Photogeneration and reactivity of naphthoquinone methides as purine selective DNA alkylating agents

Article abstract of DOI:10.1021/ja1063857

A one-step protecting-group-free synthesis of both 6-hydroxy-naphthalene-2- carbaldehyde and the bifunctional binaphthalenyl derivative afforded 6-hydroxymethylnaphthalen-2-ol, 6-methylaminomethyl-naphthalen-2-ol, [(2-hydroxy-3-naphthyl)methyl]trimethyl ammonium iodide, and a small library of bifunctional binol analogues in good yields. Irradiation of naphthol quaternary ammonium salt and binol-derivatives (X = OH, NHR, NMe₃⁺, OCOCH₃, and l-proline) at 310 and 360 nm resulted in the photogeneration of the 2,6-naphthoquinone-6-methide (NQM) and binol quinone methide analogues (BQMs) by a water-mediated excited-state proton transfer (ESPT). The hydration, the mono- and bis-alkylation reactions of morpholine and 2-ethanethiol, as N and S prototype nucleophiles, by the transient NQM (λ_max 310, 330 nm) and BQMs (λ_max 360 nm) were investigated in water by product distribution analysis and laser flash photolysis (LFP). Both the photogeneration and the reactivity of NQM and BQMs exhibited striking differences. BQMs were at least 2 orders of magnitude more reactive than NQM, and they were generated much more efficiently from a greater variety of photoprecursors including the hydroxymethyl, quaternary ammonium salt and several binol-amino acids. On the contrary, the only efficient precursor of NQM was the quaternary ammonium salt. All water-soluble BQM precursors were further investigated for their ability to alkylate and cross-link plasmid DNA and oligonucleotides by gel electrophoresis: the BQMs were more efficient than the isomeric o-BQM (binol quinone methide analogue of 2,3-naphthoquinone-3- methide). Sequence analysis by gel electrophoresis, HPLC, and MS showed that the alkylation occurred at purines, with a preference for guanine. In particular, a BQM was able to alkylate N7 of guanines resulting in depurination at the oligonucleotide level, and ribose loss at the nucleotide level. The photoreactivity of BQM precursors translated into photocytotoxic and cytotoxic effects on two human cancer cell lines: in particular, one compound showed promising selectivity index on both cell lines.

Full text of DOI:10.1021/ja1063857

Published on Web 09/23/2010
Photogeneration and Reactivity of Naphthoquinone Methides
as Purine Selective DNA Alkylating Agents
Daniela Verga,† Matteo Nadai,‡ Filippo Doria,† Claudia Percivalle,†
Marco Di Antonio,§ Manlio Palumbo,§ Sara N. Richter,*,‡ and Mauro Freccero*,†
Dipartimento di Chimica Organica, UniVersita` di PaVia, V. le Taramelli 10, 27100 PaVia, Italy,
Dipartimento di Istologia, Microbiologia e Biotecnologie Mediche, UniVersita` di PadoVa,
Via Gabelli, 63, 35121 PadoVa, Italy, and Dipartimento di Scienze Farmaceutiche,
UniVersita` di PadoVa, Via Marzolo, 5, 35131 PadoVa, Italy
Received July 19, 2010; E-mail: mauro.freccero@unipv.it (M.F.); sara.richter@unipd.it (S.R.)
Abstract: A one-step protecting-group-free synthesis of both 6-hydroxy-naphthalene-2-carbaldehyde and
the bifunctional binaphthalenyl derivative afforded 6-hydroxymethylnaphthalen-2-ol, 6-methylaminomethyl-
naphthalen-2-ol, [(2-hydroxy-3-naphthyl)methyl]trimethyl ammonium iodide, and a small library of bifunctional
binol analogues in good yields. Irradiation of naphthol quaternary ammonium salt and binol-derivatives (X
) OH, NHR, NMe3+, OCOCH3, and L-proline) at 310 and 360 nm resulted in the photogeneration of the
2,6-naphthoquinone-6-methide (NQM) and binol quinone methide analogues (BQMs) by a water-mediated
excited-state proton transfer (ESPT). The hydration, the mono- and bis-alkylation reactions of morpholine
and 2-ethanethiol, as N and S prototype nucleophiles, by the transient NQM (λmax 310, 330 nm) and BQMs
(λmax 360 nm) were investigated in water by product distribution analysis and laser flash photolysis (LFP).
Both the photogeneration and the reactivity of NQM and BQMs exhibited striking differences. BQMs were
at least 2 orders of magnitude more reactive than NQM, and they were generated much more efficiently
from a greater variety of photoprecursors including the hydroxymethyl, quaternary ammonium salt and
several binol-amino acids. On the contrary, the only efficient precursor of NQM was the quaternary
ammonium salt. All water-soluble BQM precursors were further investigated for their ability to alkylate and
cross-link plasmid DNA and oligonucleotides by gel electrophoresis: the BQMs were more efficient than
the isomeric o-BQM (binol quinone methide analogue of 2,3-naphthoquinone-3-methide). Sequence analysis
by gel electrophoresis, HPLC, and MS showed that the alkylation occurred at purines, with a preference
for guanine. In particular, a BQM was able to alkylate N7 of guanines resulting in depurination at the
oligonucleotide level, and ribose loss at the nucleotide level. The photoreactivity of BQM precursors
translated into photocytotoxic and cytotoxic effects on two human cancer cell lines: in particular, one
compound showed promising selectivity index on both cell lines.
antitumor agents, such as aziridinomitosenes4 and bizelesin,5
nitrogen mustard derivatives,6 and cis-platinum-like organome-
Introduction
tallics.7 The photoactivated agents are probably the less
developed class, with the exception of psoralens.8 Only quite
recently bifunctional quinone methides (QMs) have been
DNA interstrand cross-linking (ISC) effectively inhibits both
DNA replication and gene expression by preventing the melting
of the two strands. Such an event, shutting down the DNA
replication process, represents by far the most cytotoxic of all
the alkylations. Therefore, ISC has considerable potential in
molecular biology and human medicine for pharmacological
applications.1 The mechanism of action of interstrand cross-
linking agents, including their activation, and the design of new
types of cross-linking agents has been extensively investigated.
The compounds capable of DNA ISC include antitumor
antibiotics, such as mitomycins,2 azinomycins,3 and synthetic
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† Universita` di Pavia.
‡ Dipartimento di Istologia, Universita` di Padova.
§ Dipartimento di Scienze Farmaceutiche, Universita` di Padova.
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10.1021/ja1063857  2010 American Chemical Society
J. AM. CHEM. SOC. 2010, 132, 14625–14637 14625

A R T I C L E S
Verga et al.
Scheme 2. Synthesis of the 6-CH2X-Substituted-2-naphthol- and
Scheme 1
Binol-Analogues As Precursors of p-NQM and p-BQM Quinone
Methides (p-NQMP and p-BQMP)
successfully used to accomplish nucleoside alkylation and DNA
ISC9 by photochemical,10 fluoride-induced activations,11 thermal
digestion,12 and oxidation.13 Concerning the photochemical
activation, o-QM and p-QM have been generated by Wan,14
Kresge,15 Popik,16 and our group10c,d,12,17 using benzyl alcohols
(X ) OH), Mannich bases (X ) NMe2), and quaternary
ammonium salts (X ) NMe3+I-) (Scheme 1).
It has been shown that the efficiency of the photoactivation
process and the water solubility of the quinone methide
precursors (QMPs) are both a function of the leaving groups
(X).18 In fact, quaternary ammonium salts of phenolic Mannich
bases (Scheme 1, X ) NMe3+I-) are better and preferable
QMPs than the alcohol analogues (X ) OH, photochemical
quantum yield Φ 0.98 vs 0.23).19 The prototype o-QMPs and
p-QMPs have little absorbance above 310 nm, and therefore
they are not suitable for biological applications as photoactivated
alkylating agents under mild conditions. The absorbance of
naphthols derivatives and binol analogues as quinone methide
precursors (o-NQMPs and o-BQMPs, respectively, see Scheme
1) on the other hand, extends beyond 310 nm, and they still
exhibit excellent solubility under physiological conditions. In
fact, we have shown that o-BQMPs, such as the bis-quaternary
ammonium salt, are capable of undergoing bis-alkylation in
water and DNA cross-linking with promising potency by
UV-vis photoactivation.10d The high efficiency of the o-QM
photogeneration has been associated to the higher excited state
acidity of phenol, 2-naphthol (pKa* 2.8),20 and 3-hydroxy-2-
naphthalenemethanol (pKa* 0.77),16a which should facilitate the
excited state intramolecular proton transfer (ESIPT) to the
leaving group X at the ortho position. In the case of p-QMPs
the distance between the phenol and the hydroxyl group is too
large to undergo ESIPT, and although excited state proton
transfer (ESPT) can occur in the presence of a protic solvent
the efficiency for the p-QM photogeneration is often much
lower.21 This is probably the reason why p-QMs have been
almost completely neglected in the recent literature as photo-
activatable alkylating agents. In this paper, we report (i) the
efficient generation of 2,6-naphthoquinone-6-methide (p-NQM)
and binol quinone methide analogues (p-BQM) (Scheme 2) by
irradiation of suitable precursors in water, (ii) the original
protecting-group-free synthesis of both p-NQMPs, and p-
BQMPs, (iii) the ability of p-BQMs to efficiently alkylate and
cross-link single- (ss) and double-stranded (ds) DNA at the
purine level, and (iv) their promising photocytotoxic versus
cytotoxic properties.
(9) Rokita, S. E. In Wiley Series on Reactive Intermediates in Chemistry
and Biology. Quinone Methides; Rokita, S. E., Eds.; Wiley, Hoboken,
NJ, 2009; pp 297-323.
(10) (a) Lukeman, M. In Wiley Series on Reactive Intermediates in
Chemistry and Biology. Quinone Methides; Rokita, S. E., Eds.; Wiley:
Hoboken, NJ, 2009; pp 1-25. (b) Doria, F.; Richter, S. N.; Nadai,
M.; Colloredo-Mels, S.; Mella, M.; Palumbo, M.; Freccero, M. J. Med.
Chem. 2007, 50, 6570–6579. (c) Verga, D.; Richter, S. N.; Palumbo,
M.; Gandolfi, R.; Freccero, M. Org. Biomol. Chem. 2007, 5, 233–
235. (d) Richter, S.; Maggi, S.; Colloredo-Mels, S.; Palumbo, M.;
Freccero, M. J. Am. Chem. Soc. 2004, 126, 13973–13979. (e) Wang,
P.; Liu, R.; Wu, X. J.; Ma, H. J.; Cao, X. P.; Zhou, P.; Zhang, J. Y.;
Weng, X. C.; Zhang, X. L.; Qi, J.; Zhou, X.; Weng, L. H. J. Am.
Chem. Soc. 2003, 125, 1116–1117.
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47, 1291–1293. (b) Pande, P.; Shearer, J.; Yang, J.; Greenberg, W. A.;
Rokita, S. E. J. Am. Chem. Soc. 1999, 121, 6773–6779.
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Sissi, C.; Palumbo, M.; Freccero, M. J. Am. Chem. Soc. 2009, 131,
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Van Breemen, R. B.; Thatcher, G. R. J.; Bolton, J. L. Chem. Res.
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Results and Discussion
Synthesis of the Naphtho- and Binol-Quinone Methide
Precursors p-NQMP and p-BQMPs. Two small libraries of
compounds (3a-3d and 4a-4j, Scheme 3) have been investi-
gated as photoprecursors of the 2,6-naphthoquinone-6-methide
(p-NQM) and binol quinone methide analogues (p-BQMs) (p-
NQMP and p-BQMPs, respectively Scheme 2). The key
intermediates for the synthesis are the 6-hydroxynaphthalene-
2-carbaldehyde (1) and the 2,2′-dihydroxy-[1,1′]binaphthalenyl-
6,6′-dicarbaldehyde (2). Although these aldehydes are known
in the literature, they have always been prepared through a four-
(14) (a) Basaric, N.; Zabcic, I.; Mlinaric-Majerski, K.; Wan, P. J. Org.
Chem. 2010, 75, 102–116. (b) Flegel, M.; Lukeman, M.; Huck, L.;
Wan, P. J. Am. Chem. Soc. 2004, 126, 7890–7897. (c) Brousmiche,
D. W.; Xu, M.; Lukeman, M.; Wan, P. J. Am. Chem. Soc. 2003, 125,
12961–12970.
(15) (a) Chang, J. A.; Kresge, A. J.; Zhan, H.-Q.; Zhu, Y. J. Phy. Org.
Chem. 2004, 17, 579–585. (b) Chiang, Y.; Kresge, A. J.; Zhu, Y. J. Am.
Chem. Soc. 2003, 125, 6349–6356.
(16) (a) Arumugam, S.; Popik, V. V. J. Am. Chem. Soc. 2009, 131, 11892–
11899. (b) Kostikov, A. P.; Malashikhina, N.; Popik, V. V. J. Org.
Chem. 2009, 74, 1802–1804.
(17) Colloredo-Mels, S.; Doria, F.; Verga, D.; Freccero, M. J. Org. Chem.
2006, 71, 3889–3895.
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Mitchell, C. H.; Freccero, M.; Rokita, S. E. J. Am. Chem. Soc. 2006,
128, 11940–11947.
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DNA Cross-Linking by Photogenerated Quinone Methides
A R T I C L E S
Scheme 3. Synthesis of the 6-Hydroxynaphthalene-2-carbaldehyde (1), the 2,2′-Dihydroxy-[1,1′]binaphthalenyl-6,6′-dicarbaldehyde (2), and
the p-QMPs 3a-3d and 4a-4ja
a (i) n-BuLi 4.4 equiv, THF, -78°C, 5 h. (ii) DMF, -50°C 1 h. -50 °C f 0 °C, 1 h. Workup H2O/HCl, 0°C (iii) NaBH4, EtOH/THF ) 7:3, 5 h. (iv)
Morpholine (or proline methyl and t-butyl esters and valine methyl ester) NaBH(AcO)3, EtOH, 24 h, r.t. NH2Me (or NHMe2), CH3OH, 20 h, r.t. and then
NaBH4 r.t. 3 h. (v) CH3I, CH2Cl2 r.t. 24 h. (vi) n-BuLi 8.0 equiv, THF, -78°C, 5 h. (vii) CH3COOH, HBr, r.t.. 5 h. (viii) NMe3, THF, r.t., 15 min. (ix)
CH3COOH, ∆, 48 h. (x) CH2Cl2/CF3COOH/Et3SiH ) 20.5:10:4, r.t. 1 h.
step synthesis.22 We have been able to synthesize 1 and 2 with
a novel one-step protecting-group-free synthetic protocol, in
quantitative yields, starting from 6-bromo-2-naphthol and 6,6′-
dibromobinol (Scheme 3). The lithiation and the following
formylation by DMF have been performed on both racemic and
enantiomerically pure (R)- and (S)-6,6′-dibromobinols. The 6,6′-
dibromobinol enantiomers have been prepared starting from
enantiomerically pure [1,1′]binaphthalenyl-2,2′-diol (binols)
according to published procedures, with 98% ee.23 Both
enantiomers react with retention of the configuration giving
(R)-2 and (S)-2 at -78 °C. In fact, (R)-2 and (S)-2 have been
recovered from the formylation workup at 0 °C, with a 96%
and 94% ee, respectively, measured by chiral HPLC (chiralcel
OD-H 0.46 × 25 cm).
methylation and from an anhydrous THF solution of 6,6′-bis-
bromomethyl-[1,1′]binaphthalenyl-2,2′-diol (4k), bubbling gas-
eous NMe3.
Photoreactivity of p-NQMPs. The possibility to exploit 3a-3d
and 4a-4j as phototriggerable mono- and bis-alkylating agents,
respectively, through the generation of transient QMs has been
investigated in aqueous acetonitrile solutions (water/ACN )
7:3), irradiating at 310 nm (1 × 10-3 M, in a photoreactor with
4 or 2 lamps 15 W, ca. 25 °C, 10 and 30 min, under Ar and O2
purged solutions), in the presence of morpholine or 2-ethaneth-
iol, as prototypes of water-soluble nucleophiles.
Although the naphthol 3a was quantitatively recovered after
3 h of irradiation, photolysis of the amine 3b in the presence of
2-ethanethiol gave 3f as the main adduct (in low yield), with
traces of 3a and 3e (Table 1). The photoactivation afforded a
quantitative conversion of 3c into the hydration adduct 3a. The
same photochemical reaction performed in the presence of
2-ethanethiol produced a mixture containing the alkylated adduct
3e, together with 3a and 3f in lower yields (Table 1). The
quaternary ammonium salt was the most photoreactive naphthol,
generating the hydration adduct 3a or the alkylation adduct 3e,
without or with 2-ethanethiol, respectively.
Both the aldehydes 1 and 2 have been quantitatively reduced
to the alcohols 3a and 4a, by NaBH4 in EtOH/THF ) 7:3, or
transformed into the amines 3b-c and 4b-c by a one-pot
reductive amination performed in methanol. Similarly, 4d-i
have been synthesized in good yield using NaBH(AcO)3, in
ethanol as solvent. 4d has been recovered and purified as
hydrochloride. The binol amino acid (binolam) 4h has been
obtained as hydrochloride by acid deprotection of 4g in
quantitative yield. The reductive amination of (R)-2 using proline
methyl and t-butyl esters and valine methyl ester give the
diastereoisomers (R,S,S)-4f, 4g, and 4i in good yields (>75%).
The quaternary ammonium salts 3d and 4e have been synthe-
sized from 6-dimethylaminomethylnaphthalen-2-ol (3b) by CH3I
The product distribution was slightly affected by the presence
of oxygen. In fact, the irradiation of 3d showed a higher
conversion into the alkylated adduct 3e, in air-saturated solution,
with the generation of the aldehyde 1 as a byproduct. A longer
irradiation time (g2 h) revealed that the adduct 3e was also
photoreactive, generating 3f or 1 as the main products in sloppy
reaction mixtures, performed in the absence or in the presence
of oxygen, respectively. Therefore, the photoproducts 1 and 3f
were both secondary photoproducts arising from the irradiation
(22) Dong, C.; Zhang, J.; Zheng, W.; Zhang, L.; Yu, Z.; Choi, M. C. K.;
Chan, A. S. C. Tetrahedron: Asymmetry 2000, 11, 2449–2454.
(23) Cui, Y.; Evans, O. R.; Ngo, H. L.; White, P. S.; Lin, W. Angew. Chem.,
Int. Ed. 2002, 41, 1159–1162.
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A R T I C L E S
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Table 1. Photoreactivity of Naphthols in Water/Acetonitrile (7:3) with and without Added Nucleophiles (Morpholine and 2-Ethanethiol)
b
a [3b-3e] ) 10-3 M. [HNu] ) 5 × 10-2 M. c Measured by HPLC. d Irradiated at 310 nm in a photoreactor with 4 lamps, 15 W. H2O/CH3CN =
7:3, T = 25 °C. e Argon purged for 5 min. f Oxygen purged for 10 s.
of the primary adduct 3e. Their formation suggests the genera-
tion of 6-naphthylmethyl radical as an intermediate in the
photolysis of 3e. The product analysis of the photolyzed
mixtures has been performed by HPLC using purified samples,
isolated from preparative photolysis, as standards (Table 1).
Efficiency of the QM-Photogeneration. The quantum yields
(Φ ) photoproducts/absorbed photons) listed in Table 3 revealed
that the efficiency of the QMPs photoactivation process is
affected by the nature of both the chromophore (naphthol vs
binol) and the leaving groups (Scheme 4). Indeed, the binol
quaternary ammonium salt 4e was twice more efficient than
the naphthol analogue 3d. Furthermore, the highest photoeffi-
ciency of the binol 4a (Φ ) 0.87) contrasts to the lack of
reactivity of the naphthol 3a, which was photostable. Unexpect-
edly, 4a exhibited a higher efficiency in the generation of the
p-BQM-4a compared to the quaternary ammonium salt 4e,
which still releases the monoalkylating p-BQM-4e with high
quantum yield (Table 3). Therefore, the lower conversion of
4a in comparison to 4e, measured from product distribution
analysis as a function of the irradiation time (Table 2), has to
be ascribed to a lower absorption (see Supporting Information).
Although the generation of the second quinone methide p-
BQM-5 (Scheme 4) is always less efficient than the first one
(p-BQM-4), the QMP 5e is almost twice more efficient than
5a (Table 3).
Photoreactivity of p-BQMPs. Similarly 4a-4j were irradiated
at 310 nm in water/ACN (7:3 or 1:1) and in the presence of
2-ethanethiol or morpholine (Table 2). The irradiation of 4d-f,
4j-k, performed in the absence of an added nucleophile,
afforded the alcohol 4a from good to modest yields (Table 2).
4b and 4c are essentially photostable for 3 h. Photolysis of 4a
and 4e in aqueous solutions containing 2-ethanethiol gave the
bifunctional thioether 7, together with the monofunctional
derivatives 5a and 5e. The latter were clearly precursors of the
former, which became the main adduct at long (>2 h) irradiation
times (Table 2). Similarly, the irradiation of 4a in the presence
of morpholine gave a clean conversion (75%) into 6a and 4d,
after 1 h of irradiation. These results suggest that the 6,6′-CH2X-
bis-substituted binols 4a, 4d, 4e, 4h, and 4j act as efficient
phototriggerable mono- and bis-alkylating agents, through a
stepwise generation of two monoalkylating conjugated QMs
(such as p-BQM-4 and p-BQM-5 in Scheme 4). The binolam
4f was much less efficient as a photoalkylating agent than the
above, and although it is slightly photoreactive 4i did not
undergo a clean photohydration in aqueous solution.
Comparison between p-BQMPs and p-NQMPs. The product
distribution analysis and quantum yield measurements suggest
that in general p-BQMPs are much better photoalkylating agents
than p-NQMPs. Such a large difference in reactivity may be
related to the intramolecular H-bonding in the binol derivatives,
which is lacking in the naphthol analogues. Such interaction
should improve the efficiency of the ESIPT for the binols in
comparison to the naphthols, which can only undergo intermo-
All the QM-precursors 4a-4j investigated and the resulting
alkylation adducts 5a, 5e, 6a, 7, and 8 are thermodynamically
stable upon digestion at 40 °C for several days.
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DNA Cross-Linking by Photogenerated Quinone Methides
A R T I C L E S
Table 2. Photoreactivity of Binols in Water/Acetonitrile (7:3) with and without Added Nucleophiles (Morpholine and 2-Ethanethiol)
b
a [4a, 4d-j] ) 10-3 M. [HNu] ) 5 × 10-2 M. c Reactant conversion measured by HPLC. d Irradiated at 310 nm in a photoreactor with 2 lamps, 15
W. H2O/CH3CN = 7:3, T = 25 °C. e Argon purged for 5 min. f Oxygen purged for 10 s. g NaPi buffered water:acetonitrile (pH 6).
lecular ESPT to the solvent. This explanation is consistent with
the reduced emission of the binol derivatives in comparison to
the naphthol analogues (see Supporting Information). In addi-
tion, among the binols, 4a is more prone to act as a monoalky-
lating agent than the quaternary ammonium salt 4e, which in
turn should act as a better bis-alkylating agent.
Further differences between the photoreactivity of binols
and naphthol analogues have been provided by product
distribution analysis. In fact, although the naphthols 3c and
3d act as photoactivatable monoalkylating agents, the forma-
tion of the byproducts 1 and 3f reveals a side reactivity which
can be ascribed to the 6-naphthylmethyl radical as a reactive
intermediate. Such a side naphthylmethyl-like radical reactiv-
ity is lacking in the p-BQMPs, which act as mono- and bis-
alkylating agents, without any detectable competing reaction
pathway. The behavior of the p-BQMPs is quite similar to
the previously investigated o-BQMPs (Scheme 1). The only
remarkable difference is the efficiency of the second QM
9
J. AM. CHEM. SOC. VOL. 132, NO. 41, 2010 14629

A R T I C L E S
Verga et al.
Scheme 4
phenols and naphthols.14c,25 Such a protonation should generate
the keto-tautomer of the naphthol, which could be responsible
for the transient absorbance centered at 370 nm. The aromati-
zation of the above should lead to C-deuterated naphthols
running the photohydration at 310 nm (4 lamps, 15 W), in
deuterated protic solvents.26 1H NMR analysis of the alcohol
3a recovered after 1 h irradiation of the quaternary ammonium
salt 3d in ACN/D2O ) 7:3 did not show any detectable
deuterium enrichment on the aromatic ring. This observation
enable us to rule out the generation of keto tautomer as an
intermediate (3d-K, Scheme 5). On the other hand, p-NQM,
unlike o-NQM, cannot reversibly isomerize to benzoxete
derivative,16a,27 and therefore the latter intermediate cannot be
associated with the transient absorbance at 370 nm. In addition,
the zwitterionic specie 3d-ZW (Scheme 5) resulting from the
dissociation of the highly acidic excited states of the 2-naphthol
derivative 3d, which could be the precursors of the 2,6-
naphthoquinone-6-methide, is unlikely to be the most reactive
transient of the biexponential decay at 370 nm, since the lifetime
of 3d-ZW should be much shorter than a few microseconds in
aqueous solution. As mentioned above, the strong emission
compromises a reliable investigation of the nature of the 2,6-
naphthoquinone-6-methide precursor, which is probably embed-
ded in the fast decay of the biexponential kinetics at 370 nm.
Therefore, no further LFP investigation on the naphthols was
taken into consideration.
Table 3. Efficiency of the QM-Photogeneration Evaluated from the
Quantum Yield Measurements (Φ) at 310 nm, Measured in 7:3
H2O/CH3CN Solutions of the Naphthol 3d and Binols 4a, 5a, 4e,
and 5e at Low Conversion (<23%)a
adduct
[adduct]
conversion %
irrad time (min)
Φ
3d
4a
5a
4e
5e
1 × 10-3
8.37 × 10-4
1.08 × 10-3
1.03 × 10-3
9.92 × 10-4
21.2
22.7
15.9
18.1
18.7
5
2
8
2
5
0.28
0.87
0.15
0.61
0.24
a Photon flow ) 3.23 × 10-7 E/min, measured by potassium ferri-
oxalate actinometry (monitored by UV-vis).
generation (p-BQM-5, Scheme 4), which is always lower for
p-BQMPs than o-BQMPs.
Detection and Reactivity of Transient p-NQM and p-BQM
by Laser Flash Photolysis. Our group and others have shown
that laser flash photolysis (LFP) is an effective method for direct
detection of QMs, using Mannich bases, their quaternary
ammonium salts and o-hydroxybenzyl alcohols as phototrig-
gerable precursors.10,14,15,17,19,20 Excitation of the naphthol 3d
with a 266 nm pulses of a Nd:YAG laser resulted in the
formation of two transient absorbances centered at 370 nm, and
at 310-330 nm (Figure 1). Time evolution of the transient
absorbance monitored at 370 nm occurred by biexponential
kinetics. Neither of them was appreciably affected by O2 or
2-ethanethiol. Unfortunately, the naphthol 3d was strongly
fluorescent in the 330-480 nm spectral window and the strong
emission compromised the possibility to perform reliable kinetic
measurements on the fastest decay at 370 nm. Therefore, our
attention has been focused mainly on the second transient at
310-330 nm, in which the rise was monoexponential and
occurred within 20 µs. Such an intermediate decayed at a much
slower rate, exhibiting a t1/2 > 1 ms, which was too long-lived
to be measured by our laser equipment, but it was quenched by
2-ethanethiol, with a second-order reaction rate very similar to
that of the prototype p-QM (k2 ) 1.3 × 103 M-1 s-1).24
Unlike naphthols 3a and 3d, binols 4a and 4e were much
less fluorescent in the 320-400 nm spectral window (see
Supporting Information). Therefore, LFP measurements on both
4a and 4e had been performed without artifacts due to the
saturation of the detector. Excitation of the binols 4a and 4e
with 10 ns 266 nm pulses of a Nd:YAG laser, in argon purged
buffered solutions (pH 7), yielded the formation of a short-
lived transients (t1/2 ) 2 µs) with λmax centered at 360 and 355
nm, respectively, and a broad absorption extending to 440 nm
for the transient generated from 4e (Figure 2).
Although the molecular oxygen did not quench these transient
species generated from 4a and 4e, it slightly bleached the
shoulders stretching above 380 nm, that were more persistent
and may be associated with long-living naphthoxy radicals
generated in the photolysis. Both the transients were efficiently
quenched by both morpholine and 2-ethanethiol. In the presence
of the latter, the transients generated by flashing solutions of
4a and 4e were quenched with second-order rate constants (k2)
After 30-40 laser shots (8 mJ power) in the presence of
2-ethanethiol (10-2 M), 3e was detected as the main product of
the photolysis of 3d in aqueous solution, by HPLC. Furthermore,
the UV absorbance of the above transient was very similar to
that of the isomeric 2,3-naphthoquinone-3-methide o-NQM (λmax
330 nm).16a Therefore, we were able to identify the second
transient as 2,6-naphthoquinone-6-methide p-NQM (Scheme 2).
Since saturation of the solution with O2 did not significantly
change the distribution of the alkylation products in the
preparative experiments, we can conclude that 3d triplet excited
state was not involved in the formation of p-NQM. On the other
hand, ESPT from the phenolic OH, in the singlet excited state,
to the vicinal sp2 carbon atoms is well-documented for both for
1.95((0.1) × 107 M-1 s-1 and 2.1((0.1) × 107 M-1 s-1
,
(25) (a) Basaric, N.; Wan, P. J. Org. Chem. 2006, 71, 2677. (b) Lukeman,
M.; Veale, D.; Wan, P.; Munasinghe, V. R. N.; Corrie, J. E. T. Can.
J. Chem. 2004, 82, 240.
(26) Flegel, M.; Lukeman, M.; Wan, P. Can. J. Chem. 2008, 86, 161–169.
(27) (a) Qiao, G. G.; Lenghaus, K.; Solomon, D. H.; Reisinger, A.;
Bytheway, I.; Wentrup, C. J. Org. Chem. 1998, 63, 9806. (b) Tomioka,
H. Pure Appl. Chem. 1997, 69, 837. (c) Sauter, M.; Adam, W. Acc.
Chem. Res. 1995, 28, 289. (d) Shanks, D.; Frisell, H.; Ottosson, H.;
Engman, L. Org. Biomol. Chem. 2006, 4, 846.
(24) Freccero, M. Mini-ReV. Org. Chem. 2004, 1, 403–415.
9
14630 J. AM. CHEM. SOC. VOL. 132, NO. 41, 2010

DNA Cross-Linking by Photogenerated Quinone Methides
A R T I C L E S
Figure 1. Transient absorptions upon 266 nm excitation of an oxygen purged water solution of 3d. (a) Full spectra recorded 3, 8, 15, 30, 50, 160 µs after
the laser pulse. (b) Decay trace recorded at 370 nm (c) Decay trace recorded at 310 nm.
Scheme 5
experiment, since the QMPs 4a-4j investigated were stable in
the dark at 40 °C for several days.
Testing the binolam derivatives 4f-4i we found that 4h and
its methyl ester 4f were both able to induce XL at a concentra-
tion of 1.25 µM (Table 1). The binolams 4g and 4i were the
less active beginning to induce detectable XL at a concentration
higher than 5 µM (Table 4). It is interesting to note that the
respectively (see the effect of 2-ethanethiol in the inset of Figure
most efficient DNA XL-agents (4a and 4e) are the most efficient
2a). Considering that thiols are efficient quenchers of QMs and
mono- and bis-alkylating agents with simple nucleophiles (Table
that after 20 laser shots 5a and 5e were detected by HPLC as
2). All the 6,6′-disubstituted binol derivatives investigated, which
the main products of the photolysis of 4a and 4e in aqueous
act through the sequential generation of two p-BQMs, always
solution containing 2-ethanethiol (10-2 M), we have been able
to assign the transients centered at 360 (Figure 2a,b) and 355
display higher XL potency (by 2-16 fold) than the 3,3′-
disubstituted binol derivatives, generating the o-BQMs
isomers.10d
nm (Figure 2c) to the binol-QM structures p-BQM-4a and
p-BQM-5a, respectively (Scheme 4). It is interesting to note
that the p-BQMs exhibit a very similar maximum absorbance
to the o-BQMs10d and that both are 30 nm red-shifted in
comparison to the o-NQM and p-NQM spectra. The similarity
between p-BQMs and o-BQMs is extended to their reactivity,
since the pseudo-first-order decay with water [kobs ) 7.1((0.2
× 105 s-1)] and the second-order rate constants with thiols [k2
) 2((0.2) × 107 M-1 s-1)] are very similar. In addition, they
are both much more reactive than the o-NQMs and p-NQMs
by almost two magnitude orders, probably as a result of the
specific intramolecular acid catalysis due to the presence of the
2′-naphthol hydroxyl group.
DNA Alkylation and Cross-Linking Experiments. The water
solubility of the binols 4a, 4e-i (∼10-2 M), together with their
bis-alkylating reactivity, suggests evaluating them as DNA cross-
linking agents triggerable upon photolysis. The DNA cross-
linking ability of these compounds was investigated using a
negatively supercoiled plasmid DNA (pBR322) in an alkaline
agarose gel assay (Figure 3).
The nonreacted plasmid was mainly present (95%) in its
supercoiled (C) form and partly (5%) as open circular (OC)
DNA. Interstrand cross-linking (XL) activities of tested com-
pounds were evident as XL-bands of both initial forms (XL C
and XL OC), which run slightly slower than the open circular
form or just below gel wells, respectively (Figure 3). Activities
were measured as the minimal compound concentration required
to obtain XL-effects.
Both binols 4a and 4e showed remarkable reactivity: 4a XL
effect was evident at 1.25 µM (lane 1, Figure 3A), while 4e
induced detectable XL at concentration as low as 0.3 µM (lane
2, Figure 3B), hence being at least 4 times more potent than its
o-BQM analogue.10d In addition, both compounds were able
to cross-link more than 90% DNA at 40 µM (lanes 6 and 9,
Figure 3, panel A and B, respectively). Incubation of DNA in
the dark with QMPs was not performed as the control
Next, the alkylation properties of the most active compound
4e were tested on short ds- and ss-random oligonucleotides. As
shown in Figure 4, 4e was able to alkylate both ds- and ss-
DNA after exposure to UV light (lanes 3 and 6, respectively,
Figure 4). In more detail, 4e induced both alkylation products
and interstrand DNA cross-linking (ALK and XL band,
respectively, in Figure 4) on the ds-oligonucleotide. Both adduct
bands were multiple and slightly smeared, indicating that the
alkylation reaction likely occurred at several different sites. On
the ss-DNA, the alkylation band was extremely intense and
smeared (lane 6, Figure 4) indicating the presence of different
species such as mono- and multiple-alkylated DNA and intras-
trand alkylated oligo. In addition, a weak cleavage of both the
ds- and ss-alkylated oligos was observed. To detect the
alkylation sites, the ss-alkylated oligo was further treated with
hot piperidine, which induces cleavage at the alkylated base
according to the Maxam and Gilbert reaction mechanism.28 As
shown in Figure 4, lane 9, major cleavage sites following hot
alkali treatment were found at the guanine level and, to a lesser
extent, at adenines, indicating alkylation reaction at purines.
Adduct characterization was performed by reacting 4e with
single 2′-deoxy-oligonucleosides (dNTs) in solution upon UV
exposure. Adducts were separated by means of HPLC analysis,
which revealed one new adduct peak with dG, dA, and dC, while
no adducts were observed upon reaction with dT. Percentages
of adduct formation, with respect to total 4e amount, were 5.2%,
4.5%, and 0.8% for reaction with dG, dA, and dC, respectively.
The purine selectivity, with a preference for dG, is quite
surprising for a quinone methide, since it is known that the
prototype benzo o-QM exhibits a defined selectivity for dC.29
This could be the result of two combined effects: (i) the p-BQM
(28) Maxam, A. M.; Gilbert, W. Methods Enzymol. 1980, 65, 499–560.
(29) Veldhuyzen, W. F.; Shallop, A. J.; Jones, R. A.; Rokita, S. E. J. Am.
Chem. Soc. 2001, 123, 11126–11132.
9
J. AM. CHEM. SOC. VOL. 132, NO. 41, 2010 14631

A R T I C L E S
Verga et al.
Figure 2. Transient absorptions of p-BQM-4a in an (a) argon purged, (b) oxygen purged water solution of 4a. Transient absorption of p-BQM-5a, upon
266 nm excitation of (c) an oxygen purged water solution of 4e. Panel (a), the inset shows the quenching effect of 2-ethanethiol on the transient centered
at 360 nm [without (dark blue line) and with 2-ethanethiol 9.3 mM (fuchsia line) and 47 mM (light blue line)]. Time scale 1 µs/Div.
alkylation at N7 of purines (and as such it is routinely used in
biochemical sequencing of DNA based on the Maxam and
Gilbert protocol, by the hard electrophile dimethyl sulfate) once
again, the harder nature of the p-BQM is evident, which behaves
more like a stabilized carbocation than a prototype QM.
Photocytotoxic and Cytotoxic Effects of Binol-Amino Acid
Conjugates. 4a-b and 4e-i were investigated on two human
cancer epithelial cell lines: A549 (lung adenocarcinoma) and
HT-29 (colorectal adenocarcinoma). These two cell lines were
chosen since they represent common human tumors and are
routinely used to test the effects of anticancer drugs. Results
were reported as effective drug concentration able to kill 50%
of cell population after photoirradiation (EC50) and cytotoxic
concentration that killed 50% of cell population without
photoirradiation (CC50). Selectivity indexes (SI) were measured
as the ratio of CC50 over EC50 values. As shown in Table 4,
compounds were in general more active and less cytotoxic on
the HT-29 cell line. The most active compounds on both cell
lines were binol-derivative 4g and 4i (EC50 in the high
nanomolar range); however, they also showed remarkable
cytotoxicity prior to UV irradiation at low doses (SI ) 5-10).
Compounds 4e and 4f were slightly less active, displaying EC50
in the low micromolar range. 4f was also fairly cytotoxic (SI
10-18), while 4e was remarkably less cytotoxic (SI 22-33).
Compound 4b exhibited a high SI (SI 33) only on HT-29 cells,
but poor on the A549 cell line (SI 9). The other binolams 4a
and 4h exhibited higher EC50 paralleled by higher CC50;
however, SI remained in general low (4-18). PS (4,5′,8-
trimethylpsoralen), used as a control, in these conditions
displayed lower SI than the analyzed compounds.
Figure 3. DNA cross-linking (XL) concentration-dependent activity of
binol-amino acid conjugates. Plasmid DNA was mixed with increasing
amounts of each compound (as indicated above each gel image), in
phosphate buffer (50 mM, pH 7.5). Reaction mixtures were irradiated at
360 nm for 20 min and loaded on 1% alkaline agarose gel. Gels were stained
with ethidium bromide. Lane D is nontreated, irradiated DNA; lane PS is
a control for XL species induced by 4,5′,8-trimethylpsoralen at 360 nm.
Nonreacted circular and open circular forms of plasmid are indicated as C
and OC; their XL species are indicated as XL C and XL OC, respectively,
on the right of gel images.
nature which is a harder electrophile in comparison to the
prototype o-QM, exhibiting a much shorter lifetime in water,
and (ii) the thermodynamic stability of the resulting alkylation
adducts arising from all the binol-precursors. Such stability is
in striking contrast to the reversibility of the alkylation process
involving the prototype o-QM.9,18,29,30 Fractions corresponding
to the new peaks formed during the alkylation reactions with
dG and dA were collected and analyzed by mass spectrometry
based on electrospray ionization coupled with a time-of-flight
analyzer (ESI-TOF MS). Peak formed in the reaction with dC
was not collected due to the small amount observed. Multiple
mass peaks were obtained for each analyzed adduct: molecular
masses obtained for the two main peaks matched 1:1 covalent
adducts of 2′-deoxy-purines with 4e without proton loss and
with a double positive charge (m/z ) 319.1 and 311.1 for dG
and dA, respectively) or with one proton loss and a single
positive charge (m/z ) 637.3 and 621.3 for dG and dA,
respectively). In addition, a third major peak was detected,
corresponding to a deglycosilated adduct (m/z ) 521.2 and 505.2
for dG and dA, respectively) (Figure 5A,B). Interestingly, when
the 4e-dG HPLC-collected peak was subjected to heat treatment
(i.e., 95 °C for 30 min), this latter adduct lacking ribose became
the highly prevalent species, while the peaks retaining ribose
either disappeared or were greatly reduced (Figure 5C). A
plausible mechanism would involve the initial attack of the
p-BQM-4e to the nucleophilic N7 of the purine base followed
by the cleavage of the N-glycosidic bond at N9. Since the
depurination process is a direct and unique consequence of
Conclusion
We have described the photogeneration of new and detectable
bifunctional binol-quinone methides (p-BQMs), by laser flash
photolysis at 360 nm, which are capable of undergoing mono-
and bis-alkylation of simple nucleophiles, oligonucleotides, ss-
DNA and ds-DNA in water. The p-BQMs are more efficiently
generated and much more reactive transient electrophiles than
the monoalkylating naphtho-QM analogue (p-NQM), exhibiting
also a much harder electrophilic nature than the prototype o-QM.
Since the resulting p-BQM-conjugate adducts are very stable
and do not release free p-BQMs at T < 40 °C for several days,
unlike the o-QM, their reactivity has been exploited to achieve
stable and efficient DNA cross-linking with sub-micromolar
potency by UV-visible activation. The study highlights that
the water-soluble 6,6′-bis(trimethylaminomethyl)-[1,1′]binaph-
thalenyl-2,2′-diol (4e) is the most promising photocross-linking
(30) (a) Freccero, M.; Di Valentin, C.; Sarzi-Amade`, M. J. Am. Chem.
Soc. 2003, 125, 3544. (b) Freccero, M.; Gandolfi, R.; Sarzi-Amade`,
M. J. Org. Chem. 2003, 68, 6411. (c) Di Valentin, C.; Freccero, M.;
Zanaletti, R.; Sarzi-Amade`, M. J. Am. Chem. Soc. 2001, 123, 8366.
9
14632 J. AM. CHEM. SOC. VOL. 132, NO. 41, 2010

DNA Cross-Linking by Photogenerated Quinone Methides
A R T I C L E S
Table 4. Biological in Vitro and Cellular Properties of the Photoreactive p-BQMPs and PS (4,5′,8-trimethylpsoralen)
EC50 (µM)
cell line f A549
CC50 (µM)
SI (CC50/EC50)
photoreactive alkylating agent
DNA XL MC (µM)
HT29
A549
HT29
A549
HT29
PS
4a
4b
4e
4f
4g
4h
4i
<0.5
1.25
5
0.3
1.25
5
27 ( 14
33 ( 11
2.3 ( 0.6
9 ( 1
2 ( 0.04
0.9 ( 0.03
35 ( 11
1.2 ( 0.1
20 ( 4
>200
35 ( 7
>200
>7
>6
9
22
10
5
2
11 ( 2
>200
18
33
33
18
10
>4
7
0.6 ( 0.1
6 ( 0.5
21 ( 1
200
>200
200
1.5 ( 0.08
0.5 ( 0.02
26 ( 3
20 ( 1
4.5 ( 0.3
>200
27 ( 3
5 ( 0.2
>100
1.25
5-10
6
5
0.2 ( 0.02
6.5 ( 0.8
1.4 ( 0.03
6,6′-diBr-[1,1′]binaphthalenyl-2,2′-diol,35 2,36 4k37 are known
products. They have been synthesized via standard published
procedures, with the exception of 1 and 2.
6-Hydroxynaphthalene-2-carbaldehyde (1). A 0.500 g sample
of 6-bromo-2-naphthol (2.24 mmol) was dissolved in 26.9 mL of
dry THF under an argon atmosphere. The magnetically stirred
agents exhibiting a significant purine selectivity and promising
photocytotoxicity versus cytotoxicity.
Experimental Section
General Procedures. 2-Naphthol and [1,1′]binaphthalenyl-2,2′-
diol are commercially available products. The substituted naphthols:
6-Br-2-naphthol,31 3f,32 1,33 and 3a34 and the substituted binols:
Figure 4. Reactivity of 4e toward ds- and ss-DNA. The oligonucleotide
5′-AAC AGC CTC AGC AGC CGG TTA-3′ was 5′-end-labeled and
annealed to its complementary strand. Both the single ds- and ss-
oligonucleotides were reacted with 10 mM 4e in 50 mM phosphate buffer,
pH 7.4, and ethanol precipitated after 24 h. The ss-DNA was further treated
with piperidine 1 M at 90 °C for 30 min (Pip), vacuum-dried, and then
loaded on 20% denaturing acrylamide gels. Gels were run at 80 W for
2.5 h and autoradiographed. Control lanes: lanes 1 and 4 are the nontreated
ds- and ss-oligos, respectively; lanes 2 and 5 indicate the nonreacted oligos
exposed to UV but not to 4e. Lanes 7 and 8 represent ss-DNA treated with
piperidine and with piperidine and UV, respectively, but not exposed to
4e. Lane M is a marker for purines (G + A) obtained by the Maxam and
Gilbert reaction. Piperidine-cleaved G and A bases are indicated on the
right side with continuous and discontinuous arrows, respectively, while
the oligonucleotide resolved sequence is reported on the left.
Figure 5. ESI-TOF MS spectrum of 4e-dG and 4e-dA conjugates. The
adduct chromatographic peaks obtained in the reaction of 4e with dG (A)
or dA (B) were collected and analyzed by ESI-MS in positive ion mode to
determine the molecular masses of the reaction products. (C) The 4e-dG
HPLC-collected peak was treated at 95 °C for 30 min prior to ESI-MS
analysis. The m/z values for significant adduct ions are shown.
9
J. AM. CHEM. SOC. VOL. 132, NO. 41, 2010 14633

A R T I C L E S
Verga et al.
1
solution was cooled to -78 °C and 6.16 mL of n-BuLi in n-hexane
(1.6 M) (9.85 mmol) was added at such a rate in order to not allow
the temperature to exceed -70 °C. After 5 h of stirring at this
temperature, 1.27 mL of dry N,N-dimethylformamide (16.4 mmol)
was added so that the temperature remained below -50 °C. After
stirring for 45 min at this temperature, the reaction mixture was
poured into HCl/ice (pH < 1) under vigorous stirring. It was allowed
to reach rt overnight and extracted 3 × CH2Cl2. The combined
organic phases were washed twice with water and dried over
MgSO4, and the solvent was removed under reduced pressure after
filtration to give an oil. The oil was purified by MPLC column
chromatography (cyclohexane:ethyl acetate 8/2) to give 0.330 g of
1 (85.5%) as a white solid.33 Mp 178-180 °C: 1H NMR (DMSO):
δ 7.20-7.23 (m, 2H), 7.76-7.84 (m, 2H), 8.02 (d, J ) 9.8 Hz,
1H), 8.43 (s, 1H), 10.04 (s, 1H), 10.35 (s, 1H). 13C NMR (DMSO):
109.2, 119.8, 122.7, 126.6, 127.0, 131.3, 131.6, 134.7, 138.1, 158.5,
192.4. Anal. Calcd. For. C11H8O2: C, 76.73; H, 4.68; O, 18.58.
Found: C, 76.69; H, 4.70.
vacuum (0.190 g, 81% yield). White solid. Mp 187-189 °C: H
NMR (DMSO): δ 3.08 (s, 9H), 4.65 (s, 2H), 7.17-7.19 (m, 2H),
7.50 (d, J ) 8.3 Hz, 1H), 7.79-7.87 (m, 2H), 7.98 (s, 1H), 10.01
(bs, 1H). 13C NMR (DMSO): 51.7, 68.1, 108.5, 119.5, 122.3, 126.6,
127.1, 129.5, 130.0, 132.8 135.2, 156.7. Anal. Calcd. For.
C14H18INO: C, 48.99; H, 5.29; I, 36.98; N, 4.08; O, 4.66. Found:
C, 48.96; H, 5.31, I, 36.99; N, 4.06.
2,2′-Dihydroxy-1,1′-binaphthyl-6,6′-dicarbaldehyde (2). A 2.000
g sample of 6,6′-dibromobinol (4.50 mmol) was dissolved in 54
mL of dry THF under an argon atmosphere. The magnetically stirred
solution was cooled to -78 °C and 14.4 mL of n-BuLi in n-hexane
(2.5 M) (36.0 mmol) was added at such a rate in order to not allow
the temperature to exceed -70 °C. After 5.5-6 h of stirring at this
temperature, 2.60 mL of dry N,N-dimethylformamide (33.6 mmol)
was added so that the temperature remained below -50 °C. After
being stirred for 45 min at this temperature, the reaction mixture
was poured into HCl/ice (pH < 1) under vigorous stirring. It was
allowed to reach rt overnight. The fair yellow solid formed was
filtered. The solid was dried under vacuum, washed with CH2Cl2
and filtered. Product 2 was obtained as a pale yellow solid (1.29 g,
6-[(Dimethylamino)methyl]naphthalen-2-ol (3b). To 0.250 g
(1.45 × mmol) of 1 10 mL of a solution of dimethylamine (33%
in absolute ethanol) was added. The solution was kept under argon
atmosphere and stirred for 20 h at rt. NaBH4 (0.165 g, 4.35 mmol)
was added to the solution and stirred for 3 h at rt. The solution
was dried under a vacuum and water was added. The aqueous
solution was extracted with CHCl3 (3 × 30 mL). The organic layers
were combined and dried over Na2SO4. Solvent was removed under
reduced pressure and a white solid was obtained (0.248 g, 85%
84% yield).36 Mp: 179-181 °C. H NMR (DMSO): δ 7.16 (d, J
1
) 8.8 Hz, 2H), 7.61 (d, J ) 8.8 Hz, 2H), 7.72 (d, J ) 8.8 Hz, 2H),
8.25 (d, J ) 8.8 Hz, 2H), 8.63 (s, 2H), 10.16 (s, 2H), 10.17 (s,
2H). 13C NMR (DMSO): 115.5, 119.6, 122.7, 125.1, 127.1, 131.1,
131.2, 135.2, 137.3, 156.4, 192.4. Anal. Calcd. For. C22H14O4: C,
77.18; H, 4.12; O, 18.69. Found: C, 77.15; H, 4.15.
6,6′-Bis(hydroxymethyl)-1,1′-binaphthyl-2,2′-diol (4a). To a solu-
tion of 2 (0.500 g, 1.46 mmol) in a mixture of absolute ethanol
(20.7 mL) and THF (3.46 mL) NaBH4 (0.450 g, 11.8 mmol) was
added at rt. After being stirred for 5 h the reaction mixture was
dried under reduced pressure. The residue was taken up in 3 N
HCl and vigorous stirred until all salts were dissolved and pH
solution was acid. The white solid was filtered and washed with
CH2Cl2. The solid was dried to give 4a (0.340 g, 77% yield). White
1
yield). Mp 154-156 °C: H NMR (CDCl3): δ 2.42 (s, 6H), 3.71
(s, 2H), 6.97 (d, J ) 2.2 Hz, 1H), 7.04 (dd, J ) 8.8, 2.2 Hz, 2H),
7.34 (dd, J ) 8.4, 1.4 Hz, 1H), 7.41 (d, J ) 8.4 Hz, 1H), 7.51 (d,
J ) 8.8 Hz, 1H), 7.62 (dd, J ) 1.4 Hz, 1H), 7.91 (bs, 1H). 13C
NMR (CDCl3): 44.4, 63.6, 109.4, 119.0, 126.5, 127.7, 128.0, 128.6,
129.2, 130.2 134.3, 155.1. Anal. Calcd. For. C13H15NO: C, 77.58;
H, 7.51; N, 6.96; O, 7.95. Found: C, 77.53; H, 7.54, N, 6.98.
6-(Morpholinomethyl)naphthalen-2-ol (3c). Morpholine (0.272
g, 3.12 mmol) and 1 (0.215 g, 1.25 mmol) were mixed in dry THF
(5 mL) at rt under argon atmosphere. Sodium triacetoxyborohydride
(0.377 g, 1.70 mmol) was added and the mixture stirred at rt under
an argon atmosphere for 24 h. The reaction mixture color became
pale yellow at the end of the reaction time. The reaction mixture
was quenched by adding aqueous K2CO3 (the solution became pale
pink), and the product was extracted with Et2O. The organic phase
was dried over Na2SO4, and the solvent was removed under reduced
pressure. The oily residue was purified by column chromatography
cyclohexane/ethyl acetate 1/1. The product was obtained as a white
solid 0.304 g (90% yield). White solid. Mp 100-102 °C: 1H NMR
(CDCl3): δ 2.59 (t, J ) 4.3 Hz, 4H), 3.68 (s, 2H), 3.79 (t, J ) 4.3
Hz, 4 H), 6.00 (bs, 1H), 6.99-7.05 (m, 2H), 7.40 (dd, J ) 8.4, 1.6
Hz, 1H), 7.51 (d, J ) 8.4 Hz, 1H), 7.61-7.65 (m, 2H). 13C NMR
(CDCl3): 53.4, 63.5, 66.6, 109.4, 118.3, 126.3, 128.0, 128.2, 128.3,
129.4, 131.4 134.0, 153.9. Anal. Calcd. For. C15H17NO2: C, 74.05;
H, 7.04; N, 5.76; O, 13.15. Found: C, 74.10; H, 7.05, N, 5.68.
1-(6-Hydroxynaphthalen-2-yl)-N,N,N-trimethylmethanamini-
um Iodide (3d). Compound 3b (0.137 g, 0.681 mmol) was dissolved
in 5 mL of CH2Cl2 and 0.483 g (0.340 mmol, 0.212 mL) of CH3I
was added dropwise. The solution was stirred at rt for 5 h and a
white solid precipitated. The solid was filtered and dried under a
1
solid. Mp: 123-125 °C. H NMR (acetone d6): δ 4.25 (t, J ) 4.1
Hz, 2H), 4.73 (d, J ) 4.1 Hz, 4H), 7.04 (d, J ) 8.6 Hz, 2H), 7.24
(dd, J ) 8.7, 1.6 Hz, 2H), 7.35 (d, J ) 8.7 Hz, 2H), 7.85-7.91 (m,
6H). 13C NMR (Acetone d6): 65.2, 115.1, 119.9, 125.8, 126.5, 127.0,
130.1, 130.7, 135.0, 138.1, 154.6. Anal. Calcd. For. C22H18O4: C,
76.29; H, 5.24; O, 18.48. Found: C, 76.33; H, 5.22.
6,6′-Bis[(methylamino)methyl]-1,1′-binaphthyl-2,2′-diol (4b). Me-
thylamine (6.8 mL, 33% by weight, 8 M solution in absolute
ethanol, 54.4 mmol) was added under nitrogen atmosphere to 2,2′-
dihydroxy-1,1′-binaphthyl-6,6′-dicarbaldehyde (2) (0.200 g, 5.84
mmol), the reaction mixture was stirred at rt for 24 h. Then NaBH4
(0.332 g, 8.76 mmol) was added in one portion. The solution was
stirred for another 3 h until the Schiff base was completely reduced
to amine. The solution was concentrate under reduced pressure and
the residue was mixed with 20 mL of water. The aqueous solution
was extracted with CHCl3 (3 × 40 mL). The combined organic
phase was dried over Na2SO4 and the solvent was removed under
reduced pressure to give the amine as pale yellow solid 4b (0.142
1
g, 65% yield). Pale yellow solid. Mp > 115 °C dec. H NMR
(DMSO): δ 2.30 (s, 6H), 3.72 (s, 4H), 6.88 (d, J ) 8.6 Hz, 2H),
7.15 (d, J ) 8.6 Hz, 2H), 7.29 (d, J ) 8.8 Hz, 2H), 7.74 (s, 2H),
7.80 (d, J ) 8.8 Hz, 2H). 13C NMR (DMSO): 35.5, 55.0, 115.5,
118.5, 124.4, 126.4, 126.6, 127.9, 128.3, 133.2, 134.2, 152.7. Anal.
Calcd. For. C24H24N2O2: C, 77.39; H, 6.49; N, 7.52; O, 8.59. Found:
C, 77.43; H, 6.47; N, 7.53.
(31) Koelsch, F. C. Org. Synt. 1940, 20, 18–21.
(32) Sue, D.; Kawabata, T.; Sasamori, T.; Tokitoh, N.; Tzubaki, K. Org.
Lett. 2010, 12, 256–258.
1,1′-(2,2′-Dihydroxy-1,1′-binaphthyl-6,6′-diyl)bis(N,N-dimethyl-
methanaminium) Chloride (4c HCl). Dimethylamine (8 mL, 33%
by weight, 8 M solution in absolute ethanol, 64.0 mmol) was added
under nitrogen atmosphere to 2,2′-dihydroxy-1,1′-binaphthyl-6,6′-
dicarbaldehyde (2) (0.200 g, 5.84 mmol), and the reaction mixture
was stirred at rt for 24 h. Then NaBH4 (0.332 g, 8.76 mmol) was
added in one portion. The solution was stirred for another 3 h until
the Schiff base was completely reduced to amine. The solution was
concentrated under reduced pressure and the residue was mixed
with 20 mL of water. The aqueous solution was extracted with
(33) Schmidt, J. M.; Tremblay, G. B.; Plastina, M. A.; Ma, F.; Bhal, S.;
Feher, M.; Dunn-Dufault, R.; Redden, P. R. Bioorg. Med. Chem. 2005,
13, 1819–1828.
(34) Samat, A.; Lokshin, V.; Chamontin, K.; Levi, D.; Pepe, G.; Gugliel-
metti, R. Tetrahedron 2001, 57, 7349–7359.
(35) Yao Sogah, G. D.; Cram, D. J. J. Org. Chem. 1979, 3035–3042.
(36) Dong, C.; Zhang, J.; Zheng, W.; Zhang, L.; Yu, Z.; Choi, M. C. K.;
Chan, A. S. C. Tetrahedron: Asymmetry 2000, 11, 2449–2454.
(37) Cheng, W.; Deqing, Z.; Guaxin, Z.; Junfeng, X.; Daoben, Z.
Chem.sEur. J. 2008, 14, 5680–5686.
9
14634 J. AM. CHEM. SOC. VOL. 132, NO. 41, 2010

DNA Cross-Linking by Photogenerated Quinone Methides
A R T I C L E S
CHCl3 (3 × 40 mL). The combined organic phase was dried over
Na2SO4 and the solvent was removed under reduced pressure. The
obtained reaction mixture was purified by reverse phase MPLC
H2O/MeOH 7/3 TFA 0.1% to give the amine hydrochloride as a
white solid 4c HCl (0.200 g, 72.2% yield), after treatment with a
added and the solvent was removed in a vacuum. The residue was
suspended in diethyl ether, and the product was isolated by filtration.
The yields were almost quantitative (93%) only using triethylsilane,
1
as the carbocation scavenger. White solid. Mp > 110 °C Dec. H
NMR (CD3OD): δ 2.03-2.04 (m, 2H), 2.17-2.19 (m, 4H), 2.62
(m, 2H), 3.36-3.54 (m, 4H), 4.44 (m, 4H), 4.69 (d, J ) 12.0 Hz,
2H), 7.10-7.15 (m, 2H), 7.32 (m, 2H), 7.41 (d, J ) 8.8 Hz, 2H),
7.99 (d, J ) 8.9 Hz, 2H), 8.07 (s, 2H). 13C NMR (CD3OD): 23.58,
23.63, 29.6, 55.8, 55.9, 60.2, 67.9, 116.5, 118.5, 120.7, 125.77,
125.81, 127.2, 128.78, 128.84, 130.2, 131.4, 132.54, 132.58, 136.53,
136.56, 156.0, 171.3. Anal. Calcd. For. C34H38Cl2N2O6: C, 63.65;
H, 5.97; Cl, 11.05; N, 4.37; O, 14.96. Found: C, 63.60; H, 5.98;
Cl, 11.07; N, 4.36.
1
diluted solution of HCl. White solid. Mp > 138 °C dec. H NMR
(DMSO): δ 2.71 (s, 12H), 4.35 (d, J ) 4.1 Hz, 4H), 6.00 (bs, 2H),
6.98 (d, J ) 8.6 Hz, 2H), 7.37 (d, J ) 8.7 Hz, 2H), 7.44 (d, J )
8.9 Hz, 2H), 7.90 (d, J ) 8.9 Hz, 2H), 8.02 (s, 2H), 10.65 (bs,
2H). 13C NMR (DMSO): 41.5, 59.6, 115.52 119.3, 124.1, 124.8,
127.5, 127.9, 129.1, 131.1, 134.2, 154.1. Anal. Calcd. For.
C26H30Cl2N2O2: C, 65.96; H, 6.39; Cl, 14.98; N, 5.92; O, 6.76.
Found: C, 65.92; H, 6.43; Cl, 15.00; N, 5.90.
1,1′-(2,2′-Dihydroxy-1,1′-binaphthyl-6,6′-diyl)bis(N,N,N-trimeth-
ylmethanaminium) Bromide (4e). Compound 4k (0.250 g, 0.530
mmol) was dissolved in 9 mL of dry THF. A slow stream of dry
Et3N was bubbled through at the previously stirred solution. The
bubbled solution was stirred at rt for 15 min and a white solid
precipitated. The obtained product was filtered under nitrogen
atmosphere and dried as product 4e (0.297 g, 95% yield). White
solid. Mp > 110 °C dec. 1H NMR (DMSO): δ 3.08 (s, 18H), 4.66
(s, 4H), 7.03 (d, J ) 8.8 Hz, 2H), 7.34 (d, J ) 8.8 Hz, 2H), 7.46
(d, J ) 8.8 Hz, 2H), 7.99 (d, J ) 8.8 Hz, 2H), 8.09 (s, 2H), 9.64
(bs, 2H). 13C NMR (DMSO): 51.7, 67.9, 115.1, 119.4, 122.2, 124.8,
127.5, 129.4, 129.6, 133.2, 134.5, 154.4. Anal. Calcd. For.
C28H34Br2N2O2: C, 56.96; H, 5.80; Br, 27.07; N, 4.74; O, 5.42.
Found: C, 56.92; H, 5.84; Br, 27.08; N, 4.74.
General Procedure for the Synthesis of Binolams Derivatives.
The desired amino acid (2.63 mmol) and (R)-2 (0.300 g, 0.876
mmol) were mixed in anhydrous ethanol (20 mL) at rt under argon
atmosphere. Sodium triacetoxyborohydride (0.186 g, 2.63 mmol)
was added and the mixture stirred at rt under an argon atmosphere
for 24 h. The reaction mixture color became pale orange at the
end of the reaction time. The reaction mixture was quenched by
adding aqueous KHCO3 and the product was extracted with CHCl3.
The organic phase was dried over Na2SO4 and the solvent was
removed under reduced pressure. The oily residue was purified by
column chromatography cyclohexane/ethyl acetate.
(R,S,S)-Dimethyl-2,2′-(2,2′-dihydroxy-1,1′-binaphthyl-6,6′-diyl)bis-
(methylene)bis(azanediyl) bis(3-methylbutanoate) (4i). The oily
residue was purified by column chromatography (cyclohexane: ethyl
acetate 65/35). Product 4i was isolated as pale yellow solid (0.376
g, 75% yield). Pale yellow solid. Mp: 69 - 71 °C. 1H NMR
(CDCl3): δ 0.93-0.97 (m, 12H), 1.88-1.97 (m, 2H), 3.07 (d, J )
5.9 Hz, 2H), 3.70 (d, J ) 13.2 Hz, 2H), 3.73 (s, 6H), 3.95 (d, J )
13.2 Hz, 2H), 7.09-7.13 (m, 2H), 7.29 (dd, J ) 8.8 Hz, 2H), 7.36
(d, J ) 9.0 Hz, 2H), 7.81 (s, 2H), 7.92 (d, J ) 9.0 Hz, 2H). 13C
NMR (CDCl3): 18.6, 19.2, 31.6, 51.3, 52.2, 66.4, 111.1, 117.8,
124.3, 127.1, 128.2, 129.2, 130.9, 132.6, 135.5, 152.4, 175.6. Anal.
Calcd. For. C34H40N2O6: C, 71.31; H, 7.04; N, 4.89; O, 16.76.
Found: C, 71.33; H, 7.01; N, 4.92.
(2,2′-Dihydroxy-1,1′-binaphthyl-6,6′-diyl)bis(methylene) dietha-
noate (4j). 0.150 g (0.581 mmol) of compound 4a were dissolved
in 7 mL of glacial acetic acid. The solution was refluxed for 48 h
under nitrogen atmosphere, and after this period the solvent was
removed under reduced pressure. The oily residue was purified by
column chromatography (cyclohexane/ethyl acetate 7/3). Compound
4j was obtained as colorless oil (74 mg, 40% yield). Colorless oil.
1H NMR (Acetone d6): δ 2.05 (s, 6H), 5.20 (s, 4H), 4.73 (d, J )
4.1 Hz, 4H), 7.09 (d, J ) 8.7 Hz, 2H), 7.27 (dd, J ) 8.7, 1.6 Hz,
2H), 7.39 (d, J ) 8.9 Hz, 2H), 7.91-7.96 (m, 4H), 8.02 (s, 2H).
13C NMR (Acetone d6): 21.2, 67.0, 115.3, 120.2, 126.1, 127.8,
128.9, 130.0, 131.0, 132.2, 135.5, 155.2, 172.3. Anal. Calcd. For.
C26H22O6: C, 72.55; H, 5.15; O, 22.30. Found: C, 72.50; H, 5.17.
Photochemical Synthesis. General Procedure for the Synthesis
of Naphthol Derivatives. The typical procedure for the photochemi-
cal synthesis of 3e is as follows. A solution of naphthol derivatives
(3b-d, 1 × 10-3 M) in 300 mL of CH3CN/H2O 3:7 in the presence
of mercaptoethanol (5 × 10-2 M) was poured into Pyrex tubes,
flushed with argon for 5 min, and externally irradiated by means
of four 15 W phosphor-coated lamps (center of emission 310 nm)
for 10-120 min in a merry-go-round apparatus. The irradiated
solution was evaporated under reduced pressure, and the residue
was purified by preparative HPLC. The product was obtained as a
white solid.
(R,S,S,)-Dimethyl-1,1′-(2,2′-dihydroxy-1,1′-binaphthyl-6,6′-diyl)-
bis(methylene)dipyrrolidine-2-carboxylate (4f). The oily residue
was purified by column chromatography (cyclohexane/ethyl acetate
1
6/4). White solid. (0.458 g, 92% yield). Mp 64-66 °C: H NMR
(CDCl3): δ 1.80-2.0 (m, 6H), 2.13-2.18 (m, 2H), 2.43-2.46 (m,
2H), 3.07-3.10 (m, 2H), 3.28-3.33 (m, 2H), 3.62 (s, 6H), 3.70
(d, J ) 12.8 Hz, 2H), 4.02 (d, J ) 12.8 Hz, 2H), 7.09 (d, J ) 8.6
Hz, 2H), 7.27-7.32 (m, 2H), 7.34-7.38 (m, 2H), 7.82 (s, 2H),
7.91-7.95 (m, 2H). 13C NMR (CDCl3): 22.9, 29.2, 51.7, 53.2, 58.4,
65.2, 111.0, 117.7, 124.1, 128.1, 128.9, 129.1, 131.0, 132.6, 133.9,
152.5, 174.4. Anal. Calcd. For. C34H36N2O6: C, 71.81; H, 6.38; N,
4.93; O, 16.88. Found: C, 71.85; H, 6.37; N, 4.91.
(R,S,S)-tert-Butyl 1,1′-(2,2′-dihydroxy-1,1′-binaphthyl-6,6′-diyl)
bis(methylene) dipyrrolidine-2-carboxylate (4g). The oily residue
was purified by column chromatography (cyclohexane/ethyl acetate
6-((2-Hydroxyethylthio)methyl)naphthalen-2-ol (3e). White solid.
1
Mp: 117-119 °C. H NMR (Acetone d6): δ 2.56 (t, J ) 6.7 Hz,
2H), 3.65 (t, J ) 6.7 Hz, 2H), 3.70 (bs, 1H), 3.88 (s, 2H), 7.13
(dd, J ) 8.8, 2.4 Hz, 1H), 7.18 (d, J ) 2.4 Hz, 1H), 7.42 (dd, J )
8.5, 1.7 Hz, 1H), 7.65 (d, J ) 8.5 Hz, 1H), 7.68 (d, J ) 1.7 Hz,
1H), 7.57 (d, J ) 8.8 Hz, 1H), 8.59 (bs, 1H). 13C NMR (Acetone
d6): 34.8, 37.2, 62.6, 110.2, 119.8, 127.8, 128.4, 128.9, 129.5, 130.4,
134.5, 135.4, 156.6. Anal. Calcd. For. C13H14O2S: C, 66.64; H, 6.02;
O, 13.66; S, 13.68. Found: C, 66.68; H, 6.01; S, 13.64.
1
7/3). Pale orange solid (0.475 g, 83% yield). Mp: 92-94 °C: H
NMR (CDCl3): δ 1.43-1.44 (s, 18H), 1.67-1.78 (m, 4H),
1.87-1.97 (m, 2H), 2.06-2.10 (m, 2H), 2.38-2.46 (m, 2H),
3.03-3.08 (m, 2H), 3.17-3.22 (m, 2H), 3.67 (d, J ) 13.1 Hz, 2H),
4.10 (d, J ) 13.1 Hz, 2H), 7.10 (d, J ) 8.6 Hz, 2H), 7.28-7.38
(m, 4H), 7.83-7.85 (m, 2H), 7.93 (d, J ) 8.8 Hz, 2H). 13C NMR
(CDCl3): 22.8, 26.8, 28.0, 29.1, 30.1, 43.4, 53.1, 58.2, 65.8, 80.4,
110.9, 117.6, 124.1, 128.0, 129.0, 129.2, 131.0, 132.6, 134.5, 152.3,
173.2. Anal. Calcd. For. C40H48N2O6: C, 73.59; H, 7.41; N, 4.29;
O, 14.70. Found: C, 73.57; H, 7.45; N, 4.33.
General Procedure for the Synthesis of Binol Derivatives. The
typical procedure for the photochemical synthesis of 5a, 5e, 6a, 7,
8 is as follows. A solution of binol derivatives (4a-4j, 1 × 10-3
M) in 300 mL of CH3CN/H2O in the presence of nucleophile (5 ×
10-2 M) was poured into Pyrex tubes, flushed with argon for 5
min, and externally irradiated by means of two 15 W phosphor-
coated lamps (emission 310 nm) for 20-60 min in a merry-go-
round apparatus. The irradiated solution was evaporated under
reduced pressure, and the residue was purified by chromatography
on silica gel 60 HR or by reverse phase MPLC. The products were
(R,S,S)-1,1′-(2,2′-Dihydroxy-1,1′-binaphthyl-6,6′-diyl)bis(meth-
ylene)bis(2-carboxy-1-methyl pyrrolidinium) Chloride (4h HCl).
The t-butyl amino ester (1 mmol) was deprotected by dissolving it
in a solution of trifluoroacetic acid (1.94 mL, 13 mmol) and
dichloromethane (10 mL) in the presence of triethylsilane (0.8 mL,
2.5 mmol) at rt. After stirring for 2 h 4g, 1 M HCl (1 mL) was
9
J. AM. CHEM. SOC. VOL. 132, NO. 41, 2010 14635

A R T I C L E S
Verga et al.
obtained as solids or oils from the fractions (by repeating the
separation with HPLC in the case of unsatisfactory separation).
6-(Hydroxymethyl)-6′-(morpholinomethyl)-1,1′-binaphthyl-2,2′-
diol (6a). Compound 6a was obtained after irradiation of 4a at 310
nm, as described above, for 1 h. Compound 6a was separated by
silica gel chromatography and eluted with pure ethyl acetate. A
pale yellow oil was isolated as product (30% yield). Pale yellow
oil. 1H NMR (Acetone d6): δ 2.42 (m, 4H), 2.86 (bs, 1H), 3.59-3.62
(m, 6H), 4.72 (d, J ) 5.2 Hz, 2H), 7.02-7.06 (m, 2H), 7.21-7.27
(m, 2H), 7.31-7.36 (m, 4H), 7.80-7.91 (m, 4H). 13C NMR
(Acetone d6): 54.9, 64.3, 65.1, 67.8, 115.38, 115.40, 119.8, 125.8,
126.5, 127.0, 128.9, 129.1, 130.16, 130.66, 130.77, 133.9, 135.0,
135.1, 138.2, 152.2, 154.6, 154.6. Anal. Calcd. For. C26H25NO4:
C, 75.16; H, 6.06; N, 3.37; O, 15.40. Found: C, 75.19; H, 6.04; N,
3.36.
4,4′-(2,2′-Dihydroxy-1,1′-binaphthyl-6,6′-diyl)bis(methylene)di-
morpholin-4-ium chloride (4d HCl). Compound 4d was obtained
after irradiation of 4e at 310 nm, as described above, for 1 h 45
min. Compound 4d was separated by silica gel chromatography
and eluted with pure ethyl acetate, followed by HPLC purification:
T ) 8.43 s. Flow 1.40 mL/min; gradient elution: eluent A% MeCN,
eluent B% H2O 0.1% TFA: 0 min (5/95), 3 min (5/95), 13 min
(100/0), 16 min (100/0), 22 min (5/95). After HPLC elution,
trifluoroacetate ion was exchanged with chloride ion: the solid was
dissolved in 3 mL of HCl 1 M and dried under reduced pressure.
Compound 4d HCl was isolated as pale orange solid (60% yield).
Pale orange solid. Mp > 200 dec 1H NMR (D2O): δ 3.18-3.21 (m,
4H), 3.34-3.38 (m, 4H), 3.64-3.72 (m, 4H), 3.99-4.03 (m, 4H),
4.40 (s, 4H), 7.06 (d, J ) 8.7 Hz, 2H), 7.20 (d, J ) 8.7 Hz, 2H),
7.40 (d, J ) 8.7 Hz, 2H), 8.01-8.04 (m, 4H). 13C NMR (D2O):
51.0, 60.6, 63.5, 114.5, 118.9, 122.8, 124.7, 125.1, 128.0, 128.4,
130.6, 131.9, 134.1, 152.9. Anal. Calcd. For. C30H34Cl2N2O4: C,
64.63; H, 6.15; Cl, 12.72; N, 5.02; O, 11.48. Found: C, 64.61; H,
6.16; Cl, 12.75; N, 4.99.
J ) 6.8 Hz, 2H), 3.90 (s, 2H), 7.04 (d, J ) 8.6 Hz, 2H), 7.23-7.28
(m, 2H), 7.35 (dd, J ) 8.9, 1.9 Hz, 2H), 7.81-7.91 (m, 4H). 13C
NMR (Acetone d6): 34.8, 37.0, 62.4, 65.0, 115.3, 115.5, 119.8,
120.0, 125.8, 126.2, 126.5, 127.0, 128.8, 130.0, 130.1, 130.6, 130.8,
134.4, 134.9, 135.0, 138.2, 154.5, 154.7. Anal. Calcd. For.
C24H22O4S: C, 70.91; H, 5.46; O, 15.74; S, 7.89. Found: C, 70.94;
H, 5.42; S, 7.93.
[2,2′-Dihydroxy-6′-(2-hydroxy-ethylsulfanylmethyl)-[1,1′]binaph-
thalenyl-6-ylmethyl]-trimethyl-ammonium (5e). Compound 5e was
obtained after irradiation of 4e at 310 nm, as described above, for
20 min. Compound 5e was purified by reverse phase MPLC and
eluted with H2O/MeOH 7: 3 TFA 0.1%. Compound 5e was
1
separated as pale orange oil (53.5% yield). H NMR (CD3OD): δ
2.68 (bs, 2H), 3.20 (s, 9H), 3.74 (t, J ) 6.8 Hz, 2H), 3.96 (bs, 2H),
4.68 (s, 2H), 7.09 (d, J ) 8.6 Hz, 1H), 7.26-7.41 (m, 4H), 7.50
(d, J ) 8.6 Hz, 1H), 7.84 (s, 1H) 7.95 (d, J ) 8.0 Hz, 1H), 8.09 (d,
J ) 8.6 Hz, 1H), 8.16 (s, 1H). 13C NMR (CD3OD): 34.5, 37.2,
62.6, 71.3, 116.0, 117.0, 119.8, 120.9, 123.2, 126.3, 127.2, 128.9,
129.1, 130.1, 130.4, 130.9, 131.4, 135.0, 137.0, 154.7, 156.3. Anal.
Calcd. For. C27H30ClNO3S: C, 66.99; H, 6.25; Cl, 7.32; N, 2.89;
O, 9.92; S, 6.62. Found: C, 67.02; H, 6.22; Cl, 7.35; N, 2.85; S,
6.59.
Cross-Linking of Plasmid DNA. Compounds 4a, 4b, 4e-4i
were dissolved in DMSO to a final concentration of 20 mM. These
stock solutions were diluted in mQ-grade H2O and used for
reactions. Plasmid pBR322 (0.5 µg/sample) was mixed with
increasing amounts (0.2-40 µM, 2-fold serial dilutions, as indicated
in each figure) of each compound, in phosphate buffer (50 mM,
pH 7.5). A saturated water solution of PS was used to get a 0.5
µM concentration of drug in the control samples. Reaction mixtures
were irradiated on ice for 20 min at 360 nm at 120 W in a
photochemical multirays reactor (Helios Italquartz, Italy). Irradiated
solutions were added to alkaline agarose gel loading buffer [50
mM NaOH, 1 mM ethylenediaminetetraacetic acid (EDTA), 3%
Ficoll, and 0.02% bromophenol blue] and loaded on a 1% alkaline
agarose gel containing 50 mM NaOH and 1 mM EDTA. Gels were
run in 50 mM NaOH and 1 mM EDTA at 12 V for 15-16 h, stained
with ethidium bromide (0.5 µg/mL) for 10 min, and subsequently
washed in water for 10 min. Stained gels were visualized in Gel-
Doc 1000 (Bio-Rad, Italy), and DNA bands were quantified by
Quantity One software (Bio-Rad).
Reaction with Oligonucleotides. The ability of binol derivatives
to damage single-stranded DNA was tested at the sequencing level
using a short oligonucleotide whose sequence is 5′-AAC AGC CTC
AGC AGC CGG TTA-3′ (prF ACCM). The oligonucleotide was
obtained in desalted and lyophilized form and was gel purified
before use. It was then 5′-end-labeled with [γ-32P]ATP by T4
polynucleotide kinase and purified by Micro-Spin G-25 desalting
columns (Amersham Biosciences, Europe). For reactions with ds-
DNA, the labeled oligonucleotide prF ACCM was annealed to
equimolar amounts of its complementary cold oligonucleotide (prF
ACCM comp), whose sequence is 5′-TAA CCG GCT GCT GAG
GCT GTT-3′. Drug reactions with the labeled ss- or ds-oligonucle-
otides (4 pmol/sample) in 50 mM phosphate buffer, pH 7.4, were
irradiated on ice for 20 min at 360 nm at 120 W in a photochemical
multirays reactor (Helios Italquartz, Italy). Samples were im-
mediately ethanol precipitated to eliminate nonreacted drug, then
resuspended and split into two aliquots, one of which was kept on
ice, while the other was treated at 90 °C for 30 min with 1 M
piperidine to complete strand scission according to the Maxam and
Gilbert protocol.28 Samples were finally lyophilized twice, resus-
pended in formamide gel loading buffer, heated at 90 °C for 5 min
and cleavage products were resolved in 20% denaturing polyacry-
lamide gels. Gels were visualized by phosphorimaging analysis
(Molecular Dynamics, Amersham Biosciences Europe). Quantifica-
tion was performed by Image-Quant software (Molecular Dynam-
ics).
6,6′-Bis[(2-hydroxyethylthio)methyl]-1,1′-binaphthyl-2,2′-diol (7).
Compound 7 was obtained after irradiation of 4e at 310 nm, as
described above, for 45 min. Compound 7 was separated by MPLC
reverse phase chromatography and eluted with H2O/MeOH 6: 4
TFA 0.1%. Compound 7 was separated as white solid (73.6% yield).
White solid. Mp: 100-102 °C. 1H NMR (CD3OD): δ 2.66 (t, J )
6.9 Hz, 4H), 3.74 (t, J ) 6.9 Hz, 4H), 3.96 (s, 4H), 7.11 (d, J )
8.7 Hz, 2H), 7.33 (dd, J ) 8.7, 1.7 Hz, 2H), 7.40 (d, J ) 8.9 Hz,
2H), 7.85 (d, J ) 1.7 Hz, 2H), 7.95 (d, J ) 8.9 Hz, 2H). 13C NMR
(CD3OD): 34.5, 37.3, 62.6, 116.6, 119.8, 126.6, 128.8, 129.0, 130.4,
130.6, 134.4, 135.2, 154.5. Anal. Calcd. For. C26H26O4S2: C, 66.92;
H, 5.62; O, 13.72; S, 13.74. Found: C, 66.90; H, 5.64; S, 13.75.
6-[(2-Hydroxyethylthio)methyl]-6′-methyl-1,1′-binaphthyl-2,2′-
diol (8). Compound 8 was obtained after irradiation of 4a at 310
nm, as described above, for 2 h. Compound 8 was separated by
silica gel chromatography and eluted with CHCl3/MeOH 99/1 and
followed by HPLC purification: T ) 11.48 s. Flow 1.40 mL/min;
gradient elution: eluent A% MeOH, eluent B% H2O: 0 min (50/
50), 3 min (50/50), 13 min (100/0), 16 min (100/0), 21 min (50/
50). Compound 8 was separated as white solid (10% yield). Pale
1
green oil. H NMR (acetone d6): δ 2.43 (s, 3H), 2.59 (t, J ) 6.9
Hz, 2H), 3.68 (t, J ) 6.9 Hz, 2H), 3.90 (s, 4H), 6.96-7.05 (m,
2H), 7.10 (dd, J ) 8.6, 1.7 Hz, 1H), 7.26 (dd, J ) 8.7, 1.8 Hz,
1H), 7.30-7.36 (m, 2H), 7.66 (d, J ) 1.8 Hz, 1H), 7.81-7.90 (m,
3H). 13C NMR (acetone d6): 21.6, 34.9, 37.0, 62.4, 115.1, 119.7,
120.0, 125.8, 126.2, 128.3, 128.8, 129.6, 130.1, 130.3, 130.6, 133.2,
134.0, 134.4, 134.9, 154.1, 154.7. Anal. Calcd. For. C24H22O3S: C,
73.82; H, 5.68; O, 12.29; S, 8.21. Found: C, 73.79; H, 5.65; S,
8.25.
6-[(2-Hydroxyethylthio)methyl]-6′-(hydroxymethyl)-1,1′-binaph-
thyl-2,2′-diol (5a). Compound 5a was obtained after irradiation of
4a at 310 nm, as described above, for 1 h. Compound 5a was
purified by reverse phase MPLC and eluted with H2O/MeOH 4: 6
TFA 0.1%. Compound 5a was separated as colorless oil (53.5%
yield). 1H NMR (Acetone d6): δ 2.58 (t, J ) 6.8 Hz, 2H), 3.68 (t,
HPLC-MS and MS/MS. The following reaction mixtures were
prepared to measure reactivity of 4e with nucleosides: (i) 4e 5 mM
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14636 J. AM. CHEM. SOC. VOL. 132, NO. 41, 2010

DNA Cross-Linking by Photogenerated Quinone Methides
A R T I C L E S
and nucleosides 8.9 mM in phosphate buffer 50 mM, pH 7.4; (ii)
4e 5 mM in phosphate buffer 50 mM, pH 7.4; (iii) nucleosides 8.9
mM in phosphate buffer 50 mM, pH 7.4. Each mixture was
irradiated on ice for 30 min at 360 nm, filtrated with 0.45 mm filters
(Sartorius Stedim Biotech, Goettingen, Germany), and loaded on
a HPLC C18 reverse phase column (Eclipse XDB C18 column,
Agilent Technologies, Milan, Italy). The HPLC system consisted
of an Agilent 1200 series liquid chromatography, equipped with a
binary pump, standard autosampler, thermostatted column compart-
ment, diode-array detector, and ChemStation software (Agilent
Technologies). To separate the adducts solvents A (CH3CN) and
B (H2O/0.05% TFA) were used, delivered at a flow rate of 1 mL/
min with the following gradient: A from 10% to 48.5% in 15 min;
A from 48.5% to 10% in 1 min; A 10% for 4 min (postrun
equilibration time). Peaks were detected at 260 nm, collected in
Eppendorf tubes, dried at rt in Speed Vac UniVapo 100 H
(UniEquip, Martinsried, Germany), and stored at 4 °C if necessary.
Positive ion mass spectra were acquired on a time-of-flight (TOF)
mass analyzer (Mariner ESI-TOF, Applied Biosystems, CA) by
directly injecting 10 µL of analyte solutions in CH3CN/H2O/
HCOOH (50:49.5:0.5) at rt and with a 0.16 µL/min flow rate. The
nozzle temperature was 140 °C, while a constant flow of N2 gas
was kept at 0.35 L min-1 to facilitate the spray. A three-point
external calibration provided typical 100 ppm accuracy.
working concentrations with DMSO. Cells (1.75 × 104 cells/well)
were plated onto 96-microwell plates to a final volume of 100 µL
and allowed an overnight period for attachment. At day 1, 1 µL of
each dilution of tested compounds was added per well to get a 1%
final concentration of drug solvent per well; at day 2, medium was
removed, cells were washed with phosphate-buffered saline (PBS),
and fresh medium was added. Immediately after, QM- and TMP-
treated cells were irradiated for 20 min at 360 nm in a photochemi-
cal multirays reactor and incubated at 37 °C for an additional 24 h.
Control cells (without any compound but with 1% drug solvent)
were treated under the exact same conditions. Cell survival was
evaluated by an MTT assay: 10 µL of freshly dissolved solution
of MTT (5 mg/mL in PBS) was added to each well, and after 4 h
of incubation, 100 µL of solubilization solution [10% sodium
dodecyl sulfate (SDS) and 0.01 M HCl] was added. After overnight
incubation at 37 °C, absorbance was read at 540 nm. Data were
expressed as mean values of three individual experiments conducted
in triplicate. The percentage of cell survival was calculated as
follows: cell survival ) (Awell - Ablank)/(Acontrol - Ablank) × 100,
where blank denotes the medium without cells.
Acknowledgment. This work was supported by MIUR, Grant
FIRB-Ideas RBID082ATK, and Grant AIRC 5826 (Associazione
Italiana per la Ricerca sul Cancro).
Photocytotoxicity and Cytotoxicity Assays. Human lung ad-
enocarcinoma cells A549 and human colorectal adenocarcinoma
cells HT-29 were obtained from ATCC. A549 and HT29 cells were
grown as monolayers in Dulbecco’s Modified Eagle Medium (D-
MEM) (Invitrogen, Italy) with 10% fetal bovine serum (FBS)
supplemented with penicillin (100 units/mL) and streptomycin (100
µg/mL) in a humidified atmosphere with 5% CO2 at 37 °C.
Cytotoxic effects on tumor cell growth were determined by a MTT
assay. QM compounds and TMP were dissolved and diluted into
Supporting Information Available: General procedure and
1
materials, together with H NMR and 13C NMR, LC/MS, and
DEPT for the following 2-naphthol and binol derivatives: 1-2,
3a-f, 4a-k, 5a, 5e, 6a, 7, 9. This material is available free of
charge via the Internet at http://pubs.acs.org.
JA1063857
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J. AM. CHEM. SOC. VOL. 132, NO. 41, 2010 14637