0040 4020'89 $3.00+.00
`
`lemhrdron Vol 45. No. 19.pp,h091 to 6100. 19X9
`I'r,nrcd 11, Grat
`
`Br~taln Pergamon Press plc Synthesis of (4)-2,3-Methanoproline: A Novel Inhibitor of Ethylene Biosynthesis Frank L. Switzer, Herman Van Halbeek, Elizabeth M. Holt, Charles H. Stammer*
`
`Department
`
`of Chemistry,
`
`School
`
`of Chemical
`Georgia
`
`Sciences, University
`30602
`
`of Georgia, Athens,
`
`Department
`
`of Vegetable Crops, Mann Laboratory, University
`95616
`
`of California, Davis, California
`
`1989)
`
`1
`
`Abstract: The title compound, 2-aza-bicyclo[3.1.0]hexane-1-carboxylic acid (2) was prepared by treatment of N-benzyloxycarbonyl-2,3_dehydroproline tert-butyl ester with diazomethane followed by photolysis of the resulting pyrazoline and deprotection. Its N- acetyl-N’-methyl amide, a peptide mimic, was synthesized and the structure of was confirmed by X-ray diffraction studies. NMR spectroscopy was also used to examine the effect of the cyclopropane ring on its conformation. This 2,3-methanoamino acid (2) was found to be a weak inhibitor of ethylene biosynthesis in cucumber cotyledon strips and germinating squash seeds, The data show that 2 probably inhibits the conversion of l- aminocyclopropanecarboxylic acid to ethylene in these tissues. The biosynthesis of the plant hormone ethylene has attracted considerable interest in recent years particularly since l-aminocyclopropanecarboxylic acid (Act) was found to be the direct precursor of ethylene.
`Much of the research in this area has concerned the mechanism by which Act is degraded to ethylene, cyanide, and CO2 by the ethylene forming enzyme (EFE)2. A second area of research has involved the search for potential inhibitors of EFE.3 Early in these studies, Hoffman and coworkers4 found that (+)- nllo-coronamic acid (1, (lR, 2S )-2-ethyl-Act) was converted into 1-butene at 25% the rate at which Act yields ethylene, and that this stereoisomer of 1 was processed much more efficiently than any of the other stereoisomers (>40:1). The many inhibitors of EFE tested have led various workers3hT4 to propose a model for the active site of EFE (Figure 1) in which both the amino and carboxyl functions of Act are required for binding to occur. The active site is also sterically constrained so that large groups at the 2-position cannot be accommodated. e’ .,,m
`
`-O&f
`
`NH,+
`
`1
`
`FQure2
`
`609 1
`
`Hz+
`
`2
`
`SAXA-DEF-00430
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`Page 1 of 10
`
`AstraZeneca Exhibit 2039
` Mylan v. AstraZeneca
` IPR2015-01340
`
`Mikal E. Saltveit, Jr.
`(Receirrd in CJ’SA 7 Mm91
`

`
`6092
`
`F. L. SWITZER et Ui.
`
`Based on this model, it was clear that 2,3-methanoproline (2, Figure 2) could be accommodated by the active site of EFE. This new cyclopropane amino acid would not only be an analog of proline but also of Act; i. e., as a cycle-allo-coronamic acid, which would not be fully processed by EFE because it is a secondary amine. It might, therefore, be a tightly binding competitive or irreversible inhibitor of the enzyme. Results and Discussion. The synthesis of 2 was completed as shown (Scheme 1). Its hydrochloride salt (3) was prepared from the known5 dehydroproline (4) in 31% overall yield. When this imine was treated with excess benzyl chloroformate and pyridine, the N-benzyloxycarbonyl derivative of enamine 5 was isolated and allowed to react with excess diazomethane. The intermediate pyrazoline was not isolated, but was directly photolyzed to give the fully protected cyclopropane 6. Deprotection of 6 could be accomplished in a single step using trifluoroacetic acid and thioanisole or, more efficiently, in a two-step process involving hydrogenolysis (5% Pd/C) followed by acidolysis (2M HCI). The amino acid zwitterion was then obtained in 73% yield from the hydrochloride using Dowex-1 (acetate form).
`
`4
`
`5
`
`6
`
`d
`
`Reagents: a) CbzCl , Pyr ; b) CHzNz : c) hd ; d) Hz, 5% Pd/C ; e) HCI; 1) Dowex-1
`
`Scheme 1 The crystalline N-acetyl-N’-methylamide derivative 8 was prepared from amino ester 7 by standard methods (Scheme 2). X-ray diffraction confirmed the structure of 8 (Figure 3) and, by inference, the structure of 2. This particular derivative was chosen because it is a model peptide and its conformation should give an indication of the conformational behavior of this novel amino acid in peptides. The amide exhibited the small N2-Cl-C2-Nl (w) dihedral angle which is characteristic of the conformations taken by 2,3-methanoamino acids in the solid state6. Furthermore, the $ and w angles of 8 are quite similar to those obtained by X-ray crystallography of the analogous proline derivative7; i.e., in amide 8
`= 76” and w = 7” while in N-acetylproline-N’-methylamide the corresponding values are 76” and 16”. The slightly smaller v angle in amide 8 is probably due to conjugation of the carbonyl group with the cyclopropane ring, which is maximized at v=O”. These conformational similarities indicate that the cyclopropane containing amino acid may be an effective replacement for proline for purposes of stabilization to enzymolysis with retention of bioactivity.
`
`, Q,
`
`SAXA-DEF-00431
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`Page 2 of 10
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`

`
`Synthesis of ( 1 )—2.3~methanoproline
`
`Crystallography.
`A crystal of C9H14N2O2 (8) was mounted on a Syntex P3 automated diffractorneter. Unit
`cell dimensions (Table I) were determined by least squares refinement of the best angular
`positions for fifteen independent reflections (2(-)>l5.°) during normal allignment procedures
`using molybdenum radiation ('y=O.71069A). Data,
`(1063 points) were collected at
`room
`temperature using a variable scan rate, a 6-29 scan mode and a scan width of 1.2° below
`KOL2 and l.2° above Kotz to a maximum 26 value of 45.0°. Backgrounds were measured at
`each side of the scan for a combined time equal
`to the total scan time. The intensities of
`three
`standard reflections were
`remeasured after every 97
`reflections
`and as
`the
`intensities of these reflections showed less than 6% variation, corrections for decomposition
`
`were deemed unnecessary. Data were corrected for Lorentz, polarization and background
`effects. After removal of space group forbidden and redundant data, observed data,
`(423
`points)
`(I>3.0o(l)) were used for solution and refinement.
`The structures were solved for
`
`carbon, nitrogen and oxygen positions using direct methodss. Least squares refinement‘)
`converged with anisotropic thermal parameters.
`Hydrogen atoms were located from a
`difference Fourier synthesis.
`These positions were included in the final
`refinement with
`isotropic thermal parameters but held invariant.
`A final difference Fourier
`revealed no
`electron density of interpretable level.
`Scattering factors were taken from Cromer and
`Mann“).
`The final cycle of refinement
`-
`function minimized 2(\Fo\-\Fc\)2,
`led to final
`agreement
`factor, R=8.2%; R=(Z\\Fo\-FC\\/2\Fo\) x 100. Unit weights were used until
`the
`final cycles of refinement when weights equal
`to 1/GF were introduced. Rw=l0.8%.
`
`H
`
`H
`
`0
`7
`
`_.a:.. A
`A 0
`O
`
`L1,. [NE--H"/”\
`/K 0
`O
`8
`
`Reagents: a) AcCI , NE't3 b) TFA , CHZCIZ c) (CH3)2CHCH2OCOCl,NEl3 d) MeNH3C| , NEt3
`S c h em e
`2
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`SAXA-DEF-00432
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`Page 3 of 10
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`

`
`6094
`
`F. L. SWITZER
`
`et al. We have also carried out an NMR analysis of amide 8 in order to study its conformational properties in dilute solution and to compare them with the corresponding prolineamide derivative in order to allow interpretation of future peptide spectra containing the new amino acid. Using 1H -1I-I COSY, 1I-I -13C COSY, DEPT, steady-state NOE experiments, and a 500 MHz proton spectrum, we have been able to make preliminary assignments in D20 of the protons and carbon atoms of both the
`
`s-cis
`
`s-trans
`
`s-tram
`
`conformers of 8 (Figure 4). In general, we found that 8 exhibits a somewhat greater preference for the s-cis conformation than does the prolineamide. In contrast, a recent study of N-acetyl-2,4-methanoproline-N’-methylamide by Scheraga 11 showed that only the
`conformer was present in dilute solution. Further NMR studies of amide 8, now in progress, will be published separately. TabIe 1. Ethylene production from plant tissues treated with (f)-2,3-methanoproline (2).
`
`Apple fruit
`
`Cucumber
`cot-
`
`-_&u&seeds
`
`Carrot
`lppts
`
`Concen-
`tration
`(mM)
`
`post-
`Climae-
`t4X-k
`
`Pre-
`Climac-
`teric
`
`strips
`
`Whole
`
`Getmin-
`ating
`
`Excised
`roots
`
`cortex
`disks
`
`1.61
`
`1.69
`
`2.53
`
`1.52 aZ
`
`2.80
`
`4.10 a
`
`12.1
`
`0.231
`
`3.67 b
`
`11.6
`
`0.243
`
`2.04
`
`1.19 b
`
`2.39
`
`2.87 c
`
`13.0
`
`0.252
`
`2.41
`
`0.98 c
`
`0.75 d
`
`2.60
`
`2.76
`
`n.s.
`
`2.48 d
`
`14.6
`
`n.s.
`
`0.268
`
`0.255
`
`n.s.
`
`61.8x
`
`64.9
`
`65.0
`
`66.6
`
`64.8
`
`61.7
`
`61.3
`
`62.2
`
`0.0
`
`0.008
`
`0.024
`
`0.08
`
`0.24
`
`0.8
`
`2.4
`
`8.0
`
`80
`
`LSD 5%
`
`n.s.
`
`n.s.
`
`x Ethylene production in nl (ghr)-1 z Means followed by the same letter are not significantly different at the 5% level.
`
`SAXA-DEF-00433
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`Page 4 of 10
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`and the
`

`
`Synthesis of ( f )-2,3-methanoproline 6095
`
`1.97 dddd
`
`t
`
`s
`
`2.01 dddd J=13.1 J=9.2 J=8.6 J=7.3 2.33 dddd Jz7.1 J=6.0 1.81 ddt 2.20 dddd 228dddd J=13.0 J=92 Z3io J=2.2 J=7.1 J=7:1 J=8.7 J=5.8 J=2.2 J=3.0 H H 2.07
`
`J=5.6 2.14 dd J=9.2 J&2 29.4 26.5 26.3 33.1 53.0 22.0 21.5 Figure 4 Biology Application of 40 to 60 ~1 of 0.8 mM aqueous solutions of 2,3-methanoproline (2) to germinating squash seeds and strips of cucumber cotyledons reduced ethylene production by 30% and 20%, respectively. An 8 mM solution of 2 inhibited ethylene production by 36% and 40% in the same respective tissue. Inhibition was roughly fold increase in concentration resulted in an additional 14% production from strips of cucumber cotyledons; i. e., 22%, 36%, and 8, and 80 mM solution, respectively. loglinear and each ld- reduction in ethylene 50% reductions for 0.8, In contrast, application of from 20 to 60 ~1 of up to 8 mM 2 had no inhibitory effect on ethylene production by cortex disks of pre- and post-climacteric apple tissue, carrot cortex tissue, whole cucumber cotyledons, or excised squash roots (Table 1). Ethylene production from whole cucumber cotyledons was unaffected by 20 l.~l of the 80 mM solution. Two applications of 20 ~1 of the 80 mM solution did result in a 28% reduction in ethylene production from pre-climacteric apple cortex disks, but this extremely high concentration was phytotoxic; treated tissue became brown and water soaked (data not shown). Three other inhibitors of ethylene synthesis were more effective than 2. Applications of 40 to 60 ~1 of 0.8, 8.0 and 80 mM solutions of 2 inhibited ethylene production in receptive tissue by an average of 26%, 38% and 50%, respectively, while application of 20 ul of a 10 mM solution of aminoethoxyvinylglycine (AVG) inhibited ethylene production
`
`SAXA-DEF-00434
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`Page 5 of 10
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`1.24
`

`
`6096 F. L. SWITZER
`
`et al.
`
`from tomato pericarp disks by about 97% (11 to 0.3 nL/g.h) 12. Similarly, 20 pl applications of 5 mM aminooxyacetic acid (AOA) and 100 mM a-aminoisobutyric acid (Aib) solutions inhibited ethylene production by 80% and 95%, respectively. AVG and AOA are thought to interfere with the synthesis of Act, the immediate precursor of ethylene, while Aib is thought to block the conversion of Act to ethylene. Treating cucumber cotyledon strips with 2,3_methanoproline, before application of Act, indicated that it inhibited ethylene production by affecting the EFE. Ethylene production from the control and treated strips was very low: averaging 0.38 and 0.37 nl/g hr, respectively, and the long incubation period probably resulted in this diminished rate of ethylene production. Addition of 80 ul of 0.5 mM Act solution stimulated ethylene production over 70-fold in the control tissue, but only 11-fold in tissue treated with 80 ul of 80 mM 2,3_methanoproline solution indicating that it probably inhibited conversion of Act to ethylene through an effect on EFE. For comparison, the 80 mM solution of 2 required for 50% inhibition can be compared to the 100 mM concentration of Aib that produced a 95% inhibition of ethylene production9. Experimental Section Material and Methods. (A) Chemistry Melting points were determined on a Thomas Hoover capillary melting point apparatus and are uncorrected. The ‘H NMR spectra were recorded on a Varian EM-390 continuous wave NMR spectrometer operating at 90 MHz or a Bruker AM-250 Fourier transform NMR at 250 MHz using tetramethylsilane as the internal standard. The ’ 3C NMR spectra were recorded either on a Bruker AM-250 operating at 62.5 MHz or on a JEOL FX 90Q Fourier transform NMR spectrometer operating at 22.5 MHz with CDC13 or dioxane (for D20) as the internal standard. Anhydrous and HPLC grade solvents were used directly in the synthetic reactions. Other commercial reagents also were used without additional purification. Microanalysis was performed by Atlantic Microlab Inc., Atlanta, Georgia. All reactions were carried out in either a nitrogen or an argon atmosphere. (B) Biologv. Solution nrenaration. Aqueous treatment solutions of 2,3_methanoproline were made by diluting 80 and 0.8 mM (i.e., 10,000 and 100 ppm (wt/wt)) stock solutions prepared before each experiment. ADDIe cortex tissue. Disks (0.5 x 1 cm diam) were cut with a stainless steel razor blade from cylinders of cortex tissue excised with a cork bore from pre- and post-climacteric apples (Malus domestica Borkh., cv. Golden Delicious). The disks were soaked for 30 min in 500 ml of 0.5 M glycerol, blotted dry, and distributed among 15 x 100 mm plastic petri dishes. Twenty to 60 ul of the test solutions were applied to each disk.
`
`SAXA-DEF-00435
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`Page 6 of 10
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`

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`Synthesis of (Ifr)-2,3-methanoproline 6091 Cucumber cotvledons. Cucumber seedlings (Cucumis sativus L. cv. Poinsett 76) were germinated in vermiculite for about one week at 20°C under fluorescent light. Only fully expanded cotyledons were used. They were excised from the hypocotyl with a razor blade and a 20 pl drop of the test solutions were applied to the base of each cotyledon. Two cotyledons were enclosed in each test tube and each treatment contained 4 replicates. Cotyledons were also cut into 3 mm thick longitudinal slices to increase absorption of the solutions. Sixty ~1 of the test solutions were applied to about nine strips. Excess solution was removed after 2 hr, and the tissue was weighed and enclosed in 15 mL- glass test tubes. Ethylene forming enzyme activity was assayed using strips of cucumber cotyledons. The strips (0.1 g FW) were treated with 80 pl of water or 80 mM 2,3_methanoproline for 2 hr, blotted dry, and then treated with 80 ~1 of water or 0.5 mM Act for 16 hr. Carrot cortex disks. Disks (0.3 x 1 cm diam) were cut with a stainless steel razor blade from cylinders of cortex tissue excised with a cork bore from carrot roots. The disks were soaked for 30 min in 500 mL deionized water, blotted dry, and distributed among 15 x 100 mm plastic petri dishes. Twenty ~1 of the test solutions were applied to each disk. Germinating squash seeds. Squash seeds (CucurbitqDeDo L. cv. Table King Acorn) were germinated on moist filter paper for either 3 or 5 days at 20”. Three uniform seeds were placed into 15-ml glass tubes with 40 ~1 of the test solutions. Ethvlene measurements. After waiting for the solutions to be absorbed by the tissue, the tissue was blotted, weighed, placed in a 16 x 100 mm glass test tube and immediately capped with a rubber serum stopper. After 1 to 6 hr of incubation at 20°C in dim light, 1 mL gas samples of the headspace were taken and analyzed for ethylene by flame ionization gas chromatography as previously describedIs. Each experiment was repeated at least twice and each treatment contained from 3 to 5 replicates in a completely randomized experimental design. Synthesis. N-Benzyloxvcarbonyl-2.3-dehvdronroline-OtBu 5 To a stirred solution of 4 (16.0 g, 94.8 mmol)5 and pyridine (12 mL, 150 mmol) in CH2C12 (100 mL) at -20°C was added benzyl chloroformate (21.4 mL, 150. mmol) dropwise over a 15 min period and the solution was stirred at room temperature overnight. N,N- diethylethylenediamine (14 mL, 100 mmol) was then added dropwise over 15 min to destroy the excess chloroformate and the reaction was stirred an additional 2 h. The solution was then diluted with CH2C12 (300 mL) and washed with 10% aq citric acid (2 X 300 mL) and 10% aq Na2C 0 3 (300 mL). The organic phase was dried (K2C 0 3 ), concentrated, and chromatographed on silica gel (230-400 mesh, 65 X 270 mm column, 3 portions, hexanes to 6:4 hexanes/ethyl acetate gradient elution) to afford 22.4 g (78%) of 5 as a colorless oil; lH NMR (CDC13)
`
`: 6 7.21 (s, 5H), 5.68 (t, lH, J = 3 Hz), 5.08 (s, 2H), 3.91 (t, 2H, J = 9 Hz), 2.57 (d of t, 2H, J = 9 Hz, J = 3 Hz), 1.40 (s, 9H) ppm; 13C NMR (CDC13)
`160.8,
`
`: 6
`
`SAXA-DEF-00436
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`Page 7 of 10
`
`153.4, 137.6, 136.0, 128.3, 128.0, 119.1, 81.8, 67.3, 48.6, 28.3, 27.8 ppm.
`

`
`6098
`
`F. L. SWITZER et al. N-Benzvloxvcarbonvl-2.3-methanomoline-OtBu 6 The dehydroamino acid derivative 5 (24.0 g, 79.2 mmol) was divided into three equal portions and each was treated with excess diazomethane generated under nitogen from N- methyl-N-nitroso-para-toluenesulphonamide (Diazald, 32 g, 150 mmol) according to the published procedure14 using a preparative scale apparatus similar to the Aldrich Mini Diazald Apparatus. The resulting diazomethane/ether mixture was collected with a Dry Ice/CCl4-cooled condenser and added directly to a Dry Ice/CC14 cooled solution of 5 in CH2C12 (100 mL). This solution was then stirred at room temperature for 24 h after which CaC12 (lo-20 g) was added and stirring was continued for 16 h. The CaC12 was filtered to give a colorless solution of crude pyrazoline which was concentrated and photolyzed for 4 h in CH2C12 (500 mL) in an ice-water cooled apparatus using a 450 W Hanovia medium- pressure mercury lamp. The crude cyclopropane from these three runs was then chromatographed in two portions on silica gel (230-400 mesh, 65 X 270 mm column, hexanes to 1:l hexanes/ethyl acetate gradient elution) to afford 16.4 g (65%) of 6 as a colorless oil; 1H NMR (CDC13)
`
`: 6 7.21 (s, 5H), 5.07 (s, 2H), 3.4-4.1 (m, 2H), 1.6-2.4 (m, 4H), 1.47 (s, 9H), 0.97 (br s, 1H) ppm; 13C NMR (CDC13)
`: 6 169.5, 155.7, 136.54, 128.2, 127.5, 80.9, 66.8, 51.4 (br), 48.2, 30.5 (br), 27.7, 26.5, 26.0 (br) ppm. 2.3-Methanooroline CF3CQ2H salt
`
`2-TFA
`
`The fully protected amino acid 6 (0.5 g, 1.6 mmol) was added at room temperature to a stirred mixture of trifluoroacetic acid (30 mL), thioanisole (3 mL, 25 mmol), and m-cresol (3 mL, 28 mmol). The resulting light brown solution was then stirred at room temperature for 4 h. and the mixture was concentrated by rotary evaporation from CC4 (3 X 100 mL). The residue was triturated with hexanes (100 mL), washed with ether (100 mL), and dissolved in D20 (5 mL). The solution was passed through a small reversed-phase silica gel column (5 X 50 mm, 40~ octadecyl silica) followed by an additional 5 mL portion of D20. The combined eluate was lyophilized to afford a gummy yellow solid (0.08 g, 40%); the NMR spectrum showed it to be the trifluoroacetate salt of 2
`: 6 174.8, 49.6 (quat), 45.3 (CH2, J = 148 Hz), 28.6 (CH, J = 182 Hz), 27.0 (CH2, J = 129 Hz), 16.5 (CH2, J = 169 Hz) ppm. 2.3-Methanonroline-OtBu 7 Protected amino acid 6 was dissolved in ethyl acetate (300 mL) and the solution was flushed with argon. 5% Pd/C (-0.3 g) was added and hydrogen was bubbled into the stirred suspension until no starting material remained (2 h). The flask was then flushed with nitrogen; the catalyst was removed by filtration and washed with methanol (3 X 100 mL) and the combined filtrates were concentrated. The residue was then Kuglerohr distilled at reduced pressure to give 6.6 g (85%) of 7 as a light yellow oil
`
`; 1H NMR (D20)
`
`: 6 2.6 - 3.6 (m,
`
`2H), 2.0 - 2.2
`
`3H),
`
`2H)
`
`: bp 90-12O”C/O.8 mm; 1H NMR (C6D6)
`: 6 2.83 (m, lH), 2.32 (m, lH), 2.10 (br s, lH), 1.76 (m, lH), 1.3-1.65 (m, 3H), 1.36 (s,9H), 0.85 (t, J = 6 Hz) ppm. 13C NMR (C6D6)
`: 6 172.2 (C=O), 80.1 (quat), 50.1 (quat), 44.3 (CH2), 28.1 (CH3), 27.9 (CH2), 27.7 (CH), 16.7 (CH2) ppm.
`
`SAXA-DEF-00437
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`Page 8 of 10
`
`(m,
`1.3 - 1.6 (m,
`ppm; l3C NMR (D20)
`

`
`: 6 171.5, 46.7, 43.2, 27.0, 24.6,14.9 ppm; MS
`
`Synthesis of (k)-2,3-methanoproline 6099 2.3-Methanoproline 2 Distilled 7 (4.4 g, 24 mmol) and anisole (3.0 mL, 28 mmol) were dissolved in 3.0 M HC1 in dioxane (40 mL) and the solution was stirred at room temperature overnight during which a white precipitate formed. The suspension was diluted with hexanes (200 mL) and the precpitate was filtered and washed with ether (2 X 100 mL). The solid was dissolved in isopropanol (100 mL) and reprecipitated with ether (800 mL) giving a light orange solid which was chromatographed on reversed-phase silica gel (- 10 g, 401.1 flash chromatography, grade Clg, water elution). The aqueous solution was lyophilized and the yellow-white solid was recrystalized from isopropanol/ether and, after the removal of a small amount of highly colored precipitate, the amino acid hydrochloride hydrate (3, 3.2 g, 73%) was isolated as granular white crystals: mp 200-215°C; 13C NMR (D20)
`: m/e 127 (M+), 109 (M - H20). Anal. Calcd. for CgHgN02.HCl.H20 : C, 39.67%; H, 6.66%; N, 7.74%. Found : C, 39.73%; H, 6.72%; N, 7.68%. Zwitterion 2 was prepared from 3 by stirring an aqueous solution of 3 with Dowex 1X8-50 (OAc-,- 50 g) overnight at room temperature. The resin was removed by filtration, washed with water (3 X 100 mL) and the combined filtrates were lyophilized to afford the desired zwitterion (1.7 g, 53% from 6) as a white powder
`: 6 173.9, 48.8, 41.2, 24.8, 24.8, 13.2 ppm. Anal. Calcd. for C6HgN02.1/4 H20 : C, 54.72%; H, 6.89%; N, 10.68%. Found : C, 54.65%; H, 7.30%; N, 10.55%. N-Acetvl-2.3-methanonroline-N’-methvlamide 8 TO a stirred solution of amino ester 6 (1 .O g, 5.5 mmol) and triethylamine (1.5 mL, 11 mmol) in CH2C12 (30 mL) at room temperature was added acetyl chloride (0.7 mL, 10 mmol). After 1 h, N,N-diethylethylenediamine (1 .O mL, 7.1 mmol) was added and stirring was continued for an additional hour. The solution was then diluted with CH2C12 (300 mL), washed with 2% aq KHS04 (2 X 200 mL) and 10% aq. K2CO3 (1 x 200 mL), dried (K2CO3), and concentrated to afford 1.0 g (80%) of crude N-acetylester as a light yellow oil; 1H NMR (CDC13)
`6 171.2, 169.2, 81.8, 52.5, 48.8, 35.6, 30.5, 27.9, 26.8, 22.1 ppm. This material was dissolved in CH2C12 (30 mL) and anisole (2 mL, 18 mmol) was added. The solution was cooled to 5°C and trifluoroacetic acid (30 mL) was added. The cold bath was removed and the solution was stirred for 1 h, diluted with CC14 (100 mL) and concentrated at 35°C. After twice re-evaporation from CC14 (200 mL), the residue was dissolved in CH2C12 (50 mL), the solution was cooled to 5°C and triethylamine (3.0 mL, 21 mmol) and isobutyl chloroformate (0.8 mL, 6.2 mmol) were added. The solution was stirred for 30 min, after which methylamine hydrochloride (1.0 g, 15 mol) was added and the mixture was stirred at room temperature overnight. The solution was diluted with CH2Cl2 (200mL) and extracted with 2% aq KHS04 (2 X 200 mL). The aqueous solution was then extracted with 1O:l CHC13/methanol (4 x 150 mL) and the combined extracts were
`
`: mp 197-203°C; 1H NMR (D20)
`
`: 6
`
`: 6 3.4-4.2 (m, 2H), 2.04 (s, 3H), 0.9-2.4 (m, 5H), 1.44 (s, 9H) ppm; 13C NMR (CDC13)
`
`:
`
`SAXA-DEF-00438
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`Page 9 of 10
`
`3.58 (m, lH), 3.06 (m, lH), 2.1-2.5 (m, 3H), 1.3-1.8 (m, 2H) ppm; 13C NMR (D20)
`

`
`6100
`
`F. L.
`
`al.
`
`concentrated to dryness. The residue was reconcentrated from ethyl acetate and chromatographed on silica gel (230-400 mesh, 45 X 240 mm column, 1O:l CHClg/methanol). The crude product was crystallized from ethyl acetate/hexanes to give 0.2 g (20% from 6 ) of amide 7 as analytically pure prisms
`: mp 150-151°C; 1H NMR (D20, 250 MHz)
`: 8 3.96
`(m, lH), 3.70 (m, lH), 2.74 and 2.68 (s, 3H), 2.05 and 2.04 (s, 3H), 1.7-2.4 (m, 4H), 1.23 and 1.02 (t, lH, J = 6 Hz) ppm; 13C NMR (D20)
`: 6 175.7 (C=O), 174.9 (C=O), 172.9 (C=O), 172.3 (C=O), 53.0 (CH2). 52.9 (CH2), 50.4 (quat), 49.3 (quat), 33.1 (CH), 29.4
`
`WI),
`
`28.0 (CH2), 26.5 (CH2). 26.2 (CH2), 26.2 (CH3), 26.1 (CH3), 24.1 (CH2), 22.0 (CH3), 21.5 (CH3) ppm. Anal. Calcd. for CgHl4N202
`
`: C, 59.28%;
`
`: C, 59.28%;
`
`Sci. U.S.A.
`Proc. Natl.
`; Yang, S. F.
`J. Org.
`1979,x, 170 - 176. Pirrung, M. C.
`J. Org.
`Chem. 1987, 2, 4179 - 4184 and references cited therein. a. Wheeler,T. N.
`; Ray, J. J.
`J. Org.
`Chem. 1987,52, 4875 - 4877; b. Pirrung, M. C.; McGeehen, G. M.
`; c. Pirrung, M. C.; McGeehen, G. M.
`Angew. Chem. Int. Ed.
`Plant Physiol. 1982,70,
`Engl. 1985,&, 1044 - 1045. Hoffman, N. E.
`; Yang, S. F.
`; Ichihara, A.
`; Sakamura, S.
`195 - 199. Hausler, J.
`
`Acta Cryst.
`
`1073-1088.
`Justus Liebigs Ann. Chem. 1981,
`Znt. J. Pept. Prot. Res.
`Int. J. Pept. Prof. Res., 1988,
`1986,=, 349-359. b) Varughese, K. I.; Wang, C. H.; Kimura, H.; Stammer, C. H.
`31_, 299-300. c)
`J. Chem. Sot., Chem.
`252.
`Commun.,
`1988,
`; Iitaka, Y.
`
`1971, m, 507 - 516. Main, P., Fiske, S. J., Hull, S. E., Lessinger, L., Germain, G., DeClerq, J. P., and Woolfson, M. M., University of York, England, 1980. Stewart, J. M., Ed., The X-Ray System-Version of 1980, Technical Report TR446 of the Computer Center, University of Maryland, College Park, Maryland. Cromer, D. T. and Mann, I. B., Acta Cryst., A24, 321-324 (1968). a) Montelione, G. T.; Hughes, P.; Clardy, J.; Scheraga, H. A.
`
`H, 7.75%; N, 15.37%. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. REFERENCES Adams, D. 0.
`
`6765-6773.
`c)
`4477-4485.
`J. Am. Chem. Sot., 1987, m,
`4473-4477.
`Talluri, S.; Montelione, G. T.; van Duyne, G.; Piela, L.; Clardy, J.; Scheraga, H. A.
`J. Amer. Sot. Hoi-r.
`
`b) Piela, L.; Nemethy, G.; Scheraga, H. A.
`
`Sci..,1983, 108, 512. Saltveit, M. E. Jr., Hort. Science, 1982, 17, 878. Hudlicky, M.
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`J. Org. Chem. 1980, 45, 5377-5378.
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`J. Am Chem. Sot., 1986,108,
`J. Am. Chem. Sot. 1987,109,
`
`SAXA-DEF-00439
`
`Page 10 of 10
`
`SWITZER et
`H, 7.74%; N, 15.43%. Found
`Acad
`Chem. 1986,& 2103 - 2106.
`a) Mapelli, C.; Kimura, H.; Stammer, C. H.
`Elrod, L. F.; Holt, E. M.; Mapelli, C.; Stammer, C. H.
`Matsuzaki, T.
`Edwards, J. I., Saltveit, M. E.,Jr., and Henderson, W. R.,