Photodegradation of 2-chloro substituted phenothiazines in alcohols

Article abstract of DOI:10.1111/j.1751-1097.2008.00412.x

The mechanisms that trigger the phototoxic response to 2- chlorophenothiazine derivatives are still unknown. To better understand the relationship between the molecular structure of halogenated phenothiazines and their phototoxic activity, their photophysics and photochemistry were studied in several alcohols. The photodestruction quantum yields were determined under anaerobic conditions using monochromatic light (313 nm). Absorption- and emission-spectroscopy, ¹H- and ¹³C-NMR and GC-MS were used to characterize the photoproducts and reference compounds. An electron transfer mechanism had been previously proposed by Bunce et al. (J. Med. Chem. 22, 202-204) to explain the large difference between the photodestruction quantum yield of 2-chlorpromazine (φ = 0.46) and 2-chlorphenothiazine (φ = 0.20). According to these authors, the alkylamino chain transfers an electron to the phenothiazine moiety. Our results demonstrate that this mechanism is incorrect, because the photodestruction quantum yields of all chlorinated derivatives of this study are the same under the same conditions of solvent and irradiation wavelength. The quantum yield has no dependence on the 10-substituent, but it depends on the solvent. The percentage of each photoproduct, on the other hand, strongly depends on that substituent, but not very much on the solvent. Finally, it is demonstrated that the phototoxic effect of chlorinated phenothiazines is not related to the photodechlorination, although both processes share the same transient.

Full text of DOI:10.1111/j.1751-1097.2008.00412.x

Photochemistry and Photobiology, 2009, 85: 160–170
Photodegradation of 2-chloro Substituted Phenothiazines in Alcohols
1
Carmelo Garcıa* , Luis Pinero1,2, Rolando Oyola1 and Rafael Arce2
´
˜
1Department of Chemistry, Humacao Campus, University of Puerto Rico, Humacao, Puerto Rico
2
´
Department of Chemistry, Rıo Piedras Campus, University of Puerto Rico, San Juan, Puerto Rico
Received 14 February 2008, accepted 3 June 2008, DOI: 10.1111 ⁄ j.1751-1097.2008.00412.x
Other mechanisms consider that the biochemical damages are
ABSTRACT
produced by free radicals (5,6), ground state complexation (7,8)
or the photoaddition of CPZ to ds-DNA (9–13). Based on the
observation that the photobinding of CPZ in vivo can be induced
even with longwave UVA light, Ljunggren and Moller (14)
suggested that these adverse photobiological effects could also
be caused by the CPZ-metabolites. Therefore, the attention was
once focused on chlorpromazine sulfoxide (CPZSO), the major
metabolite of CPZ (15). According to Rosenthal et al. (16) and
Davis et al. (5), the sulfoxide can also be produced by the attack
of singlet oxygen to the ground state of CPZ. These photo-
oxidation reactions of CPZ were exclusively observed in
aqueous solutions, but never in organic solvents. Photolysis of
the sulfoxide derivative in aqueous solution further resulted in a
species capable of oxidizing ascorbate, cysteine, glutathione,
NADH and azide by single electron transfer (15). In addition,
this species can abstract hydrogen atoms from ethanol and
dimethyl sulfoxide. As the oxidation does not require the
presence of dissolved oxygen, the oxidizing species was proposed
to be the hydroxyl free radical arising from the homolytic
cleavage of the S–O bond of the sulfoxide. Nevertheless,
Schoonderwoerd et al. (17) ruled out the metabolic product of
CPZ to be responsible for the drug phototoxicity. They based
their conclusion on the bioavailability of CPZ and its oxidation
product (CPZSO) in the skin and the absorption spectrum in the
UVA spectral region. They also concluded that, in fact,
sulfoxidation of CPZ results in less photobinding.
The mechanisms that trigger the phototoxic response to
2-chlorophenothiazine derivatives are still unknown. To better
understand the relationship between the molecular structure of
halogenated phenothiazines and their phototoxic activity, their
photophysics and photochemistry were studied in several alco-
hols. The photodestruction quantum yields were determined
under anaerobic conditions using monochromatic light (313 nm).
1
Absorption- and emission-spectroscopy, H- and 13C-NMR and
GC-MS were used to characterize the photoproducts and
reference compounds. An electron transfer mechanism had
been previously proposed by Bunce et al. (J. Med. Chem. 22,
202–204) to explain the large difference between the photo-
destruction quantum yield of 2-chlorpromazine (u = 0.46) and
2-chlorphenothiazine (u = 0.20). According to these authors,
the alkylamino chain transfers an electron to the phenothiazine
moiety. Our results demonstrate that this mechanism is incor-
rect, because the photodestruction quantum yields of all chlori-
nated derivatives of this study are the same under the same
conditions of solvent and irradiation wavelength. The quantum
yield has no dependence on the 10-substituent, but it depends on
the solvent. The percentage of each photoproduct, on the other
hand, strongly depends on that substituent, but not very much on
the solvent. Finally, it is demonstrated that the phototoxic effect
of chlorinated phenothiazines is not related to the photodechlo-
rination, although both processes share the same transient.
The ground state complexes between CPZ and DNA cannot
be responsible for the phototoxicity either, because no complex
formation was detected at physiological conditions (18). The
involvement of the covalent binding of CPZ to DNA in the
phototoxic side effect has also been questioned. Rosenthal (16)
found no toxic effects on Escherichia coli attributed to CPZ-
sensitization. Our previous results showed that the triplet
quantum yield of most TCAs strongly depends on the
substituent at the 2-position and the solvent (19–21). It was
further demonstrated that the triplet state of halogenated
phenothiazine derivatives is efficiently quenched by a hydrogen
transfer process and that the most phototoxic derivatives have
a high triplet quantum yield and a short lifetime. Therefore,
it was concluded that the triplet intermediate is somehow
involved in the phototoxic mechanism of the promazines (20).
Studies on the photochemistry of the phenothiazine family
have produced a series of reports with different and, most of
the time, contradictory results. This fact is mainly due to the
spectrum of experimental conditions and initial drug concen-
tration used in each study. Felmeister and Discher (22) studied
INTRODUCTION
Chlorpromazine (CPZ, 2b, Fig. 1) is a major tricyclic anti-
depressant drug (TCA). The studies on the photochemistry of
this and other related TCAs were initially stimulated by the
observation that it produces skin rashes and ocular changes in
patients treated with large doses. The mechanism responsible
for this promazine-induced phototoxicity is still unknown,
although several proposals have been made to account for the
phototoxicity of CPZ (1,2). Kochevar and Hom (3) attributed
this response to the formation of dimers and higher multimers
of the drug produced by preirradiation of CPZ. Nevertheless,
the dimers and polymers of CPZ cannot form in concentra-
tions high enough to be toxic. The in vivo therapeutic
concentration of CPZ is only 0.03–3.0 lM, which is far less
than the critical concentration required for dimerization (4).
*Corresponding author email: carmelo.garcia@upr.edu (Carmelo Garcıa)
´
ꢀ 2008 The Authors. JournalCompilation. The AmericanSocietyofPhotobiology 0031-8655/09
160

Photochemistry and Photobiology, 2009, 85 161
detected the formation of PZ and PZOCH(CH3)2. Based on
these results, they proposed a direct homolysis of the triplet
state of CPZ to produce the free radicals. The formation of
these radicals was confirmed by irradiating an air-free CPZ
solution in methyl methacrylate, which undergoes polymeri-
zation. For the determination of the quantum yield, they used
a 3 mM solution and a Pyrex cell with a pathlength of 3.3 cm.
According to this setup, an absorption gradient is produced
within the first centimeters of the solution, rendering the
quantum yield values uncertain.
Rosenthal et al. (16) used selectively labeled methanol to
elucidate the mechanism of the photoreaction of CPZ-HCl
under anaerobic conditions at wavelength longer than 300 nm.
No hydrogen scrambling occurred along the reaction pathway
between methyl and hydroxyl hydrogen as could be expected
for the suggested all-radical mechanism proposed by Davis
et al. (5). They found that the isotopic composition of the
methoxy group in the alkoxy-derivative is identical to that of
the original methyl group in the methyl alcohol. They
concluded that the methyl group of the solvent is the exclusive
source of the hydrogen atom that replaces the chlorine.
Therefore, for the formation of the methoxy-phenothiazine
derivative, they proposed an ionic photonucleophilic substitu-
tion of the chlorine. They further found that the photolysis of
CPH (1b) under the same conditions produces 90% PH and
ꢀ2% MOPH. Similarly, the direct photolysis of CPZ-HCl
resulted in almost total conversion to PZ and MOPZ, in
addition to minor amounts of other decomposition products.
The photodestruction quantum yield and the chemical yield of
the CPZ photoproducts were not reported, but they observed
that the N-alkyl side chain is not a critical requirement for this
reaction. The concentration of CPZ-HCl in this reaction was
14 mM and the pathlength of the cylindrical Pyrex cell was not
mentioned.
Figure 1. Structure of the phenothiazine derivatives and related
compounds [1 R1 = H; 2 R1 = (CH2)3-N(CH3)2; 3 R1 = (CH2)3-
CH(CH3)2].
the photodecomposition of CPZ-HCl under aerobic and
anaerobic conditions at 253.5 nm. For the kinetic studies
and photodestruction quantum yield determination, they used
microirradiation to excite its p fi p* transition. The charac-
terization of the photoproducts, on the other hand, was done
in a concentration range of 10)2 to 10)3 M using a Hanovia UV
lamp and removable glass filters transmitting from 360 to
370 nm. These wavelengths correspond to the excitation of the
n fi p* transition. They observed that the absorption spectra
taken during the photolysis under aerobic conditions changed
in a different way than those taken under anaerobic condi-
tions, implicating the formation of different products. Among
the photoproducts, they characterized an alcohol derivative
using acetylation reactions and IR spectroscopy. The photo-
products formed in the bulk reaction appeared to be more
hydrophilic than CPZ and CPZSO. They reported photo-
destruction quantum yield values in aerobic and anaerobic
conditions at 253.5 nm of 0.18 and 0.14, respectively. The
major problem in these sets of experiments is that the
characterized photoproducts at 360 nm are not necessarily
the same ones quantified with 253.5 nm.
Until now, no direct evidence has been presented to confirm
or rule out the participation of the TCA neutral radicals in
their phototoxicity. For the dehalogenation of 2-chlorinated
promazines from the triplet state (3CPZ*), two parallel
mechanisms have been proposed: homolytic cleavage of the
C–Cl bond and nucleophilic attack of the solvent (5,23,24).
Nevertheless, data on the quantification of this process are
scarce and most of the values show very poor reproducibility.
Davis et al. found that the irradiation of the hydrochloride salt
of CPZ (CPZ-HCl) in deoxygenated propan-2-ol solution
yielded free chloride ion and a concomitant equimolar amount
of hydronium ion with a quantum yield of 0.12 (5). Therefore,
these authors proposed a direct homolysis of 3CPZ* affording
radicals, which—in turn—abstract hydrogen atoms from the
solvent. Bunce et al. proposed that electron transfer is the
major reason for the photochemical instability of CPZ and
reported a quantum yield of 0.46 for the dehalogenation of
chlorpromazine free base in degassed 4:1-acetonitrile-water
mixtures (23). They also measured the quantum yield of the
parent compound 2-chlorophenothiazine (CPH, 1b) in the same
solvent and reported a value of 0.20. Based on these findings,
they concluded that the N-alkyl substituent is not a necessary
requirement for the photodehalogenation, but may accelerate
the process of chloride removal by the intramolecular electron
transfer mechanism. Moore and Tamat reported a quantum
yield value of 0.65 for this dehalogenation process in three
different degassed solvents (propan-2-ol, methanol and water),
Davis et al. (5) found no spectral changes on the >300 nm
irradiation of CPZ-HCl in iPrOH under aerobic conditions.
Singlet oxygen is evidently incapable of oxidizing CPZ,
confirming the previous observation of Iwaoka and Kondo
(12). Under anaerobic conditions, on the other hand, they
reported a photodestruction quantum yield of 0.12 and

´
162 Carmelo Garcıa et al.
diphenyl-65% dimethylpolysiloxane, nominal length = 30 m). The
oven conditions were set to: Initial Temp = 200ꢁC, Final Temp =
300ꢁC and Ramp = 10ꢁC min)1. The detector conditions were set to:
but they did not specify whether the drug was protonated or in
the free form (24).
In this work, we report a systematic study of the photo-
physical properties and the quantum yields for the dehalogen-
ation of CPH (1b) and CPZ-HCl in methanol, ethanol,
1-propanol, 2 -propanol and t-butanol. The photolysis of the
novel 2-chloro-10-(4-methyl)-pentyl phenothiazine (CMPPH,
3b) was also performed in selected alcohols to assert the
contribution of the N-alkyl substituent to the dehalogenation
process. A general photodestruction mechanism is proposed to
account for the measured quantum yields, the characterized
photoproducts and the phototoxicity of these TCAs.
Detector = FID, Temp = 350ꢁC, Hydrogen Flow = 40 mL min)1
,
and Air Flow = 450 mL min)1. The inlet conditions were: Mode =
Split, Initial Temp = 280ꢁC, Split Flow = 10 mL min)1 and Gas
Type = Helium. The proton- and carbon-NMR spectra were taken
with an Advance 400 NMR spectrometer (TX) using the 5 mm Bruker
BioSpin BBO probe (Boston). Deuterated dimethyl sulfoxide was used
for all solutions. For the mass spectra, the separation of the products
was done with a Thermo Finnigan Trace GC ⁄ Polaris Q chromato-
graph with a capillary column model Restek 12623 (stationary phase
RTX-5MS: Crossbondꢂ 5% diphenyl ⁄ 95% dimethyl polysiloxane,
nominal length = 30 m). The oven conditions were set to: Initial
Temp = 90ꢁC, Final Temp = 250ꢁC and Ramp = 10ꢁC min)1. The
detector conditions were set to: Ion Source = 200ꢁC, Transfer
Line = 275ꢁC, Scan Mode = Full Scan (range 50–650), Electron
Impact = 70 eV and Mass Selector = Ion Trap. The inlet conditions
MATERIALS AND METHODS
were: Mode = Split, Temp = 200ꢁC, Split Flow = 26 mL min)1
Split Ratio = 17, Gas Type = Helium and Constant Flow =
1.5 mL min)1
,
Materials and chemicals. Phenothiazine (PH, 1a), chlorphenothiazine
(CPH, 1b), the hydrochloride salts of promazine (PZ, 2a) and
chlorpromazine (CPZ, 2b), anhydrous ethyl alcohol, anhydrous
2-propanol, anhydrous 1-propanol and anhydrous tert-butanol were
purchased from Sigma-Aldrich (IL). The hydrochloride salts of
2-methoxypromazine (MOPZ, 2c) and 2-trifluoromethylpromazine
were a gift from the NIH-National Cancer Institute (Drug Synthesis
& Chemistry Branch, Developmental Therapeutics Program, Division
of Cancer Treatment). CPZ and MOPZ were purified by addition of
NaOH to an aqueous solution of the protonated drug and then
extracting with diethyl ether. All other compounds were used as
received. Other HPLC-grade solvents were obtained from Fisher
Scientific (Cayey, PR). High purity helium and nitrogen were
purchased from Air Products (Humacao, PR).
.
Photodestruction quantum yields. The photolysis light source was a
Sylvania 200 W high pressure Hg-Xe lamp and the 313 nm line was
isolated with
a 1 ⁄ 8 m Spectral Physics grating monochromator
(Cincinnati, OH). The lamp intensity was determined before and after
each set of photolysis with the Packard and Hatchard method using
the potassium ferrioxalate actinometer (27). All photoreactions were
carried out in a quartz cuvette (1 · 1 · 4 cm3) for up to 10–80%
conversion of the starting material and using the same cell orientation.
Three milliliters of multiple solutions of ꢀ0.22 mM of the hydrochlo-
rinated TCA or its free base in each alcohol, previously saturated with
helium or dry nitrogen (ꢀ15 min), were irradiated with 313 nm for
different times at room temperature. The photoreaction was controlled
with an electronic shutter managed by a Labview 7.5 based program
(TX). After photolysis, 45 lL of a 20.00 mM alcohol solution of
2-trifluoromethylpromazine (TFMPZ) was added as internal standard
for the determination of the conversion percent and the yields of the
photodestruction. Then, 2 lL of this mixture was injected at least three
times in a 6850 gas chromatograph to determine the quantity of
remaining TCA. Calibration curves of amount ratio vs area ratio of
each phenothiazine derivative or the corresponding photoproduct were
prepared using a concentration range of 0.05–0.30 mM and a constant
concentration of 0.15 mM of TFMPZ. All solutions used for calibra-
tion were injected three times and the average of the integrated area
was used for the curve. An absorption spectrum was taken for all
solutions before and after irradiation. As the photodestruction of the
TCA is a zeroth order reaction for small irradiation times, its quantum
yields were determined from the linear part of the [TCA] vs time plot
using the following equation:
Synthesis of MPPH 3a, CMPPH 3b and MMPPH 3c. Compounds
3a and 3b were synthesized by a method based on literature procedures
with some minor modifications (25,26). Briefly, a solution of DMSO
(25 mL) containing 0.0051 mol of the corresponding phenothiazine (1a
or 1b) and 0.0051 mol of potassium hydroxide was stirred at room
temperature, while adding 0.084 mL (0.0056 mol) of 1-bromo-4-
methylpentane. After 4 h, 30 mL of water was added and the product
was extracted by washing the solution several times with methylene
chloride, saving the organic phase. This organic phase was then
washed with water and brine, and dried over magnesium sulfate. The
solvent was removed by rotary evaporation and the oily product was
then purified with silica gel column chromatography with a hexane ⁄
ethyl acetate mobile phase. MPPH 3a was obtained with 47% yield:
1H-NMR = 7.20–7.12 (m, 4H), 6.99–6.90 (m, 4H), 3.83–3.80
(t, J = 6.8 Hz, 2H), 1.69–1.62 (m, 2H), 1.51–1.44 (m, 1H), 1.27–1.22
(m, 2H), 0.80–0.78 (d, J = 6.4 Hz, 6H); 13C-NMR = 144.8, 127.8,
127.5, 127.0, 123.6, 122.3, 115.7, 46.6, 35.3, 27.0, 24.0, 22.4; and
MS = 284(26), 283(100), 213(18), 212(84), 199(19), 198(33), 181(11),
180(19); CMPPH 3b was obtained with a 51% yield: 1H-NMR
400 MHz in CD3SOCD3: 7.23–7.13 (m, 3H), 7.03–6.94 (m, 4H), 3.86–
3.82 (t, J = 6.9 Hz, J = 13.8 Hz, 2H), 1.53–1.44 (m, 1H), 1.28–1.22
(quartet, J = 6.9 Hz, J = 15.20 Hz, 2H), 0.80 (d, J = 6.6 Hz,
6H);13C-NMR = 146.8, 144.5, 132.9, 128.6, 127.7, 123.9, 123.2,
122.5, 116.8, 116.2, 47.1, 35.7, 27.5, 24.4, 22.9; and MS = 319(26),
318(14), 317(82), 248(36), 247(16), 246(100), 234(24), 233(25), 232(44).
MMPPH 3c was obtained by a bulk photolysis of 3a in methanol. The
product was obtained by solvent evaporation and separation with the
same column chromatography. After purification, MMPPH 3c was
obtained with a 16% yield: 1H-NMR = 7.21–7.16 (ddd, J = 8.2 Hz,
J = 7.4 Hz, J = 1.4 Hz, 1H), 7.14–7.12 (dd, J = 7.6 Hz, J = 1.2 Hz,
1H), 7.04–6.99 (m, 2H), 6.95–6.91 (ddd, J = 1.2 Hz, J = 0.80 Hz,
J = 0.80, 1H), 6.59–6.54 (q, 2H), 3.86–3.82 (t, J = 7.0), 3.74 (s, 3H),
1.69–1.66 (q, 2H), 1.53–1.49 (m,1H), 1.30–1.24 (quartet, 2H), 0.82–
0.81 (d, J = 6.4 Hz, 6H). 313(100), 243(21), 242(56), 229(35), 228(39);
13C-NMR = 160.0, 146.8, 145.1, 127.9, 127.8, 127.5, 124.8, 122.9,
116.4, 114.9, 107.9, 103.4, 55.8, 47.2, 35.9, 27.5, 24.6, 22.9; and
MS = 313(100), 243(21), 242(56), 229(35), 228(39).
ꢃ
ꢂ
ꢄ
ꢀ
ꢁ
ꢁd½TCAꢂ dt V
ꢁdn
1
ꢁkV
ꢃ
ꢄ
/
¼
¼
¼
Dehal:
I0ð1 ꢁ 10ꢁeb½TCAꢂÞ
I0 1 ꢁ 10ꢁeb½TCAꢂ ð2ꢁaÞ=2
0
dt Iabs:
ð1Þ
where k is the photodestruction rate constant (slope of the plot in
M s)1), V is the reaction volume (3 mL), I0 is the lamp intensity at
313 nm, [TCA]0 is the initial drug concentration and a is the destructed
fraction of the TCA. The division by 2 in the absorption term is in-
cluded to account for the gradient produced in the Iabs term, i.e. the
absorbed intensity is taken as the average of the initial and final
absorption. Similarly, the formation quantum yield of PZ and other
photoproducts were determined with the equation:
ꢀ
ꢁ
dn
1
kV
I0ð1 ꢁ 10ꢁeb½PZꢂ =2
/
¼
¼
ð2Þ
Form:
f
dt Iabs
Þ
where [PZ]f is the amount of PZ formed after irradiation and the factor
0.5 is introduced also to average the absorption of PZ and taking
[PZ]0 = 0. The determination of [PZ]f for small irradiation times is
difficult, as there is almost no product formed and sometimes the
regression gives nonzero intercepts. For these cases, corrections were
made by forcing a zero intercept and keeping the same value of k.
Characterization of the photoproducts. The photoproducts were
identified and characterized with GC-MS using standards. All of them
Absorption spectroscopy, gas chromatography, NMR and mass
spectroscopy. Absorption spectra were taken with a HP 8453 UV–
Vis photodiode array spectrophotometer (CA). The chromatograms
were taken with an Agilent GC 6850 gas chromatograph (CA) with a
capillary column model Restek 176832 (stationary phase = 35%

Photochemistry and Photobiology, 2009, 85 163
have a very similar mass spectrum, as they have a very similar MS
fragmentation pattern (data not shown). Typical values of m ⁄ z (%) are
the following: PH 1a: 166(27), 167(70), 168(11), 199(100), 200(15);
CPH 1b: 198(63), 199(42), 234(22), 235(49); MOPZ 2c: 299(50),
297(49), 218(100), 217(50), 185(35); EOPZ 2d: 328(100), 243(48),
86(30), 58(72); POPZ 2e: 342(100), 257(45), 86(28), 58(61); iPOPZ 2f:
299(50), 297(49), 218(100), 217(50); and TBOPZ 2g: 356(100), 300(27),
215(27), 86(27), 58(50).
In this set of equations, d is pathlength of the cell and ꢀ is the
extinction coefficient of Ai at a particular wavelength. If at a given
time t, there is any relationship between the xk (with constant
coefficients ak) or the qkk (with constant coefficients bk), i.e. if some
of the reactions are not independent from one another and there are
only s independent steps (i.e. the rank of the matrix is s < r), then
all linear relationships are equal to zero for k = 1,r (Eq. 9) and
Eq. (8) yields Eq. (10).
Theoretical calculations. All geometry optimizations were initially
performed at the semiempirical level with the Polak-Ribiere conju-
r
s
X
X
akxkðtÞ ¼ 0;
biqki ¼ 0;
akxkðtÞ ¼ LkðtÞ
gated gradient protocol with 1 · 10)5 convergence limit and 0.01
) rms limit, as previously described (28). Density
k¼1
k¼1
kcal (A mol)1
˚
ð9Þ
n
s
X
X
functional theory (DFT) with B3LYP ⁄ 6–31 G(d) was used for the
optimization refinement and the calculation of the dissociation
energies of solvents and TCA molecules, according to Eqs. (3) and (4):
biqki ¼ Qki
i¼1
i¼1
s
X
R1 ꢁ R2 ! Rꢃ1 þ Rꢃ2
DEkðtÞ ¼
QkkLkðtÞ
ð10Þ
R1 ꢁ H ! Rꢃ1 þ Hꢃ
R2 ꢁ H ! Rꢃ2 þ Hꢃ
ð3Þ
ð4Þ
k¼1
According to this equation, it is always possible to determine the value
of s by measuring absorbencies, without knowledge of the individual
absorption coefficients. For an isosbestic point, for instance, this
equation equals zero for a given wavelength at all times. Nevertheless,
isosbestic points only give limited information about the uniformity of
a chemical reaction. More reliable expressions can be made using
absorbency difference diagrams (ED-diagrams) (33). The total change
in absorbency for two different wavelengths with time is given by
Eq. (11), which yields Eq. (12) for s = 1.
ꢃ
ꢃ
DR ꢁH ¼ DHR þ DHHf ꢁ DHRf ꢁH
1
1
1
f
where DHf (X–Y) is the formation enthalpy of the species XY and
D(X–Y) is the dissociation enthalpy of the X–Y bond. For some rad-
)1
•
•
icals, including DHf(H ) = 52.1 kcal mol
and DHf(Cl ) = 28.95
kcal mol)1, the experimental values are taken from the literature (29–
32). For systems with known D(R–H), the D(R1–R2) is calculated
using the combination of all reactions given in Eq. (3), according to:
DE1ðtÞ ¼ Q11x1 þ Q12x2 þ ꢄ ꢄ ꢄ þ Q1sxs
ð11Þ
DR ꢁR ¼ DR ꢁH þDR ꢁH ꢁDHꢁH þDHðR ꢁHÞ þDHfðR ꢁHÞ ꢁDHfðR ꢁR Þ
1
2
1
2
DE2ðtÞ ¼ Q21x1 þ Q22x2 þ ꢄ ꢄ ꢄ þ Q2sxs
1
2
1
2
f
ꢀ
ꢁ
ð5Þ
Q11
Q21
DE1 ¼ DE2
ð12Þ
Evaluation of steps in the photodestruction mechanism. The number
of steps or ‘‘independent reactions’’ (s) involved in the photodestruction
of the 2-chlorinated phenothiazine derivatives is required for the proper
formulation of a mechanism. As previously mentioned, Rosenthal (16)
and Grant and Green (6) proposed a mechanism in which the first step
is the homolytic cleavage of the C–Cl bond in the triplet excited state.
The promazinyl radical then abstracts a hydrogen from the solvent to
yield the parent compound. The second proposed reaction is the
nucleophilic attack of the solvent to the 3TCA* to produce the alkoxy
derivative. Therefore, the mechanism of the photolysis of chlorinated
phenothiazines should consist of two reactions.
The evaluation of s in the TCA photodestruction was made
according to the method described by Mauser (33). Briefly, for r
parallel reaction steps involving n components A of the general form
given in Eq. (6), the total change in the concentration of the ith
component (D[A]i) is given by Eq. (7), where xk is the degree of
advancement of the kth reaction and mki is the coefficient of the ith
component in the kth reaction.
The ED-diagram results in a straight line passing through zero with the
slope Q11 ⁄ Q21. The ED-diagrams must be examined at as many
wavelength combinations as possible, because an DE1 vs DE2 plot can
be linear by chance (34,35). In this case, Eq. (11) requires three dif-
ferent wavelengths (k = 1, 2, 3), Eq. (11) is given in the same way for
the extra DE3(t) and the corresponding Kronecker-Capelli’s determi-
nant is equal to zero (Eq. 13).
ꢅ
ꢅ
ꢅ
ꢅ
ꢅ
ꢅ
ꢅ
ꢅ
DE Q11 Q12
1
DE Q21 Q22 ¼ 0
ð13Þ
ꢅ
2
ꢅ
DE3 Q31 Q32
In this case, D23DE1 + D13DE2 + D12DE3 = 0 with D23 = Q21Q32
)
Q31Q22, D13 = Q11Q32 ) Q31Q12 and D12 = Q11Q22 ) Q12Q21. These
equations result in a three-dimensional space spanned by the absor-
bency differences DE1, DE2 and DE3. They are presented graphically in
a two-dimensional plot by rearranging them into Eq. (14):
m
k1A1 þ mk2A2 þ ꢄ ꢄ ꢄ ! mknAn
ð6Þ
ð7Þ
ꢀ
ꢁ
DE3
DE1
DE2
DE1
¼ q þ r
ð14Þ
n
X
D½Aꢂi ¼ ½AꢂiðtÞ ꢁ ½Aꢂiðt0Þ ¼
mkixk
i¼1
where q = )D23 ⁄ D12 and r = )D13 ⁄ D12. Plots of DE3 ⁄ DE1 vs DE2 ⁄
DE1 are called extinction difference quotient diagram or EDQ-plots
(33). This type of diagram becomes linear if either the mechanism
consists of only two steps or, if in more than two linear independent
partial reactions, the rank of the matrix Q reduces to 2 by chance. For
a clear discrimination between these cases, as many combinations as
possible have to be chosen in a large wavelength range extended far
into the short wavelength region.
If for this system, h absorbencies Ek (t)(k = 1,…h) are measured at m
different times tj (j = 1,…m), the matrix for the difference in
absorption at a particular wavelength (DEk) can be rearranged to give
Eq. (8).
n
X
DEkðtÞ ¼ d
¼ d
ekiD½AꢂiðtÞ
i¼1
n
r
X
X
RESULTS AND DISCUSSION
eki
mkixk
i¼1
k¼1
Photophysics of CPH 1b
"
#
ð8Þ
r
n
X
X
¼
¼
xk
d
mkieki
The absorption spectra of the free base phenothiazines present
two main bands in the 250–265 and 300–320 nm wavelength
ranges (Table 1 and Fig. 2) (20). The first band is attributed to
a p fi p* transition and the other belongs mainly to an
k¼1
i¼1
r
X
xkqkk
k¼1

´
164 Carmelo Garcıa et al.
Table 1. Photophysical properties of the phenothiazines in different alcohols under anaerobic conditions, including the irradiation wavelength
(313 nm). The emission maxima were measured in methanol.
kmax (nm) (e[M)1 cm)1] · 10)4
)
kmax (nm) [f]
kemm
(nm)
Phenothiazine
Methanol
Ethanol
Theoretical
PH 1a
CPH 1b
254 [5.1], 313 [0.439], 318 [0.48]
256 [4.86], 313 [0.484],
323 [0.430]
319 [0.508], 313 [0.501]
257 [4.55], 313 [402],
325 [0.467]
208 [0.08], 253 [0.32], 262 [1.08], 323 [0.11]
206 [0.46], 216 [0.16], 262 [1.12], 321 [0.23]
442
450
Figure 2. Left: Absorption spectra of methanol solutions of PH 1a (. . .), CPH 1b (——) and their equimolar mixture (- - -). Right: Absorption
curves for the photolysis of CPH in methanol under anaerobic conditions, Irradiation wavelength = 313 nm; lamp intensity = 4.70 · 10)10E s)1
time intervals for the irradiation = 200 s with t(a) = 0 s.
;
n fi p* transition involving the sulfur lone-electron pairs (36).
The absorption spectra of PH 1a and CPH 1b show some
significant differences (Fig. 2), including the blueshift of
ꢀ5 nm in the absorption maximum of PH relative to CPH
(Table 1). This shift and the corresponding higher oscillator
strength is introduced by the chlorine atom. CPH has larger
absorption extinction coefficients than PH at wavelengths
>310 nm and smaller values for wavelengths <310 nm,
producing an isosbestic point at 310 nm. Therefore, the
resulting absorption spectrum of a mixture of CPH and PH
has a maximum wavelength at 321 nm. This behavior should
describe the spectral changes in photolyzed solutions of CPH,
if its only photoproduct is PH.
The absorption spectra of irradiated solutions of CPH
changes according to the type and amount of products formed.
In methanol, for instance, the absorption increases for
wavelengths <320 nm, indicating that the photoproducts
have larger molar absorption coefficients at these wavelengths
(Fig 2). Although the general characteristics at wavelengths
>330 nm are very similar to those expected, the formation of
the isosbestic point is observed at 328 nm and not at 310 nm.
Besides, for long irradiation times, the absorption maximum at
308 nm does not match with the maximum of the mixture at
321 nm. These new characteristics are most probably due to
the formation of several other photoproducts with different
absorption profiles.
For the determination of the quantum yield of the CPH
photodestruction and the PH photoformation, the concentra-
tion of each compound was determined as function of
irradiation time using the integration capabilities of the GC.
For very long irradiation times (t > 720 s), single exponential
behavior was observed for both processes, which is character-
istic of first-order reactions (Fig. 3). It also indicates that there
might be, among other processes, filter effects and secondary
reactions. For short irradiation times (t < 720 s), on the other
hand, there is a linear concentration ⁄ time dependence, indi-
cating a zeroth order photoreaction. The quantum yields were
calculated using the linear part of the corresponding plot,
which is maintained for up to 45% CPH photodestruction.
Table 2 summarizes the kinetic values for the photoreactions
in methanol and ethanol, which corroborate that the major
photoproduct of this reaction is PH. The fact that the
photodestruction quantum yield of CPH is slightly larger than
Photochemistry of CPH 1b
The major photoproduct found for CPH 1b in all alcohols was
PH 1a. In methanol and ethanol, a small amount of the
corresponding alkoxide (PH-OR) and other unidentified prod-
ucts was detected for long irradiation times (t > 720 s), as
illustrated in Eq. (15) for R1 = H.

Photochemistry and Photobiology, 2009, 85 165
photoproducts can be calculated to be uOther = 0.34 in
methanol and only 0.11 in ethanol. To verify this statement,
the mass balance of the photoreaction was determined in terms
of the recovered mass percent. It was found that the larger the
irradiation time, the bigger the amount of unrecovered
material. For long irradiation times, a limiting chemical yield
for PH of 68% was obtained for methanol, in disagreement of
the yield measured by Rosenthal et al. of 90% (16).
The number of independent reactions was asserted with
the ED and EDQ diagrams. For both methanol and
ethanol, excellent linear ED-plots were obtained with zero
intercept and r2 > 0.999 (Fig. 4, left). This indicates that, as
there is only one main photoproduct, the photodecomposi-
tion of CPH occurs through a single reaction, yielding the
reduction product PH (s = 1, Table 2). This was further
corroborated with the EDQ plots (data not shown), which
have r2 values smaller than 0.83, especially for wavelengths
in the 300–310 nm range and irradiation times >720 s.
Figure 3. Photokinetics of CPH 1b in methanol under anaerobic
conditions and irradiating with 313 nm light. The solid lines represent
the linear regression for 0–720 s. The photodestruction of CPH (s)
produces mostly PH (d). The linear regressions yield: [CPH]t =
0.219 ) 1.39 · 10)4t [r2 = 0.9982] and [PH]t = 0.0003 + 9.15 · 10)5
t
Photophysics of CPZ 2b and CMPPH 3b
[r2 = 0.9979].
The absorption and emission properties of CPZ 2b and similar
compounds have been previously reported (19,20) (Table 3).
The emission spectra of all 10-alkylated phenothiazines consist
of a broad band with maximum between 440 and 470 nm in all
Table 2. Isosbestic point of the PH–CPH mixtures before and after
photolysis, number of independent reactions (S), kinetic constant (k)
and quantum yield (u) for the production of PH and destruction of
CPH.
solvents. They also present Stoke’s shift larger than 104 cm)1
,
which is a considerable magnitude. The emission maxima are
more solvent dependent than the corresponding absorption
maxima. No differences were observed in the absorption
properties of CMPPH 3b. Compared to the parent PZ 2a,
chlorinated derivatives have small fluorescence quantum
yields, especially in methanol. All promazines have uf values
in the order of 10)2–10)3. Therefore, other deactivation
mechanisms for the S1 state must be competing favorably
with the fluorescence process for these molecules. The
10-alkylamino chain was found to be very flexible in solution,
given to all molecules several thermally accessible stable
conformations (37,38). The emission properties of promazines
2 are also very sensitive to the solvent and the 2-substituent,
but not to the alkylamino chain (20,39). The fluorescence
Isosbestic
point (nm)
k
(mM s)1) · 105
u
Solvent Before After
S
PH
CPH
PH
CPH
MeOH
EtOH
310
330
328
320
1
1
9.15
12.4
13.9 0.70
14.7 1.03
0.03 1.04
0.09 1.14
0.03
0.04
unity definitely indicates that other processes might be ongoing
in this reaction and ⁄ or the amount of absorbed light is
underestimated in Eq. (1). This is further confirmed by the
smaller value of uPH, compared to uCPH. The quantum yield
for the formation of the alkoxide and all other unidentified
Figure 4. Left: ED-plots for the photolysis of CPH in methanol for the following wavelength combinations (nm): DE280 vs DE290 (d); DE280 vs
DE300 (s); DE280 vs DE360 (.); DE290 vs DE300 (ꢃ); DE290 vs DE360 ( ); and DE300 vs DE360 (h). Right: ED-plots for the photolysis of CPZ-
HCl in ethanol for the following wavelength combinations (nm): DE280 vs DE290 (d); DE280 vs DE310 (s); DE280 vs DE320 ( ); DE290 vs DE310
(D); DE290 vs DE320 ( ), and DE310 vs DE320 (h).

´
166 Carmelo Garcıa et al.
Table 3. Absorption, emission and triplet-state properties of the promazine derivatives measured in methanol.
)1
Phenothiazine
derivative
kmax (nm), Stoke’s shift (cm)1),
uf · 103 and sf (ns)
kmax (nm), eT · 10)4 (M cm)1),
)1
kmax (nm) (e[M cm)1] · 10)4
)
uT and sT (ls)
PZ 2a
255 [3.3
258 [3.66
258 [3.41
0.2], 307 [0.42
0.01], 311 [0.46
0.03], 313 [0.44
0.03]
444
10 050
4.5
1.75†
449
9474
0.95
0.89†
–
460
2.65
0.41
61
460
1.95
0.90
2.2 §
–
CPZ 2b
0.01]‡
0.02]
CMPPH 3b
†Values from Garcı
257 [3.83
´
a et al. (19). ‡Corresponding values in ethanol are: 257 [3.26
0.06], 309 [0.472 0.001], 313 [0.461 0.001]; 2-propanol: 257 [3.37
0.05], 310 [0.398 0.001], 313 [0.391
0.04], 308 [0.409
0.03], 309 [0.417
´
0.001]. §Values from Garcıa et al. (20).
0.001], 313 [0.400
0.002], 313[0.408
0.001]; 1-propanol:
0.002]; and
t-butanol: 257 [3.21
lifetime (sf) shows the same substituent and solvent depen-
dency. The 10-alkyl chain has no effect on the lifetime values,
as shown by the sf values reported by these authors. The small
lifetime values (<1.0 ns) reported for the chlorinated deriva-
tives are also attributed to the chlorine atom, which can
enhance the spin-orbit coupling in the S1 fi S0 nonradiative or
the ISC processes (40).
The laser flash transient absorption spectrum of nitrogen-
saturated solutions of 10-alkylated phenothiazines 2 at high
laser intensities generally consists of an intense band with a
maximum between 460 and 480 nm, one near 530 nm and
another very broad one extending into the red region of the
spectrum (Table 3) (19–21). A self-quenching process of their
2-substitution, on the other hand, induces a larger variation
in the triplet lifetime values, as noted for the promazines in
methanol (19,20).
Photochemistry of CPZ 2b and CMPPH 3b
The photolysis of CPZ-HCl 2 was carried out in methanol,
ethanol, 1-propanol, 2-propanol and t-butanol. The photolysis
of CMPPH 3b was studied only in MeOH and EtOH. In all
these solvents the ground state molar absorption spectra of
CPZ-HCl show two bands with maxima around 258 and
312 nm (Table 4). The spectra of photolyzed CPZ-HCl in
methanol, ethanol and isopropanol present an isosbestic point
in the 304–312 nm range, although the isosbestic points of the
corresponding nonphotolyzed mixtures with PZ and the
alkoxy derivative are not the same. In methanol, the isosbestic
point is blueshifted with irradiation time. In tert-butanol, no
isosbestic point is observed, the only product is tertbutoxy-
promazine, and the photoreaction is slower than in the other
solvents. For CMPPH 3b, the isosbestic points are observed at
309 and 308 nm in MeOH and EtOH, respectively, and both
shift by 9 nm on irradiation. To be certain about the
participation of the triplet excited state in the dehalogenation
process of CPZ, some of these photoreactions were performed
in the presence of 0.35 mM the triplet quencher 1,3 cyclohex-
adiene (ET = 52.6 kcal mol)1 [42]), keeping all experimental
conditions constant. In this case, no photodestruction was
observed, confirming that the photodehalogenation occurs
triplet state was reported by Barra et al. (41). Self-quenching
s)1 were obtained
)1
rate constants in the order of 107–108
M
for several derivatives, in excellent agreement with those
previously reported for the nonsubstituted phenothiazines
(41). The triplet state molar absorption coefficients are of the
)1
order of 1.5–7.8 · 104
M
cm)1 (19,20,39). The intersystem
crossing quantum yields (uT) are in the range of 0.2–0.9 and
show some solvent dependence. For the chlorinated deriva-
tives, for instance, uT cannot be measured in aqueous
solutions, because their triplet state is rapidly quenched by a
proton transfer process (19). In methanol, on the other hand,
CPZ-triplet forms with an impressive quantum yield of 0.90.
The triplet lifetimes is a very solvent ⁄ substituent-sensitive
property too, but the 10-substitution has the least effect. Davis
et al. reported triplet lifetime values for PZ-HCl and CPZ-HCl
in isopropanol of 22.8 and 3.2 ls, respectively (5). The
3
from the CPZ*.
Table 4. Isosbestic point of the PZ-CPZ mixtures before and after photolysis, number of independent reactions (S), kinetic constant (k), percentage
of photoconversion of CPZ and formation of PZ, and quantum yield (u) for the production of PZ and destruction of CPZ.
Isosbestic point (nm)
k (mM s)1) · 105
%
u
Solvent
Before
After
S
PZ
CPZ
CPZ
PZ
PZ
CPZ
0.03†
MeOH
EtOH
1-PrOH
2-PrOH
t-BuOH
306
315
304
312
312
298–302*
306‡
303
2
1§
1
1
1
7.0
12.0
13.4
16.4
–
16.2
16.3
16.1
16.4
11.0
82
89
84
91
62
37
66
83
89
0
0.39
1.02
0.85
1.1
0.01
0.06
0.05
0.1
0.93
1.15
1.01
0.99
0.75
0.05||
0.06
0.04–
0.03
306
–
–
*Formation of a well-defined isosbestic point was not observed. For CMPPH 3b the isosbestic point is observed at 309 nm. †For CMPPH 3b,
u = 0.88
u = 1.01
0.07. ‡For CMPPH 3b, the isosbestic point is observed at 308 nm. §A value of 2 is obtained for long irradiation times. ||For CMPPH 3b,
0.02. –u = 0.12 (5).

Photochemistry and Photobiology, 2009, 85 167
The photolysis of CPZ-HCl in methanol produces PZ 1a
and MOPZ 1c in a 1:1 ratio [Eq. 6 with R1 = (CH2)3
N(CH3)2], as previously identified by Grant and Green (6).
This product distribution differs significantly from that corre-
sponding to the photochemistry of CPH 1b in the same
solvent, as only less than 2% of converts to the methoxy
derivative and other compounds. The photodestruction quan-
tum yield of 0.93 is in excellent agreement with the triplet
quantum yield of 0.90 reported by our group (19). As MOPZ is
produced in the same amount as PZ, uMOPZ = 0.39 and, using
uCPZ = uPZ + uMOPZ + uOther (Table 4), a quantum yield
of 0.15 is obtained for the formation of all other photoprod-
ucts. For CMPPH 3b, the main photoproduct in MeOH and
EtOH is the reduction product (MPPH, 3a). The correspond-
ing destruction quantum yields are slightly smaller than those
for CPZ (Table 4), but the quantum yield for the formation of
MPPH is higher than those for PZ. From the expected alkoxy
derivatives, only the methoxy one (MMPPH, 3c) formed with
a quantum yield high enough to allow its characterization
(u = 0.41). Obviously, the nitrogen at the alkyl amino chain
does not affect the photodestruction quantum yield of these
TCAs, but somehow influences the distribution of the photo-
products.
time. This is mainly due to the fact that, in these two solvents,
PZ is not the only photoproduct. This was confirmed by the
ED-EDQ analysis, which gave s = 2 for MeOH (data not
shown). In EtOH, linear ED-plots with zero intercepts and
s = 1 were obtained for irradiation times where the time–
concentration curve is linear. For longer times, on the other
hand, the linearity is lost and an s = 2 is also obtained (Fig. 4,
right). Exactly the same behavior was observed for CMPPH in
both solvents, although the linearity of the ED-plots in EtOH
is lost at very long irradiation times (t > 900 s). In the case of
1-PrOH and 2-PrOH, the destruction percentage of CPZ is
constant up to 720 and 420 s, respectively. Under this time
restriction, an s = 1 is found for both propanol isomers.
Thereafter, the percentage increases with irradiation time,
indicating that secondary processes are taking place. For
t-BuOH the calculated percentage of destruction is constant
for all irradiation intervals of 120 s for up to 960 s and no PZ
was detected. Therefore, an s = 1 value was obtained from the
Mauser analysis.
Davis et al. reported a photodestruction quantum yield of
only 0.12 for CPZ-HCl in isopropanol under anaerobic
conditions (5). They further reported the formation of PZ as
the major photoproduct and iPOPZ as a minor product. The
quantum yield determined in this work is in better agreement
with the triplet quantum yield of this drug. The large difference
between the destruction quantum yields is mainly due to
differences in the experimental conditions. For instance, Davis
The destruction percentage of CPZ-HCl in MeOH and
EtOH was not constant for a constant irradiation time interval
of 120 s. The unrecovered material percent was larger than
that observed for other alcohols and increased with irradiation
Figure 5. Mechanism proposed for the photodehalogenation of chlorinated phenothiazines in alcohols.

´
168 Carmelo Garcıa et al.
Table 5. Bond dissociation energy of the solvents used for the
photolysis of halogenated phenothiazines and the TCA-chlorine bond
(E[H•] = )0.500273 hartree; E[Cl•] = )460.136242 hartree; 1 Har-
tree = 627.5095 kcal mol)1).
alkoxy derivative, the R2O-radical formation should be
kinetically favored over the promazyl radical formation (43).
Table 5 contains the BDEs of the TCA–chlorine and the
solvent–H bonds determined with DFT. The ground and
triplet excited state BDEs for both TCA–chlorine bonds are,
respectively, ꢀ92 and ꢀ30 kcal mol)1 in all alcohols. This
indicates that the TCA–chlorine bond cleavages with the same
thermodynamic feasibility efficiency for both compounds,
especially in the excited state. These values further show that,
within the experimental and theoretical errors, the BDE for the
phenothiazine-chlorine bond is not affected by the alkylamino
chain. Therefore, the efficiency of the photodestruction of
CPH 1b, CPZ 2b, CMPPH 3b and any 2-chlorinated pheno-
thiazine derivative should be almost similar in all alcohols.
This is experimentally observed for each separate alcohol
included in this study, i.e. all TCAs have about the same
destruction quantum yield in the same solvent (Tables 2 and
4). Nevertheless, the BDE values of the TCAs cannot be used
to explain the differences in the TCA photodehalogenation
quantum yields in different solvents, nor the electron transfer
mechanism previously proposed by Bunce et al. (23) for this
process. The solvent-H BDEs, on the other hand, clearly
explain why it is easier for the alpha hydrogen to be
abstracted than the hydroxyl hydrogen, especially for alcohols
with long and branched alkyl chains. The corresponding
energy for this R2O–H bond also increases with the alkyl
chain, making the formation of the alkoxy derivative less
competitive. The BDE of the a-hydrogen also increases with
the chain length, but this increase is compensated by the
stability of the corresponding radical. In summary, the
formation of the alkoxy product is very inefficient and can
only compete with the reduction one if the solvent radicals are
not stable, as is the case of methanol.
E (UB + HF ) LYP)
hartrees
BDE (kcal mol)1
)
•
R
R–X
R–X
This work Exp.*
)115.714405
)115.714405
)115.050462
)115.062993
102.7
94.8
102
92
)155.034287
)155.034287
)154.370492
)154.375316
102.6
99.6
103
–
)194.348026
)194.348026
)193.685044
)193.688468
102.1
100.0
103
–
)194.353452
)194.353452
)193.688795
)193.697975
103.2
97.4
103
94
)233.670958
)233.670958 )232.997898†
)233.006172
103.2
108.4
102
–
)1375.239118 )914.955531
)1375.140165 )914.955531
)1627.126232 )1166.842675
)1627.027190 )1166.842675
92.5
30.4
92.4
30.3
–
–
–
–
*Values from references (28–31). †Calculated for the beta hydrogen.
et al. used 3.0 mM CPZ-HCl solutions, which have a large
absorption at wavelengths >300 nm. This obviously intro-
duces primary and secondary filter effects. Moreover, they
measured the formation of HCl assuming that the only
photoprocess is the dehalogenation with no alkoxy derivative
formation. In this work, on the other hand, the destruction of
the drug was measured using optically diluted solutions
(abs < 1.0 at 313 nm) without any assumption regarding the
mechanism. This approach allows a better quantification of the
destruction process.
The mechanism proposed for the photodehalogenation in
Fig. 5 accounts very well for all experimental results on the
photochemistry of CPH (1b), CPZ (2b) and CMPPH (3b) in
terms of the photoproducts, their photodestruction quantum
yields and the effect of the solvent on the product
distribution. Table 5 shows that the amount of decomposed
CPZ converted to PZ increases with the length of the
alcohol alky chain. The difference between the percentage of
CPZ destruction and PZ formation rapidly drops to zero,
indicating that the mechanism of formation of PZ is
definitely affected by the R-group of the alcohol. This is
explained in terms of the stability of the alcohol radicals and
the OH-BDE (see next section). t-Butanol produces no PZ,
as it has no alpha hydrogen to be abstracted.
CONCLUSIONS
To solve the controversy regarding the so many different
values reported for the photodestruction quantum yield of
halogenated phenothiazines, a methodology was developed in
this work to determine the quantum yields using a monochro-
matic 313 nm light for irradiation of optically diluted solutions
and a GC ‘‘total quantification’’ procedure for the determina-
tion of the quantum yield (44). All these parameters are very
important because: (1) the light intensity and the photoprocess
quantum yield strongly depend on the selected wavelength;
(2) by using an excitation wavelength range, several transitions
can be excited at once; (3) by using an excitation wavelength
range, there is no easy way to determine the absorbed light
intensity; (4) irradiation of concentrated solutions induces
filter effects; and (5) the total drug photodestruction can be
measured without assuming a specific mechanism or a specific
product distribution.
The results of this approach indicate that: (1) according to
the values of the s parameter, the photodestruction of
2-chlorinated TCAs consists of only one reduction reaction.
The effectiveness of this reaction is determined by the BDE
and the radical stability of the participating partner. Under
special conditions of prolonged irradiation and ⁄ or small
alcohols the formation of the corresponding alkoxide can be
observed; (2) the larger the alkyl chain of the alcohol, the lesser
the amount of alkoxide photoproduct formed, in agreement
Quantum chemical calculations
The photodestruction process of CPZ is effective and PZ is the
main product, only if both the promazyl and the solvent
radicals are relatively stable (Eq. 3) and ⁄ or the corresponding
BDEs are relative low. In other words, for the formation of the

Photochemistry and Photobiology, 2009, 85 169
17. Schoonderwoerd, S., G. Beijerbergen, V. Henegouwen and
J. Leijendijk (1988) Photobinding of chlorpromazine and its
sulfoxide in vitro and in vivo. Photochem. Photobiol. 48, 621–626.
18. Kochevar, I. E., C. Garcia and N. E. Geacintov (1998) Photo-
addition to DNA by nonintercalated chlorpromazine molecules.
Photochem. Photobiol. 68(5), 692–697.
with the calculated differences in the solvent BDEs and the
mechanism of the photonucleophilic substitution proposed by
Grant and Green (6). The biggest effect is introduced by
tBuOH, in which the photodestruction quantum yields is small
and no reduction product was obtained; (3) the photodestruc-
tion quantum yields of the TCAs depend more on the solvent
than on the alkylamino chain at the 10-position. Therefore, the
electron transfer mechanism proposed by Bunce et al. (23) is
not correct; and (4) the phototoxicity of the phenothiazine-
type drugs is not directly determined by their dehalogenation,
as their constant photodestruction quantum yield cannot so far
account for their differences in toxicity strength. Nevertheless,
these species might somehow induce this side effect through a
mechanism involving membrane components in vivo (45).
19. Garcıa, C., G. A. Smith, M. Grant, I. E. Kochevar and
´
R. W. Redmond (1995) Mechanism and solvent dependence for
the photoionization of promazine and chlorpromazine. J. Am.
Chem. Soc. 117, 10871–10877.
20. Garcı
´
V. Sa
˜
a, C., R. Oyola, L. E. Pinero, R. Arce, J. Silva and
nchez (2005) Substitution and solvent effect on the photo-
´
physical properties of several series of 10-alkylated phenothiazine
derivatives. J. Phys. Chem. A 109, 3360–3371.
21. Oyola, R. (2001) Design, construction and use of a nanosecond
laser transient absorption spectrokinetic system for the study of
the photochemical and photophysical properties of relevant bio-
logical molecules: dTpA, dApT and Tricyclic antidepressive drugs.
Ph.D. thesis, University of Puerto Rico, Rio Piedras.
22. Felmeister, A. and C. A. Discher (1964) Photodegradation of
chlorpromazine hydrochloride. J. Pharm. Sci. 53, 756–762.
23. Bunce, N. J., Y. Kumar and L. Ravanal (1979) Phototoxicity of
chlorpromazine. J. Med. Chem. 22, 202–204.
Acknowledgements—This work has been supported in part by NIH-
MBRS grant SO6GM08216 to UPR-Humacao. We specially thank
Melvin De Jesus and Jorge Castillo for the support with the NMR
spectroscopy and the GC-MS chromatography.
24. Moore, D. and S. Tamat (1980) Photosensitization by drugs:
Photolysis of some chlorine-containing drugs. J. Pharm. Phar-
macol. 32, 172–177.
REFERENCES
25. Kricka, L. J. and A. Ledwith (1972) Reactions of condensed N-
heteroaromatic molecules. Part I. Alkylation by thallium(I)eth-
oxide. J. Chem. Soc. Perkin Trans. 1, 2292–2293.
1. Kochevar, I. E. (1981) Phototoxicity mechanism: Chlorpromazine
photosensitized damage to DNA and cell membranes. J. Invest.
Dermatol. 76, 59–64.
26. Wang, Z. and W. G. McGimpsey (1993) One and two laser
photochemistry of iminodibenzyl. J. Phys. Chem. 97, 9668–9672.
27. Kuhn, H. J., S. E. Braslavsky and R. Schmidt (1989) Chemical
actinometry. Pure Appl. Chem. 61(2), 187–210.
2. Kochevar, I. E. (1987) Mechanisms of drug photosensitization.
Photochem. Photobiol. 45, 891–895.
3. Kochevar, I. E. and J. Hom (1983) Photoproducts of chlor-
promazine that cause red blood cell lysis. Photochem. Photobiol.
37, 163–168.
˜
28. Garcıa, C., R. Oyola, L. E. Pinero, N. Cruz, F. Alejando, R. Arce
´
and I. Nieves (2002) Photophysical, electrochemical and theo-
retical study of protriptyline in several solvents. J. Phys. Chem. B
106, 9794–9801.
4. Curry, S. H. (1970) Plasma protein binding of chlorpromazine.
J. Pharm. Pharmacol. 22, 193–197.
5. Davis, A. K., S. Navaratnam and G. O. Phillips (1976) Photo-
chemistry of chlorpromazine (2-chloro-N-(3dimethylamino pro-
pyl) phenothiazine) in propan-2-ol solution. J. Chem. Soc. Perkin
Trans. II 25, 25–29.
29. Benson, S. W. (1976) Thermochemical Kinetics: Methods for the
Estimation of Thermochemical Data and Rate Parameter. John
Wiley & Sons, New York.
30. McMillen, D. F. and D. M. Golden (1982) Hydrocarbon bond
dissociation energies. Annu. Rev. Phys. Chem. 33, 493–532.
31. (NISTWebBook ) (2005) NIST Standard Reference Database
Number 69. http://webbook.nist.gov/chemistry. Accessed June
2008.
32. Wedenejew, V. W., L. W. Gurwitsch, W. H. Kondratjew,
W. A. Medwedew and E. L. Frankenwitsch (1971) Energien
chemischer bindungen, Ionisationspotentiale und Elektronenaffinitae
VEB Deutcher Verlag Fuer Grundstoffindustrie. Leipzig, Germany.
33. Mauser, H. (1974) Formale Kinetic: Experimentelle Methoden der
6. Grant, F. W. and J. Green (1972) Phototoxicity and photonucle-
ophilic aromatic substitution in chlorpromazine. Toxic. Appl.
Pharmacol. 23, 71–74.
7. Epstein, S. (1968) Chlorpromazine photosensitivity, phototoxic
and photoallergic reactions. Arch. Dermatol. 98, 354–363.
8. Kahn, G. and B. P. Davis (1970) In vitro studies on longwave
ultraviolet light dependent reactions of the skin photosensitizer
chlorpromazine with nucleic acids. Purines and pyrimidines.
J. Invest. Dermatol. 55, 47–53.
9. Ciulla, T. A., G. A. Epling and I. E. Kochevar (1986) Photo-
addition of chlorpromazine to guanosine-5’-monophosphate.
Photochem. Photobiol. 43, 607–613.
10. Fujita, H., F. Yanagisawa, A. Endo and K. Suzuki (1980) Pho-
toreactivity of chlorpromazine with native DNA in an aqueous
solution. J. Radiat. Res. 21, 279–287.
11. Fujita, H., A. Endo and K. Suzuki (1981) Inactivation of bacte-
riophage lambda by near ultraviolet irradiation in the presence of
chlorpromazine. Photochem. Photobiol. 33, 215–222.
12. Iwaoka, T. and M. Kondo (1974) Mechanistic studies on the
photooxidation of chlorpromazine in water and ethanol. Bull.
Chem. Soc. Japan 47, 980–986.
13. Kochevar, I. E., F. Chung and A. M. Jeffrey (1984) Photo-
addition of chlorpromazine to DNA. Chem. Biol. Interact. 51,
273–284.
14. Ljunggren, B. and H. Moller (1977) Phenothiazine phototoxicity:
An experimental study on chlorpromazine and its metabolites.
J. Invest. Dermatol. 68, 313–317.
15. Buettner, G. R., A. G. Motten, R. D. Hall and C. F. Chignell
(1986) Free radical production by chlorpromazine sulfoxide.
An ESR spin trapping and flash photolysis study. Photochem.
Photobiol. 44, 5–10.
Physik und der Chemie. Bertelsmann Universitatsverlag 1, 297–363.
¨
34. Daniil, D., G. Gauglitz and H. Meier (1977) Kinetic investigation
of the photolysis of 2-oxo-2,5-dihydro-1,3,4-oxadiazoles. Photo-
chem. Photobiol. 26, 225–229.
35. Mauser, H. and G. Gauglitz (1998) Photokinetics: Theoretical
fundamentals and applications. In Comprehensive Chemical
Kinetics, Vol. 36 (Edited by R. G. Compton and G. Hancock), pp.
342–369. Elsevier Science Publishers, New York.
36. Aaron, J., M. Maafi, C. Kersebet, C. Parkanyi, A. M. Shafik and
´ ´
N. Motohashi (1996) A solvatochromic study of new benzo [a]
phenothiazines for the determination of dipole moments and
specific solute solvent interactions in the first excited singlet state.
J. Photochem. Photobiol. A, Chem. 101, 127–136.
37. Casarotto, M. G. and D. J. Craick (2001) Ring flexibility within
tricyclic antidepressant drugs. J. Pharm. Pharmac. Sci. 90, 713–721.
38. Casarotto, M. G., D. J. Craik and S. L. Munro (2005) 13C NMR
studies of the solution molecular dynamics of Tricyclic antide-
pressants. Magn. Reson. Chem. 28, 533–540.
39. Elisei, F., L. Latterini, G. G. Aloisi, U. Mazzucato, G. Viola, G.
Miolo, D. Vedaldi and F. Dall’Acqua (2002) Excited state prop-
erties and in vitro phototoxicity studies of three phenothiazine
derivatives. Photochem. Photobiol. 75(1), 11–21.
16. Rosenthal, I., E. BenHur, A. Prager and E. Riklis (1978)
Photochemical reactions of chlorpromazine: Chemical and bio-
chemical implications. Photochem. Photobiol. 28, 591–594.
40. Turro, N J, I. V. Khudyakov and H. v. Willigen (1995) Photo-
ionization of phenothiazine: EPR detection of reactions of the

´
170 Carmelo Garcıa et al.
polarized solvated electron. J. Am. Chem. Soc. 117(49), 12273–
12280.
43. Wright, J. S., E. R. Johnson and G. A. DiLabio (2001) Predicting
the activity of phenolic antioxidants: Theoretical method, analysis
of substituent effects, and application to major families of an-
tioxidants. J. Am. Chem. Soc. 123, 1173–1183.
41. Barra, M., G. S. Calabrese, M. T. Allen, R. W. Redmond, R.
Sinta, A. A. Lamola, R. D. Small and J. Scaiano (1991) Photo-
physical and photochemical studies of phenothiazine and some
derivatives: Explanatory studies of novel photosensitizers for
photoresist technology. Chem. Mater. 3, 610–616.
42. Toth, M. (1980) Triplet-triplet absorption of cyclohexadiene-1,3
and ergosterol-Sensitized total ground state depletion of dienes by
triplet energy transfer from the ruby laser excited 2fluorenyl-
phenylketone. Chem. Phys. Lett. 46, 437–443.
˜
44. Pinero, L. E. (2006) Synthesis, characterization, photophysics,
and photochemistry of 2chlorinated phenothiazine derivatives
in several solvents. Master thesis, University of Puerto Rico, Rio
Piedras, Puerto Rico.
45. Chignell, C. F. (1972) Application of physicochemical and analytic
techniques to the study of drug interactions with biological
systems. CRC Crit. Rev. Toxicol. 413–465.