Document akOwEz5GNYGBqJv3MaNqYjkY
(Chfnv Phorm. Bull.') UUU>)2237-- 2242(^873)J
UDC 547. 622. 09 ; 6IS. 31.015. 4.076. 9
Metabolic Studies on Polychlorinated Biphenyls. III.11 Complete Structure ' and Acute Toxicity of the Metabolites of 2,4,3',4'-Tet^achIorobiphenyl^,
Hiro-aki Yamamoto and Hidetoriii Yoshimura
f aculty of Pharmaceutical Sciences, Kyushu University'*
' (Received March 12, 1973)
The present investigation was undertaken to establish the structures of two metabo lites which were isolated from the feces of rats administered with 2,4,3',4'-tetrachloro' biphenyl (2,4,3',4'-TCB), n major component of commercial preparation of polychlorinated ' biphenyls. For this purpose six possible isomers of ntonohydroxy-2,4,3',4'-TCB were synthesised by condensation reactions between appropriate diazolized dichloroanilines and diehlorophenols. Among these isomers, 5-hydroxy- and 3-hydroxy-2,4,3',4'-TCB were shown to be identical with a major metabolite (M-At), mp l.>5--156, and a minor meta bolite (M-A,), mp 92--98, respectively.
Furl her study was made to know the acute lethal dose of 2,4,3',4'-TCU and its major mctulxilitc, 5-hydroxy-2,4,3',4'-TCB, by i.p. injection using male mice of CF-1 strain. The results showed that l.Dw of 2,4,3',4'-TCB and 5-hydroxy-2,4,3',4'-TCB were 2.15 g/kg anil 0.43 g/kg, respectively, suggesting that acute toxicity of 2,4,3',4'-TCB might be attri butable to phenolic mctnlxalitcs produced in vivo.
In preceding paper" of this series the authors reported that 2,4,3',4'-tetrachlorobiphcnyl (2,4,3',4'-TCB), one of the representative components of KC-400," was converted partly to monohydroxy-metabolites in rats. Together with unchanged compound, these metabolites were exclusively excreted into the feces, and excretion rate of a major metabolite, M-Aj, was accounted about 10% of the dose during 12 days after the treatment. Although the metabolites were well characterized to be monohydroxyl derivatives of the parent compound by various spectrometries, there were G possible isomers for satisfying the structure of monoliydroxy-2,4,3',4'-TCB and the complete structure was remained to be elucidated.
On the other hand, such an aromatic hydroxylation is generally recognized to be a detoxi cation mechanism (or foreign compounds. It is also true, however, that phenolic metabolite frequently shows activity comparable to or more than the parent compound, as exemplified in the metabolism of acetanilide51 or phenylbutazone.
The present paper will report identification of a major (M-A,) and a minor metabolite (M-A,) o( 2,4,3',4'-TCB in rats which were described in the preceding paper" and also describe acute toxicity of M-A, in mice comparing with that of the parent compound.
Method
Synthesis of 5-Hydroxy-2,4,J',4'-TCB------- To 0.4 g of 3,4-dichloroaniline, mp 71--72, was added 1.6 g of cone. HC), and this solution was diluted to 3.2 ml with H,0, being diazotized with saturated solution of
1.1.2 g of NaNO, at 0--5 under stirring for 30 min. This clear solution ol diazonium salt was then added to
1.2 g of 2,4-dichlorophenol, nip 84--65, under stirring and a mixture was heated on a boiling water bath ,>r 2 hr. Tho resulting rvnetion mixture was extracted with CHClj, and the extract was then shaken with1 2 3 4 * 6
1) Part 11: H. Yoshimura, H. Yamamoto, and S. Saeki, Chem. Pharm. Pull. (Tokyo), 21, 2231 (1973).
2) This work was presented at the 4th Symposium on Drug Metabolism and Action, Sendai, Sept. 1972;
Abstracli ot papers, p. 03.
3) Location: Katakasu, lUgashi-ku, Fukuoka.
4) S. Sueki, A, Tsutsul, K. Oguri, II. Y'oshimura, and M. Hamana, Fuhuoka Acta Med., 62, 20, (1971).
i) F.B. Flinn and B.B. Brodic, J. Pharmacol. P.xptl. Tlterap., 94, 70 (1948).
6) J.J. Burns, it.K. ltusc, S. Goodwin, J. Keichcnthal, 1L.C. Morning, and B.B. Brodie, J. Pharmacol.
Bxptl. Thcrap., 113, 481 (1955).
.
22VM
Vyl. 21 (1973)
2\' NaOH. TJic alkaline layer wn made acidic with 1IC1, and it was again extracted with CHCI*. The
extract was distilled at 200--230 under atomospheric pressure yielding 0.7 g unrcactcd 2,4-dichlorophcnoI.
The residue was then distilled under reduced pressure collecting distillate o/ bp ISO--250 (lOmmHg).
The mldishorange oil thus obtained was dissolved in a small amount of McOH and submitted to prepora-
tivc thin-layer chromatography (TLC) using solvent system of hexanc-AcOEt-AcOH (10:10:1) and silica
gvd plates described below. The band corresponding to M-A, (around /?/ 0.30) was scraped off into a flask
under ultraviolet (UV) lamp and extracted with McOH to obtain crystalline material. It was rccrystalliied
from McOH-H,0 (2: 1) to colorless needles, mp 155--150*. Yield was llOmg. The mixed melting point
was not depressed on admixture with a major metabolite of 2,4,3',4'-TCB, M-Av. Mass Spectrum itift: 300
(M4), :ms (M + 2). UV
252. .100 mu. IK
3560 cm '1 (OH).
Synthesis of 3-Hydroxy2,4,3'f4'-TCB----Similarly as described in synthesis of 5-bydroxy-2,4,3',4'
TCB. 3,4-flirhloroaniline (0.4 s') wan diazotized with cone. IIC1 (1.6 g) and satmated solution of NaNO,
(ft.2 g). and allowed to react with 2,0-dichlorophcno! {1.2 g). The reaction mixture was purified by prepara
tive TJX same as above, and crystalline material was obtained from the band corresponding to M-A, (around
Jif ft.4). It was rccrystallizeri from McOH-HtO (2: 1) to colorless needles, mp 05--08. Yield was only
ft mg. The mixed melting point was not depressed on admixture with a minor metabolite of 2,4,3',<-TCBf
M-A,. Mass Spectrum vtfe: 300 (M4), 308 (M + 2). UV *'': 252, 200 mp. IK ryB: 3500 cm'1 (OH).
Chlorination of S-Hydroxy^^^'^'-TCB----To 50.4 mg of phenol was added 41.0 mg of phosphorus
poilnchloride, and the mixture was heated at 100* for G hr on an oil bath. To this reaction mixture, after
cooling, was added 01.2 mg of 5-hyclroxy-2,4,3',4'-TCB. The mixture was again heated ut 100 for 6 hr on an
oil bath and further at 300 for 10 min on a sand bath. The reaction mixture was distilled under a reduced
pressure collecting a distillate of bp I70--230 (6 mmHg), which was 8oIidificd soon. This solid material
was purified through silisic acid (MalJjncrodt, 100 mesh) column chromatography using hexane as effluent
solvent and recrystallized from McOH to colorless needles, mp 83--85. The mixed inciting point was not
depressed on admixture with authentic sample of 2,4,5,3,,4'-pcntachlorobiphcnyl synthesized previously.ft
Mass Spectrum mfe; 324 (AH). UV/*'TM: 253 mp. IK
cm-*: 1580, 1453, 1330, 1145, 1000, 1045, 885,
823, 720, 678, 634.
-
Thin-Layer Chromatography (TLC) and Gas-Liquid Chromatography (GLC)-------These were performed
similarly as described in the preceding paper.1) TLC was conducted using silica gel plates (Wakogel B-5UA
containing fluorescent indicators, 0.25 mm thick, activated at 105 for 30 min) and a solvent system of hexane-
AcOKt-AcOH (40: 10: 1). The spots were visualized by UV lamp. Phenolic compounds were also revealed
by Folin-Ciocalteu reagent or diazotized benzidine reagent. GLC were carried out by a Shimadzu GC-
,'IAP. yns chromatograph equipped with electron capture detector. The column was a glass spiral tube
(4 mm y 2.5 m) and the column packing was 1.5% SE-30 on Chromosoib \V (GO--80 mesh). The column
temperature was maintained at 200. Nitrogen wns used as a carrier gas with flow rate of GO ml/min (1.5
klHcnv). The samples containing phenolic compounds were dissolved in dry pyridiue, trimethylsiiylated
with N,Odus(trimcthyIsUyl)acetamide and submitted to above GLC.
Animal Experiment-------Male CF-1 mice weighing 15--23 g were used for the present study. They
wore divided into 13 groups, each of which consisted of 8 mice having similar body weight and was injected
mlrnpcriloncally with 0.35, 0.70, 1.05, 1.40, 1.75, 2.10, 2.45 or 3.15 g of 2,4,3',4'-TCB, or 0.2, 0.3, 0.4, 0.5,
or 0.75 g of 5-hydroxy-2,4,3',4'-TCB per kg body weight of mice, respectively. In above injection, each
dose of 2,4,3',4'-TCB and 5-hydroxy-2,4,3',4'-TCJ3 was dissolved in 0.2--0.5 ml of soybean oil. Animals
were housed in a room maintaining at 20 and numbers of died mice were counted at 24, 48, 72, and 06 hr
after administration.
.
Result and Discussion
Complete Structure of the Metabolites of 2,4,3',4'-TCB
As already described, both M-A,, mp 155--166, and M-A,, mp 92--98, a major and minor metabolites of 2,4,3',4'-TCB in the rat, respectively, were found to be monohydroxy2,4,3',4'-TCB. All possible isomers satisfying this structure are illustrated in Fig. 1.
These monohydroxy-derivatives of 2,4,3',4'-TCB could be synthesized by coupling reac tions of appropriate diazotized dichloroanilines and dichlorophenols utilizing the method of Colbert and Lacy,71 as shown in Fig. 2.
Therefore each reaction described in Fig. 2 was firstly carried out in a small scale to examine which reaction could provide the metabolites, M-A2 and M-A, by GLC and TLC. Judging from the retention time of products yielded in above 5 reactions, M-A, was suggested to be
7) J.C. Colbert and K.M. Lacy, J, Amer. C/rm. Sac68, 270 (1946)
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Cl Cl
H6
I: 6 hydroxy-2,4,3\4'-TCB
Cl Cl
bH Hr 5-!iydrox)"2,4.3',4'-TCB
Cl
Cl
Cl OH \/
-Cl
III: 3-hydroicy-2,4,3',4'-TCB
Cl Cl
Cl Cl
Cl OH Cl
c,-<">-0-CI bn
ClHO
-C1 Cl
IV: fi'-hydroxy*2,4,3\4'-TCB
V: 5'-hydroxy-2,4,3',4'-TCB
VI: 2'-hydroxy-2,4,3',4'-TCB
Fig. 1. Possible Isomers of Monohydroxy-2,4,3',4'-TCB
reaction* obtaining tnonohydroxy gai chromatogram of expected
- 2,4,3,4 TCB
reaction products
compound*
produced only by the reaction (5). On the other hand, however, a peak
(1)
ci-gn JS?H*
Cl.
+ CI
OH
PC`~
(2) Cl-O-NH, cS'-
JV V, VI
having identical retention time with that of M-A2 could be detected in the gas chromatogram of the extract
(3) CH, >-Mlli C^H-
I
(4) Cl-O-NH. + CI^O-0H
eff
(5) Cl-O-NH. +
Met,bo!iU' of
VC*ll -
II
III
M-A.IM-Ai
2,4, 3.4 ' TCQ
10 20 30 40 fa(min)
from either reaction (4) or (5). Finally by TLC examination it was concluded that M-Aa and M-A, could be produced only by reactions (4) and (6), respectively, as shown in Fig. 3.
Fig. 2. Cas Chromatographic Patterns of Products Obtained in the Coupling Reactions
By these preliminary experi ments, M-Aj and M-A, were presumed
to correspond with the compounds
(II) and (HI), respectively, and there
reaction ( 4 ) oo o
fore an attempt to isolate these as crystalline form was then carried
reaction ( 5 )
<S25
out by larger scale of reactions (4) and (5).
The reaction (4), in which diazo-
M-A,
O
tized 3,4-dichloroaniline was con densed with 2,4-dichlorophenol, was
M-A,
found to afford three products by GLC (Fig. 2). These were also visu
alized as blue spots at Rf 0.20, 0.30
0.0 5.0 Rf
1.0
Fig. 3. Thin-Layer Chromatograms of Products
Obtained in the Coupling Reactions
and 0.45 with Folin-Ciocalteu reagent in TLC using a solvent system of bexane-AcOEt-AcOH (40: 10: 1) (see Fig. 3). Three products described
solvent ayitero: hexane-AcOEt-AcOH (40:10: ])
0: poiitivc to Folin-Ciocalteu reagent and UV Ump
above could be isolated by prepara tive TLC using the same solvent
system as above and all their mass spectra showed prominent peaks at m/e 30G (M+) and
308 (M + 2), suggesting that they should correspond to the compound (II, Ila and I lb) which
were theoretically possible to form in the reaction (4). Among these, one having Rf value
of about 0.30 gave colorless needles, mp 156--150, on recrystallization and its melting
point was not depressed on admixture with M-As.
.
The complete identity of both compounds was further shown by UV (4J**: 262, 300 m^),
infrared (IR) (Fig. 6) and Mass Spectra [m/e: 30G (M+), 308 (M+2)J. As described above,
three compounds (II, Ila and lib), were produced in the reaction (4), however only II was
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2240
Vol. 21 (1973)
Cl
Cl-^^-NH.
+ Cl
S
HO-^"^>-Cl
NaNO. HC1
PCI.
1---CC--..HH--..-OO-HH--- -
+ Cl Cl OH
Cl h0~<L)> ia
/
Cl
Cl 1 Cl
HO Cl \/ lb
Cl Cl
\--f
Cl
f
Cl
Cl
Fig. 4. Coupling Reaction of Diazotized 3,4-Dichloroaniline and 2,4-Dichlorophcnol
a derivative of 2,4,3',4'-TCB, and this indicated undoubtedly that the structure of M-A, should be
--i ..--v '(
WWV
V'"''... V/ *
i: t
6-hydroxy-2,4,3',4'-TCB (II). The
structure of II was further confirmed
by its conversion to the known
compound, 2,4,6,3',4'-pentachlorobi-
phenyl, mp 83--87,*> according to
the method of Coe, et oi.,> The product, after purification by distill ation, column chromatography and recrystallization, was obtained as colorless needles, mp83--85, and was shown to be identical with authentic
4000
2000
1500
1000
500 200 (cm'1)
Fig. J. IR Spectra ot &-Hydroxy-2,4,3',4'- TCB and M-A,1) (KBr)
----- : 5bydroy3,4,3',4-.TCB ------: M-A,
sample of 2,4,5,3',4'-pentachIoro-
biphcnyl41 by the mixedmelting point test, GLC and UV, IR and mass spectrometry.
The reaction (6), in which diazotized 3,4-dichloroaniline was condensed with 2,0-dichioro-
phenol, was then carried out to obtain compound (III). As shown in Fig. 2 and Fig. 3, two
products could be produced in this coupling reaction, and separated by preparative TLC
using hcxanc-AcOEt-AcOH (40: 10: 1) as a solvent system. They were revealed as blue
spots at Rf 0.40 and 0.35 with Folin-Ciocalteu reagent in above TLC and showed prominent
----- : 3-hydrciyJ,4.S',4'-TCB ------ : M-A,
STLCOPCB4009886
peaks at m/ 300 (M+) and 308 (M+2) in the mass spectra. These findings suggested that above two products should correspond to the compounds (III and Ilia) (see Fig. (i).
One of these two products showed identical Rf value (0.40) and retention time (14.5 min) with M-Aj on TLC and GLC (TMS derivatives). It was isolated as colorless needles, mp 9j--!)8, after purification by preparative TLC- and recrystallization. This sample showed complete identity with M-At in the mixed melting point test, TLC, GLC and UV, IR (Fig. 7) and mass spectrometry. From these findings the complete structure of M-Aj can be concluded to be 3-hydroxy-2,4,3',4'-TCB (III), because (Ilia) could not be derived from 2,4,3',4'-TCB.
It must be noticed that this is the first study by which complete structure of the meta bolite of PCB isomers having more than two chlorine atoms was established.
Acute Toxicity of 5-Hydroxy-2,4,3',4'-TCB, a Major Metabolite of 2,4,3',4'-TCB
Considering high toxicity, for example, of pentaehlorophenol, the question whether phenolic metabolites of PCB possess more toxicity than the parent compound is of very importance. Acute oral LDW of KC-400 was already reported to be about 2.0 g/kg in mice by Tanaka, et al.'> In the present study, intraperitoneal LDW of 5-liydroxy-2,4,3',4'-TCB, a major metabolite of 2,4,3',4'-TCB, was determined together with that of 2,4,3',4'-TCB using male CF-1 mice by the method of Litchfield-Wilcoxon.101 The compounds dissolved in soybean oil were injected intraperitoneally and numbers of mice died within 4 days after the injection were counted as described in Method.
As shown in Tabic I, LD60 of 2,4,3',4'-TCB and 6-hydroxy-2,4,3',4'-TCB were 2.15 and u.43 g/kg, respectively. This means that acute toxicity of 5-hydroxy-2,4,3',4'-TCB, a major metabolite of 2,4,3',4'-TCB is about 5 times as high as that of the parent compound. In addition, most of mice died at 3 rd to 4 th day after the injection of 2,4,3',4'-TCfe, while in l lie case of 5-hydro.vy-2,4,3',4'-TCB all the mice died within the first 2 days.
Table I. Mortality and Acute Lethal Dose of
2,4t.'r,4'-TCB and M-A, in Mice
Dose (fi/kg) (i ./>.) 2,4,3',4'-TC
24 hr
0.35 0.70 1.05 1.40 1.75 2.10 2.45 3.15
0 0 0 .... 0 0 0 0 1
Number 0/ dead mice
48 hr
72 hr
90 hr
000
00 0
00 0
0 '0
0
011
01 3
02 3
13 2
LD,, 2.15(1.79--2. 58) g/kg (jOCO.05)
Total
0/8 0/8 0/8 0/8 2/8 4/8 5/8 7/8
M-A, 0.20 0.30 0.40 0.50 0.75
0 l 3 5 5
Had) pump ecntiiit oJ 0 mice.
00 0 100 100 100 100 LI\o 0.43(0.34--0.55)g/kg(/><0.05)
0/8 2/8 4/8 6/8 6/8
il) K. Tnnaka, S. Fujita, F, Komatsu, and N. Tamtira, Fukuoka Arm Med., 60, 544 (1969). 1(1) J.T. Litchfield, Jr. and F. Wilcoxon, J. Pharmacol. Uxpll. Thera/-., 96, 0!) (1040).
These results suggested that the acute toxicity of 2,4,3',4'-TCB might be attributable to its phenolic metabolites produced vivo. Further studies, however, are necessary to ascertain this suggestion conclusively. Recently Brodie, el al.u' reported that single doses of chloro-, bronio- or iodobenzene (1 ml/kg, i.p.) administered to rats produced massive necrosis in the centrolobuler regions of the liver and suggested that an epoxide which produces in aroma tic hvdroxylation as a labile intermediate would be responsible for this hepatic necrosis. This hypothesis may be extended to PCB toxicity, however no evidence has been obtained (ui epoxide formation in PCB metabolism to date.
Acknowledgement This work was supported partially by a Grant-in-Aid for Scientific Research provided by the Ministry of Education and also by a research grant provided by the Ministry of Health and Welfare to which the authors are greatly indebted.
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II) B.B. Brodie, A.K. Cho, G. Krishna, and W.D. Reid, Ann. A'. Y. Acad. Sci., 179, 11 (1971).
VW