Process for preparing N6-substituted adenosine derivatives

The invention is directed to improved methods for preparing N6-substituted adenosine derivatives, to intermediates useful therefor and to methods of preparing these intermediates.

TECHNICAL FIELD
 This invention is directed to a process for preparing N6-substituted
 adenosine derivatives, to intermediates useful therefor and to methods of
 preparing these intermediates.
 BACKGROUND OF THE INVENTION
 N6-substituted adenosine derivatives, as exemplified by
 [1S-[1.alpha.,2.beta.,3.beta.,4.alpha.(S*)]]-4-[7-[[1-[(3-chlorothien-2-yl
 )methyl]propyl]amino]-3H-imidazo[4,5-b]pyrid-3-yl]
 N-ethyl-2,3-dihydroxycyclopentanecarboxamide are useful as cardiovascular
 agents, more particularly as antihypertensive and anti-ischemic agents, as
 cardioprotective agents which ameliorate ischemic injury or myocardial
 infarct size consequent to myocardial ischemia, and as an antilipolytic
 agents which reduce plasma lipid levels, serum triglyceride levels, and
 plasma cholesterol levels. See U.S. Pat. Nos. 5,364,862 and 5,561,134.
 Methods of preparing these N6 adenosine derivatives and intermediates
 thereto are disclosed in U.S. Pat. Nos. 5,364,862 and 5,561,134 and
 International Patent Application Nos. PCT/US97/11320, PCT/US97/15729 and
 PCT/US97/21439.
 SUMMARY OF THE INVENTION
 This invention is directed to a process for preparing a
 2-halo-3-nitro-4-aminopyridine compound of formula
 ##STR1##
 in which a 2,4-dihalo-3-nitropyridine compound of formula
 ##STR2##
 wherein X.sub.1 and X.sub.2 are independently Cl or F, is reacted with an
 amine of formula H.sub.2 N--X--(Y).sub.a -Z; wherein X is a straight or
 branched chain alkylene, cycloalkylene or cycloalkenylene group; Y is
 NR.sub.4, O or S; a=0 or 1; and Z is of the formula:
 ##STR3##
 where
 Z.sub.1 is N, CR.sub.5, (CH).sub.m -CR.sub.5 or (CH).sub.m -N, m being 1 or
 2; Z.sub.2 is N, NR.sub.6, O or S, n being 0 or 1;
 R.sub.4, R.sub.5 and R.sub.6 are independently H, alkyl, aryl or
 heterocyclyl; and
 R.sub.a and R.sub.b are independently H, OH, alkyl, hydroxyalkyl, alkyl
 mercaptyl, thioalkyl, alkoxy, alkoxyalkyl, amino, alkyl amino, carboxyl,
 acyl, halogen, carbamoyl, alkyl carbamoyl, aryl or heterocyclyl.
 In another aspect, this invention is directed to a process for preparing
 (R)-N-[1-[(3-chlorothien-2-yl)methyl]propyl]-2-halo-3-nitro-4-pyridinamine
 , by reacting a 2,4-dihalo-3-nitropyridine, wherein halo is Cl or F with
 (R)-1-(3-chlorothien-2-yl)-2-aminobutane, hydrochloride.
 The reaction products are process intermediate useful in the preparation of
 2,3,4-triaminopyridine compounds. The processes of this invention offers
 improved yields, purity, ease of preparation and/or isolation of
 intermediates and final product, and more industrially useful reaction
 conditions and workability over previously disclosed methods of
 preparation.
 DETAILED DESCRIPTION OF THE INVENTION
 Definitions of Terms
 As used above and throughout the description of the invention, the
 following terms, unless otherwise indicated, shall be understood to have
 the following meanings:
 "Acyl" means a straight or branched alkyl-C.dbd.O group. "Thioacyl" means a
 straight or branched alkyl-C.dbd.S group. Preferred acyl and thioacyl
 groups are lower alkanoyl and lower thioalkanoyl having from 1 to about 6
 carbon atoms in the alkyl group.
 "Alkyl" means a saturated aliphatic hydrocarbon group which may be straight
 or branched and having about 1 to about 20 carbon atoms in the chain.
 Preferred alkyl groups may be straight or branched and have about 1 to
 about 10 carbon atoms in the chain. Branched means that a lower alkyl
 group such as methyl, ethyl or propyl is attached to a linear alkyl chain.
 "Lower alkyl" means an alkyl group having 1 to about 6 carbons.
 "Cycloalkyl" means an aliphatic ring having 3 to about 10 carbon atoms in
 the ring. Preferred cycloalkyl groups have 4 to about 7 carbon atoms in
 the ring.
 "Carbamoyl" means an
 ##STR4##
 group. Alkylcarbamoyl and dialkylcarbamoyl means that the nitrogen of the
 carbamoyl is substituted by one or two alkyl groups, respectively.
 "Carboxyl" means a COOH group.
 "Alkoxy" means an alkyl-O group in which "alkyl" is as previously
 described. Lower alkoxy groups are preferred. Exemplary groups include
 methoxy, ethoxy, n-propoxy, i-propoxy and n-butoxy.
 "Alkoxyalkyl" means an alkyl group, as previously described, substituted by
 an alkoxy group, as previously described.
 "Alkoxycarbonyl means an alkoxy-C.dbd.O group.
 "Aralkyl" means an alkyl group subsituted by an aryl radical, wherein
 "aryl" means a phenyl or naphthyl. "Substituted aralkyl" and "substituted
 aryl" means that the aryl group, or the aryl group of the aralkyl group is
 substituted with one or more substituents which include alkyl, alkoxy,
 amino, nitro, carboxy, carbalkoxy, cyano, alkyl amino, halo, hydroxy,
 hydroxyalkyl, mercaptyl, alkylmercaptyl, trihaloalkyl, carboxyalkyl or
 carbamoyl.
 "Aralkoxycarbonyl" means an aralkyl-O--C.dbd.O group.
 "Aryloxycarbonyl" means an aryl-O--C.dbd.O group.
 "Carbalkoxy" means a carboxyl substituent esterified with an alcohol of the
 formula C.sub.n H.sub.2n+1 OH, wherein n is from 1 to about 6.
 "Halogen" (or "halo") means chlorine (chloro), fluorine (fluoro), bromine
 (bromo) or iodine (iodo).
 "Heterocyclyl" means about a 4 to about a 10 membered ring structure in
 which one or more of the atoms in the ring is an element other than
 carbon, e.g., N, O or S. Heterocyclyl may be aromatic or non-aromatic,
 i.e., may be saturated, partially or fully unsaturated. Preferred
 heterocyclyl groups include pyridyl, pyridazinyl, pyrimidinyl,
 isoquinolinyl, quinolinyl, quinazolinyl, imidazolyl, pyrrolyll, furanyl,
 thienyl, thiazolyl, benzothiazolyl, piperidinyl, pyrrolidinyl,
 tetrahydrofuranyl, tetrahydropyranyl, and morpholinyl groups.
 "Substituted heterocyclyl" means that the heterocyclyl group is substituted
 by one or more substituents wherein the substituents include alkoxy,
 alkylamino, aryl, carbalkoxy, carbamoyl, cyano, halo, heterocyclyl,
 trihalomethyl, hydroxy, mercaptyl, alkylmercaptyl or nitro.
 "Hydroxyalkyl" means an alkyl group substituted by a hydroxy group. Hydroxy
 lower alkyl groups are preferred. Representative groups include
 hydroxymethyl, 2-hydroxyethyl, 2-hydroxypropyl and 3-hydroxypropyl.
 Preferred Embodiments
 The preparation of N6-substituted adenosine derivatives of formula (I) is
 outlined in Scheme 1.
 ##STR5##
 In Scheme 1, X.sub.1 and X.sub.2 are independently Cl or F,
 Q is CH.sub.2 O;
 T is
 ##STR6##
 or R.sub.3 O--CH.sub.2 ;
 X is a straight or branched chain alkylene, cycloalkylene or
 cycloalkenylene group;
 Y is NR.sub.4, O or S;
 a=0 or 1;
 Z is of the formula
 ##STR7##
 or
 ##STR8##
 Z.sub.1 is N, CR.sub.5, (CH).sub.m -CR.sub.5 or (CH).sub.m -N, m being 1 or
 2;
 Z.sub.2 is N, NR.sub.6, O or S, n being 0 or 1;
 R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are independently
 H, alkyl, aryl or heterocyclyl;
 R.sub.7 and R.sub.8 are independently hydrogen, alkyl, aralkyl, carbamoyl,
 alkyl carbamoyl, dialkyl-carbamoyl, acyl, alkoxycarbonyl,
 aralkoxycarbonyl, aryloxycarbonyl, or R.sub.7 or R.sub.8 together may form
 ##STR9##
 where R.sub.c is hydrogen or alkyl,
 ##STR10##
 where R.sub.d and R.sub.e are independently hydrogen, alkyl, or together
 with the carbon atom to which they are attached may form a 1,1-cycloalkyl
 group; and
 R.sub.a and R.sub.b are independently H, OH, alkyl, hydroxyalkyl, alkyl
 mercaptyl, thioalkyl, alkoxy, alkoxyalkyl, amino, alkyl amino, carboxyl,
 acyl, halogen, carbamoyl, alkyl carbamoyl, aryl or heterocyclyl.
 As shown in Scheme 1, the preparation of N6-substituted adenosine
 derivatives of formula (I), begins with the reaction of the
 2,4-dihalo-3-nitropyridine compound (II) with an amine of formula H.sub.2
 N--X--(Y).sub.a -Z (III) provides the 2-halo-3-nitro-4-aminopyridine
 compound (IV). The reaction is carried out in the presence of a tertiary
 amine or aromatic amine base such as triethylamine, diisopropylethylamine,
 pyridine, 4-dimethylaminopyridine, 4-methylmorpholine, and the like or an
 inorganic carbonate base such as sodium carbonate, potassium carbonate,
 and the like. Preferred bases are 4-methylmorpholine, triethylamine and
 diisopropylethylamine. The reaction is carried out in a polar aprotic
 solvent such as 1-methyl-2-pyrrolidone or dimethylformamide, an aromatic
 solvent such as benzene or toluene, a higher-boiling ethereal solvent such
 as diglyme or a hindered alcohol such as isopropanol or 2-butanol at a
 temperature of from about ambient temperature to the reflux temperature of
 the solvent. A preferred solvent is 1-methyl-2-pyrrolidone, in which the
 reaction is carried out at about 35.degree. C.
 The 2-halo-3-nitro-4-aminopyridine compound (IV) is then reacted with the
 protected dihydroxyaminocyclopentane compound (V) using the reaction
 conditions described above to prepare the 2,4-diamino-3-nitropyridine
 compound (VI).
 Reduction of the 2,4-diamino-3-nitropyridine compound (VI) to the
 2,3,4-triaminopyridine compound (VII) is accomplished using methods well
 known in the art for the reduction of aromatic nitro compounds. Preferred
 methods for the reduction employ metals such as zinc, iron or tin in the
 presence of an acidic proton source such as ammonium acetate. Solvents are
 generally alcohols, preferably methanol or mixtures of alcohols and
 aromatic solvents such as toluene or benzene. The reduction is preferably
 accomplished at about ambient temperature. The nitro group is also reduced
 by catalytic hydrogenation, with platinum catalysts such as PtO.sub.2
 being preferred.
 The groups R.sub.7 and R.sub.8 are then removed using methods well known in
 the art to form the 2-N-cyclpentane diol-2,3,4-triaminopyridine compound
 (VIII). In a preferred aspect of this process, R.sub.7 and R.sub.8
 together form a dimethylacetonide, in which case the acetonide is
 hydrolyzed with acid using any solvent which is stable to the acid. A
 preferred method of acetonide hydrolysis uses HCl and methanol at about
 ambient temperature. The 2-N-cyclpentane diol-2,3,4-triaminopyridine
 compound (VIII) may be isolated as the acid solution salt, for example as
 the dihydrochloride salt, or the acid addition salt may be neutralized
 with base to form the free amine. In a preferred aspect, the
 2-N-cyclpentane diol-2,3,4-triaminopyridine compound (VIII) is isolated as
 its crystalline acid addition salt. In many instances, the properties of
 the 2-N-cyclpentane diol-2,3,4-triaminopyridine acid addition salt,
 including ease of crystallization, are controlled by the particular acid
 counterion. The counterion in the isolated acid addition salt is selected
 by treating the free amine with the desired acid, or preferably, by
 employing an acid having the desired counterion in the acid hydrolysis
 described above.
 The 2-N-cyclpentane diol-2,3,4-triaminopyridine compound (VIII) is then
 cyclized to the N6-substituted adenosine derivative (I) by reaction with
 formamidine acetate, formic acid or the ortho esters of formic acid as
 described in U.S. Pat. Nos. 5,364,862 and 5,561,134 and Intern. Pat. App.
 No. PCT/US97/21439, incorporated herein by reference. Polar aprotic
 solvents, higher boiling alcohols and esters and aromatic solvents such as
 toluene have been employed for these types of cyclizations. Esters such as
 n-propyl and n-butyl acetate or a combination of one of these esters with
 ethyl acetate are especially preferred. The N6-substituted adenosine
 derivative (I) is preferably purified by recrystallization from a suitable
 organic solvent or mixture of organic solvents. Ethyl, n-propyl and
 n-butyl acetate and propionate are preferred solvents.
 In a preferred aspect of the foregoing process,
 Q is CH.sub.2 ;
 T is
 ##STR11##
 X is a straight or branched chain alkylene;
 a=0;
 Z is
 ##STR12##
 Z.sub.1 is N, CR.sub.5, (CH).sub.m -CR.sub.5 or (CH).sub.m -N, m being 1 or
 2;
 Z.sub.2 is N, NR.sub.6, O or S, n being 0 or 1;
 R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are independently H
 or alkyl;
 R.sub.7 and R.sub.8 are independently hydrogen or alkyl, or R.sub.7 and
 R.sub.8 together may form
 ##STR13##
 where R.sub.d and R.sub.e are independently hydrogen or alkyl, or together
 with the carbon atom to which they are attached may form a 1,1-cycloalkyl
 group; and
 R.sub.a and R.sub.b are independently H, OH, alkyl, hydroxyalkyl, alkyl
 mercaptyl, thioalkyl, alkoxy, alkoxyalkyl, amino, alkyl amino, carboxyl,
 acyl, halogen, carbamoyl, alkyl carbamoyl, aryl or heterocyclyl.
 In a more preferred aspect, this invention is directed to a process for
 preparing
 [1S-[1.alpha.,2.beta.,3.beta.,4.alpha.(S*)]]-4-[7-[[1-[(3-chlorothien-2-yl
 )methyl]propyl]amino]-3H-imidazo[4,5-b]pyrid-3-yl] N-ethyl
 2,3-dihydroxycyclopentanecarboxamide as shown in Scheme 2.
 ##STR14##
 As shown in the foregoing Scheme 2, reaction of the
 2,4-dihalo-3-nitropyridine compound (II) with
 (R)-1-(3-chloro-thienyl-2-yl)-2-aminobutane hydrochloride (IX) using the
 conditions of base and solvent described in Scheme 1 above provides
 (R)-N-[1-[(3-chlorothien-2-yl)methyl]propyl]-2-halo-3-nitro-4-pyridinamine
 (X) (X.sub.2 is Cl or F). The reaction is conducted in the presence of
 excess base to neutralize the hydrochloride salt of (IX), or
 alternatively, the hydrochloride salt of (IX) is neutralized with base
 separately, and a solution of the free base (IX) is then mixed with a
 solution of the 2,4-dihalo-3-nitropyridine compound (II).
 Preferred 2,4-dihalo-3-nitropyridine compounds are the compounds of formula
 (II) wherein X.sub.1 is F and X.sub.2 is F or Cl, or a mixture thereof,
 which are prepared by reaction of 2,4-dichloro-3-nitropyridine with a
 fluorinating agent such as KF.
 The
 (R)-N-[1-[(3-chlorothien-2-yl)methyl]propyl]-2-halo-3-nitro-4-pyridinamine
 (X) is then reacted with
 3aR-[3a.alpha.,4.alpha.,6a,6a.alpha.]-6-amino-N-ethyltetrahydro-3,3-dimeth
 yl-2,4-dioxabicyclo[3.3.0]octan-8-carboxamide benzoate (XI) as described in
 Scheme 1 above to form
 [3aR-[3a.alpha.,4.alpha.,6a(R*),6a.alpha.]]-6-[4-[[1-[(3-chlorothien-2-yl)
 methyl]propyl]amino]-3-nitropyrid-2-ylamino] N-ethyl
 tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-carboxamide (XII)
 which is then reduced as described in Scheme 1 above to form
 [3aR-[3a.alpha.,4.alpha.,6a(R*),6a.alpha.]]-6-[4-[[1-[(3-chlorothien-2-yl)
 methyl]propyl]amino]-3-aminopyrid-2-ylamino] N-ethyl
 tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-carboxamide (XIII).
 The reduction is preferably accomplished using zinc, iron or tin in the
 presence of an acidic proton source such as ammonium acetate, zinc being
 especially preferred.
 Acid hydrolysis of the dimethylacetonide provides
 [1S-[1.alpha.,2.beta.,3.beta.,4.alpha.(S*)]]-4-[3-amino-4-[[1-[3-chlorothi
 en-2-yl)methyl]propyl]amino]pyrid-2-ylamino] N-ethyl
 2,3-dihydroxycyclopentanecarboxamide. The hydrolysis is preferably
 accomplished using HCl, in which case crystalline
 [1S-[1.alpha.,2.beta.,3.beta.,4.alpha.(S*)]]-4-[3-amino-4-[[1-[3-chlorothi
 en-2-yl)methyl]propyl]amino]pyrid-2-ylamino] N-ethyl
 2,3-dihydroxycyclopentane carboxamide dihydrochloride is isolated
 directly.
 Cyclization of the
 [1S-[1.alpha.,2.beta.,3.beta.,4.alpha.(S*)]]-4-[3-amino-4-[[1-[3-chlorothi
 en-2-yl)methyl]propyl]amino]pyrid-2-ylamino] N-ethyl
 2,3-dihydroxycyclopentanecarboxamide to form
 [1S-[1.alpha.,2.beta.,3.beta.,4.alpha.(S*)]]-4-[7-[[1-[(3-chlorothien-2-yl
 )methyl]propyl]amino]-3H-imidazo[4,5-b]pyrid-3-yl] N-ethyl
 2,3-dihydroxycyclopentane carboxamide (I) is accomplished as described in
 Scheme 1 above.
 In a still more preferred aspect, this invention is directed to a synthesis
 of
 [1S-[1.alpha.,2.beta.,3.beta.,4.alpha.(S*)]]-4-[3-amino-4-[[1-[3-chlorothi
 en-2-yl)methyl]propyl]amino]pyrid-2-ylamino] N-ethyl
 2,3-dihydroxycyclopentane carboxamide dihydrochloride hydrate effected in
 a concatenated manner without isolation of the intermediate compounds
 (R)-N-[1-[(3-chlorothien-2-yl)methyl]propyl]-2-halo-3-nitro-4-pyridinamine
 (X),
 [3aR-[3a.alpha.,4.alpha.,6a(R*),6a.alpha.]]-6-[4-[[1-[(3-chlorothien-2-yl)
 methyl]propyl]amino]-3-nitropyrid-2-ylamino] N-ethyl
 tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-carboxamide (XII) and
 [3aR-[3a.alpha.,4.alpha.,6a(R*),6a.alpha.]]-6-[4-[[1-[(3-chlorothien-2-yl)
 methyl]propyl]amino]-3-aminopyrid-2-ylamino] N-ethyl
 tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-carboxamide (XIII).
 In particular, 2,4-dihalo-3-nitropyridine is reacted with
 (R)-1-(3-chloro-thienyl-2-yl)-2-aminobutane hydrochloride in a reaction
 vessel using the condition of base and solvent described above to form a
 solution of
 (R)-N-[1-[(3-chlorothien-2-yl)methyl]propyl]-2-halo-3-nitro-4-pyridinamine
 (X) in the reaction vessel.
 3aR-[3a.alpha.,4.alpha.,6a,6a.alpha.]-6-amino-N-ethyltetrahydro-3,3-dimeth
 yl-2,4-dioxabicyclo [3.3.0]octan-8-carboxamide benzoate (XI) and base are
 then added to the solution in the vessel and the mixture is heated until
 conversion to
 [3aR-[3a.alpha.,4.alpha.,6a(R*),6a.alpha.]]-6-[4-[[1-[(3-chlorothien-2-yl)
 methyl]propyl]amino]-3-nitropyrid-2-yl-amino] N-ethyl
 tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-carboxamide (XII) is
 essentially complete.
 The reaction mixture contained in the vessel is the mixed with water and
 the aqueous phase, which contains water soluble solvents and impurities,
 is removed from the vessel.
 An alcoholic solvent such as methanol, followed by ammonium acetate and
 zinc powder, is then added to the organic solution remaining in the vessel
 and the reaction mixture is heated until reduction of the
 [3aR-[3a.alpha.,4.alpha.,6a(R*),6a.alpha.]]-6-[4-[[1-[(3-chlorothien-2-yl)
 methyl]propyl]amino]-3-nitropyrid-2-ylamino] N-ethyl
 tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-carboxamide (XII) to
 [3aR-[3a.alpha.,4.alpha.,6a(R*),6a.alpha.]]-6-[4-[[1-[(3-chlorothien-2yl)m
 ethyl]propyl]amino]-3-aminopyrid-2-ylamino] N-ethyl
 tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-carboxamide (XIII) is
 essentially complete.
 The zinc metal and any other insoluble impurities are then removed by
 filtration of the contents of vessel and the filtrate is collected in
 another reaction vessel. Acid hydrolysis of
 [3aR-[3a.alpha.,4.alpha.,6a(R*),6a.alpha.]]-6-[4-[[1-[(3-chlorothien-2-yl)
 methyl]propyl]amino]-3-aminopyrid-2-ylamino] N-ethyl
 tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-carboxamide (XIII) is
 then effected by adding concentrated HCl to the second reaction vessel and
 heating until conversion to
 [1S-[1.alpha.,2.beta.,3.beta.,4.alpha.(S*)]]-4-[3-amino-4-[[1-[3-chlorothi
 en-2-yl)methyl]propyl]amino]pyrid-2-ylamino] N-ethyl
 2,3-dihydroxycyclopentanecarboxamide (XIII) is essentially complete.
 Cooling of the second reaction vessel results in crystallization of
 [1S-[1.alpha.,2.beta.,3.beta.,4.alpha.(S*)]]-4-[3-amino-4-[[1-[3-chlorothi
 en-2-yl)methyl]propyl]amino]pyrid-2-ylamino] N-ethyl
 2,3-dihydroxycyclopentane carboxamide dihydrochloride hydrate which is
 isolated by filtration.
 In another still more preferred aspect, this invention is directed to the
 concatenated synthesis described above further comprising converting
 2,4-dichloro-3-nitropyridine to 2,4-difluoro-3-nitropyridine, or a mixture
 of 2,4-difluoro-3-nitropyridine and 2-chloro-4-fluoro-3-nitropyridine in
 the reaction vessel R.sub.1.
 In order to successfully concatenate reaction steps as describe above,
 solvents and reagents for accomplishing each of the individual reaction
 steps are selected such that excess reagents and reaction byproducts are
 readily removed by filtration or extraction, or can remain in the reaction
 vessel without causing detrimental effects on subsequent reactions
 performed in the vessel. Accordingly, the following reagents and solvents
 are preferred.
 (1) For conversion of 2,4-dichloro-3-nitropyridine to
 2,4-difluoro-3-nitropyridine; KF; tetraphenylphosphonium bromide;
 1-methyl-2-pyrrolidinone; toluene.
 (1) For conversion of 2,4-dihalo-3-nitropyridine to
 [3aR-[3a.alpha.,4.alpha.,
 6a(R*),6a.alpha.]]-6-[4-[[1-[(3-chlorothien-2-yl)methyl]propyl]amino]-3-ni
 tropyrid-2-ylamino]N-ethyltetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole
 -4-carboxamide (XII): 1-methyl-2-pyrrolidinone; triethylamine;
 diisopropylethylamine; 4-methyl morpholine.
 (2) For conversion of to
 [3aR-[3a.alpha.,4.alpha.,6a(R*),6a.alpha.]]-6-[4-[[(1-[(3-chlorothien-2-yl
 )methyl]propyl]amino]-3-nitropyrid-2-ylamino] N-ethyl
 tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-carboxamide (XII) to
 [3aR-[3a.alpha.,4.alpha.,6a(R*),6a.alpha.]]-6-[4-[[1-[(3-chlorothien-2-yl)
 methyl]propyl]amino]-3-aminopyrid-2-ylamino] N-ethyl
 tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-carboxamide (XIII):
 ethyl acetate, methanol, zinc, ammonium acetate.
 (3) For conversion of to
 [3aR-[3a.alpha.,4.alpha.,6a(R*),6a.alpha.]]-6-[4-[[1-[(3-chlorothien-2-yl)
 methyl]propyl]amino]-3-aminopyrid-2-ylamino] N-ethyl
 tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-carboxamide (XIII) to
 [1S-[1.alpha.,2.beta.,3.beta.,4.alpha.(S*)]]-4-[3-amino-4-[[1-[3-chlorothi
 en-2-yl)methyl]propyl]amino]pyrid-2-ylamino] N-ethyl
 2,3-dihydroxycyclopentanecarboxamide dihydrochloride hydrate: ethyl
 acetate, concentrated HCl.
 The concatenated synthesis of
 [1S-[1.alpha.,2.beta.,3.beta.,4.alpha.(S*)]]-4-[3-amino-4-[[1-[3-chlorothi
 en-2-yl)methyl]propyl]amino]pyrid-2-ylamino] N-ethyl
 2,3-dihydroxycyclopentanecarboxamide dihydrochloride hydrate described
 above, in which the intermediate compounds are not isolated but rather are
 carried forward to the subsequent reaction in solution, minimizes the four
 step process to two reaction vessels and two filtrations, resulting in a
 significant reduction in cycle time from weeks to 1-2 days, elimination of
 pressurized hydrogenation techniques not easily accommodated or handled in
 conventional reactions, elimination of the isolation of oily or tar
 products, crystallization of previously uncrystallizable materials,
 increased purity of product and significant increase in ease of handling.
 The concatenated synthesis also significantly impacts the ease of
 crystallization and increased purity of the final drug substance,
 [1S-[1.alpha.,2.beta.,3.beta.,4.alpha.(S*)]]-4-[7-[[1-[(3-chlorothien-2yl)
 methyl]propyl]amino]-3H-imidazo[4,5-b]pyrid-3-yl] N-ethyl
 2,3-dihydroxycyclopentane carboxamide.

The foregoing may be better understood by reference to the following
 Examples, which are presented for purposes of illustration and are not
 intended to limit the scope of the invention.
 EXAMPLE 1
 First Concatenated Synthesis of
 [1S-[1.alpha.,2.beta.,3.beta.,4.alpha.(S*)]]-4-[3-amino-4-[[1-[3-chlorothi
 en-2-yl)methyl]propyl]amino]pyrid-2-ylamino] N-ethyl
 2,3-dihydroxycyclopentanecarboxamide dihydrochloride hydrate.
 A reaction vessel R.sub.1 is charged with 11.7 g of
 (R)-1-(3-chloro-thienyl-2-yl)-2-aminobutane hydrochloride (prepared as
 described in PCT/US97/15729, incorporated herein by reference), 55 ml of
 1-methyl-2-pyrrolidinone and 13.5 ml of N,N-diisopropylethylamine at room
 temperature. The mixture is stirred. The reaction vessel R.sub.1 is then
 charged with 7 ml of water and 14.4 ml of triethylamine at room
 temperature. The mixture is stirred and 10.01 g of solid
 2,4-dichloro-3-nitropyridine is directly added to reaction vessel R.sub.1
 at room temperature. The resulting mixture is heated at about 35.degree.
 C. for 6 hours with stirring. The mixture is cooled to room temperature
 with stirring over 16 hours.
 The reaction vessel R.sub.1 is then charged with 19.97 g of
 3aR-[3a.alpha.,4.alpha.,6a,6a.alpha.]-6-amino-N-ethyl
 tetrahydro-3,3-dimethyl-2,4-dioxabicyclo[3.3.0]octan-8-carboxamide,
 benzoate and 32.5 ml of N,N-diisopropylethylamine. The mixture is heated
 to about 105.degree. C. and held at that temperature for 5 hours. The
 mixture is allowed to cool overnight at room temperature. The product of
 the second reaction is identified as
 [3aR-[3a.alpha.,4.alpha.,6a(R*),6a.alpha.]]-6-[4-[[1-[(3-chlorothien-2-yl)
 methyl]propyl]amino]-3-nitropyrid-2-ylamino] N-ethyl
 tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-carboxamide.
 The mixture is then cooled to below 15.degree. C. 80 mL of ethyl acetate
 and 80 mL of water are added and the mixture cooled to 5-10.degree. C. The
 mixture is stirred for 20 minutes while the temperature is maintained at
 5-10.degree. C. Stirring is stopped and the layers are allowed to
 separate, typically over 15-20 minutes. The lower aqueous layer is removed
 to a holding drum/reactor vessel.
 The ethyl acetate layer is washed once with 40 mL of cold water
 (5-10.degree. C.). 30 mL of methanol is added to the ethyl acetate
 reaction mixture in reaction vessel R.sub.1 and the mixture is stirred for
 10 minutes. 31.9 g of ammonium acetate is added to reaction vessel R.sub.1
 and the mixture is stirred for 15 minutes. An endotherm is observed upon
 ammonium acetate addition. 20.3 g of zinc powder is added to reaction
 mixture R.sub.1 in portions until no exotherm is observed. The temperature
 is maintained to about 50.degree. C.
 The mixture in reaction vessel R.sub.1 is then cooled to about 22.degree.
 C. The mixture is filtered onto Whatman filter paper #1 and the filtrate
 transferred to reaction vessel R.sub.2. Reaction vessel R.sub.1 is rinsed
 with ethyl acetate, filtered and the filtrate transferred to reaction
 vessel R.sub.2. The filter cake is washed three times with 50 mL of ethyl
 acetate and the washes are transferred to reaction vessel R.sub.2. 40 mL
 of concentrated hydrochloric acid is added to reaction vessel R.sub.2 and
 the temperature allowed to rise to about 42.degree. C.
 When the hydrolysis reaction is substantially complete, the temperature in
 reaction vessel R.sub.2 is adjusted to about 22.degree. C. and maintained
 at about 22.degree. C. for 2 hours. The reaction mixture is then filtered
 through Whatman filter paper #1. The reaction vessel R.sub.2 is rinsed
 with ethyl acetate and the rinse is filtered through the filter paper.
 17.89 g of crude
 [1S-[1.alpha.,2.beta.,3.beta.,4.alpha.(S*)]]-4-[3-amino-4-[[1-[3-chlorothi
 en-2-yl)methyl]propyl]amino]pyrid-2-ylamino] N-ethyl
 2,3-dihydroxycyclopentanecarboxamide dihydrochloride hydrate is collected
 on the filter paper. The crude product is washed three times with 35 mL of
 ethyl acetate and then transferred to a dryer and dried at about
 50.degree. C. under vacuum to give
 [1S-[1.alpha.,2.beta.,3.beta.,4.alpha.(S*)]]-4-[3-amino-4-[[1-[3-chlorothi
 en-2-yl)methyl]propyl]amino]pyrid-2-ylamino] N-ethyl
 2,3-dihydrocyclopentane carboxamide dihydrochloride hydrate (11.69 g,
 88.5% w/w assay).
 The
 [1S-[1.alpha.,2.beta.,3.beta.,4.alpha.(S*)]]-4-[3-amino-4-[[1-[3-chlorothi
 en-2-yl)methyl]propyl]amino]pyrid-2-ylamino] N-ethyl
 2,3-dihydroxycyclopentanecarboxamide dihydrochloride hydrate prepared as
 described above is converted to
 [1S-[1.alpha.,2.beta.,3.beta.,4.alpha.(S*)]]-4-[7-[[1-[(3-chlorothien-2-yl
 )methyl]propyl]amino]-3H-imidazo[4,5-b]pyrid-3-yl] N-ethyl
 2,3-dihydroxycyclopentanecarboxamide as described in PCT/US97/21439,
 incorporated herein by reference.
 Alternatively, the
 [1S-[1.alpha.,2.beta.,3.beta.,4.alpha.(S*)]]-4-[3-amino-4-[[1-[3-chlorothi
 en-2-yl)methyl]propyl]amino]pyrid-2-ylamino] N-ethyl
 2,3-dihydroxycyclopentanecarboxamide dihydrochloride hydrate prepared as
 described above is purified prior to conversion to
 [1S-[1.alpha.,2.beta.,3.beta.,4.alpha.(S*)]]-4-[7-[[1-[(3-chlorothien-2-yl
 )methyl]propyl]amino]-3H-imidazo[4,5-b]pyrid-3-yl] N-ethyl
 2,3-dihydroxycyclopentanecarboxamide using the method described below.
 11.69 g of
 [1S-[1.alpha.,2.beta.,3.beta.,4.alpha.(S*)]]-4-[3-amino-4-[[1-[3-chlorothi
 en-2-yl)methyl]propyl]amino]pyrid-2-ylamino] N-ethyl
 2,3-dihydroxycyclopentanecarboxamide dihydrochloride hydrate, 70 mL of
 isopropyl alcohol and 70 mL of methanol are added to a reaction vessel.
 The mixture is heated at about 68.degree. C. for 1 hour. The mixture is
 then hot filtered (approximately 50.degree. C.) through Whatman filter
 paper #1. The white solid product is then washed twice with 20 mL of
 isopropyl alcohol:methanol (1:1) at room temperature. The white solid
 product is dried in a vacuum oven for 16 hours at about 40.degree. C. to
 give
 [1S-[1.alpha.,2.beta.,3.beta.,4.alpha.(S*)]]-4-[3-amino-4-[[1-[3-chlorothi
 en-2-yl)methyl]propyl]amino]pyrid-2-ylamino] N-ethyl
 2,3-dihydroxycyclopentanecarboxamide dihydrochloride hydrate (6.95 g,
 97-100% w/w assay).
 EXAMPLE 2
 Second Concatenated Synthesis of [1S-[1.alpha.,2.beta.,
 3.beta.,4.alpha.(S*)]]-4-[3-amino-4-[[1-[3-chlorothien-2-yl)methyl]propyl]
 amino]pyrid-2-ylamino] N-ethyl 2,3-dihydroxycyclopentanecarboxamide
 dihydrochloride hydrate.
 A reaction vessel R.sub.1 is charged with 49.0 g of
 (R)-1-(3-chloro-thienyl-2-yl)-2-aminobutane hydrochloride and 38.2 g of
 2,4-dichloro-3-nitropyridine at room temperature. The reaction vessel
 R.sub.1 is further charged with 225 mL of 1-methyl-2-pyrrolidinone, 120.7
 mL of N,N-diisopropylethylamine and 32.6 mL of N-methylmorpholine at room
 temperature with stirring. The reaction vessel R.sub.1 is heated at about
 72.degree. C. for 8 hours with stirring. The mixture is cooled overnight
 to room temperature with stirring.
 The reaction vessel R.sub.1 is then charged with 76.3 g of
 3aR-[3a.alpha.,4.alpha.,6a,6a.alpha.]-6-amino-N-ethyl
 tetrahydro-3,3-dimethyl-2,4-dioxabicyclo[3.3.0]octan-8-carboxamide
 benzoate. The mixture is heated to about 105.degree. C. and held at that
 temperature for 7 hours. The mixture is allowed to cool overnight at room
 temperature. The product of the second reaction is identified as
 [3aR-[3a.alpha.,4.alpha.,6a(R*),6a.alpha.]]-6-[4-[[1-[(3-chlorothien-2-yl)
 methyl]propyl]amino]-3-nitropyrid-2-ylamino] N-ethyl
 tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-carboxamide.
 The reaction mixture is then cooled to about 25.degree. C. and 300 mL of
 ethyl acetate and 300 g of ammonium chloride (20%) are added to reaction
 vessel R.sub.1. The mixture is stirred for 10 minutes while at 25.degree.
 C. Stirring is then stopped and the layers are allowed to separate. The
 lower aqueous layer is removed to a holding drum/reactor vessel.
 150 mL of water is added to the ethyl acetate layer remaining in reaction
 vessel R.sub.1. The mixture is stirred for 10 minutes. Stirring is stopped
 and the layers are allowed to separate. The lower aqueous layer is removed
 to the holding drum/reactor vessel.
 200 mL of methanol and 91 g of ammonium acetate are added to reaction
 vessel R.sub.1. An exotherm is observed with ammonium acetate addition. 58
 g of zinc powder is added to reaction vessel R.sub.1 in portions until no
 exotherm is observed. The temperature in reaction vessel R.sub.1 is
 maintained at 50.degree. C.
 When the reduction reaction is substantially complete, the mixture in
 reaction vessel R.sub.1 is cooled to 0.degree. C. The mixture is filtered
 through Whatman filter paper #1 and the filtrate is transferred to
 reaction vessel R.sub.2. The filter cake is washed three times with 150 mL
 of ethyl acetate and the washes are transferred to reaction vessel
 R.sub.2. 300 mL of water is added to reaction vessel R.sub.2 and the
 mixture is stirred for 10 minutes. Stirring is stopped and the layers are
 allowed to separate. The lower aqueous layer is removed to a holding
 drum/reactor vessel.
 The ethyl acetate layer is filtered through Whatman filter paper #1.
 Reaction vessel R.sub.2 is rinsed with ethyl acetate and the rinse and
 filtrate are transferred to reaction vessel R.sub.3. 180 mL of methanol
 and 154 mL of concentrated hydrochloric acid are added to reaction vessel
 R.sub.3. The reaction is stirred for 2 hours at room temperature during
 which time an off-white precipitate forms.
 The off-white precipitate is isolated by filtration through Whatman filter
 paper #1 and is washed three times with 150 mL of ethyl acetate. The solid
 product is dried in a vacuum oven at about 45.degree. C. to provide
 [1S-[1.alpha.,2.beta.,3.beta.,4.alpha.(S*)]]-4-[3-amino-4-[[1-[3-chlorothi
 en-2-yl)methyl]propyl]amino]pyrid-2-ylamino] N-ethyl
 2,3-dihydroxycyclopentanecarboxamide dihydrochloride hydrate (68 g, 61%
 yield, 80 A % (HPLC)). MS 470 (M+H). Analysis calculated for C.sub.21
 H.sub.30 N.sub.5 O.sub.3 ClS 2HCl H.sub.2 O: C, 45.13; H, 6.13; N, 12.53;
 Cl, 19.03. Found: C, 45.14; H, 6.15; N, 12.46; Cl, 19.12.
 EXAMPLE 3
 Preparation of 2,4-difluoro-3-nitropyridine
 A reaction vessel is charged with 1 eq. of 2,4-dichloro-3-nitropyridine,
 3.5 eq. of KF, 0.05 eq. of 18-crown-6 and 1-methyl-2-pyrrolidinone. The
 mixture is heated at 100.degree. C. for 15 hours and then cooled to
 22.degree. C. Ethyl acetate is added to the reaction vessel and the
 mixture stirred for 15 minutes. The mixture is washed with water and then
 with brine. The ethyl acetate layer is dried with magnesium sulfate and
 the ethyl acetate removed under reduced pressure to provide the title
 compound.
 EXAMPLE 4
 Third Concatenated Synthesis of
 [1S-[1.alpha.,2.beta.,3.beta.,4.alpha.(S*)]]-4-[3-amino-4-[[1-[3-chlorothi
 en-2-yl)methyl]propyl]amino]pyrid-2-ylamino] N-ethyl
 2,3-dihydroxycyclopentanecarboxamide dihydrochloride hydrate.
 A reaction vessel is charged with 1 eq. of
 (R)-1-(3-chloro-thienyl-2-yl)-2-aminobutane hydrochloride, 1.05 eq. of
 2,4-difluoro-3-nitropyridine and 1-methyl-2-pyrrolidinone. The reaction
 mixture is cooled to -10.degree. C. and N-methylmorpholine added. The
 reaction is complete at the end of the addition. The reaction mixture is
 warmed to 22.degree. C. and 1.1 eq. of
 3aR-[3a.alpha.,4.alpha.,6.alpha.,6a.alpha.]-6-amino-N-ethyltetrahydro-3,3-
 dimethyl-2,4-dioxabicyclo[3.3.0]octan-8-carboxamide benzoate is added and
 the reaction mixture is heated at 100.degree. C. for 1 hour.
 The reaction mixture is cooled to about 22.degree. C. and ethyl acetate and
 20% ammonium chloride are added. The mixture is stirred, the layers are
 allowed to separate and the lower aqueous layer is removed. The organic
 layer is washed with water and then methanol followed by ammonium acetate
 are added. The mixture is stirred for 15 minutes. Zinc powder is added to
 the reaction mixture until no exotherm is observed. The temperature of the
 reaction mixture is maintained at about 50.degree. C.
 The reaction mixture is then cooled to 0.degree. C. and filtered. The
 reaction vessel and filter cake are washed with ethyl acetate. The
 filtrate is washed with water and filtered. Methanol and concentrated
 hydrochloric acid are added to the organic layer and the reaction
 temperature is allowed to rise to about 45.degree. C. The mixture is
 cooled to 22.degree. C. and left standing for two hours. The reaction
 mixture is then filtered and the reaction vessel and filter cake are
 rinsed with ethyl acetate. The solid product is dried at 45.degree. C.
 under 25 mm Hg until constant weight, typically about 3 hours, to provide
 the title compound as an off-white solid (69% yield, 94 A % (HPLC)). MS
 468, 470 (100) (M+H, Cl pattern). Elemental analysis calculated for
 C.sub.21 H.sub.30 N.sub.5 O.sub.3 ClS 2HCl H.sub.2 O: C, 45.13; H, 6.13;
 N, 12.53; Cl, 19.03; S, 5.74. Found: C, 45.11; H, 6.03; N, 12.41; Cl,
 19.16; S, 5.98.
 EXAMPLE 5
 Fourth Concatenated Synthesis of
 [1S-[1.alpha.,2.beta.,3.beta.,4.alpha.(S*)]]-4-[3-amino-4-[[1-[3-chlorothi
 en-2-yl)methyl]propyl]amino]pyrid-2-ylamino] N-ethyl
 2,3-dihydroxycyclopentanecarboxamide dihydrochloride hydrate.
 10.5 g of KF, 3.2 g of tetraphenylphosphonium bromide, 33 mL of
 1-methyl-2-pyrrolidinone and 130 mL of toluene are added to a 250 mL round
 bottom flask. The bulk of the toluene is distilled off at reduced pressure
 (80 mbar, 60-70.degree. C.) to remove residual moisture (110 mL
 collected), after which the slurry is cooled to 24.degree. C. under
 nitrogen. 10 g (0.052 mole) of 2,4-dichloro-3-nitropyridine is added and
 the mixture heated to 100.degree. C. with stirring under a nitrogen. After
 approximately 10 hours, HPLC analysis shows completely conversion to
 2,4-difluoro-3-nitropyridine.
 The reaction mixture is then cooled to 0.degree. C. and 26.2 g (0.26 mole)
 of 4-methylmorpholine added. A solution of 10.6 g (0.047 mole) of
 (R)-1-(3-chloro-thienyl-2-yl)-2-aminobutane, hydrochloride in 15 mL of
 1-methyl-2-pyrrolidinone is added dropwise to the reaction mixture while
 holding the temperature between 5 and 0.degree. C. with a salt water ice
 bath. By the end of the addition, HPLC analysis indicates complete
 conversion of 2,4-difluoro-3-pyridine to the aminated product.
 The reaction is allowed to warm to 24.degree. C. 17.0 g (0.048) of
 3aR-[3a.alpha.,4.alpha.,6.alpha.,6a.alpha.]-6-amino-N-ethyl
 tetrahydro-3,3-dimethyl-2,4-dioxabicyclo[3.3.0]octan-8-carbox-amide
 benzoate is added and the mixture heated at 90.degree. C. for 4 hours.
 After 4 hours, HPLC analysis indicates complete conversion to
 [3aR-[3a.alpha.,4.alpha.,6a(R*),6a.alpha.]]-6-[4-[[1-[(3-chlorothien-2-yl)
 methyl]propyl]amino]-3-nitropyrid-2-ylamino] N-ethyl
 tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-carboxamide. The
 reaction mixture is cooled to 24.degree. C., diluted with 80 mL of ethyl
 acetate and washed with 80 mL of 20% aqueous ammonium chloride solution.
 The aqueous phase is separated and removed. The organic phase is washed
 with 40 mL of water. Following the removal of the aqueous phase, 24 mL of
 methanol is added to the organic phase followed by the addition of 27.0 g
 (0.35 mole) of ammonium acetate. The mixture is cooled to 5.degree. C. in
 an ice bath. 17 g (0.26 mole) of zinc dust is added in portions while
 maintaining the reaction temperature below 40.degree. C. This addition is
 exothermic. Following the addition, the deep yellow color of the reaction
 mixture turns a light amber color and a grayish precipitate forms. TLC
 analysis at this point shows complete reduction.
 The mixture is cooled to 0.degree. C. and filtered to remove zinc salts.
 The filter cake is washed three times with 40 mL of ethyl acetate and the
 combined filtrate and washes are washed with 80 mL of water. Some
 additional inorganic precipitate forms during this process and is removed
 by filtration. Following phase separation, 27 mL of methanol is added to
 the organic phase, followed by 33 mL of concentrated HCl. The temperature
 of the mixture raised to 40.degree. C. The mixture is then allowed to cool
 to 24.degree. C. over 3 hours, after which HPLC analysis shows that the
 acetonide deprotection is complete.
 The mixture is cooled to 0.degree. C. and a solid tan precipitate is
 isolated by filtration. The reaction flask and filter cake are washed 3
 times with 30 mL of cold (5.degree. C.) ethyl acetate. The solid is then
 dried in a vacuum oven (45.degree. C., 27 mm Hg) to constant weight (4
 hours) to provide the title compound (22.9 g, 79% based on
 2,4-dichloro-3-nitropyridine) as an off white solid. Analysis calculated
 for C.sub.21 H.sub.34 Cl.sub.3 N.sub.5 O.sub.4 S.sub.1 ; C, 45.13; H,
 6.13; N, 12.53; S, 5.74; Cl 19.03. Found: C, 45.32; H, 6.31; N, 12.24; S,
 5.86; Cl 18.90.
 While the invention has been described in connection with what is presently
 considered to be the most practical and preferred embodiments, it is to be
 understood that the invention is not limited to the disclosed embodiments,
 but on the contrary is intended to cover various modifications and
 equivalent arrangements included within the spirit and scope of the
 appended claims.
 Thus, it is to be understood that variations in the present invention can
 be made without departing from the novel aspects of this invention as
 defined in the claims. All patents and articles cited herein are hereby
 incorporated by reference in their entirety and relied upon.