Disclosed are arylazo chromoionophores characterized by the formula: ##STR1## wherein X is hydrogen or a monovalent cation, Y is H or methoxy and R is a ringed aromatic organic structure which affects the optical absorption properties of the compounds. Also disclosed is a method for the detection of calcium ion using these compounds.

BACKGROUND OF THE INVENTION 
Calcium is one of the more important elements found in the body. It is 
necessary not only for the skeleton but also for cells. There is, on 
average, about one kilogram of calcium in the human body of which 99% is 
located in bone with the remaining 1% distributed in plasma, extracellular 
fluids and intercellular compartments. This small fraction, however, plays 
a vital role in many biochemical and physiological functions such as a 
cell regulator and messenger. These functions include bone formation and 
homostasis, maintenance of cell membrane integrity and permeability, nerve 
excitation, muscular contraction and blood coagulation together with 
regulation of many enzyme and hormone reactions. 
The concentration of calcium in body fluids, particularly in plasma, needs 
to be kept within a very narrow range. Its level is controlled by a number 
of hormones, primarily by parathyroid hormone (PTH), and calcitonin. PTH 
is released from the parathyroid gland in response to a decrease of 
calcium concentration in plasma and indirectly promotes calcium absorption 
in the intestine and renal tubules and increases the calcium mobilization 
from bone. Calcitonin, which inhibits PTH activity in bone tissue, is 
secreted by the thyroid gland in response to a rise in calcium ion. 
Deviations from normal calcium levels occur in certain diseases. Calcium 
levels significantly less than normal can be indicative of 
hypoparathyroidism, Vitamin D deficiency or nephritis. Calcium levels of 
greater than normal may indicate hyperparathyroidism, Vitamin D 
intoxication or myeloma. 
The normal value of total calcium in plasma is about 2.4 mM/L. Generally, 
infants have the highest calcium concentration which declines slightly 
with age. 
The determination of calcium in serum began with the gravimetric method in 
which calcium was precipitated with ammonium oxalate whereupon the 
precipitate was dried and weighed. This method was improved upon in 1921 
when there was reported a technique in which the calcium oxalate is 
dissolved in acid with the oxalate being determined by titration with 
potassium permanganate. A modification of this method, in which the 
washing procedure and temperature were standardized during the titration, 
was used as the primary procedure for calcium determination. While 
reasonably accurate, these procedures required large amounts of serum and 
were time consuming. A more sensitive and rapid complexometric titration 
was introduced in the 1940's which used murexide as an indicator. Several 
other indicators, e.g. calcon, calcein, methylthymol blue, eriochrome 
black T, glyoxal bis-(2-hydroxyanil) and arsenazo III, were subsequently 
introduced. Regardless of what indicator was used, these complexometric 
titration methods required a large volume of serum sample, were time 
consuming and suffered from a poor end-point as well as interferences by 
metal ions other than calcium. 
More recently, the titration method was replaced by a direct 
spectrophotometric method using various metallochromic indicators, the 
most popular of which is the ortho-cresolphthalein (CPC) complex method. 
In this method, calcium combines with CPC in an alkaline solution (pH 10.5 
to 12) to form a deep purple calcium-dye complex. The dye's absorbance 
increases at 575 nm and is proportional to the concentration of calcium in 
the sample. A disadvantage of this method is the requirement that it be 
carried out at a pH in the 10 to 12 range. At this pH level, the reagent 
can absorb carbon dioxide resulting in baseline drift. 
Arsenazo III forms colored complexes with many divalent and trivalent 
cations but can be used to determine micromolar quantities of calcium ion 
at pH 5.5 without significant interference from magnesium ion. This 
reagent has a high affinity for calcium ion at the physiological pH, a 
high extinction coefficient of the calcium-dye complex at 650 nm and 
exhibits high chemical stability in aqueous solutions. Accordingly it has 
become a useful tool for determining micromolar concentrations of calcium 
in single cells. While arsenazo III is widely used by researchers in 
studies of calcium transport in cells and cell fractions, its utility in 
clinical chemistry has been limited due to the presence of toxic arsenic 
moieties and their concomitant safety and environmental concerns. 
In Biochemistry 19, 2396 (1980) Tsien reports the preparation of 
2-[[2-bis(ethoxycarbonyl)methyl]amino]-quinoline(QUIN1) and its 6-methoxy 
analog (QUIN2). These compounds are described as having utility as 
fluorescent calcium ionophores. In a later publication, Tsien et al. 
describe monitoring the fluoroscence of QUIN2 as being the most popular 
method for measuring [Ca.sup.++ ]. They go on to point out that, while 
QUIN2 has revealed much important biological information, its use has some 
inherent limitations since its preferred excitation wavelength of 339 nm 
is too low. It is also pointed out that its extinction coefficient (&lt;5000) 
and fluorescence quantum yield (0.03 to 0.14) are also too low. In 
addition, autofluorescence from cells requires QUIN2 loadings of several 
tenths millimolar or more to obtain a satisfactory result. It is also 
pointed out that QUIN2 signals Ca.sup.++ by increasing its fluorescence 
intensity without much shift in either excitation or emission wavelengths 
and that there is a need for an indicator which responds to calcium by 
shifting wavelengths while maintaining strong fluorescence. Another 
deficiency reported for QUIN2 is that its selectivity for calcium over 
magnesium and heavy metal divalent cations could bear improvement. This 
article goes on to point out that compounds having a stilbene fluorophore 
and an octacoordinate, tetracarboxylate pattern of liganding groups 
characteristic of EGTA, [(ethylene glycol bis(.beta.-aminoethyl ether)] 
and BAPTA, [1,2-bis (o-aminophenoxy) ethanol-N,N,N'N'-tetraacetic acid] 
are preferable to QUIN2. This preference is based on several factors such 
as improved selectivity for Ca.sup.++ and the ability of BAPTA and EGTA 
to exhibit much stronger fluorescence together with wavelength shifts upon 
Ca.sup.++ binding. The preparation and utility of these compounds is also 
disclosed in U.S. Pat. 4,603,209 to Tsien et al. 
More recently Toner et al. have disclosed chromogenic derivatives of BAPTA 
and BAPTA like compounds in U.S. Pat. No. 4.795,712. They point out that 
the fluorogenic compounds of Tsien suffer from the disadvantage of 
adsorbing in the ultraviolet region of the spectrum, so that normal 
constituents of body fluids which also adsorb in the UV and short visible 
wavelengths tend to produce background interference with standard 
colorimetric equipment and procedures. They go on to say that it would be 
desirable to have highly selective calcium complexing compounds which 
would be detectable at longer wavelengths (above 400 nm) and would shift 
to other wavelengths when complexed with calcium to allow quantitative 
analysis for calcium without interference from UV and short wavelength 
visible light-absorbing species. 
The present invention is predicated on the discovery that arylazo 
derivatives of QUIN1 and QUIN2 can be effectively used for the 
colorimetric determination of Ca.sup.++ since they are highly selective 
for calcium in media which also contains magnesium ion. Furthermore, these 
compounds adsorb light at longer wavelengths than do similarly derivatized 
BAPTAs and exhibit a significantly greater shift in the maximum absorbance 
of the complexed -vs- non-complexed compound than do the corresponding 
chromogenic BAPTA compounds. 
SUMMARY OF THE INVENTION 
The present invention involves arylazo calcium chromoionophores 
characterized by formula A: 
##STR2## 
In the above formula, X is hydrogen or a monovalent cation, Y is H or 
methoxy and R is a five or six membered, substituted or unsubstituted 
aromatic or heteroaromatic ring or a fused ring system made up of five or 
six membered, substituted or unsubstituted, aromatic or heteroaromatic 
rings. 
Also included within the scope of the present invention is the use of these 
chromophores in the quantitative determination of calcium ion. 
DESCRIPTION OF THE INVENTION 
The synthesis of the chromoionophores of the present invention is 
illustrated by the following Scheme I in which the previously mentioned Y 
substituent is hydrogen. 
##STR3## 
Referring to Scheme I, calcium indicators 14-41 (Table 2) are prepared 
using either method 1 or 2 as mentioned herein. Method 1 involves coupling 
of an aromatic diazonium salt R-N.sub.2.sup.+ with 1 to afford 
arylazotetraester intermediates 2-11 (Table 1) followed by base hydrolysis 
to afford the calcium indicators 14 to 23 (Table 2). In the case of QUIN2, 
the starting material 1 will have a 6-methoxy group. 
TABLE 1 
______________________________________ 
Compound R Y 
______________________________________ 
##STR4## 
##STR5## H 
3 
##STR6## H 
4 
##STR7## H 
5 
##STR8## H 
6 
##STR9## H 
7 
##STR10## H 
8 
##STR11## H 
##STR12## 
9 
##STR13## H 
10 
##STR14## H 
11 
##STR15## H 
12 
##STR16## OCH.sub.3 
51 
##STR17## -- 
52 
##STR18## -- 
______________________________________ 
Method 2 first hydrolyses the esters of 1 to give 13 then couples this with 
the aromatic diazonium salt to afford indicators 24-41. Method 1 has an 
advantage since the arylazo-tetraester intermediates are highly 
crystalline and easily purified by simple recrystallization. Their base 
hydrolysis under preferred conditions, i.e. with a stoichiometric amount 
or slight excess of 4.0M KOH or LiOH in n-butyl alcohol, affords the 
calcium indicator compounds directly in a pure, easily collected and 
highly water soluble form which requires no additional purification. 
Method 2 has an advantage in connection with the synthesis of certain 
analogs labile to the base hydrolysis conditions. Some analogs can be made 
by either method and representative synthetic methods are given below. All 
starting materials are readily available to those skilled in the art of 
organic synthesis. 
Calcium indicator compounds 43-49 (Table 2) incorporate a 6-methoxy 
substituent into the quinolone ring and are known in the art as QUIN2 
compounds in contrast to those which are unsubstituted in the 6 position 
referred to as QUIN1 compounds. These may be prepared from commercially 
available QUIN2 free acid, compound 42, X=H, (from MTM Research Chemicals, 
Windham, N.H., USA) by reaction with an appropriate aromatic diazonium 
salt (R-N.sub.2.sup.+) using method 1. Alternatively, QUIN2 
tetraethylester, also available from MTM may be first coupled with an 
aromatic diazonium salt to give an arylazo QUIN2 tetraester (e.g. 12) 
which is hydrolyzed under basic conditions to give the calcium indicator 
compound (e.g. 48) by method 2 according to Scheme I': 
##STR19## 
The structure and wavelengths of maximum adsorption for compounds 14-41 and 
43-49 are set out in Table 2. 
TABLE 2 
__________________________________________________________________________ 
##STR20## 
.lambda..sub.max (nm) pH = 9 
Compound 
R Y X Method 
- Ca.sup.++ 
+ Ca.sup.++ 
__________________________________________________________________________ 
14 
##STR21## H K I 508 363 
15 
##STR22## H K I 510 370 
16 
##STR23## H K I 520 373 
17 
##STR24## H K I 532 365 
18 
##STR25## H K I 538 370 
19 
##STR26## H K I 544 383 
20 
##STR27## H K I 548 404 
21 
##STR28## H K I 560 387 
22 
##STR29## H K I 560 389 
23 
##STR30## H K I 580 414 
24 
##STR31## H H II 524 370 
25 
##STR32## H H II 546 380 
26 
##STR33## H H II 594 396 
27 
##STR34## H H II 584 378 
43 
##STR35## OCH.sub.3 
H II 508 404 
44 
##STR36## OCH.sub.3 
H II 510 410 
45 
##STR37## OCH.sub.3 
H II 516 410 
46 
##STR38## OCH.sub.3 
H II 518 404 
47 
##STR39## OCH.sub.3 
H II 528 -- 
48 
##STR40## OCH.sub.3 
K I 536 408 
49 
##STR41## OCH.sub.3 
H II 560 422 
28 
##STR42## H H II 460,560 
450 
29 
##STR43## H H II 575 432 
30 
##STR44## H H II 576 420 
31 
##STR45## H H II 577 422 
32 
##STR46## H H II 580 414 
33 
##STR47## H H II 580 430 
34 
##STR48## H H II 580 420 
35 
##STR49## H H II 585 430 
36 
##STR50## H H II 588 486 
37 
##STR51## H H II 590 426 
38 
##STR52## H H II 592 422 
39 
##STR53## H H II 595 428 
40 
##STR54## H H II 607 -- 
41 
##STR55## H H II 600 -- 
__________________________________________________________________________ 
Calcium indicating compounds such as 54.58 (Table 3) are arylazo 
derivatives of BAPTA as disclosed in previously mentioned U.S. Pat. No. 
4,795,712. These compounds can be prepared from BAPTA tetraester compounds 
50 (Y=5-CH.sub.3) by the method disclosed in J. Biol. Chem. 260, 3440 
(1985) or (Y=4-tert-C.sub.4 H.sub.9) by the method disclosed in said U.S. 
Pat. No. 4,795,712 as illustrated in Scheme II. 
##STR56## 
TABLE 3 
__________________________________________________________________________ 
.lambda..sub.max (nm) pH = 9 
Compound 
R Y X Method 
- Ca.sup.++ 
+ Ca.sup.++ 
__________________________________________________________________________ 
54 
##STR57## 
5-CH.sub.3 
LI 
I 468 366 
55 
##STR58## 
5-CH.sub.3 
LI 
I 506 382 
56 
##STR59## 
5-CH.sub.3 
H II 518 404 
57 
##STR60## 
5-CH.sub.3 
H II 540 388 
58 
##STR61## 
4-T-BU 
H II 576 424 
__________________________________________________________________________ 
The 5 arylazo-BAPTA analogs (54-58) were prepared in order to demonstrate 
the superior calcium indicating characteristics of the arylazo-QUIN 
compounds by direct comparison of BAPTA and QUIN analogs with the same 
substituted arylazo moieties. Table 4 summarizes the visible spectral data 
for the uncomplexed (-Ca.sup.++) and the metal-complexed (+Ca.sup.++) 
indicators in pH=9.0 borate buffer as further described under performance 
evaluation. 
TABLE 4 
__________________________________________________________________________ 
ARYLAZO-QUIN COMPOUNDS 
ARYLAZO-BAPTA COMPOUNDS 
.lambda..sub.max (nm) 
.lambda..sub.max (nm) 
.lambda..sub.max (nm) 
.lambda..sub.max (nm) 
ARYLAZO Compound 
pH = 9.0 
pH = 9.0 Compound 
pH = 9.0 
pH = 9.0 
MOIETY (R) No. - Ca.sup.++ 
+ Ca.sup.++ 
.DELTA..lambda..sub.Q (nm) 
No. - Ca.sup.++ 
+ Ca.sup.++ 
.DELTA..lambda..sub.B 
(nm) .DELTA..lambda..sub.Q 
- .DELTA..lambda..su 
b.B 
__________________________________________________________________________ 
(nm) 
##STR62## 14 508 363 145 54 468 366 102 43 
##STR63## 19 544 383 161 55 506 382 124 37 
##STR64## 20 548 404 144 56 518 404 114 30 
##STR65## 27 584 378 206 57 540 388 152 54 
##STR66## 26 594 396 198 58 576 424 152 46 
__________________________________________________________________________ 
Referring to Table 4, it is important to note that the arylazo-QUIN 
compounds exhibit a greater shift in their absorption maxima 
(.DELTA..lambda..sub.max) ) upon complexation with Ca.sup.++ than do the 
corresponding arylazo BAPTA compounds of the prior art. For the five pairs 
of compounds set out in Table 4, the increased spectral shift ranged from 
30 to 54 nm. 
One skilled in the art of dye chemistry might anticipate the 
.lambda..sub.max of an uncomplexed arylazo-QUIN compound to be at a longer 
wavelength than that of the corresponding arylazo-BAPTA analog due to the 
extended conjugation afforded by the quinoline ring system. However, one 
could not anticipate the .lambda..sub.max of the metal-complexed 
arylazo-QUIN compound to be at the same or shorter wavelength than the 
arylazo-BAPTA analog. This in part accounts for the increased spectral 
shift of the arylazo-QUIN indicators, which increase offers significant 
advantages when these compounds are used as indicators in diagnostic 
assays for calcium in biological fluids. 
It is unexpected that the arylazo-QUIN compounds are at all suitable for 
the determination of calcium in biological fluids, such as human blood or 
plasma, where calcium is present at high levels in a mixture containing 
other metal ions such as magnesium. Thus, in J. Biological Chem. 260, 3440 
(1985) Grynkeiwicz et al. state that "the high effective affinity of QUIN2 
for Ca.sup.++ is ideal for measuring levels . . . near 10.sup.-7, but also 
means that at the micromolar levels or above, the dye approaches 
saturation and loses resolution." In human blood, the levels of Ca.sup.++ 
can range from 1-20 mg/dl (2.5.times.10.sup.-4 to 5.times.10.sup.-3 M), 
and even with a 1:100 dilution of the sample on a typical clinical 
analyzer the final Ca.sup.++ concentration will still be 
2.5.times.10.sup.-6 to 5.times.10.sup.-5 M, which is well into the 
micromolar range. Unexpectedly, the arylazo-QUIN compounds of the present 
invention exhibit a linear response to calcium in serum over the range of 
0-20 mg/dL with no loss of resolution when the sample is diluted 1:100 
into the analytical reagent solution containing the indicator. 
Referring again to the Grynkeiwicz et al. reference, they point out that 
"the selectivity for QUIN2 for calcium over magnesium could bear 
improvement." This is an important consideration since Mg.sup.++ levels in 
human serum are typically higher than are the calcium levels and can be as 
high as 2.93 mg/dL (1.2.times.10.sup.-3 M). Interference by Mg.sup.++ 
would limit the utility of the arylazo-QUIN indicators in medical 
diagnostics yet we have found no significant interference at levels more 
than 3-fold higher using reagents incorporating compounds such as 14. 
Grynkeiwicz et al. also note that "Greater selectivity for binding 
Ca.sup.2+ instead of Mg.sup.2+ is observed in related tetracarboxylate 
chelators in which the rings are linked by ether linkages without any 
quinoline ring nitrogen." Contrary to the doubts raised by this reference, 
we have discovered the arylazo-QUIN compounds to be very useful indicators 
which offer substantial advantages over prior art compounds for measuring 
calcium in biological samples. As noted above, the arylazo-QUIN compounds 
of the present invention can be illustrated by general formula A in which 
Y is hydrogen or methoxy, and X represents hydroge or a monovalent cation, 
e.g. lithium, sodium, or potassium, with potassium being the preferred 
specie. The R moiety can be any of a wide variety of ringed aromatic 
organic structures unsubstituted or substituted with moieties such as, for 
example, alkyl, alkoxy, halo, cyano, nitro, aryl, heteroaryl, keto or 
mesyl which completes the structure of the azo dye and effects the optical 
absorption properties of the compounds of the present invention. Typical 
of R are: 
1. A six membered, substituted or unsubstituted carbocyclic aromatic ring. 
Examples of such six membered rings include 2-nitrophenyl; 
2-nitro-4fluorophenyl; 2-nitro-4-chlorophenyl; 
2-nitro-4trifluoromethylphenyl; 2-nitro-4-cyanophenyl; 4-nitrophenyl; 
2-fluoro-4-nitrophenyl; 2-chloro-4-nitrophenyl; 3-nitro-4-sulfophenyl; 
2,5-dichloro-4-(2'-sulfoethylsulfonamido)phenyl; 
2-methane-sulfonyl-4-nitrophenyl; 2,4-dinitrophenyl; 
2-nitro-4-fluorophenyl; 2-chloro-5-nitrophenyl; or 3,5-dinitrophenyl. 
2. A five or six membered, substituted or unsubstituted heteroaromatic 
ring, for example, 2-thiazolyl; 4-methyl-2-thiazolyl; 4-phenyl-2thiazolyl; 
4,5-dimethyl-2-thiazolyl; 4-phenyl-2thiazolyl; 5-nitro-2-thiazolyl; 
5-bromo-2-thiazolyl; 4-carboxymethyl-2-thiazolyl; 
5-nitrophenylsulfonyl-2-thiazolyl; 2-pyridyl; 4,6-dimethyl-2-pyridyl; 
5-chloro-2-pyridyl; 5-bromo-2-pyridyl; 3-methyl-2pyridyl; 
5-bromo-3-nitro-2-pyridyl; 3-chloro-5-trifluoromethyl-2-pyridyl; 
3,5-dichloro-2-pyridyl; 3-nitro-2-pyridyl; 4-pyridyl; 
2,5,6-trifluoro-3-chloro-4-pyridyl; 2-methoxy-5-pyridyl; 
2,6-dimethoxy-3-pyridyl; 5-nitro-2-pyrimidinyl; 4-methyl-2-pyrimidinyl; 
4,6-dimethyl-2-pyrimidinyl; 4,6-dimethoxy-2-pyrimidinyl; 
4-chloro-6-methyl-2-pyrimidinyl; 5-methyl-3-isoxazolyl; 
3-methyl-5-isoxazolyl; 3-methyl-5-isothiazolyl; 1-ethyl-5-pyrazolyl; 
2-(1,3,4-thiadiazolyl); 5-ethyl-2-(1,3,4-thiadiazolyl) or 
3-phenyl-5-(1,2,4-thiadiazolyl). 
3. A fused ring system made up of five or six membered, substituted or 
unsubstituted, aromatic or heteroaromatic rings, for example, 
4-trifluoromethyl-6-chloro-2-benzothiazolyl; 1-napthyl; 
6-(2'-hydroxyethyloxy)-2-benzothiazolyl; 6-tertbutyl-2-benzothiazolyl; 
4-methyl-5-chloro-2-benzothiazolyl; 4,5-dimethyl-2-benzothiazolyl; 
2-benzothiazolyl; 5-fluoro-2-benzothiazolyl; 6-sulfo-2-benzothiazolyl; 
5,6-dichloro-2benzothiazolyl; 2-.beta.-naphthothiazolyl; 
4-bromo-6-chloro-2-benzothiazolyl; 4,5-dichloro-2-benzothiazolyl; 
6-nitro-2-benzothiazolyl; 4,5,6,7-tetrachloro-2-benzothiazolyl; 
1-isoquinolinyl; 5-isoquinolinyl; 6-nitro-5-quinolinyl; 
5-chloro-2-benzoxazolyl; 5,6-dimethyl-2-benzothiazolyl; 
6-ethoxy-2-benzothiazolyl; 6-fluoro-2-benzothiazolyl; 
4-methoxy-2-benzothiazolyl; 6-methoxy-2-benzothiazolyl; 
4-methyl-2-benzothiazolyl; or 6-methyl-2-benzothiazolyl. 
The present invention is further illustrated by the following examples: