· 'Uiiiv. of Minn.
`Bio .. Medical
`
`U. . R "'I
`
`;\
`
`armaceu ic
`• c1e
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`J 0 u R N A 1
`
`0 F
`
`A publication of the
`American
`Pharmaceutical
`Association
`and the
`American
`Chemical
`Society
`
`May 1994
`Volume 83, Number 5
`JPMSAE 83(5) 611-762
`ISSN 0022-3549
`
`RESEARCH ARTICLES
`
`A Novel Skeletal Drug Delivery SY,stem Using Self-Setting Calcium Phosphate Cement. 2.
`Physicochemical Properties and Drug Release Rate of the Cement-Containing Indomethacin
`Makoto Otsuka, Yoshihisa Matsuda, Yoshiko Suwa, Jeffrey L. Fox, and William I. Higuchi .........................
`
`High-Performance Capillary Electrophoresis/Frontal Analysis for the Study of Protein Binding
`of a Basic Drug
`Akimasa Shibukawa, Yasuki Yoshimoto, Toshio Ohara, and Terumichi Nakagawa .........................................
`
`Effect of (Hydroxypropyl)-P-cyclodextrin on Flux of Morphine, Fentanyl, Sufentanil, and
`Alfentanil through the Spinal Meninges of Monkey
`Christopher M. Bernards ...............................................................................................................................................
`
`Determination of Acitretin in the Skin, in the Suction Blister, and in Plasma of Human
`Volunteers after Multiple Oral Dosing
`Jean-Philippe Laugier, Christian Surber, Hot Bun, Jean-Marie Geiger, Klaus- Peter Wilhelm,
`Alain Durand, and Howard I. Maibach ......................................................................................................................
`
`Deconvolution Method for Assessing the Absorption of a Drug with Reversible Metabolic
`Pathways
`Stephen Hwang and Mark Knowles ............................................................................................................................
`
`Sustained Release of Ferrous Sulfate from Polymer-Coated Gum Arabica Pellets
`Veena Batra, As his Bhowmick, Basanta K. Behera, and Alok R. Ray .. .. ........ .......... .... .. .. ...... .......... .............. .....
`
`In Vitro and in Vivo Evaluation of Thyrotrophin Releasing Hormone Release from
`Copoly(dl-lactic/glycolic acid) Microspheres
`Toshiro Heya, Hiroaki Okada, Yasuaki Ogawa, and Hajime Toguchi ..................................................................
`
`Release of Indomethacin from Transparent Oil-Waters Gels
`A. De Vos, L. Vervoort, and R. Kinget ........................................................................................................................
`
`Morpholinoalkyl Ester Prodrugs of Diclofenac: Synthesis, In Vitro and In Vivo Evaluation
`Vijay K. Tammara, Milind M. Narurkar, A. Michael Crider, and Mansoor A. Khan .......................................
`
`Determination of Pyrrolizidine Alkaloids in Commercial Comfrey Products (Symphytum sp.)
`Joseph M. Betz, Robert M. Eppley, Wendell C. Taylor, and Denis Andrzejewski .............................................
`
`Isoelectric Focusing and Capillary Zone Electrophoretic Studies Using Luteinizing Hormone
`Releasing Hormone and Its Analog
`·
`Mark C. Heit, Ann McFarland, Randy Bock, and Jim E. Riviere .........................................................................
`
`Radioimmunoassay for the Novel Platelet Activating Factor Receptor Antagonist ES880
`Hiromasa Suzuki, Osamu Asano, Kyoichi Tadano, and Toru Horie .....................................................................
`
`Hepatic Enzyme Induction Potential of Acitretin in Male and Female Sprague-Dawley Rats
`DavidS. Small and Patrick J. McNamara· ................................................................................................................
`
`Derivation of a Rigorous Equation for the Calculation of the F-Value in Isothermal Sterilization
`Processes
`Piero M. Armenante . . . . . . . . . ..... . . . ....... .... .... ..... . . . . . . . . . . . .. . . . ..... . ...... . ...... . ............ ....... .... . . . ...... .... . . . . .............................. ...... . .
`
`Binding of Fantofarone, A Novel Ca2+ Antagonist, to Serum Albumin: A Fluorescence Study
`Pierre Chatelain, Jean-Roger Matteazzi, and Rene Laruel ....................................................................................
`
`611
`
`616
`
`620
`
`623
`
`629
`
`632
`
`636
`
`641
`
`644
`
`649
`
`654
`
`657
`
`662
`
`668
`
`674
`
`Contents continued on V
`Journal of Pharmaceutical Sciences I I I I
`Vol. 83, No.5, May 1994
`
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`.
`A Lamellar Liquid Crystal with Fosinopril Sodium
`Stig E. Friberg, Teanoosh Mdaddel, Ke.nneth R. Morris, Robert Abramowitz, and Kent L.· Amsberry
`
`The Ferguson Principle and an Analysis of Biological Activity of Gases and Vapors
`M. H. Abraham, G. D. Nielsen, andY. Alarie ........................................................................................................
`
`Anticonvulsant Properties of N-Substituted a,a-Diamino Acid Derivatives
`Harold Kahn, Kailash N. Sawhney, David W. Robertson, and J. David Leander
`
`Kinetics of Water Vapor Sorption in Porcine Stratum Corneum
`Zvi Liron, Harvey J. Clewell, and James N. McDougal ..........................................................................................
`
`Cytotoxic Effects of Pamidronate on Monolayers of Human Intestinal Epithelial (Caco-2) Cells
`and Its Epithelial Transport
`Irene M. Twiss, Ruud de Water, Jan den Hartigh, Rolf Sparidans, Willy Ramp-Koopmanschap,
`Hadewich Brill, Monique Wijdeveld, and Pieter Vermeij .......................................................................................
`
`Effect of Diazoxide on Serum and Tissue Electrolyte Levels in Rats with Deoxycorticosterone
`Acetate-Induced Hypertension
`T. Nakai ................................. :.........................................................................................................................................
`
`Structure Evolution of Tablets during Compression Unloading
`Edward G. Rippie and William T. Morehead ...........................................................................................................
`
`Degradation Chemistry of Gemcitabine Hydrochloride, a New Antitumor Agent
`Sally L. Anliker, Michael S. McClure, Thomas C. Britton, Erwin A. Stephan, Steven R. Maple, and
`··········································································~······················································································
`Gary G. Cooke
`High-Performance Liquid Chromatographic Determination of 1,1'-Ethylidenebis(L-tryptophan) in
`L-Tryptophan Preparations
`Mary W. Trucksess, Frederick S. Thomas, and Samuel W. Page ..........................................................................
`
`Renal Handling of Tobramycin in the Isolated Perfused Rat Kidney
`Tetsuya Aiba, Yoshie !toga, Hiromasa Shimizu, Yusuke Tanigawara, and Ryohei Hori ..................................
`
`In Vitro Drug Release Behavior of D,L-Lactide/Glycolide Copolymer (PLGA) Nanospheres with
`Nafarelin Acetate Prepared by a Novel Spontaneous Emulsification Solvent Diffusion Method
`............................................................................
`T. Niwa, H. Takeuchi, T. Hino, N. Kunou, and Y. Kawashima
`
`Valproic Acid Intensifies the Depressant Action of Phenobarbital and Ethanol by a
`Pharmacodynamic Mechanism
`Amnon Hoffman and Gastav Habib ............................................................................................................................
`
`High-Performance Liquid Chromatographic Determination of Ceftibuten and its Metabolite in
`Biological Fluids: Applications in Pharmacokinetic Studies
`J. M. Kinowski, F. Bressolle, D. Fabre, F. Goncalves, R. Rouzier-Panis, and M. Galtier .................................
`
`Effect of the Addition of Electrolytes on the Partition Coefficients, Activity Coefficients, and
`Acid Dissociation Constants of Carnitine and its Acetyl and Propionyl Derivatives
`Paolo De Maria, Antonella Fontana, Sara Frascari, Giuseppe Gargaro, Domenico Spinelli, and
`Maria 0. Tinti .................................................................................................................................................................
`
`Effects of Indomethacin on the Pharmacokinetics and Pharmacodynamics of Prednisolone in Rats
`Varun Garg and William J. Jusko ..............................................................................................................................
`
`Comparison of Three New Spectrophotometric Methods for Simultaneous Determination of
`Aspirin and Salicyclic Acid in Tablets without Separation of Pharmaceutical Excipients
`Bernhard W. Glombitza and Peter C. Schmidt .........................................................................................................
`
`Pharmacokinetic Study of (S)-(-)-2-(N-Propyl-N-(2-thienylethyl)amino)-5-hydroxytetralin
`Infusion in Cynomolgus Monkeys
`Denise R. Lowe Walters, William R. McConnell, and Eugenio A. Cefali .............................................................
`
`Potent Inhibitors of Histamine Release: Polyhydroxylated Sterols from the Okinawan Soft Coral
`Sin ularia abrupta
`Noboru Shoji, Akemi Umeyama, Masao Takei, and Shigenobu Arihara .............................................................
`
`677
`
`680
`
`689
`
`692
`
`699
`
`704
`
`708
`
`716
`
`720
`
`723
`
`727
`
`733
`
`736
`
`742
`
`747
`
`751
`
`758
`
`761
`
`Journal of Pharmaceutical Sciences I V
`Vol. 83, No.5, May 1994
`
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`
`Anticonvulsant Properties of N-Substituted a,a-Diamino Acid. Derivatives
`
`HAROLD KOHNtx, KAILASH N. SAWHNEYt, DAVID W. ROBERTSON+§, AND J. DAVID LEANDER+
`
`Received April 19, 1993, from the toepartment of Chemistry, University of Houston, Houston, TX 77204-5641,
`:tUlly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285.
`Accepted for
`publication November 24, 1993®.
`§Current address: Ligand Pharmaceuticals, 9393 Towne Center Dr., Suite 100,
`San Diego, CA 92121.
`
`Abstract 0 Recent studies have demonstrated that functionalized
`a,a-diamino acids (1) display excellent activity when evaluated in the
`maximal electroshock seizure (MES) test in mice. The synthesis and
`pharmacological evaluation of 14 select analogues within this series
`of compounds are detailed.
`Included in this survey were 10 N-acyl
`derivatives in which the basic C(a) N-group in 1 was replaced by a
`neutral N-substituent and four dipeptides where the amino acid fusion
`point was the a-carbon site. N-Acylation of 1 led to decreased
`anticonvulsant activity. The importance of these findings in relation
`to the requirements of the C(a) substituent for anticonvulsant activity
`in 1 are briefly discussed.
`
`I ntroductlon
`
`Recent studies have shown that a,a-diamino acid derivatives
`(1) and related compounds are surprisingly stable and readily
`accessible materials.1-5 Moreover, we have demonstrated that
`a-amino, a-hydrazino, and a-N-hydroxylamino adducts display
`excellent anticonvulsant activity when evaluated in the maximal
`electroshock seizure (MES) test in mice.5 For example, the
`median effective dose (ED 50) values after intraperitoneal injection
`for the a-N-ethylamino (2a) (4:2.4 mg/kg) and a-.N2-(benzyloxy(cid:173)
`carbonyl)hydrazino (2b) (55.6 mg/kg) derivatives approached
`the ED50 of phenobarbital6 (21.8 mg/kg), whereas the ED50s of
`the a-methoxyamino (2c) (6.2 mg/kg) and the a-[(methoxy(cid:173)
`methyl)amino] (2d) (6.7 mg/kg) adducts exceeded the ED50 of
`phenytoin6 (9.5 mg/kg). Both 2c and 2d exhibited these potent
`anticonvulsant effects at doses much lower than those which
`produced neuromotor impairment on the horizontal screen (HS)
`test ( 46.0 and 50.5 mg/kg were the ED 50 doses for 2c and 2d on
`the HS test).5 These findings prompted our investigation of the
`pharmacological activity of the racemic N-substituted a,a(cid:173)
`diamino acid derivatives (2e-r) (Table 1). TheN-acyl derivatives
`(2e-n) were evaluated to determine the effect of conversion of
`the basic C(a)-amino group in 2a-d to a neutral C(a)-carbamate
`(2e, 2f), urea (2g-2i), thiourea (2j, 2k), amide (21, 2n), or suc(cid:173)
`cinimide (2m) substituent on anticonvulsant activity. Also
`included in our study were the unique dipeptides 2o-r, where
`the amino acid fusion point was the a-carbon site.
`
`Experimental Section
`
`Chemical Methods-Melting points were determined with a Thomas(cid:173)
`Hoover melting point apparatus and are uncorrected. Infrared spectra
`(IR) were run on a Perkin-Elmer 1330 and 283 spectrometers and
`calibrated against the 1601-cm-1 band of polystyrene. Absorption values
`are expressed in wavenumbers (cm-1). Proton (lH NMR) and carbon
`(13C NMR) nuclear magnetic resonance spectra were taken on Nicolet
`NT -300 and General Electric QE-300 NMR instruments. Chemical shifts
`( o) are in parts per million (ppm) relative to Me4Si and coupling constants
`(J values) are in hertz. Low-resolution mass spectra (MS) were recorded
`at an ionizing voltage of 70 e V from a Varian MAT CH -5 spectrometer
`at the Lilly Research Laboratories. Microanalyses were provided by the
`
`0 Abstract published in Advance ACS Abstracts, February 1, 1994.
`
`~, ,R3
`N
`I
`1
`R NH -C -C- Ff
`I
`II
`H 0
`
`1
`
`n R2 = NHC(O)CH2NHC(O)OCH2Ph
`g, R2 = NHCH2C(O)OCH3
`g, R2 = NHCH2C(O)OCH2CH3
`a R2 = NHCH2C(O)OCH2Ph
`R2 = NH2CH2CO£
`.S R2= NH2
`1 R2 =Br
`.1.1. R2 = N(CH3h· BF 4-
`y_ R2 = NHC(O)CH3
`YJ. . R2 = NHC(O)CF3
`
`2a R2 = NHCH2CH3
`b. R2 = NHNHC02CH2Ph
`~ R2 = NH(OCH3)
`g ~ = N(CH3)0CH3
`,a R2 = NHC(O)OCH3
`f ~ = NHC(O)OPh
`~ R2 = NHC(O)NHCH3
`h R2 = NHC(O)NHPh
`i R2 = NHC(O)NHS(02)Ph
`j R2 = NHC(S)NHCH3
`k R2 = NHC(S)NHPh
`1 R2 = NHC(O)Ph(2'C02H)
`0
`
`2=NO
`
`m R
`
`0
`
`Physical Chemistry Department of the Lilly Research Laboratories. All
`compounds gave satisfactory elemental analyses (C, H, N) that were
`within :±:0.4% of theoretical values. Thin-and thick-layer chromatog(cid:173)
`raphy were run on precoated silica gel GHLF microscope slides (2.5 X
`10 em; Analtech No. 21521) or silica gel GHLF (20 X 20 em; Analtech
`11187).
`Chemical Synthesis-General Procedure for the Synthesis of
`Functionalized Amino Acid Derivatives 2e-k-A tetrahydrofuran
`(THF) solution containing 2s5 and either the acylating agent (1.06-1.10
`equiv) and triethylamine (1.20 equiv) or the isocyanate (isothiocyanate)
`(l.Q-1.1 equiv) was heated. The reaction was then filtered to remove
`any salts formed and purified, and the product was recrystallized if
`necessary. The reaction temperatures, times, and recrystallization
`solvents (if appropriate) were as follows: (2e) 55-60 °C, 2 h, EtOH; (2f)
`45-50 °C, 2 h, MeOH; (2g) 45-50 °C, 2 h, MeOH; (2h) 45-50 °C, 2 h;
`(2i) 5Q-55 °C, 22 h; (2j) 65 °C, 4 h, EtOH; (2k) 65 °C, 3 h, EtOH.
`Synthesis of .N-[Acetamid~(benzylcarbamoyl)methyl]phthalamic
`Acid (21). To a warm pyridine solution (7.0 mL) containing 2s (0.63
`g, 2.83 mmol) was added phthalic anhydride (0.43 g, 2.87 mmol), and
`the reaction was stirred at 5Q-55 °C (5 h). Pyridine was removed by
`distillation in vacuo and the residue was treated with H 20 (20 mL). The
`aqueous mixture was extracted with EtOAc (2 X 20 mL) and then acidified
`with aqueous 1 N HCl solution. The white solid (0.70 g, 70%) that
`precipitated was filtered, washed with H 20 (10 mL), and dried; mp 186-
`188 °C.
`Synthsisof2-Acetamido-N-benzyl-2-(N-succinimidyl)acetamide
`(2m). A cooled (-78 °C) THF solution (150 mL) of 2t5 [prepared from
`2-acetamido-N-benzyl-2-ethoxyacetamide 7•8 (2.00 g, 8.0 mmol) and BBr3
`(2.51 g, 10.05 mmol)] was added slowly into a cooled (-78 °C) THF
`suspension (50 mL) of sodium succinimide (3.06 g, 25.25 mmol). The
`
`© 1994, American Chemical Society and
`American Pharmaceutical Association
`
`0022-354919411200-689$04.50/0
`
`Journal of Pharmaceutical Sciences I 689
`Vol. 83, No.5, May 1994
`
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`
`

`
`reaction mixture was stirred at -78 °C (30 min) and at room temperature
`(90 min), and then treated with a 10% aqueous citric acid solution (50
`mL). The resulting solution was neutralized with a saturated aqueous
`NaHC03 solution, and the reaction mixture· extracted with EtOAc (3 X
`100 mL). The combined extracts were dried (Na2S04), and the volatile
`materials were removed by distillation in vacuo. The residue was purified
`by flash column chromatography on Si02 gel (6% MeOH/CHCla) to give
`1.10 g (45%) of 2m; mp 181-183 °C (recrystallized from EtOH).
`Synthesis of N -(Benzyloxy)-N-[acetamido(benzylcarbamoyl)(cid:173)
`methyl]malondiamide (2n). 4-Methylmorpholine (0.35 g, 3.56 mmol)
`was added to a solution of N-CBZ-glycine (0.74 g, 3.55 mmol) in THF
`(75 mL) at -10 to -15 °C. The solution was stirred (5 min), and then
`isobutyl chloroformate (0.49 g, 3.55 mmol) was added and the mixture
`was stirred for an additional20 min. A cooled (-10 °C) solution of 2s
`(0.79 g, 3.55 mmol) in THF (125 mL) was then added slowly (30 min).
`The reaction mixture was stirred at this temperature (2 h) and then at
`room temperature (2 h). The insoluble materials were filtered, and the
`filtrate was concentrated in vacuo. The residue was triturated with
`EtOAc (20 mL) and the white solid (0.60 g) that remained was filtered,
`washed with H20, and dried to give 2n. The initial insoluble material
`on trituration with H20 gave an additional 0.40 g of 2n to give a combined
`yield of 1.00 g (68%); mp 177-179 °C (recrystallized from EtOH).
`Synthesis of Methyl N-[Acetamido(benzylcarbamoyl)methyl](cid:173)
`glycinate (2o). A methanolic solution (50 mL) containing 2u5 (1.00 g,
`2.85 mmol) and methyl glycinate (prepared from methyl glycinate
`hydrochloride (1.01 g, 8.55 mmol), and NaOMe (0.38 g, 7.10 mmol)) was
`heated to reflux (2 h). The reaction was concentrated in vacuo to give
`an oily residue that was purified by flash column chromatography on
`Si02 gel (3% MeOH/CHCla) to give 0.60 g (72%) of 2o; mp 144-146 °C
`(recrystallized from EtOAc).
`Synthesis of Ethyl N-[Acetamido(benzylcarbamoyl)methyl](cid:173)
`glycinate (2p). A methanolic solution (70 mL) containing 2u5 (1.50 g,
`4.28 mmol) and ethyl glycinate [prepared from ethyl glycinate hydro(cid:173)
`chloride (3.10 g, 22.2 mmol) and NaOMe (1.17 g, 21.7 4 mmol)] was heated
`to reflux (2 h). The reaction was concentrated in vacuo to give an oily
`residue that was purified by flash column chromatography on Si02 gel
`(5% MeOH/CHC13) to give 0.60 g (46%) of 2p; mp 125-127 °C
`(recrystallized from EtOAc).
`Synthesis of Benzyl N-[Acetamido(benzylcarbamoyl)methyl](cid:173)
`glycinate (2q). A suspension of benzyl glycinate hydrochloride (5.00
`g, 24.8 mmol) in THF (400 mL) containing Et3N (4.90 g, 48.5 mmol) was
`stirred (4 h) at room temperature. The reaction mixture was cooled
`(-78 °C) and then a cooled (-78 °C) THF solution (150 mL) of 2ts
`[prepared from 2-acetamido-N-benzyl-2-ethoxyacetamide 7•8 ( 4.00 g, 16.0
`mmol) and BBr3 (1 M in CH2Cb, 20.0 mL, 20.0 mmol)] was added (30
`min). The reaction mixture was stirred at -78 °C (30 min) and then at
`room temperature (16 h). The insoluble materials were filtered, the
`filtrate was concentrated in vacuo, and the residue was purified by flash
`column chromatrgraphy on Si02 gel (3% MeOH/CHC13) to give 1.56 g
`(26%) of 2q as a white solid; mp 133-135 °C (recrystallized from EtOH).
`Synthesis of N-[Acetamido(benzylcarbamoyl)methyl]glycine
`(2r). A solution of methyl N-[acetamido(benzylcarbamoyl)methyl](cid:173)
`glycinate (2o) (0.60 g, 2.05 mmol) and KOH (0.30 g, 5.36 mmol) in 90%
`aqueous EtOH (50 mL) was stirred at room temperature (48 h). The
`volatile materials were then removed in vacuo, and the residue dissolved
`in H20 (10 mL). The aqueous solution was extracted with EtOAc (2 X
`20 mL), and the aqueous layer was acidified to pH ...... 2.0 with aqueous
`1 N HCL A column containing ion-exchange resin Dowex 50 X W 4 was
`prepared using 10% aqueous pyridine. The column was thoroughly
`washed with H20. The acidic aqueous reaction solution was added to
`the top of the column, and the column was eluted with H20 (300 mL
`or until the eluate was neutral). The column was then eluted with 10%
`aqueous pyridine (400 mL). The aqueous pyridine fraction was
`concentrated in vacuo to give a white solid, dried in vacuo and then
`triturated with absolute EtOH (7 mL). The insoluble materials that
`remained were filtered and dried to give 0.29 g (50%) of 2r; mp 124-126
`°C dec.
`Pharmacological Evaluation-All tests were performed with male
`CF -1 mice from Charles River Breeding Laboratories (Portage, Ml). All
`compounds were dissolved in 30% poly(ethylene glycol) 400 and
`administered in an injection volume ofl mL/100 g of body weight. Initial
`anticonvulsant evaluation of 2e-r was conducted with two dose levels
`(30, 100 mg/kg) administered intraperitoneally. Four mice at each dose
`level were tested at 0.5, 1, and 4 h after administration, unless otherwise
`indicated in Table 1, to determine if there was protection against MES
`seizures.
`
`690 I Journal of Pharmaceutical Sciences
`Vol. 83, No. 5, May 1994
`
`Table 1-Physical and Pharmacological Data In Mice for
`N-Substituted a,a-Diamino Acid Derlvatlves8
`Ff
`0
`0
`II
`I
`II
`CH3CNH- CH- CNHCH2Ph
`
`No.
`
`R2
`
`mpb
`
`MES0 EDs0
`
`NHC(O)OCH3
`
`202-204
`
`NHC(O)OPh
`NHC(O)NHCH3
`NHC(O)NHPh
`NHC(O)NHS(02)Ph
`NHC(S)NHCH3
`NHC(S)NHPh
`NHC(O)Ph(2' -C02H)
`
`201-203
`229-230
`242-244
`188-191
`162-"163
`196-197
`186-188
`181-183
`
`NC(O)CH2CH2C(O)
`NHC(O)CH2NHC(O)OCH2Ph 177-179
`144-146
`NHCH2C(O)OCH3
`125-127
`NHCH2C(O)OCH2CH3
`NHCH2C(O)OCH2Ph
`133-135
`+NH2CH2C02-
`124-126
`
`2e
`
`2f
`2g
`2h
`21
`2j
`2k
`21
`2m
`
`2n
`20
`2p
`2q
`2r
`phenytoin'
`
`p_henobarbital'
`
`valproate'
`
`48.0
`(37. 7-56. 7)
`>100
`>100
`>100
`>100
`>100d
`>100d
`>100
`>100
`
`_,3oe
`>100
`>100
`>100
`>100d
`9.5
`(8.1-10.4)
`21.8
`(15.0-22.5)
`272
`(247-338)
`
`a The compounds were administered intraperitoneally. ED 50 values
`are in milligrams per kilogram. Numbers in parentheses are 95%
`confidence intervals. A dose-response curve was generated for all
`compounds that displayed sufficient activity. The dose-effect data
`for these compounds were obtained at 0.5 h ("time of peak effect").
`b Melting points (0 C) are uncorrected. c MES = maximal electroshock
`seizure test. All compounds were suspended in 30% PEG. d The
`compound was tested only at 0.5 and 1 h due to insufficient supply
`of sample. e Unable to definitively define an ED 50 value and confidence
`limits because of insufficient supply of sample. 'Reference 6.
`
`MES seizures were elicited by electrical current (ac, 60 cps, 50 mA,
`0.2 s) applied via corneal electrodes. A drop of 0.9% saline was instilled
`on each eye prior to application of the electrodes to ensure electrical
`contact. Abolition of the hind limb tonic extension component of the
`seizure was defined as protection in the MES test. This is the identical
`protocol used by the Antiepileptic Drug Development Program of the
`Epilepsy Branch of NINCDS, NIH.s,9
`After the time of peak anticonvulsant activity and the approximate
`dose range were determined, a dose-response curve was generated at
`the time of peak activity with at least three or four doses and 10-12 mice
`per dose. TheMES ED 50 is the calculated dose required to protect 50%
`of the mice in the MES test. For those compounds with significant
`anticonvulsant activity, the doses that caused neuromotor impairment
`on the horizontal screen (HS) test also were determined.1o Previously
`trained mice were dosed with the compound and then placed individually
`on top of a square (13 X 13 em) wire screen which was mounted on a
`vertical rod. The rod was rotated 180°, and the number of mice that
`returned to the top of the screen in one minute were counted.
`
`Results and Discussion
`
`Chemistry-Several preparative routes were utilized for the
`construction of the targeted compounds. In most cases, 2-ac(cid:173)
`etamido-N-benzyl-2-aminoacetamide5 (2s) served as the starting
`material. Treatment of 2s with the appropriate chloroformate,
`isocyanate, isothiocyanate, or anhydride, or use of the mixed
`anhydride protocol advanced for peptide synthesis, 11 led to the
`preparation of the N-acyl substituted adducts 2e-l and 2n.
`Correspondingly, the preformed a-bromo derivatives 2t was
`
`Argentum Pharm. v. Research Corp. Techs., IPR2016-00204
`RCT EX. 2055 - 6/7
`
`

`
`Table 2-Key Spectral Properties of N-Substltuted a,a-Diamino
`Acid Derivatives
`
`R2
`0
`0
`I
`II
`II
`CH3CNH-CH-CNHCH2Ph
`a
`
`1H NMRb
`
`13C NMR0
`
`No.
`
`IR8
`
`CaH
`
`CH2Ph
`
`CaH CaNRC MSd
`
`5.56 (t, 7 .8) 4.27 (d, 5.6)
`
`58.57 -9
`
`1630, 1700 5.66 (t, 7 .6) 4.29-4.35. (m) 58.69 _,
`
`.2e 1650
`279
`341
`2f
`5.59 (t, 7 .8) 4.26 (d, 5.8)
`57.92 157.30 279
`2g 1630
`5.67 (t, 7 .6) 4.30 (d, 5.9)
`2h 1600 (br)
`57.59 153.98 340
`5.47 (t, 7.7) 4.24 (d, 5. 7)
`57.14 150.36 405
`1630 (br)
`21
`61.33 _g
`4.27 (d, 5.8)
`6.10 (br, s)
`1620
`2)
`294
`61.18 180.02 356
`5.24 (t, 6.9) 4.32 (d, 5.8)
`2k 1620
`5.92 (t, 7 .2) 4.36 (d, 6.0)
`1620 (br)
`57.44 - h
`21
`370
`6.31 (d, 9.0) 4.23-4.36 (m) 55.19 176.33 3041
`2m 1620 (br)
`5.79 (t, 7.7) 4.28 (d, 5.8)
`2n 1640 (br)
`56.77 167.86 413
`2o 1610, 1710 5.00 (t, 7 .8) 4.28 (d, 6.0)
`63.98
`46.09 294
`2p 1600, 1710 5.01 (t, 8:2) 4.28 (d, 5.8)
`63.96
`46.22 342
`2q 1620, 1710 5.02 (t, 8.2) 4.27 (d, 6. 1)
`63.94
`46.22 370
`64.08 47.48 -1
`4.98 (d, 8.2) 4.29 (d, 5. 7)
`1630
`2r
`
`8 Infrared spectra were taken with KBr discs and values are reported
`in cm-1. b All spectra were recorded with DMSO-d6. The number in
`each entry is the chemical shift. value (o) observed in ppm relative to
`DMSO-d6, followed by the multiplicity of the signal and the coupling
`constant in hertz. 1H NMR spectra were recorded at 300 MHz. c 13C
`NMR spectra were obtained at 75 MHz and the values referenced to
`DMSO-d6• d All spectra were recorded using FD-MS unless otherwise
`indicated. Molecular ions reported refer to either M+ or M+ + 1 ions.
`9 The carbamate carbonyl carbon resonance was not detected. The
`attached NHC(O)OQ-13 signal was observed at o 51.46. 'The carbamate
`carbonyl carbon resonance was not detected. The attached
`NHC(O)C6H5 signals were observed at o 121.70, 125.18, 129.30, and
`150.91. g The thiocarbonyl carbon resonance was not detected. The
`attached NHC(S)NHQ-13 signal was observed at o 30.92. h The amide
`carbonyl resonance could not be readily assigned. Signals corre(cid:173)
`sponding to four carbonyl peaks were observed at o 167.85, 167.93,
`168.48, and 169.47. 'Value refers toM++ 1 ion observed by FAB(cid:173