otides in length. In vitro “evolution” was done on this region at
`30% mutagenesis. and four more rounds of in vitro selection
`followed before this second population was cloned. From these
`sequences, a consensus region was discovered. Certainly though
`this work is a pioneering achievement in the field, it is an ex-
`ample of how the conventional protocol is significantly more
`involved than that presented here.
`In summary, a novel in vitro selection protocol has been de-
`signed to take advantage of a combinatorial library of small size
`that has multiple copies of every distinct sequence. The method
`condensed the many days of a typical screening strategy to less
`than two days. This was a proof—of—concept experiment that
`showed that the new method succeeds by creating a large num-
`ber of copies of individual sequences in the initial random pool,
`consistently reducing the level of nonspecific binding sequences
`per selection round, and effectively amplifying the few surviving
`sequences.
`Since only the original synthesized sequences were used for all
`the screenings,
`the technique should allow for the iterative
`in vitro selection of modified oligonucleotides that previously
`could not undergo this powerful process.“°] Hence, this method
`should significantly increase the power of the in vitro selection
`method and is the direction that we are currently investigating.
`
`Experimental Section
`The DNA library [4] (31 mg) was labeled at the 5'—end with [y—”P]ATP, purified by
`gel chromatography, and suspended in 300 mL of folding buffer (300 mM KCl, 5 mM
`MgCl2. 20mM Tris. pH 7.5). After cooling down to room temperature following
`denaturation at 75 C, the “P-labeled DNA was loaded onto an acetate-agarose
`precolumn (300 uL), which was attached directly to a 2.5 mM ATP-agarose column
`(800 1.11.. Sigma). The precolumn was washed with 600 uL of buffer, and the eluted
`DNA was allowed to equilibrate on the ATP—agarose column for 10 min. The
`precolumn was discarded after a single use as were all subsequent columns. After
`equilibration, the ATP—agarose column was washed with 4 mL of folding buffer to
`elute unbound or weakly bound oligonucleotides. The retained DNA was eluted
`with 3 mL of the ATP elution buffer (5 mM ATP in folding buffer) and collected in
`500 uL fractions.
`In order to perform another round ofselection, the ATP had to be removed. Hence,
`the eluted fractions were collected directly into Microcon—3 microcentrifuge devices
`(3000 D cutoff. Amicon). After membrane diafiltration, about 98% of the total
`ATP was removed. The filtered fractions were then pooled, and folding buffer was
`added until a final volume of 10 mL was attained. The concentration ofcontaminat—
`ing ATP concentration was 30uM for the DNA sample, which was over 80 times
`more dilute than that of the 2.5mM ATP-agarose column. Each cycle of selection
`started with a new set of stacked affinity columns, i.e. a precolumn attached to a
`ligand column. The screening cycles for the ATP aptamers are summarized in
`Table 1.
`The rare-DNA PCR was performed as follows: On the last cycle the DNA was
`eluted from the ATP~agarose column with 3 mL of 10mM ATP in 20mM Tris,
`pH 7.5. This last fraction was precipitated twice from ethanol, and the PCR reagents
`(50mM KCI, 8mM MgSO,,,10mM(NH,,),SO4,20mM Tris, pH 8.8 at 25 °C, 200;zM
`dNTPs. 0.1 “/0 Triton X-100, 20 units of Deep Vent (exo—) DNA polymerase, 0.5 ug
`5’-primer. 0.5 ug 3'—primer) were added. Thermal cycling (94 °'C for 45 s; 42 ‘C for
`90 s: 60 ‘ C for 45 s; 45 cycles) was done in a microcentrifuge tube that had first been
`irradiated with UV light. A positive control containing a dilute solution (~20 000
`molecules) of a 52-mer. and a negative control containing no DNA also underwent
`the same amplification protocol. Gel electrophoresis after amplification showed
`DNA in all lanes except the negative control.
`
`Received: February 27, 1997 [Z10170lE]
`German version: Angew. Chem. 1997, 109, 1956-1958
`
`Keywords: aptamers - combinatorial chemistry - in vitro selec-
`tion - nucleotides - polymerase chain reaction
`
`[1] L. C. Bock, L. C. Griffin, J. A. Latham, E. H. Verrnaas, J. J. Toole, Nature
`1992, 335, 564 566; J. R. Lorsch, J. W. Szostak, Biochemistry 1994, 33, 973-
`982; D. Smith. G. P. Kirschenheuter, J. Charlton, D. M. Guidot, J. E. Repine,
`Chem. Biol. 1995, 2. 741-750; D. Jellinek, L. S. Green, C. Bell, N. Janjié,
`Bioz'hemistr}' 1994. 33, 10450—- 10456; C. Tuerk, S. MacDougal, L. Gold, Proc.
`Natl. Amd. Sci. USA 1992, 89, 6988-6992; C. Tuerk, S. MacDougal-Waugh,
`Gene1993, 137, 33-39: D. P. Bartel, M. L. Zapp, M. R. Green,J. Szostak, Cell
`1991. 67. 529; C. T. Lauhon, J.W. Szostak,
`.1 Am. Chem. Soc. 1995, I17,
`
`COMMUNICATIONS
`
`1246-1257; P. Burgstaller, M. Famulok, Angew. Chem. 1994, 33, 1163-1166;
`Angew. Chem. Int. Ed. Engl. 1994, 33, 1084-1087; M. Famulok, J. W. Szostak,
`J. Am. Chem. Soc. 1992, 114, 3990-3991; M. Famulok,ibiz1. 1994, 116, 1698-
`1706; J. G. Connell, M. lllangesekare, M. Yarus, Biochemistry 1993, 32, 5497;
`M. Sassanfar, J. W. Szostak, Nature 1993, 364, 550-553: a) M. G. Wallis, U.
`von Ahsen, R. Schroeder, M. Famulok, Chem. Biol. 1995, 2. 543-552; b) R. D.
`Jenison, S. C. Gill, A. Pardi, B. Polisky, Science 1994, 263, 1425-1429; J. F.
`Milligan, M. D. Matteucci, J. C. Martin, 1 Med. Chem. 1993. 36,1923—l937;
`J. D. Ecker, S. T.Crooke, Biotechnology 1995, I3, 351- 360.
`[2] Some exceptions include 2’-amino- and 2’-lluoro substitutions on sugar (H.
`Aurup, D. M. Williams, F. Eckstein, Biochemistry 1992, 31, 9636-9641) and
`1-pentynyl substitution on pyrimidine (J. A. Latham, R. Johnson. J. J. Toole,
`Nucleic Acids Res. 1994, 22, 2817-2822).
`[3] D. E. Huizenga. J. W. Szostak, Biochemistry 1995, 34, 656 665.
`[4] Oligonucleotides were purchased HPLC-purified from Operon. The initial se-
`quence was 5'-GAATTCCAGATCTCT-(18N)—GATATC/XGGATCCCA-3’.
`The two primers were 5’—GAATTCCAGATCTCT-3’ and 5’—TGGGATCCT-
`GATATC—3’. These sequences incorporated EQQRI and B_arnHI digestion en-
`zyme restriction sites.
`[5] D. M. Coen in Current Protocols in Molecular Biology (Eds: F. M. Ausubel, R.
`Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, K. Struhl),
`Current Protocols, New York, 1994, Chapter 154.
`[6] J. P. Katz, E. T. Bodin, D. M. Coen, J. Virol. 1990, 64. 1690-1694.
`[7] S. A. Kauffman, The Origins of0rder: Self Organization and Selection in Evo-
`lution, Oxford University Press, New York, 1993. Chapter 3.
`[8] D. H. Freeman, Anal. Chem. 1972, 44, 117-120‘, B. M. Dunn, l. M. Chaiken,
`Proc. Natl. Acad. Sci. USA 1974, 71, 2382.
`[9] J. P. Hummel, W. J. Dreyer, Biochim. Biophys. Acta 1962, 63, S30.
`[10] Another method for the use ofmodified nucleotides in the 5’-region ofa primer
`has been described recently: J. Burmeister, G. von Kiedrowski, A. D. Ellington,
`Angew. Chem. 1997, 109, 1379-1381; Angew. Chem. Int. Ed. Engl. 1997, 36,
`1321 -1324.
`
`The Synthesis of Enantiopure
`w-Methanoprolines and co-Methanopipecolic
`Acids by a Novel Cyclopropanation Reaction:
`The “Flattening” of Proline**
`
`Stephen Hanessian,* Ulrich Reinhold, and
`Gabriella Gentile
`
`Proline occupies a prominent position in the hierarchy of
`natural amino acid constituents of mammalian proteins“) As
`part of a peptidic motif, its unique structure results in secondary
`amide bonds, leading to important conformational and func-
`tional consequencesm For example, the well—known cis—trans
`isomerism in prolylamides is associated with vitally important
`biological phenomena and functions, such as protein folding,“
`hormone regulation,l"" recognition)“ and transmembrane sig-
`naling[6] to mention a few. The importance of cis— trans confor-
`mational changes is manifested by the role that peptidyl prolyl
`isomerases such as the immunophilins play in immunoregula-
`tion.m Proline has also figured prominently as a component of
`therapeutic agents,”” in drug design,[9‘ and in probing enzyme
`activity.“°]
`Conformationally constrained analogues of proline have
`been used extensively in connection with peptidomimetic re-
`search.“” Although 2,3— and 3,4-methanoprolines have been
`
`[*] Prof. Dr. S. I-Ianessian, Dr. U. Reinhold, G. Gentile
`Department of Chemistry
`Universite de Montreal
`C. P. 6128, Succ. Centre—ville
`Montreal, QC, H3C 3J7 (Canada)
`Fax: Int. code +(514) 343-5728
`[**] We thank NSERC for generous financial assistance through the Medicinal
`Chemistry Chair program. We thank Dr. Michel Simard for the X-ray analy-
`ses. U. R. acknowledges a DFG Research Fellowship from the Deutsche
`Forschungsgemeinschaft. G. G. thanks the University of Siena and the Italian
`C. N. R. for a summer fellowship.
`
`Ex. 1010
`
`Angew. Chem. Int. Ed. Engl. I997. 36,‘No. 17
`
`(cid:54)(cid:88)(cid:81)(cid:16)(cid:36)(cid:80)(cid:81)(cid:72)(cid:68)(cid:79)(cid:16)(cid:44)(cid:51)(cid:53)(cid:21)(cid:19)(cid:20)(cid:25)(cid:16)(cid:19)(cid:20)(cid:20)(cid:19)(cid:23)(cid:16)(cid:3)(cid:40)(cid:91)(cid:17)(cid:3)(cid:20)(cid:19)(cid:20)(cid:19)(cid:15)(cid:3)(cid:83)(cid:17)(cid:3)(cid:20)(cid:3)(cid:82)(cid:73)(cid:3)(cid:23)
`Sun-Amneal-|PR2016-01104- Ex. 1010, p. 1 of 4
`© WILEY-VCH Verlag Gmbl-I, D-69451 Weinheim, 1997
`0570-0833/97/3617-1881 $ 1750+ .50/0
`
`1881
`
`

`
`COMMUNICATIONS
`
`described,[”'”] the 4,5-methanoprolines are relatively unex-
`plored.“‘” Furthermore, structural investigations that study the
`consequences of introducing strain and its effects on the config-
`uration and stability of amide linkages are not available to com-
`pare such systems to their proline counterparts.
`We describe herein highly stereocontrolled syntheses of the
`diastereomeric 4,5-methano-L-prolines and 5,6-methano-L-
`pipecolic acids by a novel intramolecular cyclopropanation re-
`action of iminium ions and the extension of the methodology to
`other congeners.‘ 15* 16’
`Treatment of the readily available lactam 1”“ with lithium
`hexamethyldisilazide (LiHMDS) and Me3SnCH2I gave the oc-
`alkylated products 2 ([01],, = -15.3, c = 0.43 in CHCI3) and 3
`([01],, = -16.0, c =1.23 in CHCI3) in 63% and 23% yields, re-
`spectively (Scheme 1). The syn-isomer 3 could be easily obtained
`
`LiHMDS, THF Measn-—-._
`— 8°C, then
`'
`
`Messn
`
`O
`
`N
`Boc
`
`Me3SnCH2l
`0
`OR -30 c
`
`1
`R = mops
`
`0
`
`+ O
`
`N
`Boc OR
`2, 53°/,
`74%
`I
`1. UHMDS, THF
`2. 2,6-di—ten-butylpl'1enoI_ —78°C
`
`N
`soc OR
`3, 23%
`Q
`
`2
`
`1. LiEt3BH, THF
`2. TFA, cnzciz
`
`'-.,
`
`ea%, 4
`+14"/1,5
`
`RuCI3, Marc).
`CCL,/CH3CN/H20
`———————>
`
`OR
`
`75%
`
`N
`30¢
`
`Bu4NF, THF, 90%
`
`(U2: IIBDPS)
`' ’
`
`
`
`m.p,133-134°C
`[a]D = —2oo.o
`(c = 1.0 in CHCl3)
`
`1. LiEt3BH, THF M983“
`2. MeOH, CSA
`3 ———-—->
`9
`3. au.NF/Aeoi-1
`THF83/e
`
`M90
`
`OC
`B
`7
`
`1. TFA, CH2Cl2
`(79%) moo H
`——————»
`2
`2. ':I:a(I:Cl\>‘7.
`Eioc
`on H20 (7130)
`8
`m.p.128-129°C
`[ale = —9-7
`(c = 1.0 in CHCI,)
`
`Table 1. Selected torsion angles and root—mean-square deviations from fitted atoms
`in a given plane ofX-ray crystal structures, and “C NMR chemical shifts (CDCI3).
`
`
`
`N-Boe-L-proline I20)
`6
`8
`
`
`r(NC«)
`z(C_C,,)
`r(C,C.,)
`r(C,C,)
`e(C,N)
`e(BocNC,CO2I-I)
`ms deviation
`of fitted atoms
`
`— 17
`+ 31
`~—35
`+24
`-4
`~72
`0.018
`C,, N, C4, C,
`
`~ 5.6
`+4.8
`- 2.6
`-0.7
`+4.1
`~64.0
`0.003
`C,,, C.,, C,, N
`
`— l4.4
`+ 15.3
`— 11.4
`+2.9
`+ 7.6
`— 67.1
`0.013
`C,, C,, C), N
`
`(5(ct's/trans)
`6(cis/trans)
`6(cis/trans)
`
`
`COOH
`NC=O
`C,
`C,
`
`178.35/176.60
`153.95/155.39
`58.8
`3075/2953
`
`177,7/175.5
`157.1/154
`60.8/60.1
`32.0
`
`179.1/176.1
`155.7/154.1
`59.5/59.1
`31.5/29.1
`
`from 6 and 8, in which the out-of-plane carbon atom was the one
`bearing the carboxyl group (0.082 A and 0.235 A respectively).
`Intermediates 2 and 3 could also be subjected to further stereo-
`controlled branching leading to the or-C-allyl derivative 9, which
`upon controlled reduction and acid-catalyzed destannyl-
`ation led to the branched 4,5-methano-L-proline precursor 10
`([a],, = + 4.3°, c = 0.72 in CHCI3; Scheme 2). Treatment of 2
`
`\
`
`SHMB3
`
`1. LiHMDS, THF
`2. allyl bromide
`2, 3 ————————->0
`57 7°
`
`\___
`
`1. LiEt3BH, THF
`2. TFA, CHZCIZ
`j_____p.
`62 %
`
`N
`BOO OR
`10
`
`N
`BOC OH
`9, trans/cis >20;1
`
`Scheme 1. TBDPS = t-BuPh,Si, TFA = trifluoroacetic acid, Bee -2 Iert-butoxy-
`carbonyl, CSA = camphor-10-sulfonic acid.
`
`R = TBDPS
`
`1. allyIMgCl, THF, —78°C
`2. TFA, CH2Cl2
`- “" ' —_ ——’
`so °/
`‘’
`
`\ N
`3°C orsops
`11
`
`Scheme 2.
`
`by treatment of the enolate from 2 with the proton source 2,6-di-
`tert-butylphenol.“8' 19’ Generation of the hemiaminal from 2
`and treatment with TFA led to the (4R,5R)—methanopyrrolidine
`derivative 4 ([a],, = ~ 69.3, c = 1.41 in CHCI3), which was
`smoothly deprotected to 5, and the latter oxidized to give the
`crystalline (4R,5R)-rnethano-N-Boc-L—proline in excellent over-
`all yield.
`Similar treatment of the syn-isomer 3 gave the diastereomeric
`with allylmagnesium chloride, followed by trifluoroacetic acid
`crystalline acid 8 via its methylaminal derivative 7. The struc-
`(TFA),
`led to the (S)-5-(2-propenyl)-4,5-methano-L-proline
`tures and conformations of 6 and 8 in the solid state were unam-
`derivative 11 ([01],, = ~ 27.0, c = 0.57 in CHCI3) on migration
`biguously confirmed by single-crystal X-ray analysis. Tablel
`of the double bond. Compounds 10 and 11 represent uniquely
`lists selected torsion angles for compounds 6 and 8, where con-
`functionalized precursors to constrained a)—methan0prolines.
`siderable “llattening” of the pyrrolidine ring is observed relative
`The versatility and generality of the intramolecular carbo-
`to N-Boc-L-proline,”°1 particularly in the case of 6. The flatten-
`cyclization reaction with appended trimethylstannylalkyl
`ing of the pyrrolidine ring in 6 is also manifested in the root-
`groups via incipient iminium ions can be demonstrated in the
`synthesis of bicyclic proline congeners (Scheme 3). These com-
`mean-square value of 0.003 A for the C), and N atoms from the
`plane defined by C ,c,,c,,, and N (0.013 A in 8). The lowest
`pounds are related to the antihypertensive agent ramipril.[2” A
`highly stereoselective allylation of the enolate from 1 gave 12
`deviation of 0.018
`in the case of N-Boc prolinewas found for
`([oc]D =—45.0, c :10 in CHCI3), which was subjected to a
`C, and C, atoms in the plane C,,,N,C,,,Cy; In this 0355 Cawas
`photoinduced trimethylstannylationml to give 13 ([04,, = — 23.6,
`distinctly above the plane (0.521 A). This differs substantially
`(cid:54)(cid:88)(cid:81)(cid:16)(cid:36)(cid:80)(cid:81)(cid:72)(cid:68)(cid:79)(cid:16)(cid:44)(cid:51)(cid:53)(cid:21)(cid:19)(cid:20)(cid:25)(cid:16)(cid:19)(cid:20)(cid:20)(cid:19)(cid:23)(cid:16)(cid:3)(cid:40)(cid:91)(cid:17)(cid:3)(cid:20)(cid:19)(cid:20)(cid:19)(cid:15)(cid:3)(cid:83)(cid:17)(cid:3)(cid:21)(cid:3)(cid:82)(cid:73)(cid:3)(cid:23)
`Sun-Amneal-|PR2016-01104- Ex. 1010,
`p. 2 of 4
`0570-0833/97/3617-1882 s 1750+ .50/0
`© WILEY-VCH Verlag Gmbii, D-69451 Weinheim, 1997
`
`1882
`
`Angew. Chem. Int. Ed. Engl. 1997, 36, No. 17
`
`

`
`Me3SnH
`hv
`T;
`
`M93S"“"-)3
`"
`
`93% O
`
`Boc OR
`13
`
`Boc OR
`12, trans/cis >4o;1
`
`1. LiHMDS,THF *-.
`2. allyl bromide
`"
`1 %-?<——>
`
`87%
`
`O
`
`N
`
`R =TBDPS
`
`1. LiEt3BH, THF
`2. TFA, CH2C|2
`
`3.Bu4NF, THF ( FL‘ CrO3/H2804,
`
`83%
`
`N
`Boc OH
`14
`
`72%
`
`Scheme 3.
`
`
`
`c = 0.92 in CHCI3). Formation of the hemiaminal, followed by
`acid-catalyzed cyclization and deprotection, led to the bicyclic
`prolinol derivative 14 ([01],, = — 97.3, c = 1.38 in CHCI3). Final-
`ly, oxidation under Jones conditions gave the immediate precur-
`sor to the N-Boc-(4R,5R)-ramipril diastereomer 15 (m.p. 61-
`63 °C; [oc]D = - 126.7, c = 0.46 in CHCI3).
`It is also of interest to view compounds 6, 8, 10, and 11 as
`precursors to constrained analogues or precursors to L-pipecolic
`acid. The extension of the cyclopropanation reaction to the
`pipecolic acid series is shown in Scheme 4. Trimethylstannyl—
`methylation of the lithium enolate derived from the readily
`available 17”“ gave the anti—isomer 18. Reduction to the hemi-
`aminal and acid-catalyzed cyclization led to 20 ([11],, = — 56.0,
`c =1.02 in CHCI3), which was deprotected and oxidized to
`the crystalline (5R,6S)-methano-N-Boc-L-pipecolic acid 21
`(m.p. 138-140 °C; [o1]D = — 105.2, c = 1.17 in CHCI3). Epimer-
`
`1. UHMDS, THF, —78°C
`1_ TBDPSCI
`2. Me3SnCl-121, —2o°c
`2. BDCQO
`*—-—> —->
`
`O
`
`fi
`1 6
`
`OH
`
`78%
`
`0
`
`OR
`
`E00
`1 7
`R=TBDPS
`
`77%
`
`S M
`in 93
`"».,
`
`o
`
`N
`305
`
`1.LiEt3BH,THF
`2.p-ToISO3H
`CH2C12IMeOH
`—————->
`90%
`
`‘
`:3:
`
`1.Bu4NF,THF
`2. Rucla, NalO4
`cot,/cH3cN/H20
`N —:——>
`Boc
`65%
`
`OR
`18, trans/cis =14:1
`
`111),, = -1052 (c = 1.15 in CHC13)
`
`
`
`21, m.p. 13a—14o°c
`
`1. LiHMDS. THF, —7a°c
`2. 2,6-di-lertbutylphenol
`—100°C
`--?*-:—>
`92%
`
`18
`
`SnMe3
`
`0
`
`E00
`OR
`19, trans/cis : 19:1
`
`1 LE, 3” THF
`2: M90311, b5A
`3. Bu,NF/AcoH, THF
`——————>
`83%
`
`SW63
`
`M80
`
`gm
`
`OH
`
`22
`Scheme 4.
`
`1. p—To|SO3H, MeOH/
`TL)
`CH2C12<42°/0)
`2. RuCI;,, Na1o,,,cc1./
`CH3CN/H20 (60%)
`
`<1
`N
`Boc
`
`CO H
`2
`
`23, mp, 79-81°C
`[a]D : +12e.7 (c = 0.4 in CHCI3)
`
`COMMUNICATIONS
`
`ization of 18 to the syn—isomer 19 by diastereoselective protona-
`tion, followed by functional group manipulation as described
`above, led to the crystalline diastereomeric (5S,6S)-methano-N-
`Boc-pipecolic acid 23 (m.p. 79-81 °C; [oc]D = -126.7°, c = 0.40
`in CHCI3). The structure of crystalline 21 was unambiguously
`established by X-ray analysis. It is of interest to note that while
`the proline derivatives 6 and 8 adopt a cis-N~Boc proline orien-
`tation in the solid state (Table 1),
`the corresponding 4,5-
`methanopipecolic acid analogue 21 exhibits a trans orientation
`(Scheme 4). The presence of cis and trans isomers of 6 and 8 in
`CDCI3 was evidenced by the corresponding 13C NMR shifts, as
`in the case of N-Boc-L-proline (Table 1).
`Pipecolic acid is an important constituent of the immunosup-
`pressive agents FK-506"“ and rapamycin,”5l in which its on-ke-
`toamide portion is intimately involved in an “active complex”
`with the target enzyme.[26] It is also involved in the metabolism
`of L-lysine, an essential amino acid for mammalian growth and
`development?” Functionalized pipecolic acids are also consid-
`ered strained analogues of lysine with applications in drug de-
`sign and peptidomimetic research,[281 as well as in the inhibition
`of L—pipecolate oxidasem]
`It is our belief that the replacement of L-proline and L-pipecol-
`ic acid by conformationally altered ring variants represented by
`the methano congeners described in this work could have im-
`portant consequences in biological recognition,
`in cis- trans
`conformational changes, in the susceptibility of the secondary
`amide bonds to enzymatic cleavage, and in related processes or
`phenomena. Studies that address these issues will be reported in
`due course.
`
`Received: February 4, 1997 [Z 10076IE]
`German version: Angew. Chem. 1997, 109, 1953-1956
`
`Keywords: amino acids - asymmetric synthesis -
`strained molecules
`
`cyclizations -
`
`[1] Example: T. E. Creighton, Proteins: Structures and Molecular Principles, W H.
`Freeman, New York, 1984.
`[2] a) C. M. Deber, V. Madison, E. R. Blout, Acc. Chem. Res. 1976, 9, 106-113,’
`b) E. S. Eberhardt, S. N. Loh, R. T. Raines, Tetrahedron Lett. 1993, 34, 3055-
`3059; e) W A. Houry H. A. Seheraga, Biochemistry 1996, 35, 11719-11733.
`[3] a) J. F. Brandts, H. R. Halvorson, M. Brennan, Biochemistry 1975, I4, 4953-
`4963; for more recent references see b) K. L. Borden, F. M. Richards, ibid.
`1990, 29, 3071-3077; c) F. L. Texter, D. B. Spencer, R. Rosenstein, C. R.
`Matthews, ibid. 1992, 31, 5687-5695; cl) K. Lang, F. X. Schmid, .1 Mol. Biol.
`1990, 212, 185-196.
`[4] a) K. A. Williams, C. M. Deber, Biochemistry 1991, 30, 8919-8923; b) J. Wess,
`S. Nanavati, Z. Vogel, R. Maggio, R. EMBO J. 1993, 12. 331-338; C) T. M.
`Suchyna, L. X. Xu, F. Gao, C. R. Fourtner, B. J. Nicholson, Nature 1993, 365,
`847-849.
`[5] N. G. J.Richards, M. G. Hinds, D. M. Brennand, M. J. Glennie, J. M. Welsh,
`J. A. Robinson, Biochem. Pharmacol. 1990, 40, 119-123.
`[6] a) A. Yaron, F. Naider, F. Crit. Rev. Biochem. Mol. Biol. 1993, 28, 31-81; b)
`R. E. London, J. M. Stewart, J. R. Carin, Biochem. Pharmacol. 1990, 40, 41-
`48.
`[7] a) C. J. Andres, T. L. Macdonald, T. D. Ocain, D. Longhi. J. Org. Chem. 1993,
`58, 6609; b) M. K. Rosen, S. L. Schreiber, Angew. Chem. 1992, 104, 413-439;
`Angew. Chem. Int. Ed. Engl. 1992, 31, 384-400; c) G. Fischer, ibid. 1994, 106,
`1479-1501 and 1994, 33, 1415-1436.
`[8] D. W. Cushman, H. S. Cheung, E. F. Sabo, M. A. Ondetti, Biochemistry 1977,
`16, 5484-5491.
`[9] M. J. Genin, R. K. Mishra, R. L. Johnson, J. Med. Chem. 1993,36. 3481-3483.
`[10] J. T. Yli—Kauhaluorna,J. T.; J. A. Ashley, C.-H. L. Lo, J. Coakley, P. Wirsehing,
`K. D. Janda, J. Am. Chem. Soc. 1996, 118, 5496-5497.
`[11] A. Giannis, T. Kolter, Angew. Chem. 1993, 105, 1303-1325; Angew. Chem. Int.
`Ed. Engl. 1993, 32, 1244-1267; M. Kahn, Synlett, 1993, 821-826; Peptide
`Secondary Structure Mimetics, Tetrahedron Symposium-in—PrinI, No. 50 (Ed.:
`M. Kahn), 1993, 49, 3433-3689; J. Gante, Angew. Chem. 1994, 106, 1780-
`1802; Angew. Int. Ed. Engl. 1994, 33, 1699- 1720; A. E. P. Adang, P. H. H.
`I-Iermkens, J. T. M. Linders, H. C. J. Ottenheijm, C. J. van Staveren, Recl. Trav.
`Chim. Pays Bas 1994, 113, 63-78; see also, T. Curran. P. M. McEnancy,
`Tetrahedron Left. 1995, 36, 191-194; G. Miiller, M. Gurrath, M. Kurz, H.
`Kessler, Proteins: Strucl., Funct. and Genet. 1993, 15, 235; S. Hanessian, G.
`McNaughton-Smith, H.-G. Lombart, W. D. Lubell, Tetrahedron, in press.
`
`(cid:54)(cid:88)(cid:81)(cid:16)(cid:36)(cid:80)(cid:81)(cid:72)(cid:68)(cid:79)(cid:16)(cid:44)(cid:51)(cid:53)(cid:21)(cid:19)(cid:20)(cid:25)(cid:16)(cid:19)(cid:20)(cid:20)(cid:19)(cid:23)(cid:16)(cid:3)(cid:40)(cid:91)(cid:17)(cid:3)(cid:20)(cid:19)(cid:20)(cid:19)(cid:15)(cid:3)(cid:83)(cid:17)(cid:3)(cid:22)(cid:3)(cid:82)(cid:73)(cid:3)(cid:23)
`Angew. Chem. Int. Ed. Engl. 1997, 36, No. 17 (13gbg:Ilg—g94<.151EW)’1(:iniI1e9rii,q99e' 3o(5)7fti—‘ti833/97/3617-1833 $ 17.50 + .50/0
`
`1883
`
`

`
`COMMUNICATIONS
`
`[12] Synthesis of racernic and enantiopure 2,3-methanoproline: a) A. Hercouet, B.
`Bessiéres, M. Le Corre, Tetrahedron.‘ Asymmetry 1996, 7, 1267-1268; b) F. L.
`Switzer, H. van Halbeek, E. M. Holt, C. H. Stammer, Tetrahedron 1989, 45,
`6091-6100. Recent accounts on 2,3—methanoamino acids: C. H. Stammer,
`Tetrahedron 1990, 46, 2231-2254; K. Burgess, K. K. Ho, D. Moyl—Sherman,
`Synlett 1994, 575-583; K. Burgess, C.-Y. Ke, J. Org. Chem. 1996, 61, 8627-
`8631; J. M. Jimenez, R. M. Ortuno, Tetrahedron: Asymmetry 1996, 7, 3203-
`3208, and references therein.
`[13] Synthesis of cis- and trarz.r-3,4—methanoprolines: a) Y Fujimoto, F. lrrevere,
`J. M. Karle, I. L. Karle, B. Witkop, J. Am. Chem. Soc. 1971, 93, 3471-3477;
`recent discussion of cyclopropylpyrrolidines: D. F. Harvey, D. M. Sigano, J.
`Org. Chem. 1996, 61, 2268-2272; K. E. Brighty, M. J. Castaldi, Synlett 1996,
`1097-1099.
`[14] Synthesis of racemic 4,5-methanoproline amides: a) H. Urbach, R. Henning,
`R. Becker, DE—A 3,324,263 (Cl. C07D209/2) [Chem. Abstr. 1985, 103: P
`54461q]; b) related example involving mixtures of diastereomers: R. Pe1licia-
`ri, L. Arenare, P. De Caprariis, B. Natalin, M. Marinozzi, A. Galli, J. Chem.
`Soc. Perkin Trans. 1 1995, 1251-1257.
`[15] a) S. I-Ianessian, U. Reinhold, S. Ninkovic, Tetrahedron Lett. 1996, 37, 8967-
`8970; b) S. Hanessian. S. Ninkovic, U. Reinhold, ibid. 1996, 37, 8971 -8974.
`[16] Selected examples of carbocyclizations mediated by alkyltin(iv) intermediates:
`T. L. Macdonald, C. M. Delahunty, K. Mead, D. E. O’Dell, Tetrahedron Lett.
`1989, 30, 1573-1576; T. L. Macdonald, S. Mahalingam, D. E. O’Dell, J. Am.
`Chem. Soc. 1981, 103, 6767-6769; cyclopropane formation from y-stannyl
`alcohols: D. D. Davis, H. '1‘. Johnson ibid. 1974, 96, 7576-7577; H. G. Kuivila,
`N. M. Scarpa, ibid. 1970, 92, 6990-6991; N. Isono, M. Mori, J Org. Chem.
`1996, 61, 7867-7872; cyclopropane formation from B-stannyl ketones: T. Sato,
`M. Watanabe, T. Watanabe, Y. Onoda, E. Murayama, ibid. 1988, 53, 1894-
`1899; C. R. Johnson, J. F. Kadow, ibtd. 1987, 52, 1493-1500.
`[17] a) S. Hanessian, G. McNaughton-Smith, Bioorg. Med. Chem. Lett. 1996, 6,
`1657-1662; see also 1)) S. Saijo, M. Wada, J.-I. I-Iimizu, A. lshida, Chem.
`Pharm. Bull. 18, 28, 1449; c) T. Katoh, Y. Nagata, Y. Kobayashi, K. Arai, J.
`Minami, S. Terashima, Tetrahedron Lett. 1993, 34, 5473-5476; d) K.-H. Alt-
`mann, (bid. 1993, 34, 7721-7724. e) I. Ackermann, M. Matthes, C. Tamm,
`Helv. Chim./1cm 1990, 73, 122-132.
`[18] S. G. Davies, N. M. Garrido, O. lchihara, l. A. S. Walters, J. Chem. Soc. Chem.
`Commun. 1993, 1153-1159.
`[19] All new compounds afforded satisfactory spectroscopic and analytical data
`(‘I-I, “C NMR, IR, LRMS, HRMS). Experimental procedures are available
`upon request. Crystallographic data (excluding structure factors) for the struc-
`ture reported in this paper have been deposited with the Cambridge Crystallo-
`graphic Data Centre as supplementary publication no. CCDC—100227. Copies
`of the data can be obtained free of charge on application to The Director,
`CCDC, 12 Union Road, Cambridge CB21EZ, UK (fax: int. code + (1223) 336-
`033; e-mail: deposit@chemcrys.cam.ac.uk).
`[20] E. Benedetti, M. R. Ciajolo, A. Maisto, Acta Crystallogr. Sect. B 1974, 30,
`1783-1788.
`[21] a) V. Teetz, R. Geiger, R. l-Ienning, H. Urbach, Arzneim.—Forsch./Drug Res.
`1984, 10b, 1399; b) R. Henning, U. Lerch, H. Urbach, Synthesis 1989, 265 -268
`and references therein.
`[22] Hydrostannylation of alkenes: M. Lautens, S. Kumanovic, C. Meyer, Angew.
`Chem. 1996, 108, 1428-1429; Angew. Chem. Int. Ed. Engl. 1996, 108, 1428-
`1431, and references therein.
`[23] a) S.-B. Huang, J. S. Nelson D. D. Weller, Synth. Comm. 1989, 19, 3485-3496;
`b) S. A. Hermitage, M. G. Moloney, Tetrahedron." Asymmetry 1994, 5, 1463-
`1464.
`[24] J. J. Sierkierka, S. K. Y. Hung, M. Roe, C. S. Lin, N. H. Sigal, Nazure1989, 341,
`755-757.
`[25] a) M. W Harding A. Galat, D. E. Uehling, S. L. Schreiber, Nature 1989, 341,
`758-760; b) H. Fretz, M. W. Albers, A. Galat, R. F. Standaert, W. S. Lane, S. J.
`Burakoff, B. E. Bierer, S. L. Schreiber, J. Am. Chem. Soc. 1991. 113, 1409-
`1410.
`[26] J. Liu, J. D. Farmer, W. S. Lane, J. Friedman, L. Weissman, S. L. Schreiber,
`Cell 1991, 66, 807-815.
`[27] T. M. Zabriskie, J. Med. Chem. 1996, 39, 3046-3048, and references therein.
`[28] P. J. Murray, I. D. Starkey, Tetrahedron Lett. 1996, 37, 1875-1878; A. Claes-
`son, B.-M. Swahn, K. M. Edvinsson. H. Molin, M. Sandberg, Bioorg. Med.
`Chem. Lett. 1992, 2, 1247-1250.
`
`1,8-Bis(dimethylamino)-4,5—dihydroxy-
`naphthalene, a Neutral, Intramolecularly
`Protonated “Proton Sponge” with
`Zwitterionic Structure“
`
`Heinz A. Staab,* Claus Krieger, Gisela Hieber, and
`Klaus Oberdorf
`
`The interaction of basic groups in close proximity to each
`other may lead, as in the case of 1,8—bis(dimethylamino)-
`naphthalene (1), to unusually high basicities (“proton spon-
`ges”).‘” The influences of gradually changed distances and ori-
`entations of the basic centers as well as of inductive, mesomeric,
`and steric effects on the basicity of such compounds have been
`thoroughly studiedfzl In comparison to the basicity of 1
`[pK, x 12.1 (H20); 7.5 (DMSO)],l3‘ that of 2,7-dimethoxy-1,8-
`bis(dimethylamino)naphthalenc (2) is found to be increased by
`four powers of ten [pK, z 16.1 (H20); 11.5 (DMSO)].l3l To
`separate the mesomeric effect of the two methoxy groups from
`their steric effect on the dimethylamino groups, we were inter-
`ested in 3, an isomer of 2 in which the two methoxy groups are
`not in the 2,7—positions but in the opposite peri—positions. In
`fact, 1,8-bis(dimethylamino)-4,5—dimethoxynaphthalene (3) is
`considerably less basic [pK, z 13.9 (H10); 9.3 (DMSO)] than
`the isomer 2, indicating that the main reason for the high basic-
`ity of 2 is the steric effect of the methoxy groups in 0rtho-posi-
`tions to the dimethylamino groups. Irrespective of this primarily
`intended basicity comparison of 2 and 3, the synthesis of 3
`should allow the easy preparation of the corresponding 4,5-di-
`hydroxy compound 5, which by intramolecular proton displace-
`ment may lead to a new type of neutral, yet zwitterionic “proton
`sponge” (formula 6).
`
`Me2N
`
`NMe,
`
`Me2N
`
`NMe2
`
`Me2N
`
`NMe2
`
`”°°°”° 00
`
`M90
`
`3
`
`OM42
`
`1
`
`2
`
`Me;
`
`NMe2
`
`“
`
`MeO
`
`4
`
`0949
`
`Me2N
`
`NMa2
`
`Me2N\_v_’NMe2
`
`on
`
`H0
`
`S
`
`9
`
`of”‘p.H..
`6
`
`For the synthesis of 3, 1,8-dihydroxynaphthalene[4] was
`methylated to give 1,8-dimethoxynaphthalene, which was ni-
`trated (conc. nitric acid, glacial acetic acid/dichloromethane,
`9:5)
`to yield 1,8-dimethoxy—4,5-dinitronaphthalene (39%;
`m.p. 278 °C);
`the isomeric 2,5-dinitro product
`(m.p. 151 —
`153 °C) was separated by chromatography on silica gel with
`dichloromethane as eluent. Catalytic hydrogenation (10% Pd/
`C, tetrahydrofuran (THF), 20°C) resulted in the formation of
`1,8—diamino-4,5-dimethoxynaphthalene (97%; mp. 83-95 °C,
`decomp), which was N-methylated according to the method of
`Quast et al.[5] to give 3 (71 %; m.p. 75 °C, from n—hexane/ethyl
`
`[*] Prof. Dr. H. A. Staab, C. Krieger, Dr. G. Hieber, Dr. K. Oberdorf
`Abteilung Organische Chemie
`Max-Planck-Institut fiir medizinische Porschung
`Iahnstrasse 29, D-69120 Heidelberg (Germany)
`Fax: Int.code +(6221)486219
`e—mail: staab(a;;mixi.mpimf—heidelberg.mpg.de
`[**] New “Proton Sponges“, Part 12. Part 11: H. A. Staab, M. Diehm, C. Krieger,
`Tetrahedron Lett. 1996, 36, 2967-2970.
`
`1884
`
`(cid:54)(cid:88)(cid:81)(cid:16)(cid:36)(cid:80)(cid:81)(cid:72)(cid:68)(cid:79)(cid:16)(cid:44)(cid:51)(cid:53)(cid:21)(cid:19)(cid:20)(cid:25)(cid:16)(cid:19)(cid:20)(cid:20)(cid:19)(cid:23)(cid:16)(cid:3)(cid:40)(cid:91)(cid:17)(cid:3)(cid:20)(cid:19)(cid:20)(cid:19)(cid:15)(cid:3)(cid:83)(cid:17)(cid:3)(cid:23)(cid:3)(cid:82)(cid:73)(cid:3)(cid:23)
`© WILEY—VCl-1 Verlag GmbH,Sl5!6r94SA1r5‘Vt-!i‘r§igr|1:,||1D9 7209§79901;l3%7'Bcl'l'1SlsgP$119.’5P¥.5 /9f 4 Angew Chem» W 54- E"8*’- 1997» 36» N0 17