It?/. J . Peptide Prot~in Rm. 32, 1988. 219-291
`
`Synthesis of functionalized non-natural amino acid derivatives via
`amidoalkylation transformations
`
`PHILIPPE LEGALL*, KAILASH N. SAWHNEY, JUDITH D. CONLEY and HAROLD KOHN
`
`Department of Chemistry, University of Houston, Houston, T X , USA
`
`Received 16 February, accepted for publication 2 May 1988
`
`Synthetic routes have been developed for the preparation of functionalized amino
`acid derivatives in which the a-substitucnt at carbon 2 is either an aromatic or a
`heteroaromatic group. The a-substituent was introduced using an amidoalkylation
`reaction using boron trifluoride etherate and proceeded in moderate yield with
`excellent regioselectivity. This protocol permitted the employment of the acid sen-
`sitive heterocycles: pyrrole, benzofuran, and indole. The scope and limitations of this
`procedure have been evaluated.
`
`Key word : a-substituted; amido alkylation transformations; aromatic; heteroaromatic; non-
`natural amino acids
`
`Recent studies conducted in our laboratory
`have drawn attention to the importance of
`a-functionalized derivatives of N-acetylgly-
`cine-N-benzylamide (1, R = H) as potential
`drug candidates for the treatment of epilepsy
`In an effort to delineate the structure
`(1).
`activity relationship of this novel class of anti-
`convulsants, select derivatives of 1 were re-
`quired in which the a-substituent R was either
`an aromatic or a heteroaromatic moiety. Un-
`fortunately, relatively few methods exist for
`the preparation of the corresponding free
`amino acids** thereby diminishing the Iikeli-
`hood of employing these substrates as start-
`
`* Abstracted from the Masters dissertation of this
`author. Additional structure proof and experimental and
`spectra data may be found in this reference.
`
`**The 2- and 3-thienyl compounds are commercially
`available (Aldrich Chemical Company). For leading ref-
`erences for procedures for the preparation of non-natural
`amino acides and related studies, see ref. 2.
`
`ing materials for the synthesis of 1. In this
`paper, we describe the use and limitations of
`amidoalkylation transformations*** for the
`preparation of functionalized derivatives of
`amino acids in which the R substituent is an
`aromatic moiety.
`R O
`0
`I II
`II
`CH,CNHCC NHCH,Ph
`I H
`1
`
`RESULTS AND DISCUSSION
`Two different strategies (Scheme 1, Methods
`A and B) were investigated for the prepara-
`tion of 1. The approaches differ primarily in
`the sequence of reactions employed for the
`synthesis of the desired compound 1. In
`
`*** For excellent discussions of this reaction, see ref. 3.
`219
`
`IPR2014-01126- Exhibit 1014, p. 1
`
`

`
`trifluoride
`aromatic substrate and boron
`[he 2-.;ub-
`Method A. the initial target
`etherate. This synthetic route permitted the
`stituted alk!l
`'-acetamidoacct~ite 6. Our sqn
`preparation of compounds la-11. Moderate
`thesis of this compound \\:I> patterned after
`the procedure described b! Bun-lshai. Satat!
`J iclds (28-94%) for this step were observcd
`for
`furan.
`3-merh! Ifiiran.
`pyrrole,
`& Bet-nstein for the prcparation of iiicth! I
`1-
`m e t h y 1 p y r r o 1 e .
`.V-beiizyloxycarbonqI-r-fiiranglycinate (3).
`benzo fur an,
`t h i o p he t i e ,
`Reaction ofacetiimide (2) \4 i t h gl>ox>lic acid
`indole. phenol. p - c r e ~ ~ l . anisole and thio-
`(3) yielded 4 in near quantitati\e qield (4).
`phenol. while only ;I 4 % yield was obtained
`which upon dissolution in either methanol or
`for benzo[b]thiophenc (Table 1). Employ-
`ethanol and acid ga1.e the corresponding
`ment o f p! razole. iniitiazole, pyridine, 3- and
`a1kj.l 7-acetaniido-2-ulkoxy~icet~ites Sa and
`4-hydroxypyridine. benzene. naphthalene,
`and ,V-acetylaniline as the aromatic substrate
`Sb. Treatment o f 5 with either furan or
`pyrrole in the presence o f boron trifluoride
`in this procedure led to no detectable product
`formation. No signilic'int effort was made to
`etherate ga\.e the r-substituted product 6 in
`moderate qield (51-62?/,,). I n the case o f
`vary either the acid 01- the solvent employed
`furan. only 6a \<:is obsened in \I hich sub-
`the amidoalkylation step in order to
`in
`improve the efficiency of this transformation.
`stitution had occurred at the ?-position of the
`aromatic ring. Csrrespondingl!, . with pyrole
`Several interesting observations were noted
`a 3.4: 1 binary mixture of the aromatic 3(6c, )-
`concerning the convcrsion of the 2-ethoxy
`and 3612,)- substituted compounds. respec-
`derivative 8b to 1. Fit-st, the employed con-
`ti \.el y . was obtained . U n fo r t tin a t el y . a t t em p t s
`ditions (boron trifluoride etherate, ether) per-
`mitted the use of the acid sensitive hetero-
`to convert 6 to the corresponding benzyl
`amide adduct I pro\.ed iinsotisfactor! . Lo\\.
`cycles: pyrrole. benzofuran, and indole. These
`yields were obtained for thc condensation of
`substrates have found limited use in previous
`benzq,lamine with 6. Similarlq. unacceptable
`amidoalkylation transf'ormations. (Zaugg (3),
`o\,erall yields for 1 \\.ere eupcrienced for the
`5. 6). Second. in the rcaction of pyrrole only
`sequential coniwsion of 6 to the acid 7
`11 trace amount of rhc %substituted pyrrole
`(KOH. H1O). followed by the coupling of the
`product was detected (t.1.c. analysis). A much
`h:-protected amino acid 7 with benz) lamine
`larger percentage o f
`the corresponding
`adduct was observed when ester 5b was em-
`(i.e.. CICOzR, Et,N; DCC)-.
`ployed as the starting material (Scheme I ,
`This synthetic obstacle was expeditiously
`Method A). The high regioselectivity wit-
`circumvented by
`the use of
`the second
`pathway (Method B) outlined in Scheme 1. In
`nessed in the former transformation was also
`mirrored in the other reactions with hetero-
`this procedure, the coupling reaction ~ v a s
`aromatic substrate\. Typically, only one
`conducted prior
`to
`the amidoalkylation
`trans form at ion . Treat me tit of a 1 k y I 2 -ace t ii -
`isomer was observed. This result was par-
`ticularly surprising in the reactions involving
`\vith
`niido-2-alkoxyacetates 5a and Sb
`benzofuran and benzo[b]thiophene. With
`benzylaniine in alcoholic solution produced
`benzofuran only the ?-substituted aromatic
`the corresponding 2-acetaniido-..V-benz~l-2-
`derivative was obsened despite the known
`alkoxyacetamidcs 8a and 8b. respectively.
`tendency of this heterocycle
`to undergo
`Higher yields and cleaner product mixtures
`alkylation at both the 2- and 3-positions (7),
`were noted for the synthesis of ethoxy adduct
`Lvhile tvith benzo[b]thiophene none of the ex-
`8b kersus the methoxy derivative 8a. Com-
`pected
`hubstituled
`benzo[b]thiophene
`pound 8b was converted to 1 by treatment
`product was observed (7) but rather only a
`nith the appropriate aromatic or hetero-
`4% yield of the 2-substituted adduct was
`isolated along with iinreacted starting mat-
`erial. Third. all four substituted benzene sub-
`strates (phenol, p-cresol, anisole, and thio-
`phenol) reacted to give a single product (t.1.c.
`analysis). In the case of phenol and anisole
`
`280
`
`IPR2014-01126- Exhibit 1014, p. 2
`
`

`
`Fuiictionalized non-natural amino acid derivatives
`
`TABLE I
`ocetamirles ( I )
`Selcc,tetl pIij..sI'cd titid .spectral dritti ,for -7-~crtami~/o-N-ben~~l-2-.~ub.s~I'tute~
`
`N O
`
`R
`
`Yield
`
`M . P . ~
`
`M+/ec
`
`' H n.m.r.*
`a-CH
`
`" C n.m.r.'
`X-C
`
`58
`
`61
`
`35
`
`62
`
`37
`
`33
`
`28
`
`178-179
`
`273 (1)'
`
`5.50 (d, 7.9)
`
`50.95
`
`148- 150
`
`286 (3)
`
`5.49 (d, 8.0)
`
`53.23
`
`174- 175
`
`271 (12)
`
`5.42 (d, 6.9)E
`
`52.65g
`
`179-181
`
`285 (1 7)
`
`5.52 (d, 7.8)
`
`49.20
`
`167-169
`
`289 (2)'
`
`5.74 (d. 7.9)
`
`52.20
`
`195-196
`
`322 (5)
`
`5.77 (d, 8.1)
`
`5 1.22
`
`213-2 14
`
`321 (5)
`
`5.72 (d. 7.2)
`
`49.98
`
`4
`
`226-227
`
`338 (8)
`
`5.86 (d, 8. I }
`
`52.70
`
`56
`
`232-235
`
`299 (I)'
`
`5.34 (d, 7.4)
`
`55.90
`
`62
`
`196-198
`
`313 (2)'
`
`5.42 (d, 7.8)
`
`55.78
`
`67
`
`183-185
`
`313 (7)'
`
`5.63 (d, 7.6)
`
`5 I .54
`
`94
`
`165- 167
`
`315 (I)'
`
`5.90 (d, 9.0)
`
`57.65
`
`-
`I' Purified yields (YO) from 2-acetamido-N-benzyl-2-ethoxyacetamide (8b). Melting points ("C) are uncorrected. 'The
`molecular ion peak in the mass spectrum was obtained at an ionizing voltage of 70ev. The number in the parentheses
`indicates the relative intensity of this ion relative to the base peak in the spectrum. The 300 MHz ' H n.m.r. spectra
`were taken in DMSO-d, unless otherwise indicated. The number in each entry is the chemical shift value (6) observed
`in parts per million relative to TMS. The information in parentheses is the multiplicity of the signal, followed by the
`coupling constant (J) in Hertz. 'The 75 MHz "C n.m.r. spectra were taken in DMSO-d, unless otherwise indicated.
`The number in each entry is the chemical shift value in parts per million relative to TMS. 'The M + I peak was
`observed (McLafferty, F.W. "Interpretation of Mass Spectra," 2nd edn., W.A. Benjamin: Reading, MA, 1973).
`".m.r.
`spectrum was taken in CD,CN.
`
`28 1
`
`IPR2014-01126- Exhibit 1014, p. 3
`
`

`
`P. LeGall el a/.
`
`--b
`
`
`R
`O
`II
`I
`CH~CNHCCOOR'
`I
`H
`BF3. EtzO
`
`6
`(I R = 2- Furan. R ' = CH,
`u'R = 2- Furan. R ' = CH2CH:
`L', R = 2- Pyrrole. R' = CH,CH,
`c,R = 3- Pyrrole. R' = CHICH,
`
`<
`
`5
`
`
`R
`O
`I1
`I
`CH,C NHCCOOH
`I
`H
`
`7
`(I R = 7- Furan
`(qR = 2- Pyrrole
`c,R = 3- Pyrrole
`
`4
`
`
`R
`O
`I
`I1
`C H , C N H C C O N H C H , P h
`1
`n
`
`1
`
`RH
`
`0 OR'
`II
`I
`CHIC NHCCONHCH,Ph
`I
`H
`
`8
`(I R' = CHI
`h R' = CH,CH,
`
`0
`11
`CH,CNH,
`
`2
`
`SCHEME 1
`
`P 9"
`0 OR'
`0 0
`+ H C C O H + C H 3 C N H C C O O ~ --+ C H ~ C N H C C O O R '
`I
`II
`II I1
`I
`I
`H
`H
`5
`N R' = CH
`h R' = CH CH,
`
`3
`
`4
`
`the para-substituted adducts li and lj, respec-
`tively, were observed, while with p-cresol only
`lk was isolated in which reaction had oc-
`curred orrho- to the phenolic group. Finally,
`sulfur rather than carbon substitution was
`observed with thiophenol. Fourth, in the
`reaction involving indole, the indole trimer 9
`(8) was obtained along with the desired
`product lg. Indole is known to undergo
`trimerization in the presence of both mineral
`and Lewis acids (8, 9).
`
`9
`
`282
`
`Characteristic spectral properties were
`noted for the newly prepared functionalized
`amino acid derivatives 1 in agreement with
`the proposed structural assignments (10, 11).
`In particular, the chemical shift value for the
`r-carbon proton ranged from 65.34 to 5.90 in
`the I H n.m.r. spectra. while the correspond-
`ing methine carbon signal appeared between
`49.20 and 57.65ppm. Evidence for
`the
`proposed site of aromatic substitution was
`secured from both the ' H and I3C n.m.r.
`spectra. In each case, the proton chemical
`shift values as well as the proton-proton
`coupling patterns were in excellent agreement
`with previously reported compounds of com-
`parable substitution patterns (8, 12). More-
`over, in the 'jC n.m.r. spectra, the chemical
`shift values observed for the substituted aro-
`matic carbon atoms were always downfield
`(6.0-20.0 ppm) versus
`the corresponding
`signal in the unsubstituted heterocycle (1 1).
`In several cases (compounds li and lk) the
`" C n.m.r. assignments were aided by per-
`
`IPR2014-01126- Exhibit 1014, p. 4
`
`

`
`Functionalized non-natural amino acid derivatives
`chemicals were of the highest grade available
`forming the corresponding APT n.m.r. ex-
`and were used without further purification.
`periment (1 3).
`The mixed anhydride reactions as well as 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 vacuo prior to
`use, and the reactions were conducted under
`a positive pressure of N,. Preparative flash
`column chromatography was
`run using
`Merck silica gel, grade 60, 230-240mesh, 60
`A
`from Aldrich Chemical Company,
`Milwaukee, Wisconsin. Thin-layer chromato-
`graphic analyses were run on precoated silica
`G microscope slides (2.5 x IOcm; Analtech
`No. 01521) or on precoated silica GHLF mi-
`croscope slides (10 x 20cm; Analtech No.
`21521).
`
`CONCLUSIONS
`A facile procedure has been developed for the
`synthesis of non-natural amino acid deriva-
`tives containing an electron-rich aromatic or
`heteroaromatic a-substituent using an amido-
`alkylation
`transformation. The reaction
`proceeded with high regioselectivity and per-
`mitted the use of the acid sensitive hetero-
`cycles: pyrrole, benzofuran, and indole. Sig-
`nificantly, this approach should be applicable
`for the preparation of peptides in which the
`peptide bond is formed prior to the introduc-
`tion of the aromatic or heteroaromatic sub-
`strate. optimization of the general reaction
`conditions (i.e., Lewis acid, solvent) should
`allow the synthesis of other a-substituted
`functionalized amino acid derivatives.
`
`EXPERIMENTAL PROCEDURES
`General methods
`Melting points were determined with a
`Thomas-Hoover melting point apparatus and
`are uncorrected. Infrared spectra (i.r.) were
`run on either a Perkin-Elmer 1330 or a
`Perkin-Elmer 283 spectrophotometer and
`calibrated against the 1601cm-' band of
`polystyrene. Absorption values are expressed
`in wavenumbers (cm-'). Proton ('H n.m.r.,
`300 MHz) and carbon ("C n.m.r,, 75 MHz)
`nuclear magnetic resonance spectra were
`taken on either a Nicolet NT-300 or a
`General Electric QE300 instrument. Chemi-
`cal shifts are in parts per million (6 values)
`(TMS) and
`relative
`to
`tetramethylsilane
`coupling constants (J values) are in Hertz.
`Mass spectra were performed at the Eli Lilly
`Corporation, Indianapolis, Indiana, or by
`Dr. John Chinn at the Department of Chem-
`istry, University of Texas at Austin. Elemen-
`tal analyses were conducted at the Eli Lilly
`Corporation, Indianapolis, Indiana. Acetoni-
`trile and triethylamine were distilled from
`CaH, and tetrahydrofuran and ethyl ether
`were distilled
`from Na/benzophenone.
`Furan, pyrrole, benzofuran, ethyl chloro-
`formate, and isobutyl chloroformate were
`fractionally distilled prior to use. All other
`
`Preparation of methyt
`2-acetamido-2-methoxyacetate (5a)
`Sulfuric acid (95%, 4mL, 70mmol) was
`added to a methanolic solution (230mL) of
`2-acetamido-2-hydroxyacetic acid
`(4)
`(4)
`(1 3.30 g, 100 mmol). The solution was stirred
`at room temperature (48 h), neutralized with
`solid NaHCO,, filtered, and then the metha-
`nol was removed in vacuo. The pink oil was
`distilled under vacuum (70-120', 0.6 torr) to
`give a colorless oil which was recrystallized
`from petroleum ether (35-60") to yield 5.20 g
`(32%) of the desired product: R,0.52 (98:2
`chloroform/methanol); m.p. 44-46';
`i.r.
`(KBr) 3270,2820,1735,1650 (br), 1505,1205,
`1110, 1090, 1010, 930, 900cm-'; ' H n.m.r.
`(CDCI,) 62.08 (s, CH,CO), 3.46 (s, OCH,),
`3.81 (s, COOCH,), 5.54 (d, J = 9.3 Hz, CH),
`6.70-6.80 (br d, NH); "C n.m.r. (CDCI,)
`22.98 (CH,CO), 52.69 (COOCH,), 56.48
`(CH,O), 78.16 (CH), 168.49 (CH,CO),
`170.67 (COOCH,) ppm; mass spectrum, m/e
`(relative intensity) 162 (l), 146 (2), 131 (3),
`118 (3), 102 (46), 88 (25), 60 (100).
`Anal. calc. for C,H,, NO,: C 44.72, H 6.88, N
`8.69. Found: C 44.46, H 7.14, N 8.72.
`
`Preparation of ethyl
`2-acetamido-2-ethoxyacetate (56)
`Sulfuric acid (95%, 8mL, 140mmol) was
`added to an ice cold ethanolic solution
`
`283
`
`IPR2014-01126- Exhibit 1014, p. 5
`
`

`
`P. LeCall c't d .
`(500 mL) of 2-acetamido-7-hydroxyacetic
`acid (4) (4) (26.6 g. 200 mmol). and the solu-
`tion was stirred at room temperature for 72 h.
`The yellow colored solution was cooled (0-
`5") and neutralized with a cold aqueous sat-
`urated NaHCO, solution (4OOinL). The re-
`sulting mixture was extracted with ethyl
`acetate (3 x 500mL). The organic layers
`were combined and the volatile materials
`removed in vaciio. The remaining yellow
`liquid was extracted with ethyl acetate
`(500mL) and the extract dried (Na,SO,) and
`evaporated to dryness in IYKUO. The oily
`residue was purified by distillation under
`vacuum (70-95", 0.3-0.8 torr) to give 2 1.04 g
`( 5 5 % ) of a white waxy solid: R,0.53 (98:2
`chloroform/methanol): m.p. 35-36':
`i.r.
`(KBr) 3400 (br). 1735. 1655 (br), 1200. 1085
`(br), 1010,930. 890cm'.' ; ' H n.m.r. (CDCI,)
`61.23 (t, J = 7.3Hz. OCH,CH,), 1.32 (t.
`J = 7.3Hz, OCH,CH,), 2.08 (s. CH,CO).
`3.70 (9, J = 7.3Hz, OCH,CH,). 4.25 (9.
`J = 7.3Hz. COOCH,CH,).
`5.60
`(d.
`J = 9.6Hz. CH). 6.96 (br d, J = 9.6Hz.
`NH); "C n.m.r. (CDCI,) 13.78 (OCHICH,).
`14.75 (OCH2CH,), 22.91 (CH:CO). 61.74
`(COOCH?CH,). 64.72 (OCH,CH:). 76.85
`(CH). 168.25 (CHICO). 170.48 (COOCH2-
`CH,)ppm: mass spectrum, m e (relative in-
`tensity) 190 ( 5 ) . 160 (2). 144 (381, I 16 (98). 102
`(92), 74 (100): high resolution mass spectrum,
`for C,H,,NO,
`calc.
`190.1079.
`found
`190.1087.
`
`it iit ed-x -
`Preparation of ' alk~*l-suhst
`ucetamidoacetates ( 6 )
`General procedure. The alkyl 2-acetamido-2-
`alkoxyacetate (5) (1 equiv.) was suspended in
`anhydrous ethyl ether (60mL 10mmol). 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 of NaHCO,, stirred at ice tem-
`perature (20 min), and then extracted with
`ethyl acetate (3 x ) method. The organic
`layers were combined, dried (Na,SO,). and
`concentrated to dryness in vacim. The result-
`ing oil was purified by flash chromatography
`or recrystallization.
`284
`
`Data for all nen compounds prepared by
`this technique are IIxLed below.
`
`MCJ t hJ.1 ?-(ice fun I icfo--'-fiirunnct.tate ( 6a) . The
`desired compound M A S purified in 62% yield
`by flash chromatography (99: 1 chloroform/
`methanol): R,0.32 (09: 1 chloroform/metha-
`nol); m.p. 80-81"; i.r. (KBr) 3200, 1740, 1620
`(br). 1530 (br). 1205. 1090, 1020, 900,
`(s,
`890cm ' ; 'Hn.m.r. (CDCI,) 62.03
`CH,CO). 3.75 (s. OCH,), 5.77 (d, J =
`7.8Hz. CH), 6.35-6.36 (m, C,H, C,H), 7.02
`(d. J = 7.8 Hz. N H ) . 7.36 (br s, C,H); "C
`n.m.r.
`(CDCI,) 21.69
`(CH,CO), 50.43
`(OCH,), 52.88 (CH). 108.72 (C,), 110.78
`142.84 (C?). 148.89 (C?), 169.57
`(C,).
`(CH,CO). 169.96 (COOCH?CH,) ppm; mass
`spectrum. mie (relative intensity) 197 (14),
`165 ( 3 5 ) , 154 (78), I38 (36), 96 (IOO), 94 (93),
`69 (16).
`Anal. calc. for C,H,, NO,: C 54.82, H 5.62, N
`7.10. Found: C 54.96. H 5.40, N 7.27.
`
`x-Nc.etmiiiclo--'Tfurunacetate (6a').
`Eth.i.1
`Compound 6a' was isolated in 51% yield
`after two successive fash chromatographies
`( ( a ) 100"/0 chloroform; (b) 70:30 ethyl ether/
`pentane, then 97:3 chloroform/methanol):
`R,O. 17 (100% chloroform); m.p. 69-70';
`i.r.
`(KBr) 3200. 1750. I635 (br), 1530,1380,1335,
`1205. 1180. 1020, 890. 745, 595cm-';'H
`n.m.r.
`(CDCI,)
`(51.24
`(t,
`J = 7.2Hz,
`OCH?CH,). 2.04 (s. CH,CO), 4.14-4.32 (m,
`OCHZCH,), 5.75 (d. .I = 8.1 Hz, CH), 6.34-
`6.35 (m. C,H. C,H). 6.35-6.54 (br d, J =
`8.1 Hz. NH), 7.35-7.36 (m, C,H); I3C n.m.r.
`(CDCI,) 13.91 (OCHICH,), 22.81 (CH,CO),
`50.33 (CHI. 62.08 (OCH2CH,), 108.49 (C,),
`110.62 (C,). 142.64 ( C 5 ) , 148.85 (C2), 168.89
`(CH,CO). 169.43 (C'OOCH,CH,)ppm; mass
`spectrum. m,'e (relatiLe intensity) 21 1 (€9, 168
`(32). 138 (27), 96 (1 OO), 94 (27).
`Anal. calc. for C l o H l ~ N 0 4 : C 56.87, H 6.20,
`N 6.63. Found: C 56.98, H 6.19, N 6.83.
`
`Ethj.1 r - a ~ ~ t ~ t a n i i ~ ~ ~ - 2 - ~ ~ ~ ~ r r o l e a c r t a t e (6c, ) and
`
`( 6c2 ) .
`etii!.I ~-c~r~~tatiii~fo-3-pj~rrol~~acetate
`T.1.c. analysis at the conclusion of the reac-
`tion indicated the presence of two major com-
`pounds (R, 0.33 and K, 0.19,98:2 chloroform/
`methanol). which uere isolated by flash chro-
`
`IPR2014-01126- Exhibit 1014, p. 6
`
`

`
`Functionalized non-natural amino acid derivatives
`
`(98:2 chloroform/methanol).
`matography
`The initial fraction (R, 0.33,98:2 chloroform/
`methanol) was further purified by a second
`flash
`chromatography
`(97:3
`dichloro-
`methane/methanol) to produce 6c, in 41 YO
`yield: m.p. 104-106"; i.r. (KBr) 3310, 3200,
`1715, 1635 (br), 1515 (br), 1220, 1180, 1085,
`1010, 890cm. ' ; ' H n.m.r. (DMSO-d6) 61.16
`(t, J = 7.2 Hz, OCH,CH,), I .88 (s, CH,CO),
`(m, OCHzCH,), 5.33 (d, J =
`4.01-4.16
`6.9 Hz, CH), 5.96-5.99 (m, C,H, C,H), 6.69-
`6.72 (m, C,H), 8.48 (d, J = 6.9Hz, CONH),
`10.80-10.99 (br s, NH); "C n.m.r. (CDC1,)
`13.93 (OCH2CH,), 22.79 (CH,CO), 50.73
`(CH), 61.38 (OCH,CH,), 106.35 (C,), 107.52
`(C4), 118.07 (Cj), 125.28
`(Cz), 169.24
`(CH,CO), 170.1 1 (COOCHzCH3) ppm; mass
`spectrum, mje (relative intensity) 210 (22),
`167(36), 137(54), 121 (7), 106(7),95(100),93
`(97), 79 ( 5 ) 68 (53).
`Anal. calc. for C,,H,,N,O,: C 57.13, H 6.71,
`N 13.33. Found: C 57.20, H 6.55, N 13.13.
`The
`second
`fraction
`(R,-0.19, 98:2
`chloroform/methanol) was further purified
`by a second flash chromatography (95:5 di-
`chloroniethanejmethanol) to give 6cz in 12%
`yield: m.p. 92-93";
`i.r. (KBr) 3320, 3240,
`1720, 1640 (br), 1510, 1400 (br), 1210, 1180,
`1010, 890cm-'; ' H n.m.r. (CDCI,) 61.25 (t,
`J = 6.9H2, OCH2CH,), 2.02 (s, CH,CO),
`(m, OCH,CH,), 5.53 (d, J =
`4.10-4.30
`7.2Hz, CH), 6.17-6.30 (m, C4H), 6.25 (d,
`J = 7.2Hz, CONH), 6.70-6.75 (m, C,H),
`6.78-6.80 (m, C,H), 8.45-8.60 (br s, NH); "C
`n.m.r. (CDCI,) 13.93 (OCH,CH,), 22.79
`(CH,CO), 50.73-(CH), 61.38 (OCH2CH,),
`:06.78 (C4), 116.56 (G), 118.25 (C3), 118.63
`(Cj), 169.79 (CH,CO), 171.76 (COOCH2-
`CH,) ppm; mass spectrum, m/e (relative in-
`tensity) 210 (12), 167 (16), 152 (5), 137 (31),
`121 (3), 95 (loo), 93 (IOO), 80 (5), 68 (71); high
`resolution mass spectrum, calc. for C,,,H,,-
`N,O, 2 10.1004, found 2 10.10 15.
`
`Prc.paration of substituted
`a-acetumidoacetic acids (7) from alkyl-
`substituted-a-acetamidoacetates ( 6 )
`General procedure. The alkyl 2-substituted-a-
`acetamidoacetate (6) (1 equiv.) was dissolved
`in 90:lO ethanol/water (- 9 mL/I mmol) and
`then KOH (1.1 equiv.) was added and the
`
`tem-
`resulting solution stirred at room
`perature (48 h). The reaction was con-
`centrated in vacuo and the residue diluted
`with H 2 0 and then washed with either ethyl
`acetate or ethyl ether. The aqueous layer was
`then made acidic with 8.5% H,PO, and ex-
`tracted with ethyl acetate (3 x ). The organic
`layers were combined, dried
`(Na2S0,),
`evaporated to dryness in vucuo, and then re-
`crystallized to yield the desired product.
`Data for all new compounds prepared by
`this technique are listed below.
`
`a-Acetamido-2-jiuranacetic acid (7a). Coni-
`pound 7a was isolated in 51% yield after
`recrystallization
`from acetonitrile: R,0.37
`(8: 1 : 1 isopropanol/NH,0H/H20); m.p. 171-
`172"; i.r. (KBr) 3320, 3100, 1705, 1580(br),
`1530, 1410, 1360, 1320, 1280, 1270, 1225.
`1210, 1145, 1100, 1010, 890, 660, 640, 610,
`570, 400cm-'; 'H n.m.r. (DMSO-d,) 61.88
`(s, CH,CO), 5.45 (d, J = 7.8 Hz, CH), 6.39-
`6.45 (m, C,H, C,H), 7.65 (s, C,H), 8.69
`(d, J = 7.8Hz, NH). [The carboxyl proton
`was not detected.] ',C n.m.r. (DMSO-d,)
`22.10 (CH,), 50.16 (CH), 108.17 (C,), 110.66
`(C4), 142.83 (Cj), 149.75 (C?), 169.21
`(CH,CO), 170.01 (COOH) ppm; mass spec-
`trum, m/e (relative intensity) 183 (2), 165 (10).
`140(24), 123 (19), 109 (I), 96(100),94(43), 80
`(2), 69 (8).
`Anal. calc. for C,H,NO,: C 52.46, H 4.95. N
`7.65. Found: C 52.61, H 4.93, N 7.94.
`
`a-Acetamido-2-pyrroleacetic ocid ( 7c, ) . The
`product was
`recrystallized
`(chloroform/
`methanol/hexanes) to give 712, (29% yield):
`Rf0.55 (8: 1: 1
`isopropanol/NH,OH/HzO);
`m.p. 112-1 14"; i.r. (KBr) 3340, 3300, 1710,
`1590(br), 1530(br), 1220, 1080, 885,
`' H n.m.r.
`(DMSO-d,) 61.87
`725cm I;
`(CH,CO), 5.31 (d, J = 7.?Hz, CH), 5.96
`(s, CJH), 5.97 (s, C4H), 6.87 ( s , C,H), 8.40 (d,
`J = 7.2Hz, CONH), 10.79-10.85(brs, NH).
`[The carboxyl proron was not detected.] "C
`n.m.r. (DMSO-d,) 22.16 (CH,CO), 50.45
`(CH), 106.21 (C3), 107.45 (C4), 117.83
`(C,), 126.1 1 (G), 169.13 (CHJCO), 171.56
`(COOH) ppm; mass spectrum, mje (relative
`intensity) 182 (I), 164 (7), I5 I (49, 138 ( I 00),
`137 (25), 121 (2), 95 (98), 93 (lo), 91 (46).
`
`285
`
`IPR2014-01126- Exhibit 1014, p. 7
`
`

`
`P. LeGall et d.
`r-Acetun1ido-3-p~~rroleucetic acid i 712, ) . The
`beige residue was recrystallized (chloroform,
`methanol/hexanes) to furnish 7e2 (38% yield):
`Kf0.28 (8: 1 : 1
`isopropanol;NH,OH,'H,O):
`m.p. 135-138"; i.r. (KBr) 3340, 3300, 1700,
`1585 (br), 1525 (br). 1240(br). 920,895cm I;
`' H n.m.r. (DMSO-d,) 61.85 (s, CH,CO), 5.05
`(d, J = 7.0Hz. CH), 6.04 (s. C,H). 6.69
`(s, C2H), 6.76 (s, C,H), 8.23 (d. J = 7.0Hz.
`CONH), 10.68-10.86 (br s. NH). [The car-
`boxyl proton was not detected.] "C n.m.r.
`(DMSO-d,) 22.18 (CH3CO). 50.57 (CH),
`106.98 (C4), 116.28 (C:). 117.83 (C, and
`C5), 169.13 (CH,CO), 173.00 (COOH) ppm:
`mass spectrum, m:e (relative intensity) 182
`(4), 164 (6), 157 (I), 138 (100). 124 (3). 121
`(38), 95 (19). 93 (33), 80 (94), 68 (91): high
`resolution mass spectrum. calc. for C,H,,-
`N,O 182.0691. found 182.0688.
`
`Prrparation of
`r-Aceturiiino-N-hetrrl.l-ZT furciiiucetciriiick
`llaJ
`r-Acetamido-2-furanacetic acid (7a) (0.47 g.
`2.56mmol) was combined with acetonitrile
`(1OmL) and cooled to -5"
`(ice!salt water
`bath). Triethylamine (0.26 g. 0.36 mmol) was
`then rapidly added and the mixture stirred at
`(3 min). Ethyl chloroformate (0.28 g,
`- 5"
`0.25 mL, 2.56mmol) was added dropwise
`between -4 and -3", and the resulting sus-
`pension was stirred at - 4" (20 min). and then
`an acetonitrile solution (2 mL) of benzyla-
`mine (0.30g. 0.31 mL, 2.82mmol) was care-
`fully added. During the addition of benzyla-
`mine the temperature of the solution did not
`go above 0". The mixture was stirred at - 5"
`(1 h) and at room temperature ( 1 8 h), and
`then concentrated in iwcuo. The residue was
`then triturated with hot
`tetrahydrofuran
`(SmL), cooled at - 16" (3 h). and the result-
`ing white precipitate was filtered and iden-
`tified as triethylamine hydrochloride (' H
`n.m.r. analysis). The filtrate was evaporated
`to dryness in vucuo and the resulting oil puri-
`fied
`by
`flash
`chromatography
`(98:2
`chloroform'methanol) to give a 13% yield
`la: Ki0.30 (98:2 chloroform
`(0.09g) of
`methanol); m.p. 178-179"; i.r. (KBr) 3230.
`1625 (br), 1525 (br). 1375 (br), 1230. 1090.
`890. 740. 690cm-':'H n.ni.r. (DMSO-d(,)
`
`286
`
`cil 90(~.CH,CO),4.31 ( d , J = 6.0Hz,CH2),
`5.58 (d, J = 8.1 HI, CH), 6.27-6.33 (m,
`C,H), 6.40-6.44 (m, C,H), 7.20-7.36 (m, Ph),
`7.60-7.64 (m, C,H). 8.58 (d, J = 8.1 Hz,
`NH). 8.73 (t, J = h.OHz, NH); "C n.m.r.
`(DMSO-d,) 22.35 (CH,CO), 42.27 (CH,),
`50.95 (CH), 107.60 (C,), 110.55 (C,), 126.82
`(2C2 or X,), 127.08 (2C2 or 2C,), 128.27
`(C, ), 139.05 (C, ). 142.58 (C,), 151.16 (C,),
`168.02 (CH,CO), 169.30 (C0NH)ppm; mass
`spectrum, mle (relative intensity) 273 (I), 230
`( I ) . 139 (100). 96 (94), 91 (51), 65 (9).
`Anal. calc. for C,,H,,N,O,: C 66.16, H 5.83,
`N 10.29. Found: C 65.92, H 5.83, N 10.15.
`
`Prcparutiorr of'
`Z-ucetriniido-N-ben:!~I-2-rnethoxyacetamide
`18a)
`To a methanolic solution (1 80 mL) of methyl
`2-acetamido-2-methoxyacetate (5a) (8.73 g,
`54 mmol) was rapidly added benzylamine
`(8.68g. 8.80mL, 81 mmol) and then the
`mixture was stirred at 50" (3 days) during
`which time a beige precipitate appeared. The
`solvent was removed in ~ " c u o and the result-
`ing precipitate was recrystallized from tetra-
`hydrofuran (2 x ) to give 7.67 g (32%) of the
`desired product as beige crystals: Kf0.35 (95:5
`chloroform/methanol); m.p. 145- 146"; i.r.
`(KBr) 3260,1625 (br). 1550,1505,1435,1390,
`1370. 1230. 1120. 1060, 935, 890, 740,
`' H n.1n.r. (CDCI,) 62.06 (s,
`690cm I ;
`CH;CO). 3.39 (s. CH,O), 4.35-4.40
`(m,
`CH,), 5.S2 (d. J = 8.7Hz, CH), 7.12 (d,
`J = 8.7 Hz, NH), 7.20-7.40 (m, Ph, NH); I3C
`n.m.r. (CDCI,) 23.03 (CH?CO), 43.51 (CH,),
`55.84 (CH,O), 78.94 (CH), 127.62 (C4,),
`127.70 (2C, or 2C, ). 128.70 (2C,, or 2C,,),
`(C, ),
`137.45
`167.01
`(CH,CO), 171.57
`(CONH) ppm; mass spectrum, mje (relative
`intensity) 237 (I), 205 (2), 193 (l), 177 (2), 163
`(4). 146(l). 134(1). 121 (2), 106(26), 102(94),
`91 (95). 77 (13), 61 (100).
`Anal. calc. for C , , H , , , N 2 0 , : C 61.00, H 6.83,
`N 1 1.86. Found: C 60.91, H 6.85, N 1 I .66.
`Prcprircition of'
`~-rit~c~ttii~iir~o-N-hc~ri-,~l-Z-etfio.~~~acetnniicIe
`idhi
`An ethanolic solution (420mL) of ethyl 2-
`(27.92 g,
`(Sb)
`acetamido-2-ethoxyacetate
`
`IPR2014-01126- Exhibit 1014, p. 8
`
`

`
`147 mmol) and benzylamine (23.70 g, 24 mL,
`221 mmol) was stirred at 40-45" for 3 days.
`The reaction mixture was evaporated in vucuo
`and the residue recrystallized (3.5: 1 tetrahy-
`drofuran/hexanes [650 mL]) to yield 25.80 g
`(70%) of the desired product as beige crystals:
`R,0.59 (955 chloroform/methanol); m.p.
`153-155"; i.r. (KBr) 3260, 1630 (br), 1550
`(sh), 1505 (br), 1380, 1360, 1230, 1115, 1065,
`1015, 890, 740, 690cm-';'H n.m.r. (CDCI,)
`61.20 (t, J = 7.0Hz, OCH2CH,), 2.07 (s,
`CH,CO), 3.60-3.76 (m, OCH,CH,), 4.40-
`4.54 (m, CH,NH), 5.60 (d, J = 8.7Hz, CH),
`6.63 (d, J = 8.7Hz, NH), 7.00 (br s, NH),
`7.26-7.36 (m, Ph); I3C n.m.r. (CDCI,) 15.06
`(OCH,CH,),
`23.25
`(CH,CO),
`43.60
`(CH,NH), 64.51 (OCH,CH,), 77.43 (CH),
`127.69 (2C2 or 2C7, C,), 128.79 (2C, or
`2C, ), 137.57 (C, ), 168.13 (CH,CO), 171.29
`(CONH) ppm; mass spectrum, m/e (relative
`intensity) 251 (4), 163 (9), 116 (98), 106 (34),
`91 (98), 74 (100).
`Anal. calc. for C,,H,,N,O,: C 62.38, H 7.25,
`N 11.19. Found: C 62.49, H 7.27, N 11.24.
`
`Preparation of 2-substituted
`cc-ace famido-N-henzylacetumides ( I ) from
`2-acetamido-N-henzyl-2-ethoxyacetamide
`(86)
`General procedure. 2-Acetamido-N-benzyl-2-
`ethoxyacetamide (8b) (1 equiv.) was suspend-
`ed in anhydrous ethyl ether, and then boron
`trifluoride etherate
`(1.6-6.3 equiv.) was
`rapidly added and the resulting solution was
`stirred for 15 min. The aromatic substrate
`(! 6-1 6 equiv.) was then added and the reac-
`tion was stirred at room temperature (1-
`7 days). The. experimental workup varied
`slightly for each compound and is described
`below along with
`the observed spectral
`properties.
`
`cx- Acetumido-N-henzyl-2- furanacetamide
`( l a ) . The reaction mixture was poured into
`an ice-cold saturated aqueous solution of
`NaHCO,, and then stirred at 0" (20 min), and
`then the mixture was extracted with ethyl
`acetate (3 x ). The organic layers were com-
`bined, dried (Na,SO,), and evaporated to
`dryness in vacuo. The product was further
`purified by flash chromatography (70:30 ben-
`
`Functionalized non-natural amino acid derivatives
`zene/acetone) to yield the title compound in
`58% yield as white crystals: R,0.30 (98:2
`chloroform/methanol); m.p. 178-1 79"; mixed
`melting point with sample prepared by mixed
`anhydride method, m.p. 178-1 79".
`
`u-Acetamido-N-benzyl-2- (S-methylfuran) -
`acetamide ( l b ) . The reaction mixture was
`poured into an aqueous saturated NaHCO,
`solution and extracted with ethyl acetate
`(3 x). The ethyl acetate extracts were com-
`bined, dried (Na,SO,) and evaporated in
`vucuo to give a beige solid, which was purified
`by flash chromatography (98:2 chloroform/
`methanol) to yield 61% of the desired
`product as a white crystalline solid: Rf0.25
`(98:2 chloroform/methanol); m.p. 148-1 50";
`i.r. (KBr) 3270, 1620 (br), 1520 (br), 1440,
`1360, 1210, 1010cm-';'H n.m.r. (DMSO-d,)
`61.88 (s, CH,CO), 2.23 (s, CH,), 4.24-4.36
`(m, CH,), 5.49 (d, J = 8.0Hz, CH), 6.01 (br
`~,C,H),6.14(d,J = 2.4Hz,C,H),7.20-7.31
`(m, Ph), 8.52 (d, J = 8.0Hz, NH), 8.69 (t,
`J = 5.6Hz, NH); "C n.m.r. (DMSO-d,)
`13.44 (CH,), 22.35 (CH,CO), 44.11 (CH,),
`53.23 (CH), 107.51 (C, or C,), 110.40 (C, or
`C4>, 128.13 (C4), 128.18 (2C,. or 2C,,), 129.43
`(2C2, or 2C,,), 139.69 (Cl,), 149.18 (C, or C5),
`153.81 (C, or C,), 170.78 (CH,CO), 173.03
`(CONH) ppm; mass spectrum, m/e (relative
`intensity) 286 (3), 179 (8), 153 (57), 152 (57),
`11 1 (231, 110 (IOO), 97 (23), 91 (31).
`Anal. calc. for C,,H,,N,O,: C 67.12, H 6.34,
`N 9.78. Found: C 66.92, H 6.52, N 9.52.
`
`cx-Acetamido-N-benzyl-2-pyrroleacetaniide
`( l c , ) . Hexanes were added to the reaction,
`and the mixture was filtered and the brown
`semi-solid was
`triturated with
`95:5
`chloroform/methanol
`to furnish a green
`solid. This material was purified by flash
`chromatography (95: 5 chloroform/methanol)
`to yield the desired product in 35% yield as a
`white solid: R,0.29 (96:4 chloroform/metha-
`nol); m.p. 174-175"; i.r. (KBr) 3230, 1610
`(br), 1500, 1470 (br), 1330, 1230, 1070, 950,
`890, 860, 740, 710, 685, 655cm-'; ' H n.m.r.
`(CD,CN) 61.93 (s, CH,CO), 4.35 (d,
`J = 6.0Hz,CH2), 5.42(d,J = 6.9Hz,CH),
`6.00-6.18 (m, C3H, C4H), 6.68-6.72 (m,
`287
`
`IPR2014-01126- Exhibit 1014, p. 9
`
`

`
`P. LeGall et d.
`C5H). 7.04 (d. J = 6.9Hz. NH). 7.17 (t.
`J = 6.0Hz, NH). 7.10-7.47 (m. Ph). 9.25-
`9.35 (br s, NH); "C n.m.r. (CD,CN) 22.02
`(CH,CO), 43.83 (CH2). 52.65 (CH). 107.57
`
`(C?). 108.85 ((IJ), 119.33 (C:). 127.96 (f).
`128.01 (2C2 o