Platinum complex
This is a division of application Ser. No. 941,559 filed Sept. 12, 1978. 
    
    
     BACKGROUND OF THE INVENTION 
     The present invention relates to new platinum (II) complexes. 
     Heretofore, many reports have been made about platinum (II) complexes and the anti-tumor activity thereof, e.g. [Chem. Biol. Interaction, vol. 5, 415-424 (1972), Bioinorg. Chem. vol. 2, 187-210 (1973). Res. Commun. Chem. Pathol. Pharacol. vol. 7, 529-538, etc.]. The present inventors, while studying cis-platinum (II) complexes of 1,2-diamino-cyclohexane (hereinafter referred to as &#34;dac&#34;), succeeded to resolve dac, which is used as a starting compound, into three isomers, i.e., cis-, trans-l- and trans-d-; synthesized cis-platinum (II) complexes using these isomers and found that these complexes have anti-tumor activity. These complexes, their synthesis and utility are disclosed in U.S. Patent Application Ser. No. 775,216 filed on Mar. 7, 1977, now abandoned. 
     It has now been found that various other cis-platinum (II) complexes may be synthesized using the three isomers of dac as a starting compound; and these complexes exhibit strong anti-tumor activity. 
     SUMMARY OF THE INVENTION 
     In accordance with the present invention, novel platinum (II) complexes represented by the general formula (I) ##STR1## wherein R 1  and R 2  are the same and represent Br - , I - , NO 3   - , BrCH 2  CO 2   - , or ##STR2## and the stereoisomerism of 1,2-diaminocyclohexane is cis-, trans-l- or trans-d-. The complexes of the present invention have anti-tumor activity. 
     DESCRIPTION OF THE INVENTION 
     The novel complexes of the invention are prepared by applying known methods, such as the methods disclosed in Journal of Pharmaceutical Sciences 65, 315-328 (1976). For example: 
     (1) Pt(II)(dac)X 2  is prepared by reacting K 2  PtX 4  (X represents a halogen) with dac. 
     (2) Pt(II)(dac)I 2  and Pt(II)(dac)Br 2  are prepared by reacting Pt(II)(dac)Cl 2 , which is obtained by the method (1), with KI and KBr respectively. 
     (3) Pt(II)(dac)(NO 3 ) 2  and Pt(II)(dac)SO 4  are prepared by reacting Pt(II)(dac)X 2  with silver nitrate and silver sulfate respectively. 
     (4) Pt(II)(dac)Br 2 , glucuronate of Pt(II)(dac) and bromoacetate of Pt(II)(dac) are prepared by reacting Pt(II)(dac)(NO 3 ) 2  obtained by method (3) with KBr, sodium glucuronate and bromoacetic acid respectively. 
     The above reactions are carried out in water, if necessary, with heating (about 90° C.) or under interception of light. The reactions are usually completed in 3 to 48 hours to form generally white to yellow precipitates. The precipitates are recrystallized from 0.1 N HCl, or the like, whereby the desired products may be obtained in crystalline form. 
     This method is also described in the above-indentified U.S. Patent Application Ser. No. 775,216, which description is expressly incorporated herein by reference. 
     Diamines used in the present invention are cis-trans-l- or trans-di- isomers of dac. The process for preparation of the three isomers of dac is described in the above-mentioned U.S. Patent Application Ser. No. 775,216 which description is also expressly incorporated herein. 
     Representative compounds of the cis-platinum (II) complex of the present invention and their elementary analysis values are set forth respectively in the following Tables 1 and 2. 
     
                       TABLE 1                                                     
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Compound                        Example                                   
No.     Compound                No.                                       
______________________________________                                    
1       cis-Pt(cis-dac)Br.sub.2 1                                         
2       cis-Pt(trans-d-dac)Br.sub.2                                       
                                1                                         
3       cis-Pt(trans-d-dac)Br.sub.2                                       
                                1                                         
4       cis-Pt(cis-dac)I.sub.2  2                                         
5       cis-Pt(trans-d-dac)I.sub.2                                        
                                2                                         
6       cis-Pt(trans-d-dac)I.sub.2                                        
                                2                                         
7       cis-Pt(cis-dac) (NO.sub.3).sub.2                                  
                                3                                         
8       cis-Pt(trans-d-dac) (NO.sub.3).sub.2                              
                                3                                         
9       cis-Pt(trans-d-dac) (NO.sub.3).sub.2                              
                                3                                         
10      cis-Pt(cis-dac) (BrCH.sub.2 CO.sub.2).sub.2                       
                                4                                         
11      cis-Pt(trans-d-dac) (BrCH.sub.2 CO.sub.2).sub.2                   
                                4                                         
12      cis-Pt(trans-d-dac) (BrCH.sub.2 CO.sub.2).sub.2                   
                                4                                         
13      cis-Pt(cis-dac)SO.sub.4 5                                         
14      cis-Pt(trans-d-dac)SO.sub.4                                       
                                5                                         
15      cis-Pt(trans-d-dac)SO.sub.4                                       
                                5                                         
16      cis-Pt(cis-dac) (D-GlucH)NO.sub.3 . 2H.sub.2 O                    
                                6                                         
17      cis-Pt(trans-d-dac) (D-GlucH)NO.sub.3 . 2H.sub.2 O                
                                6                                         
18      cis-Pt(trans-d-dac) (D-GlucH)NO.sub.3 . 2H.sub.2 O                
                                6                                         
19      cis-Pt(cis-dac) (D-GlucH).sub.2 . 3H.sub.2 O                      
                                7                                         
20      cis-Pt(trans-d-dac) (D-GlucH).sub.2 . 3H.sub.2 O                  
                                7                                         
21      cis-Pt(trans-d-dac) (D-GlucH).sub.2 . 3H.sub.2 O                  
                                7                                         
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     In the foregoing Table and elsewhere herein the following abbreviations are used: ##STR3## 
     
                       TABLE 2                                                     
______________________________________                                    
Elementary analysis                                                       
Compound Found (%)       Calculated (%)                                   
No.      C       H       N     C     H     N                              
______________________________________                                    
1        15.63   3.15    6.20                                             
2        15.40   2.93    6.17  15.36 3.01  5.97                           
3        15.42   2.80    5.98                                             
4        12.70   2.56    5.05                                             
5        12.69   2.49    5.09  12.80 2.51  4.97                           
6        12.84   2.54    5.10                                             
7        16.82   3.34    12.56                                            
8        16.93   3.39    12.54 16.63 3.26  12.93                          
9        16.79   3.30    12.68                                            
10       19.60   2.99    4.90                                             
11       19.59   3.10    4.98  20.52 3.10  4.79                           
12       19.76   3.03    4.99                                             
13       18.08   3.70    7.10                                             
14       17.93   3.67    6.79  17.78 3.48  6.91                           
15       17.42   3.65    6.90                                             
16       23.21   4.45    6.82                                             
17       23.45   4.72    6.95  24.00 4.53  7.00                           
18       23.18   4.60    7.03                                             
19       27.27   4.55    3.45                                             
20       28.25   4.67    3.53  28.84 5.11  3.74                           
21       27.80   4.60    3.52                                             
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     Cis-platinum (II) complexes of the present invention exhibit anti-tumor activity upon experimental tumors induced in mice, such as P388, S180A and L1210. 
     The cis-platinum (II) complexes also exhibit anti-microbial activity and are, therefore, also useful as disinfectants for cleaning and sterilizing surfaces and the like. 
     As chemotherapeutic agents, the cis-platinum (II) complexes of the invention may be administered orally, intra-muscularly or intravenously. They may be formulated as capsules, powders, pellets or injections in combination with various known pharmaceutical lubricants, binders and excipients. 
     Suitable dosage of the cis-platinum (II) complexes is about 1 to 400 mg/kg/day in animals. 
     The anti-tumor activity of the present cis-platinum (II) complexes is illustrated by the following representative experiments. 
    
    
     EXPERIMENT 1 
     In this experiment the activity against experimental tumor P388 in mice is tested. 
     Leukemia P388 cells were injected intraperitoneally to groups of CDF 1  mice (Number of transplanted cells was 10 6  per mouse); and on the first and fifth days after transplantation, the test compound was administered by intraperitoneal injection to the mice. The dosage and test compound are set forth in the following Tables 3 and 4. 
     The effect of the test compound was evaluated by means of extension of average survival period T/C % (average survival days of the groups administered with the test compound/average survival days of the control group). When the T/C % is equal to or more than 120%, the test compound is considered to be effective. The results are also shown in Tables 3 and 4. 
     
                       TABLE 3                                                     
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        Toxic    Optimum Dose                                             
                             MED                                          
Compound                                                                  
        Dose              T/C         T/C                                 
No.     (mg/kg)  (mg/kg)  (%)  (mg/kg)                                    
                                      (%)  T.I.                           
______________________________________                                    
1       50       25       190  1.56   126  16                             
2       100      25       238  ≦1.56                               
                                      144  ≧16                     
3       50       12.5     217  1.56   130  8                              
4       ≧200                                                       
                 25       141  6.25   129  4                              
5       ≧200                                                       
                 ≧100                                              
                          182  6.25   125  ≧16                     
6       ≧200                                                       
                 ≧100                                              
                          149  12.5   121  ≧8                      
7       ≧50                                                        
                 12.5     180  ≦1.56                               
                                      144  ≧8                      
8       25       6.25     187  ≦1.56                               
                                      147  ≧4                      
9       50       12.5     198  ≦1.56                               
                                      151  ≧8                      
10      100      50       175  6.25   140  8                              
11      100      25       198  ≦6.25                               
                                      154  ≧4                      
12      ≧200                                                       
                 ≧100                                              
                          190  ≦6.25                               
                                      143  ≧16                     
13      25       6.25     202  0.78   122  8                              
14      12.5     6.25     212  1.56   140  4                              
15      25       6.25     200  0.39   124  16                             
16      50       25       208  1.56   139  16                             
17      50       25       203  0.39   124  64                             
18      50       25       188  1.56   120  16                             
19      ≧200                                                       
                 100      191  6.25   134  ≧16                     
20      100      50       224  3.12   141  16                             
21      100      50       181  6.25   123  8                              
______________________________________                                    
 MED: The lowest dose where T/C % exceeds 120%                            
 T.I.: Therapeutic Index (Optimum Dose/MED)                               
 
    
     
                       TABLE 4                                                     
______________________________________                                    
Compound     Dose (mg/kg)                                                 
No.           25       12.5      6.25                                     
______________________________________                                    
1            190       188       167                                      
2            238       201       190                                      
3            90        217       203                                      
4            141       139       129                                      
5            159       146       125                                      
6            141       121       119                                      
7            108       180       171                                      
8            65        90        180                                      
9            173       198       162                                      
10           168       164       151                                      
11           198       163       150                                      
12           172       158       141                                      
13           60        134       202                                      
14           0         83        212                                      
15           0         93        200                                      
16           208       184       170                                      
17                     203       187                                      
18           188       180       147                                      
19           178       153       134                                      
20           189       176       147                                      
21           170       133       123                                      
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     EXPERIMENT 2 
     In this experiment the activity against experimental tumor ascites-type S180A in mice is tested. 
     Ascites-type sarcoma 180A were transplanted intraperitoneally to ddN mice. The test compound was then interperitoneally injected every day from one to five days. The resulting ascites in the mice were sampled to evaulate the anti-tumor activity of the test compound by means of the growth rate of the tumor, T/C % (groups administered with the test compound/control group). 
     The results are shown in Table 5 below wherein the designations &#34;+&#34;, &#34;++&#34; and &#34;+++&#34; mean 65-41%, 40-11% and 10-0% of the T/C % respectively and the term &#34;toxic&#34; is used in the case where all the mice died. 
     
                       TABLE 5                                                     
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Compound    Dose       T/C                                                
No.         (mg/kg)    (%)        Effect                                  
______________________________________                                    
            3          41         +                                       
            10         41         +                                       
2           3          29         ++                                      
3           3          22         ++                                      
7           3          10         +++                                     
            10         --         Toxic                                   
8           3          13         ++                                      
            10         --         Toxic                                   
9           1          13         ++                                      
            3          --         Toxic                                   
10          10         43         +                                       
11          3          30         ++                                      
12          10         25         ++                                      
13          3           7         +++                                     
            10         --         Toxic                                   
14          3           5         +++                                     
            10         --         Toxic                                   
15          3          --         Toxic                                   
            10         --         Toxic                                   
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     EXPERIMENT 3 
     In this experiment, the activity against experimental tumor Leukemia L-1210 in mice is tested. 
     Leukemia L-1210 cells were transplanted intraperitoneally (Number of transplanted cells was 10 6  per mouse) to CDF 1  mice. The test compound was then administered intraperitoneally to the mice on the first, fifth and ninth day after transplantation. The effect of the test compound was evaluated by means of extension of the average survival period T/C % (average survival days of the groups administered with the test compound/average survival days of the control group). When T/C % is equal to or more than 120%, the test compound is considered to be effective. The results are shown in Tables 6 and 7. 
     
                       TABLE 6                                                     
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Com-  Toxic    Optimum Dose                                               
pound Dose              T/C   MED                                         
No.   (mg/kg)  (mg/kg)  (%)   (mg/kg)                                     
                                     T/C (%)                              
                                            T.I.                          
______________________________________                                    
1     ≧50                                                          
               25       188   6.25   151    &gt;4                            
2     ≧50                                                          
               25       229(1)                                            
                              0.78   128    &gt;32                           
3     ≧50                                                          
               6.25     252   1.56   140    4                             
7     ≧25                                                          
               12.5     253(2)                                            
                              0.39   138    &gt;32                           
8     ≧25                                                          
               6.25     335(3)                                            
                              0.39   134    16                            
9     ≧25                                                          
               6.25     287(2)                                            
                              0.39   120    16                            
13     25      6.25     180   0.78   131    8                             
14    12.5     3.12     189   &lt;0.78  156    &gt;4                            
15    12.5     3.12     219   &lt;0.78  152    &gt;4                            
16     100     50       212(2)                                            
                              6.25   130    8                             
17    ≧100                                                         
               12.5     313(4)                                            
                              1.56   136    &gt;8                            
18     50      25       313(2)                                            
                              1.56   130    16                            
19     200     25       154   6.25   129    4                             
20     200     50       330(2)                                            
                              6.25   141    8                             
21     50      25       274(1)                                            
                              6.25   191    4                             
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                                           TABLE 7                                 
__________________________________________________________________________
Compound                                                                  
      Dose (mg/kg)                                                        
No.   100                                                                 
         50    25  12.5                                                   
                       6.25                                               
                           3.12                                           
                               1.56                                       
                                   0.78                                   
                                      0.39                                
__________________________________________________________________________
1     13 --   188  184 151 115 113 110                                    
                                      --                                  
2     -- --   229 (1)                                                     
                   140 176 151 130 128                                    
                                      --                                  
3     -- --   (216)                                                       
                   146 252 142 140 115                                    
                                      --                                  
7     -- --   --   253 (2)                                                
                       166 140 319 (3)                                    
                                   148                                    
                                      143                                 
8     -- --   --   331 (4)                                                
                       335 (3)                                            
                           231 (1)                                        
                               189 144                                    
                                      134                                 
9     -- --   --   269 (2)                                                
                       287 (2)                                            
                           186 186 (1)                                    
                                   130                                    
                                      120                                 
13    -- --   --    93 (180)                                              
                           146 156 131                                    
                                      13                                  
14    -- --   --    0  (165)                                              
                           189 152 156                                    
                                      --                                  
15    -- --   --    0  197 219 167 152                                    
                                      --                                  
16    -- (212) (2)                                                        
              196  148 130 113 110 -- --                                  
17    -- (129) (1)                                                        
              284 (3)                                                     
                   313 (4)                                                
                       198 167 136 -- --                                  
18    -- 79   (313) (2)                                                   
                   221 218 (1)                                            
                           154 130 -- --                                  
19    90 139  154  125 129 --  --  -- --                                  
20    98 330 (2)                                                          
              269 (1)                                                     
                   213 (1)                                                
                       141 114 --  -- --                                  
21    74 83   274 (1)                                                     
                   212 (1)                                                
                       191 (1)                                            
                           --  --  -- --                                  
__________________________________________________________________________
 MED: Minimum Effective Dose, the lowest dose when T/C % exceeds 120%     
 T.I.: Therapeutic Index (Optimum                                         
 Figures in parenthesis are toxic by the decrease of body weight.         
 Number in parenthesis, right hand side below, indicates the survivor to 6
 mice                                                                     
 
    
     The following representative examples illustrate preparation of cis-platinum (II) complexes of the present invention. 
     EXAMPLE 1 
     Cis-dibromo(1,2-diaminocyclohexane) platinum(II) complex 
     In this example, 0.43 g (1 mmol) of [Pt(dac)(NO 3 ) 2  ] (dac is a cis-, trans-l- or trans-d-isomer) is dissolved in 20 ml of water with heating (about 90° C.) and then 1 g (8.4 mmol) of KBr is added to the solution. The mixture is stirred at room temperature to form a yellow precipitate. The reaction proceeds almost quantitatively and each platinum complex of isomers is obtained in a 90% yield. 
     EXAMPLE 2 
     Cis-diiodo(1,2-diaminocyclohexane) platinum(II) complex 
     In this example, 0.56 g (1.3 mmol) of K 2  [PtCl 4  ] is dissolved in 20 ml of water and to the solution there is added 2 g (12 mmol) of KI. The mixture is heated at 60° C. for 3 hours. To the resultant deep dark red solution 0.26 g (1.2 mmol) of cis-dac (H 2  SO 4 ), or 0.22 g (1.2 mmol) of trans-l-dac (HCl) 2  or trans-d-dac (HCl) 2  is added. Then 5% (w/v) KOH solution is added to make the pH neutral to weakly alkaline and to form yellowish brown precipitates. The precipitate is then separated, washed with water and acetone and dried. 
     Yield: 85% (each complex of isomers) 
     EXAMPLE 3 
     Cis-dinitrato(1,2-diaminocyclohexane) platinum(II) complex 
     In this example, 1.14 g (3 mmol) of [Pt(dac)Cl 2  ] (dac is a cis-, trans-l- or trans-d-isomer) is suspended in 100 ml of water and to the suspension is added 1.02 g (3 mmol) of AgNO 3 . The mixture is stirred at room temperature for 2 days under interception of light and the resultant AgCl is removed by filtration. Then, 5% (w/v) KCl solution is added to the filtrate little by little to remove unreacted AgNO 3 . The solution free of Ag +  is dried with heating under reduced pressure using an evaporator whereby the desired complexes are obtained as a yellowish white powder. 
     Yield: 80% (trans-l- or trans-d-complex) and 85% (cis-complex). 
     The [Pt(dac)Cl 2  ] used as a starting compound is prepared as follows: 
     An aqueous solution of 5 g of dac (a cis-, trans-l- or trans-d-isomer) and 18 g of K 2  (PtCl 4 ) is allowed to react at room temperature for about 12 hours to precipitate yellow needle-like crystals. The crystals are obtained by filtration and recrystallized from 0.1 N HCl. 
     Yield: 73% (cis-complex) and 78% (trans-l- or trans-d-complex). 
     EXAMPLE 4 
     Cis-bisbromoacetato(1,2-diaminocyclohexane) platinum(II) complex 
     In this example, 0.43 g (1 mmol) of [Pt(dac)(NO 3 ) 2  ] (dac is a cis-, trans-l- or trans-d-isomer) is dissolved in water with heating and the solution is cooled in a refrigerator. Then, 1 g (7.2 mmol) of bromoacetic acid is added to the solution and the mixture is allowed to stand for 3 days under interception of light in the refrigerator. A white precipitate formed in the mixture is separated by filtration, washed with water and acetone and dried under reduced pressure at 30°-40° C. 
     Yield: 40% (cis-complex) and 45% (trans-l- or trans-d-complex). 
     EXAMPLE 5 
     Cis-sulfato(1,2-diaminocyclohexane) platinum(II) complex 
     In this example, 0.38 g (1 mmol) of [Pt(dac)Cl 2  ] (dac is a cis-, trans-l- or trans-d-isomer) is added to a solution of 0.35 g of silver sulfate in 100 ml of water. The mixture is stirred at room temperature for 2 days under interception of light. AgCl formed is removed by filtration and to the filtrate there is added 5% (w/v) KCl solution, little by little, to remove Ag + . The filtrate is dried at 50°-60° C. under reduced pressure using an evaporator whereby the desired sulfato-complex is obtained as a yellowish white powder. 
     Yield: 80% (cis-complex) and 75% (trans-l- or trans-d-complex). 
     EXAMPLE 6 
     Cis-nitrato-D-glucuronato(1,2-diaminocyclohexane) platinum(II) complex 
     In this example, 0.22 g (0.5 mmol) of [Pt(dac)(NO 3 ) 2  ] (dac is a cis-, trans-l- or trans-d-isomer) is dissolved in 10 ml of water with heating and the solution is cooled to room temperature. Then, 0.12 g (0.5 mmol) of sodium glucuronate is added to the solution and the mixture is allowed to stand at room temperature for one week. The solution is then dried at 50°-60° C. under reduced pressure using an evaporator to obtain a yellow residue. The residue is washed with warm methanol to obtain the desired product. 
     Yield: 75% (cis-complex) and 70% (trans-l- or trans-d-complex). 
     EXAMPLE 7 
     Cis-bis-D-glucuronato(1,2-diaminocyclohexane) platinum(II) complex 
     In this example, 0.22 g (0.5 mmol) of [Pt(dac)(NO 3 ) 2  ] (dac is a cis-, trans-l- or trans-d-isomer) is dissolved in 10 ml of water with heating. After the solution is cooled to room temperature, 0.24 g (1 mmol) of sodium glucuronate is added thereto and the mixture is allowed to stand at room temperature for a week. The resultant solution is dried at 50°-60° C. under reduced pressure using an evaporator to obtain a yellow powder which is then washed with warm methanol and dried to obtain the desired product. 
     Yield: 70% (cis-, trans-l- or trans-d-complex). 
     Characteristic points of the infrared absorption spectra of the cis-platinum (II) complexes obtained in the above Examples are explained below. 
     Pt(dac)X 2  : X=Br - , I -   
     Trans-dac in platinum complexes exhibits peaks due to ν NH 2  at 3280, 3200 and 3020 cm -1 . Cis-dac exhibits a peak due to ν NH 2  (asym) at 3250 cm -1 , which is a lower wave number shift than that of trans complexes, and exhibits two other peaks due to ν NH 2  at a wave number (i.e. 3200 and 3020 cm -1 ) the same as trans-dac. A peak due to δ NH 2  is observed at 1570 cm -1  in both isomers. 
     Geometrical isomers of dac in the complexes are distinguishable by the rocking mode of CH 2  which is observed at 900-1000 cm -1 . That is, only one absorption peak is observed in trans-dac, whereas three peaks are observed in cis-dac which is low in symmetry. 
     The above-mentioned spectrum characteristics of dac are applied in the following platinum complexes insofar as the peaks of dac do not overlap those of a leaving group. 
     Pt(dac)(NO 3 ) 2   
     Peaks due to ν NO 3  of coordinated NO 3   -  are observed at 1500, 1385, 1260-1300 and 990 cm -1 . 
     Geometrical isomers of dac are distinguishable in that the trans-isomer exhibits a weak absorption peak at 827 cm -1 , whereas the cis-isomer exhibits two weak but clear peaks at 915 and 940 cm -1 . There is not observed such difference as in halogeno-complexes in action of ν of NH 2  in 3000-4000 cm -1 . 
     Pt(dac)(SO 4 ) 
     Sulfato-complexes exhibit a broad absorption spectrum due to ν SO 4  at 1120, 1030 and 950 cm -1 . 
     Geometrical isomers of dac are poorly distinguished by spectrum at 900-1000 cm -1  because their peaks overlap that of ν SO 4 . 
     Pt(BrCH 2  CO 2 ) 2  (dac) 
     Existence of the monobromoacetate ion in complexes may be confirmed by the fact that a broad absorption band at 1620 and 1350 cm -1  is attributable to ν CO 2 . 
     Geometrical isomers of dac are distinguishable similarly as in halogeno-complexes by spectrum at 900-1000 cm -1 . 
     Pt(GlucH)NO 3  (dac) 
     Peaks due to ν NH 2  and ν OH are observed respectively at 3100-3250 cm -1  and 3300-3600 cm -1 . Peaks due to ν CO 2  (asym) and ν CO 2  (sym) are observed respectively at 1600 cm -1  and 1400 cm -1 . Peaks due to ν NO 3  are observed at 995, 1300 and 1500 cm -1 , whereby it is concluded that NO 3   -  is included in the complex. 
     Pt(GlucH) 2  (dac) 
     A broad absorption band is observed at 3200-3400 cm -1 , which is attributed to the overlap of ν OH(GlucH) with ν NH 2  (dac). Peaks due to ν CO 2  (asym) and ν CO 2  (sym) are observed respectively at 1610 cm -1  and 1400 cm -1 .