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Progress_in_Medicinal_Chemistry._7/0408700130/files/00001___89c5ce10e8b4fc7217efe8ec81b47b93.pdfProgress in

Medicinal Chemistry 7

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Progress_in_Medicinal_Chemistry._7/0408700130/files/00003___edaf7d434a89cfbc5eb57188d475a9e4.pdfProgress in MedicinaI Chemistry 7

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In BDFl mice qd 1-1 1, observed 1-12.

[360] (as well as all other anticancer agents). However, the relative effects of these drugs on the two tissues vary. For example, 6-mercaptopurine, 2-aminoadenine, and 6-chloropurine produce similar lesions in the intestinal epithelium when given in doses that cause bone-marrow depression; the effects of thioguanine are largely limited to the bone marrow, in which most haematopoietic elements are susceptible. 6-Methylpurine appears to selectively depress erythro- genesis. The xanthine oxidase oxidation products of purine and 2-chloroadenine crystallize in the renal tubules causing kidney damage [360]. Hepatic damage occurs with many analogues, but is particularly prominent with Caminopyrazolo [3, 4-dlpyrimidine [361]. These various toxicities, and skin rashes [362, 3631 are also observed clinically along with anorexia, nause?, vomiting, and diarrhoea. The limiting clinical toxicity with 6-(methy1thio)purine ribonucleoside is gastrointestinal toxicity, particularly of the upper tract [364] , whereas bone- marrow toxicity is usually limiting with 6-mercaptopurine and thioguanine.

Administration of 3 '-amino-3 '-deoxy-N,N-dimethyladenosine (the amino- nucleoside of puromycin) to rats produces a nephrotic syndrome that is clinically indistinguishable from the nephrotic syndrome of unknown origin frequently observed in children [365] . Rats, monkeys, and humans are susceptible to this nephrotoxicity and susceptibility has been related to specie ability to demethylate the aminonucleoside [ 2 131 . N6-Methyladenosine prevents develop- ment of this syndrome [365a].

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Embryonic tissue

Because of their effects on rapidly dividing tissue, the purine analogues have a marked effect on the developing embryo. The analogues differ not only in the amounts required to produce toxicity in the embryo but in their teratogenicity. I t is possible t o produce teratogenic effects in the chick embryo with a sublethal amount of 8-azaguanine but not with 6-mercaptopurine [366]. Compounds such as 6-mercaptopurine and thioguanine affect both cellular multiplication and differentiation [367] .

The abilityofpurine analogues to affect rodent embryos in ufero at doses that are non-toxic to the mother is well documented. When 6-mercaptopurine [368] , thioguanine [369], some of their S-substituted derivatives [370, 3711, and 6-chloropurine [373] are given to the rat at the time o f implantation of fertilized ova, a high percentage of the foetuses are destroyed. To exhibit peak activity, 2-aminoadenine must be given before implantation [369] . 6-Mercaptopurine, 6-mercaptopurine ribonucleoside, h-mercaptopurine-3-oxide, and N-hydroxy- adenine are teratogenic when administered on the eleventh day of gestation [373a] . The teratogenicity of 8-azaguanine in mice depends o n the timing and amount of the dose [ 3731 .

Immune response

The finding that the administration of 6-mercaptopurine to rabbits following exposure to bovine serum albumin prevented antibody formation 13741 formed the basis for a new area of chemotherapy for purine analogues and other anti- metabolites and was soon followed by the use of these drugs for the therapy of autoimmune disease and the suppression o f homograft rejection. This subject has been reviewed in depth [ 12, 375, 375al , has occasioned a symposium [376], and has received much recent publicity as a result of human heart transplants.

Certain purines are capable of specifically inhibiting the immune response during the induction period of the response, and the inhibition is increased by increasing the antigenic stimulus. There is a close resemblance between drug- inducedand antigen-excess repression of the response, and although the mechanism by which these compounds suppress is not clear, the suggestion has been made that it is probably related to their cytotoxic nature [ 121.

Fortunately for the potential of immunosuppressive agents in the treatment of homograft rejection, they have much less effect on a secondary than on a primary immune response, although they are useful in the treatment of a number of autoimmune diseases such as psoriases, and undesirable effects have been reported [375] .

A number of thiopurines (thioguanine, 6-mercaptopurine, 6xmethylthio) purine, azathioprine (6(( l-methyl-4-nitro-5-imida~olyl)thio] purine) [ 121 ,and other derivatives o t 6-rnercaptopurine [377] ) have all been used to successfully prolong homografts, and azathioprine (Imuran) appears t o be superior in its action [ 2 6 8 ] .

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Pla tele t aggrega tion

Circulating platelets adhere to the site of damage of blood vessels aggregating into clumps or white bodies, which stop the flow of blood causing clots. Adenosine diphosphate causes the aggregation of platelets in blood plasma both in vitro [378] andin vivo [379], and this effect is antagonized by adenylic acid and even more by adenosine. Of the thirty odd purine nucleosides that have been evaluated as platelet aggregation inhibitors [38E382] only 2-aza-adenosine and the 2-haloadenosines showed significant activity. 2-Chloroadenosine was more active than adenosine, but also caused respiratory arrest in rabbits [383]. A correlation has been noted between the ability of these compounds to inhibit platelet aggregation and their vasodilator activity [382] .

Gout

The pyrazolo[3, 4-d] pyrimidines are substrates for and inhibitors of xanthine oxidase [ 266,267].4-Hydroxypyrazolo[3,4-d] pyrimidine was first investignted for its ability to protect 6-mercaptopurine and other analogues from oxidation by xanthine oxidase [384], but it also inhibits the oxidation of the natural purines, hypoxanthine, and xanthine. Its profound effect on uric acid metabolism made it an obvious choice for the treatment of gout and its utility in the control of this disease has been demonstrated [385,386].

On invading organisms

Micro-organisms (bacteria and protozoa)

Much information on the mechanism of action and cross-resistance of purine analogues has been obtained in bacteria, some of which are quite sensitive to certain of these compounds in vitro. There is a great deal of variation in response of the various bacteria to a particular agent and of a particular bacterium to the various cytotoxic purine analogues. Some, if not most, of these differences are probably due to differences in the anabolism of the various compounds. Despite the fact that certain purine analogues have quite a, spectrum of antibacterial activity in vitro, none has been useful in the treatment of bacterial infections in vivo because their toxicity is not selective-the metabolic events whose blockade is responsible for their antibacterial activity are also blocked in mammalian cells and thus inhibition of bacterial growth can only be attained at the cost of prohibitive host toxicity. In contrast, the sulpha drugs and antibiotics such as penicillin act on metabolic events peculiar to bacteria.

It is of historical interest that Tetrohyrnenagelii, whose metabolism has been described in detail [387], is inhibited by 8-azaguanine [388] and other purine analogues [389, 3901. Of more importance to chemotherapy is the fact that pathogenic protozoa such as the trypanosomes respond in vitro to a number of

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purines such as 6-mercaptopurine [39 11 , thioguanine 139 11, and 2-aminoadenine [392]. More active than these compounds, in vitro and in vivo, however, is the aminonucleoside of puromycin, 3 '-amino-3 '-deox y-N,N-dimethy ladenosine, which is more effective than puromycin itself [ 39 1,3931 . Primaquine appears to be more effeGtive than the aminonucleoside on the flagellate forms of T. cruzi; so the combination was tested in mice and found extremely effective [394]. Puromycin alone was not effective against T. cruzi in humans [395], but was effective against early infection of T. gambiense [396]. T. equiperdum and T. gumbiense in mice respond to both puromycin and the aminonucleoside [397-4001. Puromycin was also effective in suppressing T. equiperdum, T. equinum. T. Evansi, and T. rhodesiense in mice, but was ineffective against T. congolense [398]. T. equiperdum infections in mice 14011 and T. congolense, T. gambiense, and T. equinum in mice and rats are cured by treatment with nucleocidin [4021.

Although inferior to pyrimethamine plus sulphonamides, both puromycin and the aminonucleoside are active against Toxoplasma gondii in vivo [403]. These drugs are also effective against Endamoeba histolytica in vifro [404] , and puromycin is active against the infection in man [405].

The mechanism of inhibition of these protozoal infections by the most active drugs, puromycin and the aminonucleoside, is not known. Puromycin and nucleocidin both intertere with protein synthesis, but the aminonucleoside does not. It is known to be demethylated to 3'-amino-3'-deoxyadenosine, which is phosphorylated and interferes with nucleic acid metabolism (see above). Whether puromycin must be converted to the aminonucleoside before it can inhibit protozoa has not been established. Some purine analogues known to interfere with nucleic acid metabolism, however, are less effective as antiprotozoal agents, even in vifro, perhaps because their effects are primarily on the de novo pathway which many, if not all, protozoa do not use [406].

Viruses and cancer

2-Aminoadenosine, the first purine found to possess antiviral activity, inhibits vaccinia [384], spring-summer encephalitis [407], psittacosis [408], and poliomyelitis [409] viruses in cell culture. 8-Azaguanine has been reported a$ both active [410] and inactive [384] against vaccinia virus, and active against psittacosis and encephalomyocarditis. 6-Mercaptopurine interfered with the replication of both RNA and DNA viruses in Lass cells [41 I ] . 9-P-D-Arabino- furanosyladenine has a remarkable inhibitory action on the multiplication of the DNA viruses [41 l a ] , herpes, vaccinia [412, 4131, and cytomegalovirus [414], which also responds to thioguanine.

Puromycin is active against a number of viruses in cell culture. In chick embryo cells it delayed the replication of western equine encephalitis [4 151 and inhibits Venezulian equine encephalitis [4 161 . It interferes with the replication

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of poliomyelitis [417-4191 and western equine encephalitis [419] in HeLa cells. I t is reported to inhibit [420] and not inhibit [421] encephalomyocarditis and t o inhibit reovirus [422] and influenza (4231, but not herpes [424] in L cells. If added early in the eclipse stage it inhibits adenovirus in monkey kidney cells [425] . I t also inhibits polyoma virus in mouse embryo cells [426] .

Activity of an agent against a virus in cell culture is only an initial lead. Many false positives are found because there is no measure of host toxicity at viricidal levels or of the many other complicating factors. Unfortunately the purine analogues have shown minimal activity against viral infections in the whole animal. 2-Aminoadenine reduced the mortality of mice infected with spring- summer encephalitis [427] , bu t puromycin had no effect on herpes keratitis in rabbits [428] . A number of purine analogues failed t o inhibit Semliki Forest virus infections in mice [ 3721 . 9-p-D-Arabinofuranosyladenine was the only purine of a large number evaluated for in viuo activity against influenza and vaccinia viruses that was inhibitory and its effectiveness was confined to vaccinia 14291.

In vivo studies with virus-induced cancers mostly limited t o virus leukemias in mice and the Rous sarcoma in chicks, have been concerned primarily with the anticancer aspect of the problem, and have placed little emphasis on the viral aspects. 6-Mercaptopurine, its ribonucleoside, thioguanine, and azathioprine all prolong the life span of mice infected with the Friend virus leukemia [430,43 1 ] . In addition t o these compounds, 9cyclopentyl-6-mercaptopurine, 9-butyl-6- mercaptopurine, 6xbenzylthio)purine ribonucleoside, and thioguanosine are also active [432] . 6-Mercaptopurine [433] and thioguanine were active against both the Friend and Rauscher viruses in an in vitro assay system [434]. 6-Mercaptopurine showed only minimal effects against the Moloney virus leukemia [435, 4361, although other purine analogues such as thioguanine and 6chloropurine ribonucleoside are reported t o increase survival time of infected mice [ 3 6 6 , 4 3 7 ] .

Although inactive against the Kous sarcoma in the standard post-infection test, 6-mercaptopurine, 2-aminoadenine, and 8-azaguanine inhibited the develop- ment of the tumour if given prior t o infection of the chicks [417 ,418] .

Most of the adenine and adenosine analogues discussed in the precedine sections are converted to adenosine triphosphate analogues and are highlY cytotoxic. Unfortunately, their specificity for cancercells is low so that, although they show some activity in sensitive experimental animal systems such as Ehrlich ascites carcinoma, they are not useful agents; and those that have been evaluated clinically (i.e., 2-aminoadenine [363] and 4-aminopyrazolo[3, 4-d] pyrimidine [438] ) are not effective, but are toxic t o man. The ribonucleoside [439] of N-hydroxyadenine [440] , an inactive adenine analogue, may be an exception to thisstatement, since it is quite active against L1210 leukemia [439] , but haemolysis at low dosage occurred in preliminary clinical trials [439a]. Since other N-substituted adenosines are phosphorylated to the monophosphate stage only, the active form of this analogue is probably N-hydroxyadenylic acid, rather

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than the triphosphate, which may be part of the reason for its selective action on cancer cells. Trimethylpurin-6-ylammonium chloride [44 11 and some of its derivatives

[442, 4431 inhibit the growth of adenocarcinoma 755, but not sarcoma 180 or leukaemia L1210 in mice; little additional information is available on this series of compounds.

6-Chloropurine, as its ribonucleotide, is active against a number of animal neoplasms [444] and human leukaemias [363], but is less effective than the purinethiones, which it resembles in its action.

8-Azaguanine is inhibitory to several experimental animal tumour systems [444], but is not highly active. Clinically its toxicity has been more apparent than its anticancer effects [362,363].

Of the purine analogues investigated thus far, the purinethiones are by far the most effective anticancer agents. 6-Mercaptopurine remains the agent of choice clinically [363, 445-4471 , since other thiopurines, such as thioguanine, 6-(methylthio)purine, azathiopurine, 9-ethyl-6-mercaptopurine, and 6-mercapto- purine ribonucleoside, which are also active in man, appear to offer no real advantage over it. 6-Mercaptopurine is useful in the treatment of acute granulocytic, acute lymphocytic, and chronic granulocytic leukaemia and chorio- carcinoma [ 12, 363, 445-4471 . The combination of 6-mercaptopurine and 6(methylthio)purine ribonucleoside, however, is more effective in the treatment of acute adult leukaemia than either drug alone [448]. 6-Mercaptopurine is considerably less effective in the treatment of solid tumours [363] .

The reason for the selective toxicity of 6-mercaptopurine remains to be established, but two factors may be of primary importance. 6-Mercaptopurine is anabolized primarily, if not exclusively, to the monophosphate level, and it is readily catabolized by xanthine oxidase, an enzyme that is low in most cancer cells compared to normal cells. Another factor that must be considered is the metabolic state of the target cells. Actively proliferating leukaemia cells are more sensitive to 6-mercaptopurine, as they are to all antimetabolites, than cells in the so-called Go or stationary phase. Although this does not explain the difference between 6-mercaptopurine and other purine analogues, it may explain the ineffectiveness of 6-mercaptopurine against solid tumours, most of the cells of which are in the non-dividing state.

Certain derivatives of 6-mercaptopurine, such as 6-(methylthio)purine, 6-mercaptopurine-3-oxide [448a] , and 6-mercaptopurine ribonucleoside and its acylated derivatives apparently owe their activity to their in vivo conversion to 6-mercaptopurine [ 11,131. It would appear, however, that the 9-alkyl derivatives of 6-mercaptopurine, and its arabinosyl and xylosyl derivatives, are not metabolized-except in the case of the 9-alkyl derivatives, t o a limited extent to their S-glucuronides-and that their mechanism of action is quite different from that of 6-mercaptopurine.

Thioguanine is 5-30 times as toxic to rodents (depending on schedule) as 6-mercaptopurine and somewhat more effective against rodent neoplasms,

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although its therapeutic index is not greater. It would seem likely that 6-mercaptopurine and thioguanine inhibit cancerous growths in a similar manner. Changes in the structure of thioguanine also give rise to active structures. but no significant improvement in effectiveness [ 1 1,131 .

THE PROBLEM OF RESISTANCE Mechanisms

Both natural and acquired resistance constitute a serious problem to therapy witH purine analogues, particularly in the case of cancer. Why one acute leukaemia responds well to methotrexate but not to 6-mercaptopurine, whereas a morphologically identical leukaemia responds to 6-mercaptopurine but not to methotrexate, and a third, seemingly identical leukaemia responds to neither is a vexing problem that has so far defied solution [449].

In the case of acquired resistance, a patient may respond well to a drug initially and appear to be completely cancer-free, only to succumb later to a cancer that now does not respond to therapy with the same drug. Such a situation may indicate that the cancer cell population has, before resumption of treatment, multiplied to a size where it can no longer be controlled by a drug dose th?t the patient can tolerate [450], or it may indicate that a drug-resistant mutant population has replaced the original heterogeneous population containing almost entirely drug-sensitive cells.

The necessity for most purine analogues to be converted to their nucleotides to show their inhibutory effects has been discussed. Cells deficient in hypo- xanthine-guanine phosphoribosyltransferase activity cannot convert 6- mercaptopurine, thioguanine, or 8-azaguanine to their ribonucleotides and are resistant to these analogues [8,98, 101,260,451455] , but are still sensitive to adenine analogues such as 2-fluoroadenine, 2-aminoadenine, and 4-aminopyrazolo [3, 4:d] pyrimidine [456]. Conversely, cells deficient in adenine phosphoribo- syltransferase activity are resistant to the various adenine analogues, such as 2-aminoadenine [ 1 13, 3041 and 2-fluoroadenine [ 1281, but are still sensitive to cytotoxic hypoxanthine-guanine analogues [ 128, 1471 . Although mammalian cells are naturally resistant to xanthine analogues, because they are deficient in xanthine phosphoribosyltransferase activity, bacteria possess this enzyme and are sensitive to 8-azaxanthine. Bacteria that have become resistant to 8-azaxanthine were shown to have lost their xanthine phosphoribo'syltransferase activity [98]. Resistance to 2-aminoadenine and 8-azaguanine in Salmonella typhimurium is apparently due, in some cases, to genetically controlled alterations of the phosphoribosyltransferases to forms of the enzymes that can still convert the natural substrates to nucleotides but not the purine analogues [457,458]. Cells deficient in adenosine kinase fail to respond to adenosine analogues, unless they are cleaved to adenine analogues that can be converted to ribonucleotides by adenine phosphoribosyltransferase. Cells deficient in both these enzymes fail to respond to adenine and adenosine analogues, but are still sensitive to hypoxanthine-guanine analogues [ 1471 . Resistance to the various purine

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analogues due to these enzyme deletions has been observed in bacteria, mammalian cells in culture, and neoplasms in experimental animals indicating that this is a ubiquitous and an important cause of acquired resistance [8]. Since these enzymes catalyse the so-called 'salvage' pathways of purine utilization they are not essential to cellular metabolism, and their loss does not, in general, affect the ability of cells to proliferate at normal rates, which may explain why resistance by these deletions occurs so readily and frequently. However, acquired resistance is thought to result from chemical selection and overgrowth of specific drug-resistant mutant cells from a heterogeneous population, and hence it is not surprising that more than one type of resistance to a particular agent has been found. Other mechanisms of resistance to purine analogues that have been advanced are inaccessibility of the nucleotide-forming enzyme system to the analogue [459, 4601, increased rate of degradation of the analogue or its metabolities [461-463], and a decreased affinity of the activating enzyme for the analogue [ 1001 .

Circumventions

One of the first demonstrations that acquired resistance could be circumvented was the inhibition of S. faecalis resistant to 8-amguanine by 8-azaxanthine. Thus, cells that possess xanthylic acid phosphoribosyltransferase activity could form 8-azaxanthylic acid, which was then converted to 8-azaguanylic acid and incorporated in nucleic acids as such 1981 .

Early attempts to inhibit H.Ep.-2 cells resistant to 6-mercaptopurine [464J resulted in the finding that a number of 9-alkyl derivatives of 6-mercaptopurine were highly active in this test system. 9-Alkylhypoxanthines and adenines were less effective [465].

6-Mercaptopurine ribonucleotide is not active against cells resistant to 6-mercaptopurine, presumably because nucleotides cannot penetrate cell membranes intact [466] (its activity in sensitive cells is no doubt due to its cleavage back to 6-mercaptopurine [467] ).

This stumbling block led to the synthesis of esters of 6-mercaptopurine ribonucleotide [468, 4691 that might penetrate cell walls intact and then either inhibit per se or be cleaved back to the ribonucleotide. Two such derivatives, bis(thioinosine) 5 ', 5 "'-phosphate [470] and the monophenyl ester of. 6-mercaptopurine ribonucleotide [ 13, 4681 do inhibit H.Ep.-2 cells resistant to 6-mercaptopurine, but some cross-resistance was noted. Because of the relatively low therapeutic index of all known purine antagonists, this cross-resistance did not otter encouragement for in vivo activity against resistant neoplasms, and, indeed, the monophenyl ester did not inhibit leukaemia L1210 resistant to 6- mercaptopurine. More successful in this regard was the use of 6-(methylthio) purine ribonucleoside against MP-resistant cells. Thus, this nucleoside, which is converted to the nucleotide by adenosine kinase, is highly active against both resistant H.Ep.-2 cells in culture and resistant L1210 leukaemia in mice [471]. Furthermore, it is therapeutically synergistic with 6-mercaptopurine in the

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sensitive line of L1210 [471]. Similar observations were made in the Ehrlich ascites carcinoma system [472]. The clinical utility of this combination [448] has been discussed above.

AC KNOW L E DG EM EN TS

The author gratefully acknowledges the helpful criticism of Dr. Lee L. Bennett, Jr. Thanks are due to Mr. Vladimir Minic and Miss Linda Scott for checking references to the literature and to Mrs. Frances K. Hoffman for preparation of the manuscript.

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466. P. M . Roll, H. Weinfeld, E . Carroll and G . B. Brown, J. Biol. Chem.. 1956,220,439 467. J . A . Montgomery, F. M . Schabel and H. E. Skipper, Cancer Rex, 1962, 22,504 468. J . A . Montgomery, H . J . Thomas and H . J . Schaeffer,J. Org. Chem., 1961, 26,1929 469. H . J . Thomas and J . A. Montgomery, J. Med. Plzarm. Cllem.,1962,5, 24 470. J . A . Montgomery, G . J . Dixon, E. A. Dulmadge, H . T . Thomas, R . W. Brockman and

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101

Progress_in_Medicinal_Chemistry._7/0408700130/files/00132___effc57f1d614cff72a3d7379cef23a1f.pdf3 The Chemistryof Guanidines and their Actions at Adrenergic Nerve Endings G. J. DURANT, B.Sc., Ph.D., A.R.I.C. Smith Kline and French Research Institute, Welwyn Garden City, Herts.

A . M . ROE, M.A., D. Phil., F.R.I.C. Smith Kline and French Research Institute, Welwyn Garden City, Herts.

A. L. GREEN, B.Sc., Ph.D. Department of Biochemistry, University of Strathclyde, Glasgow.

INTRODUCTION THE STRUCTURE AND PHYSICAL PROPERTIES O F GUANIDINES SYNTHETIC METHODS

Guanidines Aminoguanidines

Effect on blood pressure Effect on the sympathetic nervous system

PHARMACOLOGICAL TEST PROCEDURES

STRUCTURE-ACTIVITY RELATIONSHIPS FOR ADRENERGIC NEURONE BLOCKADE

Guanethidine and close analogues Aryloxyalkylguanidines and related structures Araky lguanidines Miscellaneous guanidines Structural requirements for adrenergic neurone blockade

OTHER PHARMACOLOGICAL EFFECTS ON SYMPATHETIC NERVES BIOCHEMICAL EFFECTS

Depletion of noradrenaline Antagonism of noradrenaline depletion Effect of guanidines on enzymes involved in noradrenaline metabolism Mechanism of guanethidine-induced depletion Relationship between noradrenaline depletion and adrenergic neurone blockade

ADDENDUM REFERENCES

125 126 130 130

133

135

135

136

139

139

15 1 160

169 171

174

177

177

185 188 193 196 200 203

I24

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INTRODUCTION

Guanidines have been studied for many years as a possible source of medically useful compounds, but the recent vast increase in the literature on guanidine derivatives (from about 200 references in Chemical Abstracts in 1958 t o over 1000 in 1965) stems principally from the discovery [ I ] of the potent hypotensive properties of guanethidine (oktadin, Azetidin, Dopom, Eutensol, Guanexil, Guethine, Ipoctal, Ipoguanin, Iporal, Ismelin, Izobarin, Normorif, Octadinum, Octatensine, Pressidin, Sanotensin, Su-5864, Visutensil, I).

Around 40 years ago, the short-lived use of synthalin (11) as a hypoglycaemic drug [ 2 ] led t o numerous studies on guanidines as potential insulin substitutes [3] . Synthalin itself was withdrawn when it was reported that it can cause liver damage [4], and the widespread interest in guanidines eventually lapsed until the introduction of guanethidine nearly 30 years later.

It is chastening t o note how often during these early investigations on guanidines, marked cardiovascular actions were reported but not pursued. One such report referred t o phenethylguanidine (111) in the following terms the latter compound, in larger doses, exerts a powerful effect on the flow of blood as

Ph(CH2 ) 2 .NH.C=NH I

evidenced by the difficulty of bleeding the animals: [ 5 ] . Synthalin itself caused a fall in blood pressure, and also had curare-like effects [6] which were subse- quently rediscovered in the related compound decamethonium. However, since the object of this early work was t o find drugs which lowered blood sugar rather than blood pressure, these observations were apparently not followed up. During the 1930s several aralkylguanidines were shown by Japanese workers to lower blood pressure [7, 81, but the mechanism of this hypotensive action was not understood a t the time, and again it was not followed up.

Synthetic guanidine derivatives have been used successfully in the treatment of a variety of diseases, but the major success has undoubtedly been their exploitation as antihypertensive drugs. Guanethidine has still not been displaced,

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although it now has many competitors such as bethanidine (benzanidine, Esbatal,' Eusmanid, B.W. 467C60, IV), guanoxan (Envacar, V), guanoclor (Vatensol, VI) and debrisoquine (Declinax, VII). The comparative clinical value of some of these compounds has been reviewed recently [ 9 ] .

All of these drugs appear to lower blood pressure by blockade of sympathetic

C l

nerves, and the present review is confined to this particular aspect of the pharmacology of guanidines. The structure, physical properties and synthesis of guanidines are summarized first, and then, after an outline of the methods used for testing these drugs, the relationships between structure and adrenergic neurone blockade are discussed. The relevant biochemical effects connected with these pharmacological actions are surveyed, with particular reference to possible mechanisms of action. The review is written primarily for the medicinal chemist, and detailed pharmacology is generally included only where necessary for under- standing the structure-activity relationships.

An excellent review of the pharmacological actions of adrenergic neurone blocking agents has been given by Boura and Green [ l o ] . The biochemistry of guanethidine itself has been reviewed by Furst [ 1 I ] , with particular emphasis on tissuz distribution and metabolism; consequently these two topics are not discussed i n detail.

THE STRUCTURE AND PHYSICAL PROPERTIES OF GUANIDINES

A prerequisite t o a full understanding of tlie nature of tlie interactions of guanidines with living tissue is an accurate expression of the molecular and physico- chemical behaviour of both interactants. The molecular arrangement, and hence

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the physicochemical properties, of the biological surfaces with which this review is concerned are still unknown; but the position with respect to the guanidine partner in these interactions is reasonably well understood.

Guanidine (VIII) and those substituted guanidines with which we shall be concerned are strong bases [12-191, and form stable salts with relatively weak acids [ 201 . Table 3.1 lists the published pK, values of some simple guanidines. The increased stability of symmetrical guanidinium ions is reflected in the high basic strength of guanidine and in the still higher basicity of N,N:N':trimethyl- guanidine. In strong acid, both the guanidinium ion and the aminoguanidinium ion can accept a second proton. The second pK, of guanidine has been estimated [ 151

Table 3.1. THE BASlCITY OF SOME SIMPLE GUANIDlNES*

R' I t 2 R3 R4 RS P K ,

H H H H H H Me H H H Me H Me Me H H Me H Me Me Me Me Me H Me Me Me H H H H H H Me H H H Me H H H H H H H H Ph H H H H

Guanethidine ( I )

H H H H H Me H Me Me H H H H H H H H

13.65 (131 13.6 (141 13.74 [18] 13.4 1141 13.4 1141 13.6 [14] 13.6 [ 141 13.9 [ 141 13.6 1141 13.9 [ 141 13.8 [ 141 10.8 (171 11.0 1381 10.6 [ 3 7 ] 10.5 (381 12.6 (371 10.85 [ 371 11.4 1381 11.5 i38j

* 9.9 [417] 9.8 (4181 11.04 (4191 11.97 1211 11.3-11.4 [420]

'The references should be consulted for the temperature and other conditions under which these measurements were made.

to be -10.9 on the Ho scale. The very large difference between the first and second pK, values of guanidine is thought to be associated with the loss of symmetry which arises when the guanidinium ion accepts another proton [ 151. The second pK, of (benzy1amino)guanidine is -3.2 on the Ho scale [21].

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When the pK, of a guanidine is greater than 13, the ratio of guanidinium ion to guanidine is greater than 1 O6 : 1 under physiological conditions [22] ; amino- guanidine will exist as the protonated ion to the extent of at least lo4 : 1, relative to the unprotonated species. In view of the foregoing, the ensuing discussion will concentrate on the structure and properties of the mono-protonated species, that is, on guanidinium ions.

There is ample crystallographic evidence that the guanidinium ion is planar and symmetrical [23, 241. Both ions in the crystal of methylguanidinium nitrate exhibit almost perfect trigonal symmetry [25] , and the aminoguanidinium ion has also been shown to be planar [26]. The formal similarity between the guanidinium ion, the carbonium ion, and the carbonate anion has long been recognized [27], and many papers have been devoted to defining the precise electronic structure of guanidinium ions [28-361.

Infra-red studies [31] and molecular orbital calculations [33,34] have led to the description of the guanidinium ion as a tri-amino carbonium ion with the nelectron charge distribution shown (IX) Most of the positive charge is located in the vicinity of the central carbon atom. The relevance of this description to the pharmacological properties of guanidinium ions will be discussed later. For typographical convenience, guanidines will be formulated in this review in the unprotonated form.

, tO.086 Y

H\ rH H2N.C-H N- C+O .7L 2

/ 1 .318 \ N 4 /

I H N H2 (VIII)

Some aspects of the structure and properties of amidines, including guanidines, in relation to their biological properties have been discussed by Fastier [36a] . The formation of relatively rigid amidinium carboxylate ion-, pairs, formulated as eight-membered rings stabilized by two hydrogen bonds, is thought to precede the formation of crystalline salts and is suggested [36b, 36c] as the basis for their biologcal activity. An alternative formulation of a guanidinium carboxylate ion-pair has been proposed [36d]. The affinity of amidinium groups for anionic sites, such as carboxylate and phosphate, has been stressed [36a].

The similarity of the guanidinium ion, in which the distance between the carbon and the hydrogen atoms is about 2.1A, to the hydrated sodium ion [Na(OH,),]+, in which the distance between the sodium and the hydrogen atoms is about 2.3A, has been pointed out [33, 36a], and the physiological

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properties of these ions have been compared [33, 36al. A comparison, which is more relevant to this review and is pursued later, may be made with the trimethyl- ammonium group. Finally, it has been suggested [21] that the activities of certain aryloxyalkylguanidines depend on their ability to adopt an internally hydrogen-bonded conformation such as (LXXVIII) and (LXXIX) (p. 172 ). The strength of the hydrogen bond in these systems is related to the basicity of the oxygen atom, which in turn is markedly affected by the position and nature of the substituents in the aromatic ring.

Although not strictly relevant to the present discussion, some other physico-chemical aspects of guanidines are summarized here, since no review of this subject has been published previously.

The structures of aromatic guanidines and their conjugate acids, in which the T-electrons of the ring can interact with the delocalized electrons of the guanidine system, have been studied. From the ultra-violet absorption spectra and acid dissociation constants of pchlorophenylguanidine, structure (X) was preferred [ 371 over structure (XI); in contrast, the most probable structures for phenylguanidine and its conjugate acid were considered [ 171 to be (XI) and (XII). A more extensive study of some para-substituted phenylguanidines, which includes an estimate of the

Ar N H - G N H

1 ArN=C-NH2 ArNH-C=hH,

I I

Hammett p constant (+ 2.30) for the formation of the conjugate acid and the Up constant of the guanidinium substituent (+0'317 to + 0.443), supported the conclusion [ 3 M ] that the base has the structure (X). A different view [ 391 was based on the fact that since p for substituted anilines is + 2-77, dissociationof the phenylguanidinium ion most likely takes place from the nitrogen atom which bears the aromatic ring. This argument, however, neglects the delocalization of charge in the ion. The directing effect of the guanidinium substituent in electrophilic aromatic substitution is a measure of the interaction of this substituent with an aromatic ring. The guanidinium substituent is not such a powerful meta- directing group as those substituents in which the positive Charge is localized on the atom which bears the aromatic ring [40] .

The ultra-violet spectra of guanidines have often been determined in connection with the measurements of dissociation constants [ 17, 38, 411, and other studies have been reported [42-441. There have been many reports on the infra-red spectra [31, 35, 42, 45-48], the nuclear magnetic resonance spectra [ 38, 49-5 1 ) (see also p. 134). the Raman spectra [52-541, force constants [ 35, 361, and mass spectra [SS] of substituted guanidines.

Guanidine forms salts with such relatively weak acids as nitromethane, phthalimide, phenol and carbonic acid [20] . lnteractions between carboxylate anions of proteins and added guanidinium ion are thought [19, 561 to be weaker than the interactions with ammonium ions; the role of guanidinium-carboxylate interactions in stabilizing natural protein conformations has been discussed [36c]. A few reports of metal complex formation by guanidines 157-601, and aminoguanidines [61] have appeared.

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Much attention has been given recently to the chromatographic behaviour of guanidines [ 62-69), and various techniques for the detection and determination of guanidines are described there and elsewhere [ 70-761.

SYNTHETIC METHODS

Guanidines

Guanidines have been prepared by a wide variety of methods, of which two are of much greater importance than the others. These two methods, (a) the addition of amines to cyanamides and (b) the displacement of an alkylmercaptan by an amine from an alkylisothiouronium salt, together with close variants, are discussed first, and this discussion is followed by a survey of less frequently used procedures.

Since an excellent summary of some of the methods that have been used to obtain guanidines is available [77] , the following discussion concentrates on those aspects of synthetic chemistry that are likely to be of interest to medicinal chemists. Patents are only cited when the experimental methods described supplement those available elsewhere.

It is worth stressing at this point that although many statements have been made concerning the relative merits of one method of synthesis compared with another, there is little consistency and much contradiction to be found. Except where a comparative study by the same workers has been carried out, it is unwise to rely on these generalizations as a guide for preparing novel guanidines. It is probable that much of the confusion arises from the use of inappropriate methods of isolation rather than from anomalous reactivities.

Method (a) RzNH + RZN-CN + RZNGNH I

Guanidine itself was obtained by Erlenmeyer [78] in 1868 from the action of ammonia on cyanamide, a method that soon led to the synthesis of phenyl- guanidine from aniline hydrochloride and cyanamide [79,80]. This method has been used to prepare many arylguanidines, and the use of substituted cyanamides has given NJdiary1- [81] , N-aryl-N-alkyl- (or N-aryl-N:N:dialkyl-) [82], and N-aryl-Nbenzoyl-guanidines [83] . Arylguanidines are sometimes advantageously prepared by using the benzoylguanidine as an intermediate [83].

Reaction between an amine salt and cyanamide has been used successfully for the synthesis of many mono- and poly-alkylguanidines [84-95], and also of alkoxyguanidines [96, 971 and aryloxyguanidines [98]. The reaction is usually carried out in boiling water or ethanol for from 1 to 24 hours. Higher temperatures have been employedusingsealed tubes [99, 1001 or butanolasasolvent [82,101].

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Very dilute acetic acid [lo21 and ethyl acetate [lo31 have also been used as solvents. The fusion of amine salts with cyanamide or a substituted cyanamide is often more satisfactory than using a solvent [ 107-109, 2281, particularly for sterically hindered guanidines such as t-butylguanidine [228] .

The preparation of alkylguanidines by fusing amine salts with dicyandiamide at 180" for three hours has been advocated [104], however it has been shown that, depending on the conditions, a guanidine or a biguanide can result [105, 106).

Odo studied the formation of methylguanidine from cyanamide and aqueous mixtures of methylaminc and methylamine hydrochloride in various proportions [ 1101 . He concluded that the reaction occurred by a reversible nucleophilic attack of the free amine on cyanamide, and that an acid was required to shift the equilibrium in the direction of the guanidine.

This method of formation of guanidines is considered especially suitable for arylguanidines [77] . The dihydrochloride of p-aminobenzylamine (XIII) reacted with cyanamide to give a mono-guanidine which was assumed [ 11 11 to be the arylguanidine (XlV) on the grounds that benzvlamine hydrochloride did not react under the same conditions, whereas aniline hydrochloride did. An isomer was obtained [ 1 1 1 ] when p-aminobenzylamine reacted with S-methylisothiouronium sulphate, and this isomer was formulated as (XV). However, these structural

assignments need verification. It is relevant to note that the free base (XIII) reacted with cyanamide to give the bis-guanidine [ 11 I ] . Further, guanidines have been obtained by reaction of cyanamide with salts of benzylamine [82] and p-nitrobenzylamine [87, 1011. The product from the latter was reduced [87, 1011 to authentic p-aminobenzylguanidine (XV) dihydrochloride, which decomposed only 3-5" above the reported [ 1 1 1 ] decomposition point of the dihydrochloride of the isomer assigned structure (XiV).

Method (b) RZNH + RzN.C=NR R2 N G N R I - I + H X

X NR2

The most frequently used synthesis of guanidines involves the displacement by an amine of a suitable group, X, from the amidine-type compound shown.

The preparation of phenylguanidine from ammonia and phenylthiourea was claimed [ 1 121 in 1879 but the authenticity of the product has been questioned

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[79] . The generally recognized originator of this synthesis of guanidines is Rathke who showed that ammonia reacts with S-ethyl-N,N'-diphenylisothiourea to form N,N-diphenylguanidine [ 1131 . That this product can be formulated in two ways: PhNH.C(:NPh)NH, or PhNH.C(:NH)NHPh, was clearly recognized at that time. Similarly, guanidine itself was obtained from S-ethylisothiourea and ammonia [ 1141.

Although 0-ethylisourea was reported not t o react with ammonia or with aniline [ 7 9 ] , the synthetic potential of this method became apparent when various polymethylguanidines were prepared from S-alkylisothiouronium hydr- iodides [ 14, 115, 1161 or, more conveniently, from S-methylisothiouronium sulphate [ 14, 1171. Aniline was reported not t o react with the latter salt [ 1171, but a later paper [ 1181 described its conversion into phenylguanidine.

Many different alkvl- and awl-guanidines have been obtained by this procedure [SS, 86, 1 1 1, 119-1321, as have alkoxyguanidines [97, 1331, and benzoylguanidines [ 134, 1351 .

It is usual t o carry out the reaction in water or ethanol, or in mixtures of the two, a