Syntheses, properties and cellular studies of metallo-isoporphyrins

Journal of Porphyrins and Phthalocyanines

J. Porphyrins Phthalocyanines 2011; 15: 918–929

DOI: 10.1142/S108842461100380X

Published at http://www.worldscinet.com/jpp/

Syntheses, properties and cellular studies of metallo-

isoporphyrins

Sandra C. Mwakwari, Haijun Wang,Timothy J. Jensen, M. Graça H. Vicente^and

Kevin M. Smith*^

Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803, USA

Dedicated to Professor Karl M. Kadish on the occasion of his 65^thbirthday

Received 8 May 2011

Accepted 3 June 2011

ABStrAct: b-Bilene hydrochlorides are shown to be improved intermediates for the synthesis of

metallo-isoporphyrins in enhanced yields (28% vs. 6%). Several new diamagnetic zinc(II) and a

novel paramagnetic copper(II) isoporphyrin salts were also obtained using this approach. Metal-free

isoporphyrins were also isolated. In vitro studies using human carcinoma HEp2 cells show that all

metallo-isoporphyrins accumulate within cells and localize partially in the mitochondria. The zinc-

isoporphyrins were found to be moderately phototoxic while the copper complex showed the lowest

phototoxicity, maybe as a result of its paramagnetic nature.

KEYWordS: intracellular localization, metalloisoporphyrins, porphyrins, synthesis, toxicity.

H

IntroductIon

Isoporphyrins (1) are tautomers of porphyrins (2)

which exhibit interrupted macrocyclic conjugation due

to the presence of a saturated (sp³) bridging carbon atom.

They contain three sp²and one sp³hybridized meso car-

bons and one NH. The existence of this type of compound

was ﬁrst suggested by Woodward [1] in 1961, whose pre-

diction was conﬁrmed about a decade later when Dolphin

et al. [2] reported the ﬁrst synthesis of a metalloisoporphy-

rin (4) from zinc 5,10,15,20-tetraphenylporphyrin by way

of electrochemical oxidation. Other methods for directly

generating metallo-isoporphyrins since then include per-

oxide oxidation [3, 4] and photooxidation [5–8].

Due to their absorption at long wavelengths (~800 nm),

metallo-isoporphyrins are potential candidates as pho-

tosensitizers in photodynamic therapy (PDT), a ternary

modality for cancer treatment. At long wavelengths, light

penetrates deeper through tissue, making it possible to

treat large or deep-seated tumors. Metallo-isoporphyrins

are also of biological interest due to their unique redox

N

NH

N

HN

1

2

properties among porphyrin derivatives with potential as

a new class of near-IR dyes [4, 9–13].

All early examples of metallo-isoporphyrins were

derived from tetraphenylporphyrin metal complexes, and

they are usually not stable with regard to reconversion

into the corresponding fully conjugated porphyrins [2, 4].

This problem was overcome when Smith and co-workers

[14, 15] reported the total synthesis of the ﬁrst thermody-

namically stable zinc isoporphyrin bearing a geminal-di-

methyl meso-motif; it was prepared using the MacDonald

“2+2” method [16]. The availability of this stable zinc

isoporphyrin immediately initiated a series of studies on

this compound, including electrochemical [17], crystal-

lographic [18], and photophysical investigations [19].

However, using the MacDonald approach, zinc iso-

porphyrin products were isolated in low yields. Herein,

^SPP full member in good standing

*Correspondence to: Kevin M. Smith, email: kmsmith@lsu.

edu, tel:+225-578-7442, fax: +225-578-3458

SyntheSeS, PrOPertIeS anD Cellular StuDIeS Of metallO-ISOPOrPhyrInS

919

we report more efﬁcient syntheses of metallo-isoporphy-

Synthesis of metal complexes other than zinc(II)

rins from b-bilene salts, and studies of their chemical,

physical and biological properties to probe the potential

for applicability in photodynamic therapy.

To date, only the zinc(II) isoporphyrin complexes and

metal-free derivatives have been isolated [14, 15], and

the latter only with difﬁculty [15]. We are now able to

synthesize a copper(II) isoporphyrin in 23% yield; meta-

lation of the metal free isoporphyrin intermediate with

cuprous chloride yielded a novel copper isoporphyrin (7)

(Scheme 1) with an observed bathochromic shift of both

the Soret and the Q-band in its optical spectrum (Fig. 1)

to 428 and 842 nm (compared with zinc isoporphyrin

at 416 and 804 nm). Since the copper(I) salt was used,

rESultS And dIScuSSIon

Synthesis of zinc(II) isoporphyrin (6)

The synthetic route investigated involves cyclization of

open chain tetrapyrrole intermediates, notably b-bilenes,

by ring closure using a suitable carbon-linking unit as the

ﬁnal step.

The precursor b-bilene hydrochloride (5) was synthe-

sized by condensation of dipyrromethane-1-carboxylic

acid (3) [20] with 1-formyldipyrromethane (4) to yield

crystalline b-bilene hydrochloride in 70% yield; in initial

studies this was followed by cyclization using 2,2-dime-

thoxypropane as an acetone surrogate and carbon linking

unit, then addition of zinc salt to afford the zinc isopor-

phyrin cation (6) in 28% yield (Scheme 1). These yields

were higher than previously reported (6%) [14, 15] and

the reaction time was dramatically reduced from six days

to one day. Other ketones, for example cyclohexanone or

the 1,1-dimethoxycyclohexane, proved too bulky for the

cyclization reaction.

Fig. 1. UV-visible spectrum of copper isoporphyrin (7) in

dichloromethane

Me

CO₂t-Bu

t-BuO₂C

CO₂t-Bu

t-BuO₂C

HN

OHC

1. pTsOH, CH₂Cl₂

50 min

2. HCl gas, 10 s

Me

NH

CO₂H

Me

NH

HN

+

-

Cl

HN

NH

Me

3

4

5

1. TFA

2. Zn(OAc)₂,

O

Me

Me Me

Me

O

Me

24 h, rt

N

HN

Me

CH₂Cl₂, CuCl

30 min, rt

CH₂Cl₂, Zn(OAc)₂

30 min, rt

Me

Me Me

Me

Me Me

Me

N

+

Zn

-

+

Cu

-

Cl

N

Me

6

7

Scheme 1. Syntheses of zinc (6) and copper (7) isoporphyrin salts via b-bilene (5)

J. Porphyrins Phthalocyanines 2011; 15: 919–929

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S.C. mWakWarI et al.

Fig. 2. WinEPR spectrum of copper(II) isoporphyrin (7) in dichloromethane. Frequency: 9.634 GHz, Mod. Frequency: 100.00 kHz,

Power: 20.170 mW, Mod. Amplitude: 4.00 G, Temperature: 295 K

formation of a neutral copper(I) isoporphyrin complex

was expected, but qualitative EPR studies (Fig. 2) con-

ﬁrmed the presence of a paramagnetic copper(II) species,

a copper(II) cationic isoporphyrin complex with a chlo-

the already developed b-bilene synthetic route, the metal-

free compound was directly synthesized by cyclization of

b-bilene hydrochloride (5) in the presence of zinc acetate

with 2,2-dimethoxypropane as the carbon-linking unit at

room temperature in the presence of air. After 24 h, the

UV-visible spectrum of the reaction mixture showed

absorption peaks at 420 and 700 nm, characteristic of

metal-free isoporphyrin. Attempts to purify and charac-

terize the compound remained challenging because of

product decomposition. Being aware of previous reports

of decomposition in the presence of sodium bicarbonate,

working under basic conditions was avoided, and suc-

cessful puriﬁcation of the compound was performed on

an alumina chromatography column using acid treated

solvents for elution. It was discovered that the metal-free

complex was relatively stable under acidic conditions.

Scheme 2 shows the synthesis of the protonated metal-

free complex (8) obtained as a green compound in 23%

yield, with absorptions at 432 nm and 700 nm (Fig. 3).

Mass spectrometry conﬁrmed the formation of the metal-

free product (8) but, as had previously been observed

[15], ¹H NMR spectroscopy showed all expected signals

except for the 5,5-dimethyl protons (expected to appear

between 1–2 ppm) plus a strong (impurity?) signal at 1.26

ppm which did not integrate to the expected 6-methyl

protons. The missing signal was assumed [15] to be

obscured beneath the impurity peak at 1.26 ppm. The

1

ride counterion. As expected, H NMR spectroscopy of

this compound in CDCl₃showed no signals for the mac-

rocycle; the product was therefore characterized using

spectrophotometry and low resolution/high resolution

mass spectrometry. Though the analysis conﬁrmed that

the metal complex was copper(II), attempts to synthe-

size the copper isoporphyrin using copper(II) salts were

unsuccessful. In addition, attempts to obtain other metal

complexes using CuCl₂, Ni(acac)₂, FeCl₂, AgCl, and

CdCl₂salts, failed.

Synthesis and characterization of metal-free isopor-

phyrins

It is known that metal-free isoporphyrins are not very

stable [15], and therefore no such isoporphyrins have ever

been fully isolated and characterized. Two approaches

have previously been attempted to obtain a metal-free

isoporphyrin: (i) direct synthesis utilizing a variation of

the MacDonald 2+2 procedure, and (ii) demetalation of

stable zinc isoporphyrins [14, 15]. However, the expected

metal-freeisoporphyrincouldnotbeisolatedandalthough

demetalation was successful, following several trials the

product was unstable and decomposed readily. Utilizing

CD₃

Me

CD₃

Me

1. TFA, 10 min, Ar

1. TFA-d, 10 min, Ar

2. CH₂Cl₂, Zn(OAc)₂

N

5

2. CH₂Cl₂, Zn(OAc)₂

O

HN

D₃C

O

D₃C

O

Me

rt, air, 24 h

9

8

Scheme 2. Synthesis of metal-free isoporphyrin (8) and deuterium labeled metal-free isoporphyrin (9)

J. Porphyrins Phthalocyanines 2011; 15: 920–929

SyntheSeS, PrOPertIeS anD Cellular StuDIeS Of metallO-ISOPOrPhyrInS

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DEPT 135, for non-deuterated metal-free isoporphyrin.

On the other hand, deuterium labeled metal-free isopor-

phyrin would be expected to show positive signals for

2-CH carbons in DEPT 90, and positive 2-CH carbons

and 4-CH₃carbons in DEPT 135, due to deuterium label-

ing of the 5,5-dimethyl motif. Both samples were dis-

solved in CDCl₃and four different experiments were run

overnight to obtain results which complied precisely with

the theoretical speculations/predictions. These results

conﬁrmed the location of the 5,5-dimethyl signal at 1.70

ppm in acetone and 1.26 ppm in chloroform.

Synthesis of meso-monosubstituted porphyrins via

isoporphyrins

Fig. 3. UV-visible spectrum of protonated metal-free isopor-

phyrin (8) in dichloromethane

Inanattempttofurtherimprovetheyieldoftheb-bilene

cyclization step to give isoporphyrins, linking carbon

units other than those related to acetone were investi-

gated. Cyclization of b-bilene salts with α-ketoesters

and α-diketones afforded new isoporphyrin macrocycles.

The isoporphyrins from the α-ketoesters (e.g. 10d) were

subsequently transformed with KOH in methanol into

the corresponding zinc(II) meso-monosubstituted por-

phyrins, and after demetalation into the meso-substituted

free-base porphyrin (11d; Scheme 3). Figure 4 shows

the absorption spectral changes as zinc(II) isoporphy-

rin (10d) was transformed via the zinc(II) complex into

metal-free meso-substituted porphyrin (11d).

During the macrocyclization process (Scheme 3),

a novel transformation from b-bilene through a,b-biladiene

to a,c-biladiene was observed; for a preliminary publica-

tion of this work see [22]. Use of these new α-di-carbonyl

linking units resulted in signiﬁcant improvements in the

yields of isoporphyrins obtained.

missing signal was also initially associated with some

unusual dynamic processes which was ruled out after per-

forming a variable-temperature ¹H NMR study. To locate

the missing NMR signal, a deuterium labeling strategy of

the 5,5-dimethyl substituents by chemical synthesis was

employed. This was achieved by cyclizing the b-bilene

hydrochloride (5) with acetone-d₆as the carbon-linking

unit to introduce deuterium into the macrocycle (Scheme

2). It was necessary to ensure that the reaction condi-

tions did not allow D/H isotopic exchange by carrying

out the synthesis using TFA-d and acetone-d₆. The reac-

tion was followed by spectrophotometry and after 24 h

the characteristic absorption peak at 700 nm was at its

maximum. Puriﬁcation on neutral alumina and silica gel

under acidic conditions provided the green target deuteri-

um-labeled compound (9). Using ¹H NMR spectroscopy

of this sample in chloroform-d the signal at 1.26 ppm was

still present, with the same intensity as in the spectrum of

compound (8).

Table 1 shows the various α-ketoesters (entries a–f)

and the α-diketone (entry g) used in the b-bilene cycli-

zation reactions to afford the corresponding zinc isopor-

phyrin cation complexes in yields (33–56%) signiﬁcantly

higher than previously reported [14, 15]. The reactions

were carried out at room temperature and were complete

Solvent stability studies were performed [21] on the

deuterated product and the NMR studies were carried out

in a different solvent hoping to obtain better solubility

and resolution. The solvent of choice was acetone-d₆con-

taining a trace of TFA-d to stabilize the metal-free isopo-

rphyrin (8). In this solvent, a signal at ~1.7 ppm which

integrated to 6-H was observed, this corresponding to

the 5,5-dimethyl protons. To conﬁrm the identity of this

signal, the deuterium labeled metal-free isoporphyrin (9)

1

was analyzed under the same conditions by H NMR in

acetone-d₆and the signal was absent.Another observation

made was the reduced intensity of the signal at 1.26 ppm

in acetone-d₆compared with chloroform-d, possibly due

to better solubility/reduced aggregation in the acetone/

TFA mixture and stability of the metal-free macrocycle

in this solvent mixture. To further verify the structure of

metal-free isoporphyrin and to conﬁrm presence and loca-

tion of the 5,5-dimethyl protons, more sensitive ¹³C NMR

techniques (DEPT 135 and DEPT 90) were employed.

The metal-free macrocycle, which has symmetry through

the 5,15-meso-positions, would be expected to yield

positive signals corresponding to 2-CH carbons in DEPT

90, while positive 2-CH carbons and 5-CH₃carbons in

Fig. 4. Electronic absorption spectra in dichloromethane of: (A)

zinc isoporphyrin (10d) before (dashed line) and (B) after (full

line) after transformation to zinc meso-monosubstituted por-

phyrin (11d), and then (C) after demetalation (dotted line)

J. Porphyrins Phthalocyanines 2011; 15: 921–929

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S.C. mWakWarI et al.

R²

O

R¹

Me

R¹

Me

Cl

R³

N

NH

N

1. TFA

1. KOH, MeOH

2. TFA

5

Zn

O

2.

N

HN

R²

Me

R¹

Me

O

Me

3. CH₂Cl₂, Zn(OAc)₂,

DDQ or air

10

11

d: R¹= Ph; R³= Me

a: R¹= R³= Me; R²= OMe

b: R¹= R³= Me; R²= OEt

d: R¹= Ph; R²= OEt; R³= Me

e: R¹= i-Bu; R²= OEt; R³= Me

g: R¹= R²= R³= Me

Scheme 3. Cyclization of b-bilene to isoporphyrins (10) followed by transformation to meso-monosubstituted porphyrins (11)

table 1. Cyclization of b-bilene (5) with various 1,2-diketones

to yield zinc isoporphyrin (10)

reaction. It was discovered that when KOH was dissolved

in water the reaction did not proceed as expected. The

reaction occurred at room temperature and formation of

the product was monitored by spectrophotometry, moni-

toring the Soret band of the product (around 400 nm)

until it reached its maximum intensity. Puriﬁcation was

performed on an alumina (Grade III) column. The driv-

ing force for the decarboxylation reaction is the con-

siderable thermodynamic stabilization gained upon the

formation of a fully conjugated isomer (porphyrin 11)

in comparison to zinc isoporphyrin (10) that exhibits

an interrupted macrocyclic conjugation owing to the

presence of a sp³-hybridized meso carbon. Subsequent

attempts to functionalize/conjugate the geminal ester

function (for example with amino acids), via the alkyl

ester, have been thwarted by this facile decarboxylation

after hydrolysis, and attempts to circumvent this process

are in progress.

Owing to the success of the methodology whereby

zinc metal was incorporated into the intermediate before

cyclization and oxidation to zinc isoporphyrin, it was

speculated that incorporation of other transition met-

als may allow for the isolation of metallo-isoporphyrins

that may otherwise be inaccessible by direct metalation

of metal-free macrocycles. Several transition metal salts

including CuCl, CuCl₂, Ni(acac)₂, Fe(II), Co(II), Ag(I),

Cd(II), were incorporated into the intermediate followed

by addition of DDQ at room temperature with no success-

ful cyclization to the corresponding metallo-isoporphyrin.

Mostly, decomposed products were obtained (monitored

by UV-vis spectroscopy).

Cmp

R¹

R²

Reaction time,

min

% yield (10)

10a

10b

10c

10d

10e

10f

Me

CF₃

Ph

OMe

OEt

OMe

OEt

Me

10

—

30

60

—

10

55

54

0

35

33

0

i-Bu

i-Pr

Me

10g

56

in about one hour depending upon the carbonyl sub-

strate. The high electrophilicity of the carbonyl on

the α-ketoesters and α-diketone substrates compared

with simple ketones (such as acetone) facilitated the

enhanced rate of the reaction. The results indicate that

the more bulky is the substrate the lower is the yields

of isoporphyrin. Surprisingly (Table 1, entry c), no

cyclization occurred with methyl 3,3,3-triﬂuorometh-

ylpyruvate to give zinc isoporphyrin, despite the highly

electrophilic character of the carbonyl due to the pres-

ence of the electronegative ﬂuorine atoms. For entry

(f), steric factors may have played a role in the lack of

cyclization.

Having successfully synthesized a library of target

zinc(II) isoporphyrins (10), their potential as intermedi-

ates in the synthesis of meso-substituted porphyrins was

studied. Saponiﬁcation of the 5-alkyl ester substituent

to give carboxylic acid followed by decarboxylation on

the sp³-hybridized meso carbon led to a rapid transfor-

mation of the cationic complex into the corresponding

zinc(II) meso-monosubstituted porphyrin, which was

then demetalated to give 11d in 32% yield, (Scheme 3).

10% KOH dissolved in dry methanol was used for this

cellular studies

Figure 5 shows the results of time dependent cellular

uptake of zinc-isoporphyrins 6, 10a, 10b, 10d, and 10e,

investigated in HEp2 cells at a concentration of 20 μM

over a time period of 24 h. All the compounds exhib-

ited a rapid uptake within the ﬁrst 2–4 h. Compounds

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923

isoporphyrins generally showed higher cytotoxicities,

especially upon light activation (~1 J/cm²light dose);

the most phototoxic was zinc-isoporphyrin 10e, prob-

ably as a consequence of its high cellular uptake (Fig.

5) and multiple sites of intracellular localization (Table

2). These results reﬂect the profound effect of the nature

of the metal ion on the macrocycle. It is to be expected

that the zinc-isoporphyrins will be more efﬁcient than the

copper complexes at generating singlet oxygen upon light

activation, due to the paramagnetic nature of the d⁹Cu(II)

ion compared with the diamagnetic d¹⁰Zn(II) complexes.

Observation of PDT activity in copper tetrapyrroles is

unusual but not unique [23, 24].

The intracellular localizations of zinc-isoporphy-

Fig. 5. Time-dependent cellular uptake of compounds 6 (brown

rins (6, 7, 10a, 10b, 10d, and 10e) were investigated

in HEp2 cells using ﬂuorescence microscopy. The

organelle-speciﬁc ﬂuorescent probes ERtracker Green

(ER), Lysotraker Green (lysosomes), Mitotracker Green

(mitochondria) and BODIPY Ceramide (golgi) were

used in overlay experiments. The results obtained are

shown in Figs 6–10 and Table 2. Our results indicate

that all metallo-isoporphyrins localize to some extent in

cell mitochondria, a critically important organelle and

PDT target [25]. Such mitochondrial localization might

be favored for these hydrophobic and cationic mol-

ecules for reasons that have been previously observed

[25, 26]. Cationic compounds tend to accumulate in the

mitochondria in part due to the highly negative electro-

chemical potential of the inner mitochondrial system.

Mitochondrial localization is important because por-

phyrin-induced apoptosis in tumors has been correlated

with mitochondrial photodamage, and usually occurs

rapidly as a result of a cascade-like cell killing process,

leading to a rapid loss of treated tissue [27, 28]. On the

other hand, the most phototoxic compounds were those

that were found in multiple organelles, in addition to

mitochondria, speciﬁcally in Golgi, lysosomes and ER.

Photodamage to organelles other than mitochondria has

also been shown to lead to apoptotic pathways [29];

therefore photodamage to multiple organelles might

trigger several apoptotic pathways that induce effective

cell destruction.

open square), 10a (black square), 10b (red triangle), 10d (green

inverted triangle), and 10e (blue diamond), in HEp2 cells at

20 µM, for 24 h

6 and 10d reached a plateau after 4 or 8 h, respectively,

while all other compounds continued to accumulate

within cells during the 24 h period investigated. Among

the zinc-isoporphyrins studied, compounds 10d and 10e

accumulated the most within cells at all time points, and

after 24 h the amount of 10e found in cells was twice that

found in 10a and about 5-fold that of 6. It is interesting

to note the signiﬁcant differences observed in the cel-

lular uptake of this series of structurally similar isopo-

rphyrins; in particular, the presence of a bulky group at

the tetrahedron carbon, such as an isobutyl (as in 10e)

or phenyl (as in 10d) rather than a methyl as in all other

compounds, enhanced cellular uptake, probably due to

decreased aggregation.

The dark and phototoxicity assays of isoporphyrins 6,

7, 10a, 10b, 10d, and 10e, in HEp2 cells at increasing

concentrations of up to 100 μM were evaluated and the

results obtained are summarized in Table 2. All metallo-

isoporphyrins, especially the copper isoporphyrin 7,

showed low dark toxicities. The IC₅₀value, which deﬁnes

50% cell viability, for the dark toxicity of the copper

isoporphyrin was the highest found among all isopor-

phyrins, 85 μM (Table 2). On the other hand, the zinc

table 2. Dark toxicity, phototoxicity, and intracellular localization of metallo-isoporphyrins

Metal ion [M]²⁺

5,5-geminal

R³

Dark toxicity

IC₅₀, μM

Phototoxicity

IC₅₀, μM

Intracellular

localization

disubstituents R¹, R²

6

Zn

Cu

Zn

Me, Me

Me

a

85^a

95^a

≥ 20^b

15^b

35

85

M, L

7

M, L

10a

10b

10d

10e

a,b

Me, CO₂Me

Me, CO₂Et

Ph, CO₂Et

iBu, CO₂Et

17

G, M, L, ER

G, M, L ER

L, M, ER

G, L, M, ER

19

≥20

11

b

Experiment carried out at concentrations up to: 100 μM and 20 μM. ER = endoplasmic reticulum;

G = Golgi; L = lysosomes; M = mitochondria.

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S.C. mWakWarI et al.

Fig. 6. Intracellular localization of zinc isoporphyrin (6) at

10 μM in HEp2 cells. (a) phase contrast; (b) overlay of phase

contrast with ﬂuorescence of zinc isoporphyrin, (6); (c, e, g and

i) show the ﬂuorescence of BODIPY Ceramide, Lysotracker

Green, Mitotracker Green, and ER-Tracker Green, organ-

elle tracers for the Golgi apparatus, lysosomes, mitochondria,

and endoplasmic reticulum, respectively; (d, f, h and j) show

the overlay of the ﬂuorescence of zinc isoporphyrin (6) and the

organelle tracers

Fig. 7. Intracellular localization of zinc isoporphyrin (10a) at

10 μM in HEp2 cells. (a) phase contrast; (b) overlay of phase

contrast with ﬂuorescence of zinc isoporphyrin, (10a); (c, e, g

and i) show the ﬂuorescence of BODIPY Ceramide, Lysotracker

Green, Mitotracker Green, and ER-tracker Green, organelle

tracers for the Golgi apparatus, lysosomes, mitochondria, and

endoplasmic reticulum, respectively; (d, f, h and j) show the

overlay of the ﬂuorescence of zinc isoporphyrin (10a) and the

organelle tracers

deactivated with 6% water (grade III) or non-deactivated

(grade 0) and Merck silica gel 60 (70–230 mesh) were

used for column chromatography. ¹H NMR spectra were

obtained on a Bruker DPX-250, Bruker ARX-300, and

Bruker DPX-400 spectrometers. Chemical shifts (δ) are

given in ppm. Electronic absorption spectra were mea-

sured on PerkinElmer Lambda 35 UV/VIS Spectrometer.

Low and high resolution mass spectra were obtained on

a Bruker ProFlex III MALDI-TOF and Hitachi M8000

ESI mass spectrometer. The compounds were dissolved

in dichloromethane or chloroform using dithranol as the

ExpErImEntAl

materials

All commercially available starting materials were

used without further puriﬁcation. Solvents were puriﬁed

and dried from a specially designed solvent puriﬁcation

system from Innnovative Technology, Inc. Analytical TLC

using Sorbent Technologies 200 μm silica gel or alumina

neutral plates with UV 254 was used to monitor all reac-

tions. E. Merck neutral alumina (70–230 mesh) either

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SyntheSeS, PrOPertIeS anD Cellular StuDIeS Of metallO-ISOPOrPhyrInS

925

Fig. 9. Intracellular localization of zinc isoporphyrin (10d) at

10 μM in HEp2 cells. (a) phase contrast; (b) overlay of phase

contrast with ﬂuorescence of zinc isoporphyrin, (10d); (c, e, g

and i) show the ﬂuorescence of BODIPY Ceramide, Lysotracker

Green, Mitotracker Green, and ER-tracker Green, organelle

tracers for the Golgi apparatus, lysosomes, mitochondria, and

endoplasmic reticulum, respectively; (d, f, h and j) show the

overlay of the ﬂuorescence of zinc isoporphyrin (10d) and the

organelle tracers

Fig. 8. Intracellular localization of zinc isoporphyrin (10b) at

10 μM in HEp2 cells. (a) phase contrast; (b) overlay of phase

contrast with ﬂuorescence of zinc isoporphyrin, (10b); (c, e, g

and i) show the ﬂuorescence of BODIPY Ceramide, Lysotracker

Green, Mitotracker Green, and ER-tracker Green, organelle

tracers for the Golgi apparatus, lysosomes, mitochondria, and

endoplasmic reticulum, respectively; (d, f, h and j) show the

overlay of the ﬂuorescence of zinc isoporphyrin (10b) and the

organelle tracers

matrix and in acetonitrile for HR-ESI. The EPR data

were acquired on Bruker WinEPR.

two portions to the solution and stirring was continued

for 2 h, after which TLC showed no starting material

and the UV-visible spectrum showed a strong absorp-

tion at 502 nm. The dark red solution was washed

with 5% sodium carbonate solution and water and

dried over magnesium sulphate. Evaporation of sol-

vent under reduced pressure afforded the tetrapyr-

rolic intermediate (b-bilene). The dark residue was

then dissolved in 5 mL dichloromethane and hydro-

gen chloride gas was bubbled through the yellowish-

orange solution for 10 s; the color changed to dark

Syntheses

di-tert-butyl 2,3,7,8,12,13,17,18-octamethyl-b-bilene

hydrochloride (5). 50 mg (0.14 mmol) of dipyr-

romethane monocarboxylic acid (15) and formyldipyr-

romethane (16) (40 mg, 0.12 mmol) were dissolved in

10 mL of dry dichloromethane and stirred under argon.

p-toluenesulfonic acid (53 mg, 2 equiv.) was added in

J. Porphyrins Phthalocyanines 2011; 15: 925–929

926

S.C. mWakWarI et al.

1.55 (t-butyl, 18H). HRMS (MALDI-TOF): calcd. for

C₃₇H₅₀N₄O₄614.3827, found m/z 614.3832 [M]⁺.

Zinc(II) 2,3,5,5,7,8,12,13,17,18-decamethylisopor-

phyrin chloride (6). Without further puriﬁcation, the

bilene (5) (50 mg, 0.08 mmol) was dissolved in 0.2 mL

of cold TFA in a round bottomed ﬂask and stirred for

10 min under argon. The mixture was diluted with dry

dichloromethane followed by addition of zinc(II) acetate

(50 mg) dissolved in dry methanol (2 mL), and excess

2,2-dimethoxy propane. The mixture was left to stir in air

for 28 h. TLC and UV-visible showed formation of prod-

uct. The electronic absorption spectrum showed peaks

at around 440 and 690 nm characteristic of metal-free

isoporphyrin. Work-up was carried out by washing with

water twice, drying over anhydrous Na₂SO₄and evaporat-

ing the solvent. The residue was immediately dissolved

in dichloromethane and zinc(II) acetate in methanol was

added. After 15 min, the absorption spectrum showed

successful insertion of zinc ions with absorptions red

shifted to 810 nm. The product was chromatographed on

a silica gel column, eluting with 1–3% methanol/dichlo-

romethane. The appropriate fractions were collected and

the solvent was removed. The product was dissolved in

dichloromethane, washed with saturated sodium chloride

solution and dried over Na₂SO₄. Recrystallization using

dichloromethane/petroleum ether afforded the product as

a green solid (12 mg, 28%). mp > 300 °C (dec); Lit[15].

>300 °C (dec.). UV-vis (CH₂Cl₂): λ_max, nm (ε × 10³,

1

M^-1.cm^-1) 420 (48.9), 807 (47.5). H NMR (CDCl₃, 300

MHz): δ, ppm 7.70 (s, meso-H, 1H), 7.60 (s, meso-H,

2H), 2.47, 2.43 (s, β-CH₃, 24H), 1.96 (s, 5-CH₃, 6H).

HRMS (MALDI-TOF): calcd. for C₃₀H₃₃N₄Zn 513.1996,

found m/z 513.1990 [M]⁺. MS (MALDI-TOF): calcd.

514.999, found m/z 514.868 [M]⁺(dithranol).

copper(II) 2,3,5,5,7,8,12,13,17,18-decamethyliso-

porphyrin chloride (7). In a round-bottomed ﬂask

equipped with a stirrer under argon, was added 50 mg

(0.08 mmol) of b-bilene (5) and 0.2 mL of cold TFA. The

mixture was left to stir for 10 min, after which dry dichlo-

romethane (20 mL) was added followed by an excess of

zinc(II) acetate dissolved in dry methanol, then excess

2,2-dimethoxypropane. The reaction mixture was left to

stir in air for 24 h. UV-visible (abs. at 700 nm) and TLC

on alumina indicated completion of reaction. Excess TFA

and dichloromethane were removed by evaporation and

the mixture was puriﬁed on a column of neutral alumina

using acidic solution of chloroform as eluant (two drops

of TFA were added to 200 mL of chloroform) to collect

a fraction of mixed isoporphyrin and porphyrin. A sec-

ond column using silica gel and chloroform/ethylacetate

4:1 and a few drops of TFA was performed to separate

the isoporphyrin from the porphyrin. The pure isopor-

phyrin was metalated using excess cuprous chloride in

chloroform at room temperature for 2 h. The mixture

was ﬁltered through a Celite plug, and the product was

crystallized from chloroform/petroleum ether to give

the title compound in 23% yield (10 mg). mp > 300 °C

Fig. 10. Intracellular localization of zinc isoporphyrin (10e) at

10 μM in HEp2 cells. (a) phase contrast; (b) overlay of phase

contrast with ﬂuorescence of zinc isoporphyrin, (10e); (c, e, g

and i) show the ﬂuorescence of BODIPY Ceramide, Lysotracker

Green, Mitotracker Green, and ER-tracker Green, organelle

tracers for the Golgi apparatus, lysosomes, mitochondria, and

endoplasmic reticulum, respectively; (d, f, h and j) show the

overlay of the ﬂuorescence of zinc isoporphyrin (10e) and the

organelle tracers

red, forming the hydrochloride salt. Immediately, the

solvent was evaporated and the residue was taken up

twice in dry toluene and evaporated in order to remove

any traces of water and HCl. The residue was recrystal-

lized from DCM/hexane and left in the freezer over-

night. Filtration of solvent yielded orange-red prisms

of the b-bilene hydrochloride (5) (54 mg, 70%). mp >

300 °C (dec). UV-vis (CH₂Cl₂): λ_max, nm (ε × 10⁵,

1

M^-1cm^-1) 502 (1.11). H NMR (CDCl₃, 250 MHz): δ,

ppm 13.8 (br, NH⁺, 2H), 10.4 (br, NH, 2H), 7.08 (1H),

4.2 (CH₂, 4H), 2.23, 2.18, 2.04, 2.02, (each CH₃, 6H),

J. Porphyrins Phthalocyanines 2011; 15: 926–929

SyntheSeS, PrOPertIeS anD Cellular StuDIeS Of metallO-ISOPOrPhyrInS

927

(dec). UV-vis (CH₂Cl₂): λ_max, nm (ε × 10³, M^-1.cm^-1) 428

under argon for 10 min. The mixture was diluted with

dry dichloromethane (20 mL) followed by addition of

α-ketoester (1 equiv. of methyl, ethyl pyruvate or 1,2-

diketone, and excess of phenyl or isobutyl pyruvates). The

mixture was left to stir under argon for 1 h after which

the UV-visible spectrum showed no starting material,

but instead a new product absorbing at 450 and 520 nm.

Excess TFA was removed by washing with aqueous

Na₂CO₃(product color changed from reddish to green).

The UV-visible absorption for the green product showed

peaks at 430 and 790 nm. Zn(OAc)₂(20 mg) dissolved

in 1 mL of dry methanol was added to the green prod-

uct in dry dichloromethane (20 mL) and stirred under

argon. The reaction mixture immediately changed color

to reddish, and after stirring for 5 min the UV-visible

spectrum indicated new absorptions at around 470 and

540 nm. 50 mg (0.22 mmol) of DDQ dissolved in dry

dichloromethane (0.3 mL) was added to oxidize the

product. After 15 min, the UV-visible spectrum of the

mixture showed absorptions at 430 and 840 nm, sug-

gesting formation of a zinc isoporphyrin. The mixture

was washed with water, then brine, and dried over

anhydrous Na₂SO₄. Puriﬁcation on an alumina column

(Grade III) eluting with dechloromethane separated the

main fraction which absorbed at 430 and 830 nm, as

expected for a zinc isoporphyrin. Further puriﬁcation

was carried out on silica gel using dichloromethane/

ethyl acetate (7:3) to yield a pure product which was

further recrystallized using dichloromethane/petroleum

ether.

(10.98), 842 (9.75). HRMS (MALDI-TOF): calcd. for

C₃₀H₃₃CuN₄512.2001, found m/z 512.2006 [M]⁺. MS

(MALDI-TOF): calcd. 513.155, found m/z 513.339 [M]⁺

(dithranol).

2,3,5,5,7,8,12,13,17,18-decamethylisoporphyrin

(8). b-Bilene hydrochloride (5) (50 mg, 0.08 mmol) was

dissolved in 0.2 mL of cold TFA in a round-bottomed

ﬂask and stirred for 10 min under argon. The mixture was

diluted with dry dichloromethane followed by addition of

zinc(II) acetate (20 mg) dissolved in dry methanol (0.3 mL)

and 0.1 mL of 2,2-dimethoxy propane (excess). The mix-

ture was left to stir in air for 24 h. TLC and spectropho-

tometry showed formation of the product. The electronic

absorption showed peaks at around 440 and 690 nm

characteristic of a metal-free isoporphyrin. Excess TFA

and solvent were evaporated to dryness. The product was

chromatographed on a neutral (Brockmann Grade 0) alu-

mina column (approximately two inches long), prepared

and eluted with slightly acidiﬁed chloroform (CHCl₃/

TFA: pH= 4–5) to collect the major green fraction. The

solvent was evaporated to dryness and dried under vac-

uum to yield 28% (10 mg) of the target compound. UV-

1

vis (CH₂Cl₂): λ_max, nm 432, 700. H NMR ((CD₃)₂CO,

300 MHz): δ, ppm 8.43 (s, meso-H, 2H), 7.50 (s, meso-H,

1H), 2.86, 2.77, 2.61, 2.49 (s, β-CH₃, 24H), 1.64 (s,

5-CH₃, 6H). ¹³C DEPT 90 (CDCl₃, 300 MHz) δ, ppm

106.38, 84.20. ¹³C DEPT 135 (CDCl₃, 300 MHz): δ, ppm

106.38, 84.20, 28.29, 13.06, 11.26, 11.14, 10.79. HRMS

(MALDI-TOF): calcd. for C₃₀H₃₆N₄452.2929, found m/z

452.2875 [M]⁺(dithranol). MS (MALDI-TOF): calcd.

452.61, found m/z 452.59 [M]⁺(dithranol).

Zinc(II) 2,3,5,7,8,12,13,17,18-nonamethyl-5-meth-

oxycarbonylisoporphyrin chloride (10a). 25 mg, 55%

yield. UV-vis (CH₂Cl₂): λ_max, nm (ε × 10⁴, M^-1.cm^-1)

5,5-di(trideuteromethyl)-2,3,7,8,12,13,17,18-octa-

methylisoporphyrin (9). b-Bilene hydrochloride (5)

(50 mg, 0.08 mmol) was dissolved in 0.2 mL of cold

TFA-d in a round-bottomed ﬂask and stirred for 10 min

under argon. The mixture was diluted with dry dichlo-

romethane (20 mL) followed by addition of zinc(II)

acetate (20 mg) dissolved in dry methanol (0.3 mL) and

0.07 mL of acetone-d₆(excess). The rest of the procedure

is similar to that described above for compound (5).The

solvent was evaporated to dryness and the residue dried

under vacuum to yield 22% (8 mg) of the target com-

1

430 (3.37), 830 (2.63). H NMR (CDCl₃, 300 MHz): δ,

ppm 7.69 (s, meso-H, 1H), 7.62 (s, meso-H, 2H), 3.73

(s, 5-OCH_3,3H), 2.58, 2.47, 2.45, 2.42 (s, β-CH₃, 24H),

2.01 (s, 5-CH₃, 3H). HR ESI calcd. for C₃₁H₃₃N₄O₂Zn

557.1889, found m/z 557.1895 [M]⁺. MS (MALDI-TOF):

calcd. 559.01, found m/z 559.80 [M]⁺(dithranol).

Zinc(II) 2,3,5,7,8,12,13,17,18-nonamethyl-5-ethoxy-

carbonylisoporphyrin chloride (10b). 25 mg, 54%

yield. UV-vis (CH₂Cl₂): λ_max, nm (ε × 10⁴, M^-1.cm^-1) 429

(3.58), 826 (2.89). ¹H NMR (CDCl₃, 400 MHz): δ, ppm

7.68 (s, meso-H, 1H), 7.61 (s, meso-H, 2H), 4.10–4.15

(q, OCH₂CH₃, 2H), 2.45, 2.43, 2.40, 2.31 (s, β-CH₃,

24H), 1.96 (s, 5-CH₃, 3H), 1.07–1.04 (t, OCH₂CH_3,3H).

HR ESI calcd. for C₃₂H₃₅N₄O₂Zn 571.2051, found m/z

571.2041 [M]⁺. MS (MALDI-TOF): calcd. 573.04, found

m/z 573.70 [M]⁺(dithranol).

1

pound. UV-vis (CH₂Cl₂): λ_max, nm 429, 692. H NMR

(CDCl₃, 300 MHz): δ, ppm 8.19 (s, meso-H, 2H), 7.33

(s, meso-H, 1H), 2.80, 2.72, 2.59, 2.47 (s, β-CH₃, 24H).

²H NMR (CHCl₃, 300 MHz): δ, ppm 1.85. ¹³C DEPT 90

(CDCl₃, 300 MHz): δ, ppm 106.383, 84.204. ¹³C DEPT

135 (CDCl₃, 300 MHz): δ, ppm 106.33, 84.20, 13.06,

11.26, 11.14, 10.79. HRMS (MALDI-TOF): calcd. for

C₃₀H₃₀D₆N₄458.3299, found m/z 458.3262 [M]⁺(dithra-

nol). MS (MALDI-TOF): calcd. 458.6, found m/z 456.9

(M-D⁺) (dithranol).

Zinc(II) 5-ethoxycarbonyl-2,3,7,8,12,13,17,18-octa-

methyl-5-phenylisoporphyrin chloride (10d). 18 mg,

35% yield. UV-vis (CH₂Cl₂): λ_max, nm (ε × 10⁴, M^-1.cm^-1)

1

439 (4.84), 842 (4.27). H NMR (CDCl₃, 300 MHz): δ,

General procedure for cyclization of b-bilene with

dicarbonyl compounds (10). Cold TFA (0.2 mL) was

added to 50 mg (0.08 mmol) of b-bilene hydrochloride

(5) in a 50 mL round-bottomed ﬂask and left to stir

ppm 8.35, 7.70 (m, 5H, Ph), 7.77 (s, meso-H, 1H), 7.66

(s, meso-H, 2H), 4.27–4.24 (q, OCH₂CH₃, 2H,), 2.50,

2.44, 2.34, 1.94 (s, β-CH₃, 24H), 1.14 (t, OCH₂CH₃, 3H).

HR ESI calcd. for C₃₇H₃₇N₄O₂Zn 633.2207, found m/z

J. Porphyrins Phthalocyanines 2011; 15: 927–929

928

S.C. mWakWarI et al.

633.2202 [M]⁺. MS (MALDI-TOF): calcd. 635.10, found

m/z 635.61 [M]⁺(dithranol).

10e, and 10g stocks were prepared in DMSO at a con-

centration of 10 mM and then diluted into medium to

ﬁnal working concentrations. Cells were then exposed

to 20 µM of ZnIP in medium for 0, 1, 2, 4, 7, and 24 h.

At the end of the loading period, the loading medium

was removed; cells were washed with PBS (200 µL),

and solubilized with 100 µL 0.25% Triton X100 in PBS.

Intercellular accumulation of ZnIP derivatives was deter-

mined by measuring the compound’s ﬂuorescence emis-

sion using a BMG FluoStar Optima plate reader using

excitation/emission wavelengths of 410 nm and 840 ±

40 nm respectively. Cell numbers were measured using

the CyQuant Cell Proliferation Assay (Promega).

Cytotoxicity. Dark toxicity. HEp2 cells were plated as

described above and allowed 36–48 h to grow. Two fold

serial dilutions of zinc isoporphyrin 6 and copper isopor-

phyrin 7 ranging from 0–100 8 µM, and concentrations

of 0.0, 2.5, 5.0, 10.0 and 20.0 µM for zinc isoporphyrins

10a, 10b, 10d, 10e, and 10g were prepared in the plate

and exposed to the cells. After 24 h incubation, the load-

ing medium was removed and viability was determined

using Cell Titer Blue Cell Viability Assay Kit (Promega).

Results were read using a BMG FluoStar Optima plate

reader with an excitation/emission wavelength of 520 nm

and 584 nm respectively.

Phototoxicity. HEp2 cells were prepared as per the

cytotoxicity experiment. Compound was loaded into the

cells and incubated overnight at a concentration range of

0.0, 6.25, 12.5, 25.0, 50.0 and 100.0 µM for zinc isopor-

phyrin 6 and copper isoporphyrin 7, and 0.0, 2.5, 5.0,

10.0 and 20.0 µM for zinc isoporphyrins 10a, 10b, 10d,

10e, and 10g. Loading medium was then removed and

replaced with growth medium containing 50 mM HEPES

pH 7.2. Cells were then exposed to light from a 100 Watt

halogen light source ﬁltered through a 610 nM long pass

ﬁlter. To protect cells from overheating, the plate was

cooled by placing on a metal block in an ice water bath

and IR radiation was ﬁltered by placing 10 mm of water

in the light path. Exposure to the light source was the

equivalent of 1 J.cm^-2. After exposure, the cells were

incubated overnight and viability was assayed using Cell

Titer Blue as in the cytotoxicity assay above.

Fluorescence microscopy. HEp2 cells were seeded

onto Lab-Tek II, 2-chamber cover glass and incubated

for 48 h. Metallo-isoporphyrins 6, 7, 10a, 10b, and 10d,

at a concentration of 10 µM, and 2.5 µM for compounds

10e and 10g was then added to the cells and incubated for

24 h. The cells were then washed with drug-free medium

and fed medium containing 50 mM HEPES pH 7.2.

Coverslips were examined using a Zeiss Axiovert 200M

inverted ﬂuorescent microscope ﬁtted with standard

FITC and Texas Red ﬁlter sets (Chroma Technologies).

The images were acquired with a Zeiss Axiocam MRM

CCD camera ﬁtted to the microscope.

Zinc(II) 13,17-diethyl-5-ethoxycarbonyl-5-isobutyl-

2,3,7,8,12,18-hexamethylisoporphyrin chloride (10e).

16 mg, 33% yield. UV-vis (CH₂Cl₂): λ_max, nm (ε × 10⁴,

1

M^-1.cm^-1) 431 (2.75), 822 (2.28). H NMR (CDCl₃, 400

MHz): δ, ppm 7.89 (s, meso-H, 1H), 7.80 (s, meso-H, 2H),

4.01–3.89 (q, OCH₂CH_3,2H), 3.04–2.96 (q, -CH₂CH₃,

4H), 2.55, 2.51, 2.43 (s, β-CH₃, 18H), 1.96 (s, 5-CH₃, 3H),

1.32–1.20 (m, –CH₂–CH–, 3H), 1.01–0.96 (t, OCH₂CH_3,

3H), 0.94–0.86 (t, –CH₂CH₃, 6H), 0.44 (d, -CH (CH₃)₂,

6H). HR ESI calcd. for C₃₇H₄₅N₄O₂Zn 641.2833, found

m/z 641.2832 [M]⁺. MS (MALDI-TOF): calcd. 643.17,

found m/z 643.56 [M]⁺(dithranol).

Zinc(II) 5-acetyl-2,3,5,7,8,12,13,17,18-nonameth-

ylisoporphyrin chloride (10g). 24 mg, 54% yield. UV-

vis (CH₂Cl₂): λ_max, nm (ε × 10⁴, M^-1.cm^-1) 430 (4.23),

812 (3.87). ¹H NMR (CDCl₃, 400 MHz): δ, ppm 8.10 (s,

meso-H, 1H), 8.07 (s, meso-H, 2H), 2.60, 2.57(s, β-CH₃,

24H), 2.30 (s, COMe, 3H), 1.91 (s, 5-CH₃, 3H). HR ESI

calcd. for C₃₁H₃₃N₄OZn 541.1940, found m/z 541.1943

[M]⁺. MS (MALDI-TOF): calcd. 543.01, found m/z

542.80 [M]⁺(dithranol).

2,3,7,8,12,13,17,19-octamethyl-5-phenylporphyrin

(11d). Zinc(II) isoporphyrin (10d) was dissolved in dry

dichloromethane followed by addition of 5% KOH dis-

solved in dry methanol. The reaction mixture was left

to stir under argon for 1 h after which the color of the

mixture turned purple. The UV-visible spectrum indi-

cated no starting material. Excess KOH was neutralized

by washing with acetic acid (pH = 5), then with water

several times. The organic phase was dried over anhy-

drous Na₂SO₃, and puriﬁed by column chromatography

on alumina (Grade III) using dichloromethane as eluent,

to give Zn11d after evaporation. The product was dis-

solved in TFA, and allowed to stand at room temperature

for 30 min before being poured into water and extracted

with dichloromethane. The organic phase was washed

with aqueous NaHCO₃, the with water, and evaporated to

give 11d, 8 mg, 32% yield. ¹H NMR (CDCl₃, 300 MHz):

δ, ppm 10.17 (s, meso-H, 2H), 9.96 (s, meso-H, 1H),

8.10, 7.79 (m, Ph, 5H), 3.65, 3.62, 3.56 (s, β-CH_3,24H),

-3.15 (br, NH, 2H). HR ESI calcd. for C₃₄H₃₄N₄498.2783,

found m/z 498.2767 [M]⁺. MS (MALDI-TOF): calcd. for

C₃₄H₃₄N₄498.28, found m/z 498.29 [M]⁺.

cell studies

Tissue culture. All tissue culture media and reagents

were obtained from Invitrogen. The human HEp2 cell

line was purchased from ATCC. Cells were maintained

in a 50:50 mix of DMEM:Advanced MEM (Gibco) + 5%

Fetal Bovine Serum (FBS) (Gibco) in a humidiﬁed, 5%

CO₂incubator at 37 °C.

Time dependent cellular uptake. HEp2 cells were

seeded at 7500 cells per well in a 96 well plate (Costar)

and allowed to attach for 36 h. ZnIP’s 6, 10a, 10b, 10d,

For co-localization experiments, the organelle tracers,

MitoTracker Green (mitochondria), LysoSensor Green

(lysosomes), ER-Tracker Green (ER), and BODIPY FL

J. Porphyrins Phthalocyanines 2011; 15: 928–929

SyntheSeS, PrOPertIeS anD Cellular StuDIeS Of metallO-ISOPOrPhyrInS

929

C₅-ceramide (Golgi) were introduced into the cells for

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isoporpyrins, before being washed and prepared for

microscopy as described above. Organelle tracers were

obtained from Molecular Probes (Invitrogen) and used as

per the manufactures instructions.

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concluSIon

The b-bilene route is shown to be a successful path-

way for the synthesis of metallo-isoporphyrins in high

yields (28% vs. 6% previously reported) and employ-

ing a much shorter reaction time (24 h) than previously

reported (6 days). Through this route, a novel copper

isoporphyrin was obtained. In addition, metal-free and

zinc isoporphyrins were isolated and characterized, and

could be transformed into their corresponding meso-

monosubstituted porphyrins. Cell studies show that all

metallo-isoporphyrins accumulate within HEp2 cells

and localize in mitochondria; in addition they were also

found in other organelles, including the lysosomes and

the ER. The zinc-isoporphyrins were moderately photo-

toxic (IC₅₀= 11–35 µM at 1 J/cm²) while the copper-

isoporphyrin showed only low cytoxicity (IC₅₀> 85 µM),

both in the dark and after exposure to 1 J/cm²light dose.

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Acknowledgements

This work was supported by grants from the US

National Institutes of Health (CA 132861, KMS; CA

139385, MGHV).

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J. Porphyrins Phthalocyanines 2011; 15: 929–929

Article Doi

Syntheses, properties and cellular studies of metallo-isoporphyrins

DOI: 10.1142/S108842461100380X

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Article abstract of DOI:10.1142/S108842461100380X

Full text of DOI:10.1142/S108842461100380X

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