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On the mixed complexes of tetracycline metal chelates
ANALYTICA
452
ON
THE
MIXED
CHIMICA
COMPLEXES
OF
ACTA
TETRACYCLINE
METAL
CHELATES JlOI~I%O ISII1I~A7’1~.TAKEIC1-I1
SAKAGUCHI, S/\CHIKO KANAO
I~ncirlly o/ Plrcwnrucea~t~cuL Sctenccs,
Unavcrsz(y
(Iiccc~vcd
of
J uly 8th.
ICIYOMI
T0hyo.
TAGUCHI
motkyO-krc.
AND
*rokyO
(f
upun)
I gsg)
ISHIIIATE AND SAKAGUCIII~ and SAKAGUCHI AND TAGUCIII~ hnve studied the tctracyclinc chelates ancl showed that only the phenolic P-dikctonc group in the molecule is rcsponsiblc for chelate formation with metallic ions. Further investigations revealed that metal cliclates of tctracyclmcs arc always cationic chclatcs in which the molar ratio i5 I to I. It sccmccl likely that the metallic ion in this cationic chclate retains the ability to combine with other ligands. Conscqucntly, tetracycline chelates should tend to combine further wit!1 chelating agents such as isomazld, penicillin, etc. We have shown that several mixed complex compounds can be prepared; tllcse may be termed multiligand or polyligand complexes. ~~ITlEH3 and DALE AND ISENNETT~ obtained tetracycline-metal complexes with organic acids or penicillin-aluminium-sulfanilamide but no detailed information was given.
Isonicrzid c/date
(INAH-cldate)
have described INAH chelate. We have modified their procedure slightly. Copper-INAH &elate was obtained as blue crystals, its molar ratio being I to I, Cobalt-INAH chclate was obtained as a light carmine-red crystalline powder with a 2 to I ratio of ligand to metal (Table I). A fall In prr during metal chelate formation appeared in both cases (Table II). Since the p~r decrease with cobalt chelates appcarccl above phi 7 but not below pIr 7,
Foua
AND
DUVALL~
IN/U-l-Cu(l1) INAH-Co(II)
20.7
24 1
12.g
=2
34
INAH-Cu-CT
‘4 4 7.55
12
po
INAI-I -Co-C; IN AI-1 - lFc+ 3 - CT a.
IYohItCd
at
PH 4.0
0
3.5 r1.G
7.16 7 62 7 73 G 87
7 90
7.79 G ‘3 b.
As
4.26
5 75 G 57 9.14 CT
INAI-I-Cu(N03) (INt\H)a-Co(N03)a-n
3:,8 II 8 4.06
G 4g 7 07 8 85
57 o
G5.3b 05.9 67.9
57 9
58.6 68 2 63 4
(INAH)a-Co&-jaq. (INAI-l)l-Cu -CT-HCl-2aq.
LNAEI-Cu-CT-HC104-3aq. lNAH-Co-CT-I-El-3aq
LNAH-l?cCI-CT-HCl-4nq
biuc
Anal.
Chiitt.
Actu,
22
(rg6o)
452-457
TETRACYCLINE
METAL
453
CHELATES
it seems likely that copper-INAH chelate has the structure (I) while cobalt-INAH has the equilibrium formulae shown as structures (II) and (III):
3
C---N
N
I
C --
I
0
NH -- NH2
II 0
NH2
I co*
“Co’ / .
2
I
P C--
NH-NH2
ffJH2
m
II
TABLE DCCHRASL
IS
~-~~-------_~___--_---____
2 79
Co(lI)
4 30 5 82 6
I I)
---____-* Conccntratlon
complex
FOKMAlION
OF
-_----
.? 97 5 11
of chlovotetvucycline
Ctll:LATLS*
fyfg u,*t*
solutron
metal
was
1>8f--trdnc~
m,r,“g
__ ---
.? OH
2 79 4 34 5 87 0 87 .? 79 5 03 _.-__-
go
of each
INAH
INAH
hfdd
Cll(II)
CT-Cu(
IN
PII ..-- -__-_----
---^
nfrlnlJ
Mixed
l>H
II
--_
3 88 5 Hz 6 rs 2 92 4-44
----o II -0.42 fo
00
-0 6p + 0 13 ---
.--
59 . . -_ --
I/ZOO &I
with isohazid
A mixecl chelate of INAH-Metal-Chlorotetracyclmc (CT) was obtained by addmg IHAH solution to an alcol~ol~c solution of the chlorotetracyclinc-metal chclatc. The mixed chelates formed wcrc INAH-Fe-CT (brown precipitate) and INAH-CoCT (orange powder) which were soluble in water and hot methanol, but insoluble in ether and chloroform. Both complexes had the molar ratio of I : I. I (Table I). Only IHAH-Cu-CT (yellow-green precipitate) had a molar ratio of nearly 2 : I : I but it was slightly unstable on storage. In some of these cases a fall in pH drop occurred (Table II). The structure of the mixed &elate is postulated as follows:
INAH
-Fe-Cl
muted
chalote
Isoniazid was destroyed by ferric iron without forming any chelates but a mixed chelate of INAH-Fe-CT could be obtained if previously prepared CT-Fe chelatc was added to the INAH solution. --
Anal.
Cl&n.
Acta,
22
(1960) 452-457
MI.ISHIDATE
454
et cd.
Pe9taciLli9achelnles Penicillin G forms precipitates with several metallic ions. Only copper(I1) causes cleavage of penicillina. Thonum(lV) forms a vcry_sensltive prectpitate from aqueous or methanolic solution with penicillin but not with methyl penicillin-G. This shows that the carboxylic acid group of penicillin G is important for chelatc formatlon with metalhc ions such as thorium. On the other hand, the infrared spectrum of the cobalt-, -cnicillin G chclate showed a remarkable decrease of absorbance at 1786 cm-l, which was due to the p-lactam of penlcillin C; (Fig. I) ; this incllcates that the@-lactam group 1scsscntlal for chelatc formation. --
---
-_---
-
COWlplCW
Mclal uA, -_ -_/mml call.
TABI,I<
III
--_------_
---
CT y” _- .-.. - _- . . --talc. fnu,ul
H110 y, _-/uumf talc.
_--
I~ortnulac ---.-.--~
_----_---__-----.s..---..----
G-l ll(l\‘) l’cn. G-Co(l I) l’on. G- Fc(llL) I’cn G-I I1-CL-i PC11 G-‘l’hI,hC”l’ Pen G - 1:~ I J _ (,‘I‘ Pen. G-C+u-C-1 Pm G-c o-Cl’ I’m.
27 33 7 86 G.00 210 22 3
6 10 5 58 5 83
6.41 4.46 6 48 8 88 8 .W 5 95 7 19 4 67
28 7 6 19 21
70 74 87 5 00 5 63
6 67 4 74 0.65 8.99 fl ‘4 5 45
43 0
42 7
594
519
62
I’wIG- L‘h(NOa)*,-3nq (IknG)z-Co-znq (I’cnC;)zx;cc1- 3cq I’ct~G-Tl~O-C’1’-~1CI-C~;rc~ l’cnG-ThO - I)A\c’L’- 5aq I+nG-FcC l-<:‘~-~t<:I-3;rq.
n*
prr
at
6
0
at 1Wi 0 0
67.7
_-__
* il.\ c”I’ I,asc.
Co(NO,Iz’ 6H@
1
750
lco0
1250
1500
1750
20,po
12,50
1500
1750
2090
2500 .
,
3000,
,
,
3500
4000,
,
2
750
Ptg
I. Infixcd
1000
,
25po
spectrum of l~en~cdlm C-cobalt(II) chclatc (I nnc’ 3) In NuJd mctllum with scdium
,
3oqo
35po
,
comp:Llccl with ctiloridc prlem
tllc components
(2)
Anal
ChLWd./1&Z,
4090
22
(1960)
452-457
”
TETRACYCLIXE
METAL
CHEL.4’l-ES
455
Analysis showed that the thoriam and cobalt penicillin G chclatcs,have the molecular formulae Pen G-Th(XO&-?,aq. and (Pen. ‘G)2-CO-zaq. recpccttvely (Table ITT).
The Th-CT chelatc was prcparccl from CT-IX1 and thorium mtrate solution ; it was dissolved in z ml of water and I ml of an aqueous solution of pcnlclllin G was added gradually. An orange-yellow powder ptcclpitateci m a short tlmc, and was washed wth ethanol and cthcr ancl dncd. It was slightly soluble m water but insoluble in methanol, ethanol, acetone, and dloxanc. Penicillin G forms a shghtly soluble Salt with chlorotctracychnc (KUSKIN’). The fact that dcsdimethylammochlorotetracycline (DACT) 15 able to form a chelatc with thorium (Fig. 2) provcq the cxlstence of mlscd complexes cl-en in the CT-Th -Pen. G.
1~csclit~~ctliylnii~it~ochlorotctr;rc~-cliclc nwtnl ci&tc 1 IO pg/lld 1 Ku*7 10 mrlllll -I- ‘I’h(l\‘~. II , .., I)ACT111 IO pg/ml 1)ACl CH~O~I -i- ztjiv)',. Cl-13011 -IIlO*( I\’ 10 pg/nrl l~/\C’i c1~1~011 -f i’c(li1). \’ 10 pjq/tnl 7)Arl Fin
I’iK 3 l~il~~tirc~~trc~~~tc~ri~~ciri-C’ri(i
2
(Ij,\CT) CHO11
Cii7OI-1
$
-
RIM d-jqlucos,rinlnc Cu(l1)
-
0
-
Glu
-
1’11
0 0125 nr. 2 Gill
001
-Cu(ll) 3
5,
0 .I
1s
--Cn(1 Al,
7 7,
I’14
-Cu(Il) 001
nf,
r-*/n
I) corllplcu
I) coniplcx
I’
0 0125
3
i
At,
CU(NC)D)
1 ins04
0
1 WI
-
l)s’l--~‘ll(li)
I
Al,
prt
0
7
2,
02, f”i
2’ Iv,
7 0.
--
Dcsdimctl~ylntninoclllorot~tracyclinc (m.p. x57- x60”) was prcparcd by tile method of !SrlsrWEss cl al.8. It can form a chclntc with thorium nncl other metallic ions In the same way us CT (Fig. 2). Thorium-DACT was obtained by mixing DACT rind thorium(N) in mcthanolic solution; the potassium salt af penicillin C in methanolic solution (if it does not dissolve in pure methanol, ;Llittle water is added) was then added to form a precipitate which was dissolved in dlmethylformamide. The solution was filtered and the metal chclqte, DACT-Th-Pen. G, was precipitated by adding’ ether . I to- the solution. The ‘A’kal. Ch&
Ada,
22
(xc@)
452-&
M.
456
ISHIDATE
et d.
chclate could bc purified by repreclpitation. The product which was insoluble in water and alcohol (Table III). I)t~~ydrostreptomycin
chclate
was a yellowish
powder
(DST-chelute)
FOYE et al.0 reported on the stre+tomycin chelates of copper, cobalt and mckel. l’hc chclatcs of dihydrostreptomycin and metal ions were also studied. The chelates were prepared by mixing clihydrostleptomycm solution with a slight excess of metallic ion solution; excess copper was removed by precipitation of the hydroxide by adjusting the prr of the solution to 7. Ethanol was then added to give a concentration of less than 40% of alcohol. In the case of copper, the precipitate obtained was deep blue (Table IV). This precipitate was soluble in water and 0.x N potassium hydroxide, but insoluble m organic solvents. When the DST-chelate was prepared at pn II, it -.-
.---____.--
CWlpll’%C~
--__--
--_--_-
Ix’r-cll(
11)
IXW-NI(
II)
Isr-‘I’I1-cI
n
fr,wrrl
0
pcrccnt to the
20 3 tlcllytlr,~tctl
CT - iILl
talc
790
59 14.4 14 0 HI
6 39 14 5 13.8 821
26 G
G 4
6.25
23.6
H I,(
/ouall
cult
_
767 *7 50
7 GG
18.03 23. I
DS1’-Crr -CT
Ha0 y:,
hf da1 y:,
snmplcs
I
54 Sh
” calculated
1~-1~mulac ursunwd
ralc --__
/ounJ
GO
‘:L’
G4.0 55 0
PH
1)s r-Cu(~is3~)a-3rLq IHI--(CuOf-l)a-7aq ~I)ST-(NIOH)~]~NIO-~Z~~~~ I~S’I‘(C’~-Cu-I-1Cl)~-SO,-r2acl Ixx(CI’-‘rho)
as a. C’L’ lxuic
,-
7 I I
I I G-7 G-7
r3;rr1
‘? OS’1’-ItNO?
wa’~ usutl
was of a different type from that formed at plr 7 ; at pi II the chelates contained more copper (Table IV). The strcptidmc moiety of strcptomycm did not form chelates below prr 7, for the pn drop of streptidine was negative (Table V) ; but dihyclrostrcptomycin could form a chelatc in the streptidinc moiety above 1)~ II (Table IV). Copper-DST chelate showed all absorption maximum at 670 my& at prr 7.7 and
DI:CRI>r\SIC IN t’H IN
----I WI
Strcplitllnc
GlucosnrlllIlc
_-_--_-
---. --
IrOHMATION
01’
CIll.Lhl’l:S
MET,\L
-------Cu(II) N1(11) Co(Il) ‘l’h( IV)
__-5 G G 3
IO 30 25 00
5 6 G 3
4 75 0 10 6 20 3.93
20 30 30 87
--0.35 --0.20
-0 05 -l-o 03
Cu(l1)
5.20
5 20
5.28
+o.os
Nl(LI) Co(Il) ‘Th( IV)
G 30 5.70 3 7.5
0 30 5 70 3 70
6 25 G 00 3 80
05 4-0.3 +o 0.5
Cu(l1) Nl(II) Co(II) Th(IV)
5 6 G 3
5 40 6.00 6.40 3 85
4 80 5 80 6.40 3 80
-0.60 -0.20 fo.00 -
40 00 40 75
Anal.
Chim.
Acta,
22 (1960)
452-457
TETRACYCLINE
METAL
457
CIiELATXS
copper-glucosamme chelate also showed a peak at 670 ml& at I_‘H 7.2 (Fig. 3). The molar ratio of copper-glucosamine at 650 rnp is I to I and both showed marked pn decreases below PH 7 (Table V). Therefore, it may bc assumed that dihyclrostrcptomycin combined with copper(I I) at the N-methylglucosaminc moiety in copper -DST chelate prepared at about pri 7.
filzxed chelates of DW-naeLal-CT The affinity of copper for chlorotetracycline is larger than that for DST, and DST in the mixed chelate tends to separate on washing with ethanol and then water. Consequently, correct analytical values for the mixed chelatc with DST could not bc obtained. Qualitative tests for CT2 or DST* showed that a mlscd chclatc must have been formed. The mixed chelate was prepared as follows. ‘l’ho copper-C’I chelatc was separated as mentioned above. A mcthanolic solution of this chclatc was then added to an aqueous solution of DST to give a concentration of 40%, methanol or less since 40% methanol did not cause any precipitation of DST. The solubility of copper-CT chelate in water decreases at pH 5-q The mixed chelatc could thus bc separated by adding excess DST solution to the copper-CT solution, and adjusting the PH to 6-7. This chelate could be washed with water and ethanol without any danger of decomposing. The preparation of mixed chelates suggests that enzyme proccsscs may be clarified in the near future, for it is possible that enzyme functions arecffectcd in mixed chelates. SUMMARY Tlic nnxctl complcxca of clllorotctracycl~ncmetal chcl,Ltcs with i~oni~~~id, pcntcillin G, and drhydrostrcptomycm wcrc cxamincd wtll ‘l’h+J. Cu+a, Fc+a, Co+“, and Nr+s tons. A mcchamsm IS proposed that accounts for the formation of the mnccd complcxcr, cationIc chelates of tctracychnc have a molar ratio of I : I of metal to chlorotctracychnc and rnixcd complcxcs arc formed by combmlng with otlrcr ligancls through the rcmainrng covalcncic*s. l’hc propcrtms and prcparatron of the mtxctl complcxcs arc fqvcn
Lcs autcurs ont cffcctuc’ unc Gtudc sur Its complcxcr ct 1~; cliClatc3 form& cntrc la ChkmJt& tracpcl~nc:. quclques cations (Co+‘. Cu+n, T;c+~, Nt+“. ‘IX+“) ct Its trois c*mrpfAs su:vantlc ~rotii,rtlclc, p&ticrlhnc G ct dihydrostrqtomycmc. ZUSA;CIhfENI;ASStJSG Es wrrd crnc Untcrsuchun~ bcschrmben uber die fWdunfC und E+ynschaftcn tlcr KompIcxc*, tlic aus Chlorotctracyclm-Jlctallchelatcn. Co +2, Cu+2, FcfJ, Nt+2, Th+J mlt Isornazrd, Pcnicrllin C und Dlhpdrostreptomycm cntstehcn 1 1 3 4 a
6 7
8 9
REFERENCES 31 ISHIDATl’ Ahn T !%KACUCH1, ~/WPFZ, Bld~. (Tokyo). 3 (rr).55) I47 T SAKACUCHI AXD Ii TAGUCHI. Plrarnt. Bull. (Tokyo). 3 (1955) 303. 1,. RITTER, U.S Pal 2, 736. 725, C A ., 50 (1956) 10348. J K DALE AND M E BENNETT, CJ S. Pa: 2, 752, 335: C A . 50 (1956) r5029 \V 0. Fove AND R N DUVALL, J Ant. Pllurm. Asroc , SC; Ed , 47 (rcj.58) 285 G. GUNTHER. Pho*mat~e, 5 (1950) 577.C A., 45 (r95r) 7566 S. L RUSKIS, Brat Pa: 772, 573. C. A., 51 (1957) 10848. C.R STCPHESS, L H CONOVER, R PASTBRNACK,~: A. CIootsra~x, IV. 'I-MoR~~~AF~~, I'. I'. REGNA. F J. PILGR~%I, K. j URUNISCS AND R B WOODWARD, J. Am Chcm SOC , 76 (rr)jg) 3568 W 0. FOYE, W.E LANGE,J. V. SWINTDSKY. R E. CHAMBERLAIN AND J R. GUARINI,~. Am. Pharm Assoc., Scr Ed , 44 (rg55) 261.
* Sulfosalicyhc
aad -oxlnc-NaOH-NaOBr
or didcctyl-fl-naphthol
reactron.
Anal. Chim. Ada,
22 (1960) 452-457
452
ON
THE
MIXED
CHIMICA
COMPLEXES
OF
ACTA
TETRACYCLINE
METAL
CHELATES JlOI~I%O ISII1I~A7’1~.TAKEIC1-I1
SAKAGUCHI, S/\CHIKO KANAO
I~ncirlly o/ Plrcwnrucea~t~cuL Sctenccs,
Unavcrsz(y
(Iiccc~vcd
of
J uly 8th.
ICIYOMI
T0hyo.
TAGUCHI
motkyO-krc.
AND
*rokyO
(f
upun)
I gsg)
ISHIIIATE AND SAKAGUCIII~ and SAKAGUCHI AND TAGUCIII~ hnve studied the tctracyclinc chelates ancl showed that only the phenolic P-dikctonc group in the molecule is rcsponsiblc for chelate formation with metallic ions. Further investigations revealed that metal cliclates of tctracyclmcs arc always cationic chclatcs in which the molar ratio i5 I to I. It sccmccl likely that the metallic ion in this cationic chclate retains the ability to combine with other ligands. Conscqucntly, tetracycline chelates should tend to combine further wit!1 chelating agents such as isomazld, penicillin, etc. We have shown that several mixed complex compounds can be prepared; tllcse may be termed multiligand or polyligand complexes. ~~ITlEH3 and DALE AND ISENNETT~ obtained tetracycline-metal complexes with organic acids or penicillin-aluminium-sulfanilamide but no detailed information was given.
Isonicrzid c/date
(INAH-cldate)
have described INAH chelate. We have modified their procedure slightly. Copper-INAH &elate was obtained as blue crystals, its molar ratio being I to I, Cobalt-INAH chclate was obtained as a light carmine-red crystalline powder with a 2 to I ratio of ligand to metal (Table I). A fall In prr during metal chelate formation appeared in both cases (Table II). Since the p~r decrease with cobalt chelates appcarccl above phi 7 but not below pIr 7,
Foua
AND
DUVALL~
IN/U-l-Cu(l1) INAH-Co(II)
20.7
24 1
12.g
=2
34
INAH-Cu-CT
‘4 4 7.55
12
po
INAI-I -Co-C; IN AI-1 - lFc+ 3 - CT a.
IYohItCd
at
PH 4.0
0
3.5 r1.G
7.16 7 62 7 73 G 87
7 90
7.79 G ‘3 b.
As
4.26
5 75 G 57 9.14 CT
INAI-I-Cu(N03) (INt\H)a-Co(N03)a-n
3:,8 II 8 4.06
G 4g 7 07 8 85
57 o
G5.3b 05.9 67.9
57 9
58.6 68 2 63 4
(INAH)a-Co&-jaq. (INAI-l)l-Cu -CT-HCl-2aq.
LNAEI-Cu-CT-HC104-3aq. lNAH-Co-CT-I-El-3aq
LNAH-l?cCI-CT-HCl-4nq
biuc
Anal.
Chiitt.
Actu,
22
(rg6o)
452-457
TETRACYCLINE
METAL
453
CHELATES
it seems likely that copper-INAH chelate has the structure (I) while cobalt-INAH has the equilibrium formulae shown as structures (II) and (III):
3
C---N
N
I
C --
I
0
NH -- NH2
II 0
NH2
I co*
“Co’ / .
2
I
P C--
NH-NH2
ffJH2
m
II
TABLE DCCHRASL
IS
~-~~-------_~___--_---____
2 79
Co(lI)
4 30 5 82 6
I I)
---____-* Conccntratlon
complex
FOKMAlION
OF
-_----
.? 97 5 11
of chlovotetvucycline
Ctll:LATLS*
fyfg u,*t*
solutron
metal
was
1>8f--trdnc~
m,r,“g
__ ---
.? OH
2 79 4 34 5 87 0 87 .? 79 5 03 _.-__-
go
of each
INAH
INAH
hfdd
Cll(II)
CT-Cu(
IN
PII ..-- -__-_----
---^
nfrlnlJ
Mixed
l>H
II
--_
3 88 5 Hz 6 rs 2 92 4-44
----o II -0.42 fo
00
-0 6p + 0 13 ---
.--
59 . . -_ --
I/ZOO &I
with isohazid
A mixecl chelate of INAH-Metal-Chlorotetracyclmc (CT) was obtained by addmg IHAH solution to an alcol~ol~c solution of the chlorotetracyclinc-metal chclatc. The mixed chelates formed wcrc INAH-Fe-CT (brown precipitate) and INAH-CoCT (orange powder) which were soluble in water and hot methanol, but insoluble in ether and chloroform. Both complexes had the molar ratio of I : I. I (Table I). Only IHAH-Cu-CT (yellow-green precipitate) had a molar ratio of nearly 2 : I : I but it was slightly unstable on storage. In some of these cases a fall in pH drop occurred (Table II). The structure of the mixed &elate is postulated as follows:
INAH
-Fe-Cl
muted
chalote
Isoniazid was destroyed by ferric iron without forming any chelates but a mixed chelate of INAH-Fe-CT could be obtained if previously prepared CT-Fe chelatc was added to the INAH solution. --
Anal.
Cl&n.
Acta,
22
(1960) 452-457
MI.ISHIDATE
454
et cd.
Pe9taciLli9achelnles Penicillin G forms precipitates with several metallic ions. Only copper(I1) causes cleavage of penicillina. Thonum(lV) forms a vcry_sensltive prectpitate from aqueous or methanolic solution with penicillin but not with methyl penicillin-G. This shows that the carboxylic acid group of penicillin G is important for chelatc formatlon with metalhc ions such as thorium. On the other hand, the infrared spectrum of the cobalt-, -cnicillin G chclate showed a remarkable decrease of absorbance at 1786 cm-l, which was due to the p-lactam of penlcillin C; (Fig. I) ; this incllcates that the@-lactam group 1scsscntlal for chelatc formation. --
---
-_---
-
COWlplCW
Mclal uA, -_ -_/mml call.
TABI,I<
III
--_------_
---
CT y” _- .-.. - _- . . --talc. fnu,ul
H110 y, _-/uumf talc.
_--
I~ortnulac ---.-.--~
_----_---__-----.s..---..----
G-l ll(l\‘) l’cn. G-Co(l I) l’on. G- Fc(llL) I’cn G-I I1-CL-i PC11 G-‘l’hI,hC”l’ Pen G - 1:~ I J _ (,‘I‘ Pen. G-C+u-C-1 Pm G-c o-Cl’ I’m.
27 33 7 86 G.00 210 22 3
6 10 5 58 5 83
6.41 4.46 6 48 8 88 8 .W 5 95 7 19 4 67
28 7 6 19 21
70 74 87 5 00 5 63
6 67 4 74 0.65 8.99 fl ‘4 5 45
43 0
42 7
594
519
62
I’wIG- L‘h(NOa)*,-3nq (IknG)z-Co-znq (I’cnC;)zx;cc1- 3cq I’ct~G-Tl~O-C’1’-~1CI-C~;rc~ l’cnG-ThO - I)A\c’L’- 5aq I+nG-FcC l-<:‘~-~t<:I-3;rq.
n*
prr
at
6
0
at 1Wi 0 0
67.7
_-__
* il.\ c”I’ I,asc.
Co(NO,Iz’ 6H@
1
750
lco0
1250
1500
1750
20,po
12,50
1500
1750
2090
2500 .
,
3000,
,
,
3500
4000,
,
2
750
Ptg
I. Infixcd
1000
,
25po
spectrum of l~en~cdlm C-cobalt(II) chclatc (I nnc’ 3) In NuJd mctllum with scdium
,
3oqo
35po
,
comp:Llccl with ctiloridc prlem
tllc components
(2)
Anal
ChLWd./1&Z,
4090
22
(1960)
452-457
”
TETRACYCLIXE
METAL
CHEL.4’l-ES
455
Analysis showed that the thoriam and cobalt penicillin G chclatcs,have the molecular formulae Pen G-Th(XO&-?,aq. and (Pen. ‘G)2-CO-zaq. recpccttvely (Table ITT).
The Th-CT chelatc was prcparccl from CT-IX1 and thorium mtrate solution ; it was dissolved in z ml of water and I ml of an aqueous solution of pcnlclllin G was added gradually. An orange-yellow powder ptcclpitateci m a short tlmc, and was washed wth ethanol and cthcr ancl dncd. It was slightly soluble m water but insoluble in methanol, ethanol, acetone, and dloxanc. Penicillin G forms a shghtly soluble Salt with chlorotctracychnc (KUSKIN’). The fact that dcsdimethylammochlorotetracycline (DACT) 15 able to form a chelatc with thorium (Fig. 2) provcq the cxlstence of mlscd complexes cl-en in the CT-Th -Pen. G.
1~csclit~~ctliylnii~it~ochlorotctr;rc~-cliclc nwtnl ci&tc 1 IO pg/lld 1 Ku*7 10 mrlllll -I- ‘I’h(l\‘~. II , .., I)ACT111 IO pg/ml 1)ACl CH~O~I -i- ztjiv)',. Cl-13011 -IIlO*( I\’ 10 pg/nrl l~/\C’i c1~1~011 -f i’c(li1). \’ 10 pjq/tnl 7)Arl Fin
I’iK 3 l~il~~tirc~~trc~~~tc~ri~~ciri-C’ri(i
2
(Ij,\CT) CHO11
Cii7OI-1
$
-
RIM d-jqlucos,rinlnc Cu(l1)
-
0
-
Glu
-
1’11
0 0125 nr. 2 Gill
001
-Cu(ll) 3
5,
0 .I
1s
--Cn(1 Al,
7 7,
I’14
-Cu(Il) 001
nf,
r-*/n
I) corllplcu
I) coniplcx
I’
0 0125
3
i
At,
CU(NC)D)
1 ins04
0
1 WI
-
l)s’l--~‘ll(li)
I
Al,
prt
0
7
2,
02, f”i
2’ Iv,
7 0.
--
Dcsdimctl~ylntninoclllorot~tracyclinc (m.p. x57- x60”) was prcparcd by tile method of !SrlsrWEss cl al.8. It can form a chclntc with thorium nncl other metallic ions In the same way us CT (Fig. 2). Thorium-DACT was obtained by mixing DACT rind thorium(N) in mcthanolic solution; the potassium salt af penicillin C in methanolic solution (if it does not dissolve in pure methanol, ;Llittle water is added) was then added to form a precipitate which was dissolved in dlmethylformamide. The solution was filtered and the metal chclqte, DACT-Th-Pen. G, was precipitated by adding’ ether . I to- the solution. The ‘A’kal. Ch&
Ada,
22
(xc@)
452-&
M.
456
ISHIDATE
et d.
chclate could bc purified by repreclpitation. The product which was insoluble in water and alcohol (Table III). I)t~~ydrostreptomycin
chclate
was a yellowish
powder
(DST-chelute)
FOYE et al.0 reported on the stre+tomycin chelates of copper, cobalt and mckel. l’hc chclatcs of dihydrostreptomycin and metal ions were also studied. The chelates were prepared by mixing clihydrostleptomycm solution with a slight excess of metallic ion solution; excess copper was removed by precipitation of the hydroxide by adjusting the prr of the solution to 7. Ethanol was then added to give a concentration of less than 40% of alcohol. In the case of copper, the precipitate obtained was deep blue (Table IV). This precipitate was soluble in water and 0.x N potassium hydroxide, but insoluble m organic solvents. When the DST-chelate was prepared at pn II, it -.-
.---____.--
CWlpll’%C~
--__--
--_--_-
Ix’r-cll(
11)
IXW-NI(
II)
Isr-‘I’I1-cI
n
fr,wrrl
0
pcrccnt to the
20 3 tlcllytlr,~tctl
CT - iILl
talc
790
59 14.4 14 0 HI
6 39 14 5 13.8 821
26 G
G 4
6.25
23.6
H I,(
/ouall
cult
_
767 *7 50
7 GG
18.03 23. I
DS1’-Crr -CT
Ha0 y:,
hf da1 y:,
snmplcs
I
54 Sh
” calculated
1~-1~mulac ursunwd
ralc --__
/ounJ
GO
‘:L’
G4.0 55 0
PH
1)s r-Cu(~is3~)a-3rLq IHI--(CuOf-l)a-7aq ~I)ST-(NIOH)~]~NIO-~Z~~~~ I~S’I‘(C’~-Cu-I-1Cl)~-SO,-r2acl Ixx(CI’-‘rho)
as a. C’L’ lxuic
,-
7 I I
I I G-7 G-7
r3;rr1
‘? OS’1’-ItNO?
wa’~ usutl
was of a different type from that formed at plr 7 ; at pi II the chelates contained more copper (Table IV). The strcptidmc moiety of strcptomycm did not form chelates below prr 7, for the pn drop of streptidine was negative (Table V) ; but dihyclrostrcptomycin could form a chelatc in the streptidinc moiety above 1)~ II (Table IV). Copper-DST chelate showed all absorption maximum at 670 my& at prr 7.7 and
DI:CRI>r\SIC IN t’H IN
----I WI
Strcplitllnc
GlucosnrlllIlc
_-_--_-
---. --
IrOHMATION
01’
CIll.Lhl’l:S
MET,\L
-------Cu(II) N1(11) Co(Il) ‘l’h( IV)
__-5 G G 3
IO 30 25 00
5 6 G 3
4 75 0 10 6 20 3.93
20 30 30 87
--0.35 --0.20
-0 05 -l-o 03
Cu(l1)
5.20
5 20
5.28
+o.os
Nl(LI) Co(Il) ‘Th( IV)
G 30 5.70 3 7.5
0 30 5 70 3 70
6 25 G 00 3 80
05 4-0.3 +o 0.5
Cu(l1) Nl(II) Co(II) Th(IV)
5 6 G 3
5 40 6.00 6.40 3 85
4 80 5 80 6.40 3 80
-0.60 -0.20 fo.00 -
40 00 40 75
Anal.
Chim.
Acta,
22 (1960)
452-457
TETRACYCLINE
METAL
457
CIiELATXS
copper-glucosamme chelate also showed a peak at 670 ml& at I_‘H 7.2 (Fig. 3). The molar ratio of copper-glucosamine at 650 rnp is I to I and both showed marked pn decreases below PH 7 (Table V). Therefore, it may bc assumed that dihyclrostrcptomycin combined with copper(I I) at the N-methylglucosaminc moiety in copper -DST chelate prepared at about pri 7.
filzxed chelates of DW-naeLal-CT The affinity of copper for chlorotetracycline is larger than that for DST, and DST in the mixed chelate tends to separate on washing with ethanol and then water. Consequently, correct analytical values for the mixed chelatc with DST could not bc obtained. Qualitative tests for CT2 or DST* showed that a mlscd chclatc must have been formed. The mixed chelate was prepared as follows. ‘l’ho copper-C’I chelatc was separated as mentioned above. A mcthanolic solution of this chclatc was then added to an aqueous solution of DST to give a concentration of 40%, methanol or less since 40% methanol did not cause any precipitation of DST. The solubility of copper-CT chelate in water decreases at pH 5-q The mixed chelatc could thus bc separated by adding excess DST solution to the copper-CT solution, and adjusting the PH to 6-7. This chelate could be washed with water and ethanol without any danger of decomposing. The preparation of mixed chelates suggests that enzyme proccsscs may be clarified in the near future, for it is possible that enzyme functions arecffectcd in mixed chelates. SUMMARY Tlic nnxctl complcxca of clllorotctracycl~ncmetal chcl,Ltcs with i~oni~~~id, pcntcillin G, and drhydrostrcptomycm wcrc cxamincd wtll ‘l’h+J. Cu+a, Fc+a, Co+“, and Nr+s tons. A mcchamsm IS proposed that accounts for the formation of the mnccd complcxcr, cationIc chelates of tctracychnc have a molar ratio of I : I of metal to chlorotctracychnc and rnixcd complcxcs arc formed by combmlng with otlrcr ligancls through the rcmainrng covalcncic*s. l’hc propcrtms and prcparatron of the mtxctl complcxcs arc fqvcn
Lcs autcurs ont cffcctuc’ unc Gtudc sur Its complcxcr ct 1~; cliClatc3 form& cntrc la ChkmJt& tracpcl~nc:. quclques cations (Co+‘. Cu+n, T;c+~, Nt+“. ‘IX+“) ct Its trois c*mrpfAs su:vantlc ~rotii,rtlclc, p&ticrlhnc G ct dihydrostrqtomycmc. ZUSA;CIhfENI;ASStJSG Es wrrd crnc Untcrsuchun~ bcschrmben uber die fWdunfC und E+ynschaftcn tlcr KompIcxc*, tlic aus Chlorotctracyclm-Jlctallchelatcn. Co +2, Cu+2, FcfJ, Nt+2, Th+J mlt Isornazrd, Pcnicrllin C und Dlhpdrostreptomycm cntstehcn 1 1 3 4 a
6 7
8 9
REFERENCES 31 ISHIDATl’ Ahn T !%KACUCH1, ~/WPFZ, Bld~. (Tokyo). 3 (rr).55) I47 T SAKACUCHI AXD Ii TAGUCHI. Plrarnt. Bull. (Tokyo). 3 (1955) 303. 1,. RITTER, U.S Pal 2, 736. 725, C A ., 50 (1956) 10348. J K DALE AND M E BENNETT, CJ S. Pa: 2, 752, 335: C A . 50 (1956) r5029 \V 0. Fove AND R N DUVALL, J Ant. Pllurm. Asroc , SC; Ed , 47 (rcj.58) 285 G. GUNTHER. Pho*mat~e, 5 (1950) 577.C A., 45 (r95r) 7566 S. L RUSKIS, Brat Pa: 772, 573. C. A., 51 (1957) 10848. C.R STCPHESS, L H CONOVER, R PASTBRNACK,~: A. CIootsra~x, IV. 'I-MoR~~~AF~~, I'. I'. REGNA. F J. PILGR~%I, K. j URUNISCS AND R B WOODWARD, J. Am Chcm SOC , 76 (rr)jg) 3568 W 0. FOYE, W.E LANGE,J. V. SWINTDSKY. R E. CHAMBERLAIN AND J R. GUARINI,~. Am. Pharm Assoc., Scr Ed , 44 (rg55) 261.
* Sulfosalicyhc
aad -oxlnc-NaOH-NaOBr
or didcctyl-fl-naphthol
reactron.
Anal. Chim. Ada,
22 (1960) 452-457