Xenocoumacins
The instant application is a continuation-in-part of U.S. application Ser. No. 819,894, filed Jan. 16, 1986, which issued as U.S. Pat. No. 4,672,130 on June 9, 1987, and which is a continuation of U.S. application Ser. No. 623,969, now abandoned, filed June 25, 1984. 
    
    
     TECHNICAL FIELD 
     The present invention relates to a new class of compounds, known as xenocoumacins, which comounds may be isolated from the culture of strains of bacteria of the genus Xenorhabdus. 
     BACKGROUND ART 
     Insect pathogenic nematodes of families Heterorhabitidae and Steinernematidae are known to be symbiotically associated with bacteria of the genus Xenorhabdus. It has been observed that these bacteria have the ability to inhibit the activity of other bacterial growth. 
     International Application No. PCT/AU83/00156 (WO84/01775) discloses certain lipid soluble antibiotics isolated and characterised from cultures of the genus Xenorhabdus. The disclosures of that application are incorporated herein by reference. 
     DISCLOSURE OF INVENTION 
     The compounds of the present invention include those isolated from the water soluble component of the culture supernatant of Xenorhabdus nematophilus and Xenorhabdus luminescens and their derivatives and have been found to have the structure of fomula I. ##STR2## wherein 1 R 1  is hydrogen, straight or branched chain, alky or acyl 
     R 2  is straight or branched chain alkyl of at least 2 carbon atoms, unsubstituted or substituted by one or more substituents selected from hydroxy, acyl, acyloxy, halogen; 
     R 3  and R 4  are hydrogen, hydroxy, alkoxy or acyloxy 
     R 5  is hydrogen hydroxy, alkoxy or acyloxy; 
     A is --(CH 2 ) m  --B, wherein 
     m is 0, 1, 2 or 3 and 
     B is an amino-containing radical, unsubstituted or substituted by acyloxy; or ##STR3## taken together represents a 5 to 7 membered heterocyclic ring which may contain a further nitrogen, oxygen or sulfur heteroatom, 
     and the phamaceutically acceptable salts and other derivatives thereof. 
     Particularly preferred compounds of the present invention have the structure of formulae II and III below, and are designated xenocoumacin 1 and xenocoumacin 2 respectively. ##STR4## 
     The invention also provides a process for preparing xenocoumacin 1 and xenocoumacin 2 which comprises culturing an antibiotic producing strain of Xenorhabdus nematophilus, or Xenorhabdus luminescens, in a suitable culture medium and separating the compounds of the invention and their precursors from the culture broth. 
     Preferably, the process is a continuous process wherein culture medium is continuously added to the fermenter and culture medium is continually removed from the fermenter at a rate to maintain the volume of the culture within predetermined limits. The compounds of the invention and their precursors are separated from the collected culture. 
     The invention also provides for preparing compounds of formula 1 as defined above, which process comprises 
     (a) to produce compounds of formula 1 wherein 
     (i) R 1  is hydrogen, R 2  is 2-methylpropyl, R 3  and R 4  are hydroxy, R 5  is hydrogen, n is O and A is --(CH 2 ) m  --B wherein m is 3 and B is guanidino; and 
     (ii) R 1  is hydrogen, R 2  is 2-methylpropyl, R 3  and R 4  are hydroxy, R 5  is hydrogen, n is O and ##STR5## is a pyrrolidinyl ring, culturing a xenocoumacin producing strain of Xenorhabdus nematophilus or Xenorhabdus luminescens in a suitable culture medium and separating the desired compounds from the culture broth; 
     (b) to produce compounds of formula 1 wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , n, A, m and B are other than as defined under (a) 
     (i) oxidising a compound of formula 1 as defined above, wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , n, A, m and B are as defined under (a) to produce an aldehyde of the formula 2 ##STR6## (ii) reacting the compound of formula 2 with a phosphane of formula 3: ##STR7##  wherein Ar is aryl and R 4 , R 5 , and A are as defined above to produce a compound of formula 4: ##STR8## (iii) oxidising the compound of formula 4 to produce a compound of formula 1 in which R 3  is hydroxy; 
     (c) to produce compounds of formula 1 wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , n, A, m and B are as defined under (b), alkylating compound of formlua 1 as defined above wherein R 1  is hydrogen, R 3  and/or R 4  are hydroxy, to produce compounds of formula 1 as defined above wherein one or more of R 1 , R 3  and/or R 4  are alkyl; or 
     (d) to produce compounds of formula 1 wherein R 1 , R 2 , R 3  R 4 , R 5 , R 6 , n, A, m and B are as defined under (c), acylating a compound of formula 1 as defined above wherein R 1  is hydrogen, R 2  and/or R 4  is hydroxy, or R 5  is hydrogen to produce compounds of formula 1 as defined above wherein R 1  is acyl, one or more of R 3 , R 4  and R 5  is acyloxy and/or B is substituted by acyloxy. 
     In a further aspect of the invention provides pharmaceutical formulations characterised in that the active ingredient comprises a compound of the invention. 
     Xenocoumacins have been found to possess antibacterial, antifungal, acaricidal, anti-inflammatory and antiulcerogenic properties. The invention therefore also provides a method for the prevention or control of such conditions in a mammal requiring said prevention or control, which method comprises administering to said mammal a therapeutically effective amount of at least one compound of the invention. 
     Preferably, the compounds of the invention are isolated from cultures of Xenorhabdus using typical extraction techniques such as liquid chromotography and subsequent extraction by an organic or aqueous solvent. 
     Suitable culture media for the antibiotic strains of Xenorhabdus include materials containing suitable carbon and energy sources such as glucose or other carbohydrates, glycerol, or lipids, suitable nitrogen sources such as ammonia, urea, amino acids, peptides or proteins, appropriate quantities of inorganic nutrients such as phosphate, potassium, magnesium, calcium and trace elements, and preferably a source of vitamins and growth factors, e.g. yeast extract. 
     The continuous culture processes of the invention are preferably carried out between 23° C. and 37° C., most preferably at about 28° C. They are preferably carried out at a pH of between 6.3 and 7.5, most preferably about 6.8. In continuous culture, fresh culture medium is preferably added to give a dilution rate of between 0.01 hr -1  and 0.5 hr -1 , most preferably between 0.04 hr -1  and 0.1 hr -1 . 
    
    
     MODES FOR CARRYING OUT THE INVENTION 
     Notwithstanding other forms which may fall within the scope of the present invention, preferred forms will now be described with references to the following examples: 
     Examples 1. 
     Culture of Xenorhabdus 
     Xenorhabdus nematophilus strain All/1 (ATCC 53200) can exist in two morphologically different forms known as primary (1°) and secondary (2°) as reported by R. J. Akhurst in J. Gen. Micro, 121, 303-309, (1980). These forms can be distinguished by their gross cell morphology, the fact that only the primary forms elicit zenorhabdins and zenocoumacins, and by their colour when grown on 0.004% triphenyl tetrazolium chloride and 0.0025% bromothymol blue, as secondary colonies appear red since they do not absorb bromothymol blue whereas colonies of the primary form have a red core overlaid by blue. 
     Culture 1 
     X. nametophilus pk strain A11/1 (ATCC 53200) (symbiont of Steinernema feltiae A11) was cultured in the following medium (5L) for 48 hr. 
     glycerol 5 gL -1  ; yeast extract 15 gL -1  ; MgSO 4  5 mL -1  (1M); (NH 4 ) 2  SO 4  2 gL -1  ; KH 2  PO 4  5 mlL -1  (1M); K 2  HPO 4  5 mlL -1  (1M); Na 2  SO 4  10 mlL -1  (1M). 
     The cells were harvested by centrifugation (9000 RMP, 0.25 h) and the supernatant was decanted. A sintered Pyrex funnel was uniformly packed with dry octadecyl silica (50-70 μm, 6 cm×12.5 cm) and covered with filter paper. The solvent flow through the silica was induced by a vacuum (10 kPa) and the eluate collected in a filter flask. The silica was washed with methanol (1L), water (4L) and then the supernatant (5L) was applied followed by water (2L) and acetonitrile: ammonium acetate (1:1) (0.2M), (pH4.5,2L). This latter fraction was evaporated in vacuo to yield a crude mixture of zenocoumacins (3%, 21g). 
     A solution of this mixture 5g) in water (20 ml) was chromatographed on Sephadex G10 (84×5 cm, 1650 ml) in aqueous acetic acid (0.5%) at a flow rate of 3.2 ml min -1 . The eluate was monitored continuously at 254 nm and absorbances corresponding to xenocoumacin I and II occurred at 1150-1400 ml and 1300-1600 ml respectively. A total of 3.7 g (20%) of xenocoumacins was recovered as a brown solid. 
     Culture 2 
     The following medium is found to be suitable for the culture of Xenorhabdus nematophilus ATCC 39497. 
     Glycerol 20 gL -1  ; yeast extract 10 gL -1  ; (NH 4 ) 2  SO 4 , 20 gL -1  ; KH 2  PO 4 , 10 gL -1  ; MgSO 4 .7H 2  O, 2.5 gl -1  ; CaCl 2 .2H 2  O.0.29 gL FeSO   4 .7H 2  O, 27.8 mgL -1  ; MnSO 4 .H 2  O, 8.45 mgL -1  ; ZnSO 4 .7H 2  O, 14.4 mgL -1  ; CoClhd 2.6H 2  O, 0.10 mgL -1  ; and CuSo 4 .5H 2  O, 0.19 mgL -1 . 
     The medium was innoculated and growth proceeded for 6 days. 
     The supernatant (136L) was applied to a column (150 mm by 1 m) of Amberlite XAD-2 resin (17.1L) at 500 ml.min -1 . After washing the column with 20 L of water, methanol was pumped onto the column at a rate of 500 ml/min and the eluent collected in 20 L aliquots. 
     500 ml of the first aqueous methanolic fraction was lyophilised, extracted with ethylacetate (3×200 ml) and then chromatographed on Sephadex G25 in water. 
     The column (2.5 cm×76 cm) was washed thoroughly with water, and then eluted with aqueous acetic acid (10% V/V). The eluent was lyophilised. 
     Substantially pure xenocoumacin 1 was obtained by subjecting the acetic acid fraction to HPLC on a Whatman Partisil M9 10/50 ODS column with acetonitrile: ammonium acetate (0.2 M,pH4.5) 30:70 as eluant. At 4 ml/min on a 254 mm column the retention time was 19 minutes for xenocoumacin I. 
     Culture 3 
     High yields of zenocoumacins 1 and 2 can be extracted from the supernatant of cultures of Xenorhabdus nematophilus strain All/1 grown on tryptone soya broth as shown in Table 1. 
     
                       TABLE 1                                                     
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Yields of Xenocoumacins During Fermentation                               
Time (hrs)   Concentration mgL.sup.-1                                     
from inoculation                                                          
             Xenocoumacin I                                               
                          Xenocoumacin II                                 
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  4.5         8.7         0.3                                             
23           52.6         40.8                                            
  28.5       72.5         89.4                                            
27           72.2         115.9                                           
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     Example 2. Recovery of Xenocoumacins 
     The raw xenocoumacins recovered by the process of Example 1 were subjected to preparative, isocratic phase HPLC on a Whatman Partisil 10 ODS column using 0.2 M acetonitrile:ammonium acetate (pH 4.5) in the ratio 40:60 as the mobile phase delivered by 12 ml/min. The compounds of formulae II and III, xenocoumacin 1 and xenocoumacin 2 respectively were eluted at 26-30 min and 32-36 min respectively. 
     Various compounds within the scope of the present invention were isolated as derivatives of xenocoumacin 1 and xenocoumacin 2. NMR and mass spectra data follows: 
     Xenocoumacin 1 and 2  13  C NMR 100.62 MHz (D 2  O.) 
     Referenced to dioxan 67.8 ppm 
     
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Carbon     Xenocoumacin 1                                                 
                        Xenocoumacin 2                                    
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1          170.9        171.1                                             
3          82.5         82.6                                              
4          30.4         30.4                                              
4a         141.2        141.2                                             
5          120.5        120.4                                             
6          138.3        138.3                                             
7          116.8        116.9                                             
8          161.4        161.6                                             
8a         109.1        109.3                                             
1&#39;         22.2         22.2                                              
2&#39;         23.6         23.8                                              
3&#39;         25.5         25.6                                              
4&#39;         40.0         40.0                                              
5&#39;         50.9         50.8                                              
7&#39;         174.5        174.4                                             
8&#39;         73.7         74.4                                              
9&#39;         72.1         70.7                                              
10&#39;        55.1         62.8                                              
11&#39; (12&#39;)  25.7         25.8                                              
12&#39; (11&#39;)  26.0         24.6                                              
13&#39;        42.0         46.9                                              
15&#39;        158.00                                                         
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 ##STR9##
 
    
     Xenocoumacin 1 Hydrochloride 
     400 MHz  1  H spectrum (D 2  O) ref. Dioxan 3.70 wrt TMS. 8.22, 0.90, 2×d, J6.6 Hz, 1&#39;-2&#39;-Me; 1.41. q of d, J4a&#39;,4&#39;b 13.2 Hz J4&#39;a,3&#39;7.2 Hz, J4&#39;a,5&#39; 4.0 Hz, H4&#39;a; 1.58, m, H3&#39;; 1.64, m, H12&#39;a; 1.70, m, H4&#39;b; 1.74, m, H11&#39;a; 1.79, m, H12&#39;b; 1.83, m, H11&#39;b; 2.96, m, (H4) 2  ; 3.19, t, J6.4 Hz, (H13) 2  ; 3.47, sextet, J10&#39;,9&#39;, 4 Hz, J10&#39;11&#39;a 3.5 Hz, J10&#39;11&#39;b 8.4 Hz, H10&#39;; 4.11, dd, J9&#39;,10&#39; 4 Hz, J9&#39;,8&#39;, 6.1 Hz H9&#39;,4.20, dt, J5&#39;,4 &#39;a 4.0 Hz, J5&#39;,4&#39;b 9.8 Hz, J5&#39;,3 4.0 Hz, H5&#39;; 4.59, dt, J3,4a 8.1 Hz, J3,4b 4.5 Hz, J3,5&#39;4.0 Hz, H3; 6.8, 6.82, 2×d, J8 Hz, H5, H6; 7.45, t, J8 Hz, H6 ##STR10## 
     Xenocoumacin 1 Hexaacetate 
     400 MHz  1  H NMR CDCl 3  :C 6  D 6 , 3:2 referenced to TMS. 0.89, 0.91, 2×d, 1&#39;-,2&#39;-Me; 1.20, dq, H4&#39;a; 1.37, H11&#39;a; 1.5, m, H11b, (H12) 2  ; 1.66, m, H3&#39;; 1.81, m, H4&#39;b; 1.80, 1.85, 1.89,1.92, 2.10, 2.20, 6×s, OAc; 2.53, dd, J4a,4b 14.1 Hz J4a,3 2.8 Hz. H4a; 2.97, dd, J4a4b 14.1 Hz, J4b, 3 12.6 Hz, H4b; 3.39, q, J13.14 6 Hz, J13&#39;,12&#39;6 Hz, (H13&#39;)2; 4.09, dq, J3,4b 12.6 Hz, J3,4a 2.8 Hz, J3,5&#39;3.5 Hz, H3; 4.24, dt. J5&#39;.4&#39;a 9.1 Hz, J5&#39;,4&#39;b, 9.1 Hz, J5&#39;,3 3.5 Hz, H5&#39;; 4.43, qd, J10&#39;,9&#39;7.2 Hz, J10&#39;,11&#39;a 7.2 Hz, J10&#39;11&#39;b 2Hz, H10&#39;; 5.09, dd, J9&#39;,10&#39;7.2 Hz, J9&#39;,8&#39;1.7 Hz, H9&#39;; 5.32, d, J8&#39;,9&#39;1.7 Hz H8&#39;; 6.59, d, J8 Hz. 10&#39;NH; 6.84, 6.86, 2×d, J 8 Hz, H5, H7; 7.06, d. J 9 Hz, H6&#39;; 7.2, t, J 8 Hz, H7; 9.0, t, J 6 Hz, H14&#39;. ##STR11## 
     Xenocoumacin 2 Tetraacetate 
       1  H NMR 400 MHz (CDCl 3 ) referenced to TMS. 0.93, 0.97, 2×d, J 6.5 Hz, 1&#39;-,2&#39;-Me; 1.48, qd, J4&#39;a,4&#39;b 13.5 Hz, J4&#39;a,3&#39;8.5 Hz, J4&#39;a,5&#39;5.5 Hz, H4&#39;a; 1.63, dd (b), J 6 Hz, 13.5 Hz, H11&#39;a; 1.7, m, 3H, H3&#39;, H12&#39;a, H11&#39;b; 1.88, qd, J4&#39;b,4&#39;a 13.5 Hz, J4&#39;b,5&#39;10 Hz, J4&#39;b,3 5.5 Hz, H4; 1.95,s, OAc; 2.01, m, H12&#39;a; 2.10, 2.11, 2.40, 3 x s, OAc; 2.89, dd, J 16.7 Hz, 2.5 Hz, H4a; 3.30, dd. J 16.7, 13 Hz, H4b; 3,45, q(b), J gem  10 Hz, J13&#39;a, 12a, J13&#39;a,12b 10 Hz, H13&#39;a; 3.54, td, J gem  10 Hz, J13&#39;b,12a 3 Hz, J13&#39;b,12b 10 Hz, H13&#39;b; 4.34, m, H5&#39;; 4.51, dt, J 12.5, 1.5 Hz, H3; 4.56, m, H10&#39;; 4.14, dd, J9&#39;,10&#39;9.9 Hz, J9&#39;,8&#39;1.5 Hz, H9&#39;; 5.21, d, J8&#39;,9&#39;1.5 Hz, H8&#39;; 7.03, 7.12, 2 d, J 8 Hz, H5, H7; 7.52, t, J 8 Hz, H6; 8.69, d, J 8.5 Hz, H6&#39; ##STR12## 
     Xenoucoumacin 2 
       1  H NMR 400 MHZ (D 2  O) referenced to Dioxan 3.70. 0.82, d, J 6.5 Hz, 1&#39;-Me; 0.89, d, J 6.5 Hz, 2&#39;-Me; 1.39, qd, H4&#39;a; 1.55, m, H3&#39;; 1.70, m, H4&#39;b; 1.92, m, 2H, H11&#39;a, H12&#39;a; 2.06, m, H12&#39;b; 2.1, m, H11&#39;b; 2.96, m, (H4) 2  ; 3.27, t, J 8 Hz, (H13&#39;) 2  ; 3.61, m, H10&#39;; 4.10, dd, H9&#39;; 4.16, m, H8&#39;, H5&#39;; 4.58, m, H3; 6.78, 6.81, 2 x d, J 8 Hz, H5, H7; 7.44, t, J 8 Hz, H6. ##STR13## 
     Xenocoumacin 2 Monoacetate 
       1  H NMR 400 MHz (CDCl 3  /D 2  O) referenced to TMS. 0.93, 0.96, 1&#39;-,2&#39;-Me; 1.50, qd, J 13.5, 8.6, 5 Hz, H4&#39;a; 1.68, m, H3&#39;; 1.79, m, H4&#39;b; 1.88, m, H12&#39;a; 1.91, m, H11&#39;a; 2.04, m, H12&#39;b 2.12, s, OAc; 2.27, m, H11&#39;b; 2.83, dd. J 16.7, 2.5 Hz, H4a; 3.11, dd, J 16.7, 13 Hz, H4b; 3.53, m, H13&#39;a, H13&#39;b; 3.70, dd, J 7.8, 4.4 Hz, H9&#39;; 3.90, d, J 7.8 Hz, H8&#39;; 4.21, m, H10&#39;; 4.35, m, H5&#39;; 4.60, dt, J 12.7, 2.4 Hz, H3; 6.71, 6.90, 2×d, H5, H7; 7.42, t, H 6; 7.9, d, 9Hz, H6&#39;; 10.8,×, 8-OH. 
     MASS SPECTRA 
     Xenoucoumacin 1 Hexaacetate 
     Negative Ion Chemical Ionisation (isobutane, gold probe, 200°) 
     m/z: 717M -  (25), 675 (52) (M-CH 2  CO), 657 (42), 615 (52), 403 (5), 3611 (20), 254 (10), 163 (38), 150 (100). 
     Electron Ionisation 
     70 EV, 200°, gold probe m/z: 717 M +  (2), 702 (4), 675 (15), 658 (10), 632 (10), 616 (10), 512 (20) 470 (15), 341 (5), 267 (28), 184 (90), 170 (28), 112 (30), 86 (50), 70 (100). Observed Mass 184.1076. Calculated for C 8  H 14  N 3  O 2  184.1086 
     Positive Ion Chemical Ionisation (ammonia, gold probe, 200°) 
     m/z: 718 (M +H, 75), 676 (MH-CH 2  CO, 100), 658 (15) 634 (10), 617 (12), 593 (10), 557 (7), 513 (15), 471 (12), 267 (22), 184 (40), 170 (15), 112 (15). 
     Xenocoumacin 2 Hexaacetate 
     Negative Ion Chemical Ionisation (isobutane, gold probe, 200°) 
     mz: 573 (M--H), 531 (M--CH 3  CO, 100), 514 (8), 454 (18), 412 (12), 394 (12), 265 (10), 222 (10), 163 (8), 130 (10). 
     Electron Ionisation (70 EV, gold probe, 200°) 
     m/z: 574 (M + , 
     10), 369 (10), 242 (11), 196 (10), 154 (12), 135 (5), 112 (35), 70 (100). 
     Positive Ion Chemical Ionisation (ammonia, gold probe, 200°) 
     m/z: 575 (M+H, 100) 533 (MH--CH 2  O, 12), 370 (10), 242 (8), 196 (12), 154 (8), 112 (15), 70 (17). 
     Xenoucoumacin 1 
     Fast Atom Bombardment: observed Mass 464.2574. 
     Theoretical Mass for C 22  H 34  N 5  O 6  (M--H) 464.2509. 
     Example 3. Production of Derivatives 
     The following synthetic pathways serve to illustrate how derivatives of xenocoumacins may be produced. ##STR14## Ar is an aryl and A,R 4  and R 5  are as defined above. As will be understood by those skilled in the art, the stereospecificity may be altered or controlled by altering the oxidant used, e.g. H 2  O 2  /SeO 2  will confer a different stereochemistry on the vicinal diol to OsO 4 . 
     R 1 , R 3  and R 4  of formula I may be varied by known processes of alkylation and acylation as per the following reactions: 
     (1) Alkylation ##STR15## wherein R is alkyl 
     (2) Acylation ##STR16## wherein R is alkyl. 
     Example 4. Antiulcerogenic activity 
     The antiulcerogenic activity of the xenocoumacins of the present invention was demonstrated by the following tests: 
     Test 1 
     Male Wistar rats were dosed perorally (p.o.) dosed with varying amounts of xenocoumacin 1. The animals were then exposed to ulcer inducing stress conditions, killed and examined for ulcer induction. The results were compared to a control using the Wilcoxon rank sum test. Significance is taken as p&lt;0.05. 
     Xenoucoumacin 1 was shown to be 74% effective when a dosage of 25 mg/kg.po was administered, but only 8% effective for a dosage of 10mg/kg.po. 
     Test 2 
     Test 1 was repeated using xenocoumacin 2, which was found to be 70% effective for a dosage of 25 mg/kg.po., 61% effective for a dosage of 10mg/kg.po and 26% effective for a dosage of 5mg/kg.po. 
     The xenocoumacins of the present invention may be used in the treatment of bacterial or fungal infections or in the treatment or prophylaxis of inflammatory diseases or ulcers. 
     The compounds or mixtures of them may be administered in standard pharmaceutical formulations, in admixture with known pharmaceutically acceptable excipients, adjuvants and diluents. Formulations of the invention may also contain other active substances. 
     Example A 
     Xenoucoumacins 1 and 2 can be used independently or in admixture as the active ingredient in the manufacture of the following pharmaceutical formulations: 
     
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(a)   Tablets            1 tablet contains                                
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Active ingredient    200 mg                                               
Microcrystalline cellulose                                                
                     155 mg                                               
Maize Starch         25 mg                                                
Talc                 25 mg                                                
Hydroxypropylmethylcellulose                                              
                     20 mg                                                
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(b)   Capsules           1 capsule contains                               
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Active substance     100 mg                                               
Maize starch         20 mg                                                
Lactose              95 mg                                                
Talc                 4.5 mg                                               
Magnesium Stearate   0.5 mg                                               
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     The xenocoumacins of the invention may also be administered topically or intravenously. 
     Preferred dosage rates are within the range of 0.1mg/kg to 50mg/kg when administered periorally, and 0.01 to 20 mg/kg when administered intravenously. Topical formulations can contain up to approximately 10% active substance.