Synthesis and evaluation of a cyclic imine derivative conjugated to a fluorescent molecule for labeling of proteins

Article abstract of DOI:10.1016/j.bmcl.2008.12.071

A cyclic imine conjugated to a fluorescent dansyl group was synthesized and used for covalent labeling of proteins. The covalent attachment to proteins was confirmed by gel electrophoresis and mass analysis.

Full text of DOI:10.1016/j.bmcl.2008.12.071

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1210–1213
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of a cyclic imine derivative conjugated
to a fluorescent molecule for labeling of proteins
Hai-Ming Guo a, Maki Minakawa a, Lynn Ueno b, Fujie Tanaka a,
*
a Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
b Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A cyclic imine conjugated to a fluorescent dansyl group was synthesized and used for covalent labeling of
proteins. The covalent attachment to proteins was confirmed by gel electrophoresis and mass analysis.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 12 November 2008
Revised 15 December 2008
Accepted 17 December 2008
Available online 25 December 2008
Keywords:
Imine
Tyrosine
Protein modification
Covalent labeling reactions of proteins are required for prepara-
tion of protein conjugates that are useful in research and medicine.
For example, antibody–drug conjugates are more efficient thera-
peutics than antibody alone or drug alone.1 When conjugated to
protein, small molecule drugs can be selectively delivered to tar-
gets, reducing side effects and extending the half-life of the
drugs.1,2 Fluorescently labeled proteins can be traced by detection
of the fluorescence.3 Currently available labeling methods include
labeling at lysine with succinimidyl ester derivatives, labeling at
cysteine with maleimide derivatives, and labeling of an unnatural
amino acid residue incorporated in protein sequence during its
expression.1c,d,4,5 Small peptide tag-based strategies for labeling
of proteins have also been developed.6 Most of the methods have
advantages and disadvantages.
To provide labeling reactions complementary to the existing
reactions, we have recently developed cyclic imines that covalently
react with phenols, including tyrosine residues, in aqueous solu-
tions7 (Scheme 1). The reactions of the imines with phenols pro-
ceeded over a wide pH range without the need of additional
catalysts.7 The imines were relatively stable and were resistant
to hydrolysis in aqueous solutions but efficiently reacted with
phenols.7
can be added by usual site-directed mutagenesis or by usual pep-
tide synthesis. Although imines prepared from formaldehyde and
aniline derivatives in situ have been used for labeling of proteins
at tyrosine,8 the optimal pH range for this reaction is 5.5–6.5 and
the reaction does not proceed above pH 8.8a In addition, formalde-
hyde cannot be used in many cases; for example, use of formalde-
hyde is not suitable for labeling of proteins at tyrosine inside living
cells or in the presence of living cells. Reactions at tyrosine using
reagents other than imines typically require additional catalysts
or multistep conversions after the first reaction step on the phenol
moiety of tyrosine.9a–c Use of the imine shown in Scheme 1 may
bypass the limitations and disadvantages of the existing labeling
methods.
To demonstrate the use of the cyclic imines shown in Scheme 1
for covalent attachment of non-native molecules to proteins and
peptides, here, we have synthesized a conjugate of the imine with
a fluorescent dansyl group and examined the reactions of the dan-
syl-conjugated imine with proteins and peptides.
H
N
H
N
Tyrosine residues are observed less often on the surface of
folded proteins than lysines or carboxylic acid-containing resi-
dues,8 which are commonly used as labeling sites. Thus, accessible
tyrosine is an attractive covalent labeling site.7–9 For proteins and
peptides that do not have an accessible tyrosine, a tyrosine residue
or
H
H
OH
OH HN
NH
NH
NH
O
O
O
aqueous buffer
pH 2–10
* Corresponding author.
E-mail address: ftanaka@scripps.edu (F. Tanaka).
Scheme 1.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.12.071

H.-M. Guo et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1210–1213
1211
O
O
HO
N
O
COOH
COOMe
Mn(OAc)3, NaN3
COOMe
LiOH
O
O
N
EDC, DMAP
O
TFA
THF-H2O
8%
CH2Cl2
N3
N3
CH3CN
N3
85%
N3
N3
1
85%
O
N3
S
9
2
4
3
H
N
O
O
N
H
H2N
S
O
N
N
O
O
5
N
O
H
O
O
MeOH-CH3CN
46%
N3
N3
6
O
H
O
N
N
Pd-C, H2
N
O
S
H
O
O
EtOAc-MeOH
quant
H2N
7
NH2
O
H
N
O
N
O
N
O
S
H
O
O
H
COOEt
N
CF3CH2OH-toluene
60%
NH
N
8a
O
O
H
O
N
N
N
O
S
H
O
O
HN
8b
Scheme 2.
O
The imine derivative linked to a fluorescent dansyl group was
synthesized as shown in Scheme 2.10 Diazidation of 1 afforded a
mixture of stereoisomers of 2; the major isomer was expected to
have trans-diazide groups due to the procedures used.11 Hydrolysis
of the ester with LiOH afforded 3, which was converted to succin-
imidyl ester 4. Reaction of 4 with amine 5, prepared from the cor-
responding diamine and dansyl chloride, afforded compound 6.
Diamine 7 was obtained by reduction of the azide groups by hydro-
genation with Pd–C under H2 in EtOAc–MeOH (3:1). The solvents
for this reduction were critical; reduction of 6 with Pd–C under
H2 but in MeOH mainly afforded monoamine, which might have
been generated via elimination of HN3. Diamine 7 was then trans-
formed to imine 8 as a mixture of regioisomers (8a + 8b). To con-
firm the imine functionality of 8, reaction of 8 with p-cresol was
performed in the presence of TFA (Scheme 3).12 Formation of addi-
tion product 9 confirmed the presence of the imine functionality in
compound 8.
Imine 8 was used for modification of proteins and peptides.
Reaction of peptide was performed using GlyTyr or AlaTyrAla
(90 mM) with 8 (20 mM) in 10% DMSO/100 mM sodium phos-
phate, pH 6.0 for 48 h at 37 °C. Imine 8 was completely consumed
as judged by TLC analysis of the reaction mixture. Formation of the
addition products were confirmed by mass analysis.13
To test reaction of 8 with proteins, lysozyme from chicken egg
white, a-chymotrypsinogen A type II from bovine pancreas, myo-
globin from equine skeletal muscle, carbonic anhydrase isozyme
II from bovine erythrocytes, and cytochrome C from horse heart
were used. Reactions of proteins (475 lM) were performed in the
presence of 8 (5 mM) in 5% DMSO/100 mM sodium phosphate,
pH 6.0 for 4 days at 37 °C and were analyzed by gel electrophoresis
(Fig. 1). Gel bands corresponding to modified proteins were fluo-
H, R
OH
H, R
a
a
b
b
c
c
d
d
e
e
f
f
OH HN
N
NH
Me
TFA, CH2Cl2
54%
NH
O
O
8
9
Me
O
H
N
Figure 1. Analysis of proteins modified with 8 after separation by gel electropho-
resis. Lane a, protein standard marker; b, lysozyme; c, chymotrypsinogen (see text);
d, myoglobin; e, carbonic anhydrase; f, cytochrome C. (A) Analysis of fluorescence of
dansyl group attached to proteins under UV 312 nm. (B) Analysis of proteins by
Coomassie staining.
O
N
N
H
O
S
R
=
O
O
Scheme 3.

1212
H.-M. Guo et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1210–1213
4. (a) Prescher, J. A.; Bertozzi, C. R. Nat. Chem. Biol. 2005, 1, 13; (b) Antos, J. M.;
Francis, M. B. Curr. Opin. Chem. Biol. 2006, 10, 253.
rescent under a UV lamp (312 nm). Unmodified proteins were not
fluorescent on a gel under the UV lamp (data not shown). Gel anal-
ysis showed that lysozyme was most easily modified among pro-
teins tested (lane b). Mass analysis of the reaction mixture of
lysozyme with 8 showed the formation of the mono-addition prod-
uct.14 We previously demonstrated that the imine derivative
shown in Scheme 1, prepared from trans-diaminocyclohexane,
selectively reacted at tyrosine and did not react with other natural
amino acid residues except cysteine thiol.7 Thus, reaction of 8 with
lysozyme probably occurred at tyrosine. Cytochrome C was not la-
beled with 8 under the conditions used (lane f). Myoglobin and car-
5. (a) Link, A. J.; Tirrell, D. A. J. Am. Chem. Soc. 2003, 125, 11164; (b) Agard, N. J.;
Prescher, J. A.; Bertozzi, C. R. J. Am. Chem. Soc. 2004, 126, 15046; (c) Saxon, E.;
Bertozzi, C. R. Science 2000, 287, 2007; (d) Dirksen, A.; Hackeng, T. M.; Dawson, P. E.
Angew. Chem., Int. Ed. 2006, 45, 7581; (e) Crich, D.; Banerjee, A. J. Am. Chem. Soc.
2007, 129, 10064; (f) Tanaka, K.; Masuyama, T.; Hasegawa, K.; Tahara, T.; Mizuma,
H.; Wada, Y.; Watanabe, Y.; Fukase, K. Angew. Chem., Int. Ed 2008, 47, 102.
6. (a) Tanaka, F.; Fuller, R.; Asawapornmongkol, L.; Warsinke, A.; Gobuty, S.;
Barbas, C. F., III Bioconj. Chem. 2007, 18, 1318; (b) Tanaka, F.; Fuller, R. Bioorg.
Med. Chem. Lett. 2006, 16, 4059.
7. Minakawa, M.; Guo, H.-M.; Tanaka, F. J. Org. Chem. 2008, 73, 8669.
8. (a) Joshi, N. S.; Whitaker, L. R.; Francis, M. B. J. Am. Chem. Soc. 2004, 126, 15942;
(b) McFarland, J. M.; Joshi, N. S.; Francis, M. B. J. Am. Chem. Soc. 2008, 130, 7639.
9. (a) Hooker, J. M.; Kovacs, E. W.; Francis, M. B. J. Am. Chem. Soc. 2004, 126, 3718; (b)
Tilley, S. D.; Francis, M. B. J. Am. Chem. Soc. 2006, 128, 1080; (c) Hass, J. A.; Frederick,
M. A.;Fox,B.G.ProteinExpr. Purif.2000, 20, 274;(d)Koshi,Y.;Nakata, E.;Miyagawa,
M.; Tsukiji, S.; Ogawa, T.; Hamachi, I. J. Am. Chem. Soc. 2008, 130, 245.
10. Compound 2. To a mixture of Mn(OAc)3Á2H2O (16.1 g, 60 mmol) and NaN3 (6.50 g,
100 mmol) in CH3CN (180 mL), compound 1 (2.75 mL, 20 mmol) was added
followed by trifluoroacetic acid (TFA, 20 mL) at À20 °C under Ar.11 After 5 min, the
mixturewas added tosaturated NaHSO3 and extracted withCH2C12. Usualworkup
and purification by silica gel flash column chromatography afforded 27 (3.83 g,
85%).Compound 3. To a solution of 2 (3.38 g, 17 mmol) in THF (20 mL) was added a
solution of LiOH (1.43 g, 34 mmol) in water (20 mL) at room temperature and the
mixture was stirred for 15 h. After THF was evaporated in vacuo, 3 N HCl was
added to adjust the pH to 3 and the mixture was extracted with CH2C12. Usual
workup and purification by silica gel flash column chromatography (EtOAc/
hexane) afforded 3 (3.27 g, 98%). 1H NMR (400 MHz, CDCl3): d 11.1 (br s, 1H), 3.94
to 3.49 (m, 1H), 3.42 to 3.21 (m, 1H), 2.82 to 2.68 (m, 1H), 2.46 to 2.27 (m, 1H), 2.16
to 1.85 (m, 2H), 1.83 to 1.40 (m, 3H).
bonic anhydrase were slightly modified (lanes
d
and e).15
Chymotrypsinogen was hydrolyzed into small fragments by autol-
ysis and by catalysis of generated chymotrypsin under the condi-
tions used (lane c). Due to this hydrolysis, we were unable to
evaluate labeling of chymotrypsinogen with 8. These results indi-
cated that 8 did not inhibit the serine protease activity under the
conditions used; this implies that the nucleophilic serine in the ac-
tive site of chymotrypsin did not efficiently react with imine 8.
Mass analysis of the reaction of 8 with lysozyme at 48 h sug-
gested that the reaction of 8 was slower than that of the imine syn-
thesized from trans-cyclohexanediamine shown in Scheme 1. We
previously observed that reaction of the trans-isomer of the imine
shown in Scheme 1 was faster than the corresponding cis-isomer.7
Relative stereochemistries of the two nitrogen functionalities and
the amide carbonyl group on the cyclohexane ring of 8 may affect
the reactivity of the imine moiety. In order to increase the reaction
rate, stereoselective synthesis of 8 may be required. Alternatively,
introduction of moieties that provide noncovalent interactions
with the target protein may be used to enhance the reaction rate
of the imine with the target.9d
Compound 4. A mixture of 3 (2.64 g, 12.6 mmol), N-hydroxysuccinimide (2.17 g,
18.9 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(EDC) (3.62 g, 18.9 mmol), and DMAP (5.0 mg, 0.04 mmol) in CH2Cl2 (40 mL) was
stirred at room temperature for 24 h. The reaction mixture was added to H2O and
extracted with CH2Cl2. Usual workup and purification by silica gel flash column
chromatography (EtOAc/hexane) afforded 4 (3.28 g, 85%). 1H NMR (400 MHz,
CDCl3):d 3.73to 3.69(m, 1H), 3.44 to3.28(m, 1H), 3.12 to3.08 (m, 1H), 2.85(s, 4H),
2.41 to 2.35 (m, 1H), 2.24 to 2.14 (m, 1H), 2.04 to 1.92 (m, 1H), 1.83 to 1.66 (m, 3H).
Compound 5. To a solution of 1,2-bis(2-aminoethoxy)ethane (2.5 mL, 17.0 mmol)
in CH2Cl2 (20 mL), a solution of dansyl chloride (467 mg, 1.73 mmol) in CH2Cl2
(20 mL) was added dropwise at 0 °C. After stirring at roomtemperature for 1 h, the
mixture was acidified with 1 N HCl and washed with CH2Cl2. The aqueous layer
was basified (pH 9) with 3 N NaOH and extracted with CH2Cl2. Organic layers were
combined, washed with brine, dried over Na2SO4, filtered, and concentrated in
vacuo to afford 5 (630 mg, 95%). 1H NMR (400 MHz, CDCl3): d 8.52 (d, J = 8.8 Hz,
1H), 8.34 (d, J = 8.8 Hz, 1H), 8.23 (dd, J = 7.2 Hz, 1.2 Hz, 1H), 7.56 to 7.48 (m, 2H),
7.17 (d, J = 7.6 Hz, 1H), 3.50 to 3.46 (m, 4H), 3.41 to 3.39 (m, 4H), 3.11 to 3.09 (m,
4H), 2.87 (s, 6H).
In these reactions, excess of compound 8 was used compared to
protein (5 mM vs 475 lM). After protein was separated by gel fil-
tration or C18 column, compound 8 was recovered by extraction
with organic solvent (such as CH2Cl2). This recoverable feature of
the imine should be beneficial when a toxic drug or an expensive
molecule is covalently attached to protein.
In summary, we have synthesized a fluorescent conjugate of cyc-
lic imines that we have recently developed for covalent reactions
with phenols, including accessible tyrosine. We have demonstrated
that the imine derivative can be used to fluorescently label proteins,
although the reaction of the imine was relatively slow. The imine
derivative was stable in aqueous solutions and recoverable after
reactions. Reactions of the imine derivative proceeded without the
need of additional catalysts. This system does not require formalde-
hyde or multistep conversions after the first reaction step on tyro-
sine and thus is an improvement over previously reported labeling
systems. Furtherstudiesandimprovementsontheiminederivatives
described here are under investigation.
Compound 6. A mixture of 5 (612 mg, 1.60 mmol) and 4 (493 mg, 1.60 mmol) in
CH3CN (0.5 mL)–MeOH (1.5 mL) was stirred for 15 h at room temperature.
Solvents were evaporated in vacuo and the residue was purified by silica gel
flash column chromatography (CH2Cl2/EtOAc, 4:1) to afford 6 (322 mg, 47%). 1
H
NMR (400 MHz, CDCl3): d 8.55 (d, J = 8.8 Hz, 1H), 8.30 (d, J = 8.8 Hz, 1H), 8.24 (d,
J = 7.2 Hz, 1H), 7.57 to 7.50 (m, 2 H), 7.19 (d, J = 7.6 Hz, 1H), 6.58 (br s, 1H), 5.90 (t,
J = 5.6 Hz,1H),3.85to3.80(m,1H),3.59to3.57(m,2H),3.54to3.45(m,8H),3.37to
3.32 (m, 1H), 3.12 to 3.08 (m, 2H), 2.89 (s, 6H), 2.56 to 2.54 (m, 1H), 2.22 to 2.16 (m,
1H), 1.90 to 1.81 (m, 2H), 1.76 to 1.71 (m, 1H), 1.61 to 1.53 (m, 2H). 13C NMR
(100 MHz, CDCl3): d 174.7, 152.1, 134.0, 130.8, 129.8, 129.6, 129.3, 128.6, 123.1,
118.2, 115.2, 115.2, 63.7, 63.6, 60.1, 45.3, 43.0, 39.1, 38.5, 29.8, 26.9, 25.3, 24.6.
HRMS: calcd for C25H36N9O5S (MH+) 574.2555, found 574.2561.
Compound 7. A mixture of 6 (322 mg, 0.56 mmol) and 10% Pd/C (32 mg) in MeOH
(5 mL)–EtOAc (15 mL) was stirred under H2 for 24 h at room temperature. The
reaction mixture was filtered through Celite and concentrated in vacuo to afford 7
(290 mg, quant). 1H NMR (400 MHz, CD3OD): d 8.52 (d, J = 8.8 Hz, 1H), 8.31 (d,
J = 8.8 Hz, 1H), 8.17 (dd, J = 7.2 Hz, 1.2 Hz, 1H), 7.56 to 7.51 (m, 2H), 7.23 (d,
J = 7.6 Hz,1H),3.78to3.74(m,1H),3.46to3.36(m,2H),3.36to3.33(m,2H),3.30to
3.25 (m, 7H), 3.02 to 2.98 (m, 2H), 2.83 (s, 6H), 2.55 to 2.52 (m, 1H), 2.09 to 2.03 (m,
1H), 1.83 to 1.76 (m, 2H), 1.64 to 1.51 (m, 3H). 13C NMR (100 MHz, CDCl3): d 174.6,
151.8, 135.1, 130.3, 130.2, 129.8, 129.6, 129.1, 128.18, 128.15, 128.12, 128.0, 123.1,
118.9, 115.16, 115.14, 70.23, 70.20, 70.2, 70.1, 70.0, 69.4, 56.1, 53.4, 45.3, 42.8, 39.5,
39.2, 26.1. HRMS: calcd for C25H40N5O5S (MH+) 522.2745, found 522.2745.
Compound 8 (8a + 8b). To a solution of 7 (406 mg, 0.78 mmol) in CF3CH2OH
Acknowledgments
This study was supported by NIH Grant R21 GM078447. We
thank Prof. Peter K. Vogt for helpful technical discussions.
References and notes
(15 mL), a solution of ethyl glyoxylate polymer form (45–50% in toluene, 218 lL,
0.78 mmol) in CF3CH2OH (15 mL) was added dropwise over 24 h at room
temperature. The mixture was further stirred for 6 days. After solvents were
removed in vacuo, the residue was purified by silica gel flash column
1. (a) Schrama, D.; Reisfeld, R. A.; Becker, J. C. Nat. Rev. Drug Discov. 2006, 5, 147;
(b) Wu, A. M.; Senter, P. D. Nat. Biotechnol. 2005, 23, 1137; (c) Hamann, P. R.;
Hinman, L. M.; Hollander, I.; Beyer, C. F.; Lindh, D.; Holcomb, R.; Hallett, W.;
Tsou, H.-R.; Upeslacis, J.; Shochat, D.; Mountain, A.; Flowers, D. A.; Bernstein, I.
Bioconj. Chem. 2002, 13, 47; (d) Lillo, A. M.; Sun, C.; Gao, C.; Ditzel, H.; Parrish, J.;
Gauss, G.-M.; Moss, J.; Felding-Habermann, B.; Wirsching, P.; Boger, D. L.;
Janda, K. D. Chem. Biol. 2004, 11, 897.
2. Vincent, S.; Thomas, A.; Brasher, B.; Benson, J. D. Nat. Biotechnol. 2003, 21, 936.
3. Keppler, A.; Pick, H.; Arivoli, C.; Vogel, H.; Johnsson, K. Proc. Natl. Acad. Sci. U.S.A.
2004, 101, 9955.
chromatography (CH2Cl2/EtOH, 20:1) to afford
8
(259 mg, 60%). 1H NMR
(400 MHz, CDCl3): d 8.56 to 8.53 (m, 1H), 8.31 to 8.29 (m, 1H), 8.25 to 8.22 (m,
1H), 7.72 to 7.68 (m, 1H), 7.56 to 7.50 (m, 2H), 7.19 to 7.17 (m, 1H), 6.80 to 6.39 (m,
1H), 6.30 (m, 1H), 6.28 to 6.25 (m, 1H), 3.72 to 3.40 (m, 12H), 3.13 to 3.07 (m, 2H),
2.89 (s, 6H), 2.75 to 1.30 (m, 7H). 13C NMR (100 MHz, CDCl3): d 174.5, 173.8, 157.8,
157.5, 156.49, 156.47, 151.97, 151.93, 134.8, 134.4, 130.5, 130.4, 129.8, 129.58,
129.55, 129.4, 129.3, 128.16, 128.13, 123.1, 118.8, 118.7, 115.2, 115.1, 70.4, 70.3,
70.2, 70.1, 69.3, 69.2, 62.8, 59.2, 54.0, 50.4, 45.3, 42.9, 42.8, 39.4, 39.2, 39.1, 38.1,

H.-M. Guo et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1210–1213
1213
33.0, 32.2, 28.0, 27.5, 27.4, 25.5. HRMS: calcd for C27H38N5O6S (MH+) 560.2537,
found 560.2543.
11. Snider, B. B.; Lin, H. Synth. Commun. 1998, 28, 1913.
3.09 to 3.04 (m, 3H), 2.88 (s, 6H), 2.70 to 1.30 (m, 8H), 2.26 (s, 3H Â 1/2), 2.23 (s,
3H Â 1/2). HRMS: calcd for C34H46N5O7S (MH+) 668.3112, found 668.3115.
13. GlyTyr labeled with 8. HRMS: calcd for C38H51N7O10S (MH+) 798.3491, found
12. Compound 9. A mixture of compound 8 (136 mg, 0.24 mmol) and 4-methylphenol
(39.4 mg, 0.36 mmol) in CF3COOH (1 mL)–CH2Cl2 (1 mL) was stirred for 24 h at
roomtemperature. After solventswereremovedinvacuo, residue was dissolved in
CH2Cl2 and was washed with saturated NaHCO3. The aqueous layer was extracted
with CH2Cl2. Usual workup and purification by silica gel flash column
798.3495. AlaTyrAla labeled with 8. HRMS: calcd for C42H58N8O11S
(MH+)
883.4018, found 883.4015.
14. Lysozyme labeled with 8. ESI-MS: 14,865 (mono-addition product); unmodified
14306.
15. Myoglobin labeled with 8. ESI-MS: 17,512 (mono-addition product); unmodified
16,953. Modification of myoglobin, which lacks surface-accessible tyrosine,8,9b
may have occurred when the protein was denatured during the 4-day reaction
time. We previously showed that myoglobin was not modified with the cyclic
imine derivative shown in Scheme 1 in aqueous buffer, pH 7.0 at 37 °C for
48 h.7
chromatography (CH2Cl2/MeOH, 30:1) afforded
9
(88 mg, 54%). 1H NMR
(400 MHz, CDCl3): d 8.54 (d, J = 8.8 Hz, 1H), 8.30 to 8.27 (m, 1H), 8.24 to 8.20 (m,
1H), 7.55 to 7.49 (m, 2H), 7.17 (d, J = 7.2 Hz, 1H), 7.06 (s, 1H), 6.99 to 6.93 (m, 1H),
6.78 to 6.75 (m, 1H + 1H Â 1/2), 6.65 (m, 1H Â 1/2), 6.45 (s, 1H), 6.36 (br s, 1H Â 1/
2), 6.20 (br s, 1H Â 1/2), 4.96 (s, 1H Â 1/2), 4.91 (s, 1H Â 1/2), 3.62 to 3.42 (m, 12H),