throbber
·1;~·
`/.f.''-:'·•
`,~·::·' ."
`
`..,..,.
`
`.t~;..---~
`
`'
`
`,G.9..b
`
`1'~- 22.3.il
`·~~71
`
`1'
`
`I
`
`I'
`
`168,43c
`
`52.70
`
`169, t 2C
`
`42.38
`
`142.58
`
`127.271,h
`12a.271,h
`
`126.89
`
`122.15
`(C4• or Cr)
`122.32
`(C4• or C7)
`123.45 (C3•)
`124.37
`(Cs• or Ca1
`124.41
`(C5• or Cs•)
`138.84
`(C3•a ·or C7'a)
`138.95
`(C3•a or C1•a)
`142.58 (C2'1
`
`_.
`0 _.
`
`i]ak OCH3
`
`23.03
`
`187.91
`
`78.94
`
`171.57
`
`.43,51
`
`1-37.45
`
`afilzk
`
`OCH2CH323.25 16B. 13
`
`77.43
`
`t7L29
`
`43.60
`
`137.57
`
`127.701,h
`128.701,h
`
`127,69h,1
`12a,79l,h
`
`127.62
`
`55.84 (CH3)
`
`127.691
`
`15.0S(CH3)
`64.51 (CH2)
`
`8 Tho 13c NMR spootra were la.ken In DMSO-d6 unless otherwise lodlca!9d. The number -ln each entry ls the c:horfilc:al shlrt Vl'l!Uo In pe.rls P4f
`rolaHve lo JMS, The number Jn par1~nthoE1ses Jn si;ilocl cates Is 1he proposed asslgnmen\, b Rel, GB, e,d,o ThosG sols ol poaks rnay bGo
`mllfion
`the lnlens~y of noa-rby peaks. 0 These poaks may bo lnlerChangeablo. h Tho close pro:d·
`f The pe-ak had approximately lwk:e
`Interchangeable.
`I The remalrilng aromaHc airbon algnal.s (125,50, 126.00,
`the ~ peaks did net permit
`ihe assJ;Jnment or these rl)sonal"ICas,
`lmtty ol
`I R11f. 104,
`126.10, 126.30, 126.SO. 127.50, 127.70, 127.90, 132.40, 132.SO, 136.50) we1re not a.sa!Qnetd due lo their alrn~lar chemk::al shift veluos, k The 13 C
`I ThEt peak had approxlmately 1hroo 1lmo 1he
`lnlensh:y of. naarby peak.s.
`NMA speclfa were
`taken
`In CDCl3,
`
`

`
`102
`
`The chemical shifts values observed
`
`for
`
`the heterocyclic carbon
`
`atoms provided strong support
`
`for
`
`the proposed substitution site in
`
`this portion of
`
`the molecule.
`
`In all cases, the chemical shift value
`
`for
`
`the substituted aromatic carbons was downfield (6.0 - 20.0 ppm)
`
`versus
`
`the corresponding signal in
`
`the unsubstituted heterocycle.
`
`This
`
`trend was consistent with previous observations.158
`
`3.
`
`Pharmacological Evaluation.
`The 2-substituted-2-acetamido·N-benzylacetamides .6..9. and fill.
`
`prepared in this study were submitted
`
`to
`
`the Eli Lilly Corporation,
`
`Indianapolis,
`
`Indiana,
`
`for evaluation of
`
`their anticonvulsant activity.
`
`The previous
`
`results obtained by Kohn and co-workers68, 102,
`
`for
`
`related compounds suggested
`
`that
`
`these substances ·would be most
`
`active
`
`in · the MES seizure test.
`
`Accordingly, only
`
`this anticonvul-
`
`sant screening procedure was conducted. Each of
`
`the substances
`
`were administered
`
`intraperitoneally at
`
`three doses (300, 100 and 30
`
`mg/kg)
`
`to groups of
`
`four mice (male albino, CF-1 strain).
`
`Those
`
`compounds which were
`
`found
`
`to be effective
`
`in . the protection of
`
`seizures
`
`induced by electrical stimulation were
`
`further tested with
`
`better monitoring of dosages in groups of twelve mice. This pro(cid:173)
`
`cedure permitted
`
`the determination of
`
`the Effective Dose (ED) 50
`
`value for the drug candidate. This value
`
`is the dose which is effec(cid:173)
`
`tive
`
`in protecting 50 % of
`
`the animals
`
`tested against seizures.
`
`In
`
`addition
`
`to
`
`this
`
`test.
`
`the neurologic toxicity (TOX) of some of the
`
`biologically active compounds was evaluated by means of
`
`the
`
`

`
`103
`
`horizontal screen
`
`test using
`
`the same conditions
`
`(dosages, number
`
`of
`
`animals
`
`tested) employed
`
`in
`
`the ED 50 determination.
`
`This
`
`procedure permitted
`
`the calculation of
`
`the Toxic Dose (TD) 50
`
`value which corresponds
`
`to the dose
`
`that
`
`leads to toxic manisfes(cid:173)
`
`tations
`
`in 50 % of the animals screened. Tables 31 - 34 summarize
`fill., rui and
`
`results observed
`
`for compounds
`
`the pharmacological
`
`some clinically used anticonvulsant agents.
`
`For
`
`the purpose of
`
`the discussion of
`
`the
`
`results, several
`
`assumptions have been made. First,
`
`it is assumed that each sub(cid:173)
`
`strate
`
`is in'fluenced
`
`to
`
`the same degree by
`
`the many processes
`
`(i.e., absorption, distribution, metabolism, elimination) which affect a
`
`drug's
`
`action
`
`from
`
`the
`
`time
`
`it
`
`is administered to
`
`the time the
`
`biological response occurs.
`
`Second,
`
`it is assumed
`
`that
`
`the active
`
`agent is
`
`the substrate itself and not a metabolite of the com-
`
`pound.
`
`No
`
`tests were conducted
`
`to verify
`
`the validity of these
`
`assumptions.
`
`The 2-acetamido-N-benzylacetamides bearing a
`
`five mem(cid:173)
`
`bered
`
`ring heteroaromatic at
`
`the a.-carbon
`
`(69 a-d) (Table 31) all
`
`displayed outstanding anticonvulsant properties
`
`in the MES seizure
`
`test. Within
`
`this series,
`
`derivatives were more potent than
`
`the 2-pyrryl {fill.)
`the 2-furyl (fil!a) and
`the parent aromatic substrate fill.!:!,
`
`and 3-thienyl {filld.) adducts were
`
`less
`
`while
`
`active
`
`the 2-thienyl (fill.lj
`than filill.
`
`activity similar
`
`to phenytoin
`
`under comparable conditions.
`
`(Table 34)
`
`Of particular note, ~ and
`.6..a,b. exhibited
`tu.al and diazepam (2.$)
`Of these two medicinal agents,
`
`

`
`104
`
`Table 31. Pharmacological Evaluation of 2-Substituted-2-acetamido-N(cid:173)
`benzylacetamides (fill.) Containing a Monocyclic Heterocyclic Moiety.a
`
`0 YH
`
`R
`-~ N~Ph
`0
`
`69
`
`No.
`
`fillli.d
`
`2.9.a
`
`69b
`
`filkf
`
`.!ill.d.f
`
`R
`
`Phenyl
`
`2-Furyl
`
`MESb, ED50
`
`ToxC, TD50
`
`32.1 (27.5 - 40.2)
`
`10.3 ( 9.1 - 11.6)
`
`> 4oe
`
`-40
`
`2-Pyrryl
`
`16.1 (13.2-19.9)
`
`30 - 100
`
`2-Thienyl
`
`44.8 (38.9-51.4)
`
`30 - 100
`
`3-Thienyl
`
`87.8 (69.9 - i 50.0)
`
`> 100
`
`and TOSO
`a The compounds were administered intraperitoneally. ED50
`are in mg/kg. The number
`in parentheses are the 95 % confidence
`0 Maximal electroshock seizure
`intervals.
`test.
`c
`Horizontal
`screen
`test
`(neurotoxicity) unless otherwise
`indicated. d Ref. 162.
`eRotarod ataxia test. f Ref. 104.
`
`;, ..
`_:~ .
`
`' '
`
`' i
`. ' !
`
`

`
`105
`
`Table 32. Pharmacological Evaluation
`benzylacetamide
`(Jill) Containing a
`Moiety.a
`
`of 2-Substituted-2-acetarnido-N(cid:173)
`Benzo-fused Heteroaromatic
`
`69
`
`No.
`
`R
`
`MESb, EDSO
`
`ToxC, TOSO
`
`~d
`
`~-Naphtyl
`
`> 300
`
`> 300
`
`filli
`
`£.9.Q.
`
`fillli
`
`2-Benzoluryl
`
`> 100, < 300
`
`> 100, < 300
`
`3-lndolyl
`
`>300
`
`< 300
`
`2-Benzo[b]thienyl
`
`> 100, < 300
`
`> 100, < 300
`
`aThe compounds were administered intraperitoneally. EDSO and. TOSO
`are
`in mg/kg.
`b Maximal electroshock seizure
`test. c Horizontal
`screen
`test
`(neurotoxicity). d Ref. 104.
`
`

`
`106
`
`;:i;:i. Pharmacological Evaluation of 2-Alkoxy-2-acetamido-.~.-
`Tabla
`benzylacetamide ®).a
`
`R
`0 ~H
`~N . N~h
`H
`0
`
`86
`
`R
`
`CH3
`
`MESb, ED50
`
`ToxC, TD50
`
`51.0 ( 46.6 - 58.6}
`
`> 1ooe
`
`CH2CH2SCH3
`
`> 100, < 300
`
`CH(CH3}2
`
`> 100, <300
`
`g
`
`g
`
`OCH3
`
`OCH2CH3
`
`98.3
`
`< 300
`
`..
`
`62.0( 51.1 - 78.4}
`
`> 112
`
`No.
`
`68ad
`
`filkf
`
`filillf
`
`1lli.9.
`
`.!llib.
`
`aThe compounds were administered intraperitoneally. ED50 and TD50
`in mg/kg. The numbers
`in parentheses are the 95 % confidence
`are
`intervals.
`b Maximal electroshock seizure
`test. c Horizontal . screen
`test (neurotoxicity) unless otherwise indicated. d Ref. 104. e Rota rod
`f Ref. 68.
`g These values were not determined.
`ataxia test.
`
`

`
`107
`
`Table 34. Pharmacological Activity of Some Proven Anticonvulsants.a
`
`No.
`uact
`~d
`
`.u12e
`
`15.ae
`
`2.6.e
`
`Name
`
`MEsb •. EDSO
`
`ToxC, TDSO
`
`Phenytoin
`
`Diazepam
`
`Metphenytoin
`
`14.0
`
`18.7
`
`61.0
`
`Phenobarbital
`
`20.1
`
`Valproic Acid
`
`664.8
`
`86
`
`57
`
`154
`
`97
`
`1264
`
`aThe compounds were administered intraperitoneally. EDSO and
`TOSO
`in mg/kg. b Maximal electroshock seizure test. c Rota rod ataxia
`are
`test (neurotoxity). d Ref. 162. e Ref. 163.
`
`

`
`108
`
`phenytoin
`
`(1.aa)
`
`is the most widely prescribed drug
`
`today for the
`
`treatment of . epilepsies.
`
`Examination of
`
`the TD 50 values
`
`for
`
`the
`
`rnonocyclic
`
`heteroaromatic adducts
`
`!2.9_
`
`indicated
`
`that neurologic
`
`toxicity closely
`
`paralleled
`
`their activity
`
`in
`
`the MES
`
`·seizure
`
`test.
`
`· The TD 50 value
`
`for
`
`the most active compound,
`
`the furyl
`
`derivative .fillll , was approximately 40 mg/kg.
`
`The observed data
`
`permitted several
`
`tentative conclusions.
`
`First, compounds 69 a-d
`
`like filll1. all contain an electron
`
`rich
`
`it-aromatic system.
`
`Second,
`
`all of the heteroaromatic substituents at
`
`the u'carbon were rela-
`
`tively small. Moreover, within
`
`this series of
`
`five compounds
`
`the
`
`smallest analogues (fill.a, Jillb.) were
`the most active.
`ring heteroaromatic compounds Jill.1:h, as
`
`All of
`
`the
`
`fused
`
`well as
`
`the parent naphtyl adduct fill.a (Table 32) were considerably
`
`less active than their monocyclic counterparts (Table 31 ).
`
`None
`
`displayed significant activity at dosages below 100 mg/kg. Of
`
`these
`
`compounds,
`
`the 2-benzofuryl
`
`(filill and the 2-benzo[b]thienyl (fillh)
`
`derivatives were
`
`the most potent agents.
`
`The dramatic decrease
`
`in biological activity versus
`
`their monocyclic counterparts suggested
`
`that stringent steric
`
`requirements may exist
`
`for maximal biological
`
`activity.
`
`The 2-acetamido-N-benzyl-2-alkoxyacetamides Jill. intermediates
`
`utilized
`
`in
`
`the
`
`synthesis
`
`of many
`
`of
`
`the
`
`heteroaromatic
`
`functionalized amino acids derivatives (Method B) were also sub(cid:173)
`
`mitted
`
`for
`
`pharmacological
`
`testing
`
`(Table 33).
`
`Included
`
`in
`
`this
`
`table
`
`are
`
`several alkyl and substituted alkyl analogues which
`
`

`
`109
`
`serve as suitable
`
`reference eompounds.
`
`Both
`
`the 2-methoxy (Jlfua)
`
`and
`
`the 2-ethoxy (.flfil2) derivatives exhibited notable activity
`
`in
`
`the
`
`MES
`
`seizure
`
`test.
`
`The pharmacological
`
`activity of ruill. was
`
`comparable
`
`to metphenytoin (1.:ib.) under similar · conditions.
`
`The
`
`limited
`
`results
`
`secured
`
`from
`
`this series of compounds do not
`
`permit us to make any tentative conclusions concerning
`
`the role of
`
`the oxygen atom in modulating
`
`the biological activity of
`
`these
`
`derivatives.
`
`The composite set of data (Tables 31 • 33) suggests that
`
`stringent
`
`steric
`
`constraints exist at
`
`the
`
`receptor site
`
`for drug
`
`binding.
`
`Moreover, among
`
`the
`
`four monocyclic heteroaromatic
`
`compounds,
`
`the pharmacological
`
`. activity
`
`increased with decreasing
`
`resonance stability of
`
`the aromatic moiety.
`
`This trend suggests
`
`that
`
`the biological activity
`
`is modulated
`
`by
`
`the ability of
`
`the
`
`drug candidate· to bind with an electrophilic site at
`
`the
`
`receptor.
`
`:r:r:r.
`
`Experimental Section. General Methods. Syntheses. Melting
`
`points were determined with a Thomas-Hoover melting point apparatus and
`
`are uncorrected.
`
`Infrared spectra (IR) were run on a Perkin-Elmer 1330
`
`spectrophotometer and calibrated against the 1601 cm·1 band of polystyrene.
`
`Absorption values are expressed in wavenumbers (cm· 1 ). Proton nuclear
`
`magnetic resonance (1 H NMR) and carbon nuclear magnetic resonance
`
`(13c NMR) were _taken on a Nicolet NT-300 instrument. Chemical shifts are
`
`in parts per million {o values) relative to tetramethylsilane (TMS) or sodium
`
`2,2·dimethyl-2·silapentane-5-sulfonate (DSS) and coupling constants (J
`
`

`
`110
`
`values) are in Hertz. Mass spectra were performed atthe Eli Lilly Corporation,
`
`Indianapolis, Indiana ,or by Dr. John Chinn at the Department of Chemistry,
`
`University of Texas at Austin. Elemental analysis was conducted at the Eli
`
`Lilly Corporation, Indianapolis,
`
`Indiana. Furan (ZQa), pyrrole (ll), ethyl
`
`chloroformate, isobutyl chloroformate, benzylamine (a.3.). methanesulfonic
`
`acid, boron trifluoride etherate, benzo[b]thiophene (.Z.!i), indole (12) and
`
`triethylamine were purchased from Aldrich Chemical Company, Milwaukee,
`
`Wisconsin; ethyl acetamidocyanoacetate and NaBH4 were obtained from
`
`Sigma Chemical Company, SL Louis, Missouri; . benzofuran (25.) was
`
`obtained from Chemicals Procurement Laboratories Inc., College Point, New
`
`York; and benzothienylglycine (Z3.ll.) was provided by Eli Lilly Corporation,
`
`Indianapolis, Indiana. Acetonitrile and triethylamine were distilled from CaH2
`and tetrahydrofuran and ethyl ether were distilled from Na/benzophenone.
`
`Furan (I.Q..a), pyrrole (ZA.). benzofuran (li). acetic anhydride (.!&). ethyl
`
`chloroformate, isobutyl chloroformate and methanesulfonic acid were
`
`fractionally distilled prior to use. All other chemicals were of the highest
`
`grade available and were used without further purification. The mixed
`
`anhydride reactions and the amidoalkylation transformations using boron
`
`trifluoride etherate were run under anhydrous conditions. In these cases, all
`
`glassware was flame-dried under N2, the solid starting materials were dried
`in yacuo immediately prior to use, and the reactions were conducted under a
`
`positive pressure of N2. Preparative flash column chromatography was run
`using Merck silica gel, grade 60, 230-240 mesh, 60 A from Aldrich Chemical
`
`Company, Milwaukee, Wisconsin.
`
`

`
`111
`
`Method A.
`
`Preparation of Methyl 2-Acetamldo-2-methoxyacetate
`
`(llllli}. Sulfuric acid (95%, 4 ml, 70 mmol} was added to a methanolic
`
`solution (230 ml) of 2-acetamido-2-hydroxyacetic acid (Ia) (13.30 g, 100
`
`mmol}. The solution was stirred at room temperature (48 h}, neutralized with
`solid NaHC03, filtered, and then the methanol was removed in vacuo. The.
`pink oil was distilled under vacuum (70-120 °C, 0.6 torr) to give a colorless oil
`
`which was recrystallized from petroleum ether (35-60 °C) to yield 5.20 g
`
`(32%) of the desired product: R1 0.52 (98:2 chloroform/methanol): mp 44-46
`°C; 1H NMR (300 MHz, CDCl3) o 2.08 (s, CH3CO), 3.46 (s, OCH3), 3.81 (s,
`COOCH3). 5,54 (d, J = 9.3 Hz, CH), 6.70-6.80 (br d, NH); 13c NMR (75
`
`MHz, CDCl3) 22.98 (CH3CO). 52.69 (COOCH3), 56.48 (CH30), 78.16
`
`(CH), 168.49 (CH3CO). 170.67 (COOCH3) ppm; IR (KBr) 3270, 2820, 1735,
`1650 (br), 1505, 1205, 1110, 1090, 1010, 930, 900 cm·1; mass spectrum, m/e
`
`(relative intensity) 162 (1 ), 146 (2), 131 (3), 118 (3). 102 (46), 88 (25), 60
`
`(100).
`
`Anal. Calcd for C6H11N04: C, 44.72; H, 6.88; N, 8.69. Found: C,
`
`44.46; H, 7.14; N, 8.72.
`
`Preparation of Ethyl 2-Acetamldo·2-ethoxyacetate (fil!.12).
`
`Sulfuric acid (95%, 4 ml, 70 mmol) was added to an ethanolic .solution (250
`
`ml) of 2-acetamido-2-hydroxyacetic acid (Zil.) (13.30 g, 100 mmol). The
`
`solution was stirred at room temperature (48 h) and then poured into a chilled
`
`saturated aqueous solution of NaHC03 (250 ml) and extracted with ethyl
`acetate (3 x 100 ml). The organic layers were combined, dried (MgS04) and
`
`

`
`112
`
`evaporated to dryness in vacuo. The oily residue was purified by distillation
`
`under vacuum (70-95 °C, 0.3-0.8 torr) to give 10.52 g (55%) of a white waxy
`
`solid: Rt 0.53 (98:2 chloroform/methanol): mp 35-36 °C; 1 H NMR (300 MHz,
`CDCl3) o 1.23 (t, J = 7,3 Hz, OCH2CH3), 1.32 (t, J = 7.3 Hz, OCH2CH3), 2.08
`(s, CH3CO), 3.70 (q, J = 7.3 Hz, OCH2CH3), 4.25 (q, J = 7.3 Hz,
`COOCH2CH3), 5.60 (d, J = 9.6 Hz, CH), 6.96 (br d, J = 9.6 Hz, NH) ; 13c
`NMR (75 MHz, CDCl3) 13.78 (OCH2CH3), 14.75 (OCH2CH3), 22.91
`
`(CH3CO), 61.74 (COOCH2CH3), 64.72 (OCH2CH3), 76.85 (CH), 168.25
`
`(CH3CO), 170.48 (COOCH2CH3) ppm; IA (KBr) 3400 (br), 1735, 1655 (br),
`
`1200, 1085 (br), 1010, 930, 890 cm·1; mass spectrum, m/e (relative intensity)
`
`190 (5), 160 (2), 144 (38), 116 (98), 102 (92), 74 (100); high resolution mass
`spectrum, calcd for c8H15N04 190.1079, found 190:1087.
`
`Preparation of Alkyl 2-Substituted-2-acetamidoacetates
`
`fil).General Procedure.The alkyl 2-acetamido-2-alkoxyacetate (fill) (1
`
`equiv) was suspended in anhydrous ethyl ether (60 mU10 mmol), and then
`
`boron trifluoride etherate (1.6 equiv) was added in one portion followed by
`
`the heterocycle (4 equiv). The solution was stirred at room temperature (72 h)
`
`and then poured into an ice-cold saturated aqueous solution (50 ml) of
`
`NaHC03, stirred at ice temperature (20 min), and then extracted with ethyl
`acetate (3 x 40 ml). The organic layers were combined, dried (Na2so4),
`and concentrated to dryness in vacuo. The resulting oil was purified by flash
`
`chromatography or recrystallization.
`
`Preparation of Methyl o:-Acetamldo-2-furanacetate (SJ.JD.
`
`The preceeding procedure was employed using methyl 2-acetamido-2-
`
`

`
`113
`
`methoxyacetate (fill.a) (1-68 g, 10.4 mmol), boron trifluoride etherate (2.71 g,
`
`2.35 ml, 16.6 mmol) and furan CZQil) (2.81 g, 3 ml, 41.6 mmol) to produce a
`
`black oil which was purified by flash chromatography
`
`(99:1 chloroform/
`
`methanol) to give1.28 g (62%} of the desired product as a beige solid: Rt
`
`0.32 (99:1 chloroform/methanol); mp 80-81 °C; 1H NMR (300 MHz, CDCl3} l>
`2.03 (s, CH3CO), 3.75 (s, COOCH3), 5.77 (d, J = 7.8 Hz, CH), 6.35-6.36 (m,
`C3>H, C4·H), 7.02 (d, J = 7.8 Hz, NH}, 7.36 (br s, c5.H); 13c NMR (75 MHz,
`CDCl3) 22.69 (CH3CO), 50.43 (COOCH3), 52,88 (CH), 108.72 (C3•),110.78
`
`(C4o), 142.84 (C5•), 148.89 (C2·). 169.57 (CH3CO}, 169.96 (COOCH2CH3)
`
`ppm; JR (KBr} 3200, 1740, 1620 (br), 1530 (br), 1205, 1090, 1020, 900, 890
`
`cm-1; mass spectrum, m/e (relative intensity) 197 (14), 165 (35), 154 (78),
`
`138 (36), 96 (100}, 94 (93}, 69 (16).
`
`Anal. Calcd for C9H11 N04: C, 54.82; H, 5.62; N, 7.10. Found: C,
`
`54.96; H, 5.40; N, 7.27.
`
`Preparation of Ethyl a·Acetarnldo-2-furanacetate (lt!.Q).
`
`The preceeding procedure was employed using ethyl 2-acetamido-2-
`
`ethoxyacetate (l.l.ll.12) (3.72 g, 19.7 mmol}, boron trifluoride etherate (3.75 g,
`
`3.25 ml, 26.4 mmol) and furan (1.Qg) (5.35 g, 5.70 ml, 78.6 mmol) to produce
`
`a black oil which was purified by two successive flash chromatographies ((a)
`
`100% chloroform, (b) 70:30 ethyl ether/pentane ,then 97:3 chloroform/
`
`methanol) to give 2.11 g (51 %) of the desired product as a white solid: R1
`0.17 (100% chloroform); mp 69-70 °C; 1H NMR (300 MHz, CDCl3) l> 1.24 (t, J
`
`= 7.2 Hz, COOCH2CH3). 2.04 (s, CH3CO), 4.14 - 4.32 (m, COOCHzCH3).
`5.75 (d, J = 8.1 Hz, CH), 6.34-6.35 (m, C3°H, C4•H), 6.35-6.54 (br d, J = 8.1
`
`Hz, NH), 7.35-7.36 (m, c 5 °H); 13c NMR (75 MHz, CDCl3 ) 13.91
`
`

`
`114
`
`(COOCH2CH3), 22.81 (CH3CO), 50.33 (CH), 62.08 (COOCH2CH3), 108.49
`
`(C3•), 110.62 (C4•), 142.64 (C5•). 148.85 (C2•), 168.89 (CH3CO), 169.43
`
`(COOCH2CH 3) ppm; IR (KBr) 3200, 1750, 1635 (br), 1530, 1380, 1335,
`1205, 1180, 1020, 890, 745, 595 cm-1 ;mass spectrum,m/e(relative intensity)
`
`211 (8),168 (32), 138 (27), 96 (100), 94 (27).
`
`Anal. Calcd for C10H13N04: C, 56.87; H, 6.20; N, 6.63. Found: C,.
`
`56.98; H, 6.19; N, 6.83.
`
`Preparation of Ethyl a·Ace.tamldo-2-pyrroleacetate
`
`(.!UJU
`
`and a·Acetamido-3-pyrroleacetate (~ .The preceeding procedure
`
`was employed using ethyl 2-acetamido-2-ethoxyacetate (fillll) (3. 78 g, 20
`
`mmol), boron trifluoride etherate (4.53 g, 4.84 ml, 32 mmol) and pyrrole (11)
`
`(5.36 g, 5.54 ml, 80 mmol) to produce a black oil. TLC analysis indicated the
`
`presence of
`
`two major compounds (Rt 0.33 and Rt 0.19, 98:2
`
`chloroform/methanol) which were isolated by flash chromatography (98:2
`
`chloroform/ methanol).
`
`The initial fraction (Rf 0.33, 98:2 chloroform/methanol) was further
`
`purified by another flash chromatography (97:3 dichloromethane/metha-
`
`nol) to produce 1.74 g (41%) of the desired product as a light brown solid: mp
`104-106 °C; 1 H NMR (300 MHz, DMSO-d6) o 1.16 (t, J =7.2 Hz, CH2CH3),
`1.88 (s, CH3CO), 4.01-4.16 (m, CH2CH3), 5.33 (d, J = 6.9 Hz, CH), 5.96-5.99
`(m, C3•H, C4•H), 6.69-6.72 (m, C5•H), .8.48 (d, J =6.9 Hz, CONH).
`
`10.80-10.99 (br S, NH); 13c NMR (75 MHz, CDCl3) 13.93 (CH2CH3) 22.79
`
`(CH3CO), 50.73 (CH), 61.38 (CH2CH3), 106.35 (C3•), 107.52 (C4•), 118.07
`
`(C5•), 125.28 (C2•), 169.24 (CH3CO), 170.11 (COOCH2CH3) ppm; IR (KBr)
`
`3310, 3200, 1715, 1635 (br), 1515 (br), 1220, 1180, 1085, 1010, 890 crn-1;
`
`

`
`115
`
`mass spectrum, m/e (relative intensity) 210 (22), 167 (36), 137 (54), 121 (7),
`
`106 (7), 95 (100), 93 (97), 79 (5), 68 (53).
`
`Anal. Calcd for C10H14N203: C, 57.13; H, 6. 71; N, 13.33. Found: C,
`
`57.20; H, 6.55; N, 13.13.
`
`The second fraction (Rf 0.19, 98:2 chloroform/methanol) was further
`
`purified by a second flash chromatography (95:5 dichloromethane/
`
`methanol) to give 0.52 g (12%) of the desired product as a beige solid: mp
`
`92-93 °C; 1 H NMR (300MHz, CDCl3) l> 1.25 (t, J =6.9 Hz, CH2CH3), 2.02 (s,
`
`CH3CO), 4.10-4.30 (m, CH2CH3 ), 5.53 (d, J =7.2 Hz, CH), 6.17-6.30 (m.
`C4•H), 6.25 (d, J = 7.2 Hz, CONH), 6.70-6.75 (m. C5·H), 6.78-6.80 (m, C2·H),
`8.45-8.60 (brs, NH); 13c NMR (75 MHz, CDCl3). 13.93 (CH2CH3). 22.79
`
`(CH3CO), 50.73 (CH), 61.38 (CH2CH3). 106.78 (C4•). 116.56 (C2·). 118.25
`
`(C3-}, 118.63 (C5•), 169.79 (COCH3), 171.76
`
`(COOCH2CH3) ppm; IR (kBr)
`
`3320, 3240, 1720, 1640 (br), 1510, 1400 (br), 1210, 1180, 1010, 890 cm-1;
`
`mass spectrum, m/e (relative intensity) 210 (12), 167 (16), 152 (5), 137 (31),
`
`121 (3), 95 (100), 93 (100), 80 (5), 68 (71 ); high resolution mass spectrum,
`
`calcd for C10H14N203 210.1004, found 210.1015.
`
`Preparation of 2-Substltuted 2-Acetamldoacetlc Acids
`(ll). Preparation of a.-Acetamldo-2-furanacetlc Acid (!:!2fil. Ethyl
`
`a.-acetamido-2-furanacetate (fill2) (2.93 g, 13.8 mmol) was dissolved in 90:10
`
`ethanol/water (11 O ml), and KOH (0.85 g, 15.2 mmol) was added and the
`
`solution was stirred at room temperature (48 h). The solution was
`
`concentrated in vacuo and the residue diluted with H20 (100 ml) and then
`
`extracted with ethyl acetate (3 x 100 ml). The aqueous layer was then
`
`acidified with 8.5% H3P04 (25 ml) and extracted with ethyl acetate (3 x 200
`
`

`
`116
`
`ml). The organic layers were combined, dried (Na2so 4) and then
`evaporated to dryness in yacyo to produce a beige solid (2.00 g) which was
`
`recrystallized (acetonitrile) to yield 1.31 g (51%) of the desired product as
`
`beige crystals: Rf 0.37 (8:1 :1 isopropanol/NH40HIH20); mp 171-172 °C; 1H
`NMR (300 MHz, DMSO~d6) o 1.88 (s, CH3), 5.45 (d, J = 7.8 Hz, CH),
`6.39-6.45 (m, C3•H, C4·H), 7.65 (s, C5·H), 8.69 (d, J = 7.8 Hz, NH). [The
`carboxyl proton was not detected.]. 13c NMR (75 MHz, DMSO- d6) 22.10
`(CH3), 50.16 {CH), 108.17 (C3•), 110.66 (C4•), 142.83 (C5>), 149.75 (C2-)
`
`169.21 {CH3CO), 170.01 (COOH) ppm; IR (KBr) 3320, 3100, 1705, 1580 (br).
`
`1530, 1410, 1360, 1320, 1280, 1270, 1225, 1210, 1145, 1100, 1010, 890,
`
`640, 660, 610, 570, 400 cm-1; mass spectrum, m/e {relative intensity) 183
`
`(2), 165 (10), 140 (24), 123 (19), 109 (1), 96 (100), 94 (43), 80 (2), 69 (8).
`
`Anal. Calcd for c 8H9No4: C, 52.46; H, 4.95; N, 7.65. Found: C,
`52.61; H, 4.93; N, 7.94.
`
`Preparation of a-Acetamldo-2-pyrroleacetic Acid
`
`(!l£QJ.
`
`Ethyl a-acetamido-2-pyrroleacetate (JilQ) (2.11 g, 1 O mmol) was dissolved in
`
`90:1 O ethanol/water (88 ml), and then KOH (0.61 g, 11 mmol) was added
`
`and the resulting solution stirred at room temperature (48 h). The solution
`
`was concentrated in vacuo and the residue diluted with H20 (80 ml) and
`
`washed with ethyl ether (3 x 80 ml). The aqueous layer was then made
`
`acidic with 8.5% H3Po4 (20 ml) and extracted with ethyl acetate (3 x 100
`ml). The organic layers were combined, dried (Na2S04) and evaporated in
`vacuo to give a brown solid which was successively triturated with ethyl
`
`acetate (50ml) and petroleum ether (35-60 °C) (650 ml), and then
`
`recrystallized (chloroform/methanol/ hexanes) to give 0.53 g (29%) of the
`
`

`
`117
`
`desired product as beige crystals: Rt 0.55. (8:1 :1 isopropanoVNH40H/H20);
`mp 112-114 °C; 1 H NMR (300 MHz, DMSO-d5) S 1.87 (CH3), 5.31 (d, J = 7.2
`
`Hz, CH), 5.96 (s, C3•H), 5.97 (s, C4•H), 6.87 (s, C5•H), 8.40 (d, J = 7.2 Hz,
`
`CONH), 10.79-10.85 (br s, NH). [rhe carboxyl proton was not detected.]. 13c
`
`NMR (75 MHz, DMSO-d5) 22.16 (CH3), 50.45 (CH), 106.21 (C3•), 107.45
`
`(C4•), 117.83 (C5•), 126.11(C2•),169.13 (CH3CO), 171.56 (COOH) ppm; IR
`
`(KBr} 3340, 3300,1710, 1590 (br), 1530 (br), 1220, 1080, 885, 725 cm·1 ;mass
`
`spectrum, m/e (relative intensity)182 (1), 164 (7), 151 (45), 138 (100). 137
`
`(25), 121 (2), 95 (98), 93 (10), 91 (46).
`
`Preparation of a-Acetamldo-3-pyrroleacetlc Acid (.!l.2fil,
`
`Ethyl a-acetamido-3-pyrroleacetate (fil.d) (0.81 g, 3.85 mmol) was dissolved
`
`in 90:10 ethanol/water (33 ml), and then KOH (0.24 g, 4.2 mmol) was added
`
`and .the resulting solution stirred at room temperature (48 h). The solution
`
`was concentrated in vacuo and the residue diluted with water (30 ml), and
`
`then washed with ethyl ether (3 x 30 ml). The aqueous layer was then made
`
`acidic with 8.5% H3P04 (8 ml) and extracted with ethyl acetate (4 x 30 ml).
`The organic layers were combined, dried (Na2S04) and evaporated in
`
`vacuo,
`
`The
`
`resulting beige
`
`solid was
`
`recrystallized
`
`(chloroform/methanol/hexanes) to give 0.27 g (38%) of the desired product
`
`as beige crystals: R1 0.28 (8:1:1 isopropanol/NH40HIH20); mp 135-138 °C;
`
`1 H NMR (300 MHz, DMSO-d6) I) 1.85 (s, CH3), 5.05 (d, J = 7.0 Hz, CH), 6.04
`(s, C4•H), 6.69 (s, C2•H), 6.76 (s, C5•t1). 8.23 (d,.J = 7.0 Hz, CONH),
`10.68-10.86 (br s, NH). [the carboxyl proton was not detected). 13c NMR (75
`
`MHz, DMSO-d5) 22.18 (CH3), 50.57 (CH), 106.98 (C4•), 116.28 (Cz•),
`
`- - - - - - - - -
`
`

`
`1 18
`
`117.83 (C3• and C5•), 169.13 (CH3CO), 173.00 (COOH) ppm; IA (KBr) 3340,
`
`3300, 1700, 1585 (br), 1525 (br), 1240 (br), 920, 895 cm-1; mass spectrum,
`
`m/e (relative intensity) 182 (4), 164 (6), 157 (1), 138 (100), 124 (3), 121 (38),
`
`95 (19), 93 (33), 80 (94), 68 (91 ); high resolution mass spectrum, calcd for
`
`C8H10N203 182.0691, found 182.0688.
`
`Preparation of a-Acetamldo-2-benzofuranacetlc Acid
`(f!..2.!!).2-Acetamido-2-hydroxyacetic acid CZ.a) (6.65 g, 50 mmol) was
`dissolved in glacial acetic acid (12 ml) and the resulting yellow solution was
`
`chilled in an ice bath, benzofuran (15.) (11.80 g, 11 ml, 100 mmol) was then
`
`added in one portion, followed by the rapid dropwise addition of
`
`methanesulfonic acid. The orange solution was stirred at room temperature
`
`(16 h) and the solution was poured into an ice-water mixture (100 ml) and
`
`then triturated. The resulting semi-solid was separated from the aqueous
`
`layer and recrystallized from petroleum ether {35-60 °C) and then triturated
`
`with ethyl acetate. The product was further purified by recrystallization
`
`(acetonitrile) and then dissolved in a 5% aqueous NaOH solution, washed
`
`with ethyl acetate, and then made acidic (pH 1) with 10% aqueous HCI and
`
`filtered to give 2.38 g (20%) of the desired product as white crystals: Rf 0.62
`(8:1:1 isopropanol/NH40H/H20); mp 203·204 °C; 1H NMR (300 MHz,
`
`DMSO-d6) Ii 1.91 (s, CH3), 5.66 (d, J = 7.8 Hz, CH), 6.90 (s, C3'H), 7.23-7.35
`(m, C5•H, C5·H), 7 .. 55-7.65 (m, C4°H, C7°H), 8.85 (d, J = 7.8 Hz, NH). [The
`carboxyl proton was not detected]. 13c NMR (75 MHz, DMSO-d6) 22.11
`(CH3), 50.51 (CH), 104.93 (C3•), 111.06 (C?'), 121.22 (C4•), 123.02 (C5•).
`
`124.51 (C5•), 127.65 (C3·8 ), 152.92 (C7'8 ), 154.10 (C:z·), 169.20 (COCH3).
`
`

`
`119
`
`169.62 (COOH) ppm; IR (kBr) 3340, 3090, 1720, 1590 (br), 1535 (br), 1240,
`
`1200, 1090, 890, 750 cm-1; mass spectrum, m/a (relative intensity) 233 (12),
`
`. 215 (2), 190 (38), 173 (15), 146 (100), 144 (92), 130 (14), 91 (32).
`
`Anal. Calcd for C12H11N04: C, 61.80; H, 4.75; N, 6.01. Found: C,
`
`61.55; H, 4.76; N, 5.77.
`
`Preparation of a.-Acetam ldo-2-benzo [b ]thlophenea cetlc
`Acid (l!Zru. a.-Amino-2-benzo[b]thiopheneacetic acid (Uf.) (5.18 g, 25
`
`mmol) was suspended in H20 (60 ml), and then NaOH (1.50 g, 37 mmol)
`
`was added and the suspension stirred until a homogeneous solution was
`
`obtained. The aqueous solution was cooled to 5 °C, and then acetic
`
`anhydride (.fili) (5.08 g, 4.70 ml, 50 mmol) was added slowly so as to
`
`maintain the temperature of the solution below 7 °C. The solution was stirred
`
`an additional 5 min and then an aqueous NaOH (2.50 g, 63 mmol) solution
`
`(15 ml) was added and the solution stirred 30 min. The reaction was then
`
`f:
`l~--
`
`made acidic (pH 1) with 10% aqueous HCI and the resulting precipitate (5.93
`
`g) was filtered and washed with H20 (2 x 15 ml). The beige crystals were
`
`dissolved in an aqueous NaOH (1.00 g, 25 mmol) solution (100 ml) and the
`
`solution was extracted with ethyl acetate (2 x 50 ml) and then the aqueous
`
`solution was made acidic with 10% aqueous HCI. The precipitate which
`
`formed was washed with H20 to yield 5.23 g (83%) of the desired product as
`beige crystals: mp 224-226 °C; iH NMR (300 MHz, NaOD/020) o 2.08 (s,
`CH3 ), 7.36-7.45 (m, C3·H, C5'H, C7'H), 7.81· 7.84 (m, C4•H or C5·H)
`7.88-7.92 (m, C4'H or Cs·H) ppm. [rhe a-proton was not observed in the
`employed NMR solvent and is presumed to have undergone C-H to C-D
`
`exchange.]. 13c NMR (75 MHz, NaODID20) 26.33 (CH3).59.85 (t, CH),
`
`

`
`120
`
`126.86 (C4• or Cr). 127.18 (C4• or CrJ. 128.12 (C3•), 129.08 (Cs• or
`C5•), 129.09 (C5• or C5•), 143.66 (C3•a or Cy•8 ). 143.71 (C3•8 or Cr8 ),
`146.03 (C2·); IR (KBr) 3305, 1710, 1580 (br), 1525 (br), 1275, 1200, 1090,
`
`890cm·1; mass spectrum, m/e (relative intensity) 249 (22), 231 (20),206 (33),
`
`189 (6), 162 (100),160 (89), 135 (38), 89 (26).
`
`Anal. Calcd for C12H11N03S: C, 57.82; H, 4.45; N, 5.62. Found:
`
`C, 57.55; H, 4.38; N, 5.35.
`
`Pre para ti on of 2-Subs tituted-2-acetamldo-N.-be nzyl-ace(cid:173)
`tamides (fil!. w. Preparation of o:-Acetamido-N.-benzyl-2-fura(cid:173)
`acetamide (fill.l!.). o:-Acetamido-2-furanacetic acid (fl..2..a) (0.47 g, 2.56
`
`rnmol) was combined with acetonitrile (10 ml) and cooled to -5 °C (ice/salt
`
`water bath). Triethylamine (0.26 g, 0.36 ml, 2.56 mmol) was then rapidly
`
`added and the mixture stirred at -5 °c (3 min). Ethyl chloroformate (0.28 g,
`
`0.25 ml, 2.56 mmol) was added dropwise between -4 and -3 °C, and the
`
`resulting suspension was stirred at -4 °C (20 min). and then an acetonitrile
`
`solution (2 ml) of benzylamine (.!U) (0.30 g, 0.31 ml, 2.82 mmol) was
`
`carefully added. During the addition of benzylamine the temperature of the
`
`solution did not go above O °C. The mixture was stirred at -5 °C (1 h) and at
`room temperature (18 h), and then concentrated in vacuo. The residue was
`
`then triturated with hot tetrahydrofuran (5 ml), cooled at -16 °C (3 h), and the
`
`resulting white precipitate was filtered and identified as triethylamine
`hydrochloride (1 H NMR (60 MHz, DMSO-d6) o 1.00 (t, J = 7.5 Hz, CH3), 2.82
`(q, J,;, 7.5 Hz, CH2), 3.83 (s, NH)) 164. The filtrate was evaporated to dryness
`in vacuo and the resulting oil purified by flash chromatography (98:2
`
`

`
`121
`
`chloroform/ methanol) to give 0.09. g (13%) of the desired product as a white
`
`solid: Rt 0.30 (98:2 chloroform/methanol); mp 178-179 °C; 1 H NMR (300
`MHz, DMSO-d5) o 1.90 (s, CH3), 4.31 (d, J = 6.0 Hz, CH2). 5.58 (d, J = 8.1
`Hz, CH), 6.27-6.33 (m, C3·H), 6.40-6.44 (m, C4•H), 7.20-7.36 (m, Ph),
`. .
`. 13
`7.60-7.64 (m, C5·H), 8.58 (d, J = 8.1 Hz, NH), 8.73 (t, J = 6.0 Hz, NH), C
`NMR (75 MHz, DMSO-d5) 22.35 (CH3), 42.27 (CH2). 50.95 (CH), 107.60
`
`(C3•), 110.55 (C4•), 126.82 (2C2" or 2C3"), 127.08 (2C2" or 2C3u),
`
`128.27 (C4"), 139.05 (C1"), 142.58 (C5•), 151.16 (C2•), 168.02 (CH3CO),
`
`169.30 (NHCO) ppm; IR (KBr) 3230, 1625 (br), 1525 (br), 1375 (br), 1230,
`1090, 890, 740, 690 cm·1; mass spectrum, m/ e (relative intensity) 273 (1 ),
`
`230 (1 ), 139 (100), 96 (94), 91 (51 ), 65 (9).
`
`Anal. Calcd for C15H1sN203: C, 66.16; H, 5.83; N, 10.29. Found:
`
`C, 65.92; H, 5.83; N, 10.15.
`
`Preparation of u·Acetamldo-li-benzyl-2-benzofuranacet(cid:173)
`
`amide(.6.fil).u-Acetamido-2-benzofuranacetic acid (.82..d) (2.38 g, 10.2 mmol)
`
`was combined with acetonitrile (100 ml) and then triethylamine (1.01 g, 1.39
`
`ml, 10.2 mmol) was added. The mixture was stirred at room temperature (20
`
`min) and then cooled to -5 °C (ice/salt water bath) and isobutyl chloroformate
`
`(1.37 g, 1.30 ml, 10.2 mmol) was added rapidly. The mixture was stirred
`
`between -5 and -8 °C (20 min) and then an acetonitrile solution (5 ml) of
`
`benzylamine (.8.:2) (1.18 g, 1.19 ml, 11.0 mmol) was added dropwise. The
`
`resulting suspension was stirred at -5 °C (1 h) and at room temperature (17
`h). The mixture was then concentrµted in yacuo and the yellowish residue
`
`triturated with tetrahydrofuran (150 ml) and cooled at -16 °C (4 h). The
`
`resulting precipitate was filtered and identified as triethylamine hydrochloride
`
`

`
`122
`
`(1 H NMR ( 60 MHz, DMSO-d5) S 1.00 (t, J = 7.5 Hz, CH3), 2.82 (q, J 7.5 Hz,
`CH2). 3.83 (s, NH}) 164. The remaining filtrate was concentrated to (- 5 ml)
`in vacuo and refrigerated (-16 °C) resulting in the formation of a precipitate
`
`which was
`
`filtered and purified by
`
`flash chromatography
`
`(98:2
`
`chloroform/methanol) to give 0.42 g (13%) of the desired product as a white
`solid: Rf 0.30 (98:2 chloroform/methanol); mp 195-196 •c; 1H NMR (300
`MHz; DMS0-06) S 1.94 (s, CH3), 4.34 (d, J = 5. 7 Hz, CH2), 5. 77 (d, J = 8.1
`Hz. CH), 7.24-7.32 (m, C3°H, C5°H, C5•H, Ph), 7.54 (d, ../ = 7.0 Hz. C4·H or
`CTH), 7.62 (d, J = 7.0 Hz, C4'H or. C7'H), 8.74 (d, J = 8.1 ·Hz, NH), 8.86 (t, J =
`5.7 Hz, NH); i 3c NMR (75 MHz, DMSO-d5) 22.27 (CH3), 42.30 (CH2).
`
`51.22 (CH),

This document is available on Docket Alarm but you must sign up to view it.


Or .

Accessing this document will incur an additional charge of $.

After purchase, you can access this document again without charge.

Accept $ Charge
throbber

Still Working On It

This document is taking longer than usual to download. This can happen if we need to contact the court directly to obtain the document and their servers are running slowly.

Give it another minute or two to complete, and then try the refresh button.

throbber

A few More Minutes ... Still Working

It can take up to 5 minutes for us to download a document if the court servers are running slowly.

Thank you for your continued patience.

This document could not be displayed.

We could not find this document within its docket. Please go back to the docket page and check the link. If that does not work, go back to the docket and refresh it to pull the newest information.

Your account does not support viewing this document.

You need a Paid Account to view this document. Click here to change your account type.

Your account does not support viewing this document.

Set your membership status to view this document.

With a Docket Alarm membership, you'll get a whole lot more, including:

  • Up-to-date information for this case.
  • Email alerts whenever there is an update.
  • Full text search for other cases.
  • Get email alerts whenever a new case matches your search.

Become a Member

One Moment Please

The filing “” is large (MB) and is being downloaded.

Please refresh this page in a few minutes to see if the filing has been downloaded. The filing will also be emailed to you when the download completes.

Your document is on its way!

If you do not receive the document in five minutes, contact support at support@docketalarm.com.

Sealed Document

We are unable to display this document, it may be under a court ordered seal.

If you have proper credentials to access the file, you may proceed directly to the court's system using your government issued username and password.


Access Government Site

We are redirecting you
to a mobile optimized page.





Document Unreadable or Corrupt

Refresh this Document
Go to the Docket

We are unable to display this document.

Refresh this Document
Go to the Docket