CHAPTER IIA SYNTHESIS OF 2-[BIS(2- '_CH LOR OETHY L) AMIN 0 ] - 3,6- D I ARY L-3 ,4-DIHYDRO-1,3,2- CKAZAPH OSPHORIN-2- OX.IDES ! 6 INTRODUCTION Alkylating agents of various types are widely used as immunosuppressive drugs. Alkylating agents contain a variety of functional groupingsj A: Mustards e.g. HN2 1, mannomustine 2, melphalan 3, chlorambucil 4, cyclophosphamide 6. B: Sulfonic acid esters e.g, busulfan 6. C: Epoxides e.g. 1,2,3,4-diepoxybutane 7. D: Ethyleneimines e.g. triethylenephosphoramide 8 , triethylenethiophosphoramide 9, triethylene- melamine W (Chart 1 ). Their biological activity is influenced to a large meaaiire by the nature of the carrier; a variety of chemical substances such as amino acids, carbohydrates, steroids and various heterocyclic molecules have been employed for this purpose. In vitro alkylation proceeds through a second order nucleophilic substitution^ HgO R - N X RNHCHgCHgA + OH - HoO R-GHe,-CH^, + A r c h ( oh) ch2A + ' oh Aliphatic N rausterds act similarly after the formation of a cyclic immonium ion, the unimolecular conversion to the immonium ion is relatively fast, and once formed, it reacts 17 CHgNHCHgCHgCI HOCH I ^CHgCHgCI HOCH H3C-N I CHgCHgCI HCOH I HCOH CHgNHCHgCHgCl H (CICHgCHglgN—-^^^^^CHgCHCOOH /\ 0 N(CH2CH2CI)2 NH2 3 OSOoCH, I (C1CH2CH2)2N—^ V-CH2CH2C00H (CH2)4 OSO2 CH3 l > V CH 1 r ^ N —p =R NY ^ N CH 1 N r—” N'' '•N —7 ^CH2 V V 7 8 , R = 0 10 9 , R = S CHART 1 ■'18 by an SNg laechanism RgNCHgCHgOP ^ R g B ^ + CP i RgNCHgClgA The Host widely used alkylating agent is cy clo- phosphaniide 5 which has a broad spectrum of a ctivity in animal and human malignancies. It was first synthesised by Arnold et al^ in 1968, together with a number of related N-phosphorylated derivatives of N-mustard, As one of the most widely used chemotherapeutic agents for the treatment of many types of cancer, much effort has been directed towards landerstanding o f the mode of action of cyclophosphamide and to develop analogues with improved action. In coi^parison with other alkylating agents it interferes with cell growth only after- activation in tissues. It is capable of inhibiting both the huaoral and cell medicated immune responses. Unlike many other cytostatic agents used as immunosuppressants, i t also prevents the development of c e ll medicated auto-immune diseases in animals. The concept of latenation - the in vivo release of a biologically active compound from a suitably derivatised 2 form - has been invoked to explain the action of certain nitrogen mustards and has influenced the design of others. kn early example of such a m odification was the oxidation of 2-chloro-N-(2-chloroethyl)-N-niethylethanamine (mechlor ethamine) to a less basic, therefore less reactive, form. 4'J which was obtained as the hydrochloride, 2-chloro-N- *5 4 (2-chloroethyl)-I'I-methylethanamine-N-oxide hydrochloride 0 (CH2CH2C1 )2 .HC1] . The oxide was reported to be less tox ic and more active in certain tumor systems than the parent compound ’ and i t may be a latent form that is 6 7 activated by i^ vivo reduction ’ . The search for suitable substrates for the phosphoramidases that were reportedly more abundant in neoplastic c e lls than in normal c e lls , resulted in the synthesis of numerous acyclic and cyclic phosphorylated nitrogen mustard derivatives “ , which showed a high degree o f activity against animal tumors 9 ’ 11 ’ 12 The most successful latenation of this type is exemplified by the development of cyclophosphamide [N,N-bis(?-chloroethyl) tetrahydro-2H-l,3,2-oxazaphosphorin-?-amlne-?.-oxlde] 6 as a widely used clin ic a l alkylating agent. 1 10 14 ,A,rnold et al ’ ’ reported the synthesis of various cyclic N ', 0-alkylene-N,N-bis(f3-chloroethyl)diamido- phosphates by treating dichloro-h,N-bis(3-chloroethyl) amidophosphate with alkanolamines, NH2 (CH2 )j^0H in the presence of equimolar amounts of trimethylamine in dioxane as solvent (Chart 2). Gt) The synthesis of cyclophosphamide 5 vas achieved by condensation of (CICH2CH2 (0)fH2 0Ph 13 with -hydroxy- propyl halides lA and triethylamine with dioxane as solvent in four hours^^ (Chart 3). Various solvents used for the synthesis of cy clo ­ phosphamide 5 from -alkanolamines and N ,N -bis(2-chloro- e thyl)phosphor amide dichloride were dioxane, di chi or ome thane , ethyl acetate-water,17 Bases used in the synthesis were trimethylamine, triethylamine and pyridine 18 /Another pathway for the synthesis of 6 is the reaction of 2-chlorotetrahydro-l,3 , 2-oxa2aphosphorln-2-oxlde 15 with N,N-bls(2-chloroethyl)8mine hydrochloride 1^ in the 1 9 presence of Et^N in diehiororaethane as solvent (Chart 4). Proposed as a potentially latent form of nor- nitrogen mustard, 2_chloro-N-(^-chloroethyl)ethanamlne 23, cyclophosphamide 6 emerged from the synthesis of more than 500 phosphamides^*"’ ^'^’ '^'^ as the most e ffe ctiv e congener in evaluations against Yoshida ascitic sarcoma of the rat. ^ Was definitely superior to nornitrogen mustard 21 and other nitrogen mustards 22 ’ 23^ in a number of animal tumor systems. 5 Which is itself non-toxic even at high concentrations to tumor cells growing in culture is converted to a high: f cytotoxic form by prior incubation with liver homogenate" Gi 0 Cl NHp(CHp)-OH + ^P-N(CHpCHpCl)p (CHp)n Cl N(CH2 CH2CI)2 n = 1- 6 11 12 CHART 2 0 .NH, OH (CICHpCHglgNPv^ OPh X N "N(CH2CHpCI)2 Where X= Br,CI,I 13 14 CHART 3 Q 0 0 0 + (CICHpCH^l^NH^CI / \ N ' n ( CHpCHpCDg 16 CHART 4 ' i 2W The metabolism of 5 has been confusing for about 16 years. Various concepts were proposed since the first 2*' 26 27 synthesis in 196S. These efforts have been reviewed ’ and provide a basis for the metabolic scheme shown in Chart 5. Cyclophosphamide 5 is metabolised in the liv er at a species dependent rate, fast in rodents, but slower in man; the resulting 4-hydroxycyclophosphamide r? tautomerises to unstable aldophosphamide ^ which spontaneously loses acrolein ^ to form phosphoramide mustard v iz . N,N-bis (2-chloroethyl)phosphoramidic acid ^ Which is also unstable decomposes to nornitrogen mustard 23 which cyclises to 1-(2-chloroethyl)aziridine 25, or is detoxified in the blood as 3-(2-chloroethyl)2-oxazolidine 24. The alkylating form ^ of nornitrogen mustard is 28 uncharged at physiological pH and thus d iffe rs from the aziridinium ion formed from mechlorethamine. The essentially inactive urinary metabolities, 4-ketocyclophosphamide ^ and carboxyphosphamide 20 are detoxification products but among the other metabolites (17;;:? 19, 22, 2^) the alkylating species ultimately responsible for the anticancer activity of 5 may not be unequivocally decided. If 17 ~ 19 is the important transport form that enters tumor cells, as is widely speculated, then detoxification by cellular aldehyde oxidase in resistant cells must be considered. OJ o CM CM O X o 0) CM X a O X M O T 3 X X o o o o 0) X CJ - a o / \ >> o x: (\J _CM 0) X TJ 00 CM X x z '' CJ 7j o X) X o X CL o Q O: Xi < 2 c 172 CM mi CM CM 0) rvj CJ CM CJ o *d X X «s o o o X O CM X o <\J X CD o £ •D X o o c m o CM o X X o o X) X o k . o o d q: CDl o < o X o / \ o 0) X 2 '' _0J > CM CM X "1m <o X X O' o O d X CM T) o X X o (U £ d o CM - o w X £ o O x: V d s : CL X o 2 a. CVJ X X o X o x: o O CJ ro x : Q_ CVi X CJ Cl X o + o O o >> CM CM ro X (J X X CM X o 2 3 u o o 0) + 4 - CM c X £ d in M- X 4— x z ^ '^O o C CM 0 o X CL o a o ro Xi d <A o X 0) CJ o o V CJ o 1 CM X x" ^ J The slower rate of hydroxylation in man suggested the syntheses of r? and stabilised derivatives thereof, 4-hydroperoxycyclophosphamide 2^ and 4-peroxycyclophosphamide Pq 30 2 7 " ’ . These compounds, which spontaneously yield under physiological conditions , were at least as active as cyclophosphamide in animal tumor systems. The 4-hydroperoxy derivative ^ of Isophosphamide, N,3-bis(2-chloroethyl) tetrahydr0- 2H-1 ,3,2-oxazaphosphorin-2-amine-2-oxide i.e . ifosfam ide 29,, an analog 32 ’ 33o f 5, appeared to have superior . a ctiv ity in leukemia L1210 system in a comparison with phosphoramide mustard, the parent compounds and other peroxidised derivatives and is undergoing clinical trials 34 in Japan (Chart 6). Clinically, 5 is the most widely useful alkylating agent, effectively inhibiting a variety of human carcinomas and sarcomas as well as leukemias and lymphomas. It can be effectively administered in a number of ways and used in qc oo combination with a number of agents"" ’ , Its principal side effects are leukopenia, alopecia and cystitis, the latter being produced by a urinary metabolite. I-6s range o f therapeutic applications has widened steadily to include qg it s present use as an immunosuppressive agent"" . In recent years, the enantiomeric forms of the family of known anticancer drugs based on the 1,3,2-oxaza- phosphorlnane skeleton, which are chiral by virtue of the 5b asymmetric phosphorus atom, were synthesised.