Phenol quaternary ammonium derivatives: Charge and linker effect on their DNA photo-inducible cross-linking abilities

Article abstract of DOI:10.1039/b604552a

We report here that phenol derivatives with two and four quaternary ammoniums were synthesized and their abilities to bind and cross-link DNA were investigated. Thermal denaturizing studies indicated that derivatives possess similar DNA binding abilities and gel electrophoresis revealed that more charges (series B) and electronic donation substitute linkers (like -S-) dramatically increase the DNA cross-linking abilities. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b604552a

View Article Online / Journal Homepage / Table of Contents for this issue
PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Phenol quaternary ammonium derivatives: charge and linker effect on their
DNA photo-inducible cross-linking abilities
Yang Song,a Tian Tian,a Ping Wang,a Hanping He,a Wulue Liu,a Xiang Zhou,*a,b Xiaoping Cao,d
Xiao-Lian Zhang*c and Xin Zhou*e
Received 29th March 2006, Accepted 7th July 2006
First published as an Advance Article on the web 1st August 2006
DOI: 10.1039/b604552a
We report here that phenol derivatives with two and four quaternary ammoniums were synthesized and
their abilities to bind and cross-link DNA were investigated. Thermal denaturizing studies indicated
that derivatives possess similar DNA binding abilities and gel electrophoresis revealed that more
charges (series B) and electronic donation substitute linkers (like –S–) dramatically increase the DNA
cross-linking abilities.
Introduction
The DNA double helical structure contains the richest source
of information within a living organism. Its sequence codes
for protein synthesis, via the process of translation, and for
RNA synthesis.1 Therefore, DNA remains one of the major
targets for cytotoxic anticancer drugs. DNA interstrand cross-
links (ISC) induced by chemical agents play a very important
role in antitumor treatment.2 Bifunctional alkylating agents may
cause different lesions, show the most toxic of all alkylation events
and, ultimately, inhibit the processes of translation and replication.
Several important clinical drugs (e.g. cisplatin, chlorambucil, and
mitomycin C) are known to induce DNA ISC formation which
can disrupt cell maintenance and replication.2 The design of novel
Fig. 1 Structures of compound 2, 3, 4 and o-quinone methide intermedi-
ate 1.
dimeric agents targeting DNA has been developed by several
groups.2,3 Photochemical cross-linking agents are of particular
interest because such agents could be selectively activated both in
In our previous study, we initially reported on a potent, water-
time and space, and then be released precisely when and where a
soluble and photo inducible DNA cross-linking agent (Fig. 1b).12
desired biological effect is wanted.4 The psoralens are the only class
We found that cross-linking was achieved by mixing DNA with
of compounds known to induce ISC on photolysis.5 They have
the quaternary ammonium salt derivatives upon illumination. In
been employed in clinics for the treatment of psoriasis, vitiligo,
succession to our study, Freccero’s group had found compound
and cutaneous T-cell lymphoma.6 Among these anti-tumor agents,
4 as an important precursor to cross-link DNA after photoac-
one acting mechanism was involved in o-quinone methide (o-QM,
tivation. However, the structure relationship of the quaternary
Fig. 1a) intermediate. o-QM is a derivative of quinone methide
ammonium derivatives’ binding to DNA has not yet been estab-
which has played important roles in organic syntheses and as well
lished.
as in chemical and biological processes.7 o-QMs are significant for
To gain a better understanding of the quaternary ammonium
their nucleic acid bases and DNA alkylation.8 More recently, Wan’s
derivative cross-linking of DNA under the prototype of compound
group,9 Freccero’s group10 and Kresge’s group11 have reported
2, we synthesized two series of quaternary ammonium phenol
significant findings on the generation of o-QMs by photochemical
derivatives, and investigated the effect of different charges and
and thermal activation in aqueous solution.
linkers on the DNA binding profile and cross-linking activity of
these new compounds.
aCollege of Chemistry and Molecular Sciences, Wuhan University, Hubei,
Wuhan, 430072, P. R. of China. E-mail: xzhou@whu.edu.cn
Results and discussion
bKey Laboratory of Bioorganic Chemistry and Molecular Engineering of
Ministry of Education, Peking University, Beijing, 100871, P. R. of China
Synthesis of phenol derivatives with two quaternary ammonium
salts (series A)
cCollege of Medicine, Wuhan University, Hubei, Wuhan, 430071, P. R. of
China
dNational Applied Organic Chemistry, Lanzhou University, Gansu Lanzhou,
730000, P. R. of China
Different biphenol derivatives were mixed with formaldehyde
and dimethylamine via a Mannich reaction (Scheme 1, series A)
and purification could be finished by silica gel chromatography
eZhongnan Hospital, Wuhan University, Hubei, Wuhan, 430071, P. R. of
China
3358 | Org. Biomol. Chem., 2006, 4, 3358–3366
This journal is
The Royal Society of Chemistry 2006
©

View Article Online
Table 1 X-ray crystallographic data for 7k
7k
Empirical formula
Fw
C21H30I2N2O3·2.5H2O
657.31
Space group
Orthorhombic, Fdd2
21.868 (4)
25.240 (4)
9.6164 (16)
90
˚
a/A
˚
b/A
˚
c/A
deg
V/D3
5307.8 (15)
8
Z
T/K
Wavelength
Final R indices
293 (2)
˚
0.71073 (A)
R1 = 0.0468, wR2 = 0.1192
X-Ray analysis of compound 7k
The crystal data and structure of compound 7k are shown in
Table 1 and Fig. 2.† The angle between C(2)–C(1)–C(2A) (which
makes the whole molecule look like a claw) is around 119◦ and is
mainly due to the lone electron pair on the carbonyl between two
phenyl rings. The dihedral angle between two phenyl rings plane
is around 55◦, this twist is owed to the steric effect of the two
phenol rings, and makes the molecule fit better with the double
stranded DNA backbone. Two quaternary ammonium salt groups
are seated in the opposite direction. The distance between N (1)
Scheme 1 Synthesis of two and four quaternary ammonium derivatives.
using ethanol solvent; the appropriate white or buff powder
was obtained. Quaternary ammonium compounds 7a–m were
prepared by adding methyl iodide in a solvent of CH3CN and
then recrystallized from ether. All methylated compounds were
obtained in good yields.
˚
to N (1A), O (2) to O (2A) are 11.458 and 10.105 A, respectively.
From the crystal structure of compound 7k, we suggest that the
distance between two quaternary ammonium salt groups is long
enough to access the DNA base on the different strand. Also, there
is little possibility to block 7k interacting with DNA.
Synthesis of phenol derivatives with four quaternary ammonium
salts (series B)
Compound 2 has two quaternary ammonium arms on different
phenol rings, and compound 3 has two quaternary ammonium
salts on the same phenol ring. They both have the ability to
cross-link DNA after photoactivation, via the intermediate of o-
quinone methide. Therefore, adding more quaternary ammonium
by increasing the number of positive charges might favour their
interaction with DNA and increase their ability to cross-link
DNA. Based on this ideas, we designed and synthesized a series of
compounds with four quaternary ammonium salts on the two
phenol rings, each ring has two quaternary ammonium arms
(Scheme 1, series B). This strategy has three advantages: (1)
increase the solubility of compound in water; (2) increase the
ability to bind the DNA backbone; (3) increase the yields to
produce o-quinone methide. Series B was synthesized by improv-
ing the reaction conditions. Precursors were treated with more
formaldehyde and dimethylamine (>10 equiv.), and the Mannich
reaction occurred on the both ortho-positions of hydroxyl to
obtain the compounds 8a–n.
Fig. 2 ORTEP representation of 7k with hydrogen atoms omitted for
clarity. Thermal ellipsoids are drawn at the 30% probability level.
DNA cross-linking assay
The DNA cross-linking abilities of different compounds are
illustrated in Fig. 3 and 4. In our previous paper,12 compound
2 could cross-link DNA at a 1 lM level. In this paper, we
designed a series of compounds 7a–m, which have different linkers
between the phenol rings, DNA cross-links could be induced upon
illumination. Even at the concentration of 1 mM, compounds 7a–
m had poor DNA cross-linking abilities. Only 7b, 7d (line 3 and 5)
could cross-link linear DNA above 50% under these conditions, no
cross-linked DNA was observed in the case of compounds 7a, 7c,
7e, 7f, 7g, 7i and 7l (line 2, 4, 6, 7, 8, 10 and 13) (Fig. 3). Compounds
9a–n had been further modified with four quaternary ammonium
Due to their prodigious polarity, 2,5,2ꢀ,5ꢀ-tetra(trimethylamine
methylene)-biphenol derivatives can only be separated in a co-
solvent of ethanol and triethylamine (10 : 1). The methylation
step was rather time-consuming, for the sake of gaining pure final
product; all the reactions were carried out over two weeks.
All the new compounds were fully characterized by 1H NMR,
13C NMR, HRESIMS or EI.
† CCDC reference number 230785. For crystallographic data in CIF
format see DOI: 10.1039/b604552a
This journal is
The Royal Society of Chemistry 2006
Org. Biomol. Chem., 2006, 4, 3358–3366 | 3359
©

View Article Online
Fig. 3 Agarose gel electrophoresis of two biphenol quaternary ammo-
nium salts derivatives; (A) lane 1 1.5 lg lambda DNA/Hind III (molecular
weight standard); lane 2–14 1.0 lg pBR322 + 1 mM 7a–m + hv, 30 min.
(B) Cross-linking percents of different compounds (columns).
salts based on the structure of compounds 7a–m and 2. Apparently,
our design for compounds 9a–n with four positive charges could
increase the ability to cross-link DNA (Fig. 4). Compared to 2,
compound 9n slightly increased the DNA cross-linking ability
from 64.7 to 75.0%. Excepting compound 9a (line 4, 0%), all
the four quaternary ammonium salts dramatically increased the
DNA cross-linking ability compared to the derivatives with two
quaternary ammonium salts, respectively, even at a much lower
concentration (10 lM). Compound 9b induced 98.2% cross-
linking efficiently upon exposure to light for 30 min (lane 5).
From Fig. 4c, we can see that in most cases the compounds with
electron donating linkers have higher DNA cross-linking abilities
than those with electron withdrawing linkers, which suggests that
high electronic density might enhance the formation of o-quinone
methide formation. Among these factors, the number of charges
is the main factor to induce DNA cross-linking. Most of the
compounds with four quaternary ammonium salts can induce
more than 50% cross-linking at 10 lM, while most of the two
quaternary ammonium salts induced less than 50% cross-linking,
even at 1 mM. These results suggested that increasing the number
of quaternary ammonium salts could increase the chance to
generate o-QM, which is beneficial to DNA cross-linking ability.
From Fig. 4c, we can see that electron withdrawing substitute
linkers reduced the cross-linking efficiency. Compounds 9a, 9i
and 9k only induced 0, 1.1 and 0.8% cross-linking, respectively.
Electron donating linkers might have two advantages, one is to
favour the quaternary ammonium salts removal to generate o-
QM; the other might be to stabilize o-QM intermediate.16
Fig. 4 Agarose gel electrophoresis of compound 2 and four biphenol
quaternary ammonium derivatives; (A) lane 1 1.5 lg lambda DNA/Hind
III (molecular weight standard); lane 2–16 1.0 lg pBR322 + 10 lM 2, 9n,
9a–9m + hv, 30 min. (B) Cross-linking percents of different compounds
(columns). (C) The sequence of cross-linking ability of compound 2 and
different linker four biphenol quaternary ammonium salts in decreasing
order.
phosphate backbone quite well, and all compounds possess similar
DNA binding abilities.
Conclusion
Two series of different biphenol quaternary ammonium derivatives
with different linkers between the two phenyl rings have been
designed and prepared. We found that derivatives with four
quaternary ammonium salts, derivatives 9a–n, could efficiently
cross-link DNA compared to the compounds with two quaternary
ammoniums, derivatives 7a–m. DNA cross-linking by quaternary
ammonium salts can be obtained in two steps; first, compound
binding to DNA because of the electrostatic attraction between
quaternary ammonium ion and phosphate groups of the DNA
backbone, the second step is that the compound generates
o-QM intermediates by photo-irradiation and induces DNA
cross-linking. Thermal denaturation studies suggested that these
quaternary ammonium derivatives might possess similar DNA
binding abilities. In other words, the binding step contribution or
Thermal denaturizing study
The DNA binding abilities for these quaternary ammonium salts
derivatives have been determined by thermal denaturation studies
using calf thymus DNA (CT-DNA).13 Interestingly, in this assay,
all the compounds elevate the helix melting temperature of CT-
DNA up to about 10 ◦C after incubation with drugs for 1 h.
(Table 2). The result indicated that all compounds fit DNA
3360 | Org. Biomol. Chem., 2006, 4, 3358–3366
This journal is
The Royal Society of Chemistry 2006
©

View Article Online
Table 2 Thermal denaturizing data of series A and series B
ethanol. After evaporation of the solvents, the appropriate white
or buff powder was obtained.
a,b
Series A
DTm
Series B
DTm
ꢀ
ꢀ
2,2-Dimethylaminemethylene-4,4-sulfonyldiphenol (6a). Yield:
125 mg, 34%; Rf = 0.2 (ethanol) (the ratio of the distance of
migration by a substance compared with the solvent (ethanol
here) front); 1H NMR (CDCl3, 300 MHz) d 7.68 (d, J = 9.0 Hz,
2 H, ArH), 7.53 (s, 2 H, ArH), 6.84 (d, J = 8.4 Hz, 2 H, ArH),
3.67 (s, 4 H, –CH2–), 2.33 (s, 12 H, –CH3); 13C NMR (CDCl3,
150 MHz) d 162.53, 131.86, 128.61, 127.53, 121.93, 116.58, 62.22,
44.35; HRESIMS: m/z 365.1525 (calcd for C18H24O4N2S + H,
365.1535).
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
2
10.3
7.4
8.4
10.6
7.8
8.6
7.4
6.2
8.2
8.8
6.4
7.5
6.9
11.5
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
10.4
8.9
10.5
10.4
9.3
8.4
8.2
8.1
8.5
8.1
6.2
2,2ꢀ-Dimethylaminemethylenebis(4-hydroxyphenyl)sulfide (6b).
Yield: 240 mg, 14%; Rf = 0.2 (ethanol); 1H NMR (CDCl3,
300 MHz) d 7.12 (d, J = 4.2 Hz, 2 H, ArH), 6.96 (s, 2 H, ArH),
6.74 (d, J = 4.2 Hz, 2 H, ArH), 3.57 (s, 4 H, –CH2–), 2.31 (s,12 H,
–CH3); 13C NMR (CDCl3, 150 MHz) d 157.84, 132.26, 131.63,
125.48, 122.93, 117.16, 62.71, 44.68; HRESIMS: m/z 333.1625
(calcd for C18H24O2N2S + H, 333.1637).
6.6
6.4
11.3
a For CT-DNA alone at pH 7.40
phosphate buffer [10 mM sodium phosphate + 1 mM EDTA, pH =
7.40
0.01]. b For a 1 : 10 molar ratio of [drug] : [DNA], where CT-
0.01, Tm = 72.9
0. 1, sodium
DNA concentration = 10 lM and drug concentration = 1 lM in aqueous
sodium phosphate buffer [10 mM sodium phosphate +1 mM EDTA, pH =
7.40 0.01].
2,2ꢀ -Dimethylaminemethylene-a,aꢀ-bis(4-hydroxyphenyl)-1,4-di-
isopropylbenzene (6c). Yield: 310 mg, 13%; Rf = 0.2 (ethanol);
1H NMR(CDCl3, 300 MHz) d 7.07 (s, 4 H, ArH), 7.02 (d, J =
8.7 Hz, 2 H, ArH), 6.81 (s, 2 H, ArH), 6.70 (d, J = 8.7 Hz, 2 H,
ArH), 3.57 (s, 4 H, –CH2–), 2.30 (s, 12 H, –CH3),1.60 (s, 12 H,
–CH3); 13C NMR (CDCl3, 150 MHz) d 155.63, 147.96, 141.12,
127.11, 126.49, 126.12, 121.01, 115.27, 63.13, 44.53, 41.71, 30.94;
HRESIMS: m/z 461.3160 (calcd for C30H40O2N2 + H, 461.3168).
structure–activity relationship study in these types of compounds
is small. The structure of compounds 9a and 9b were alike,
and 9a held the higher DTm. 9b could cross-link 98.2% DNA,
but 9a could not induce DNA cross-linking at all. This result
suggests that the structure of the linker is less important than
the electronic effect. Crystal analysis of 7k predicted a good
interaction potential, which was not verified by experiment. This
confirms that the electron effect might play the major role in
the formation of o-quinone methide. Our results indicated that
the number of quaternary ammonium salts and the linkers are
important factors to influence the DNA cross-linking efficiency.
These studies might raise new opportunities to design and
search more potent anti-tumor drugs based on their abilities to
DNA cross-link.
2, 2ꢀ - Dimethylaminemethylene - 1,3 - bis(4 - hydroxyphenyloxyl)-
benzene (6d). Yield: 202 mg, 50%; Rf = 0.2 (ethanol); 1H NMR
(CDCl3, 300 MHz) d 7.15 (t, J = 8.1 Hz, 1 H, ArH), 6.88–6.84 (q,
2 H, ArH), 6.78 (d, J = 9.0 Hz, 2 H, ArH), 6.69 (d, J = 2.4 Hz, 2 H,
ArH), 6.56–6.53 (q, 2 H, ArH), 6.49 (t, J = 2.4 Hz,1 H, ArH), 3.60
(s, 4 H, –CH2–), 2.33 (s,12 H, –CH3), 13C NMR (CDCl3, 150 MHz)
d 160.27, 154.69, 148.14, 130.24, 123.00, 120.68, 120.48, 117.16,
110.95, 106.62, 62.68, 44.67; HRESI-MS: m/z 409.2124 (calcd for
C24H28O4N2 + H, 409.2127).
Experimental
2,2ꢀ - Dimethylaminemethylene - 2,2 - bis(4 - hydroxyphenyl)hexa-
1
1H and 13C NMR spectra were recorded on Varian Mercury 300
and 600 spectrometers. Chemical shifts were reported as values
relative to internal standards as CHCl3 or D2O. Mass spectra
were determined using Bruker Daltonics APEXII 47e. Elemental
analysis was recorded on PE-240 C. Super coiled pBR322 and CT
DNA was purchased from Dafeng Co. (China). All the drugs
and DNA were performed in 0.1 M phosphate buffer (pH =
7.7). 4,4ꢀ-Dihydroxyldipenyl and other derivatives were purchased
from Fischer Co. and TCI. All other chemicals and solvents were
commercial available.
fluoropropane (6e). Yield: 120 mg, 14%; Rf = 0.2 (ethanol); H
NMR (CDCl3, 300 MHz) d 7.14 (d, J = 8.1 Hz, 2 H, ArH),
6.96 (s, 2 H, ArH), 6.77 (d, J = 8.4 Hz, 2 H, ArH), 3.61 (s,
4 H, –CH2–), 2.32 (s, 12 H, –CH3); 13C NMR (CDCl3, 150 MHz)
d 158.46, 130.80, 129.93, 125.42, 123.77, 121.36, 115.59, 114.53,
62.88, 44.43; HRESIMS: m/z 451.1818 (calcd for C21H24O2N2F6 +
H, 451.1820).
2,2ꢀ -Dimethylaminemethylene-9,9-bis(4-hydroxyphenyl)fluorene
(6f). Yield: 464 mg, 50%; Rf = 0.2 (ethanol); 1H NMR (CDCl3,
300 MHz) d 7.73 (d, J = 7.2 Hz, 2 H, ArH), 7.36–7.30 (m, 4 H,
ArH), 7.26–7.22 (m, 2 H, ArH), 7.04–6.92 (m, 2 H, ArH), 6.72
(s, 2 H, ArH), 6.65 (d, J = 8.7 Hz, 2 H, ArH), 3.48 (s, 4 H,
–CH2–), 2.25 (s, 12 H, –CH3); 13C NMR (CDCl3, 150 MHz) d
156.80, 152.20, 140.12, 136.79, 128.81, 128.11, 127.77, 127.35,
126.27, 121.43, 120.22, 115.87, 64.33, 63.01, 44.65; HRESIMS:
m/z 465.2537 (calcd for C31H32O2N2 + H, 465.2542).
General procedure 1: synthesis of two Mannich base phenol
derivatives 6a–m
Appropriate 4,4ꢀ-dihydroxydiphenol was dissolved in ethanol in
a round bottom flask and aqueous solutions of dimethylamine
(3.0 equiv., 33%) and formaldehyde (4.0 equiv., 37%) were added.
The reaction mixture was stirred overnight at room temperature.
The solvents were then evaporated under reduced pressure and
the crude product was subjected to silica gel chromatography with
2,2ꢀ - Dimethylaminemethylene - 4,4ꢀ - (1 - a - methylbenzylidene)-
bisphenol (6g). Yield: 250 mg, 21%; Rf = 0.2 (ethanol);
1HNMR(CDCl3, 300 MHz) d 7.27–7.18 (m, 3 H, ArH), 7.09–7.06
This journal is
The Royal Society of Chemistry 2006
Org. Biomol. Chem., 2006, 4, 3358–3366 | 3361
©

View Article Online
(dd, J = 1.5, 7.5 Hz, 2 H, ArH), 6.85–6.83 (t, 2 H, ArH), 6.72 (s,
2 H, ArH), 6.69 (d, 2 H, ArH), 3.55 (s, 4 H, –CH2–), 2.31 (s, 12 H,
–CH3), 2.11 (s, 3 H, –CH3); 13C NMR (CDCl3, 150 MHz) d 156.13,
150.23, 140.19, 129.23, 128.88, 128.65, 127.94, 125.94, 121.22,
115.31, 63.30, 51.28, 44.73, 30.95; HRESIMS: m/z 405.2530 (calcd
for C26H32O2N2 + H, 405.2542).
General procedure 2: synthesis of 2,2ꢀ-bis(trimethylammoniummeth-
ylene)-4,4ꢀ-diphenol iodide derivatives 7a–m
6a–m was dissolved in CH3CN and CH3I (0.5 mL, 8.0 mmol)
added to the mixture. The reaction mixture was stirred in the
dark for one night. After adding 40 mL of absolute ether and the
product was precipitated and crude product was obtained. Finally,
pure product was obtained by crystallizing from ether.
2,2ꢀ -Dimethylaminemethylene-1,1-bis(4-hydroxyphenyl)cyclo-
1
2,2ꢀ -Bis(trimethyl-ammoniummethylene)-4,4ꢀ-sulfonyldiphenol
iodide (7a). Yield: 98 mg, 76%; 1H NMR (D2O, 300 MHz)
d 7.82 (s, 2 H, ArH), 7.73 (d, J = 9.0 Hz, 2 H, ArH), 6.95(d,
J = 9.0 Hz, 2 H, ArH), 4.34 (s, 4 H, –CH2–),2.91 (s, 18 H,
–CH3); 13C NMR (D2O, 150 MHz) d 162.11, 134.05,131.97,
130.81,117.62,115.78, 63.26, 52.69; HRESIMS: m/z 521.0967
(calcd for C20H30O4N2SI2–I, 521.0971).
hexane (6h). Yield: 203 mg; 13%, Rf = 0.2 (ethanol); H NMR
(CDCl3, 300 MHz) d 7.04 (d, J = 8.4 Hz, 2 H, ArH), 6.79 (s, 2 H,
ArH), 6.72 (d, J = 8.4 Hz, 2 H, ArH), 3.57 (s, 4 H, –CH2–), 2.29
(s, 12 H, –CH3), 2.16 (s, 4 H, –CH2–), 1.49 (s, 6 H, –CH2–); 13C
NMR (CDCl3, 150 MHz) d 155.31, 139.46, 127.36, 127.08, 121.26,
115.41, 63.16, 44.95, 44.47, 37.52, 26.48, 22.96; HRESIMS: m/z
383.2689 (calcd for C24H34O2N2 + H, 383.2698).
2,2ꢀ -Bis(trimethylammoniummethylene)bis(4-hydroxyphenyl)-
sulfide iodide (7b). Yield: 75 mg, 82%; 1H NMR (D2O, 300 MHz)
d 7.26 (s, 2 H, ArH), 7.23 (d, J = 8.4 Hz, 2 H, ArH), 6.80 (d,
J = 8.4 Hz, 2 H, ArH), 4.25 (s, 4 H, –CH2–), 2.88 (s, 18 H, –
CH3); 13C NMR (D2O, 150 MHz) d 156.48, 137.55, 135.84, 126.36,
117.62, 115.80, 63.74, 52.60; HRESIMS: m/z 489.1065 (calcd for
C20H30O2N2SI2–I, 489.1073).
2,2ꢀ -Dimethylaminemethylenephenolphthalein
(6i). Yield:
301 mg, 35%; Rf = 0.2 (ethanol); 1H NMR (CDCl3, 300 MHz) d
7.89 (d, J = 7.8 Hz, 1 H, ArH), 7.65 (m, 1 H, ArH), 7.53–7.46 (m,
2 H, ArH), 7.04–7.00 (m, 2 H, ArH), 6.91 (s, 2 H, ArH), 6.71 (d,
J = 4.5 Hz, 2 H, ArH), 3.55 (s, 4 H, –CH2–), 2.28 (s, 12 H, –CH3);
13C NMR (CDCl3, 150 MHz) d 170.20, 158.34, 152.86, 134.04,
131.30, 129.03, 127.82, 127.18, 125.83, 125.48, 124.04, 121.71,
115.71, 92.10, 62.67, 44.44; HRESIMS: m/z 433.2126 (calcd for
C26H28O4N2 + H, 433.2127).
2,2ꢀ -Bis(trimethylammoniummethylene)-a,aꢀ -bis(4-hydroxyphe-
1
nyl)-1,4-diisopropylbenzene iodide (7c). Yield: 88 mg, 78%; H
NMR (D2O, 300 MHz) d 7.00 (d, J = 7.5 Hz, 4 H, ArH), 6.89 (s,
4 H, ArH) 6.66 (d, J = 7.2 Hz, 2 H, ArH) 4.16 (s, 4 H, –CH2–), 2.80
(s, 18 H, –CH3), 1.35 (s, 12 H, –CH3), 13C NMR (D2O, 150 MHz)
d 154.20, 148.31, 142.89, 132.93, 130.92, 126.53, 115.99, 113.89,
64.22, 52.46, 41.47,29.85; HRESIMS: m/z 617.2595 (calcd for
C32H46O2N2I2–I, 617.2604).
2,2ꢀ-Dimethylaminemethylene-4,4ꢀ-dihydroxydiphenylether (6j).
Yield: 149 mg, 24%; Rf = 0.2 (ethanol); 1H NMR (CDCl3,
300 MHz) d 7.12 (d, J = 6.6 Hz, 2 H, ArH), 6.74 (d, J = 6.6 Hz, 2 H,
ArH), 6.60 (s, 2 H, ArH), 3.57 (s, 4 H, –CH2–), 2.31 (s, 12 H, –CH3);
13C NMR (CDCl3, 150 MHz) d (ppm) 153.62, 150.20, 122.98,
118.76, 118.74, 116.72, 63.12, 44.96. HRESIMS: m/z 317.1864
(calcd for C18H24O3N2 + H, 317.1860).
2,2ꢀ -Bis(trimethylammoniummethylene)-1,3-bis(4-hydroxyphe-
nyloxyl) benzene iodide (7d). Yield: 28 mg, 67%; 1H NMR (D2O,
300 MHz) d 7.10 (t, J = 9.0 Hz, 1 H, ArH) 6.98 (d, J = 6.9 Hz,
4 H, ArH), 6.81 (d, J = 9.6 Hz, 2 H, ArH), 6.52 (d, J =
6.0 Hz, 2 H, ArH), 6.30 (s, 1 H, ArH), 4.23 (s, 4 H, –CH2–), 2.87
(s, 18 H, –CH3); 13C NMR (D2O, 150 MHz) d 159.44, 153.38,
148.34, 125.70, 124.54, 117.71, 115.56, 112.70, 111.74, 106.38,
63.79, 52.54; HRESIMS: m/z 565.1553 (calcd for C26H34O4N2I2–I,
565.1563).
2,2ꢀ-Dimethylaminemethylene-4,4ꢀ-dihydroxybenzophenone (6k).
Yield: 279 mg, 42%; Rf = 0.2 (ethanol); 1H NMR (CDCl3,
300 MHz) d 7.58 (d, J = 8.4 Hz, 2 H, ArH), 7.51 (s, 2 H, ArH),
6.83 (d, J = 8.1 Hz, 2 H, ArH), 3.72 (s, 4 H, –CH2–), 2.38 (s,
12 H, –CH3). 13C NMR (CDCl3, 150 MHz) d 161.99, 131.86,
130.53, 129.13, 121.65, 115.42, 63.09, 45.16, 30.55. HRESIMS:
m/z 329.1867 (calcd for C19H24O3N2 + H, 329.1860).
2,2ꢀ -Bis(trimethylammoniummethylene)-2,2-bis(4-hydroxyphe-
nyl)hexafluoropropane iodide (7e). Yield: 86 mg, 88%; 1H NMR
(D2O, 300 MHz) d 7.27 (d, J = 9.0 Hz, 2 H, ArH) 7.21 (s, 2 H,
ArH), 6.83 (d, J = 8.7 Hz, 2 H, ArH), 4.26 (s, 4 H, –CH2–), 2.85 (s,
18 H, –CH3); 13C NMR (D2O, 150 MHz) d 155.51, 134.17, 132.19,
122.99, 122.28, 121.10, 114.37, 112.63, 61.74, 50.54; HRESIMS:
m/z 607.1249 (calcd for C23H30O2N2F6I2–I, 607.1256).
2,2ꢀ-Dimethylamine methylene-4,4ꢀ-dihydroxydiphenylmethane
(6l). Yield: 197 mg, 31%; Rf = 0.2 (ethanol); 1H NMR (CDCl3,
300 MHz) d 6.96 (d, J = 8.1 Hz, 2 H, ArH), 6.76–6.69 (q, 4 H,
ArH), 3.77 (s, 2 H, ArH), 3.59 (s, 4 H, –CH2–), 2.32 (s, 12 H, –CH3).
13C NMR (CDCl3, 150 MHz) d 155.94, 131.91, 128.92, 128.68
121.82, 115.92, 63.49, 45.27, 40.92. HRESIMS: m/z 315.2069
(calcd for C19H26O2N2 + H, 315.2067).
2,2ꢀ -Bis(trimethylammoniummethylene)-9,9-bis (4-hydroxyphe-
nyl)fluorene iodide (7f). Yield: 63 mg, 65%; 1H NMR (D2O,
300 MHz) d 7.73 (d, J = 7.5 Hz, 2 H, ArH), 7.30–7.25 (m,
4 H, ArH), 7.19–7.12 (m, 4 H, ArH), 6.84 (s, 2 H, ArH), 6.71
(d, J = 8.4 Hz, 2 H, ArH), 4.06 (s, 4 H, –CH2–), 2.78 (s, 18 H,
–CH3); 13C NMR (D2O, 150 MHz) d 155.49, 150.86, 139.56,
137.26, 133.36, 132.32, 128.37, 125.77, 120.79, 116.46, 114.12,
63.83, 63.37, 52.37; HRESIMS: m/z 621.1966 (calcd for
C33H38O2N2I2–I, 621.1978).
2,2ꢀ-Dimethylaminemethylene-4,4ꢀ-isopropylidenediphenol (6m).
Yield: 82 mg, 12%; Rf = 0.2 (ethanol); 1H NMR (CDCl3, 300 MHz)
d 7.02–6.98 (m, 2 H, ArH), 6.74 (d, J = 8.1 Hz, 2 H, ArH),
6.70 (d, J = 8.1 Hz, 2 H, ArH), 3.55 (s, 4 H, –CH2–), 2.29 (s,
12 H, –CH3), 1.58 (s, 6 H, –CH3). 13C NMR (CDCl3, 150 MHz) d
155.62, 141.71, 126.97, 126.83, 121.25, 115.32, 63.49, 44.89, 41.89,
31.64. HRESIMS: m/z 343.2371 (calcd for (C21H30O2N2 + H),
343.2380).
3362 | Org. Biomol. Chem., 2006, 4, 3358–3366
This journal is
The Royal Society of Chemistry 2006
©

View Article Online
2,2ꢀ -Bis(trimethylammoniummethylene)-4,4ꢀ -(1-a-methylbenzy-
lidene)bisphenol iodide (7g). Yield: 197 mg, 89%; 1H NMR (D2O,
300 MHz) d 7.09–7.05 (m, 3 H, ArH), 7.03–6.69 (m, 4 H, ArH),
6.82 (s, 2 H, ArH), 6.70 (d, J = 8.7 Hz, 2 H, ArH), 4.13 (s, 4 H,
–CH2–), 2.75 (s, 18 H, –CH3), 1.88 (s, 3 H, –CH3); 13C NMR (D2O,
150 MHz) d 153.81, 148.20, 140.07, 133.69, 131.65, 127.56, 127.44,
125.60, 115.07, 113.01, 63.24, 51.51, 49.98, 28.58; HRESIMS: m/z
561.1967 (calcd for C28H38O2N2I2–I, 561.1978).
General procedure 3: synthesis of four Mannich base phenol
derivatives 8a–n
Appropriate 4,4ꢀ-dihydroxydiphenyl was dissolved in a solvent
of ethanol in a round bottom flask, and two kinds of aqueous
solutions of dimethylamine (10.0 equiv., 33%) and formalaldehyde
(10.0 equiv., 37%) were added, respectively. The reaction mixture
was kept at reflux for about 3 h and reaction was monitored by
TLC. After finished reaction, the solvents were evaporated under
reduced pressure and the crude product was subjected to silica
gel chromatography with ethanol and triethylamine (10 : 1). After
evaporated the solvent, the appropriate white or buff powder was
obtained.
2,2ꢀ -Bis(trimethylammoniummethylene)-1,1-bis(4-hydroxyphe-
nyl)cyclohexane iodide (7h). Yield: 159 mg, 91%; 1H NMR (D2O,
300 MHz) d 7.18 (d, J = 8.4 Hz, 2 H, ArH), 7.10 (s, 2 H, ArH),
6.72 (d, J = 8.4 Hz, 2 H, ArH), 4.20 (s, 4 H, –CH2–), 2.82 (s, 18 H,
–CH3), 2.05 (s, 4 H, –CH2–), 1.27 (s, 6 H, –CH2–);13C NMR (D2O,
150 MHz) d 154.23, 132.92, 131.22, 116.34, 114.31, 64.28, 52.36,
44.40, 36.21, 25.74, 22.53; HRESIMS: m/z 539.2184 (calcd for
C26H40O2N2I2–I, 539.2135).
2,5,2ꢀ,5ꢀ-Tetra(trimethylaminemethylene)-4,4ꢀ-sulfonyldiphenol
(8a). Yield: 370 mg, 77%; Rf = 0.15 (ethanol–triethylamine =
10 : 1); 1H NMR (CDCl3, 300 MHz) d 7.58 (s, 4 H, ArH), 3.55 (s,
8 H, –CH2–), 2.28 (s, 24 H, –CH3); 13C NMR (CDCl3, 150 MHz)
d 161.73, 131.57, 128.45, 124.14, 60.26, 45.04; HRESIMS: m/z
479.2686 (calcd for C24H38O4N4S + H, 479.2692).
2,2ꢀ-Bis(trimethylammoniummethylene)phenolphthalein iodide
1
(7i). Yield: 65 mg, 78%; H NMR (D2O, 300 MHz) d 7.83 (d,
2,5,2ꢀ,5ꢀ -Tetra(trimethylaminemethylene)bis(4-hydroxyphenyl)-
J = 3.6 Hz, 1 H, ArH), 7.72–7.68 (m, 1 H, ArH), 7.57–7.51 (m,
2 H, ArH), 7.24–7.21 (m, 2 H, ArH), 7.16 (d, J = 2.4 Hz, 2 H,
ArH), 6.84 (d, J = 8.4 Hz, 2 H, ArH), 4.26 (s, 4 H, –CH2–),2.88 (s,
18 H, –CH3); 13C NMR (D2O, 150 MHz) d 157.91, 151.99, 135.78,
133.21, 131.64, 131.23, 130.19, 126.13, 124.02, 116.87, 114.75,
92.51, 63.84, 55.22, 52.49, 35.81; HRESIMS: m/z 589.1558 (calcd
for C28H34O4N2I2–I, 589.1563).
sulfide (8b). Yield: 512 mg, 57%; Rf
=
0.15 (ethanol–
1
triethylamine = 10 : 1); H NMR(CDCl3, 300 MHz) d 6.96 (s,
4 H, ArH), 3.43 (s, 8 H, –CH2–), 2.22 (s, 24 H, –CH3); 13CNMR
(CDCl3, 150 MHz) d 156.55, 132.10, 124.76, 124.33, 60.44, 45.05;
HRESIMS: m/z 447.2792 (calcd for C24H38O2N4S + H, 447.2794).
2,5,2ꢀ,5ꢀ -Tetra(trimethylaminemethylene)-a,aꢀ -bis(4-hydroxy-
phenyl)-1,4-diisopropylbenzene (8c). Yield: 210 mg, 63% Rf =
0.15 (ethanol–triethylamine = 10 : 1); 1H NMR (CDCl3, 300 MHz)
d 7.05 (s, 4 H, ArH), 6.92 (s, 4 H, ArH), 3.61 (s, 8 H, –CH2–), 2.33
(s, 24 H, –CH3),1.62 (s, 12 H, –CH3);13CNMR (CDCl3, 150 MHz)
d 154.82, 148.23, 140.77, 128.09, 126.33, 121.55, 60.40, 44.64,
41.93, 31.11; HRESIMS: m/z 575.4323 (calcd for C36H54O2N4 +
H, 575.4325).
2,2ꢀ-Bis(trimethylammoniummethylene)-4,4ꢀ-dihydroxydiphenyl-
ether iodide (7j). Yield: 78 mg, 90%; 1H NMR (D2O, 300 MHz) d
6.85–6.78 (m, 4 H, ArH), 6.78 (s, 2 H, ArH), 4.22 (s, 4 H, –CH2–),
2.87 (s, 18 H, –CH3); 13C NMR (D2O, 150 MHz) d 152.60, 150.19,
124.12, 122.96, 117.87, 115.74, 64.11, 52.91. HRESIMS: m/z
173.1119 (calcd for (C20H30O3N2I2 − 2I)/2), 173.1123).
2,2ꢀ -Bis(trimethylammoniummethylene)-4,4ꢀ -dihydroxylbenzo-
phenone iodide (7k). Yield: 349 mg, 62%; 1H NMR (D2O,
300 MHz) d 7.73 (s, 2 H, ArH), 7.68 (d, J = 8.7 Hz, 2 H, ArH),
6.99 (d, J = 8.1 Hz, 2 H, ArH), 4.43 (s, 4 H, –CH2–), 3.01 (s,
18 H, –CH3); 13C NMR (D2O, 150 MHz) d 161.16, 137.08, 135.12,
128.85, 116.19, 114.88, 64.13, 53.19. HRESIMS: m/z 179.1120
(calcd for (C21H30O3N2I2 − 2I)/2), 179.1123).
2,5,2ꢀ,5ꢀ -Tetra(trimethylaminemethylene)-1,3-bis(4-hydroxyphe-
nyloxyl)benzene (8d). Yield: 170 mg, 88%; Rf = 0.15 (ethanol–
1
triethylamine = 10 : 1); H NMR (CDCl3, 300 MHz) d 7.13 (t,
J = 8.7 Hz, 1 H, ArH), 6.78 (s, 4 H, ArH), 6.53 (d, J = 1.2 Hz,
2 H, ArH) 6.50 (s, 1 H, ArH), 3.55 (s, 8 H, –CH2–), 2.31 (s, 24 H,
–CH3); 13C NMR (CDCl3, 150 MHz) d 160.28, 153.55, 147.61,
130.22, 123.92, 121.12, 110.89, 106.77, 60.14, 44.85; HRESIMS:
m/z 523.3274 (calcd for C30H42O4N4 + H, 523.3284).
2,2ꢀ-Bis(trimethylammoniummethylene)-4,4ꢀ-dihydroxydiphenyl-
methane iodide (7l). Yield: 69 mg, 71%; 1H NMR (D2O,
300 MHz) d 7.13 (s, 2 H, ArH), 7.08 (m, 2 H, ArH), 6.78 (d, J =
8.7 Hz, 2 H, ArH), 4.22 (s, 4H, –CH2–), 3.64 (s, 2 H, –CH2–),
2.90 (s, 18H, –CH3). 13C NMR (D2O, 150 MHz) d 154.90, 134.65,
133.98, 132.97, 116.89, 114.69, 64.24, 52.77, 39.02. HRESIMS:
m/z 172.1222 (calcd for (C21H32O2N2I2 − 2I)/2), 172.1226).
2,5,2ꢀ,5ꢀ-Tetra(trimethylaminemethylene)-2,2-Bis(4-hydroxyphe-
nyl)hexafluoropropane (8e). Yield: 300 mg, 72%; Rf = 0.15
1
(ethanol–triethylamine = 10 : 1); H NMR (CDCl3, 300 MHz)
d 7.01 (s, 4 H, ArH), 3.56 (s, 8 H, –CH2–), 2.30 (s, 24 H, –CH3);
13C NMR (CDCl3, 150 MHz) d 157.62, 131.13, 125.62, 123.72,
123.06, 122.68, 60.57, 44.87; HRESIMS: m/z 565.2977 (calcd for
C27H38O2N4F6 + H, 565.2977).
2,2ꢀ -Bis(trimethylammoniummethylene)-4,4ꢀ -isopropylidenedi-
phenol iodide (7m). Yield: 67 mg, 70%; 1H NMR (D2O, 300 MHz)
d 7.18 (d, J = 8.1 Hz, 2 H, ArH), 7.11(s, 2 H, ArH), 6.78 (d,
J = 8.7 Hz, 2 H, ArH), 4.31 (s, 4 H, –CH2–), 2.93 (s, 18 H, –
CH3), 1.56 (s, 6 H, –CH3); 13C NMR (D2O, 150 MHz) d 154.45,
143.09, 132.77, 131.20, 116.26, 114.23, 64.43, 52.63, 41.36, 29.93.
HRESIMS: m/z 172.1222 (calcd for (C23H36O2N2I2 − 2I)/2),
172.1226).
2,5,2ꢀ,5ꢀ -Tetra(trimethylaminemethylene)-9,9-bis(4-hydroxyphe-
nyl)fluorene (8f). Yield: 653 mg, 57%; Rf = 0.15 (ethanol–
1
triethylamine = 10 : 1); H NMR (CDCl3, 300 MHz) d 7.74 (d,
J = 8.4 Hz, 2 H, ArH), 7.34 (t, J = 8.1 Hz, 4 H, ArH), 7.24 (t,
J = 7.2 Hz, 4 H, ArH), 6.78 (s, 4 H, ArH), 3.43 (s, 8 H, –CH2–),
2.23 (s, 24 H, –CH3); 13C NMR (CDCl3, 150 MHz) d 155.65,
152.02, 139.89, 135.71, 129.00, 127.53, 127.12, 126.00, 121.97,
This journal is
The Royal Society of Chemistry 2006
Org. Biomol. Chem., 2006, 4, 3358–3366 | 3363
©

View Article Online
120.01, 63.97, 60.31, 44.59; HRESIMS: m/z 579.3697 (calcd for
C37H46O2N4 + H, 579.3699).
ArH), 3.69 (s, 8 H, –CH2–), 2.37 (s, 24 H, –CH3); 13C NMR
(CDCl3, 150 MHz) d 156.00, 131.21, 127.66, 122.13, 59.65,
44.215; HRESIMS: m/z 415.3062 (calcd for C24H38O2N4 + H,
415.3073).
2,5,2ꢀ,5ꢀ -Tetra(trimethylaminemethylene)-4,4ꢀ -(1-a-methylben-
zylidene)bisphenol (8g). Yield: 620 mg, 60%; Rf = 0.15 (ethanol–
triethylamine = 10 : 1); 1H NMR (CDCl3, 300 MHz) d 7.26–7.18
(m, 3 H, ArH), 7.05 (d, J = 7.2 Hz, 2 H, ArH), 6.66 (s, 4 H, ArH),
General procedure 4: synthesis of 2,5,2ꢀ,5ꢀ - tetra ( trimethylammo-
niummethylene)-4,4ꢀ-dihydroxydiphenyl iodide derivatives 9a–n
3.42 (s, 8 H, –CH2–), 2.22 (s, 24 H, –CH3), 2.09 (s, 3 H, –CH3); 13
C
NMR (CDCl3, 150 MHz) d 155.00, 150.29, 139.32, 129.61, 128.85,
127.87, 125.86, 122.22, 60.90, 51.21, 44.99, 30.90; HRESIMS: m/z
519.3692 (calcd for C32H46O2N4 + H, 519.3699).
8a–n was dissolved in CH3CN and CH3I (5.0 mL, 80 mmol) was
added to the mixture. The reaction mixture was stirred in the
dark for about two weeks. After removed the solvent by reduced
pressure evaporated and crude product was obtained. Finally, pure
product was obtained by crystallizing from ether.
2,5,2ꢀ,5ꢀ -Tetra(trimethylaminemethylene)-1,1-bis(4-hydroxyphe-
nyl)cyclohexane (8h). Yield: 743 mg, 75%; Rf = 0.15 (ethanol–
1
triethylamine = 10 : 1); H NMR (CDCl3, 300 MHz) d 6.78 (s,
2,5,2ꢀ,5ꢀ -Tetra(trimethylammoniummethylene)-4,4ꢀ-sulfonyldi-
phenol iodide (9a). Yield: 86 mg, 82%; 1H NMR (D2O, 300 MHz)
d 7.97 (s, 4 H, ArH), 4.47 (s, 2 H, –CH2–), 4.40 (s, 6 H, –CH2–
), 3.01 (s, 9 H, –CH3), 2.95 (s, 27 H, –CH3); 13CNMR (D2O,
150 MHz) d 137.29, 136.54, 127.34, 125.08, 119.32, 64.12, 63.31,
53.07, 52.74; anal. found: C, 31.39; H, 4.74; N, 5.18; calcd for
C28H50O4N4I4S·H2O: C, 31.59; H, 4.92; N, 5.26%.
2,5,2ꢀ,5ꢀ-Tetra(trimethylammoniummethylene)bis(4-hydroxyphe-
nyl)sulfide iodide (9b). Yield: 123 mg, 90%; 1H NMR (D2O,
300 MHz) d 7.47 (s, 4 H, ArH), 4.34 (s, 8 H, –CH2–), 2.88 (s,
36 H, –CH3); 13C NMR (D2O, 150 MHz) d 157.36, 140.50, 140.11,
119.64, 63.74, 52.77; anal. found: C, 31.56; H, 5.18; N, 5.36; calcd
for C28H50O2N4I4S·3H2O: C, 31.47; H, 5.28; N, 5.24%.
4 H, ArH), 3.41 (s, 8 H, –CH2–), 2.18 (s, 24 H, –CH3), 2.11 (s,
4 H, –CH2–), 1.42 (s, 6 H, –CH2–); 13C NMR (CDCl3, 150 MHz)
d 154.31, 138.88, 127.98, 122.62, 61.03, 45.04, 37.58, 26.71, 23.25;
HRESIMS: m/z 497.3853 (calcd for C30H48O2N4 + H, 497.3856).
2,5,2ꢀ,5ꢀ -Tetra(trimethylaminemethylenephenolphthalein (8i).
Yield: 460 mg, 45%; Rf = 0.15 (ethanol–triethylamine = 10 : 1);
1H NMR (CDCl3, 300 MHz) d 7.90 (d, J = 7.8 Hz, 1 H, ArH),
7.66 (t, J = 7.5 Hz, 1 H, ArH), 7.52 (t, J = 6.6 Hz, 2 H, ArH),
6.90 (s, 4 H, ArH), 3.44 (s, 8 H, –CH2–), 2.22 (s, 24 H, –CH3);
13C NMR (CDCl3, 150 MHz) d 170.49, 157.26, 153.20, 134.25,
130.72, 129.18, 128.05, 126.03, 125.67, 124.22, 123.27, 92.47,
60.75, 45.21; HRESIMS: m/z 547.3284 (calcd for C32H42O4N4 +
H, 547.3284).
2,5,2ꢀ,5ꢀ-Tetra(trimethylammoniummethylene)-a,aꢀ-bis(4-hydro-
xyphenyl)-1,4-diisopropylbenzene iodide (9c). Yield: 96 mg, 87%;
1H NMR (D2O, 300 MHz) d 7.20 (s, 4 H, ArH), 7.01 (s, 4 H, ArH),
4.28 (s, 8 H, –CH2–), 2.80 (s, 36 H, –CH3), 1.42 (s, 12 H, –CH3);13C
NMR (D2O, 150 MHz) d 157.61, 148.32, 136.07, 126.85, 117.95,
64.42, 52.64, 41.80, 29.68; anal. found: C, 39.62; H, 6.37; N, 5.20;
calcd for C40H66O2N4I4.4H2O·0.5CH3CN: C, 39.87; H, 6.16; N,
5.10%.
2,5,2ꢀ,5ꢀ -Tetra(trimethylaminemethylene)-4,4ꢀ -dihydroxydiphe-
nylether (8j). Yield: 282 mg, 88%; Rf = 0.15 (ethanol–
1
triethylamine = 10 : 1); H NMR (CDCl3, 300 MHz) d 6.71 (s,
4 H, ArH), 3.58 (s, 8 H, –CH2–), 2.34 (s, 24 H, –CH3); 13C NMR
(CDCl3, 150 MHz) d 152.61, 149.69, 123.22, 119.61, 59.89, 44.58;
HRESIMS: m/z 431.3025 (calcd for C24H38O3N4 + H, 431.3022).
2,5,2ꢀ,5ꢀ -Tetra(trimethylaminemethylene)-4,4ꢀ -dihydroxybenzo-
phenone (8k). Yield: 371 mg, 72%; Rf = 0.15 (ethanol–
triethylamine = 10 : 1); 1H NMR (CDCl3, 300 MHz) d 7.51 (s, 4 H,
ArH), 3.57 (s, 8 H, –CH2–), 2.30 (s, 24 H, –CH3); 13C NMR (CDCl3,
150 MHz) d 195.13, 161.40, 131.78, 128.76, 123.09, 60.49, 45.09;
HRESIMS: m/z 443.3012 (calcd for C25H38O3N4 + H, 443.3022).
2,5,2ꢀ,5ꢀ -Tetra(trimethylammoniummethylene)-1,3-bis(4-hydro-
xyphenyloxyl)benzene iodide (9d). Yield: 55 mg, 88.5%; 1H NMR
(D2O, 300 MHz) d 7.03 (s, 4 H, ArH), 6.96 (t, J = 8.7 Hz, 1 H,
ArH), 6.52 (s, 1 H, ArH), 6.39 (d, J = 7.5 Hz, 2 H, ArH), 4.19
(s, 8 H, –CH2–), 2.74 (s, 36 H, –CH3), 13C NMR (D2O, 150 MHz)
d 158.56, 152.81, 149.94, 131.24, 128.07, 120.06, 113.17, 109.03,
63.80, 52.75; anal. found: C, 36.35; H, 5.31; N, 4.64; calcd for
C34H54O4N4I4·3H2O·0.5C2H5OC2H5: C, 36.59; H, 5.54; N, 4.74%.
2,5,2ꢀ,5ꢀ -Tetra(trimethylaminemethylene)-4,4ꢀ -dihydroxydiphe-
nylmethane (8l). Yield: 118 mg, 69%; Rf = 0.15 (ethanol–
1
triethylamine = 10 : 1); H NMR (CDCl3, 300 MHz) d 6.79 (s,
4 H, ArH), 3.74 (s, 2 H, –CH2–), 3.48 (s, 8 H, –CH2–) 2.26 (s, 24 H,
–CH3); 13C NMR (CDCl3, 150 MHz) d 154.77, 131.44, 129.22,
122.94, 60.34, 44.80, 40.14; HRESIMS: m/z 429.3228 (calcd for
C25H40O2N4 + H, 429.3229).
2,5,2ꢀ,5ꢀ -Tetra(trimethylammoniummethylene)-2,2-Bis(4-hydro-
1
xyphenyl)hexafluoropropane iodide (9e). Yield: 45 mg, 74%; H
NMR (D2O, 300 MHz) d 7.51 (s, 4 H, ArH), 4.42 (s, 8 H, –CH2–),
2.88 (s, 36 H, –CH3);13C NMR (D2O, 150 MHz) d 161.35, 141.82,
139.67, 127.28, 121.47, 121.16, 66.29, 55.57; anal. found: C, 31.71;
H, 4.61; N, 4.82; calcd for C31H50O2N4I4F6·2H2O: C, 31.86; H,
4.66; N, 4.79%.
2,5,2ꢀ,5ꢀ -Tetra(trimethylaminemethylene)-4,4ꢀ -isopropylidenedi-
phenol (8m). Yield: 230 mg, 77%; Rf
=
0.15 (ethanol–
1
triethylamine = 10 : 1); H NMR (CDCl3, 300 MHz) d 6.81 (s,
4 H, ArH), 3.46 (s, 8 H, –CH2–), 2.24 (s, 24 H, –CH3), 1.59 (s, 6 H,
–CH3); 13C NMR (CDCl3, 150 MHz) d 154.53, 141.09, 127.59,
122.39, 60.86, 45.00, 41.63, 31.38; HRESIMS: m/z 457.3530 (calcd
for C27H44O2N4 + H, 457.3543).
2,5,2ꢀ,5ꢀ-Tetra(trimethylammoniummethylene)-9,9-bis(4-hydrox-
yphenyl)fluorene iodide (9f). Yield: 64 mg, 81%; 1H NMR (D2O,
300 MHz) d 7.74 (d, J = 7.5 Hz, 2 H, ArH), 7.36–7.25 (m,
8 H, ArH), 7.15 (m, 2 H, ArH), 4.26 (s, 8 H, –CH2–), 2.83
(s, 36 H, –CH3); 13C NMR (D2O, 150 MHz) d 155.89, 150.11,
139.72, 138.55, 137.01, 128.78, 128.63, 125.55, 121.17, 118.66,
2,5,2ꢀ,5ꢀ -Tetra(trimethylaminemethylene)-4,4ꢀ -dihydroxydiphe-
nyl (8n). Yield: 331 mg, 24%; Rf = 0.15 (ethanol–triethyl-
1
amine = 10 : 1); H NMR (CDCl3, 300 MHz) d 7.26 (s, 4 H,
3364 | Org. Biomol. Chem., 2006, 4, 3358–3366
This journal is
The Royal Society of Chemistry 2006
©

View Article Online
63.67, 63.26, 52.79; anal. found: C, 40.07; H, 5.80; N, 4.60; calcd
for C41H58O2N4I4·5H2O: C, 39.82; H, 5.54; N, 4.53%.
64.18, 53.03; anal. found: C, 32.94; H, 5.41; N, 5.42; calcd for
C28H50O2N4I4. 2H2O: C, 33.02; H, 5.34; N, 5.50%.
2,5,2ꢀ,5ꢀ-Tetra(trimethylammoniummethylene)-4,4ꢀ-(1-a-methyl-
benzylidene)bisphenol iodide (9g). Yield: 111 mg, 66%; 1H NMR
(D2O, 300 MHz) d 7.18 (s, 4 H, ArH), 7.14–7.07 (m, 5 H, ArH),
Crystallographic data collection
Compound crystals were grown up by slow evaporation of
methanol for one month. A single crystal was picked up for X-
ray analysis. Data for the X-ray structures was recorded using
a Bruker Smart APEX equipped with a CCD detector in the
c range of 2.36 to 26.00◦. The structure was solved by the
direct method in conjunction with standard difference Fourier
techniques. Hydrogen atoms were placed in calculated positions
using a standard riding model and were refined isotropically.
4.32 (s, 8 H, –CH2–), 2.83 (s, 36 H, –CH3), 2.00 (s, 3 H, –CH3); 13
C
NMR (D2O, 150 MHz) d 155.09, 148.00, 142.02, 137.65, 128.58,
128.40, 126.93, 118.01, 64.06, 52.67, 51.16, 29.49; anal. found: C,
37.63; H, 5.49; N, 4.81; calcd for C36H58O2N4I4·3.5H2O: C, 37.61;
H, 5.69; N, 4.87%.
2,5,2ꢀ,5ꢀ -Tetra(trimethylammoniummethylene)-1,1-bis(4-hydro-
xyphenyl)cyclohexane iodide (9h). Yield: 98 mg, 91%; 1H NMR
(D2O, 300 MHz) d 7.34 (s, 4 H, ArH), 4.34 (s, 8 H, –CH2–), 2.84 (s,
36 H, –CH3), 2.10 (s, 4 H, –CH2–), 1.28 (s, 6 H, –CH2–);13C NMR
(D2O, 150 MHz) d 154.37, 141.89, 136.49, 118.32, 64.19, 52.65,
44.93, 36.37, 25.61, 22.52; anal. found: C, 36.68; H, 5.71; N, 4.99;
calcd for C34H60O2N4I4·3H2O: C, 36.51; H, 5.94; N, 5.00%.
2,5,2ꢀ,5ꢀ - Tetra ( trimethylammoniummethylene ) phenolphthalein
iodide (9i). Yield: 46 mg, 53%; 1H NMR (D2O, 300 MHz) d 7.79
(d, J = 3.6 Hz, 2 H, ArH) 7.64 (d, J = 3.6 Hz, 2 H, ArH), 7.49–7.44
(m, 4 H, ArH), 4.34 (s, 8 H, –CH2–), 2.84 (s, 36 H, –CH3); 13C NMR
(D2O, 150 MHz) d 171.85,157.66, 151.37, 136.50, 136.18, 132.92,
130.67, 126.56, 124.13, 123.66, 118.76, 90.89, 63.52, 52.80; anal.
found: C, 35.52; H, 5.50; N, 4.59; calcd for C36H54O4N4I4·6H2O:
C, 35.37; H, 5.44; N, 4.58%.
Alkaline agarose gel electrophoresis assay
To test DNA–DNA cross-linking ability of these compounds, we
used linear plasmid DNA by denaturing alkaline agarose gel
electrophoresis reported by Cech14 and William.15 The duplex
DNA linearized by restriction endonuclease digestion with EcoR
I. DNA cross-linking experiments were carried out in phosphate
buffer (pH = 7.7). Samples were exposed to a 50 W high pressure
mercury lamp placed 20 cm away at 25 ◦C. The crude reaction
mixtures were loaded onto a denaturing 0.9% alkaline agarose
gel. Lambda Hind III was employed as a molecular weight marker.
The gel was subsequently stained in an ethidium bromide solution
(100 lL of a 10 mg mL−1 ethidium bromide solution in 1 L of 1 M
Tris–1.5 M NaCl buffer at pH = 7.5) for 1 h. Gels were visualized
by UV and photographed using Vilber Lourmat video system.
2,5,2ꢀ,5ꢀ-Tetra(trimethylammoniummethylene)-4,4ꢀ-dihydroxydi-
1
phenylether iodide (9j). Yield: 311 mg, 89%; H NMR (D2O,
300 MHz) d 7.53 (s, 4 H, ArH), 4.42 (s, 8 H, –CH2–), 2.95 (s,
36 H, –CH3); 13C NMR (D2O, 150 MHz) d 152.93, 150.73, 127.67,
120.46, 63.94, 53.10; anal. found: C, 31.80; H, 5.51; N, 5.16; calcd
for C28H50O3N4I4·3.5H2O: C, 31.68; H, 5.54; N, 5.28%.
2,5,2ꢀ,5ꢀ -Tetra(trimethylammoniummethylene)-4,4ꢀ - dihydroxy-
benzophenone iodide (9k). Yield: 356 mg, 78%; 1H NMR (D2O,
300 MHz) d 7.95 (s, 4 H, ArH), 4.54 (s, 8 H, –CH2–), 3.03 (s,
36 H, –CH3); 13C NMR (D2O, 150 MHz) d 195.58, 162.17, 139.84,
129.89, 118.42, 63.92, 53.03; anal. found: C, 32.56; H, 5.40; N,
5.01; calcd for C29H50O3N4I4·3.5H2O: C, 32.45; H, 5.35; N, 5.22%.
2,5,2ꢀ,5ꢀ-Tetra(trimethylammoniummethylene)-4,4ꢀ-dihydroxydi-
phenylmethane iodide (9l). Yield: 160 mg, 86%; 1H NMR (D2O,
300 MHz) d 7.38 (s, 4 H, ArH), 4.44 (s, 8 H, –CH2–), 3.94 (s,
2 H, –CH2–), 2.97 (s, 36 H, –CH3); 13C NMR (D2O, 150 MHz) d
154.97, 137.97, 134.95, 118.76, 64.17, 52.83; anal. found: C, 35.02;
H, 6.04; N, 4.87; calcd for C29H52O2N4I4. 3H2O·C2H5OC2H5: C,
35.25; H, 6.09; N, 4.98%.
Thermal denaturation study
The concentrations of CT DNA were determined spectrophoto-
metrically from appropriate molar absorptivity values and calcu-
lated using a molar absorptivity of 6600 mol L−1 cm−1 at 260 nm.
In order to test the interaction between drug molecules with DNA,
the melting curves were recorded at different compounds to CT
DNA by following the absorption chang◦e at 260 nm as a function
of temperature with a heating rate of 1.0 C min−1. Tm values were
determined from the maximum of the first derivative or from the
graphs at the mid point of the transition curves. DTm values were
calculated as usual13 by subtracting the Tm of the free nucleic acid
from the Tm of the complex.
Acknowledgements
X. Z. thanks for financial support by the National Science of
Foundation of China (No. 20272046) and the Trans-Century
Training Programme Foundation for the Talents by the Ministry
of Education of China. X.-L. Z. thanks for financial support by
the National Science of Foundation of China (No. 30370310).
X.-P C. thanks for TQ program 20021001 by the National Science
of Foundation of China.
2,5,2ꢀ,5ꢀ-Tetra(trimethylammoniummethylene)-4,4ꢀ-isopropylide-
1
nediphenol iodide (9m). Yield: 207 mg, 77%; H NMR (D2O,
300 MHz) d 7.38 (s, 4 H, ArH), 4.45 (s, 8 H, –CH2–), 3.035–2.955
(d, 36 H, –CH3), 1.58 (s, 6 H, –CH3); 13C NMR (D2O, 150 MHz)
d 154.58, 144.04, 136.20, 118.26, 64.20, 52.83, 41.86, 30.03; anal.
found: C, 34.36; H, 6.11; N, 4.90; calcd for C31H56O2N4I4. 4H2O.
0.25C2H5OC2H5: C, 34.47; H, 6.01; N, 5.02%.
References
2,5,2ꢀ,5ꢀ-Tetra(trimethylammoniummethylene)-4,4ꢀ-dihydroxydi-
phenyl iodide (9n). Yield: 76 mg, 71%; 1H NMR (D2O, 300 MHz)
d 7.59 (s, 4 H, ArH), 4.44 (s, 8 H, –CH2–), 2.93 (s, 36 H, –CH3);
13C NMR (D2O, 150 MHz) d 156.28, 136.17, 132.98, 119.05,
1 G. M. Blackburn and M. J. Gait, Nucleic Acids in Chemistry and
Biology, 1996, Oxford University Press, New York.
2 (a) E. R. Jamieson and S. J. Lippard, Chem. Rev., 1999, 99, 2467–2498;
(b) S. R. Rajski and R. M. Williams, Chem. Rev., 1998, 98, 2723–2796.
This journal is
The Royal Society of Chemistry 2006
Org. Biomol. Chem., 2006, 4, 3358–3366 | 3365
©

View Article Online
3 (a) D. S. Bose, A. S. Thompson, J. Ching, J. A. Hartley, M. D. Berardini,
T. C. Jenkins, S. Neidle, L. H. Hurley and D. E. Thurston, J. Am.
Chem. Soc., 1992, 114, 4939–4941; (b) J. A. Hartley, A. Hazrati, T. J.
Hodgkinson, L. R. Kelland, R. Khanim, M. Shipman and F. Suzenet,
Chem. Commun., 2000, 23, 2325–2326; (c) Y. Na, V.-S. Li, Y. Nakanishi,
K. F. Bastow and H. Kohn, J. Med. Chem., 2001, 44, 3453–3462; (d) F.
Nagatsugi, N. Tokuda, M. Maeda and S. Sasaki, Bioorg. Med. Chem.
Lett., 2001, 11, 2577–2579.
4 (a) Biological Applications of Photochemical Switches, ed. H. Morrison,
John Wiley & Sons, Inc., New York, NY, 1993; (b) T. Mohammad, C.
Chen, P. Guo and H. Morrison, Bioorg. Med. Chem. Lett., 1999, 9,
1703–1708.
5 J. Piette, H. B. Gamper, A. Van de Vorst and J. E. Hearst, Nucleic Acids
Res., 1988, 16, 9961–9977.
6 F. P. Gasparro, Photochem. Photobiol., 1996, 63, 553–557.
7 (a) V. N. Iyer and W. Szybalski, Science, 1964, 145, 55–58; (b) H. W.
Moore and R. Czerniak, Med. Res. Rev., 1981, 1, 249–280; (c) N. W.
Gaikwad and W. J. Bodell, Chem.-Biol. Interact., 2001, 138, 217–
229.
Turnbull, J. Org. Chem., 2000, 65, 2022–2029; (c) Q. Zhou, P. Pande,
A. E. Johnson and S. E. Rokita, Bioorg. Med. Chem., 2001, 9, 2347–
2354; (d) H. Amouri and J. L. Bras, Acc. Chem. Res., 2002, 35, 501–510;
(e) W. F. Veldhuyzen, P. Pande and S. E. Rokita, J. Am. Chem. Soc.,
2003, 125, 14005–14013.
9 D. Brousmiche and P. Wan, Chem. Commun., 1998, 4, 491–492.
10 S. N. Richter, S. Maggi, S. C. Mels, M. Palumbo and M. Freccero,
J. Am. Chem. Soc., 2004, 126, 13973–13979.
11 Y. Chiang, A. J. Kresge and Y. Zhu, J. Am. Chem. Soc., 2001, 123,
8089–8094.
12 P. Wang, R. P. Liu, X. J. Wu, H. J. Ma, X. P. Cao, P. Zhou, J. Y. Zhang,
X. C. Weng, X.-L. Zhang, J. Qi, X. Zhou and L. H. Weng, J. Am. Chem.
Soc., 2003, 125, 1116–1117.
13 L. Kibler-Herzog, B. Kell, G. Zon, K. Shinozuka, S. Mizan and W. D.
Wilson, Nucleic Acids Res., 1990, 18, 3545–3555.
14 T. R. Cech, Biochemistry, 1981, 20, 1431–1437.
15 J. J. Tepe and R. M. Williams, J. Am. Chem. Soc., 1999, 121, 2951–
2955.
16 E. E. Weinert, R. Dondi, S. Colloredo-Melz, K. N. Frankenfield, C. H.
Mitchell, M. Freccero and S. E. Rokita, J. Am. Chem. Soc., 2006, in
press.
8 (a) P. Pande, J. Shearer, J. Yang, W. A. Greenberg and S. E. Rokita,
J. Am. Chem. Soc., 1999, 121, 6773–6779; (b) Q. Zhou and K. D.
3366 | Org. Biomol. Chem., 2006, 4, 3358–3366
This journal is
The Royal Society of Chemistry 2006
©