H, N.

DMSO-d₆): d 1.57 (18H, s), 5.00 (4H, s), 7.45 (4H, d,

J = 8.4 Hz), 7.92 (4H, d, J = 7.6 Hz), 8.19 (4H, s), 8.21

(4H, s), 8.61 (4H, d, J = 4.4 Hz), 8.65 (4H, d,

J = 4.4 Hz), 9.75 (4H, d, J = 6.8 Hz), 13.25 (2H, br s).

¹³C NMR (75 MHz, DMSO-d₆): d 32.0, 35.3, 65.6,

114.5, 125.1, 133.8, 134.2, 134.5, 135.2, 135.8, 136.3,

138.1, 147.6, 147.9, 151.3, 156.3, 156.5, 158.8, 170.8.

High-resolution ESI MS: m/z 909.3027 (calcd for

C₅₆H₄₈N₂O₆S₂+ H, 909.3029). Anal. C, H, N.

4.2.42. 20-(4-tert-Butylphenyl)-5-phenyl-10,15-bis(4-carb-

oxylatomethoxyphenyl)-21,23-dithiaporphyrin (7). Dith-

iaporphyrin 43 (0.50 g, 0.55 mmol) in 100 mL of THF

was treated with 50 mL of 1 M aqueous NaOH as

described for the preparation of 2 to give 0.42 g (90%)

of dithiaporphyrin 7; mp 227–230 ꢁC. ¹H NMR

(500 MHz, 1:1 CDCl₃/DMSO-d₆): d 1.57 (9H, s), 4.85

(4H, s), 7.39 (3H, m), 7.71–7.87 (9H, m), 8.11–8.17

(4H, m), 8.21–8.27 (2H, br s), 8.61–8.71 (4H, m), 9.63–

9.77 (4H, m), 12.75–13.33 (2H, br s). ¹³C NMR

(75 MHz, 1:1 CDCl₃/DMSO-d₆): d 31.7, 35.2, 65.6,

114.2, 114.9, 121.0, 124.8, 124.8, 127.8, 127.8, 128.3,

128.5, 128.8, 133.7, 133.8, 134.5, 134.6, 134.6, 134.7,

134.8, 134.9, 135.1, 135.8, 135.9, 136.2, 138.4, 138.4,

141.4, 141.4, 142.8, 147.9, 148.0, 148.2, 148.3, 151.5,

156.7, 156.8, 156.9, 157.0, 157.0, 157.4, 158.6, 171.6.

High-resolution ESI MS: m/z 853.2401 (calcd for

C₅₂H₄₀N₂O₆S₂+ H, 853.2403). Anal. C, H, N.

4.2.39. 5,20-Bis(2,4,6-trimethylphenyl)-10,15-bis(4-carb-

oxylatomethoxyphenyl)-21,23-dithiaporphyrin (4). Core-

modiﬁed porphyrin 51 (0.41 g, 0.46 mmol) in 80 mL of

THF was treated with 40 mL of 1 M aqueous NaOH

as described for the preparation of 2 to give 0.35 g

(87%) of dithiaporphyrin

4 as a purple solid;

1

mp > 300 ꢁC. H NMR (500 MHz, 1:1 CDCl₃/DMSO-

d₆): d 1.83 (12H, s), 2.57 (6H, s), 4.91 (4H, s), 7.28

(4H, s), 7.38 (4H, d, J = 8.5 Hz), 8.14 (4H, d, J =

8.5 Hz), 8.39 (2H, d, J = 4.0 Hz), 8.59 (2H, d, J =

4.0 Hz), 9.31 (2H, s), 9.69 (2H, s). ¹³C NMR

(126 MHz, 1:1 CDCl₃/DMSO-d₆): d 21.7, 21.8, 65.6,

114.4, 128.6, 132.8, 133.6, 133.9, 134.0, 135.0, 135.5,

135.6, 135.9, 137.4, 138.3, 138.4, 147.2, 147.9, 156.4,

156.5, 158.7, 170.8. High-resolution Q-TOF MS: m/z

881.2747 (calcd for C₅₄H₄₄N₂O₆S₂+ H, 881.2719).

Anal. C, H, N.

4.2.43. 5,20-Bis(4-methylphenyl)-10,15-bis(4-carboxyla-

tomethoxyphenyl)-21,23-dithiaporphyrin (8). Core-modi-

ﬁed porphyrin 30 (0.18 g, 0.20 mmol) in 40 mL of

THF was treated with 16 mL of 1 M aqueous NaOH

as described for the preparation of 2 to give 0.16 g

(95%) of dithiaporphyrin 8; mp > 300 ꢁC. ¹H NMR

(500 MHz, DMSO-d₆): d 2.65 (6H, s), 4.89 (4H, s),

7.37 (4H, d, J = 7.0 Hz), 7.61 (4H, d, J = 7.0 Hz), 8.06

(4H, d, J = 7.0 Hz), 8.12 (4H, d, J = 7.5 Hz), 8.61 (4H,

s), 9.66 (2H, s), 9.70 (2H, s), 13.07 (2H, s). ¹³C NMR

(126 MHz, 1:1 CDCl₃/DMSO-d₆): d 14.5, 21.8, 61.8,

66.0, 114.0, 128.4, 133.6, 134.4, 134.4, 134.6, 134.8,

135.0, 135.5, 135.6, 135.6, 138.0, 138.6, 148.1, 156.7,

156.8, 158.2, 169.2. High-resolution ESI MS: m/z

825.2052 (calcd for C₅₀H₃₆N₂O₆S₂+ H, 825.2098).

Anal. C, H, N.

4.2.40. 5-Phenyl-20-(2,4,6-trimethylphenyl)-10,15-bis(4-

carboxylatomethoxyphenyl)-21,23-dithiaporphyrin

(5).

Core-modiﬁed porphyrin 50 (0.50 g, 0.56 mmol) in

100 mL of THF was treated with 50 mL of 1 M aqueous

NaOH as described for the preparation of 2 to give

0.42 g (90%) of dithiaporphyrin 5 as a purple solid;

mp > 300 ꢁC. ¹H NMR (500 MHz, 1:1 CDCl₃/CD₃OD):

d 1.84 (6H, s), 2.58 (3H, s), 4.89 (4H, s), 7.27 (2H, s),

7.37 (4H, d, J = 6.5 Hz), 7.78 (3H, s), 8.11–8.17 (4H,

m), 8.19 (2H, br s), 8.47 (1H, d, J = 4.5 Hz), 8.62 (2H,

s), 8.64 (1H, s), 9.42 (1H, d, J = 2.5 Hz), 9.62 (1H, d,

J = 2.5 Hz), 9.70 (2H, s). ¹³C NMR (126 MHz, 1:1

CDCl₃/CD₃OD): d 21.2, 65.4, 113.9, 127.6, 128.2,

128.3, 133.3, 134.0, 134.1, 134.2, 134.5, 135.1, 135.5,

4.2.44. 5,20-Bis(4-ethylphenyl)-10,15-bis(4-carboxylato-

methoxyphenyl)-21,23-dithiaporphyrin (9). Core-modi-

ﬁed porphyrin 31 (0.35 g, 0.38 mmol) in 40 mL of

THF was treated with 40 mL of 1 M aqueous NaOH

as described for the preparation of 2 to give 0.25 g

1

(76%) of dithiaporphyrin 9; mp 245–247 ꢁC. H NMR

Y. You et al. / Bioorg. Med. Chem. 13 (2005) 2235–2251

2249

(500 MHz, 1:1 CDCl₃/CD₃OD):

d

1.51 (6H, t,

tained by transplantation in the abdominal region of

100–120 g Fischer female rats, using the sterile trochar

technique described earlier by Hilf et al.⁵¹R3230AC

cells were cultured from tumor homogenates using the

method described earlier.⁵²All cell lines were main-

tained in passage culture on 35 mm diameter polystyr-

ene dishes (Becton Dickinson, Franklin Lakes, NJ) in

3.0 mL of minimum essential medium (a-MEM) supple-

mented with 10% FBS, 50 units/mL penicillin G,

50 lg/mL streptomycin, and 1.0 lg/mL Fungizone

(complete medium). Only cells from passages 1–10 were

used for experiments and cells from passages 1–4, stored

at À86 ꢁC, were used to initiate cultures. Cultures were

maintained at 37 ꢁC in a 5% CO₂humidiﬁed atmosphere

(Forma Scientiﬁc, Marietta, OH). Passage was accom-

plished by removing the culture medium, adding a

1.0 mL solution containing 0.25% trypsin, incubating

at 37 ꢁC for 2–5 min to remove the cells from the surface

followed by seeding new culture dishes with an appro-

priate number of cells in 3.0 mL of a-MEM. Cell counts

were performed using a particle counter (model ZM,

Coulter Electronics, Hialeah, FL).

J = 7.5 Hz), 2.99 (4H, q, J = 8.0 Hz), 4.91 (4H, s), 7.41

(4H, d, J = 8.0 Hz), 7.66 (4H, d, J = 8.0 Hz), 8.11

(4H, d, J = 7.5 Hz), 8.14 (4H, d, J = 8.5 Hz), 8.64

(2H, d, J = 4.5 Hz), 8.66 (2H, d, J = 4.5 Hz), 9.70 (2H,

m), 9.71 (2H, s). High-resolution ESI MS: m/z

853.2401 (calcd for C₅₂H₄₀N₂O₆S₂+ H, 853.2394).

Anal. C, H, N.

4.2.45. 5,20-Bis(4-butylphenyl)-10,15-bis(4-carboxylato-

methoxyphenyl)-21,23-dithiaporphyrin (10). Core-modi-

ﬁed porphyrin 33 (0.35 g, 0.36 mmol) in 40 mL of

THF was treated with 40 mL of 1 M aqueous NaOH

as described for the preparation of 2 to give 0.27 g

(82%) of dithiaporphyrin 10; mp 250–252 ꢁC. ¹H

NMR (500 MHz, 1:1 CDCl₃/CD₃OD): d 1.07 (6H, t,

J = 7.0 Hz), 1.56–1.62 (4H, m), 1.87–1.94 (4H, m), 2.95

(6H, t, J = 7.5 Hz), 4.91 (4H, s), 7.43 (4H, d, J =

8.0 Hz), 7.66 (4H, d, J = 7.5 Hz), 8.14 (4H, d,

J = 7.5 Hz), 8.19 (4H, d, J = 8.0 Hz), 8.68 (2H, d,

J = 4.5 Hz), 8.70 (2H, d, J = 4.5 Hz), 9.75 (2H, s), 9.76

(2H, s). High-resolution ESI MS: m/z 909.3027 (calcd

for C₅₆H₄₈N₂O₆S₂+ H, 909.3037). Anal. C, H, N.

4.4.2. Incubation of cell cultures with dithiaporphyrins.

For experiments designed to determine the amount of

intracellular porphyrin after incubation with core-mod-

iﬁed porphyrins, R3230AC cells were seeded on 96 well

plates as above. Compounds 1–10 were added at the

appropriate concentrations in the complete medium

24 h after cell seeding. Cells were incubated at 37 ꢁC in

the dark for various periods, the medium removed,

monolayers washed once with 0.9% NaCl, and 200 lL

of a 25% solution of Scintigest were added to solubilize

the cells. The intracellular porphyrin content was deter-

mined using a ﬂuorescence multi-well plate reader

(Molecular Devices, Sunnyvale, CA) set at the appropri-

ate excitation and emission wavelengths. Intracellular

dye concentration was determined by comparing ﬂuo-

rescence values obtained from solubilized cells with

dye standards dissolved in 25% Scintigest. Data are ex-

pressed as fmol porphyrin/cell.

4.3. Photophysical properties

4.3.1. Determination of quantum yields for the generation

of singlet oxygen. The quantum yields for singlet oxygen

generation [/(¹O₂)] of 21,23-dithiaporphyrins, 2–10,

were measured by direct methods in MeOH in a manner

identical to the determination of /(¹O₂) for 1.^38,42

A

SPEX 270M spectrometer (Jobin Yvon) equipped with

InGaAs photodetector (Electrooptical Systems Inc.,

USA) was used for recording singlet oxygen emission

spectra. A diode-pumped solid state laser (Millenia X,

Spectra-Physics) at 532 nm was the excitation source.

The sample solution, in a quartz cuvette, was placed di-

rectly in front of the entrance slit of the spectrometer,

and the emission signal was collected at 90ꢁ relative to

the exciting laser beam. An additional long-pass ﬁlter

(850LP) was used to attenuate the excitation laser and

the ﬂuorescence from the photosensitizer.

For experiments designed to determine cell viability in

the presence of individual core-modiﬁed porphyrins

(1–10) in the dark or after light exposure, R3230AC

cells were seeded on 96 well plates at 1–1.5 · 10⁴cells/

well in complete medium. Cultures were then incubated

for 24 h after which the appropriate concentrations of

1–10 were added directly to the wells in the complete

medium.

4.3.2. Determination of quantum yields for ﬂuorescence.

The quantum yields for ﬂuorescence (/_F) of the core-

modiﬁed porphyrins were measured in MeOH as de-

scribed previously ³⁸and were compared to the standard

rhodamine 6G (/_F= 1.0).

4.3.3. Determination of octanol/water partition coeﬃ-

cients at pH 7.4. The octanol/water partition coeﬃcients

were determined at pH 7.4 using the absorbance of the

core-modiﬁed porphyrins. A ꢁshake ﬂaskꢀ direct mea-

surement⁵¹with 3–5 min mixing followed by 4 h settling

period was used. Equilibration and measurements were

made at 23 ꢁC using a Perkin–Elmer Lambda 12 spec-

4.4.3. Irradiation of cultured cells. Following incubation

of R3230AC cells with porphyrins, the medium was

removed, cells were washed once with 0.2 mL of 0.9%

NaCl and 0.2 mL of medium minus FBS, and phenol

red (clear medium) was added. Plates, with lids

removed, were positioned on a orbital shaker (LabLine,

Melrose Park, IL) and exposed for various times to

broadband visible light (350–750 nm) delivered at

1.4 mW cm^À2from a ﬁltered 750 W halogen source

defocused to encompass the whole 96 well plate. The

culture plates were gently orbited on the shaker in

order to ensure uniform illumination of all of the wells

trophotometer. Values are reported as logD_7.4

.

4.4. Photophysical properties

4.4.1. Cells and culture conditions. Cells cultured from

the rodent mammary adenocarcinoma (R3230AC) were

used for these studies. The R3230AC tumors were main-

2250

Y. You et al. / Bioorg. Med. Chem. 13 (2005) 2235–2251

on the plate. The clear medium was then removed,

0.2 mL of fresh complete medium was added and cul-

tures were incubated at 37 ꢁC for 24 h in the dark. Cell

monolayers were also maintained in the dark undergo-

ing the same medium changes and addition of dyes as

those that were irradiated. Cell counts, as above, were

performed on irradiated cells, cells maintained in the

dark or cells exposed to neither porphyrins nor light

(control cells). Cell viability, obtained for experimental

samples is expressed as the percent of control cell

counts.

1.0 h irradiation period. Aliquots (10 lL) were removed

at various times during irradiation for determination of

cytochrome c oxidase activity. A portion of the mito-

chondria/dye suspension was maintained in the dark,

and determinations of cytochrome c oxidase activity

were performed on 10 lL aliquots from these samples

as dark controls. Measurement of cytochrome c oxidase

activity was performed according to a method described

earlier.^32,48Initial enzyme activity was adjusted to ob-

tain a decrease in the reduced cytochrome c oxidase

absorbance at 550 nm of 0.4–0.6 OD units/min. Data

are expressed as the % of initial, preirradiation cyto-

chrome c oxidase activity.

4.4.4. Fluorescence microscopy with dithiaporphyrin 3.

R3230AC cells were seeded at 3–5 · 10⁵cells/dish in

35 mm diameter culture dishes containing sterilized

12 mm diameter cover slips in 3 mL complete medium

for 24 h at 37 ꢁC as described above. After the 24 h incu-

bation the medium was removed and fresh complete

medium containing dye 3 at 5 · 10⁶M was added for

24 h. Following the incubation period the medium

was removed, the dish was washed once with 0.9%

NaCl and 3 mL clear medium was added. The cover

slips with the cells attached were removed immediately

and mounted in a holder and images were captured

using a Nikon inverted ﬂuorescence microscope ﬁtted

with a Dage CCD 72 camera (Dage MTI, Michigan

City, IN), and a Genisys image intensiﬁer and Image

I/AT processing software (Universal Imaging, Media,

PA).

4.4.6. Measurement of cytochrome c oxidase activity in

cultured, whole cells. To determine whether dithiaporph-

yrin 3 aﬀected mitochondrial cytochrome c oxidase

activity in whole cells, thus taking into account cellular

uptake and distribution of the dyes, R3230AC cells were

plated on 12 well culture plates as described above until

they reached 4–7 · 10⁵cells/well, a number when the

cells were still in log phase growth. Stock solutions of

core-modiﬁed porphyrin 3, were diluted with sterilized

doubly distilled water to give appropriate concentra-

tions, and 50 lL of the diluted solutions was added to

the each well for ﬁnal concentrations ranging from 0

to 1.0 · 10^À7M. Cells were incubated for 24 h in the

dark at 37 ꢁC, the medium was removed, and the cells

were washed with 0.9% NaCl, and 1.0 mL of clear med-

ium was added. Monolayers were then exposed to ﬁl-

tered, 350–750 nm light from a 750 W tungsten source

(1.4 mW cm^À2) for a total ﬂuence of 5 J cm^À2. Immedi-

ately following irradiation, the cell number was deter-

mined by counting, cells were harvested and the

activity of cytochrome c oxidase was determined accord-

ing to the method described earlier.^38,48,53Data are ex-

pressed as DOD/min/10⁵cells at 550 nm and compared

to that determined from cells not exposed to dye or

light.

4.4.5. Preparation of mitochondrial suspensions and

measurement of cytochrome c oxidase activity. Prepara-

tion of isolated mitochondrial suspensions from homog-

enized rat liver followed a method described earlier.⁴⁸

Mitochondrial suspensions were divided into 0.5 mL ali-

quots (6–10 mg of mitochondrial protein/mL) and

stored at À86 ꢁC until used for experiments.

Mitochondrial suspensions were removed from storage

and thawed on ice. Stock solutions, 2 · 10^À3M, of 3

or 4 were prepared in DMSO and diluted with phos-

phate buﬀer (pH 7.4) to give 1 · 10^À3M. Dyes 3 or 4

were added to 1 mL of mitochondrial suspension at a

ﬁnal concentration of 5 · 10^À6M and allowed to incu-

bate in the dark on ice for 5 min. Dye containing suspen-

sions were then centrifuged at 8000g for 3 min using an

Eppendorf microcentrifuge (Model 3200, Brinkmann

Ind., Westbuty, NY), the supernatant was discarded

and the pellet was resuspended in 1.0 mL of mitochon-

drial preparation buﬀer containing 0.33 M sucrose,

1.0 mM dithiothreitol, 1.0 mM EDTA, 0.3% BSA, and

100 mM KCl (pH 7.1). The suspension was then trans-

ferred to a 3.0 mL quartz cuvette, which was positioned

in a focused, 1.0 cm diameter, ﬁltered (530–750 nm) light

beam emitted from a 750 W tungsten source. The inten-

sity of the beam was uniform over the wavelength

band used and was delivered at a ﬂuence rate of

100 mW cm^À2. Beam intensity was measured using a

radiometer (Model 210, Coherent Inc., Palo Alto,

CA). The light was cooled by passing it through a water

ﬁlter, eliminating thermal eﬀects as the sample tempera-

ture did not rise above 25 ꢁC. The mitochondrial suspen-

sions were magnetically stirred continuously during the

4.4.7. Statistical analyses. All statistical analyses were

performed using the Studentꢀs t-test for pairwise com-

parisons. A P value of <0.05 was considered signiﬁcant.

Acknowledgements

We thank Dr. Patricia Hinkle of the Department of

Pharmacology and Physiology at the University of

Rochester for help in obtaining the ﬂuorescence images.

This research was supported by the Department of

Defense [Breast Cancer Research Program] under

award number (W81XWH-04-1-0500). Views and opin-

ions of, and endorsements by the author(s) do not

reﬂect those of the US Army or the Department of

Defense.

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Article Doi

Core-modified porphyrins. Part 4: Steric effects on photophysical and biological properties in vitro

DOI: 10.1016/j.bmc.2004.12.048

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