CHEMICAL COMPOUND

This invention relates to a novel chemical compound, the application of the compound as an active component in a calcium sensitive sensor, and a calcium sensitive sensor containing the compound. More particularly, the compound is a derivative of calix[4]arene.

Calixarenes comprise a class of cyclic compounds pre- pared from p-alkylphenols and formaldehyde in the pres¬ ence of a catalytic amount of a base. Calixarenes are disclosed in Gutsche CD . Calixarenes . Ace Chem Res 1983 ; 16 : 161-70. The synthesis procedures for ca- lix[4]arene, calix[6]arene and calix[8]arene suggested by Gutsche CD are disclosed in Organic Synthesis 1989; 68 : 234-46.

Calix[4]arene is usually represented as follows:

or

or

or

and the systematic IUPAC term for calix[4]arene is:

pentacyclo[19,3,1,l ³ - ⁷ ,l ⁹ - ¹³ ,l ¹⁵ - ¹⁹ ]-octacosa-1(25) ,3,5,7 (28) ,9,11,13(27) ,15,17,19(26) ,21,23-dodecaene-25,26,27, 28-tetrol.

The ion binding properties of calixarenes have recently been recognized, see e.g. Arduini A et al . The prepara¬ tion of a new lipophilic sodium selective ether ester ligand derived from p-t-butylcalix[4]arene . Tetrahedron 1986; 42 : 2089-100 and Arduini A et al . p-t-butyl- calix[4]arene tetra-acetamide : a new strong receptor for alkali cations . J Inclu Phenom 1988; 6 : 119-34. The use of calixarenes in ion selective electrodes is dis¬ closed in the following scientific papers and patents:

Kimura K et al . Lipophilic calix[4]arenes ester and amide derivatives as neutral carriers for sodium ion- selective electrodes . Chem Lett 1988; 615-16;

Cadogan A et al . Sodium-selective polymeric membrane electrodes based on calix [ 4 ] arene ionophoreε . Analyst 1989; 114 : 1551-54;

Cunningham K et al . Sodium-selective poly (vinyl chlori- de) membrane ion-selective electrode based on a novel calix [4] arene ionophore . Analytical Proceedings 1991; 28 : 294-96;

Harris SJ et al . European Patent Application No . EP 0490631 . Ion selective electrodes; and

Shono et al . Japanese Patent Publication 1-250750 (1989) . Sodium ion-selective membrane electrode .

To particular applications optical ion selective sen¬ sors are preferred over ion selective electrodes. Opti¬ cal sensors based on calixarenes and/or the ion binding properties of calixarenes are disclosed in the follow¬ ing scientific papers:

Deng G et al . Light-responsive metal encapsulation in calix [4] arene . Chem Lett 1992; 1287-90;

Shimizu et al . Chromogenic calix [4] arene . Chem Lett 1991; 2147-50;

Kubo Y et al . New chromoionophores based on indoaniline dyes containing cali [4] arene . Tetrahedron Lett 1991; 32 : 7419-20;

Jin T et al . A fluorescent calix[4]arene as an intramo¬ lecular eximer- forming Na ⁺ sensor in nonaqueous solu¬ tion . J Chem Soc Chem Commun 1992 : 499-501;

McCarrick M et al . Novel chromogenic ligands for lit¬ hium and sodium based on calix[4] arene tetraesters . J Chem Soc Chem Commun 1992 : 1287-89;

King AM et al . A highly selective chromoionophore for potassium based upon a bridged calix [4] arene . J Chem Soc Chem Commun 1992 : 582-84 ; and

Kubo Y et al . Synthesis of a 1, 3 bis (indoaniline) -de¬ rived calix[4]arene as an optical sensor for calcium ion . J Chem Soc Chem Commun 1993 : 305-307.

The only published work so far dealing with a calcium sensitive calixarene based optical sensor is thus Kubo's above-mentioned 1993 paper.

From the data disclosed by Kubo it is obvious that the selectivity for calcium ions towards potassium and sodium ions is inadequate in case the optical sensor is to be used for measurement of physiological fluids such as blood, plasma, serum, etc.

Further, Kubo's calixarene compound cannot stand ster¬ ilization. The compound will be destroyed when subject¬ ed to radiation sterilization or ETO sterilization. Due to the fact that in some physiological applications, particularly the invasive application, it is essential to use sterilized sensors, sensors based on the calix¬ arene compounds of Kubo are unsuitable for these appli¬ cations.

It is an object of the present invention .to provide a novel calix[4]arene compound having improved selectivi¬ ty properties for calcium ions and being more stable during sterilization than present calcium sensitive calix[4]arene derivatives.

The object is accomplished by the cali ^' x[4]arene com¬ pound according to the invention, said compound being characterized by the general formula

wherein

X is -OH, -OR ¹ , -NR ² ₂ or morpholino.

whereby R ¹ is straight chain or branched alkyl of 1-22 C-atoms and R ² is straight chain or branched alkyl of 1-12 C-atoms, and

Z is -N=N-Ar, -CH=CH-Ar, -CH=CZ'Z" or

whereby either of Z ¹ and Z ² are selected from -H, -N0 ₂ , -CN, -CF ₃ , -SOR ³ , -S0 ₂ R ³ , -S0 ₂ OR ³ , -S0 ₂ NHR ³ , -S0 ₃ H, -COOR ³ , -C00NR ³ ₂ , -COONHR ³ , -COOH, -CHO, -COR ³ , -F, -Cl and -Br, R ³ is straight chain or branched alkyl of 1-4 C-atoms and both of Z ¹ and Z ² are not -H;

either of Y ¹ and Y ² are selected from =0, =N-CN and =C(CN) ₂ ; and

Ar is

(Substituted phenyl) ,

_R ll _R 12

(Substituted 1 - naphtyl) ,

_R 17 _R 18

(Substituted 2 -naphtyl ) or

_R 23

(4 - Pyridylium) ,

and R,R ⁵ ,...R ²² are each selected from -H, N0 ₂ ,

-CN , -CF ₃ , -F , -Cl , -Br , S0 ₂ R .24 -S0 ₂ CH ₂ CH ₂ OR ²⁵ , -S0 ₂ 0R ²⁴ , -S0 ₂ NHR ²⁴ , -S0 ₃ H, -COOR ²⁴ , -CONR ⁴ ₂ , -CONHR ²⁴ , -COOH , -CHO and -COR ²⁴ , wherein R ²⁴ is straight chain or branched alkyl of 1-4 C- atoms , and R ²⁵ is -H , -S0 ₃ H , -S0 ₃ Li , -S0 ₃ Na or -S0 ₃ K ,

with the proviso that when Z is -CH=CHAr and Ar is phenyl, at least one of the substituents R ⁴ ,R ⁵ ,...R ⁸ of the phenyl group must be different from H, and when Z is -CH=CHAr and Ar is 1-naph- thyl, at least one of the substituents R ⁹ ,R ¹⁰ , ...R ¹⁵ of the 1-naphtyl group must be different from H, and when Z is -CH=CHAr and Ar is 2-naphtyl, at least one of the substituents R ^l6 ,R ¹⁷ , —R ²² of the 2-napthyl group must be different from H.

Preferred compounds are compounds of the type (I) wherein Ar is a phenyl group having at least one sulph- oxylate substituent, particularly compounds of the general formula

(ID

wherein R ⁴ ,R ⁵ ,...R ⁸ are each selected from.-H and -S0 ₂ CH ₂ CH ₂ OR ²⁵ ; R ⁴ ,R ⁵ ,...R ⁸ not all being H, R ²⁵ has the meaning stated above, and X is -OH or -OR ¹ , whereby R ¹ has the meaning stated above.

Particularly preferred compounds are compounds of the type (II) wherein R ²⁵ is -S0 ₃ H, -S0 ₃ Li, -S0 ₃ Na or -S0 ₃ K, as said compounds are suitable for being bound cova- lently to polymers with available -OH groups, e.g. cel¬ lophane compounds.

Other preferred compounds are compounds of the type (I) wherein Ar is a phenyl group having a least one substi¬ tuent of the type -N0 ₂ , -CN, -Cl, particularly com¬ pounds of the general formula (II) mentioned above

wherein R ⁴ ,R ⁵ , ...R ⁸ are each selected from -H, -N0 ₂ , -CN and -Cl; R ⁴ ,R ^S ,...R ⁸ not all being H, and X is -OH or -OR ¹ , whereby R ¹ has the meaning stated above.

Particularly preferred compounds are compounds of the type (II) wherein at least one of the substituents R ⁴ ,R ⁵ , R ⁸ of the phenyl group is -N0 ₂ and the others

are -H, particularly 4-nitrophenyl and 2,.4-dinitro- phenyl.

The invention also relates to application of any of the compounds mentioned above of the general formulae (I) and (II) and the particularly preferred compounds men¬ tioned above as an active component in a calcium sensi¬ tive sensor.

The invention also relates to a calcium sensitive sen¬ sor having a calcium sensitive area containing an immo¬ bilized calcium sensitive active component, said calci¬ um sensitive sensor being characterized in that the calcium sensitive active component is a compound of the general formula

wherein

X is -OH, -OR ¹ , -NR ² ₂ or morpholino,

whereby R ¹ is straight chain or branched alkyl of 1-22 C-atoms and R ² is straight chain or branched alkyl of 1-12 C-atoms, and

Z is -N=N-Ar, -CH=CH-Ar, -CH=CZ'Z ² or

whereby either of Z ¹ and Z ² are selected from -H, -N0 ₂ , -CN, -CF ₃ , -SOR ³ , -S0 ₂ R ³ , -S0 ₂ OR ³ , -S0 ₂ NHR ³ , -S0 ₃ H, -COOR ³ , -COONR ³ ₂ , -COONHR ³ , -COOH, -CHO, -COR ³ , -F, -Cl and -Br, R ³ is straight chain or branched alkyl of 1-4 C-atoms and both of Z ¹ and Z ² are not -H;

either of Y ¹ and Y ² are selected from =0, =N-CN and =C(CN) ₂ ; and

Ar is

R ⁶

ΎV ^R7 (Substituted phenyl) ,

(Substituted l -nap tyl) ,

(Substituted 2 -naphtyl) or

(4 - Pyridylium) ,

and R ⁴ ,R ⁵ ,...R ²² are each selected from -H, N0 ₂ , -CN, -CF ₃ , -F, -Cl, -Br, -SOR ²⁴ , S0 ₂ R ²⁴ , -S0 ₂ CH ₂ CH ₂ OR ²⁵ , -S0 ₂ OR ²⁴ , -S0 ₂ NHR ²⁴ , -S0 ₃ H, -COOR ²⁴ , -CONR ⁴ ₂ , -CONHR ²⁴ , -COOH, -CHO and -COR ²⁴ , wherein R ²⁴ is straight chain or branched alkyl of 1-4 C- atoms, and R ²⁵ is -H, -S0 ₃ H, -S0 ₃ Li, -S0 ₃ Na or -S0 ₃ K,

with the proviso that when Z is -CH=CHAr and Ar is phenyl, at least one of the substituents

R ⁴ ,R ⁵ , R ⁸ of the phenyl group must be different from H, and when Z is -CH=CHAr and Ar is 1-naph- thyl, at least one of the substituents R ⁹ ,R ¹⁰ , R ¹⁵ of the 1-naphtyl group must be different from H, and when Z is -CH=CHAr and Ar is 2-naphtyl, at least one of the substituents R ¹⁶ ,R ¹⁷ , —R ²² of the 2-napthyl group must be different from H.

Particularly preferred calcium sensitive sensors con- tain as an active component any of the preferred com¬ pounds mentioned above.

The calcium sensitive area must be located such that it will contact the sample when using the sensor. Thus, the calcium sensitive area must be located on the sur¬ face of the sensor facing the sample.

For practical applications the calcium sensitive active component will most often be immobilized in a polymeric membrane.

To ensure good contact between a sample whose calcium content is to be determined and the calcium sensitive active component, the polymeric membrane is preferably a hydrophilic polymeric membrane, especially a membrane provided from one of the following compounds: cellu- loseacetate, cellophane, cuprophane, polyvinylacetate, polyhy _d roxyethylmethacrylate (poly-HEMA) or another hydrogel.

In another preferred embodiment the calcium sensitive area comprises a calcium permeable membrane, and the calcium sensitive active component is located in a compartment of the sensor adjacent the membrane.

The sensor may be constituted by a so-called dipping sensor, usually rod-shaped, the calcium sensitive area of which is located at one end of the sensor on the surface of the sensor facing the surroundings. The sensor may also constitute a part of a measuring cu- vette designed for containing a sample. In the latter case, the sensor will most often constitute a measuring cuvette wall part. The measuring cuvette may be de¬ signed for disposable use or may be provided as an integral component of an analyzer for the determination of the calcium content in samples, preferably physio¬ logical samples.

The invention will be further described by the follow¬ ing experiments and in connection with the drawing where:

Fig. 1 shows absorption spectra for a preferred ca- lix[4]arene compound according to the invention in the abscence of metal ions and with the addition of potas- sium, sodium and calcium ions;

Fig. 2 shows absorption spectra for the same calix- [4]arene compound having a varying content of calcium ions.

The spectra shown in Fig. 1 and Fig. 2 are recorded on an absorption spectrophotometer of the type Kontron UVIKON-860. Both figures show a spectrum of a solution of 5.5«10" ⁵ mol/L of compound (4) described below, i.e. 5,17-bis(4-nitrophenyldiazo)-26,28-dihydroxy-25,27- bis(ethoxycarbonylmethoxy)calix[4]arene in 96% etha- nol/tetrahydrofuran (2:1 v/v) . In the figure the com¬ pound (4) is designated "ligand". In Fig. 1 is also shown spectra of the same solution to which is added 6.67-10" ³ mol/L of sodium, potassium and calcium per- chlorate, respectively. It is seen that addition of calcium ions displaces the absorption peak by 100 nm from 397 nm to 497 nm, whereas addition of sodium and potassium ions only results in a negligible change of the absorption spectrum.

Fig. 2 shows, apart from the spectrum of the pure li¬ gand solution, spectra of the same solution to which is added varying quantities of calcium ions corresponding to calcium concentrations of 3.33'10 ^-0 ; 6.67«10 ^* °; 3.33-10" ⁵ ; 6.67 _' 10" ⁵ ; 3.33-10 ^"4 ; 6.67-10 ^"4 ; 3.33-10 ^"3 and

6.67-10" ³ mmol/L. As seen, the absorbance varies clearly with varying calcium concentrations at the absorption peak. Thus, it will be possible to establish a mathe¬ matical model or a standard curve from which the con- . tent of calcium ions in an unknown sample may be deter¬ mined.

Experimental

Qualitative determination of calcium with a glass sen¬ sor

2 mg of compound (6) described below, i.e. 5,17-bis-(2,4-dinitrophenyldiazo)-26,28-dihydroxy- 25,27-bis(hydroxycarbonylmethoxy)calix[4]arene, is added to a mixture of 2.3 L water, 2.5 mL methanol and 2.5 L tetramethoxysilane. 5 drops of 0.1 M KOH is added with stirring. The reaction mixture was left for 4 days in order to gel (formation of a glass) for 4 days in a beaker (6 cm diameter) and was then vacuum- dried for 2 hours at 40°C.

The glass formed was then washed thoroughly with dilut- ed HC1 and distilled water.

A piece of the glass was brought in contact with 0.1 M aqueous solutions of sodium chloride, potassium chlo¬ ride and calcium chloride. In the solution of calcium chloride the colour of the glass changed. In the solu¬ tions of sodium chloride and potassium chloride there was no visually detectable change of colour.

Preparation of calixf41arene compounds and inter edi- ates therefor

The compounds prepared are characterized by data for melting point, NMR, IR, by molecular weight determined by mass-spectrophotometry and by the result of a funda¬ mental analysis.

The melting points were measured by means of a digital thermometer.

NMR data were recorded on the following instruments: Bruker AM-100, Bruker AM-250 and Varian Unity 400 spec- trometer.

IR spectra were recorded using KBr technique on a Per- kin Elmer FT-IR 176OX spectrometer.

UV/Vis spectra were measured at room temperature on a Kontron UVIKON-860 and a Perkin Elmer Lambda-9. Some of the microanalyses differ more than one would normally accept. This is due to incomplete removal of small neutrale molecules included in the lipophilic cavity of calix[4]arene, e.g. solvent molecules like: CH ₂ C1 ₂ , EtOAc, toluene, etc.

25,27-dihydroxy-26,28-bis(ethoxycarbonylmethoxy) ca- lix[4] rene (1)

1 g (2.4 mmol) calix[4]arene, 0.33 g (2.4 mmol) anhy¬ drous K ₂ C0 ₃ and 0.79 g (0.53 mL; 4.7 mmol) ethylbromo- acetate are mixed together in a 100 mL roundbottom flask, and 50 L dry CH ₃ CN is added. The reaction mix¬ ture is heated to reflux for 18 h. The solvent is evap¬ orated and the residue is extracted with CH ₂ Cl ₂ /5% HC1. The organic layer is separated and dried with MgS0 ₄ . After evaporating the solvent the residue is triturated with MeOH and heated to boiling and cooled to 5°C, then filtered and washed with MeOH. Yield: 1.0 g (71%) . Melting point [176-177] °C ¹³ C NMR (CDC1 ₃ ) 14.19, 31.54, 61.42, 72.40, 119.16,

125.82, 128.22, 128.54, 129.20, 133.18, 152.40, 153.04,

169.90

Η NMR (CDC1 ₃ )5 (100 MHz) 1.35(t ,6H,J=7.20 Hz), 3.39

(d,4H,J=13 HZ) , 4.38(q,4H,J=7.20 Hz) , 4.48 (d,4H,J=13 HZ), 4.72(S,4H), 6.71-7.42 (m, 12H) , 7.62(s,2H) M ⁺ (e/z)=597

Anal. Calcd. for C ₃₆ H ₃₆ O _g (596, 36) : C,72.50; H,6.04 Found: C,72.67; H,6.26.

5,17-diformyl-26,28-dihydroxy-25,27-bis(ethoxycarbonyl- ethoxy)calix[4]arene (2)

2 g (3.4 mmol) 1 and 6.0 g (4.6 mL; 52 mmol) α,α-di- chloromethyl ethylether are dissolved in 100 mL CHC1 ₃ . 20 g (11.6 mL; 105 mmol) TiCl ₄ is added slowly from a dripping funnel while keeping the temperature below 30°C. The solution turns dark red and after 30-45 min¬ utes (followed by thin layer chromatography) at room temperature, the reaction mixture is quenched with 5% HCl/ice and extracted with 2 x 50 mL CH ₂ C1 ₂ . The organic phase is purple, probably due to formation of titanium complexes which can be decomposed by several extrac¬ tions with semi-concentrated HCl. The organic phase is dried with MgS0 ₄ , filtered, and the solvent is evapo- rated to give a yellowish compound. Yield: 2.5 g (100%) . Melting point: [180-182] °C

Η NMR (CDC1 ₃ ) (100 MHZ) 1.35(t,6H,J=7.20 Hz) , 3.50 (d,4H,J=13.0 HZ), 4.35(q,4H,J=7.20 Hz) , 4.45(d,4H, J=13.0 HZ), 4.71(s,4H), 6.75-7.25(m,6H) , 7.61(s,4H), 8.70(S,2H), 9.77(S,2H)

IR (KBr) : 1682 cm ^_1 (s,C=0 formyl) , 1752 cm ^"1 (s,C=0 ester), 3364 cm ^"1 (b,-OH) M ⁺ (m/e)=653 Anal. Calcd. for C ₃₈ H _3g O ₁₀ (654.38) : C,69.74; H,5.81 Found: C,65.03; H,5.50 (+ an uncombusted restl).

Diesterdiquinone (3)

2.1 g (4.7 mmol) T1(N0 ₃ ) ₃ '3H ₂ 0 is placed in a 500 mL flask under N ₂ and dissolved in a mixture of 150 mL absolute EtOH and 100 mL dry MeOH. A solution of 0.50 g (0.84 mmol) 1 in 50 L CHC1 ₃ is added quickly. The so¬ lution turns yellow immediately and after 2-3 minutes a precipitate is formed. Upon standing for 15-30 minutes with stirring followed by quenching with 20 mL H ₂ 0, 10%

HC1 is added dropwise until the precipitate is dis¬ solved. The reaction mixture is transferred to a sepa¬ ration funnel together with 100 mL CHC1 ₃ and 50 mL H ₂ 0. The organic phase is isolated and dried with Mg ₂ S0 ₄ and the solvent is evaporated. Purification is performed on silica with 2% MeOH in CH ₂ C1 ₂ as eluent, and the yellow band with a R _j =0.45 is collected. Yield: 0.340 g (66%) . Melting point [203-206] °C ¹³ C NMR (CDC1 ₃ ) 13.54, 29.84, 62.07, 70.64, 70.78,

124.93, 129.32, 129.77, 132.87, 147.38, 170.39, 186.76,

187.76

^> H NMR (CDC1 ₃ ) δ (250 MHz) 1.21(t,6H,J=7.1 Hz), 3.05

(d,4H,J=12.9 HZ) , 3.88(d,4H,J=12.9 HZ) , 4.02(s,4H), 4.25(q,4H,J=7.1 HZ) , 6.61(s,4H), 6.66(s,4H)

IR (KBr) : 1677 cm"'(s,C=0 quinone) , 1738 cm ^_1 (s,C=0 es¬ ter)

M ⁺ (m/e)=625 Anal. Calcd. for C ₃₆ H ₃₂ 0 ₁₍₎ T1C1 (864.18): C,50.03; H,3.70 Found: C,47.21; H,3.56 (+ an uncombusted rest!).

5,l7-bis(4-nitrophenyldiazo)-26,28-dihydroxy-25,27- bis(ethoxycarbonylmethoxy)calix[4]arene (4)

0.50 g (0.84 mmol) 1 is dissolved with stirring in 50 mL THF and 3 mL pyridine. The reaction mixture is cooled on ice. 0.58 g (2.45 mmol) 4-nitrophenyldiazoni- u tetrafluoroborat is added in small portions to en¬ sure that the temperature does not exceed 5°C. After stirring and cooling for 2 hours the temperature is allowed to rise to room temperature and the reaction is left for another 14 hours. The solvent is evaporated and the red solid is dissolved in 50 mL CH ₂ C1 ₂ and ex¬ tracted with 2 x 50 mL 5% HC1. The organic phase is dried with MgS0 ₄ , and the solvent is evaporated to give a red semi-solid. The solid is purified on a short

silica column with CH ₂ C1 ₂ as eluent and isolated as a foam after removing the solvent. The foam is dissolved in a small amount of CH ₂ C1 ₂ and precipitated with EtOH, filtered and washed with EtOH. The resulting substance is airdried.

Yield: 0.30 g (40%) .

Melting point [256-258] °C

¹³ C NMR (CDC1 ₃ ) 14.06, 31.33, 61.53, 72.38, 122.76,

124.59, 124.62, 125.87, 128.65, 129.55, 132.27, 145.67, 147.87, 152.13, 156.24, 157.84, 168.70

'H NMR (CDCI ₃ ) S (250 MHZ) 1.37(t,6H,J=7.2 Hz), 3.56 (d,4H,J=13.3 HZ), 4.37(q,4H,J=7.2 Hz), 4.52 (d.4H,J=13.3 HZ), 4.76(S,4H), 6.81(t,2H,J=7.5 Hz) , 7.03 (d,4H,J=7.5 Hz), 7.79(S,4H), 7.94(d,4H,J=9.0 HZ) , 8.34(d,4H,J=9.0 HZ), 8.58(S,2H)

IR (KBr) : 1522 cm" ¹ og 1343 cm ^"1 (s,-N0 ₂ ) , 1751 cm- ¹ (s,C=0 ester), 3392 CUT ¹ (b,-OH)

M ⁺ (m/e)=895

Anal. Calcd. for C ₄₈ H ₄₂ N ₆ O _l2 (894.48) : C, 64.45; H,4.70; N,9.39

Found: C,62.32; H _f 4.76; N,8.66.

5,17-bis(2,4-dinitrophenyldiazo)-26,28-dihydroxy-25,27- bis(ethoxycarbonylmethoxy)cali [ ]arene (5) 0.67 g (1.1 mmol) 3 is dissolved in a mixture of 20 mL CHCI ₃ and 20 mL MeOH. 1.0 g (2.5 mmol) 2,4-dinitrophe- nylhydrazine (50% in H ₂ 0) is dissolved in about 80 mL MeOH/CHCl ₃ and added with stirring to the solution of 3. Then the reaction mixture is heated at reflux for 2 hours and left for 14 hours at room temperature. The solution is filtered to give red crystals. The crystals are dissolved in a small amount of CHC1 ₃ and triturated with MeOH to give glistening crystals. Yield: 0.65 g (61%) . Melting point [254-256] °C

¹³ C NMR (CDCI ₃ ) 12.73, 29.73, 60.19, 71.13., 118.77, 119.09, 124.24, 124.85, 126.64, 127.69, 128.29, 131.16, 144.58, 145.52, 147.67, 150.93, 157.61, 167.27 Η NMR (CDCI ₃ ) δ (250 MHz) 1.38 (t, 6H,J=7.2 Hz) , 3.56(d,4H,J=13.3 Hz) , 4.37 (q,4H,J=7.2 Hz),

4.52(d,4H,13.3 Hz) , 4.76(S,4H) , 6.81(t,2H,J=7.5 Hz) , 7.03(d,4H,J=7.5 HZ) , 8.45(S,4H), 8.49 (d,4H,J=9.0 Hz) , 8.76(d,4H,J=9.0 HZ) , 8.84(S,2H) IR (KBr): 1346 cm" ¹ og 1535 cm ^-1 (s,-N0 ₂ ) , 1747 cm ^"1 (s,C=0 ester), 3401 cm ^_I (b,-OH) M ⁺ (m/e)=985

Anal. Calcd. for C ₄₈ H ₄₀ N ₈ O ₁₆ (984.48) : C,58.56; H,4.06; N,11.38 Found: C,57.69; H,3.85; N,11.12.

5,17-bis(2, -dinitrophenyldiazo)-26,28-dihydroxy-25,27- bis(hydroxycarbonylmethoxy) calix[4]arene (6) 0.10 g (0.1 mmol) 5 is dissolved in 20 mL EtOH and 10 mL H ₂ 0 and then heated to reflux. 0.07 g (0.6 mmol) potassium tert.butoxide is added and the reaction is refluxed for 30 minutes. After cooling to room tempera¬ ture 30 mL 5% HCl is added, and the reaction mixture is then cooled to 5°C. The red precipitate is collected by centrifugation and washed twice with H ₂ 0. The precipi- tate is transferred to a roundbottom flask with EtOH and the solvent is removed to give a red powder. Yield: 0.091 g (97%) . Melting point > 345°C IR (KBr): 1345 cm" ¹ og 1510 cm ^"1 (s,-N0 ₂ ) , 1730 cm ^'1 (s,C=0 acid), 3425 cm ^'I (b,-OH)

Anal. Calcd. for 0^ ₃ ^0, ₆ (928.44): C,56.92; H,3.45;

N,12.06

Found: C,54.73; H,3.49; N,11.40.

5,17-bis(l-dicyanovinylenindan-3-one) -26,28-dihydroxy- 25,27-bis(ethoxycarbonyl ethoxy) calix[4]arene (7) 0.36 g (0.55 mmol) 2 and 0.25 g (1.3 mmol) 1-dicyanovi- nylenindan-3-one are dissolved in 20 mL absolute EtOH with heating. The solution turns red and after 2 hours at reflux the reaction mixture is allowed to cool to room temperature, then the precipitate is filtered off and washed with EtOH. Yield: 0.45 g (80%) . Melting point [286-289] °C

¹³ C NMR (CDC1 ₃ ) 14.05, 31.10, 61.60, 72.39, 114.35, 114.57, 123.82, 124.80, 124.95, 125.63, 126.12, 126.29, 128.54, 129.56, 129.76, 130.86, 132.03, 134.39, 134.89, 136.99, 137.28, 139.44, 148.183, 151.80, 160.16, 162.83, 168.53, 190.72

*H NMR (CDCI ₃ ) δ (250 MHZ) 1.38 (t, 6H,J=7.1 Hz), 3.57(d,4H,J=13.4 Hz), 4.38 (q,4H,J=7.1 Hz) , 4.46 (d,4H,J=13.4 HZ), 4.75(S,4H), 6.89 (t,2H,J=7.7 Hz) , 7.10(d,4H,J=7.7 Hz) , 7.23 (t,2H,J ₀ =7.2 Hz, J _m =1.28 Hz), 7.76(t,2H,J ₀ =7.2 Hz, J _n =1.28 Hz), 7.93 (d,2H,J ₀ =7.2 Hz, J _m =1.28 Hz), 8.24(S,4H), 8.67 (d, 2H,J ₀ =7.2 Hz, J _m =1.28 HZ), 9.10(S,2H)

IR (KBr): 1704 cm- ¹ (s,C=0 indan) , 1747 cm'(s,C=0 ester), 2221 cm-'(m,CN), 3387 cm ^'l (b,-OH) M ⁺ (m/e)=1005

Anal. Calcd. for 0^ ₄₄ ^,0, ₀ (1004.62): C,74.12; H,4.38;

N,5.57

Found: C,72.29; H,4.48; N,4.88.