USEFUL AS NEURAMINIDASE INHBITORS

Field of the Invention This invention relates to novel substituted benzene compounds and derivatives thereof useful as neuraminidase inhibitors, to pharmaceutical compositions containing said compounds, and to methods of using said compounds, useful for the prevention, treatment, or amelioration of viral and other infections.

Background of the Invention

Influenza viruses consist of eight pieces of single stranded R A, packaged in orderly fashion within the virion. Each piece codes for one of the major viral proteins. The replication complex is enclosed within a membrane composed of matrix protein associated with a lipid bilayer. Embedded in the lipid bilayer are two surface glycoproteiπ spikes, hemagglutinin (HA) and the enzyme neuraminidase (NA). All of the viral genes have been cloned and the three- dimensional structures of the surface glycoproteins have been determined.

Despite the wealth of information available, influenza remains a potentially devastating disease of man, lower mammals, and birds. No effective vaccine exists and no cure is available once the infection has set in.

Influenza viruses continually undergo antigenic variation in the two surface antigens, HA and NA, toward which neutralizing antibodies are directed. For this reason, vaccines and a subject's natural immune system have not been very effective. Attention is now being directed to finding other potential antiviral agents acting at other sites of the virion. This invention is directed to novel compounds which are useful in inhibiting the viral surface enzyme NA.

Furthermore, many other organisms carry NA. Many of these NA-possessing organisms are also major pathogens of man and/or mammals, including Vibrato choleral, Clostridium perfringes, Streptococcus pneumonia, Arthrobacter sialophilas, and other viruses such as parainfluenza virus, mumps virus, Newcastle disease virus, fowl plague virus, and Sendai virus. Compounds of this invention are also directed to inhibiting NA of these organisms.

In viruses, NA exists as a tetramer made of four roughly spherical subunits and a centrally-attached stalk containing a hydrophobic region by which it is embedded in the organism's membrane. Several roles have been suggested for NA. The enzyme catalyzes cleavage of the o-ketosidic linkage between terminal sialic acid and an adjacent sugar residue. Removal of the sialic acid lowers the viscosity and permits access of the virus to the epithelial

cells. NA also destroys the HA receptor on die host cell, thus allowing elution of progeny virus particles from infected cells. In general, the role of NA is thought to be for the mobility of the virus both to and from the site of infections. Compounds that inhibit neuramiπidase's activity may protect a subject from infection and/or cure a subject once infection has set in. It is a further object of this invention to provide a method of using compounds of this invention for treating and/or curing a viral infection.

Analogs of neuraminic acid, such as 2-deoxy-2,3-didehydro-N-_cery_neu__π__nic acid (DANA) and its derivatives are known to inhibit HA in vitro; however, these compounds are inactive in viva. Palese and Schuiman, in Chemoprophvlaxis and Virus Infection of the Upper Respirator. Tract. Vol. 1 (J. S. Oxford, Ed.), CRC Press, 1977, at PS 189-205.

Von ltzstein et al. describes cyclohexane analogs of a-D-neuraminic acid of the formula:

(a) (b) wherein:

A is O, C, or S in Formula (a), and N or C in Formula (b); R ¹ is C0 ₂ H, P0 ₃ H ₂ , N0 ₂ , S0 ₂ , S0 ₃ H, tetrazolyl-, CH-CHO, CHO, or CH(CHO)-; R ² is H, OR ⁶ , F, Cl, Br, CN, NHR ⁶ , SR ⁶ or CH ₂ X, where X is NHR ⁶ halogen, or OR ⁶ ; R ³ and R ³ ' are H, CN, NHR ⁶ , N ₃ , SR ⁶ , -NOR ⁶ , OR ⁶ , guanidino, NR ⁶ ; R ⁴ is NHR ⁶ , SR ⁶ , OR ⁶ , C0 ₂ R ⁶ , N0 ₂ , CfR ⁶ )^ CH ₂ C0 ₂ R ⁶ , CH ₂ N0 ₂ , or CH ₂ NHR ⁶ ; R ⁵ is CH ₂ YR ⁶ , CHYR ⁶ CH ₂ YR ⁶ or CHYR ⁶ CHYR ⁶ CH ₂ YR ⁶ ; R ⁶ is H, acyl, alkyl, allyl, or aryl;

Y is O, S, NH, or H, and pharmaceutical salts thereof; useful as antiviral agents. This reference does not disclose aromatic derivatives of the present invention.

Summary of the Invention An embodiment of the invention is directed to novel benzene compounds useful for the treatment of influenza. These compounds have the formula:

ll 26

(D or pharmaceutically-suitable salts or prodnig forms thereof, wherein: n is 0-1; m is 0-3; p is 0-1;

R ¹ is selected from the group consisting of -C0 ₂ H, -S0 ₂ H, -S0 ₃ H, -P0 ₃ H ₂ , and tetrazolyl; R ² is selected from the group consisting of H, -OH, and -NH ₂ ; R ³ is selected independently at each occurrence from the group consisting of -OR, -N(R)2, -N _3> -NHC(R ⁶ )NH ₂ , -CN,

-C(R ⁶ )NH ₂ , H, -C(RX-N)NHC(-NH)NH ₂ , -C(-N)RC(-NH)NH ₂ . NHCN, and CH-NOH; R ⁴ is selected from the group consisting of H, -OR, -(CH-) _p NHR ⁷ and -C(0)NHR ⁸ ; R ⁵ is selected from the group consisting of H, -P? , -(CHO* ³ )^ _m CH ₂ ^ _> -NHCO? ⁶ )NH _2> -CH-CHCH ₂ R? , NHCN, and CH-NOH; R ⁶ is selected from the group consisting of -NH, -NOH, -NCN, -O, and -S;

R ⁷ is selected from the group consisting of -C(-Y)R ⁸ , -S^R, -C(0)OR ⁸ , -C(-Y)NHR ⁸ , and -CH ₂ C(-Y)R ⁸ ; R ⁸ is selected from the group consisting of C,-C linear or branched alkyl substituted with 0-3 halogens on each carbon; R is selected from the group consisting of H, Cι-C ₄ linear or branched alkyl, C,-C linear or branched alkyl-OH, C _j -C ₄ linear or branched a_kyl-NH ₂ ; Y is o or S; with the following provisos:

1. When R ¹ is CO _j H, S0 ₃ H, or P0 ₃ H ₂ , and R ⁴ is (CH-) _p NHR ⁷ , Λen R ² , R ³ , and R ⁵ cannot all be H;

2. The number of substituents on the aromatic ring cannot exceed four.

Preferred compounds of this invention are compounds of Formula (I) or pharmaceutically-suitable salts or prodnig forms thereof, wherein: R ¹ is CO2H;

SUBSTT TE SHEET (ROIE 26

R ² is H;

R ³ is selected from the group consisting of H, -OR, -NHC(R ⁶ )NH ₂ , NHCN, and CH-NOH; R ⁴ is selected from the group consisting of H, -OR, -<CH2)pNHR ⁷ , and -C(0)NHR ⁸ ; R ⁵ is selected from the group consisting of H, -NHCN, and CH-NOH; R ⁶ is selected from the group consisting of-NH, -NOH, -NCN, -O, and -S;

R ⁷ is selected from the group consisting of -C(-Y)R ⁸ , -S(0 )R, -C(0)OR ⁸ , -C(-Y)NHR ⁸ , and -CH ₂ C(-Y)R ⁸ ; R ⁸ is selected from the group consisting of C _t -C ₄ linear or branched alkyl substimted with 0-3 halogens on each carbon; m is 0-2; and p is 0, with the following provisos:

1. When R ^l is C0 ₂ H, and R ⁴ is (CH ₂ ) _p NHR ⁷ , then R ² , R ³ , and R ^J cannot all be H; and

2. The number of substituents on the aromatic ring cannot exceed four.

More preferred compounds are compounds of Formula (I) or pharmaceutically-suitable salts or prodnig forms thereof, wherein: R ¹ is CO2H; R ² is H;

R ³ is selected from the group consisting of H, -OH, -NHC(-NH)NH ₂ , NHCN, and CH-NOH; R ⁴ is H, -OH, -OCH ₃ , -NHCOCH _j , -NHSθ2CH ₃ , and C(0)NHCH ₃ ;

R ⁵ is H, -CH ₂ CH(OH)CH ₂ OH, -CH ₂ OH, -NHC(-NH)NH ₂ , and -CH-CHCH ₂ OH, -NHCN, CH-NOH; and m is 0-2; with the following provisos:

1. When R ¹ is C0 ₂ H, and R ⁴ is (CH ₂ ) -NHR ⁷ , then R ² , R ³ , and R ^J cannot all be H; and

2. The number of substituents on the aromatic ring cannot exceed four.

Specifically preferred compounds of mis invention are d e following, or pharmaceutically-suitable salts or prodnig forms thereof: Phenylguanidine; 1 -Phenylbiguanide; 4-Acetylam.nobenzo.c acid; 4-Acetvlaminobenzenesulfonic acid;

4-Acety_am_3ophenyipho_pho_ic acid;

4-(Trif_ucHoace_amido)benzoic acid;

4-T- oacs-_midobe_ E0tc acid;

4-[(Meώyl_>ulfony a ino]beπzoic acid;

3-Gtαnid_αoben_sic acid;

3^A_ι_i_ϊc _y-noi_oiιιo)m_tfayI]aπιinobeι_2oic acid;

3-Cyanoaminoben-sic acid;

3^-Aa_in_-2-ir_ino)e__yIb____3ic acid;

4-(Acst__nino)pheny___-tic acid;

4-{Metfay__α_inocarboπyI ben2oic acid;

4-Acetylam_no-3-_ydroxym_tby_ e_αDic acid;

P^-N-A-Stylamino-5-<__π)θxyph_πy0e___nol -Ac__y__t__inc)-3-{Ξ'^'-d_ayd_Oxypropyl)_«n2oic acid; 4-Acsiy_______o-3-_n_inobei__oic acid;

4-Acety_2_π__mo-3-[(ammoimin∞ acid;

3_[(Am_αoiminom-tby -xn_αo}^^-methyipnpiony_amino)be__» acid; 4-Aceτy__ mc>-3-[(hydr_xyl_α_ino)met_;yI]b___»ic acid; 3-[(Amiao_minomet__y _mino]- -[(metfayl_u_53nyI ___i^ acid;

_^(N-Hydroxyimino)raedιyπ- -[(m-Λy_sulfόny _mino]beπ2Dic acid; 3^((Ammoimino)m-thyI)-_mino}-4-__e_Boxybe-aoic acid; 3^(Amino_m__om_t__y_)aι_ino}- -^ιydιτxybeπzDic acid; 3^B-w[(aminoiminor_tetfay-)-mino]be_goic acid; 3-Am_m_-5-{I(amino_n__o)__e_ayn__nino}benzoic acid; _>[(AminoimmomeώyI)--_ino]-5-[(N-hydnxyi_m__ιo)meώy_]b e_3s acid; and acid.

In the present invention, it has been discovered that the compounds ofFormula (I) above are useful as inhibitors of vinl neuraminidase and neuraminidase from other pathogenic organisms, and fbr the prevention, .i miifπt _, or amelioration of vinl and other infections.

A further embodiment of this invention ts directed to ph_uum_.Hii.ra compositions contatnins a compound of

Formula (I) useful for the prevention, treatment, or amelioration of a viral infection.

Still a further embodiment of this invention is directed to methods for preventing, treating, or ameliorating viral infections comprising administering to a host infected with such vims a therapeutically effeαive amount of one or more compounds of Formula (I) as described above.

The compounds herein described may have asymmetric centers. All chiral, diastereomeric, and racemic forms are included in die present invention. Many geometric isomers of olefins, C-N double bonds, and die like can also be present in die compounds described herein, and all such stable isomers are contemplated in the present invention.

When any variable (for example, R through R ⁸ , m, n, p, etc.) occurs more than one time in any constituent or in Formula (I) or any other formula herein, its definition on each occurrence is independent of its definition at every other occurrence. Also, combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.

As used herein and in the claims, "alkyl" is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms; "alkoxy" represents an alkyl group of indicated number of carbon atoms attached through an oxygen bridge; "cycloalkyr is intended to include saturated ring groups, such as cyclopropyL, cyclobutyl, cyclopentyl, cyclohexyl, cyclohepryL, and cyclooctyl; and "bicycloalkyF is intended to include saturated bicyclic ring groups such as [3.3.0] bicyclooctane, [4.3.0] bicyclononane, [4.4.0] bicyclodecane (decalin), [22.2] bicyclooctane, and so forth. "Halogen," as used herein and in the claims, refers to fluoro, chloro, bromo, and iodo; and "counterion" is used to represent a small, negatively-charged species such as chloride, bromide, hydroxide, acetate, sulfate, and the like.

As used herein and in the claims, "aryl" or "aromatic residue" is intended to mean phenyl or naphthyl; "carbocyclic" is intended to mean any stable 5- to 7-membered monocyciic or bicyclic or 7- to 14-membered bicyclic or tricyclic carbon ring, any of which may be saturated, partially unsaturated, or aromatic; for example, indanyl or tetrahydronaphthyl (tetralin).

The term "substituted," as used herein and in the claims, means that a one or more hydrogen on the designated atom is replaced with a selection from me indicated group, provided that the designated atom's normal valency is not exceeded, and tiiat the substitution results in a stable compound.

By "stable compound" or "stable structure" is meant herein a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent As used herein and in die claims, "therapeutically effective amount" refers to tiiat amount necessary to

SUBSTITUTE SHEET (MLE 26)

administer to a host to achieve the desired result of inhibiting die enzyme neuraminidase to prevent, treat, or ameliorate a viral or other infection.

As used herein and in the claims, "pharmaceutically-suitable salts and prodrugs" refers to derivatives of die disclosed compounds that are modified by making acid or base salts, or by modifying functional groups present in die compounds in such a way tiiat the modifications are cleaved, eidier in routine manipulation or in vivo, to the parent compounds. Examples include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; acetate, formate, and benzoate derivatives of alcohols and amines; and the like.

Pharmaceutically-suitable salts of the compounds of the invention can be prepared by reacting d e free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, 2-propanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences. 17th Ed., Mack Publishing Company, Easton, PA, 1982, p. 1418, the disclosure of which is hereby incorporated by reference in its entirety.

Synthesis

The compounds of the present invention may be synthesized using die general synthetic procedures described below. Each of the references cited below are hereby incorporated herein by reference.

Compounds of the Formula (I) where X is C; n is I; m is 0; R ² , R ³ , and R ⁵ are H; R ¹ is C0 ₂ H, S0 H, S0 ₃ H, and P0 ₃ H ₂ ; and R ⁴ is as defined above may be prepared by die following methods. When R ⁴ is (CH ₂ ) _p NH ₂ , it may be reacted in the presence of a base and solvent under standard conditions with a halide, acid anhydride, or sulfonyl halide to produce die corresponding amide or sulfonamide derivatives. Useful bases for this procedure include pyridine, triethylamine, dietiiyiamine, or sodium bicarbonate. Useful solvents include methylene chloride, diethyl ether, methanol, ethanol, benzene, and water. Thioamides of the amides produced above may be prepared under standard conditions using phosphorus pentasutfide in benzene. In the case where R ¹ is tetrazole, n is 0, and R ⁴ is NHCOCH ₃ , conversion of the cyano derivative using sodium azide in dimetiiylformam.de affords die tetrazole derivative.

Compounds of the Formula (I) where X is C; n is 1, m is 0; R ² and R ³ are H; R ⁴ is (CH ₂ ) _p NHR ⁷ and R ⁵ is as defined above may be prepared according to Scheme I. Compound ( 1 ) may be mono-nitrated by treatment witii fuming nitric acid or other standard metiiods to give compound (2). The nitro group may be reduced to die a ine (3) by

hydrogen in the presence of 10% palladium on carbon or platinum oxide. Alternatively, the nitro group in (2) may be reduced witi tin chloride or iron chloride in such solvents as ethanol, methanol, benzene, or toluene. Compound (3) may be treated witii cyanogen bromide to give (4) or witii sodium dicyanamide to give (7). Compound (4) can also be reacted with hydroxylamine hydrochloride to give die hydroxyguanidine adduct (6). The amine (3) may be reacted with cyanamide, NH ₂ C(-NH)S0 ₃ H, or pyrazole carboxamidine in methanol, ethanol, or tetrahydrofuran, respectively, to yield die guanadinium derivative (5). (See G. Wagner, H. Vieweg, and H. Kohmstedt, Pharmazie. 28, 293 (1973); A. E. Miller and J. J. Bischoff, S ^y nthesis. 777 (1986), and M. S. Bematowicz, Y. Wu, and G. R. Matsueda, J. Org. Chem . 57, 2497 (1992).) Treatment of compound (3) witii ammonium isothiocyanate in methanol affords the thiourea (8, S) while treatment with sodium isocyanate yields die corresponding urea (8, 0).

Compounds of the Formula (I) as defined above may be mono- or bis-functionalized according to Scheme II. Reaction of compound (1) witii excess N-bromo succinimide and bromine in carbon tetrachloride containing a trace of azoisobutyronitrile yields die mono- or dibromide (9). Displacement of die bromide with copper cyanide in ditnethylfoπmamide or dimethylsulfoxide yields die cyano derivative (10). Compound (10) can be hydrolyzed with strong mineral acid to yield die amide (11) which can conveniently be transformed to die tiiioamide (12) with phosphorus pentasulfide in benzene. Alternatively, the cyanide (10) can be converted to the amidine derivative (13) by die addition of ammonia in ethanol.

Compounds of the Foπnula (I) where R ⁵ is C(R)(-N)NHC(-NH)NH ₂ , CH-NOH or C(R)(-N)C(-NH)NH ₂ may be prepared according to Scheme III. Reaction of (14) with chlorine and didiiane in the presence of sodium methoxide yields die rearrangement product (IS). Treatment of (IS) with an acyl chloride or acid anhydride yields die N-acyl derivative (16). Removal of the didiiane protecting group is accomplished by treatment with mercuric oxide in the presence of boron trifiuoride etherate in a mixture of tetrahydrofuran and water which yields the aldehyde (17). Compound (17) is tiien condensed widi guanidine to form the guanidine derivative (19), condensed witii hydroxylamine to form the oxime (18), or condensed with aminoguanidine to yield die iminoguanidine derivative (20). These reactions can be performed in water or ethanol.

For compounds of d e Formula (I) where R ⁵ is (CH(R ³ ) _n ) _m CH R ³ , homologation reactions may be performed on compound (14). One-carbon homologation can be effected on (14) through the Gassman reaction [Gassman, P. G.; Drews, H. R. J. Am. Chem. Soc. 1978, 100, 7600-7610] to yield an aldehyde such as (17). The aldehyde function can be reduced to the alcohol under standard conditions using sodium borohydride in methanol. Treatment of the alcohol widi phosphorus tribromide affords d e benzyl bromide while further treatment with potassium cyanide in the presence

of 18-crown-6 yields a benzyl cyanide. Attematively, die aldehyde (17) can be converted to the corresponding benzyl amine by treatment with hydroxylamine hydrochloride followed by reduction of the intermediate oxime (18) with hydrogen over 10% palladium on carbon in ethanol.

A two-carbon homologation of ( 14) may be performed by reduction of die benzyl cyanide above widi hydrogen in the presence of 10% palladium on carbon to yield the phenediylamine derivative. Alternatively, two-carbon homologations could be performed on a compound of Formula (I) where R ¹ is C0 ₂ R, n is 0, and R ⁴ is NH _; with all other positions on the aromatic ring being hydrogen as shown in Scheme IV. Treatment of compound (21) with acetonitrile, boron trichloride, and aluminum chloride yielded die corresponding substituted acetophenone (22). N- acylation of (22) with acetic anhydride in pyridine followed by bromination with N-bromosuccinimide yielded the bromoketone (23). Displacement of me bromide widi hexamethylenetetramine (HMTA) followed by reduction of the ketoπe afforded die amino alcohol (24).

Two-carbon homologations may also be performed according to Scheme V. Treatment of (25) widi ethylene in the presence of palladium acetate in toluene afforded die vinyl derivative (26). Catalytic osmylation of (26) with osmium tetroxide affords die racemic diol (27). Treatment of (27) with triflic anhydride or mesylchloride affords d e intermediate epoxide. Treatment of mis epoxide widi sodium cyanide in dimethylsulfoxide yields d e compound (28). Alternatively, treatment of the epoxide widi sodium azide followed by reduction with hydrogen over 10% palladium on carbon yielded the amine (24).

Three-carbon homologation is also possible via palladium-assisted allylation as described in Scheme VI. Treatment of (25) with allyl iodide in the presence of palladium acetate in toluene afforded die allyl derivative (29). Catalytic osmylation of (29) with osmium tetroxide yielded the diol (30). Triol (32) may be prepared according to Scheme VII. Treatment of (25) with palladium acetate and acrolein dietiiyl acetal yielded (31). Reduction of the aldehyde witii sodium borohydride in methanol followed by catalytic osmylation afforded a diastereomeric mixture of triols (32). The primary alcohol of (32) may be converted to an amino group or cyano group as previously described.

A fourth substituent may be added onto a trisubstituted compound (33) by reaction with fuming nitric acid as shown in Scheme VIII to give (34). The nitro group can tiien be reduced by methods previously described to yield the amine (35). One-carbon homologation of die amine may be performed by starting with die appropriately-substituted dialkyl ethylbenzoate by methods previously described.

Compounds of the type where R ⁴ - C(0)NHR ^s can be readily prepared by die mediods described in Scheme IX. Treatment of (36), which can be purchased, with oxalyl chloride followed by a primary aliphatic amine gives

intermediates (37). Basic hydrolysis selectively cleaves d e ester function to give the acids (38). The preparations of nisubstituted analogues is described in Scheme X. Purchased compound (39) can be selectively esterified by literature procedures to give (40). Treamient of (40) witii diionylchloride followed by a primary aliphatic amine gives die amide (41). Reduction of the nitro substituent gives the amines (42). The amino group can be converted to a guanidino substituent by treamient of (42) with cyanamide to give (43). Hydrolysis of die esters of (43) under basic conditions give the acid (44).

Compound (42) can be hydrolyzed to give the acid (45) or it can be reacted with cyanogen bromide to give the intermediate (46). This intermediate can be converted to the hydroxyguanidino derivative (47) with hydroxylamine or converted to die cyanoguanidino derivative (49) with dicyanamide. Hydrolysis of (47) and (49) gives die acids (48) and (50), respectively.

Scheme I

I ΠUTE SHEET iani 26)

Scheme II

11 12

Scheme III

S0BSππi!ESSE_T__BIE2

Scheme IV

24

Scheme V

28 24

SUBSTTI SHEET BUIE 26

Scheme VI

Scheme VII

32

SUBSTITUTE SHEET R

Scheme VIII

Scheme IX

BSmUTE SHEET HR£ 26

Scheme X

39 40 41

SnCI ₂

Example 1

Phenvleuanidine carbonate hvdrate (2: 1 : IV

A 5-g sample of phenylguanidine carbonate was obtained from Parish Chemical Company and submitted as a white solid, mp 136-142 *C (dec, depends upon rate of heating).

Analysis: Calculated for C ₇ H ₉ N ₃ ^• 0.5 H ₂ C0 ₃ ^• 0.5 H ₂ 0 (175.19): C, 51.42; H, 6.33; N, 23.99

Found: C, 51.04; H, 6_ 6; N, 23.99

Example 2

Benzvlsuanidine hydrochloride.

A 0.1-g sample of benzylguanidine, mp 171-173 *C, was purchased from Maybridge Chemical Company via Ryan Chemical Company, as a white crystalline solid.

Analysis: Calculated for CgH„N ₃ - HCI (185.65): C, 51.75; H, 6.52; N, 22.63

Found: C, 51.78; H, 6.56; N, 22.61

MRS SHEET BUlf 26

Example 3

I-Phenvlbjguanide.

A sample of 1-phenylbiguanide, mp 139-140 'C, was purchased from Research Biochemical International and submitted.

Analysis: Calculated for C _g H, ,N ₅ : C, 54.22; H, 626; N, 39.52

Found: C, 54.18; H, 6_26; N, 39.47

4-Acetylaminobenzoic acid.

A mixture of 4-amtnobenzoic acid (Aldrich, 50.0 g, 0J65 mol) and anhydrous sodium acetate (35.0 g. 0.427 mol) in glacial acetic acid (150 mL) was heated to reflux for 15 h. The mixture was poured into cold water (1 L). The precipitate was separated by filtration and washed several times with cold water. The cake was dried in oven at 70 *C and recrystallized from 2:1 waterethanol to give 43.0 g (68%) of die title compound as off-white flakes, mp 258 *C.

Analysis: Calculated for C9H9NO3. C, 6032; H, 5.06; N, 7.82

Found: C, 60.51; H, 5.14; N, 7.81

Example 5

4-Acetvlaminobenzenesulfιnic acid, sodium salt, hvdrate f 1.1:2V

A 25-g sample of p-acetylaminobenzenesulfinic acid sodium salt dihydrate was purchased from TCI America as a white solid, mp 313-316 'C, and was submitted.

Analysis: Calculated for CgHgN0 ₃ S ^• Na ^• 2 H ₂ 0 (25724): C, 37.35; H, 4.70; N, 5.46

Found: C, 37.24; H, 4.68; N, 5.37

SUIISTIΪI. SHEET RULE 26

Example 6

4-Acetvlaminobenzenesulfonic acid hvdrate (4: 1V ¹

Acetanilide (Aldrich, 6.75 g, 50 mmol) was suspended in 20 mL of anhydrous 12-dichloroetiιane and cooled to 0-5 *C. Trimethylchlorosulfonate was added dropwise in about 10 min and die resulting mixωre was stirred at room temperature for 30 min followed by 1 h at 50 *C. The volatiles were evaporated on a rotoevaporator and the residue was diluted witii 70 mL of anhydrous acetone. The brown solution was stirred at room temperature for 2 h and the precipitate collected through filtration, washed widi 15 mL of acetone, dried in a pistol at acetone reflux in vacuo to give the title compound as a mauve solid (1.65 g, 15%), mp 235-238 ^* C.

Analysis: Calculated for CgH ₉ N0 ₄ S ^• 025 H ₂ 0: C, 43.72; H, 435; N, 6.37

Found: C, 43.48; H, 4_51; N, 626

Ηofmann, K. F.; Simchen, G. Liebigs Ann. Chem., 282-297 (1982).

Example 7

4-Acetvlaminonhenvlphosphonic acid.

To a suspension of (4-_minophenyl)phosphonic acid (Aldrich, 0.00058 mol) in water (1.5 mL) was added sufficient sodium bicarbonate to make a clear solution. To the mixture, acetic anhydride (02 mL, 0.002 mol) was added and stirred for 0.5 h. Again, acetic anhydride (0.2 mL, 0.002 mol) was added and stirred for 0.5 h. The mixture was let stand at room temperature for 2 h and then concentrated hydrochloric acid (0.5 mL) was added. Fine white needles separated out which were collected by filtration, washed widi water and dried to give 0.048 g (38.5%) of die title compound, mp 228 *C (rep. mp 229 ^* C - Bauer, H. J. Am. Ctiem. Soc.1947, 63, 2137-2138).

Analysis: Calculated for C ₈ H, ₀ NO ₄ P ^• H ₂ 0: C, 4121; H, 5.19; N, 6.01

Found: C, 4126; H, 524; N, 5.98

S-BSm.Ξ SHEET (RULE 26

Example 8

H ₃

S-r4-fAc_tvlamino .nhenvn-i rH1-tetra2θle.

A 10.0-g (0.062 mol) sample of 4-cyanoacetaniIide (Aldrich), sodium azide (4.6 g, 0.07 mol), and ammonium chloride (3.75 g, 0.07 mol) were added to 100 mL of dimethylformamide in a 250-mL round-bottom flask with vigorous stirring. The mixture was stirred for 20 h at 120 *C and was then poured into 800 mL of ice water with vigorous stirring. The solid precipitate was collected by filtration and was added to 200 mL water in a 500-mL Erienmeyer flask with vigorous stirring. The solid was again collected and dried in vacuo to give 3.4 g (27%) of the pure title compound as a white solid, mp 287 *C.

Analysis: Calculated for (20320): C, 5320; H, 4.46; N, 34.46

Found: C, 53.33; H, 4.54; N, 34.44

Example 9

4- Trifluoroacetamidolbenzoic acid.

To a 500-mL round-bottom flask (argon atmosphere) equipped with a dropping funnel and calcium chloride drying tube were added 220 mL anhydrous pyridine, 4-aminobenzoic acid (Aldrich, 5 g, 0.036 mol), and triethylamine (7.5 g, 0.072 mol). The solution was cooled on ice and trifluoracetic anhydride was added over 45 min. The yellow solution was removed from the ice and allowed to come to room temperature with stirring. The volume was reduced in vacuo by -60% and die solution was acidified with 3 N HC1. A red solid was removed by filtration. The yellow solution was extracted with ethyl acetate three times with a total volume of -600 mL. The organic phase was washed with 5% HC1 (x 5), water (x 3), brine, and dried with magnesium sulfate. The drying agent was removed by filtration and die filtrate concentrated to a tan solid. This solid was recrystallized from ethyl acetate to give an off-white solid. This solid was dissolved in a minimum of ethyl acetate and applied to a flash filtration TLC grade silica gel (-125 g) column which had been previously eluted wid dichloromethane. The column was eluted with 20% ethyl acetate in dichloromethane (DCM) until TLC (5% meuianoI DCM) indicated die absence of impurities (1.5 L). The product was then eluted widi ediyl acetate. The solvent was again concentrated to an off-white solid which, after drying in vacuo, was recrystallized from THF DCM (1 :8 respectively). This ..crystallization was repeated and activated carbon was added just before die hot filtration step. The off-white solid was collected on a filter and dried in vacuo to give 1.5 g (18%) of the title compound, mp 277-278 *C.

Analysis: Calculated for C ₉ H ₆ F ₃ N0 ₃ (233.145): C, 4637; H, 2.59; N, 6.01

Found: C, 46.46; H, 2.64; N, 6.00

Example 10

4-Thioacetamidobenzoic acid.'

To a solution of diioacetylthioglycolic acid ² ( 1.5 g, 0.010 mol), sodium hydroxide (0.4 g, 0.010 mol), and water (10 mL) was added a suspension of p-aminobenzoic acid (135 g, 0.010 mol) in water (10 mL). The reaction was stirred for 1 h at room temperature but did not go into solution. Additional 1/ZNaOH (-1.0 mL, 0.001 mol) was added until everything went into solution and the reaction was stirred for 1 h more. The reaction was then acidified with \N HC1 and die precipitate which formed was collected by filtration and washed widi cold water. The product was air-dried and gave 0.85 g (44%) of die title compound as a yellow solid. The material was recrystallized from ethanokwater (1 : 1) and the crystals collected by vacuum filtration and dried in vacuo at acetone reflux to give the title compound as tan flakes, mp 220 *C (lit ¹ mp 291 ^# C).

'Nishiguchi; Iwakura. J. Org. Chem. 35, 1591-1593 (1970). ² Jensen; Pederson. Acta Chemica Scandinava IS, 1087-1096 (1961).

Analysis: Calculated for C^ NC^S: C, 5536; H, 4.64; N, 7.17

Found: C, 5523; H, 4.64; N, 7.08

Example 11

4-f2-Methvlpropionvlamino.beπzoic acid. ¹

A sample of ethyl 4-(2-methylpropionylamino)benzoate ² (100 g, 0.0043 mol) was suspended in 1-V aOH (8 JO mL, 0.0085 mol). After stirring for 24 h at room temperature, the reaction was heated at 50 *C for 2 h. Thin-layer chromatographic analysis (Si0 ₂ , 1:1 ethyl acetate-hexane) indicated that starting material had reacted to form a new, lower-running spot (Rf - 0.1). The pH was adjusted to 7.0 with 1_V HCI causing a white precipitate to form. The material was collected by filtration and reciystallized from methanol-ether. The residue was collected by filtration and dried in vacuo at acetone reflux to give 0.44 g (50%) of die title compound as a white solid, mp 238-239 *C.

Analysis: Calculated for C, ,H ₁₃ N0 ₃ : C, 63.77; H, 628; N, 6.76

Found: C, 63.50; H, 6.40; N, 6.79

'CA 112(13) 118467s

PATENT:Austria; AT 388913 B DATE:890925

^A: 114(11) 101280u

Vagaonescu, Marisa, N-Acyl Derivatives of Anesthesin. Stud Univ. Banes-Bolyai, Chem. 1989, 34, 12-14.

Example 12

4-π-Ureido'foenzoic acid.

Urea (17.5 g, 0292 mol), 4-aminobenzoic acid (Aldrich, 10 g, 0.073 mol), 6 rnL glacial acetic acid, and 6 mL concentrated hydrochloric acid were added to 30 mL of water in a 250-mL round-bottom flask. The mixture was heated to reflux for 45 min and was then cooled. The off-white solid was removed by filtration and was washed with water twice and air dried. The pink solid was powdered and dissolved in 500 mL boiling 1:1 ethanoI:THF in a I-L Erienmeyer flask. The solution was filtered while hot and was again boiled to reduce the volume to 200 mL. Cooling to room temperature produced an off-white solid which was removed by filtration and washed widi ethanol. This solid was dried in vacuo to give 32 g (25%) of the title compound, mp >400 *C.

Analysis: Calculated for CgHgN ₂ 0 ₃ (180.162): C, 53.33; H, 4.47; N, 15.55

Found: C, 53.42; H, 4.52; N, 1535

Example 13

4~fMethylaminocarbonylamino.benzoic cid.

A mixture of 3.0 g (022 mol) of 4-aminobenzoic acid (Aldrich) in 100 mL of chloroform was treated widi 10 g (0.175 mol) of mediylisocyanate (Aldrich), and die mixmre was stirred at ambient temperatures for three days. The mixnire was concenσated and the residue was triturated witii diethyl ether/petroleum ether. The solid was collected by filtration, air-dried, and then slurried widi 100 mL of water. The solid was collected by vacuum filtration and die cake was sucked dry. The cake was then recrystallized from acetonitrile/water to yield 1.8 g (43%) of die title compound as a fluffy white solid, mp >400 *C (lit ¹ mp >300 ^β C).

Analysis: Calculated for C^H^N^ (194.19): C, 55.66; H, 5.19; N, 14.43

Found: C, 55.76; H, 5.18; N, 14.44

'Johnston, T. P.; McCaleb, G. S.; Montgomery, J. A. J. Med Chem. 6, 669 (1963).

Example 14

4-tN.N-Dimethvlcarbamvllaminobenzoic acid.

Methyl 4-aminobenzoate (Aldrich, 5.0 g, 33 mmol) was dissolved in 120 mL of anhydrous mediylene chloride containing pyridine (32 mL, 39.6 mmol), and dimethylaminopyridine (DMAP, 500 mg) was added. To die ice-cooled mixture was added dimethylcarbamyl chloride (3.34 mL, 363 mmol) while stirring. The stirring was continued for 16 h at room temperaωre. The reaction was quenched widi water (50 mL) and die organic layer separated. The aqueous layer was extracted witii chloroform (50 mL). The combined organic extract was dried over sodium sulfate. The volatiles were evaporated on a rot-evaporator to give a residue, which was applied to a column of silica gel (2 cm dia, 16 _5 cm high, 42 g). Chloroform (12 L) eluted die starting material (3 g) while the desired material was washed wid 1% methanol in chloroform (700 mL) and crystallized from chlorofoπn-metiianol-hexane. The white solid was separated, dried in air followed by drying in a pistol at acetone reflux in vacuo to give 885 mg (30% based on starting material consumed) of methyl 4-(N,N-dimethylcarbamyl)am_nobenzoate, mp 169-170 *C.

Analysis: Calculated for C, ,H _u N ₂ θ3: C, 59.44; H, 634; N, 12.60

Found: C, 59.42; H, 634; N, 12.55

To a suspension of methyl 4-(N,N-dimethyicarbamyl)aminobenzoate (475 g, 2.13 mmol) in 5 mL of deionized water was added 1 N sodium hydroxide (2-5 mL, aqueous) and die mixωre was heated to reflux for 4 h. The reaction mbcture was filtered and die filtrate was neutralized with 6/V HCI. The precipitated solid was separated through filtration, crystallized from methanol-water, dried in air followed by drying in a pistol at acetone reflux in vacuo to give 246 mg (55.5%) of the title compound as a white solid, mp 237-239 ^* C.

Analysis: Calculated for C, ₀ H, ₂ N2θ ₃ : C, 57.67; H, 5.81; N, 13.45

Found: C 57.65; H, 5.81 ; N, 13.44

Example 15

4 Methoxycarbonyl .aminobenzoic acid.

Methyl 4-aminobenzoate (Aldrich, 5.0 g, 33 mmol) was dissolved in 120 mL of anhydrous methyiene chloride containing pyridine (32 mL, 39.6 mmol) and dimethylaminopyridine (DMAP, 500 mg) was added. To die ice-cooled mixture was added methyl chloroformate (2.80 mL, 363 mmol) while stirring. The stirring was continued for 16 h at room temperature. The reaction was quenched with water (50 mL) and die organic layer separated. The aqueous layer was extracted with chloroform (50 mL). The combined organic extract was dried over sodium sulfate. The volatiles were evaporated on a rotoevaporator to give a residue, which was crystallized from chloroform-methanol. The white solid was separated, dried in air followed by drying in a pistol at acetone reflux in vacuo to give 4.95 g (71.7%) of methyl 4-(methoxycarbonyl)aminobenzoate, mp 177-178 'C.

Analysis: Calculated for C| ₀ Hι )N0 ₄ : C, 57.41; H, 5.30; N, 6.69

Found: C, 57.16; H, 528; N, 6.64

To a suspension of methyl 4-(methoxycar_onyl)_minobenzoate (1.5 g, 7.17 mmol) in 10 mL of deionized water was added \N sodium hydroxide (7 mL, aqueous) and die mixture was heated to 60-70 *C for 8 h. The reaction mixture was filtered and die filtrate was neutralized with 6N HCI. The precipitated solid was separated through filtration, dried in air followed by drying in a pistol at acetone reflux in vacuo to give 754 mg (54%) of die title compound as a white solid, mp 195-196 ^* C.

Analysis: Calculated for C, ₀ H ₉ NO ₅ - 025 H ₂ 0: C, 5538; H, 4.64; N, 7.17

Found: C, 5535; H, 4.67; N, 7.16

SUBST E SHEET Rfll£26

Example 16

4-fnvtethvlsulfonvl.a inolbenzoic cid.

A mixture of ethyl 4-[(methylsulfonyl)amino]benzoate (Lumma, W. C. et al. J. Med Chem. 1987, 30, 758- 763) (2.43 g, 0.01 mol) in \N sodium hydroxide (22 mL, 0.022 mol) was stirred at room temperamre for 24 h and then at 48 ^* C for 24 h. The mixture was acidified with concentrated hydrochloric acid to give a white precipitate which was separated by filtration. The cake was washed witii water, dried, and recrystallized widi etiianol-water to give 1.4 g (65%) of the title compound as white flakes, mp 248 *C.

Analysis: Calculated for C _g H ₉ N0 ₄ S: C, 44.64; H, 421; N, 6_50

Found: C, 44.71; H, 426; N, 6 JO

Example 17

3-Guanidinoben∞ic acid hydrochloride f2:lV

A mixture of 3-aminobenzoic acid (Aldrich, 2.74 g, 0.02 mol), hydrochloric acid (12 V, 1.66 g, 0.02 mol), and cyanamide (1.64 g, 0.04 mol) in absolute ethanol (50 mL) was heated at reflux for 16 h. The mixture was cooled and die precipitate separated by filtration. The cake was washed several times with ether and dried under vacuum to give

1.05 g (26.6%) of the title compound as an off-white amorphous powder, mp 278 *C.

Analysis: Calculated for CgH ₉ N ₃ 0 ₂ ^• 0 J HCI: C, 48.67; H, 4.62; N, 2128

Found: C, 49.10; H, 4.90; N, 21.01

Example 18

3-f Aminofcvanoimino'Unethvllaminobeπzoic acid hvdrate f4:lV

To sodium dicyanamide in 5 mL of deionized water was added dropwise a solution of methyl 3-aminobenzoate (Aldrich, 1.30 g, 8.6 mmol) in 15 mL of 1/V HCI. The resulting clear solution was heated at 80-90 *C for 2 h. The precipitate was collected through filtration, crystallized from chloroform-methanol, dried in air followed by drying in a pistol at acetone reflux in vacuo to give 1.84 g (quantitative) of methyl 3-[amino(cyanoiminomedιyl)]aminobenzoate as a white solid, mp 203-204 ^* C.

Analysis: Calculated for C _I0 H, ₀ N ₄ O2 - 020 CH ₃ OH: C, 54.53; H, 4.84; N, 24.94

Found: C, 5431 ; H, 4.71 ; N, 2530

To a suspension of methyl 3-[amino(cyanoiminomethyl)]aminobenzoate (73 g, 33.47 mmol) in 20 mL of deionized water was added 1/V sodium hydroxide (34 mL, aqueous) and die mixωre heated at reflux for 2 h. The resulting mixωre was filtered and the filtrate neutralized widi 6/V HCI. The turbid solution was filtered and the clear filtrate further acidified using 6N HCI to pH 4.0-4.5. The white solid was separated dirough filtration, washed with 30 mL of water, dried in air followed by drying in a pistol at toluene reflux in vacuo to give 1 6 (18%) of die title compound, mp >300 *C. Further acidification gave more solid 3.96 g (58%) which was slightly impure.

Analysis: Calculated for C ₉ H ₈ N ₄ 0 ₂ ^■ 025 H ₂ 0: C, 51.79; H, 4.10; N, 26.84

Found: C, 51.86; H, 3.97; N, 26.66

Example 19

1 -f 3-Carboxvnhenvl .urea.

Urea (17 g, 0292 mol), m-aminobenzoic acid (Aldrich, 10.0 g, 0.073 mol), water (30 mL), and concentrated HCI (3 mL) were added to a 125-mL Erienmeyer flask and boiled for 45 min. The brown suspension became a purple solution on boiling and a brown precipitate formed after 15 min. The pH of the solution was 7 after 45 min. The mixture was cooled on ice and a mauve solid was collected by filtration, slurried with water, and again collected by filtration. The solid was added to 50 mL boiling ethanol and dimethylformamide (DMF, 25 mL) was added by drop until the solid dissolved. No precipitate formed on cooling. Water was added widi stirring until turbidity and then a few drops of DMF were added to give a clear solution. The solution was cooled on ice and a light-purple solid was collected by filtration. Water was added to die filtrate to precipitate a tan solid which was also collected by filtration. The tan solid was dissolved in 5% sodium bicarbonate and neutralized with a 10% solution of potassium bisulfate. The tan solid was collected by filtration, washed with water, and dried in vacuo. The tan solid was then recrystallized from 700 mL methanol and 300 mL water. After cooling, a purple solid was collected by filtration. The product was precipitated from tiie filtrate with die addition of 1 L of water. This light-tan solid was collected on a filter and dried in vacuo. The product was dissolved in 175 mL boiling ed anol and die volume was reduced to 75 mL and filtered while hot This was slowly cooled to room temperaωre and then on ice to give an off-white solid. This solid was collected on a filter, washed, witii cold edianol, and dried in vacuo to give 2.5 g (20%) of the title compound, mp 276-280 *C.

Analysis: Calculated for CgHgNjOj ( 180.162): C, 5333; H, 4.47; N, 15.55

Found: C, 53.55; H, 4J8; N, 15.42

34 Example 20

1 -f 3-Carboxvphenvl V2-thiourea.

This material was purchased from Transworld Chemicals and submitted as a light-gray solid, mp 183-185 *C. It is pure by MS and *H and ¹³ C NMR.

Analysis: Calculated for CgH _g N ₂ 0 ₂ S (19623): C, 48.97; H, 4.11; N, 1428

Found: C, 48.79; H, 4.17; N, 1422

Example 21

3-Cvanoaminobenzoic acid hvdrate (5: 1 ).

To a 3-aminobenzoic acid (Aldrich, 9.05 g, 66 mmol) in acetic acidnrater (70 mL, 1:1) was added sodium acetate (8.12 g, 99 mmol) and the mbcture was cooled in an ice-water bath. Cyanogen bromide (838 g, 792 mmol) was added in two batches in 10 min and the stirring was continued for 18 h at room temperaωre. The reaction mixωre was poured into 350 g of an ice-water mixωre widi stirring. The precipitate was collected through filtration, washed wid cold water (about 70 mL), dried in air, and crystallized from methanol-water. The white solid was separated through filtration, dried in air followed by drying in a pistol at toluene reflux in vacuo to give 10.40 g (95%) of the title compound, mp 238-240 "C.

Analysis: Calculated for CgH ₆ N ₂ θ2 ^• 0.20 H ₂ 0: C, 57.97; H, 3.89; N, 16.90

Found: C, 58.19; H, 3.87; N, 16.90

i

Example 22

^^ .C^NH-

(Aj ^{■ •' ■ ■■ <} °

C0 ₂ H 3-Aminomethylbenzoic acid hvdrate (4: 1 V

Methy.-3-bromomethylbenzoate (5.72 g, 0.025 mol, Ryan Chemicals (Maybridge)), sodium azide (325 g, 0.05 mol), and tetra n-butylbromide (0.4 g, 0.00125 mol) were added to 100 mL dry benzene in a 250-mL round-bottom flask equipped widi a condenser and calcium chloride drying ωbe in an atmosphere of argon. The mixωre was then heated at reflux for 24 h and monitored by TLC (Bakerflex) using dichloromethane, 2% methanol, and 5% methanol/dichloromethane as solvents. The mixωre was cooled, die inorganic salts were removed by filtration, and washed with benzene. The filtrate was added to a 250-mL separator/ funnel and was then washed three times with water, once with brine, and dried with magnesium sulfate. The drying agent was removed by filtration, and die volume of die clear colorless filtrate was reduced to -50 mL in a 100-mL round-bottom flask. The solution was saturated with argon and 3.1 g (0.025 mol) trimethylphosphite was added by drop over 10 min. The reaction bubbled during the addition (loss of nitrogen) and was mildly exodier ic (once it was necessary to cool on ice with a calcium chloride drying ωbe in place to keep die temperamre below 30 *C). The reaction was followed in the TLC system above and after 18 h, it was necessary to add -200 mg of additional trimethylphosphite. The reaction was complete after stirring overnight. Anhydrous HCI was then bubbled into die solution with vigorous stirring. After 2 h, a precipitate formed and after 4 h, the reaction was complete. The white solid was removed by filtration. It was tiien added to 200 mL ether in a 500-mL Erienmeyer flask and vigorously stirred fbr 5 min. After filtration, the solid was dissolved in a minimum amount of boiling methanol and etiier was added until the solution was slightly turbid. Cooling, filtering, and drying in vacuo gave 42 g (83%) of methyl-3-aminomethylbenzoate hydrochloride, mp 177-178 ^* C.

Analysis: Calculated for C ₉ H , ,N0 ₂ • HCI ^• 025 H ₂ 0: C, 52.44; H, 6.10; N, 6.79

Found: C 52.28; H, 6.02; N, 6.75

A mixture of methyl 3-aminomethylbenzoate hydrochloride (2.014 g, 0.0098 mol) in 1/V sodium hydroxide (25 mL, 0.025 mol) was stirred at room temperaωre for 4 h. The mixωre was neutralized with Dowex-H+ resin. The

S-BSTrrUTE SHEET KILE

mixnire was filtered and acetone (400 mL) was added to the filtrate. The white precipitate was collected by filtration and recrystallized from water-acetone to give 0.80 g of the title compound as a white powder, mp 278-280 •C.

Analysis: Calculated for C _g H ₉ N0 ₂ ^• 025 H ₂ 0: C, 61.72; H, 6.15; N, 8.99

Found: C, 6137; H, 6.08; N, 9.03

Example 23

3-t2-Amino-2-imino.ethylbenzoic acid complex with water (5:1 V

MethyI-3-bromomethylenebenzoate (5.0 g, 0.022 mol, Ryan Chemicals (Maybridge)), dry potassium cyanide (2.9 g, 0.044 mol), and 18-crown-6 (0.5 g, 0.0018 mol) was added to -50 mL acetonitrile in a 100-mL round-bottom flask. The mixωre was vigorously stirred for 24 h at room temperaωre. The reaction was monitored by TLC (Bakerflex) in dichloromethane, 2% methanol, and 5% methanol/dichloromethane. A heavy white precipitate (KBr) was removed by filtration. The filtrate was concentrated to 1/3 of the original volume and water was added until die final volume was -100 mL. The yellow solution was added to a 250-mL separatory funnel and was extracted with dichloromethane until die organic phase was colorless and tiien twice more. The organic layers were combined and dried with magnesium sulfate. The drying agent was removed by filtration and die solvent was concentrated to a light yellow oil. The oil was dissolved into an equal volume of ether and was added to a 15 x 2.5 cm column of TLC grade silica gel and eluted widi ether. The progress of die column was followed with a hand-held UV lamp. When the faster- running impurities had eluted, the product was collected and die solvent was concentrated leaving a light yellow oil. Though this oil was pure by TLC, the product was distilled (101.5 ^* C at 0.05 mm Hg) leaving 3.5 g (92%) of methyl-3- cyanomethylbenzoate.

Analysis: Calculated for C, ₀ H ₉ NO_: C, 68.56; H, 5.18; N, 7.99

Found: C, 6836; H, 5.12; N, 7.97

A sample of methyl 3-(cyanomethyl)benzoate (1.87 g, 0.011 mol) was dissolved in anhydrous CH ₂ C1 ₂ (100 mL) containing absolute EtOH (15 mL). The mixωre was cooled to 0 ^* C and a stream of HCI gas passed through die solution for 10 min. The mixωre was then refrigerated for 6 days. The mixωre was transferred to a 500-mL flask and concentrated in vacuo (0.05 mm Hg) at 0 ^* C to give a white solid. The solid material was dissolved in anhydrous methanol (200 mL) and anhydrous ammonia passed through the solution for 20 min. The mixture was warmed to 50 *C for 18 h. The solution was cooled and filtered to remove solids. The filtrate was concentrated at room temperaωre in vacuo to give a white foam (2.5 g, quantitative). A portion (ca. 1 g) of die material was dried over toluene to give methyl 3-[(aminoiminomedιyl)methyI]benzoate hydrochloride, mp 139-140 *C.

Analysis: Calculated for C^H^N^ ^■ HCI: C, 52.52; H, 5.73; N, 1225

Found: C, 52.82; H, 5.92; N, 12.00

_*

A mbcture of methyl 3-(2-amino-2-imino)ethyl_enzoate hydrochloride (02 g, 0.00087 mol) and ammonium hydroxide (5.0 mL) was stored at room temperature for 24 h. The mixωre was evaporated to dryness and the residue was suspended in water (5 mL). The solid was collected by filtration, washed with water, and dried under vacuum over acetone for 24 h to give 0.14 g (88%) of the title compound as a white powder, mp 219-221 *C.

Analysis: Calculated for C^H. _Q NA ^• 02 H ₂ 0: C, 59.46; H, 5.76; N, 15.41

Found: C, 59.83; H, 5.74; N, 15.12

Example 24

0

II H M_NNCCt_.H- _j3 NH II ,NHCNH ₂

CF ₃ CC_H

l-Acetvlamino-2-f(amiπoiminomethvl.amino1benzene trifluoroacetate M:1V

Mercury(II) chloride (3.97 g, 0.0146 mol) was added to a mixture of 2-aminoacetaniIide (Morgan, K. J.; Turner, A. M. Tetrahedron 1966, 22, 1175-1181) (2.0 g, 0.0133 mol), bis-Boc-thiourea (3.67 g, 0.0133 mol), triethylamine (6.12 mL, 0.0439 mol), and N.N-dimetiiylfoπnamide (26.6 mL) at 0 *C with stirring. After 20 min at 0 *C, thin-layer chromatographic analysis (Si0 ₂ , etiiyl acetate:hexane 1 :1) showed formation of a compound widi a higher Rf and disappearance of the starting material. The mixωre was tiien diluted widi ethyl acetate (350 mL) and filtered through a pad of Celite. The filtrate was washed widi water (75.0 mL) and brine (75.0 mL), dried over Na ₂ S0 ₄ , and concentrated in vacuo. After column chromatography (Si0 ₂ , ethyl acetate:hexane 1:1), the resulting solid was dried in vacuo to give 3.0 g (57.4%) of the title compound as a white solid, mp 165-166 *C (ethyl acetate).

Analysis: Calculated for C _I9 H _2g N ₄ 0 ₃ : C, 58.15; H, 7.19; N, 1428

Found: C, 58.18; H, 7.16; N, 1434

A sample of l-acetylam_no-2-{[t-butoxycarbonylamino-t-butoxycarbonylimin omethyI]amino}benzene (1.10 g, 0.0028 mol) in dichloromethane (10.0 mL) containing trifluoroacetic acid (0.5 mL, 0.0062 mol) was stirred 16 h at room temperature. Thin-layer chromatographic analysis (Si0 ₂ , ethyl acetate:hexane 1:1) showed formation of a compound (80%) as a lower R _f spot in addition to die remaining starting material (20%). The reaction contents were concentrated in vacuo. Additional trifluoroacetic acid ( 13 mL, 0.0186 mol) and dichloromethane ( 10.0 mL) were added to the flask and die reaction was allowed to stir for 2 h. Thin-layer chromatographic analysis (Si0 ₂ , ethyl acetate:hexane 1:1) showed only one spot MS analysis indicated die presence of a mono-Boc intermediate and die guanidino product. After concentration, trifluoroacetic acid (03 mL, 0.0062 mol) and dichloromediane (10.0 mL) were added to the syrup. The reaction was complete after 16 h according to thin-layer chromatographic analysis (Si0 ₂ , chloroform:methanol 4.1 ). The contents were concentrated and d e residue was triturated widi dichloromethane. The resulting solid was collected

by filtration and dried in vacuo to give 0.5 g (58%) of the title compound as an off-white solid, mp 160-162 'C.

Analysis: Calculated for CnH^F _j N^: C, 43.14; H, 428; N, 1829

Found: C, 42.97; H, 421; N, 18.13

Example 25

4-rfAcetvlaminomιethvnbeπzoic acid.

A mixture of 4-aminomethylbenzoic acid (Aldrich, 1.51 g, 0.01 mol) and anhydrous sodium acetate (1.5 g, 0.018 mol) in glacial acetic acid (5 mL) was heated at reflux for 18 h. On cooling, die mixωre was poured into cold water (50 mL). The precipitate was separated by filtration and washed several times with cold water. The cake was recrystallized from water to give 1.4 g (72.5%) of die title compound as a white powder, mp 195 *C.

Analysis: Calculated for C, ₀ H _π Nθ3: C, 62.17; H, 5.74; N, 725

Found: C, 62.14; H, 5.75; N, 720

Example 26

4-f Acetamido.phenylacetic acid.

To 4-aminophenylacetic acid (Aldrich, 5 g, 33 mmol) in 50 mL of anhydrous methanol was added 2.5 mL of concentrated sulfiiric acid cautiously. The mixωre was heated to reflux for 4 h. The reaction was cooled to room temperature and neutralized widi sodium carbonate. The volatiles were evaporated on a rotoevaporator and die residue triturated widi chloroform (80 mL) and saturated sodium bicarbonate (100 mL). The chloroform layer was separated and filtered through a small pack of silica gel. The solvent was evaporated and die residue purified through vacuum distillation at 130-150 *C to give 2.7 g (50%) of methyl 4-aminophenyiacetate as a light brown syrup.

Analysis: Calculated for C ₉ H _u N0 ₂ : C, 65.43; H, 6.71; N, 8.47

Found: C, 65.41 ; H, 6.79; N, 8.42

Methyl 4-aminophenylacetate (1.0 g, 6.05 mmol) was dissolved in 5 mL of anhydrous pyridine containing dimethylaminopyridine (DMAP, 100 mg) and acetic anhydride (5 mL). The stirring was continued for 16 h at room temperaωre. The volatiles were evaporated on a rotoevaporator and the residue diluted widi chloroform-water (50 mL, 1:1). After shaking, die chloroform layer was separated, washed with 20 mL of 1 N HCI, dried over sodium sulfate, and die chloroform evaporated. The residue was crystallized from chloroform-hexane, separated through filtration, dried in air followed by drying in a pistol at acetone reflux in vacuo to give methyl (4-acetamido)phenyl acetate as an opalescent solid, 1.10 g (88%), mp 104-106 ^* C.

Analysis: Calculated for C, ,H,3Nθ3: C, 63.75; H, 632; N, 6.75

Found: C, 63.56; H, 634; N, 6.65

To a suspension of methyl (4-acetamido)phenyl acetate (830 mg, 4 mmol) in 2 mL of deionized water was added IN sodium hydroxide (4 mL, aqueous) and die mixture was heated to 60-70 "C for 3 h. The reaction mixture was filtered and the filtrate neutralized widi 6/VΗC1. The solid was separated through filtration, dried in air followed by drying in a pistol at acetone reflux in vacuo to give 530 mg (69%) of the title compound (CAS Registry No. 18699- 02-0) as a cream-colored solid, mp 158-160 *C.

Analysis: Calculated for C^ ,N0 ₃ : C, 6221; H, 5.74; N, 725

Found: C, 6230; H, 5.70; N, 721

Example 27

4-fMethvlaminocarbonvnbenzoic acid.

Monomethylterephdialate (Aldrich, 5.0 g, 0.028 mol) was added to 80 mL of dry benzene in a 250-mL round- bottom flask under argon and fitted with a calcium chloride drying ωbe. Dry pyridine (4.4 g, 0.056 mol) was added and die mixωre was stirred for 15 min. Oxalyl chloride (3.66 g, 0.029 mol) was added by drop over 10 min. The mixture bubbled vigorously during die addition and a heavy white precipitate formed. The mixωre was vigorously stirred for 40 min and followed by TLC (three solvent systems: dichloromethane, 2% methanol, and 5% methanol). Anhydrous methyl-mine was bubbled into die mixωre for 10 min. The precipitate dissipated somewhat and die solution color changed from colorless to green and finally to yellow. The mixωre was then stirred overnight The remaining white precipitate was collected on a filter and washed with benzene. The filtrate was washed widi water and dried over sodium sulfate. The drying agent was removed by filtration, washed widi benzene, and die solvent concentrated to give a white solid. The solids were combined and dissolved in 300 mL dichloromethane. The solution was washed 3 times widi 5% HCI, 3 x 5% sodium bicarbonate, 3 x water, 1 x brine, and tiien dried over sodium sulfate. The drying agent was removed by filtration and die filtrate concentrated to give a white solid which was dried in vacuo. This white solid was recrystallized from methanol to give 2 g (37%) of methyl 4-(mediyiaminocarbonyl)benzoate, mp 126 *C.

Analysis: Calculated for C _l0 H, ,NO3 (193202): C, 62.17; H, 5.74; N, 725

Found: C, 62.16; H, 5.74; N, 725

Mediyl 4-(methylaminocarbonyI)benzoate (1.9 g, 0.0098 mol) was added to lO mL 1.5 NaOH (1.5 eq) and 30 mL water in a 125-mL Erienmeyer flask. The suspension was stiπred for 1 h at room temperaωre. The solid gradually dissolved during this time. The solution was filtered and acidified to produce a white solid. The solid was removed by filtration, washed twice with water, and dried in vacuo to give 1.60 g (90%) of the title compound, mp 270

^• c.

Analysis: Calculated for C^NO _j (179.175): C, 60.03; H, 5.06; N, 7.82

Found: C, 60.07; H, 5.03 ; N, 7.77

Example 28

2-Hvdroxv-S-thvdroxvmethvnbenzoic acid.

To a solution of methyl-5-foππylsalicylate (Kadaba, J Pharm. Sci. 1974, 63, 1333-1335) (1.80 g, 0.01 mol) in methanol (20 mi) at 0 *C was added sodium borohydride (038 g, 0.01 mol) in 4 portions during 0.5 h. The solution was further stirred at 0 *C for 2 h and tiien at room temperaωre for 03 h. It was neutralized with hydrochloric acid and concentrated under vacuum. The residue was passed through a column of silica gel using 1 : 1 ethyl acetate:hexane as the eluent to give 1.0 g (55%) of methy_-2-hydroxy-5-hydroxymethylbenzoate as a pale yellow powder, mp 66-67 *C.

Analysis: Calculated for C^HJ _Q O^ C, 5934; H, 5.49

Found: C, 59.40; H, 5.58

A solution of medιyl-2-hydroxy-5-(hydroxymethyl)benzoic (0.364 g, 0.002 mol) in 1/V sodium hydroxide (4 mL) was stirred at room temperaωre for 16 h. The solution was acidified with hydrochloric acid and concentrated under vacuum. The residue was dissolved in methanol (5 mL) and filtered. The filtrate was concentrated and the residue was dissolved in water (4 mL). The resultant mixmre was extracted with 10:1 ethyl acetateanethano! (5 L). The extract was concentrated to give 0.18 g (54%) of the title compound as an off-white powder, mp 108- 110 ^* C.

Analysis: Calculated for C ₈ H ₈ 0 ₄ : C, 57.14; H, 4.76

Found: C, 56.88; H, 4.76

SUBSTΠUTE SHEET RULE 26

Example 29

2-Hvdroxv-3-π ^, J..3 ^, -dihvdroxvpropvl.benzoic acid.

To a solution of 1 : 1 water and tert-butanol (50 mL), AD-mix-α (Aldrich, 7.0 g) was added and stirred for 0.1 h. The mixmre had two layers; die lower layer was yellow. To this mixmre, methy!-2-hydroxy-3-ailylbenzoate (Claisen, Chem. Ber. 1912, 45, 3157-3166) (0.96 g, 0.005 mol) was added in one portion and stirred at room temperature for 48 h. The reaction mbcωre was cooled to 0 *C and sodium sulphite (7.5 g) was added. The reaction was stirred at 0 *C for 03 h and tiien at room temperature for 0.5 h. It was extracted with ethyl acetate (3 x 50 mL), the combined organic layers were dried over sodium sulphate, filtered, and concentrated. The residue was passed through a column of silica gel fast using 1 : 1 ethyl acetate:hexane as eluent to recover the starting material and men pure ethyl acetate to give 0.96 g (85%) of methyl-2-hydroxy-3-(2 ^, R,3'-dihydroxypropyI)benzoate as a white powder, mp 72-73 *C.

Analysis: Calculated for C, ,H, ₄ O _j : C, 58.40; H, 6.19

Found: C, 58.60; H, 630

A solution of metiiyW-hydroxyPT '-dihydroxypropyObenzoate (0.80 g, 0.0035 mol) in 1/V sodium hydroxide (6 mL) was stirred at room temperature for 16 h. The solution was acidified witii hydrochloric acid and concentrated under vacuum. The residue was washed with cold water (5 mL) and filtered. The cake was washed widi cold water and then recrystallized from water to give 0.48 g (64%) of die title compound as a white powder, mp 145-147 ^* C.

Analysis: Calculated for C, ₀ H , ₂ OJ: C, 56.60; H, 5.66

Found: C, 56.58; H, 5.74

S96/04421

45 Example 30

2.Hvdmxv-3-f2^.T-dihvdroxvprepvl.benzoic acid.

To a solution of 1 : 1 water and tert-butanol (50 mL), AD-mix-β (Aldrich, 7.0 g) was added and stirred for 0.1 h. The mixωre had two layers; tiie lower layer was yellow. To this mbcωre, methyl-2-hydroxy-3-allylbenzoate (Claisen, Chem. Ber. 1912, 45, 3157-3166) (0.96 g, 0.005 mol) was added in one portion and stirred at room temperature for 48 h. The reaction mixωre was cooled to 0 "C and sodium sulphite (73 g) was added. The reaction was stirred at 0 *C for 0.3 h and then at room temperature for 03 h. It was extracted widi ethyl acetate (3 x 50 mL), the combined organic layers were dried over sodium sulphate, filtered, and concentrated. The residue was passed dirough a column of silica gel first using 1:1 ethyl acetate:hexane as eluent to recover the starting material and then pure etiiyl acetate to give 0.96 g (85%) of methyl-2-hydroxy-3-(2 ^, S,3'-dihydroxypropyl)ben_oate as a white powder, mp 66-67 *C.

Analysis: Calculated for C, ιH, ₄ 0 ₅ : C, 58.40; H, 6.19

Found: C, 58.40; H, 626

A solution of methyI-2-hydroxy-2'53'-dmydroxypropyl)benzoate (0.82 g, 0.0036 mot) in 1/V sodium hydroxide (6 mL) was stirred at room temperature for 16 h. The solution was acidified with hydrochloric acid and concentrated under vacuum. The residue was washed widi cold water (5 mL) and filtered. The cake was washed with cold water and tiien recrystallized from water to give 0.46 g (60%) of the title compound as a white powder, mp 147-149 ^* C.

Analysis: Calculated for C, ₀ H ₁₂ O ₅ : C, 56.60; H, 5.66

Found: C, 56.58; H, 5.70

/US96/04421

46 Example 31

4-Acervlamino-3-hvdroxvmethvlbenzoic acid, complex with NaOH T5: 1.

Methy_-4-acetylrø_no-3-fo_τnylbenzoate (Gassman, P. G.; Drews, H. R. J. Am. Chem. Soc. 1978, 100, 7600- 7610) (0.331 g, 0.0015 mol) was dissolved in methanol (5 mL) and cooled to 0 *C. Sodium borohydride (0.057 g, 0.0015 mol) was added to the above solution at 0 ^* C and stirred for 025 h. The reaction was complete by TLC during this time. It was neutralized with H+ resin, filtered, and die filtrate concentrated. The residue was dried under vacuum over phosphorus pentoxide at 56 *C to give 033 g (99%) of medιy_-4-_cetylamino-3-hydroxymethylbenzoate as a pink solid, mp 60 *C.

Analysis: Calculated for C, ιH ₁₃ N0 ₄ ^■ 0.1 NaOH: C, 58.14; H, 5.77; N, 6.14

Found: C, 57.93; H, 5.64; N, 6.08

Metiiyl-4-acetylamino-3-hydroxymethylbenzoate (0215 g, 0.001 mol) was stirred in 2 mL of water and 1 mL of 1/V sodium hydroxide was added. The reaction mixωre was stirred for 4 h at room temperaωre (complete by TLC, ethyl acetate). It was neutralized witii H+ resin, filtered, and the filtrate concentrated under vacuum to give 02 g (100%) of the title compound as a white powder, mp 175-180 *C.

Analysis: Calculated for C, ₀ H , ,N0 ₄ ^• 0.6 NaOH: C, 51.50; H, 4.97; N, 6.00

Found: C, 51.60; H, 4.82; N, 5.98

Example 32

rrg -4-Ac-tvlamino-3-r2 ^, -propen- 1 '-oHbenzoic acid.

A mbcture of ethyl 4-acetylaminobenzoate (Coneglio, L. Rend Acad Sci. Napoli [3] 1931, 36, 56-60) (828 g, 0.04 mol) and palladium acetate (4.50 g, 0.02 mol) in dry toluene (100 mL) was heated at reflux for 2 h under nitrogen. The solution was cooled to 50 'C, the toluene decanted, and the residue washed witii fresh toluene. The combined filtrates were heated at reflux for 3 h, the precipitate was collected by filtration and mixed with the previous residue. The combined greenish residues were dried under vacuum for 3 h. To this residue, toluene (100 mL), acrolein diethyl acetal (17.5 g, 0.135 mol), and triethylamine (112 g, 0.11 1 mol) were added and die mixωre was heated at reflux for 3 h. On cooling, tiie palladium was removed by filtration through Celite and die filtrate was concentrated. The residue was passed through a column of silica gel using 1:1 ethyl acetate exane as eluent to give 1.1 g (20.8%) of ethyl 4-acetylamino-3-[3'-(2'-propen-r-aI)]beπzoate as an off-white flocculent solid, mp 171 "C.

Analysis: Calculated for C ₁₄ H ₁₅ N0 ₄ ^• 02 H ₂ 0: C, 63.48; H, 5.86; N, 529

Found: C, 63.61; H, 5.88; N, 522

A mixωre of ethyl 4-acetylamino-3-(3'-(2'-propen-r-al)]benzoate (0322 g, 0.002 mol) in methanol (20 mL) was cooled to 0 *C. To this mixωre, sodium borohydride (0.091 g, 0.0024 mol) in methanol (2 mL) was added and die mixωre stirred for 0.25 h. The mixωre was neutralized with H+ resin (Dowex-50W) and filtered. The filtrate was concentrated and the residue passed through a column of silica gel using 19:1 chloroform:methanol as eluent to give 0.410 g (76%) of ethyl 4-acetylamino-3-[3 ^, -(2'-propen-r-oI)]benzoate as a pale yellow powder, mp 1 18-119 ^* C.

Analysis: Calculated for C, ₄ H, ₇ N0 ₄ - 0.5 H ₂ 0: C, 61.75; H, 6.66; N, 5.15

Found: C, 61.68; H, 6.38; N, 5.12

SUBSπTt ^f T

48 A mixture of /raw-ethyl 4-acetylamino-3-(2'-propen-l ^, -oI)benzoate (0272 g, 0.001 mol) in 0.5/V sodium hydroxide (5 mL, 0.0025 mol) was stirred at room temperature for 8 h. The mixture was neutralized widi H+ resin and quickly filtered. The filtrate on cooling gave pale yellow crystals. The crystals were separated by filtration and washed witii cold water to give 0.065 g (28%) of the title compound, mp 214-215 *C. The filtrate on acidification with hydrochloric acid gave a pink precipitate. The pink solid was separated by filtration and washed widi cold water to give 0.130 g (56%, second crop) of the title compound, mp 207-208 *C.

Analysis: Calculated for C ₁₂ H ι ₃ N0 ₄ : C, 6127; H, 5.57; N, 5.95

Found: C, 61.12; H, 5.62; N, 5.88

Example 33

β-r2-V-Acetvlamino-5-carboxvphenvnethanol.

To a mbcωre of methyl 4-aminobenzoate (Aldrich, 15.1 g, 0.1 mol) in methylene chloride (400 mL) at -70 *C was added a solution of t-butyl hypochlorite (12.0 g, 0.011 mol in 50 L methylene chloride) over a period of 1 min. The mbcture was stirred at -70 *C for further 10 min and ethyl metiiylthioacetate (13.42 g, 0.1 mol in 50 mL methylene chloride) was added to it over a period of 10 min. The mixωre was further stirred for 1 h at the same temperaωre and subsequently, triethylamine (112 g, 0.11 mol in 40 mL methylene chloride) was added to it over a period of 10 min. The cooling bath was removed and the mixωre was brought to room temperaωre. Water (120 mL) was added, die organic layer separated, dried over sodium sulphate, filtered, and concentrated to give a brown viscous oil. The oil was taken in etiier (200 mL) and triethylamine (40 L) and cooled to 0-5 ^* C in an ice bath. Acetyl chloride (8.6 g. 0.1 1 mol) in etiier (50 mL) was added dropwise over a period of 15 min. The reaction mixωre was stirred for 1 h. Water (200 mL) was added, die organic layer separated, washed with water, dried over sodium sulphate, filtered, and concentrated to give a syrup. This syrup was purified by passing through a column of silica gel using ethyl acetate:hexane (12) as eluent. The appropriate fractions were pooled, combined, and concentrated. The residue of the

St_-STrrUTE SHEET RULE 26

desired product was recrystallized from ethyl acetate-hexane to give 9.5 g (29.3%) of ethyl α-(2-acetylamino-5- methoxycarbonylphenyl)-α-methyltiιio acetate as a white solid, mp 95-96 *C.

Analysis: Calculated for C, ₅ H ₁₉ N0 ₅ S: C, 55.37; H, 5.88; N, 431

Found: C, 55.43; H, 5.91; N, 428

To a mixωre of ethyl α-(2-acetyiamino-5-methoxycarbonylphenyl)-α-methylthio acetate (6.5 g, 0.02 mol) in tetrahydrofuran (150 mL), Raney nickel (about 20 mL slurry in tetrahydrofuran, washed several times with water and tetrahydrofuran) was added in portions over a period of 30 min. The reaction mixωre was stiired for 15 min and Raney nickel was removed by filtration through Celite. The filtrate was concentrated to give a white residue which was recrystallized from ethyl acetate-hexane to give 5.2 g (93%) of ethyl o-(2-acetylamino-5- methoxycarbonylphenyl)acetate as a white solid, mp 119 *C.

Analysis: Calculated for C ₁₄ H _I7 N0 ₅ : C, 6021; H, 6.13; N, 5.02

Found: C, 60.12; H, 6.13; N, 4.93

To a mixmre of ethyl α-(2-acetylamino-5-methoxycarbonylphenyl)acetate (4.18 g, 0.015 mol) in methanol (60 mL), I /V sodium hydroxide ( 15.0 mL, 0.015 mol) was added over a period of 10 min. The mixωre was stirred at room temperaωre for 1 h. After dilution with water (50 mL), the mixωre was filtered and the filtrate neutralized with concentrated hydrochloric acid. The precipitate obtained was collected by filtration and washed with water. The cake was recrystallized from ethyl acetate and dried to give 23 g (61%) of α-(2-acetyiamino-5- methoxycarbonylphenyl)acetic acid as a white solid, mp 194 ^* C.

Analysis: Calculated for C _I2 Hι ₃ N0 ₅ : C, 5737; H, 522; N, 5.58

Found: C, 57.10; H, 5.26; N, 5.50

To a mixωre of o-(2- V-acetylamino-5-methoxycarbonylphenyl)acetic acid (1 g, 4 mmol), tetrahydrofuran (10 mL), and trietiiylamine (0.56 mL, 4 mmol), ethyl chloroformate (038 mL, 4 mmol) was added over a period of 5 min at 0 "C. After stirring for an additional 20 min at the same temperature, the mixωre was filtered widi suction, and the

SUBSTI

cake was washed with tetrahydrofuran (3 x 10 mL). Sodium borohydride (0.48 g, 12.6 mmol) was added to die filtrate in one portion. Then methanol (2.6 mL) was added dropwise over a period of 1 h at 10 *C. After stirring for an additional 30 min, the reaction mbcture was quenched carefully with 1/V HCI close to pH - 7. To this mixωre was added water (25 mL) and etiier (25 mL). The organic layer was separated and the aqueous layer extracted witii methylene chloride (3 x 10 mL). The organic layers were combined, dried widi sodium sulfate, and the solvent removed in vacuo to give 0.91 g of crude product as a white residue. The crude product was purified by flash column chromatography (75-100% ethyl acetate in hexane) to obtain β-(2-V-acetylamino-5-methoxycarbonylphenyl)edιanol [Rf 0.16 in ethyl acetate:hexane (3:1)] 0.55 g (58%) as a white solid, mp 134-136 *C.

Analysis: Calculated for C ₁₂ H,5N0 ₄ : C, 60.75; H, 637; N, 5.90

Found: C, 60.70; H, 637; N, 5.81

A solution of β-(2- V-acetylamino-5-methoxycarbonylphenyI)ethanol (0.71 g, 3 mmol) in 1/V NaOH (6 L) was stirred at room temperaωre for 1 h. The reaction mixture was filtered and die pH was adjusted between 2-3 using 1/V HCI. The product obtained on standing for 1 h was collected by filtration, washed with small amounts of water and ether, and dried in a pistol at toluene reflux overnight in vacuo to furnish 0.58 g (87%) of the title compound as a light brown solid, mp 206-208 *C (dec).

Analysis: Calculated for C,,H ₁₃ N0 ₄ : C, 59.19; H, 5.87; N, 627

Found: C, 59.05; H, 5.88; N, 622

Example 34

4-Acetvlamino-3-f2-hvdroxvethoxv.benzoic acid.

To mediyl 3-hydroxy-4-nitπ.benzoate (Dictionary of Organic Compounds, Heilbron, I.; Brunburg, H. M., Eds.; Vol.3; 1953, Oxford University Press: New York, NY; p 700) (5.0 g, 0.0254 mol) dissolved in acetone (50 mL) was added potassium carbonate (4.3 g, 0.0300 mol), sodium iodide (0375 g, 0.0025 mol), and 2-bromoetiιaπol (3.44 g, 0.0275 mol). After refluxing for 60 h, thin-layer chromatographic analysis (Si0 ₂ , 32 hexane:ethyl acetate) showed partial disappearance (50%) of die starting material along with the formation (50%) of a new, lower-running spot (Rf - 02). To tiie reaction mbcture, additional potassium carbonate (4.3 g, 0.0300 mol), sodium iodide (0.375 g, 0.0025 mol), and 2-bromoethanol (3.44 g, 0.0275 mol) were added. After refluxing for 24 h more, ώin-layer chromatographic analysis (Si0 ₂ , 32 hexanerethyl acetate) showed increased disappearance of die starting material along with the formation (70%) of a lower-running spot (Rf- 02). One-half of the reaction was poured into water (200 mL) and then extracted three times with 100-mL portions of ethyl acetate. The organic layer was concentrated and die residue reciystallized from ethyl acetate-hexane. The material was collected by filtration and dried in vacuo at acetone reflux to give 0.878 g (14%) of methyl 3-(2-hydroxyethoxy>4-nitrobenzoate as an orange solid, mp 97-98 *C.

Analysis: Calculated for C _l0 H _u NO ₆ : C, 49.79; H, 4.56; N, 5.81

Found: C, 49.58; H, 5.64; N, 5.78

To a sample of methyl 3-(2-hydroxyethoxy)-4-nitrobenzoate (328 g, 0.0136 mol) dissolved in edianol was added platinum oxide (50 mg). The mixture was hydrogenated for 40 min at 35 psi. Thin-layer chromatographic analysis (SiC_, 32 hexane:ethyl acetate) showed disappearance of starting material along with the formation of a new, lower-running spot (Rf- 01) indicating die reaction was complete. The mixωre was then filtered through a pad of Celite and d e pad washed with fresh ethanol. The filtrate was concentrated and dried in vacuo to give 2.9 g ( 100%) of methyl 4-amino-3-(2-hydroxyedιoxy)beπzoate as an off-white solid.

To memyl 4-amino-3-(2-hydroxyethoxy)benzoate (2.98 g, 0.0141 mol) dissolved in dichloromethane (200 mL) was added acetic anhydride (1.49 mL, 0.0156 mol) and pyridine (1.34 mL, 0.0156 mol). After stirring for 3 h at room temperature, thin-layer chromatographic analysis (Si0 ₂ , etiiyl acetate) showed disappearance of the starting material along with the appearance of a new lower-running spot (Rf- 03) indicating die reaction was complete. The reaction was poured into water (70 mL) and d e organic layer washed three times with water (50 mL). The organic layer was concentrated and then dried in vacuo. Thin-layer chromatographic analysis (Si0 ₂ , ethyl acetate) showed formation of some material as a higher-running spot (R _f - 0.8) in addition to die product Methanol (50 mL) and a catalytic amount of sodium metiioxide was added to die residue. After 1 h, thin-layer chromatographic analysis (Si0 ₂ , etiiyl acetate) showed only die desired product. The solution was neutralized with H+ resin, filtered, and tiien concentrated. The residue was recrystallized from ethyl acetate to give 1.88 g (53%) of methyl 4-acetylamino-3-(2- hydroxyethoxy)benzoate as an off-white solid.

A sample of methyl 4-acetylamino-3-(2-hydroxyethoxy)benzoate (1.51 g, 0.0060 mol) was suspended in 1/V NaOH (12.0 mL, 0.0119 mol). After stirring for 1 h, thin-layer chromatographic analysis (Si0 ₂ , etiiyl acetate) showed disappearance of starting material along with appearance of a new lower-running spot (Rf- 0.1) indicating the reaction was complete. The reaction mbcture when neutralized with concentrated HCI formed a white precipitate. The material was collected by filtration, washed widi water, and tiien dried in vacuo at acetone reflux to give 0.47 g (34%) of the title compound as a tan solid, mp 207.5-208.5 ^* C.

Analysis: Calculated for C, ,H ₁₃ NO _j : C, 5523; H, 5.44; N, 5.86

Found: C, 55.07; H, 533; N, 5.87

Example 35

4-Acetvlamino-3-t2 ^, .3'-dihvdroxvpropvl .benzoic acid, solvate with water S: (Isomer A

A solution of ethyl-4-acetylaminobenzoate (Coneglio, L. Rend Acad Sci. Napoli [3] 1931, 36, 56-60) (2.07 g, 0.01 mol) and palladium acetate (1.12 g, 0.005 mol) in dry toluene (17 mL) was heated at reflux for 0.75 h under nitrogen. The solution was cooled to 60 *C, the toluene was decanted, and die residue was washed widi fresh toluene. The combined filtrates were heated at reflux for 3 h, the precipitate was collected by filtration, and mixed widi the previous residue. The combined greenish residues were dried under vacuum for 3 h. To this residue, glacial acetic acid (40 mL) and allyl iodide (8.40 g, 0.05 mol) were added and die mbcture was stirred at room temperature for 16 h. The palladium was removed by filtration tiirough Celite and die filtrate was concentrated. The residue was passed tiirough a column of silica gel using 1:1 ethyl acetate:hexane as eluent to give 0.85 g (69%) of ethyl-4-acetylamino-3- allylbenzoate as a white powder, mp 127-128 *C.

Analysis: Calculated for C, ₄ H ₁₇ N0 ₃ : C, 68.01; H, 6.88; N, 5.67

Found: C, 67.88; H, 6.93 ; N, 5.66

To a solution of 1:1 water and tert-butanol (10 mL), AD-mix-o (Aldrich, 1.4 g) was added and die mixωre stirred fbr 0.1 h. The mixωre had two layers; die lower layer was yellow in color. To tiiis mbcture, ethyM-acetylamino- 3-aIlylbenzoate (0.1 g, 0.0004 mol) was added in one portion and stirred at room temperaωre for 48 h. The reaction mixωre was cooled to 0 *C and sodium sulphite (13 g, 0.0119 mol) was added. The reaction was stirred at 0 *C for 0.3 h and tiien at room temperaωre for 0.5 h. It was extracted witii ethyl acetate (3 x 15 mL), the combined organic layers were dried over sodium sulphate, filtered, and concentrated. The residue was passed through a column of silica gel eluting first with 1 : 1 ethyl acetate:hexane to recover die starting material, and men with pure etiiyl acetate to give 0.075 g (66%) of etiιyl-4-acetylamino-3-(2\3^-dihydroxypropyl)ben_oate (isomer A) as a white powder, mp 1 14- 1 16 *C. The optical rotation of this solid was 0 ^* at 589 nm.

Analysis: Calculated for C ₁₄ H _j9 N0 _j : C, 59.78; H, 6.76; N, 4.98

Found: C, 59.70; H, 6.79; N, 4.94

A solution of ethyl-4-acetyl-_mino-3-(2'3'-dihydroxypropyl)benzoate (0.120 g, 0.00043 mol) in 1/V sodium hydroxide (2 mL) was stirred at room temperaωre for 15 h. The solution was acidified widi hydrochloric acid and concentrated under vacuum. The residue was dissolved in methanol (5 mL) and filtered. The filtrate was concentrated and the residue was passed tiirough a column of silica gel using 85 : 15 : 1 mixωre of eth l acetate, methanol, and acetic acid to give 0.060 g (54%) of the title compound as an off-white powder, mp 181-183 *C.

Analysis: Calculated for C ₁₂ H ₁₅ N0 ₅ ^• 02 H ₂ 0: C, 56.10; H, 6.05; N, 5.46

Found: C, 56.09; H, 5.94; N, 5.41

Example 36

4-Acervlamino-3-(2'.3'-dihvdroxvpropvnbenzoic acid, solvate with water f4: , (Isomer BV

A solution of ethyl-4-acetyiaπιinobenzoate (Coneglio, L. Rend Acad ScL Napoli [3] 1931, 36, 56-60) (2.07 g, 0.01 mol) and palladium acetate (1.12 g, 0.005 mol) in dry toluene (17 mL) was heated at reflux for 0.75 h under nitrogen. The solution was cooled to 60 *C, die toluene was decanted, and die residue was washed widi fresh toluene. The combined filtrates were heated at reflux for 3 h, the precipitate was collected by filtration, and mixed with the previous residue. The combined greenish residues were dried under vacuum for 3 h. To this residue, glacial acetic acid (40 mL) and ally] iodide (8.40 g, 0.05 mol) were added and the mixωre was stirred at room temperaωre for 16 h. The palladium was removed by filtration through Celite and die filtrate was concentrated. The residue was passed through a column of silica gel using 1:1 ethyl _cetate:hexane as eluent to give 0.85 g (69%) of ethyl-4-a_etyiamino-3- allylbenzoate as a white powder, mp 127-128 *C.

Analysis: Calculated for C ₁₄ H _I7 N0 ₃ : C, 68.01; H, 6.88; N, 5.67

Found: C, 67.88; H, 6.93; N, 5.66

To a solution of 1: 1 water and tert-butanol (10 mL), AD-mix-β (Aldrich, 1.4 g) was added and die mbcture stirred for 0.1 h. The mixture had two layers; the lower layer was yellow in color. To this mbcture, ethyl-4-acetylamino- 3-allylbenzoate (9.092 g, 0.00037 mol) was added in one portion and stiired at room temperature for 48 h. The reaction mixωre was cooled to 0 *C and sodium sulphite (13 g, 0.01 19 mol) was added. The reaction was stirred at 0 *C for 03 h and tiien at room temperaωre for 0.5 h. It was extracted witii ethyl acetate (3 x 15 mL), the combined organic layers were dried over sodium sulphate, filtered, and concentrated. The residue was passed through a column of silica gel eluting first with 1 : 1 ethyl acetate:hexane to recover the starting material, and tiien with pure ethyl acetate to give 0.068 g (65%) of ethyl-4-acetylamino-3-(2'3'-dihydroxypropyl)benzoate (isomer B) as a white powder, mp 116-117 *C. The optical rotation of this solid was 0 *C at 589 run.

Analysis: Calculated for C, ₄ H, ₉ N0 ₅ : C, 59.78; H, 6.76; N, 4.98

Found: C, 59.73 ; H, 6.80; N, 5.01

A solution of ethyl-4-acetylamino-3-(2'3'-dihydroxypropyl)benzoate (0.090 g, 0.00032 mol) in 1/V sodium hydroxide (2 mL) was stirred at room temperaωre for 15 h. The solution was acidified with hydrochloric acid and concentrated under vacuum. The residue was dissolved in methanol (5 mL) and filtered. The filtrate was concentrated and die residue was passed through a column of silica gel using a 85: 15: 1 mbcture of etiiyl acetate, methanol, and acetic acid to give 0.055 g (67%) of die title compound as an off-white powder, mp 180-182 *C.

Analysis: Calculated for C^H^NO _j ^• 0.75 H ₂ 0: C, 54.03; H, 6.19; N, 525

Found: C, 54.19; H, 5.94; N, 532

Nl tt

Example 37

4-A.etylamino-3-aminobeπzoic acid hydrochloride f2 V

A mbcωre of etiiyl 4-acetyiamino-3-aminobenzoate (Blackburn, ., et al. J. Org. Cx em. 1961, 26, 2805-2809) (1.0 g, 0.0045 mol) in 1/V sodium hydroxide (10 mL, 0.01 mol) was stirred at 50 *C for 2 h. The mixωre was filtered and acidified widi concentrated hydrochloric acid. On standing overnight a brown precipitate formed. The precipitate was separated by filtration and dried to give 0.7 g (73%) of die title compound as a brown powder, mp 272 *C (dec).

Analysis: Calculated for C ₉ H ₁₀ N ₂ O ₃ ^• 0.5 HCI: C, 50.89; H, 4.98; N, 13.18

Found: C, 50.54; H, 4.82; N, 13.01

Example 38

3-Amino-4-f2-methvlpropionvlamino.benzoic acid.

Ethyl 4-(2-methylpropionylamino)benzoate (Chemical Abstract 114(1 1) I0l280u; Vagaonescu; Marisa, N- Acyl Derivatives of Anesthesin. Stud Univ. Banes-Bolyai. Chem. 1989, 34, 12-14) was added slowly over a 50-min period to fuming nitric acid at 10 *C. The reaction was stirred for 30 min at room temperaωre. Thin-layer chromatographic analysis (Si0 ₂ , 1 : 1 ethyl acetate:hexane) showed disappearance of die starting material along with the formation of a new higher-running spot (R _f « 0.9). The reaction was poured into water and then extracted with ethyl acetate (3 x 500 mL). Next the organic layer was washed widi 1% sodium bicarbonate (500 mL) solution and men water (500 mL). The ethyl acetate layer was concentrated and die residue chromatographed (Si0 ₂ , 1 :5 ethyl

acetate exane). The fractions containing product were concentrated to give 17 g (49%) of ethyl 4-(2- methylpropionylamino)-3-nitrobenzoate as a yellow solid, mp 74-76 *C. An analytical sample was prepared by recrystaliization from hexane. This material was collected by filtration and dried in vacuo at acetone reflux.

Analysis: Calculated for C ₁₃ H _I6 N ₂ 0j: C, 55.71; H, 5.71; N, 10.00

Found: C, 55.45; H, 5.85; N, 1020

A sample of ethyl 4-(2-methylpropionylamino)-3-nitrobenzoate (8.0 g, 0.0286 mol) was suspended in edianol (75 mL) containmg platinum oxide (50 mg). The reaction mixωre was hydrogenated for 2 h at 35 psi. Thin-layer chromatographic analysis (Si0 ₂ , 1:1 hexane:ethyl acetate) showed disappearance of staπing material along with die formation of a new lower-running spot (R _f - 02). The mixωre was then filtered through a pad of Celite and die pad washed with fresh edianol. T he filtrate was concentrated and the residue reciystallized from ethyl acetate-hexane. The solid was collected by filtration to give 5.0 g (70%) of ethyl 3-amino-4-(2-methylpropionylamino)benzoate. An analytical sample was prepared by recrystaliization twice from ethyl acetate-hexane. The residue was collected by filtration and then dried in vacuo at acetone reflux to give a beige solid, mp 147-149 *C.

Analysis: Calculated for C _I3 H _l gN ₂ 0 ₃ : C, 62.40; H, 720; N, 1120

Found: C, 62.51; H, 728; N, 11.15

A sample of ethyl 3-amino- -(2-methylpropionyl_mino)benzoate (230 g, 0.0092 mol) was suspended in 1/V NaOH (13.80 mL, 0.0138 mol)- After stirring fbr 24 h at room temperature, die reaction was tiien heated at 50 ^* C fbr 6 h. The reaction was filtered and the filtrate acidified widi 1 V HCI forming an orange precipitate. The solid was collected by filtration and thin-layer chromatographic analysis (SiC^, 9:1 ethyl acetate:met_ιanol) indicated formation of a new lower-running spot (Rf- 0.1). This material was recrystallized from \-butanol. The residue was collected by filtration and then suspended in etiier. The product was filtered and dried in vacuo at acetone reflux to give 0.12 g (5%) of the title compound as a beige solid, mp 159-160 ^* C.

Analysis: Calculated for C, ,H, ₄ N ₂ 0 ₃ : C, 59.46; H, 6.31; N, 12.61

Found: C 5924; H, 6.43; N, 12.55

Example 39

4-Acetvlamino-3-f(aminoiminomcthvl .amino1benzoic acid solvate with water ti :1V

A mbcωre of ethyl 4-acetylamino-3-aminobenzoate (Blackburn, W. era/. J. Org. Chem. 1961, 26, 2805-2809) (5.5 g, 0.025 mol), concentrated hydrochloric acid (2.1 mL, 0.025 mol), and cyanamide (21.0 g, 0.5 mol) in etiiyl acetate (500 mL) was stirred at room temperature for 48 h and tiien heated at reflux for 2 h. On cooling, the white precipitate obtained was separated by filtration, washed with ethyl acetate, and dried to give 5.5 g (73%) of crude etiiyl 4- acetylam_no-3-[(aminoiminometiιyl)amino]benzoate hydrochloride as a white powder. A mixture of ethyl 4- acetylamino-3-[(am_ιoiminomethyl)amino]benzoate hydrochloride (4.5 g, 0.015 mol) in sodium hydroxide (2.1 g in 35 mL water) was stirred at room temperature for 48 h. The mixture was filtered tiirough Celite to remove suspended particles. Concentrated hydrochloric acid was added to the filtrate to bring die pH close to 9. The precipitate was collected by filtration, washed with water, and dried to give 1.6 g (42%) of die title compound as a white powder, mp 260 *C.

Analysis: Calculated fbr C, ₀ H, ₂ N ₄ O ₃ • H ₂ 0: C, 4724; H, 535; N, 22.04

Found: C, 47.00; H, 5.63; N, 21.94

Example 40

3-lϊ AminoiminomethvI.aminol-4-(2-methvlDropionvlamino'lbenzoic acid hvdrate r2:11.

Etiiyl 4-(2-metiιylpropionylamino)benzoate was added slowly over a fifty-minute period to fuming nitric acid at 10 "C. The reaction was stirred for 30 min at room temperature. Thin-layer chromatographic analysis (Si0 ₂ , 1: 1 ethyl acetate:hexane) showed disappearance of the starting material along with die formation of a new higher-running spot (Rf - 0.9). The reaction was poured into water and tiien extracted widi ethyl acetate (3 x 500 mL). Next th- organic layer was washed witii 1% sodium bicarbonate (500 mL) solution and then water (500 mL). The ethyl acetate layer was concentrated and the residue chromatographed (Si0 ₂ , 1:5 ethyl acetate:hexane). The fractions containing product were concentrated to give 17 g (49%) of ethyl 4-(2-methylpropionylamino)-3-nitrobenzoate as a yellow solid, mp 74-76 *C. An analytical sample was prepared by recrystaliization from hexane. This material was collected by filtration and dried in vacuo at acetone reflux.

Analysis: Calculated for C _l3 H ₁₆ N ₂ 0 ₅ : C, 55.71 ; H, 5.71 ; N, 10.00

Found: C, 55.45; H, 5.85; N, 1020

(Chem. Abstract 114(11) 101280u; Vagaonescu; Marisa. N-Acyl Derivatives of Anesthesin. Stud Univ. Banes-Bolyai, Chem. 1989, 34, 12-14)

A sample of ethyl 4-(2-methylpropionylamino)-3-nitrobenzoate (8.0 g, 0.0286 mol) was suspended in edianol (75 mL) containing platinum oxide (50 mg). The reaction mixωre was hydrogenated for 2 h at 35 psi. Thin-layer chromatographic analysis (Si0 ₂ , 1 : 1 hexane:ethyl acetate) showed disappearance of starting material along widi the formation of a new lower-running spot (R _r - 02). The mixωre was then filtered tiirough a pad of Celite and the pad washed witii fresh ethanol. The filtrate was concentrated and die residue recrystallized from ethyl acetate-hexane. The solid was collected by filtration to give 5.0 g (70%) of ethyl 3-amino-4-(2-methylpropionylamino)ben_oate. An

analytical sample was prepared by recrystaliization twice from ethyl acetate-hexane. The residue was collected by filtration and then dried in vacuo at acetone reflux to give a beige solid, mp 147-149 *C.

Analysis: Calculated for C ₁₃ HιgN ₂ θ3: C, 62.40; H, 720; N, 1120

Found: C, 62.51; H, 728; N, 11.15

To ethyl 3-amino-4-(2-methylpropionylamino)ben_o_te (1.7 g, 0.0068 mol) dissolved in etiiyl acetate (50 mL) was added concentrated HCI (0.57 mL, 0.0068 mol) and cyanamide (5.71 g, 0.136 mol). The solution was heated at reflux for 48 h. Thin-layer chromatographic analysis (Si0 ₂ , 9: 1 ethyl acetate:methanol) showed staπing material (35%) along with the formation of a new lower-running spot (R ^» 0.1). The material was concentrated and dried in vacuo overnight The resulting solid was passed tiirough a 350-mL frit funnel (Si0 ₂ ) using ethyl acetate to elute the higher- running spots. Next the solvent concentration was stepped up to 17:3 ethyl acetate:methanol in order to elute die product The filtrates were concentrated and the residue suspended in ethyl acetate. The resulting solid was collected by filtration to give 0.76 g (34%) of ethyl 3-[(__mino_minomethyl)amino]-4-(2-methylpropionylamino)benzo ate hydrochloride as a pale yellow solid. An analytical sample was prepared by recrystallizing a portion of the residue from methanol-ether. This sample was collected by filtration and dried over toluene in vacuo to give a white solid, mp 175- 177 *C.

Analysis: Calculated for C _|4 H ₂₀ N ₄ O ₃ ^• HCI ^• 0.5 H ₂ 0: C, 49.79; H, 632; N, 16.60

Found: C, 49.97; H, 6.57; N, 16.68

A sample of e yl 3-[(aminoiminomethyI)amino]-4-(2-medιylpropionylamino)benzo ate hydrochloride (036 g, 0.0012 mol) was suspended in 1 VNaOH (1.85 mL, 0.0019 mol). After stirring for 1 h at 38 "C, additional 1/V NaOH (03 mL, 0.0005 mol) was added and die reaction was allowed to stir for 1 h more. Thin-layer chromatographic analysis (Si0 , 4:1 etiiyl acetate-methanot) indicated that staπing material had reacted to form a new lower-running spot (R _r ^« 0.1). The pH was adjusted to 7.0 witii 1/V HCI causing formation of a white precipitate. The solid was collected by filtration. It was purified by dissolving again in a minimum amount of 1/V NaOH and adjusting the pH to 7.0 with dilute HCI causing formation of a white precipitate. The solid was collected by filtration and dried in vacuo at toluene reflux to give 0.15 g (52%) of the title compound as a white solid, mp 238-239.5 ^* C.

___S SHEET (Rtfll 26)

Analysis: Calculated for C ι ₂ H ₁ N ₄ 0 ₃ ^• 03 H ₂ 0: C, 52.75; H, 623 ; N, 20.51

Found: C, 52.70; H, 630; N, 20.44

Example 41

4-Acetvlamino-3-(rCaminoiminomethvnhvdra2ino1methvlenelhe nzoic acid dihvdrate.

A mixωre of methyl 4-_cetyl__mino-3-foπny_benzoate (Gassman, P. G.; Drews, H. R. J. Amer. Chem. Soc. 1978, 100, 7600-7610) (221 g, 0.01 mol), aminoguanidine bicarbonate (1.40 g, 0.0102 mol) and concentrated hydrochloric acid (1.68 mL, 0.02 mol) in ethanol (50 mL) was heated to reflux for 4 h. The reaction mbcture was cooled and die white precipitate was collected by filtration. The filtrate on concentration and cooling gave more precipitate which was again collected by filtration. Both precipitates were combined and dried to give 2.1 g (69%) of methyl 4- __cetylam_no-3-{[(aminoiminomethy hydrazino]methylene}benzoate hydrochloride as a fluffy white powder, mp 278

^• c

Analysis: Calculated for C, ₂ H ₁₅ Ν _j 0 ₃ ^• HCI: C, 45.94; H, 5.14; N, 2232

Found: C, 4624; H, 5.18; N, 22.46

A mixωre of methyl 4-acetylamino-3-{[(aminoiminomethyI)hydrazino]methylene}benz oate hydrochloride (0.54 g, 0.0017 mol) and 1/V sodium hydroxide (5.0 mL, 0.005 mol) was stirred at room temperaωre for 24 h. After filtration, die mixωre was neutralized with concentrated hydrochloric acid. The precipitate was collected by filtration, washed widi water, and dried over toluene under vacuum to give 0.41 g (80%) of die title compound as a white powder, mp 326-327 *C.

Analysis: Calculated for CH H^NJOJ - 2 H ₂ 0: C. 44.15; H, 5.73; N, 23.40

Found: C, 4420; H, 5.48; N, 23.16

SUBSTITUTE SHEET RUlf 26

62 Example 42

4-Acetvlamino-3-rthvdroxvlimino methvnbenzoic acid hvdrate (4:1 V

A mixture of metiiyi 4-acetylamino-3-formylbenzoate (Gassman, P. G.; Drews, H. R. J. Am. Chem. Soc. 1978, 700, 7600-7610) (0.442 g, 0.002 mol) and hydroxylamine hydrochloride (0.174 g, 0.0025 mol) in etiiyl acetate (10 mL) was heated at reflux for 16 h. White precipitate formed which was removed by filtration and the filtrate was concentrated to dryness. The residue was recrystallized from ethyl acetate-hexane to give 0.25 g (53%) of methyl 4- acetylam__io-3-[(JV-hydroxylimino)methyl]benzoate as a pale yellow powder, mp 200-203 *C.

Analysis: Calculated for C _π H, ₂ N ₂ 0 ₄ : C, 55.93; H, 5.12; N, 11.86

Found: C, 56.08; H, 5.19; N, 11.72

A mixωre of methyl 4-acetylamino-3-[(hydroxyiimino)methyl]benzoate (142 mg, 0.6 mmol) and 1/V sodium hydroxide ( 1.0 mL, 1.0 mmol) was stirred at room temperaωre for 4 h. The mixωre was filtered through a pad of cotton and neutralized with dil. hydrochloric acid. The precipitate obtained was collected by filtration, washed with water, and dried under vacuum over acetone for 24 h to give 95 mg (71 %) of the title compound as a white powder, mp 215-218 ^• C (dec).

Analysis: Calculated for C _l0 H, ₀ N ₂ O ₄ ^• 025 H ₂ 0: C, 52.98; H, 4.67; N, 12.36

Found: C, 52.92; H, 4.66; N, 1226

Example 43

3-r ⁽ Aminoiminomethvhamino1-4-rfmethvlsulfonvl.amino1ben?oi c acid, solvate with water (7-AY

Ethyl 4-[(methylsulfonyl)amino]benzoate (Lumma, W. C. et al. J. Med. Chem. 1987, 30, 758-763) (2.0 g, 0.0082 mol) was added poπionwise to fuming nitric acid (10 mL) at 5-10 *C over 025 h. The mbcωre was further stirred for 0.5 h at 10- 15 "C and then poured into cold water (50 mL). The yellow precipitate was separated by filtration and washed several times with cold water. The cake was recrystallized from ethyl acetate to give 1.7 g (72%) of ethyl 3-nitro-4-[(methylsulfonyl)amino]benzσate as yellow crystals, mp 124 *C.

Analysis: Calculated for C, ₀ H ₁₂ N ₂ O ₆ S: C, 41.66; H, 420; N. 9.72

Found: C, 41.30; H, 4.15; N, 9.86

Ethyl 3-nitro-4-[(methylsulfonyl)amino]benzoate (1.0 g, 0.0035 mol) was dissolved in ethanol (40 mL) and hydrogenated at 20 psi in die presence of platinum oxide (Aldrich, 0.02 g) for 1 h. The catalyst was removed by filtration through Celite and the filtrate was concentrated to 20 mL. On cooling, a precipitate formed which was separated by filtration, washed widi cold ethanol, and dried to give 0.75 g (84%) of ethyl 3-amino-4- [(medιylsulfonyl)amino]benzoaie as a white powder, mp 151 *C.

Analysis: Calculated for C, ₀ H _|4 N ₂ O ₄ S: C, 46.50; H, 5.46; N, 10.84

Found: C, 46.52; H, 530; N, 10.92

A mixture of ethyl 3-ammo-4-[(methylsulfonyl)amino]ben_oate (238 g, 0.01 mol), concentrated hydrochloric acid (0.84 mL, 0.01 mol), and cyanamide (42 g, 0.1 mol) in ethyl acetate (100 mL) was heated at reflux for 4 h. The reaction mbcωre was cooled and die white precipitate separated by filtration. The cake was washed several times witii cold ethyl acetate and dried under vacuum to give 2.95 g (85.5%) of ethyl 3-[(aminoiminomethyl)amino]-4-

[(methylsulfonyl)amino]benzoate hydrochloride as a white powder, mp 160-164 *C.

Analysis: Calculated for C _M H ₁₆ N ₄ 0 ₄ S ^• HCI ^• 0.5 H ₂ 0: C, 38.21; H, 525; N, 1620

Found: C, 38.42; H, 532; N, 16.00

A mbcture of ethyl 3-[(aminoiminomedιyI)amino]-4-[(metiιylsulfonyl)amino]benz oate (1.80 g, 0.0052 mol) in 1.5 V sodium hydroxide (15 mL, 0.0225 mol) was stirred at room temperaωre for 4 h. The mbcωre was neutralized widi concentrated hydrochloric acid to give an off-white precipitate. The precipitate was separated by filtration, washed with water, and dried to give 1.19 g (81%) of the title compound as an off-white powder, mp 200 *C.

Analysis: Calculated for C ₉ H _l2 N ₄ 0 ₄ S ^• 0.5 H ₂ 0: C, 38.43; H, 4.66; N, 19.92

Found: C, 38.60; H, 4.64; N, 19.90

Example 44

3-rrΛt-HvdroxviτninoVιnethv»-_-r/m-thvlsulfonv1.»ι� �ιino1henynie »cid

To 2-amino-5-(me_ioxyc_rbonyI)ben_aldehyde trimethylene mercaptal (Gassman, P. G.; Drews, H. R. J. Am. Chem. Soc. 1978, 700, 7600-7610) (5.0 g, 0.019 mol) in dichloromethane (90 mL) was added pyridine (4.51 mL, 0.055 mol). Medianesulfonyl chloride (431 mL, 0.055 mol) was tiien added over a five-minute period and the reaction mbcωre heated at reflux overnight Thin-layer chromatography analysis (Si0 ₂ , EtOA hexaπe 1:1) showed the disappearance of die starting material along with die appearance of a new lower-running spot (R _f - 0.56) indicating the reaction was complete. Upon cooling, tiie reaction was washed tiioroughly with 10% acetic acid followed by water. The organic layer was dried (Na ₂ S0 ₄ ), filtered, and concentrated. The crude residue was placed on a Si0 ₂ column and eluted with 1:1 EtOA hexane. Fractions containing die desired product were combined and concentrated to yield 5.0 g (77%) of 2-(methanesulfonylamino)-5-(methoxycart>onyl)benzaldehyde trimethylene mercaptal as a yellow solid.

An analytical sample, mp 168-168.5 ^* C, was recrystallized from ethanol.

Analysis: Calculated for C,3H _j7 N0 ₄ S ₃ : C, 44.94; H, 4.93; N, 4.03

Found: C, 44.98; H, 4.94; N, 4.01

A sample of 2-(methanesulfonylamino)-5-(methoxyc__rbonyl)benzaldehyde trimethylene mercaptal (7.66 g, 0.022 mol) dissolved in JV./V-dimethylformamide (435 mL) and acetone (39.13 mL) was added over a 5-min period through a dropping funnel to a solution of copper (II) oxide (2.09 g, 0.026 mol), copper (II) chloride (7.0 g, 0.055 mol) in acetone (174.0 mL) and heated at reflux. After 2 h at reflux, thin-layer chromatography analysis (Si0 , EtOA hexane 1:1) showed disappearance of die staπing material and the appearance of a new slightly higher R _f spot which gave a positive result when sprayed witii 2,4-DNP. Upon cooling, the reaction was filtered through Celite and die cake was washed with 10% ethanol in dichloromethane. Water (125 mL) and dichloromethane (150 mL) were added to die filtrate. The layers were partitioned and die water layer extracted 2 x 100 mL dichloromethane. The combined organic layers were washed twice with water (100 mL), dried (Na-SO^, and filtered. Concentration of the filtrate gave a beige solid which was dried in vacuo to give 4.48 g (79%) of 2-(methanesulfonylamino)-5- (methoxycarbonyl)benzaldehyde, mp 161-163 *C (ethanol).

Analysis: Calculated for C _I0 H, ,NO _j S: C, 46.69; H, 428; N, 5.44

Found: C, 46.67; H, 435; N, 5.46

A mixωre of 2-(methanesulfonylamino)-5-(methoxycarbonyl)benzaldehyde (2.56 g, 0.01 mol) and hydroxylamine hydrochloride (0.87 g, 0.0125 mol) in ethanol (40 mL) was heated at reflux for 2 h. The mixωre was filtered hot and die filtrate was concentrated to dryness. The residue was suspended in water. The solid was collected by filtration and recrystallized from etiiyl acetate-hexane to give 2.42 g (89%) of metiiyl 3-[(_V-hydroxyimino)metfιyl}-4- [(methylsulfonyl)amino]benzoate as an off-white powder, mp 160 'C.

Analysis: Calculated for C, ₀ H, ₂ N2θ ₅ S: C, 44.1 1; H, 4.44; N, 1029

Found: C 44.07; H, 4.45; N, 1020

SUBSmUTE SHEE

Example 45

3-π-Amin_-1-t/? .SVhvdroxvethvn-4-rrmethvlsulfonvl .amino1benzoic acid hvdrate f5: lV

To 2-amino-5-(medιoxyc_rbonyI)benzaldehyde trimethylene mercaptal (Gassman, P. G.; Drews, H. R. J. Am. Chem. Soc. 1978, 100, 7600-7610) (5.0 g, 0.019 mol) in dichloromethane (90 mL) was added pyridine (4.51 mL, 0.055 mol). Methanesulfonyl chloride (431 mL, 0.055 mol) was then added over a 5-min period and the reaction mixωre heated at reflux overnight Thin-layer chromatography analysis (Si0 ₂ , EtOAc:hexane 1:1) showed the disappearance of die starting material along with the appearance of a new lower-running spot (R - 0.56) indicating the reaction was complete. Upon cooling, die reaction was washed thoroughly with 10% acetic acid followed by water. The organic layer was dried filtered, and concentrated. The crude residue was placed on a Si0 ₂ column and eluted widi 1:1 EtOAchexane. Fractions containing the desired product were combined and concentrated to yield 5.0 g (77%) of 2-(methanesulfonylamino)-5-(methoxycarbonyl)benza]dehyde trimethylene mercaptal as a yellow solid. An analytical sample, mp 168-168.5 *C was recrystallized from ethanol.

Analysis: Calculated for C,3H ₁₇ N0 ₄ S ₃ : C, 44.94; H, 4.93; N, 4.03

Found: C, 44.98; H, 4.94; N, 4.01

A sample of 2-(methanesulfonylamino)-5-(medιoxycarbonyl)benzaldehyde trimethylene mercaptal (7.66 g. 0.022 mol) dissolved in _V, V-dimethylfoπnamide (4.35 mL) and acetone (39.13 mL) was added over a 5-min period tiirough a dropping funnel to a solution of copper (II) oxide (2.09 g, 0.026 mol), copper (II) chloride (7.0 g, 0.055 mol) in acetone (174.0 mL), and heated at reflux. After 2 h at reflux, diin-layer chromatography analysis (Si0 ₂ , EtOAchexane 1:1) showed disappearance of die staπing material and the appearance of a new slightly higher R _f spot which gave a positive result when sprayed widi 2,4-DNP. Upon cooling, the reaction was filtered tiirough Celite and the cake was washed widi 10% ethanol in dichloromethane. Water (125 mL) and dichloromethane (150 L) were added to die filtrate. The layers were paπitioned and the water layer extracted 2 x 100 mL dichloromethane. The combined

TE SHEET RULE 26)

organic layers were washed twice widi water (100 mL), dried (Na-SO^), and filtered. Concentration of the filtrate gave a beige solid which was dried in vacuo to give 4.48 g (79%) of 2-(methanesulfonylam_no)-5- (methoxycarbonyl)benzaldehyde, mp 161-163 ^* C (edianol).

Analysis: Calculated for C ₁₀ H, ,NO5S: C, 46.69; H, 428; N, 5.44

Found: C, 46.67; H, 435; N, 5.46

Nitrometiiane (122 g, 0.02 mol) was added to an ice-cooled mixωre of 2-(metiιanesulfonylamino)-5- (m_tiioxycarbonyI)benzaldehyde (2.57 g, 0.01 mol) in edianol and die mixωre stirred for 10 min. To this mixωre was added potassium hydroxide (1 3 g, 0.022 mol in 20 mL 90% ethanol) at 0 *C over a period of 20 min. The reaction mbcture was further stirred at 0 *C for 0.5 h and then at room temperaωre for 4 h. The mbcωre was neutralized widi 2 V HCI and poured in water (100 mL). The mbcωre was extracted with chloroform, dried over sodium sulfate, filtered, and concentrated. The residue was passed tiirough a column of silica gel eluting with chioroformtmethanol (99:1 to 953). The desired pure fractions were combined, concentrated, recrystallized from ethyl acetate-hexane, and dried to give 0.8 g (25%) of metiiyl 3-[l-(Λ5)-hydroxy-2-nitroetiιyI]-4-[(medιylsulfonyl)ammo] benzoate as off-white crystals, mp 148 *C.

Analysis: Calculated for C, ,H, ₄ N2θ ₇ S: C, 4131; H, 4.43; N, 8.80

Found: C, 41.68; H, 4.50; N, 8.78

Metiiyl 3-[l-(Λ.5)-hydroxy-2-nh_roethyI]-4-[(methylsulfonyl)amino]b enzoate (200 mg, 0.63 mmol) was dissolved in ethanol (10 L) and hydrogenated at 50 psi in die presence of platinum oxide (Aldrich, 20 mg) for 8 h. The catalyst was removed by filtration through Celite and the filtrate concentrated to 5 mL. To the concentrate, etiiyl acetate (5 mL) and hexane (10 mL) were added and die mixmre cooled to 0 *C. A white precipitate formed which was collected by filtration. The cake was washed widi ethyl acetateihexane ( 1 : 1 ) and dried to give 100 mg (55%) of metiiyl 3-[2-amino-l-(7?,S)-hydroxyedιyπ-4-[(πιetiιylsulfonyl)a mino]benzoate as a white powder, mp 172-173 *C (dec).

Analysis: Calculated for C, ,H, ₆ N ₂ 0 _j S: C, 45.82; H, 539; N, 9.72

Found: C, 45.84; H, 5.72; N, 930

A mixture of methyl 3-r2-amino-l-(ΛS)-hydroxyethyl]-4-[(m_thylsulfonyl)amino3be nzoate (50 mg, 0.17 mmol) in 1/V sodium hydroxide (44 μL, 0.44 mmol) was stirred at room temperaωre for 4 h. The mixture was neutralized widi dilute hydrochloric acid and let stand overnight at room temperaωre. A white precipitate accumulated which was collected by filtration, washed with water, and dried to give 38 mg (81%) of the title compound as a white powder, mp 274-276 'C.

Analysis: Calculated for C, ₀ H ₁₄ N ₂ O ₅ S ^• 02 H ₂ 0: C, 43.22; H, 522; N, 10.08

Found: C, 43.13; H, 526; N, 9.95

Example 46

3-ffAminoiminotτιethv1.aminol-4-methoxvbenzoic acid hydrochloride (1:1 V

A mixture of 3-amino-4-methoxybenzoic acid (Aldrich, 1.67 g, 0.01 mol), hydrochloric acid (12/V, 0.84 mL, 0.01 mol), and cyanamide (0.84 g, 0.02 mol) in absolute ethanol (20 mL) was heated at reflux for 2 h. The mixωre was cooled and die precipitate separated by filtration. The cake was washed several times with ether and dried under vacuum to give 1.60 g (65%) of the title compound as a white powder, mp 322 ^* C (dec).

Analysis: Calculated for C,H , ,N ₃ 0 ₃ ^• HCI: C, 44.00; H, 4.95; N. 17.10

Found: C, 4430; H, 4.94; N, 16.86

SUBSTITUTE SHEET

Example 47

3-f(Aminoiminomethyl .amino1-4-hvdroxyben2oic acid hydrochloride (1 :1V

A mixωre of 3-amino-4-hydroxybenzoic acid (Aldrich, 1.53 g, 0.01 mol), hydrochloric acid (127V, 0.84 mL, 0.01 mol), and cyanamide (0.84 g, 0.02 mol) in absolute ethanol (20 mL) was stirred at room temperature for 48 h and then heated at reflux for 6 h. The mixωre was allowed to stand in die refrigerator for 4 weeks during which time a brown solid began to form. The solid was collected by filtration and washed several times with ether. The solid was then dried under vacuum to give 032 g (14%) of the title compound as a brown powder, mp 254 *C.

Analysis: Calculated for ^• HCI: C, 41.48; H, 435; N, 18.14

Found: C, 41.66; H, 4.58; N, 18.12

Example 48

3.5-Dicvanaminobenzoic acid.

To a solution of 3,5-diaminobenzoic acid (Aldrich, 3.8 g, 25 mmol) in acetic acid:water (1 : 1 , 30 mL) was adde sodium acetate (4.1 g, 50 mmol). The resulting mixωre was cooled to 0 *C and cyanogen bromide (6.5 g, 60 mmol was added in two ponions over a period of 10 min. The reaction mixωre was allowed to warm to room temperaω and stiired overnight at this temperature. The reaαion mixture was poured into 130 g ice. The precipitate was collecte by filtration and washed with cold water and dried in vacuo to give 4.43 g crude product The product was dissolve in methanol and concentrated to half its volume and cooled. The product obtained was collected by filtration to giv 2.51 g (50%) of d e title compound as a grayish-white solid, mp >300 ^* C (dec).

Analysis: Calculated for CyH^O _j : C, 53.46; H, 3.00; N, 27.70

Found: C, 5328; H, 3.17; N, 27.43

C0 ₂ H

3.5-Bis-, .aminoiminomethvl.amino.bcnzoic acid hydrochloride. solvate with ethanol (10:I5:2V

A mixωre of 3,5-diaminobenzoic acid dihydrochloride semihydrate (Aldrich, 232 g, 0.01 mol) and cyanamide (232 g, 0.06 mol) in absolute ethanol (50 mL) was heated at reflux for 8 h. The mixture was cooled and die precipitate separated by filtration. The cake was washed several times with ethanol and dried under vacuum to give 220 g (73%) of the title compound as an off-white powder, mp 280 *C (dec).

Analysis: Calculated for C ₉ H ₁₂ N ₆ 0 ₂ ^• 1.5 HCI ^■ 02 EtOH: C, 37.62; H, 4.94; N, 28.00

Found: C, 37.96; H, 5.06; N, 27.75

72 Example 50

F.W. 234

Sodium 3-amino-5-f(aminoimino .methvnaminobenzoate hvdrate.

To a solution of ethyl 3,5-diaminobenzoate (Pfaltz & Bauer, 1.8 g, 10 mmol) in DMF (8 mL) was add trietiiylamine (4.91 mL, 35 mmol) and bisboc thiourea (2.76 g, 10 mmol). The resulting mbcωre was cooled to 0 and mercuric chloride (2.99 g, 11 mmol) was added. The reaction mbcωre was stirred at 0 *C for 30 min and tiien room temperaωre for 24 h. Ethyl acetate (75 mL) was added and the slurry filtered through Celite. The filtrate w washed witii water (4 x 15 mL) and die organic layer dried (Na ₂ S0 ₄ ), filtered, and solvent removed in vacuo to gi 4.95 g of crude product The crude product was purified by flash column chromatography (10-20% ethyl acetate hexane) to obtain 2.51 g (60%) of ethyl 3-_mino-5-[(r-butoxycarbonylamino-;-butoxycarbonylimino)meth yI]amin benzoate as a white solid, mp 126-128 *C.

Analysis: Calculated for C ₂ oH3 ₀ N ₄ 0 ₆ : C, 56.85; H, 7.17; N, 1326

Found: C, 56.83; H, 721; N, 13.19

To a solution of ethyl- -_un_no-5•[( butoxyca_bonylammo- bl-mxyca_bonylimino)methyl]-m_lo_enzoate (1 g, 425 mmol) in methylene chloride (20 mL) at 0 *C was added dropwise trifluoroacetic acid (327 mL, 423 mm and die solution stirred at room temperamre for 6 h. After 6 h, additional trifluoroacetic acid (327 mL, 423 mmol) w added and die reaction stirred overnight at room temperaωre. The solvent was removed in vacuo and the exce trifluoroacetic acid was removed by co-distilling twice with methylene chloride (20 mL). The resulting oily residue w dissolved in 3 M NaOH (67.5 mmol, 22.5 mL) and stirred at room temperature for 45 min. The pH was adjust between 7-8 using 1/V HCI solution. The clear solution was wanned and filtered and concentrated to half its volum The product which was obtained on standing at room temperaωre for 2 h was collected by filtration, washed with small amount of methanol and ether, and dried in a pistol at toluene reflux overnight in vacuo to furnish 0.66 g (66 of die title compound as a white solid, mp 198-203 ^* C.

Analysis: Calculated for C ₈ H ₉ N ₄ Na0 ₂ ^• H ₂ 0: C, 41.02; H, 4.74; N, 23.93

Found: C, 4123; H, 5.06; N, 23.75

Example 51

3-f(Aminoiminomethvl ■aminol-5-rf V-hvdroxvlimiπo.methvllbeπzoic acid hvdrate (4:3V

Oxalyl chloride (5.0 mL, 0.057 mol) was added to a solution of dimethylformamide (1.46 g, 0.02 mol) in dichloromethane (30 mL) at 0 *C and the reaction mixture was stirred at this temperature for 1 h. The solvent was removed under a reduced pressure. The residual white powder was suspended in tetrahydrofuran (50 mL) and acetonhrile (30 mL) and to this mixωre was added a solution of 3-carbomethoxy-5-nitrobenzoic acid (Holy, G. B. J. Med Chem. 1963, 6, 24-26) (43 g, 0.02 mol) and pyridine (1.58 g, 0.02 mol) in tetrahydrofuran (10 mL) at -30 ^* C over a period of 10 min. The reanion mixωre was stirred further for 1 h at -30 ^* C and a suspension of copper (I) iodide (0.381 g, 0.002 mol) in tetrahydrofuran (10 mL) was added to it. The reaction mixωre was cooled to -78 °C and a solution of lithium tri-rerr-butoxyaluminohydride (Aldrich, 1.0 M in tetrahydrofuran, 40.0 mL, 0.04 mol) was added over a period of 10 min. It was further stirred for 10 min at -78 *C. To this mixture was added aqueous hydrochloric acid (2 V, 50 mL) and me organic layer separated, washed with sodium bicarbonate solution, dried over sodium sulphate, filtered, and concentrated to dryness. The residue was passed through a column of silica gel using ethyl acetate:hexane (1:2) as eluent Fraction 1 [R _f - 0.55 in ethyl acetate:hexane (12)] gave 025 g (6%) of methyl 3-formyl-5- nitrobenzoate as an off-white solid, mp 85 *C.

Analysis: Calculated for C ₉ H ₇ NO _j : C, 51.68; H, 337; N, 6.70

Found: C, 51.63; H, 3.48; N, 6.57

A mixmre of methyl 3-formyl-5-nitrobenzoate (0.418 g. 2.0 mmol), hydroxylamine hydrochloride (0.209 g, 3.0 mmol) in ethanol (5.0 L) was heated at reflux for 2 h. On cooling, the mixture was poured into water and the

74 precipitate collected by filtration. The cake was recrystallized from ethyl acetate-hexane and dried to give 022 g (49% of methyl 3-[( V-hydroxylimino)methyl]-5-nitrobenzoate as an off-white solid, mp 144-145 "C.

Analysis: Calculated for G^N^: C, 4822; H, 3.60; N, 12.50

Found: C, 4822; H, 3.61 ; N, 12.41

A mixture of methyl 3-[(_V-hydroxylimmo)memyI]-5-nitrobeπzoate (2.0 g, 0.009 mol) in ethanol (150 mL) wa hydrogenated in the presence of platinum oxide (0.25 g) at 30 psi for 0.5 h. The catalyst was removed by filtratio tiirough Celite and die filtrate was concentrated. The residue was recrystallized from ethyl acetate-hexane to give 1. g (64%) of methyl 3-amino-5-[( V-hydroxylimino)medιyl]benzoate as an off-white solid, mp 160-162 ^# C.

Analysis: Calculated for C ₉ H ₁₀ N ₂ O ₃ : C, 55.67; H, 5.19; N, 14.42

Found: C, 55.55; H, 5.33; N, 14.00

A mixωre of metiiyl 3-amino-5-[(/V-hydroxylimino)methyl]benzoate (0.97 g, 0.005 mol), N,N-b s(tert butoxycarbonyI)thiourea (132 g, 0.0055 mol) and trietiiylamine (1.77 g, 0.0175 mol) in dimethylformamide (10.0 mL was cooled to 5 °C. Mercuric chloride (1.49 g, 0.0055 mol) was added to the mixture and stirred at 5 "C for 0.5 h an tiien at room temperature for 18 h. The mixωre was diluted with ethyl acetate (40 mL) and filtered tiirough Celite. The filtrate was washed with water and die organic layer dried over sodium sulphate. The mixωre was filtered, the filtrate concentrated, and the residue was passed through a column of silica gel using ethyl acetate:hexane (1:3 to 1:1). The appropriate fractions were pooled, combined, and concentrated to give methyl 3-([(r_r/-butoxycarbonyl_mino-r_rf- butoxyc_rbonylimino)methyl]amino}-5-[( V-hydroxylimino)methyl]benzoate [R _f - 0.7 in ethyl acetate:hexane (1 :1)]. An analytical sample was recrystallized from ethyl acetate:hexane to give 0.7 g (30%) as a white solid, mp 158- 160 ^* C (dec).

Analysis: Calculated for C ₂₀ H _2g N ₄ O ₇ : C, 55.04; H, 6.47; N, 12.84

Found: C, 55.48; H, 6.65; N, 12.72

A mixωre of methyl 3-{[(«r/-butoxycarbonylamino- βrr-butoxycarbonylimino)meth> ]amino}-5-[( V-

hyαroxylimino)methyl]benzoate (360 mg, 0.83 mmol) and trifluoroacetic acid (1.5 mL) in dichloromethane (15 mL) was stiired at room temperature for 24 h. The mixture was concentrated and die residue was repeatedly evaporated witii dichloromethane.

The residue was suspended in sodium hydroxide (1 V, 3.0 mL, 3.0 mmol) and stiired for 6 h. The pH of the mixωre was adjusted to close to 8 with dilute hydrochloric acid. White solid separated out, which was colleαed by filtration, washed with water, and dried under vacuum over toluene to give 125 mg (64%) of the title compound as an off-wh e solid, mp 248-253 "C (dec).

Analysis: Calculated for C ₉ H ₁₀ N ₄ O ₃ ^• 0.75 H ₂ 0: C, 45.86; H, 4.92; N, 23.77

Found: C, 46.00; H, 4.92; N, 23.72

Example 52

3-tAminoiminomethvl .amino-5-hvdroxvmethvl-4- methvl.ωlfonvl .aminobenzoic acid hvdrate complex with diisonronyl ether (10:10:1V

To 2-amino-5-(methoxyc_r_onyl)benzaldehyde trimethylene mercaptal (Gassman, P. G.; Drews, H. R. J. Am. Chem. Soc. 1978, 700, 7600-7610) (5.0 g, 0.019 mol) in dichloromethane (90 mL) was added pyridine (4.51 mL, 0.055 mol). Methanesulfonyl chloride (4 1 mL, 0.055 mol) was then added over a 5-min period and the reaction mbcωre heated at reflux overnight Thin-layer chromatography analysis (Si0 ₂ , EtOAc:hexane 1:1) showed the disappearance of die starting material along with the appearance of a new lower-running spot (Rf- 0.56) indicating die reaction was complete. Upon cooling, the reaction was washed thoroughly with 10% acetic acid followed by water. The organic layer was dried (Na ₂ S0 ₄ ), filtered, and concentrated. The crude residue was placed on a Si0 ₂ column and eluted with 1:1 EtOAc-hexane. Fractions containing the desired product were combined and concentrated to yield 5.0 g (77%) of 2-(metiι_nesulfonylamino)-5-(methoxycarfoonyl)benzaldehyde trimethylene mercaptal as a yellow solid. An analytical

B-Tf

sample, mp 168-168.5 *C, was recrystallized from ethanol.

Analysis: Calculated for C ₁₃ H _I7 N0 ₄ S ₃ : C, 44.94; H, 4.93; N, 4.03

Found: C, 44.98; H, 4.94; N, 4.01

A sample of 2-(methanesulfonylamino)-5-(methoxycarbonyI)benzaIdehyde trimethylene mercaptal (7.66 g, 0.022 mol) dissolved in sV. V-dimethylformamide (435 mL) and acetone (39.13 mL) was added over a 5-min period through a dropping funnel to a solution of copper (ϋ) oxide (2.09 g, 0.026 mol), copper (II) chloride (7.0 g, 0.055 mol) in acetone (174.0 mL), and heated at reflux. After 2 h at reflux, thin-layer chromatography analysis (Si0 ₂ , EtOAc:hexane 1:1) showed disappearance of die stanmg material and the appearance of a new slightly-higher R _f spot which gave a positive result when sprayed widi 2,4-DNP. Upon cooling, the reaction was filtered through Celite and the cake was washed widi 10% edianol in dichloromethane. Water (125 mL) and dichloromethane (150 mL) were added to die filtrate. The layers were partitioned and die water layer extracted 2 x 100 mL dichloromethane. The combined organic layers were washed twice with water (100 mL), dried (Na ₂ S0 ), and filtered. Concentration of the filtrate gave a beige solid which was dried in vacuo to give 4.48 g (79%) of 2-(methanesulfonylamino)-5-(methoxycarbonyI)benz- aldehyde, mp 161-163 *C (ethanol).

Analysis: Calculated for C ₁₀ H- JNOJS: C, 46.69; H, 428; N, 5.44

Found: C, 46.67; H, 4.35; N, 5.46

Methyl 3-foιmyl-4-[(methylsulfonyl)amino]ben_oate (3.85 g, 0.015 mol) was added portionwise to fuming nitric acid (45 mL) at 5-10 *C over 025 h. The mixωre was further stirred for 1.0 h at room temperaωre and then poured into cold water (200 mL). The yellow precipitate was collected by filtration and washed several times with cold water. The cake was recrystallized from ethyl acetate-hexane to give 2.5 g (553%) of methyl 3-formyl-4- [(methylsulfonyI)amino]-5-nitrobenzoate as light yellow needles, mp 158-160 ^* C.

Analysis: Calculated for C _I0 H, ₀ N ₂ O ₇ S: C, 39.74; H, 3.33; N, 927

Found: C, 39.56; H, 3.30; N, 923

Methyl 3-formyl-4-[(methylsulfonyl)am_ιo]-5-nitrobenz_ate (2.0 g, 0.0066 mol) was dissolved in ethanol (50 mL) and hydrogenated at 30 psi in the presence of platinum oxide (Aldrich, 0.1 g) for 4 h. The catalyst was removed by filtration through Celite and die filtrate was concentrated to dryness. The residue was recrystallized from metiianol- ethyl acetate-hexane to give 0.8 g (44%) of methyl 3-ammo-5-hydroxymetiιyl-4-[(medιylsulfonyl)amino]benzoate as a light brown powder, mp 150-155 *C.

Analysis: Calculated for C ₁₀ H ₁₄ N ₂ O _j S: C, 43.79; H, 5.14; N, 1021

Found: C, 44.03 ; H, 5.14; N, 10.08

A mixωre of methyl 3-amino-5-hydroxymethyl-4-(methylsulfonyi)aminobenzoate (0.548 g, 0.002 mol), 4/V hydrochloric acid in dioxane (0.6 mL, 0.0024 mol), and cyanamide (0.84 g, 0.020 mol) in ethyl acetate (20 mL) was heated at reflux for 16 h. A brown-colored cake was deposited at die bottom of the flask. The supernatant was removed by decantation and die cake was stirred widi fresh ethyl acetate (20 mL) at room temperature for 4 h. The brown solid was collected by filtration and dried to give 0.52 g (73%) of methyl 3-(ammoiminomethyl)amino-5-hydroxymetiιyl-4- (methylsulfonyl)aminobenzoate hydrochloride.

The above ester (9.5 g, 0.0014 mol) was stirred with 1/V sodium hydroxide (3.0 mL, 0.003 mol) at room temperaωre for 16 h and die mbcωre filtered through a cotton plug. The filtrate was neutralized with concentrated hydrochloric acid. A fine yellow precipitate was obtained which was removed by filtration and die filtrate was evaporated to dryness. The residue was dissolved in methanol, insolubles removed by filtration, and die filtrate evaporated to dryness. The residue was again dissolved in methanol and 2-propanol was added. The precipitate which formed was collected by filtration and dried under vacuum over acetone reflux for 24 h to give 0.06 g (13%) of the title compound as a fluffy solid, mp 300-307 *C.

Analysis: Calculated for C, ₀ H, ₄ N ₄ O _j S ^• H ₂ 0 ^• 0.1 C, 38.52; H, 5.30; N, 16.95

Found: C, 38.66; H, 5.16; N, 1730

Example 53

3-f(Aminoiminomethvl .amino1-4-r(methvlamino carbonvnbenzoic acid hydrochloride. complex with water and methanol (4:4:4 1 and 5:5:5:1V

A mbcture of nitroterephtiialic acid (Aldrich, 20 g, 0.095 mol) in anhydrous methanol (100 mL) was heated to reflux in the presence of concentrated sulphuric acid (10 mL) for 1 h. The solvent was evaporated and the residue poured into saturated sodium bicarbonate (200 mL). The aqueous layer was washed with chloroform and 2 V hydrochloric acid added to the aqueous layer to bring the pH close to 2. The mixture was extracted twice with ethyl acetate and die combined organic layers were washed with water and brine. The organic extract was concentrated after drying over sodium sulphate and the residue was recrystallized from ethyl acetate-hexane to give 11.8 g (55%) of 4- m_thoxycarbonyl-2-nitrobeπzoic acid as cream-colored crystals, mp 131-132 "C.

Analysis: Calculated for C ₉ H ₇ N0 ₆ : C, 48.01; H, 3.13; N, 622

Found: C, 47.97; H, 321; N, 6.16

A mixture of 4-metiioxycar_onyI-2-_itro__nzoic acid (3.6 g, 0.016 mol) in thionyl chloride (32 mL) was heated to reflux in die presence of two drops of dimetiiylformamide for 3 h. Thionyl chloride was distilled and the concentrate was left under vacuum for 0.5 h. The residue was dissolved in dichloromethane (10 mL) and added to a solution of aqueous methylamine (40%, 2.0 g, 0.026 mol) in water (5 mL) over a period of 5 min. The mixture was further stirred for 10 min and the white precipitate was collected by filtration. The cake was washed witii water, dried, and recrystallized from ethyl acetate-hexane to give 2.1 g (55%) of metiiyl 4-(methylamino)carbonyl-3-nitrobenzoate as white needles, mp 155 *C.

Analysis: Calculated for C _I0 H, ₀ N ₂ O _j : C, 50.42; H, 423; N, 11.76

Found: C, 50.54; H, 420; N, 11.89

____

A mixture of methyl 4-(medιylanιino)c_ιbonyl-3-nitrobenzoate (3.84 g, 0.0185 mol) and tin (ii) chloride (153 g, 0.08 mol) in ethanol (80 mL) was heated at 70 ^* C for 1 h. The reaction mbcωre was diluted with water (150 mL) and sodium bicarbonate solution was added to it to bring the pH close to 8. The mixωre was extracted with ethyl acetate and the organic layer washed witii water and brine and dried over sodium sulphate. After filtration, the solution was concentrated and die residue was recrystallized from ethyl acetate-hexane to give 2.16 g (64%) of methyl 3-amino-4- (methylamino)carbony_benzoate as tan needles, mp 167-168 *C.

Analysis: Calculated for C ₁₀ H, ₂ N ₂ O ₃ : C, 57.68; H, 5.81; N, 13.45

Found: C, 57.81; H, 5.83; N, 13.50

A mixture of methyl 3-amino-4-(methylamino)carbonylbenzoate (0.624 g, 0.003 mol) and 1/V sodium hydroxide (4.5 mol, 0.0045 mol) was stirred at room temperaωre for 4 h. The mbcωre was neutralized with concentrated hydrochloric acid and then acetic acid was added to bring the pH close to 3. The precipitate obtained was collected by filtration, washed with water, and dried under vacuum over acetone for 24 h to give 0.4 g (69%) of 3- amino-4-(methylamino)carbonylbenzoic acid as a pale yellow powder, mp 215-217 *C.

Analysis: Calculated for C ₉ H _I0 N ₂ O ₃ : C, 55.67; H, 5.19; N, 14.43

Found: C, 55.75; H, 525; N, 14.43

A mixωre of 3-amino-4-(methylamino)carbonyibenzoic acid (0.194 g, 0.001 mol), concentrated hydrochloric acid (0.084 g, 0.001 mol), and cyanamide (0388 g, 0.014 mol) in ethyl acetate (10 mL) was stirred at 37-38 *C for 16 h. A white solid formed which was collected by filtration. Thin-layer chromatography showed die presence of tiie starting material. The white solid was again stiired at 37-38 "C with cyanamide (0.42 g, 0.01 mol) in ethyl acetate ( 10 mL). A white solid was again collected by filtration and washed widi etiiyl acetate. It was recrystallized from methanol- ether to give 0.03 g (10%, first crop) of the title compound as white crystals, mp >300 ^* C. A second crop (0.09 g, 30%) as white crystals, mp >300 *C, was collected on allowing the filtrate to stand in the refrigerator for 2 days.

Analysis of first crop: Calculated for C, ₀ H ₁₂ N ₄ O ₃ ^• HCI ^• H ₂ 0 ^• 025 MeOH: C, 41.21; H, 5.40; N, 18.76

Found: C, 41.14; H.5.42; N, 18.98

Analysis of second crop: Calculated for C ₁₀ H ₁₂ N ₄ O ₃ ^• HCI ^• H ₂ 0 ^• 02 MeOH: C, 4123; H. 5.36; N, 18.86

_Found: C, 41.48; H, 5.47; N, 1920

LE 26

Utility

The compounds of Formula (I) possess neuraminidase inhibitory activity and are therefore useful as antiviral and anti-microbial agents for the prevention, treatment or amelioration of infections. More particularly, the compounds of Formula (I) possess influenza virus neuraminidase inhibitory activity and are effective as inhibitors of influenza infections. The neuraminidase inhibitory activity of the compounds of the present invention is demonstrated using standard assays of neuraminidase activity; for example, using tiie assay described below.

A fluorimenic assay was used to measure influenza virus neuraminidase activity. It utilized a substrate (2'-(4- methylumbelliferyl)-α-D-acetylneuraminic acid), which was cleaved by neuraminidase to yield a fluorescent product which was quantified. The assay mbcture contained a compound of Formula (I) at various concentrations and an amount of neuraminidase in the form of whole virus or crystals in an appropriate buffer, at pH 6.5. The reaction was started by the addition of the substrate to a final concentration of 75 μM. After 5 min at 37 "C, giycine and NaOH, pH 102, was added to terminate the reaction. A blank was run using die same substrate but no enzyme. Fluorescence was read using a fluorescence spectrophotometer (excitation: 360 nm and emission: 450 nM) and substrate blanks were subtracted from the readings. A compound was considered to be active if it had a percent inhibition value of more than about 10% for the inhibition of neuraminidase activity. The neuraminidase inhibitory activity of representative compounds of the invention is shown below in Table 1:

$

Table 1. Neuraminidase Inhibition as Determined by In Vitro Enzyme Assay

Dosage and Formulation

The antiviral compounds of this invention can be administered as treatment for viral infections by any means tiiat produces contact of the active agent witii the agent's she of action, the viral neuraminidase, in the body of a mammal They can be administered by any conventional means available for use in conjunction with phaimaceuticals, eidier as individual therapeutic agents or in a combination of therapeutic agents. They can be administered alone, but generally administered with a pharmaceutical carrier selected on the basis of die chosen route of administration and standard pharmaceutical practice.

The dosage administered will, of course, vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; tiie age, health, and weight of the recipient; die nature and extent of the symptoms, the land of concurrent treatment; die frequency of treatment; and the

effeα desired. A daily dosage of aαive ingredient can be expected to be about 0.001 to 1000 milligram (mg) per killigram (kg) of body weight with the preferred dose being 0.1 to about 30 mg/kg.

Dosage forms (compositions suitable for administration) contain from about 1 mg to about 100 mg of active ingredient per unit In these pharmaceutical compositions, die active ingredient will ordinarily be present in an amount of about 0.5-95% by weight based on tiie total weight of the composition.

The active ingredient can be administered orally in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elbcirs, syrups, and suspensions. It can also be administered parenterally, in sterile liquid dosage forms. Other dosage forms are potentially possible such as administration transdermally, via a patch mechanism or ointment.

Gelatin capsules contain the aαive ingredient and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar-coated or film-coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in tiie gastrointestinal tract.

Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.

In general, water, a suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions for parenteral administration preferably contain a water-soluble salt of the aαive ingredient suitable stabilizing agents, and, if necessary, buffer substances. Antioxidizing agents such as sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also used are citric acid and its salts and sodium EDTA. In addition, parenteral solutions can contain preservatives, such as benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol.

Suitable pharmaceutical carriers are described in Remington . Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field.

Useful pharmaceutical dosage forms for administration of the compounds of this invention can be illustrated as follows:

Capsules

A large number of unit capsules are prepared by filling standard two-piece hard gelatin capsules each with 100 mg of powdered active ingredient 150 mg of lactose. 50 mg of cellulose, and 6 mg of magnesium stearate.

Soft Gelatin Capsules

A mbcωre of active ingredient in a digestible oil such as soybean oil, cottonseed oil, or olive oil is prepared and injeαed by means of a positive displacement pump into gelatin to form soft gelatin capsules containing 100 mg of the active ingredient The capsules are washed and dried. Tablets .

A large number of tablets are prepared by conventional procedures so that die dosage unit was 100 mg of active ingredient 02 mg of colloidal silicon dioxide, 5 mg of magnesium stearate, 275 mg of microcrystalline cellulose, 11 mg of starch, and 98.8 mg of lactose. Appropriate coatings may be applied to increase palatability or delay absorption.

Various modifications of die invention in addition to those shown and described herein will be apparent to those skilled in the an from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

The foregoing disclosure includes all the information deemed essential to enable those skilled in the an to praαice the claimed invention. Because the cited applications may provide further useful information, these cited materials are hereby incorporated by reference in their entirety.

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