Activation of the aryl hydrocarbon receptor by methyl yellow and related congeners: structure-activity relationships in halogenated derivatives.

Article abstract of DOI:10.1248/bpb.25.466

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the biological action of many environmental compounds. Methyl yellow (4-dimethylaminoazobenzene; MY) is a principal azo-dye, and structurally related compounds were subjected to analysis of structure-activity relationships as AhR ligands by using a yeast AhR signaling assay. The effects of halogen-substitution among 23 halogenated MYs on the AhR ligand activity can be summarized as follows: enhancement by halogen-substitution at the ortho-position (2'- and 6'-position), and reduction by substitution at the para-position (4'-position). The greatest enhancement of the ligand activity was observed in 2',6'-dichlorinated MY (13.5-fold of MY), and its AhR ligand activity was very close to that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the present assay system. In the study of compounds structurally related to MY, benzanilide (BA) showed almost the same AhR ligand activity as azobenzene and trans-stilbene. Furthermore, 4'-chlorobenzanilide, in which the length of the molecule is similar to that of MY, enhanced the AhR ligand activity by ortho(2')-chlorine-substitution, and the AhR ligand activity of 2',4'-dichlorobenzanilide was similar to that of 2'-chloro-MY. These results suggest that the amide bond is equivalent to the -N=N- or -CH=CH- double bond for recognition as the ligand by AhR in 1,2-diphenyl-1,2-ene derivatives.

Full text of DOI:10.1248/bpb.25.466

466
Biol. Pharm. Bull. 25(4) 466—471 (2002)
Vol. 25, No. 4
Activation of the Aryl Hydrocarbon Receptor by Methyl Yellow and
Related Congeners: Structure–Activity Relationships in
Halogenated Derivatives
a
b
b
a
,a
*
Taka-aki KATO, Tomonari MATSUDA, Saburo MATSUI, Takaharu MIZUTANI, and Ken-ichi SAEKI
Faculty of Pharmaceutical Sciences, Nagoya City University,a 3–1 Tanabedori, Mizuho-ku, Nagoya 467–8603, Japan and
Department of Environmental Engineering, Kyoto University,b Sakyo-ku, Yoshida-honmachi, Kyoto 606–8501, Japan.
Received October 11, 2001; accepted December 29, 2001
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the biological
action of many environmental compounds. Methyl yellow (4-dimethylaminoazobenzene; MY) is a principal azo-
dye, and structurally related compounds were subjected to analysis of structure–activity relationships as AhR
ligands by using a yeast AhR signaling assay. The effects of halogen-substitution among 23 halogenated MYs on
the AhR ligand activity can be summarized as follows: enhancement by halogen-substitution at the ortho-posi-
tion (2؅- and 6؅-position), and reduction by substitution at the para-position (4؅-position). The greatest enhance-
ment of the ligand activity was observed in 2؅,6؅-dichlorinated MY (13.5-fold of MY), and its AhR ligand activity
was very close to that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the present assay system. In the study of
compounds structurally related to MY, benzanilide (BA) showed almost the same AhR ligand activity as azoben-
zene and trans-stilbene. Furthermore, 4؅-chlorobenzanilide, in which the length of the molecule is similar to that
of MY, enhanced the AhR ligand activity by ortho(2؅)-chlorine-substitution, and the AhR ligand activity of 2؅,4؅-
dichlorobenzanilide was similar to that of 2؅-chloro-MY. These results suggest that the amide bond is equivalent
to the –N؍N– or –CH؍CH– double bond for recognition as the ligand by AhR in 1,2-diphenyl-1,2-ene derivatives.
Key words aryl hydrocarbon receptor; azocompound; structure–activity relationship; methyl yellow; halogenated derivative
The aryl hydrocarbon receptor (AhR), also called the lyzed in most cases by the CYP1A family, and especially by
dioxin receptor, is a transcriptional factor that mediates the the CYP1A2 protein.16—18) The CYP1A2 protein is expressed
toxicity of a variety of compounds, most notably, of 2,3,7,8- in the liver of both animals and humans, and is the primary
tetrachlorodibenzo-p-dioxin (TCDD) and structurally related tumor site of most azocompounds.19) It has been reported
polycyclic aromatic hydrocarbons (PAHs) and polyhalo- that AAB derivatives induce CYP1A2.20) The induction of
genated PAHs.1—3) This receptor is a member of the basic CYP1A proteins is regulated by AhR.
helix-loop-helix superfamily of DNA-binding proteins.4)
Numerous studies of the structure–activity correlation of
Upon ligand-binding, the AhR is transformed and dissociates ligands for AhR have been reported.21) The effects of halo-
from a heat shock protein dimer with which it is complexed gen-substitution, especially chlorine-substitution, on the AhR
in the cyotsol, that it enters the nucleus where it joins with a ligand activity have been reported. However, most of these
nuclear protein, the AhR nuclear translocator (ARNT). Inter- reports are on dibenzodioxin, dibenzofuran and biphenyl,
action of this AhR/ARNT heterodimer with specific DNA and there but few reports on other aromatic nuclei.
sequences, called dioxin response elements (DREs), in the
Therefore, in order to explore the characteristics of the
regulatory regions of responsive genes can result in activa- general structure of AhR ligands, we investigated the AhR
tion of a certain group of genes, most of which are involved ligand activities of MY and its halogen-substituted analogs
primarily in xenobiotic metabolism, including CYP1A1, using a yeast AhR signaling assay.22—25) Additionally, struc-
CYP1A2, CYP1B1, the glutathione S-transferase Ya subunit, tural analogs of MY, azobenzenes, stilbenes, and ben-
quinone oxidoreductase, UDP-glucuronosyltransferase, and zanilides were also subjected to the analysis of their AhR lig-
aldehyde-3-dehydrogenase.5—11)
and activities.
Azocompounds are used extensively as dyes in a variety of
industries, including the cosmetics, food, leather, paper and MATERIALS AND METHODS
textile industries. However, some azocompounds manifest
carcinogenicity through highly reactive metabolic intermedi-
Materials TCDD was purchased from Wako Pure Chemi-
ates that can interact covalently with DNA and cause muta- cal Industries, Ltd. (Osaka). MY, azobenzene, 4-aminoazo-
tions.12) Methyl yellow (4-dimethylaminoazobenzene; MY) benzene, benzanilide, p-dimethylaminostilbene, trans-stil-
is a basic structure of those azo-dyes which have been used bene, cis-stilbene, halogenated anilines and N,N-dimethylani-
as dyes in food, but the use of MY is now prohibited because line were purchased from Aldrich. 2Ј-Fluoro-4-dimethyl-
of its carcinogenic activity in rodents. It was suggested that aminoazobenzene (2Ј-F-MY, CAS Registry No. 331-91-9),
MY is metabolically converted to a procarcinogen, N-hy- 3Ј-fluoro-4-dimethylaminoazobenzene (3Ј-F-MY, 332-54-7),
droxy-N-methyl-4-aminoazobenzene, by demethylation fol- 4Ј-fluoro-4-dimethylaminoazobenzene (4Ј-F-MY, 150-74-3),
lowed by N-hydroxylation.13—15) In the case of 4-aminoazo- 2Ј,3Ј,4Ј,5Ј,6Ј-pentafluoro-4-dimethylaminoazobenzene
benzene (AAB), metabolic activation may proceed through (2Ј,3Ј,4Ј,5Ј,6Ј-penta-F-MY, 970-150-0), 2Ј-chloro-4-dimethyl-
N-hydroxylation by the microsomal cytochrome P450-depen- aminoazobenzene (2Ј-Cl-MY, 3010-47-7), 3Ј-chloro-4-di-
dent mixed-function oxidase and flavin monooxygenase sys- methylaminoazobenzene (3Ј-Cl-MY, 3789-77-3), 4Ј-chloro-
tem.16,17) The N-hydroxylation of AAB appears to be cata- 4-dimethylaminoazobenzene (4Ј-Cl-MY, 2491-76-1), 2Ј,4Ј-
To whom correspondence should be addressed. e-mail: saeki@phar.nagoya-cu.ac.jp
© 2002 Pharmaceutical Society of Japan

April 2002
467
Table 1. 1H-NMR Chemical Shifts and UV Absorbance of MY and Its Halogenated Derivatives Examined
1H-MMR chemical shifts (ppm)
UV absorbance
Compounds
N-CH3
3.08
H-2, 6
7.88
H-3, 5
6.75
H-2Ј
H-3Ј
H-4Ј
H-5Ј
H-6Ј
l
max (nm)
406.2
e
MY
7.83
7.47
7.37
7.47
7.83
29700
Monofluorinated derivatives
2Ј-F-
3Ј-F-
3.09
3.09
3.08
7.90
7.87
7.86
6.75
6.75
6.76
F
7.53
7.84
7.14—7.25 7.33 7.14—7.25 7.73
415.8
416.0
406.2
28320
24600
25500
F
7.60
F
7.42
7.14
7.65
7.84
4Ј-F-
7.14
Difluorinated derivatives
2Ј,4Ј-diF-
2Ј,5Ј-diF-
2Ј,6Ј-diF-
3Ј,5Ј-diF-
3.09
3.11
3.10
3.13
7.88
7.90
7.89
7.87
6.74
6.74
6.75
6.75
F
F
F
6.88—7.00
F
6.88—7.00 7.77
415.8
429.0
409.2
426.0
28740
25440
22980
28380
7.17
6.99
F
7.01
7.20
6.81
F
6.99
F
7.47
F
7.38
7.38
Polyfluorinated derivatives
2Ј,3Ј,4Ј,5Ј,6Ј-pentaF-
Monochlorinated derivatives
2Ј-Cl-
3Ј-Cl-
4Ј-Cl-
3.13
7.87
6.74
F
F
F
F
F
432.6
29700
3.10
3.10
3.10
7.93
7.88
7.86
6.74
6.76
6.76
Cl
7.83
7.78
7.50
Cl
7.43
7.28 or 7.30
7.34
Cl
7.67
7.74
7.78
421.4
421.4
416.6
28440
28440
30480
7.40
7.43
Dichlorinated derivatives
2Ј,3Ј-diCl-
2Ј,4Ј-diCl-
2Ј,6Ј-diCl-
3Ј,5Ј-diCl-
3.11
3.11
3.12
3.12
7.93
7.95
7.92
7.87
7.79
6.76
6.75
7.37
6.75
6.79 (H-3)
Cl
Cl
Cl
Cl
7.52
7.37
Cl
7.45
Cl
7.11
7.34
7.24
7.27
7.37
Cl
7.56
7.52
Cl
431.2
433.8
397.0
432.2
26133
31860
21840
30600
7.73
7.73
2,3Ј-diCl-
3.10
7.87
Cl
7.35
7.41
7.78
419.6
(H-6) 6.62 (H-5)
31620
Polychlorinated derivatives
2Ј,4Ј,5Ј-triCl-
3Ј,4Ј,5Ј-triCl-
2Ј,3Ј,5Ј,6Ј-tetraCl-
2Ј,3Ј,4Ј,5Ј,6Ј-pentaCl-
Monobrominated derivatives
2Ј-Br-
3.12
3.12
3.13
3.13
7.91
7.86
7.91
7.90
6.75
6.76
6.77
6.76
Cl
7.88
Cl
7.62
Cl
Cl
Cl
Cl
7.50
Cl
Cl
Cl
Cl
Cl
7.83
7.88
Cl
449.4
445.0
410.2
414.8
16680
29520
22333
20800
Cl
Cl
Cl
3.13
3.10
3.09
7.93
7.78
7.86
6.76
6.76
6.72
Br
7.98
7.72
7.64
Br
7.59
7.35
7.49
Br
7.21
7.34
7.59
7.70
7.78
7.72
421.6
419.4
418.4
29340
30360
33420
3Ј-Br-
4Ј-Br-
Dibrominated derivatives
2Ј,4Ј-diBr-
2Ј,6Ј-diBr-
3.11
3.12
7.92
7.94
6.75
6.78
Br
Br
7.86
7.60
Br
6.98
7.55
7.60
7.47
Br
419.2
400.2
32580
24780
dichloro-4-dimethylaminoazobenzene (2Ј,4Ј-diCl-MY, 10286- 2,4-Difluoroaniline (1.42 g) was dissolved in 20 ml of 1 N
76-7), 2Ј-bromo-4-dimethylaminoazobenzene (2Ј-Br-MY, HCl, and sodium nitrite (954 mg) was added to this solu-
17128-10-8), 3Ј-bromo-4-dimethylaminoazobenzene (3Ј-Br- tion at 0 °C. After stirring for 5 min, N,N-dimethylaniline
MY, 17576-88-4) and 4Ј-bromo-4-dimethylaminoazobenzene (1.40 ml, 1 eq) was added. The resulting precipitate was
(4Ј-Br-MY, 3805-65-0) were synthesized from 4-dimethyl- washed with water and dried. 2Ј,4Ј-DiF-MY was obtained by
aniline and the corresponding halogenated anilines by the recrystallization from methanol as brown plates in 50%
diaza coupling reaction. 2Ј-Chlorobenzanilide (1020-39-9), yield. mp 135—137 °C. MS m/z: 261 (Mϩ). Anal. Calcd for
4Ј-chlorobenzanilide (2866-82-2), 2Ј-methylbenzanilide (584- C14H13F2N3: C, 64.36; H, 5.02; N, 16.08. Found: C, 64.31; H,
70-3) and 2Ј-ethylbenzanilide (78987-16-3) were synthesized 4.96; N, 15.85.
from benzoic anhydride and the corresponding chlorinated or
3Ј,5Ј-Difluoro-4-dimethylaminoazobenzene (3Ј,5Ј-diF-
alkylated anilines. Melting points were determined with a MY): 3,5-Difluoroaniline (227 mg) and N,N-dimethylaniline
Yamato MP-500D micro melting point apparatus without (2.15 g) were dissolved in 30 ml of methanol, and 5 ml of
correction. Mass spectra were measured with a JEOL c-HCl was added to this solution. Sodium nitrite aq. (4.31 g
1
AX505HA spectrometer. H-NMR spectra were recorded of sodium nitrite in 10 ml of water) was added to the solution
with a JEOL JNM-GSX 400 spectrometer in CDCl3 using at 0 °C. After stirring for 15 min at room temperature, the
1
tetramethylsilane as an internal standard. H-NMR chemical reaction mixture was poured into water and extracted with
shifts and the UV absorbance of MY and its halogenated de- CHCl3. The organic layer was dried over anhydrous MgSO4
rivatives examined are summarized in Table 1.
and evaporated. Purification of the extract by column chro-
The following compounds were newly synthesized for this matography (silica gel, CHCl3 : n-hexaneϭ2 : 3) and recrys-
study.
tallization from methanol yielded 3Ј,5Ј-diF-MY as red
2Ј,4Ј-Difluoro-4-dimethylaminoazobenzene (2Ј,4Ј-diF-MY): brown plates in 32% yield. mp 114—116 °C. MS m/z: 261

468
Vol. 25, No. 4
(Mϩ). High resolution (HR)-MS m/z: 261.1077, Calcd for
C14H13F2N3: 261.1078.
trichloroaniline and N,N-dimethylaniline by the same method
as for the synthesis of 2Ј,4Ј-diF-MY. 2Ј,4Ј,5Ј-TriCl-MY was
obtained by recrystallization from methanol as brown nee-
dles in 26% yield. mp 190—193 °C. MS m/z: 327 (Mϩ), 329
(Mϩϩ2). HR-MS m/z: 327.0086, Calcd for C14H12Cl3N3:
327.0097.
3Ј,4Ј,5Ј-Trichloro-4-dimethylaminoazobenzene (3Ј,4Ј,5Ј-
triCl-MY): 3Ј,4Ј,5Ј-TriCl-MY was synthesized from 3,4,5-
trichloroaniline and N,N-dimethylaniline by the same method
as for the synthesis of 3Ј,5Ј-diF-MY. 3Ј,4Ј,5Ј-TriCl-MY was
obtained by recrystallization from methanol as red brown
needles in 67% yield. mp 163—167 °C. HR-MS m/z:
327.0106, Calcd for C14H12Cl3N3: 327.0097.
2Ј,3Ј,5Ј,6Ј-Tetrachloro-4-dimethylaminoazobenzene
(2Ј,3Ј,5Ј,6Ј-tetraCl-MY): 2Ј,3Ј,5Ј,6Ј-TetraCl-MY was synthe-
sized from 2,3,5,6-tetrachloroaniline and N,N-dimethylani-
line by the same method as for the synthesis of 2Ј,4Ј-diF-
MY. 2Ј,3Ј,5Ј,6Ј-TetraCl-MY was obtained by recrystalliza-
tion from methanol as red brown needles in 38% yield. mp
247—249 °C. HR-MS m/z: 360.9709, Calcd for C14H11Cl4N3:
360.9707.
2Ј,3Ј,4Ј,5Ј,6Ј-Pentachloro-4-dimethylaminoazobenzene
(2Ј,3Ј,4Ј,5Ј,6Ј-pentaCl-MY): 2Ј,3Ј,4Ј,5Ј,6Ј-PentaCl-MY was
synthesized from pentachloroaniline and N,N-dimethylani-
line by the same method as for the synthesis of 3Ј,5Ј-diF-
MY. 2Ј,3Ј,4Ј,5Ј,6Ј-PentaCl-MY was obtained by recrystal-
lization from methanol as red brown needles in 15%
yield. mp 224—228 °C. HR-MS m/z: 394.9317, Calcd for
C14H10Cl4N3: 394.9317.
2Ј,4Ј-Dibromo-4-dimethylaminoazobenzene (2Ј,4Ј-diBr-
MY): 2Ј,4Ј-DiBr-MY was synthesized from 2,4-dibromoani-
line and N,N-dimethylaniline by the same method as for the
synthesis of 2Ј,4Ј-diF-MY. 2Ј,4Ј-DiBr-MY was obtained by
recrystallization from methanol as red brown prisms in 54%
yield. mp 127—128 °C. Anal. Calcd for C14H13Br2N3: C,
43.89; H, 3.42; N, 10.97. Found: C, 43.92; H, 3.48; N, 11.01.
2Ј,6Ј-Dibromo-4-dimethylaminoazobenzene (2Ј,6Ј-diBr-
MY): 2Ј,6Ј-DiBr-MY was synthesized from 2,6-dibromoani-
line and N,N-dimethylaniline by the same method as for the
synthesis of 3Ј,5Ј-diF-MY. 2Ј,6Ј-DiBr-MY was obtained by
recrystallization from methanol as red brown prisms in 46%
yield. mp 127—128 °C. MS m/z: 381 (Mϩ), 383 (Mϩϩ2),
385 (Mϩϩ4). Anal. Calcd for C14H13Br2N3: C, 43.89; H,
3.42; N, 10.97. Found: C, 44.01; H, 3.50; N, 11.08.
2Ј,4Ј-Dichlorobenzanilide (2Ј,4Ј-diCl-BA): 2,4-Dichloroani-
line (685 mg) was dissolved in 30 ml of pyridine, and benzoic
anhydride (1.90 g) was added to this solution. After stirring
for 3 h at 80 °C, the solvent was evaporated. The resultant
residue was dissolved in CHCl3, and this solution was
washed with water and 5% Na2CO3. The organic layer was
dried over anhydrous MgSO4 and evaporated. Purification of
the extract by recrystallization (n-hexane–acetone) yielded
2Ј,4Ј-dichlorobenzanilide as white needles in 58% yield. mp
120—121 °C. 1H-NMR (CDCl3) d: 7.32 (dd, 1H, H-5Ј), 7.44
(d, 1H, H-3Ј), 7.53 (t, 2H, H-3, H-5), 7.60 (t, 1H, H-4), 7.91
(d, 2H, H-2, H-6), 8.39 (br, 1H, NH), 8.55 (d, 1H, H-6);
J3Ј—5Јϭ2.1, J5Ј—6Јϭ8.9, J2—3ϭJ3—4ϭJ4—5ϭJ5—6ϭ7.2 Hz.
Anal. Calcd for C13H9Cl2NO: C, 58.67; H, 3.41; N, 5.26.
Found: C, 58.60; H, 3.44; N, 5.20. HR-MS m/z: 265.0056,
Calcd for C13H9Cl2NO: 265.0061.
2Ј,5Ј-Difluoro-4-dimethylaminoazobenzene (2Ј,5Ј-diF-
MY): 2Ј,5Ј-DiF-MY was synthesized from 2,5-difluoroani-
line and N,N-dimethylaniline by the same method as for the
synthesis of 2Ј,4Ј-diF-MY. 2Ј,5Ј-DiF-MY was obtained by
recrystallization from methanol as brown plates in 70%
yield. mp 137—139 °C. MS m/z: 261 (Mϩ). Anal. Calcd for
C14H13F2N3: C, 64.36; H, 5.02; N, 16.08. Found: C, 64.12; H,
4.86; N, 16.06.
2Ј,6Ј-Difluoro-4-dimethylaminoazobenzene (2Ј,6Ј-diF-
MY): 2Ј,6Ј-DiF-MY was synthesized from 2,6-difluoroani-
line and N,N-dimethylaniline by the same method as for
the synthesis of 2Ј,4Ј-diF-MY. 2Ј,6Ј-DiF-MY was obtained
by recrystallization from methanol as brown needles in
56% yield. mp 96—97 °C. MS m/z: 261 (Mϩ). HR-MS m/z:
261.1074, Calcd for C14H13F2N3: 261.1078.
2Ј,3Ј-Dichloro-4-dimethylaminoazobenzene (2Ј,3Ј-diCl-
MY): 2Ј,3Ј-DiCl-MY was synthesized from 2,3-dichloroani-
line and N,N-dimethylaniline by the same method as for the
synthesis of 2Ј,4Ј-diF-MY. 2Ј,3Ј-DiCl-MY was obtained by
recrystallization from methanol as brown plates in 35%
yield. mp 200—209 °C. MS m/z: 294 (Mϩ). Anal. Calcd for
C14H13Cl2N3: C, 57.16; H, 4.45; N, 14.28. Found: C, 56.99;
H, 4.39; N, 14.02.
2Ј,6Ј-Dichloro-4-dimethylaminoazobenzene (2Ј,6Ј-diCl-
MY): 2Ј,6Ј-DiCl-MY was synthesized from 2,6-dichloroani-
line and N,N-dimethylaniline by the same method as for the
synthesis of 2Ј,4Ј-diF-MY. 2Ј,6Ј-DiCl-MY was obtained by
recrystallization from methanol as brown needles in 59%
yield. mp 134—137 °C. MS m/z: 294 (Mϩ). HR-MS m/z:
293.0490, Calcd for C14H13Cl2N3: 293.0487.
3Ј,5Ј-Dichloro-4-dimethylaminoazobenzene (3Ј,5Ј-diCl-
MY): 3Ј,5Ј-DiCl-MY was synthesized from 3,5-dichloroani-
line and N,N-dimethylaniline by the same method as for the
synthesis of 2Ј,4Ј-diF-MY. 3Ј,5Ј-DiCl-MY was obtained by
recrystallization from methanol as brown needles in 64%
yield. mp 134—137 °C. MS m/z: 294 (Mϩ). HR-MS m/z:
293.0490, Calcd for C14H13Cl2N3: 293.0487.
2,3Ј-Dichloro-4-dimethylaminoazobenzene (2,3Ј-diCl-
MY): m-Chloroaniline (2.96 g) was dissolved in 2.3 ml of
methanol, and 60% H2SO4 was added to this solution. After
keeping the solution at 200 °C for 16 h, the reaction mixture
was poured into water and neutralized with Na2CO3, then ex-
tracted with CHCl3. The organic layer was dried over anhy-
drous MgSO4 and evaporated. Purification of the extract by
column chromatography (silica gel, n-hexane→n-hexane :
AcOEtϭ19 : 1→n-hexane : AcOEtϭ1 : 1) yielded m-chloro-
N,N-dimethylaniline as a colorless oil in 77% yield. 1H-NMR
(CDCl3) d: 2.94 (s, 6H, N(CH3)2), 6.58 (ddd, 1H, H-6), 6.66
(m, 2H, H-2, H-4), 7.13 (dd, 1H, H-5); J5—6ϭ8.4 Hz. HR-MS
m/z: 155.0491, Calcd for C8H10ClN: 155.0502. 2,3Ј-DiCl-
MY was synthesized from m-chloroaniline and m-chloro-
N,N-dimethylaniline by the same method as for the synthesis
of 2Ј,4Ј-diF-MY. 2,3Ј-DiCl-MY was obtained by recrystal-
lization from methanol as red brown columns in 59%
yield. mp 93—94 °C. MS m/z: 294 (Mϩ). Anal. Calcd for
C14H13Cl2N3: C, 57.16; H, 4.45; N, 14.28. Found: C, 57.44;
H, 4.48; N, 14.38.
2Ј,4Ј,5Ј-Trichloro-4-dimethylaminoazobenzene (2Ј,4Ј,5Ј-
triCl-MY): 2Ј,4Ј,5Ј-TriCl-MY was synthesized from 2,4,5-
2-Chlorobenzanilide (2-Cl-BA): Aniline (208 mg) was

April 2002
469
added to 10 ml of pyridine, and m-chlorobenzoyl chloride assessed by b-galactosidase activity. Expression of the LacZ
(339 ml) was added to this solution. After stirring for 3 h at reporter plasmid, pTXRE5-Z, is directed by AhR-ARNT
80 °C, the solvent was evaporated. The resultant residue was complex binding to five response elements in the promoter
dissolved in CHCl3 and this solution was washed with water region.
and 5% Na2CO3. The organic layer was dried over anhydrous
The assay procedure was essentially the same as described
MgSO4 and evaporated. Purification of the extract by column by Miller et al.22—25) The yeast strain YCM3 was grown
chromatography (CHCl3 : n-hexaneϭ1 : 4→2 : 3) and recrys- overnight at 30 °C in a synthetic glucose medium lacking
tallization from n-hexane–acetone yielded 2-chlorobenz- tryptophan. One microliter of test chemicals (dissolved in
1
anilide as white needles in 64% yield. mp 121—124 °C. H- DMSO), 20 ml of the overnight culture and 1 ml of the
NMR (CDCl3) d: 7.18 (t, 1H, H-5), 7.37—7.48 (m, 5H, H-3, synthetic medium containing 2% galactose were mixed and
-4, -3Ј, -4Ј, H-5Ј), 7.65 (d, 2H, H-2Ј, H-6Ј), 7.78 (d, 1H, H- incubated for 18 h at 30 °C. Cell density was determined by
6), 7.87 (br, 1H, NH); J2Ј—3ЈϭJ5Ј—6Јϭ7.8, J4—6ϭ2.2, J5—6ϭ reading the absorbance at 595 nm. Ten microliters of each
7.1 Hz. Anal. Calcd for C13H10ClNO: C, 67.40; H, 4.35; N, cell suspension was added to 700 ml of Z-buffer (60 mM
6.05. Found: C, 67.48; H, 4.44; N, 6.03. HR-MS m/z: Na2HPO4, 40 mM NaH2PO4, 1 mM MgCl2, 10 mM KCl, 2 mM
231.0448, Calcd for C13H10NO: 231.0451.
dithiothreitol and 0.2% sarcosyl, adjusted to pH 7), and the
2Ј-tert-Butylbenzanilide (2Ј-tert-Bu-BA): 2Ј-tert-Butyl- reaction was started by adding 200 ml of o-nitrophenol-b-D-
benzanilide was synthesized from o-tert-butylaniline and galactopyranoside (4 mg/ml solution in Z-buffer), with sub-
benzoic anhydride by the same method as for the synthesis of sequent incubation for 60 min at 37 °C. Absorbances of the
2Ј,4Ј-dichlorobenzanilide. 2Ј-tert-Bu-BA was obtained by b-galactosidase assay were read at 405 nm. b-Galactosidase
recrystallization from methanol as white needles in 50% activity (referred to as lacZ units) was calculated by the fol-
1
yield. mp 194—196 °C. H-NMR (CDCl3) d: 1.46 (s, 9H, lowing formula: absorbance at 405 nmϫ1000/(absorbance at
C(CH3)3), 7.29 (dt, 1H, H-4Ј or H-5Ј), 7.21 (dt, 1H, H-4Ј or 595 nmϫml cell suspension addedϫmin reaction time).
H-5Ј), 7.45 (dd, 1H, H-3Ј), 7.20 (dt, 2H, H-3, H-5), 7.58 (dt,
1H, H-4), 7.75 (d, 1H, H-6Ј), 7.88 (br, 1H, NH), 7.92 (dd, RESULTS
2H, H-2, H-6); J3Ј—5Јϭ2.1, J5Ј—6Јϭ8.9, J2—3ϭJ3—4ϭJ4—5
ϭJ5—6ϭ7.2 Hz. Anal. Calcd for C17H19NO: C, 80.60; H,
7.56; N, 5.53. Found: C, 80.60; H, 7.47; N, 5.58. HR-MS with the lacZ reporter gene assay using Saccharomyces cere-
m/z: 253.1471, Calcd for C17H19NO: 253.1467.
visiae strain YCM3, in which the human AhR and ARNT
Yeast Assays for AhR Ligand Activity In the yeast genes are co-expressed, as previously reported.22—25)
strain YCM3,22) the human AhR and ARNT genes are inte-
TCDD, an exogenous potent AhR ligand, showed an ap-
AhR ligand activities of the test compounds were assessed
grated into chromosome III. AhR and ARNT are expressed preciable increase in the lacZ unit in a dose-dependent man-
from the galactose-regulated GAL 1,10 promoter. Transcrip- ner in this assay system, and the EC50 value of TCDD was
tional activation mediated by the AhR/ARNT heterodimer is determined to be 70 nM from the dose-response curve (Fig.
Fig. 1. AhR Ligand Activities of TCDD, MY, and Its Structural Analogs
AhR ligand activities of TCDD (closed diamonds), Methyl Yellow (open squares), and MY structural analogs were assessed with the reporter gene assay using Saccharomyces
cerevisiae strain YCM3, in which the human AhR and ARNT genes were co-expressed. Each point represents the mean of at least three analyses. Concentrations were plotted on a
log scale. ECTCDD50 is the concentration producing lacZ units equal to 50% of the maximal response to TCDD.

470
Vol. 25, No. 4
1). To compare the activation potency of MY and related order, diCl (13.5)ϾdiBr (11.1)ϾdiF (10.8). The ECTCDD50
congeners with TCDD, the concentrations of test compounds value of 2Ј,6Ј-diCl-MY (0.12 mM), which showed the highest
producing lacZ units equivalent to 50% of the maximal lacZ AhR ligand activity among the halogenated MYs tested in
units produced by TCDD were calculated from the dose-re- this study, was close to that of TCDD (0.07 mM).
sponse curve, and expressed as ECTCDD50 values, according to
As shown in Fig. 1, the AhR ligand activities of structural
the previously reported method.26,27) Furthermore, to evaluate analogs of MY decreased in the following order: MYϷ
the relative activity of each compound, the value of lacZ AABϾDMASϾBAϷABϷtrans-stilbeneϾϾcis-stilbene. BA,
units per mM of the compound was also calculated from the AB and trans-stilbene, which have the same skeleton, i.e.,
slope of the initial linear portion of each dose-response curve two phenyl groups connected with two linker atoms, showed
because a few compounds showed levels of maximal induction almost the same activity, but showed lower activities than
of lacZ units unsatisfactory to calculate the ECTCDD50 value.
those of MY, AAB and DMAS, in which the amino or di-
Table 2 shows the AhR ligand activities of the MY deriva- methylamino group is located at the para-position of one
tives. Among all the monohalogenated derivatives, 2Ј-halo- phenyl moiety. cis-Stilbene, in which two phenyl groups are
genated MYs (2Ј-F, 2Ј-Cl, and 2Ј-Br-MY) showed higher liga- not in the same plane because of steric hindrance by each
nd activities than that of MY, and the halogen-substitution ef- ortho-proton, did not show the ligand activity up to 100 mM.
fects on the ligand activity (relative activity) decreased in the
Chlorine- and alkyl-substitution effects on the AhR ligand
following order, Cl (7.8)ϾBr (5.0)ϾF (3.1). On the other activity were investigated in BA to give the results shown in
hand, all the 3Ј- and 4Ј-halogenated MYs showed lower liga- Table 3. The ligand activity of benzanilide was increased by
nd activities than that of MY, in which the relative activity ortho- or para-chlorine substitution, and the relative activity
ranged from 0.09- to 0.9-fold. In the case of 16 di- and ranged from 2.4- to 100-fold. With regard to ortho-alkyl-sub-
polyhalogenated derivatives, only 2Ј,6Ј-dihalogenated MYs stitution on BA, methyl and ethyl group substitution showed
showed more than 10-fold enhancement of the ligand activi- a slight decrease in the AhR ligand activity. However, tert-
ty, and the enhancing effects decreased in the following butyl group substitution on the ortho-position of BA was de-
void of AhR ligand activity in the whole dose range exami-
ned (up to 300 mM).
Table 2. AhR Activation Potencies of MY and Its Halogenated Derivatives
a)
ECTCDD50
(mM)
lacZ
units/mM
Relative
activityc)
DISCUSSION
Chemicals
b)
We measured the AhR ligand activity of 24 MYs, 6 BAs, 3
stilbenes, and 2 azobenzenes, which have the same skeleton
in the molecule, i.e., two benzene moieties connected with
two linker atoms, using the yeast AhR signaling assay to
investigate their structure–activity relationships as an AhR
ligand. MY was about two orders less active as an AhR
ligand than TCDD, but showed the same ligand activity
as benzo[a]pyrene in the present study (EC50 of TCDDϭ
0.07 mM, ECTCDD50 of MYϭ1.94 mM, and ECTCDD50 of
BaPϭ1.81 mM). This result indicates that MY may be a mode-
rate AhR ligand having the capacity to alter AhR-mediated
gene expression, such as induction of the CYP1A family. In
TCDD
MY
0.07
1.94
6373.5
898.4
—
1.00
Monofluorinated derivatives
2Ј-F-
3Ј-F-
0.63
4.87
2.72
2755.8
323.9
817.6
3.07
0.36
0.91
4Ј-F-
Difluorinated derivatives
2Ј,4Ј-diF-
2Ј,5Ј-diF-
2Ј,6Ј-diF-
3Ј,5Ј-diF-
Polyfluorinated derivatives
2Ј,3Ј,4Ј,5Ј,6Ј-pentaF-
Monochlorinated derivatives
2Ј-Cl-
3Ј-Cl-
4Ј-Cl-
1.95
1.29
0.34
353.6
1239.8
9674.9
41.4
0.39
1.38
10.77
0.05
25.40
1.99
1031.2
1.15
0.15
4.41
6.71
6997.6
591.5
189.5
7.79
0.66
0.21
Table 3. AhR Activation Potencies of BA and Its Derivatives
Dichlorinated derivatives
2Ј,3Ј-diCl-
2Ј,4Ј-diCl-
2Ј,6Ј-diCl-
3Ј,5Ј-diCl-
a)
ECTCDD50
(mM)
lacZ
units/mM
Relative
activityc)
1.50
4.09
0.12
3661.3
338.2
12079.5
216.1
4.08
0.38
13.45
0.24
Chemicals
Benzanilide
b)
34.80
34.2
1.00
3.02
2,3Ј-diCl-
21.30
43.2
0.05
Polychlorinated derivatives
2Ј,4Ј,5Ј-triCl-
3Ј,4Ј,5Ј-triCl-
2Ј,30019,5Ј,6Ј-tetraCl-
2Ј,3Ј,4Ј,5Ј,6Ј-pentaCl-
Monobrominated derivatives
2Ј-Br-
44.23
1.80
Ͼ10
259.1
432.7
325.3
237.7
0.29
0.48
0.36
0.26
Chlorine-substituted derivatives
2Ј-Cl-BA
4Ј-Cl-BA
2Ј,4Ј-diCl-BA
2-Cl-BA
Alkyl-substituted derivatives
2Ј-Me-BA
Ͼ10
2.30
2.12
0.38
536.0
695.0
3427.0
82.7
15.67
20.32
100.20
2.42
0.25
3.00
Ͼ30
4530.5
597.0
80.0
5.04
0.66
0.09
3Ј-Br-
4Ј-Br-
11.40
Dibrominated derivatives
2Ј,4Ј-diBr-
2Ј,6Ј-diBr-
52.90
52.90
Ͼ300
24.7
12.7
Negative
0.72
0.37
—
4.53
0.36
200.2
9987.6
0.22
11.12
2Ј-Et-BA
2Ј-tert-Bu-BA
Each value is the mean of at least three analyses. a) Concentrations producing
lacZ units equal to 50% of the maximal response to TCDD. b) Calculated from the
slope of the linear portion of each dose-response curve near the origin. c) Relative
values of lacZ units/mM of each compound compared to MY.
Each value is the mean of at least three analyses. a) Concentrations producing
lacZ units equal to 50% of the maximal response to TCDD. b) Calculated from the
slope of the linear portion of each curve near the origin. c) Relative values of lacZ
units/mM of each compound compared to BA.

April 2002
471
fact, Cheung et al. reported that 4-aminoazobenzene (4-
The present study confirmed that the suitable size and pla-
AAB) derivatives induced CYP1A2.20) Considering that most nar structure of a molecule are important for the AhR ligand
of the MYs tested in this study showed higher ligand activi- activity in MY and related congeners. It is expected that de-
ties than that of 4-AAB, MYs may act as inducers of the termination of the characteristic structure of AhR ligands de-
CYP1A family.
scribed in the present study will be useful for prediction of
The effect of halogen-substitution in monohalogenated the toxicity of some structurally related compounds.
MYs on the AhR ligand activity can be summarized as fol-
lows: enhancement by substitution at position 2Ј, and reduc-
Acknowledgments We wish to thank Dr. Charles A.
tion by substitution at position 3Ј and 4Ј. Gillner et al. sug- Miller III of the Department of Environmental Health Sci-
gested that all potent AhR ligands could fit into a rectangle ences and Tulane-Xavier Center for Bioenvironmental Re-
of 6.8ϫ13.7 Å, on the basis of a ball-and-stick model.28,29) search, Tulane University School of Public Health and Tropi-
The length of the MY molecule is about 14 Å and the MY cal Medicine, for kindly supplying us with the YCM3 strain.
molecule fits into this rectangle. However, the length of the This work was supported in part by the Japan Society for the
MY molecule will exceed 14 Å by halogen substitution at po- Promotion of Science and the Ministry of Education, Cul-
sition 4Ј, in particular with Cl and Br. It might be possible to ture, Sports, Science, and Technology of Japan.
explain the decrease in the AhR ligand activity of MY by de-
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halogenated MYs. All the compounds with very high activi-
ties (ECTCDD50Ͻ1 mM and relative activityϾ3) had a halogen
atom at position-2Ј, and all the compounds having a Cl or Br
atom at position-4Ј showed lower AhR ligand activities than
that of MY.
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The enhancing effect of 2Ј-halogen substitution on the
AhR ligand activity of MY and BA was not explained by the
size or planarity of a molecule. With regard to MYs, the pla-
narity of 2Ј-mono and 2Ј,6Ј-di-halogenated derivatives may
not be lost because their lmax values of UV absorption are al-
most the same as that of MY (Table 1). The enhancement of
AhR ligand activity by halogen-substitution at the ortho posi-
tion of the –NϭN– or –NH–CO– bond in MY and BA might
be due to an electronic effect (i.e., inductive effect) of the
halogen substituent(s) on ligand recognition by AhR. How-
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