design and synthesis of nonpeptide mimetics of jaspamide

hi. J. Pepride Proiein Rex 38, 1991, 324-334

Design and synthesis of nonpeptide mimetics of jaspamide

MICHAEL KAHN', HIROSHI NAKANISHI'32, TING SU1s3, JESSIE Y.-H. LEEzz4 and MICHAEL E. JOHNSON2

'Department of Chemistry, and 'Department of Medicinal Chemistry & Pharmucognosy, University of Illinois at Chicago, Chicago, Illinois, USA

Received 25 June 1990, accepted for publication 10 May 1991

Jaspamide is a novel metabolite of mixed peptide/polyketide biosynthesis that was isolated from sponges of the genus Juspis, and that has been reported to exhibit both insecticidal and antifungal activity. We have evaluated three nonpeptide mimetic designs, and have synthesized a nonpeptide mimic of the proposed bioactive region to investigate the structure activity relationship. Structural investigations of potential mimetics, utilizing molecular modeling in conjunction with spectroscopic and crystallographic data, indicate that positioning of the critical functional groups in two mimetics corresponds closely to that observed in jaspamide, and that the flexibility of mimetic 4 approximates that of jaspamide. Initial biological evaluation suggests that lactam mimetic 4 exhibits a biological profile similar to jaspamide.

Key words: 8-turn; jaspamide; molecular modeling; NMR; peptide mimetics; synthesis

Jaspamide, 1, shown in Fig. 1, is a novel metabolite of mixed peptide/polyketide biosynthesis which was isolated from sponges of the genus Jaspis. Clardy and colleagues ( I ) recently reported the X-ray structure of jaspamide. Simultaneously, Crews et al. (2) published their structure of jaspamide based upon NMR. An elegant total synthesis of jaspamide has recently been completed by Grieco and coworkers (3). Zabriskie et al. (1) reported that this unusual depsipeptide contains two uncommon amino acids, B-tyrosine, previously observed in the edeine peptides (4), and 2-bromo- abrine; both are in the unnatural D configuration. Jaspamide has been reported to exhibit potent insecticidal activity against Heliothis virescens (tobacco budworms) with an LC, of 4 ppm (1); by comparison, the extremely potent insecticide, azadirachtin, was reported to exhibit an LCso of lppm in this assay (5 ) . Additionally, jaspamide exhibited a minimum inhibitory concentration and a minimum lethal concentration of 25 pg/mL each against Candida albicans, and an in vitro ED, < 1 pg/mL against the nematode Nippostrongylus braziliensis (2). The in vivo topical

Current addresses: 'Department of Chemistry, Northwestern Uni- versity, Evanston, IL 60201; 'Abbott Laboratories, Abbott Park, IL 60064. USA.

activity of a 2% solution of jaspamide against a Candida vaginal infection in mice was comparable to that of miconazole nitrate (2). However, it was completely inactive against a variety of Gram positive and Gram negative bacteria. In an effort to determine the key structural elements that contribute to the biological activity of jaspamide, we have designed and synthesized nonpeptide mimetics of what we hypothe- size to be the essential core of this molecule.

Inspection of the X-ray structure of jaspamide indicates that the two probable key pharmacophoric units, Le., the bromoindole and the phenol, are contained approximately within a type I1 B-turn. Based on this analysis, on the previously demonstrated role 06 turns in ligand receptor interactions (6, 7), and our interest in further exploring the role of turns in molecular recognition (8-lo), we designed three nine-membered ring mimetics, the bicyclic indolizidine, 2, and- the lactams, 3 and 4, of the presumed bioactive fragment of jaspamide, as shown in Fig. 2. These three mimetic frameworks can be related both conceptually and syn- thetically via transannular cyclization.

Jaspamide is a relatively large cyclic structure, and should exhibit substantial conformational flexibility. Thus, in evaluating mimetic design, and before embark- ing on the full synthesis of mimetics 2, 3, and 4, we have compared both the correspondence of the low

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energy ring conformers utilized the Batchmin program, retaining only beta carbons in the side-chains. Each conformation was minimized using the MacroModel- MM2 force field by the block diagonal Newton- Raphson method, and compared with the energy- minimized crystal structure of jaspamide at the two alpha and two beta carbon positions.

Full modeis of the three jaspamide mimetics were then built from the lowest energy ring conformations by attaching an indole side chain at the C: position, and a phenol moiety at the C, position. The bromine atom in jaspamide was replaced by a hydrogen atom to simplify the calculation. Molecular dynamics (MD) simulations were performed at constant temperatures of 300K and 400K for 20ps for each structure to obtain more realistic comparisons of the average side- chain orientations between jaspamide and its mimetics and to compare their relative flexibilities. The solvation effect was roughly taken into account by employ- ing a distance-dependent dielectric constant with a cutoff distance of 20 A, and by computing a solvation energy from the approximate water accessible surface area. During the simulation, the C;C:C, C, dihedral angle was monitored every femtosecond and tabulated as a histogram. Due to the inability of the Batchmin program to monitor atomatic distances greater than 6A, the distance between C; and C, was calculated from 2000 structures sampled every 0.01 ps during each of the simulations. Atomic coordinates were averaged during the simulations, and superimposed to estimate the flexibility. The four-atom C;C:C,C, RMS deviations were also calculated between the archived structures and the corresponding positions of the energy-minimized jaspamide crystal structure and the averaged positions of jaspamide over the 20ps simulation.

F G

0 H.. 1

JRE 1 Skeletal structure of jaspamide, as reported by Clardy and colleagues (1).

energy conformers of the mimetics with the X-ray conformation of jaspamide, and the comparative flexibilities of jaspamide and the mimetics with respect to positioning of the two key pharmacophores. In this comparison, we have investigated the conformational similarities between the three mimetic frameworks and the fl-turn containing region of jaspamide through molecular modeling and molecular dynamics simulations, and through spectroscopic studies.

Methods Models of the nine-membered ring mimetics studied were built using the MacroModel molecular modeling program (1 1). Conformational searches for low

OH

&.*, Br

2

OH

4 3

FIGURE 2 Skeletal structures of three nine-membered ring mimetin of the presumed bioactive fragment of jaspamide.

Results and discussion

Jaspamide structure and flexibility. Much of the literature to date on peptide mimetics has concentrated on static comparisons between the low energy conformation(s) of the mimetic with the crystal structure or low energy conformation of the native peptide. In receptor interactions, however, the flexibility of the mimetic, in its ability to adopt a range of conformations similar to that of the native molecule, is likely to be important in overcoming conformational barriers to binding. In developing jaspamide mimetics, we+ have thus concentrated on the design of a mimetic that exhibits both a low energy conformation similar to that of jaspamide, and a range of flexibility in the positioning of substituents that is also similar to that of jaspamide.

In evaluating the flexibility of jaspamide, and in comparing mimetic structures with that of jaspamide, we have concentrated on three parameters that par- ticularly reflect the positioning of the key substituents:

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6.0 s.5 6.0 6.5 1.0

Cp-Cp Distance (&

n s v

40

4 20

0 0 0.25 0.5 0.75

rms of Cpa*ap Positions (& FIGURE 3 (a) Histogram of the C,C:C,C, dihedral angle during a 20ps molecular dynamics run at 300K. (b) Histogram of the Ch-C, distance during a 20 ps molecular dynamics run at 300 K. (c) Histogram of the rms difference between the energy minimized jaspamide crystal structure and that of the three mimetics at the C;C:C,C, positions at 300 K. (-) jaspamide; (-- - - -) indolizidine; (- - -) double bond lactam; (. . . .) saturated lactam.

the distance between the two beta carbons, Ci-C,, the torsional angle between the C,C, and ClC; bonds, and the four-atom root-mean-square distance between the mimetic and jaspamide atoms, C;, C:, C, and C, . The crystal structure of jaspamide appears to be somewhat strained, presumably from crystal pack- ing forces: the phenyl ring is significantly non-planar, the H-N-C=O amide group between C, and C: is twisted about 12" from the trans configuration, the carbonyl oxygen and amino hydrogen between the C, and Ci are placed unnaturally on the horizontal plane formed by the ring, and the next amide group is vertical (perpendicular to the plane of the ring). Energy minimization of the crystal structure does not significantly change the relative orientations of the two indole and phenol substituents, but does increase the Ci-C, distance from 5.65 to 6.25 8 due to relaxation of the amide group to the trans orientation and its rotation by about 40". The rms deviations between the full X-ray crystal structure and the Macromodel- MM2 minimized structure is about 0.66& a reasonable value for relaxation from a constrained environ- ment.

The flexibility of jaspamide itself was characterized by following the substituent positioning parameters, described above, during the time course of the jaspamide molecular dynamics simulations. At 300 K, the C;CLC,C, torsional angle for jaspamide exhibits a relatively broad distribution (Fig. 3a), peaked at about 130". At 300 K, it can be seen from Fig. 3b that the Ch-C, distance for jaspamide exhibits an approximately gaussian distribution about a mean of N 6.2 A during the simulation. The C;C:C,C, rms deviation between the energy-minimized crystal structure and the molecular dynamics simulation exhibits a relatively narrow distribution (Fig. 3c), with a peak at about 0. I A and a full width at half maximum of about 0.1 8, indicating that the structure probably just exhibits local oscillations about the minimum energy conformation during the simulation. At 400 K, however, the jaspamide torsional angle distribution is strongly bimodal (Fig. 4a), with major peaks at about 80" and I S " , minor peaks at about 55", 125", and a shoulder at about 185". Fig. 4b indicates that the jaspamide Cb-C, separation also exhibits a much broader distribution at 400K, ranging from less than 58 up to about 6 .68 , and with at least four peaks, probably corresponding to local oscillations within four different low energy conformers. The C;C:C,C, nns deviation exhibits a relatively broad distribution, with peaks at about 0.12, 0.25, 0.35, 0.45 and 0.578, also indicating that the simulation is probably sampling multiple low energy conformers.

Indolizidine mimetic - modeling, Jlexibility and structure. A conformational search of the indolizidine structure, 2, indicates that there are four low energy

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conformers with conformational energies within 3 kcal/mol of the lowest energy conformer. Table 1 lists the conformational energies, the CbC;C,C, torsional angles, the Cb-C, distances, and the four-atom CbCiC,C, rms differences between the low energy indolizidine conformers and the energy-minimized jaspamide crystal structure. From the values listed in Table 1, it can be seen that the torsional angle between the C,C, and CkCb bonds is relatively close to that observed in the energy-minimized jaspamide crystal structure only for the lowest energy conformer. It also appears that the indolizidine Ci-C, distances are relatively close to the Cb-C, distance for the unmodified jaspamide crystal structure, but are about 0.6 A smaller than the energy-minimized crystal structure. The rms deviations between the energy-minimized jaspamide crystal structure and the indolizidine conformers are relatively small for all conformers. A comparison of the low energy indolizidine conformer with the energy-minimized jaspamide j-turn region is shown in Fig. 5.

From the 300 K molecular dynamics simulation, the indolizidine torsional angle is peaked at about 95", and exhibits shoulders at about 115" and 135" that probably correspond to local oscillations within low energy conformers; the distribution overlaps with that of jaspamide, the the peak is about 35" less (Fig. 3a). The Cb-C, separation for indolizidine exhibits a slightly broader distribution than that for jaspamide, with a mean of about 5.6 A, but overlaps very little with the jaspamide distribution. The rms deviation between indolizidine and the energy-minimized jaspamide crystal conformation exhibits a range of about 0.25- 0.5 A, with a maximum at about 0.37 A and a strong shoulder at about 0.43A at 300K. At 400K, the indolizidine CiCLC,C, torsional angle exhibits a distribution that is qualitatively similar to that at 300 K, while the Ci-C, separation exhibits a bimodal distribution, with most of the conformers exhibiting separations within the 5.3 to 6 A range. The distribution of rms deviations between indolizidine and the energy- minimized jaspamide crystal conformation at 400 K is slightly broader than that at 300K, but is otherufise similar.

To experimentally evaluate the indolizidine conformation, the model system, 5, shown in Fig. 6 , was synthesized following procedures outlined in (1 0). The 3JHrH vicinal proton coupling constants for 5 were derived from the 'H-NMR 1D spectrum in chloroform solution using the spectral simulation program in the Bruker software. Kopple et al. ( 1 2 ) have reported a modified Karplus equation suitable for the fragment, C-CH,-CH2-C, as

J = 11.0cos28 - 1 . 4 ~ 0 ~ 8 + 1.6sin28 The coupling constants calculated from the dihedral angles of the four low energy indolizidine conformers using this relationship are listed in Table 2. Individu-

321

a 0 s 20 t g 10

0 5.0 6.5 6.0 6.5 7.0

Cp-Ce Distance (&

0 Oi5 0.5 0.75

rms of ~ g ' ~ l ~ p Positions (& FIGURE 4 (a) Histogram of the C;C;C,C, dihedral angle during a 20ps molecular dynamics run at 400K. (b) Histogram of the C;-C, distance during a 20 ps molecular dynamics run at 400 K. (c) Histogram of the rms difference between the energy minimized jaspamide crystal structure and that of the three mimetics at the C;C;C,C, positions at 400 K. (-) jaspamide; (-- - --) indolizidine; (- - -) double bond lactam; (. . . .) saturated lactam.

M. Kahn et al.

TABLE 1 Conformations ofjaspamide mimetic with an indolizidine within 3 kcallmol from the lowest conformer as calculated with MMZ force fields; relative populations of each conformer, based on the Boltzmann distribution, are also tabulated. For comparison, the C, C, C, torsional

angle is 13P8 and the q - C , distance is 6.2.5R in the energy minimized jaspamide crystal structure

c,c, c: c; RMS (A) Conformer Energy e@ Population c; c; c, c, C F ,

(kcal/mol) torsional distance angle (deg) ('4

1 0.00 1 .OO 0.34 130.3 5.66 0.27 2 0.50 0.82 0.27 91.9 5.82 0.27 3 0.76 0.74 0.25 86.1 5.74 0.31 4 2.13 0.43 0.14 158.3 5.82 0.27

ally, the observed and calculated coupling constants do not compare very well for any of the conformers. However, a Boltzmann distribution-weighted average of the coupling constants from the four conformers shows good agreement with the experimentally observed coupling constants, suggesting that there is effective averaging of all conformers at room temperature. Averaging of the four conformers would also be consistent with the CiCLC,C, torsional angle distribution observed in Fig. 4a.

Double bond lactam mimetic - modeling, structure and flexibility. A conformational search of the lactam structure, 3, indicates that there are three low energy conformers with trans amide bonds, and with conformational energies within 4 kcal/mol of the lowest energy conformer. (There are two additional low energy conformers with cis conformations for the central amide bond; these have not been considered since jaspamide has a trans amide bond.) Table 3 lists

.

the conformational energies, the CiC: C,C, torsional angles, the Ci-C, distances and the four-atom CiC; C, C, rms differences between the low energy indolizidine conformers and the energy-minimized jaspamide crystal structure. From the values listed, it can be seen that the torsional angle between the C,C, and CiC; bonds is about 40" less than that observed in the energy-minimized jaspamide crystal structure for the lowest energy conformer, and 20" higher and 60" less for the next two higher energy conformers, respectively. It appears that the double bond lactam Ci-C, distances for all of the low energy conformers are relatively close to that for the energy-minimized crystal structure. The rms deviation between the energy- minimized jaspamide crystal structure is relatively small for all of the low energy lactam conformers.

From the 300 K simulation, the C;CLC,C, torsional angle distribution exhibits a major peak at about IOO", and two minor peaks at about 145" and 165" (Fig. 3a). The Ch-C, separation peaks about 6.1 A, and overlaps

FIGURE 5 Stereo diagram with a superposition of the lowest energy mimetic conformers against MM2-Macromodel energy- minimized jaspamide cr;stal structure, for which only the turn region is shown. (-) Energy- minimized jaspamide crystal structure; (-.-.) indolizidine; (---) double bond lactam; (....) saturated lactam. Super- position based on minimum rms difference of C;, C;, C, and C, atom positions.

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TABLE 2 Coupling constants for each of the four conformers listed in Table 1, as calculated from the Karplus relalion (1). the popularion-weighted average coupling constants, and the observed coupling constants. The numbering system is shown in Fig. 6 . Relative populations for each

conformer are listed in Table 1

Protons Calculated J(Hz) for each minimum energy conformer Population Observed averaged J(Hz) J W )

1 2 3 4 ~

HI A-H, 12.1 2.1 12.2 4.9 8.4 9.5

HZA-H] 10.0 1.6 9.6 I .6 6.5 7.1 HIB-HI 7.3 7.0 7.6 7. I 7.3 7.9 H7A-HS 10.6 12.4 2.2 12.4 9.2 10.0 H7B-H, 4.2 3.2 8.9 3.5 5.0 4.3

H6B-H, 5.9 5.8 2.2 6.3 5.0 3.4

HI B-H, 4.8 8.6 4.4 9.6 6.4 5.6

H+A-H, 1.8 10.8 12.2 1.7 6.8 7. I

substantially with that for jaspamide (Fig 3b). The four-atom rms deviation ranges from about 0.05 to 0.45A, with a peak at about 0.25A (Fig. 3c). At 400 K, the torsional angle exhibits a bimodal distribution very similar to that of jaspamide, with peaks at about 90 and 145" (Fig. 4a), while the Ci-C, separation exhibits a distribution qualitatively similar to that at 300 K (Fig. 4b). At 400 K, the rms deviation is qualitatively similar to that at 300 K, but with a slightly broader distribution (Fig. 4c).

Previous ' H NMR and X-ray crystallographic anal- yses have shown that the double bond lactam mimetic with slightly different substituents (a phenyl, rather than the phenol, and with the indole side chain trun- cated at the C, position with a methyl) exists in a single conformation equivalent to the low energy conformation (1 3). The low energy conformer of 3 is shown superpositioned with the /3-turn fragment of jaspamide in Fig. 7, from which it can be seen that there is good correspondence in the positioning of the substituents with respect to the ring backbones. The X- ray structure of the double bond lactam mimetic, reported in previous work (13), is also shown superpositioned with the &turn fragment of jaspamide in Fig. 7, from which it can be seen that there is also reasonably good correspondence in the positioning of

FIGURE 6 Indolizidine model 5 and numbering system for coupling system for coupling constants.

the substituents with respect to the ring backbones for the experimental structure. The molecular dynamics simulations suggest that 3 exists primarily in a single conformation at 300 K, in agreement with the experimental studies (13), but that the system will exhibit increased conformational flexibility at higher temperatures.

Saturated lactam mimetic - modelling, structure and flexibility. The conformational search of the saturated ring structure, 4, indicates that there are 1 1 low energy conformers with trans amide bonds, and with conformational energies within 3 kcal/mol of the lowest energy conformer. Table 4 lists the conformational energies, the C; C:C,C, torsional angles, the Cb-C, distances and the four-atom ChCL C,C, r m s differences between the low energy conformers and the energy-minimized jaspamide crystal structure. From the values listed in Table 4, it can be seen that the torsional angle between the C,C, and C:Ci bonds is within 20" of that observed in the energy-minimized jaspamide crystal structure only for conformers 3,4,6, and 8. It also appears that the C;-C, separations for all conformers except 11 are relatively close to that for the energy-minimized crystal structure. Tht rms deviations between the energy-minimized jaspamide

TABLE 3 Conformations ofthe double bond lactam mimetic ofjaspamide within 4 kcallmol from the lowest conformer as calculated with MM2 force

fields

Conformer Energy C;C;C,C, Cb-C, C,C,C;C; (kcal/mol) torsional distance RMS (A)

angle (deg) (A)

1 0.00 96.9 6.10 0.18 2 2.88 156.9 6.24 0.1 I 3 3.61 77.0 6.00 0.27

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M. Kahn et al.

crystal structure and the mimetic conformers are relatively small for all conformers.

From the 300 K simulation, the CiCkC,C, torsion angle distribution exhibits a major peak at about 11 5" and a minor peak at about 155' (Fig. 3a). The Ci-C, separation peaks about 6.1 A, and overlaps quite closely with that for jaspamide (Fig. 3b). The 4-atom rms deviation ranges from about 0.05 to 0.25 A, with a peak at about 0.1 5 A (Fig. 3c). At 400 K, the torsional angle exhibits a bimodal distribution with a somewhat narrower range than that of jaspamide, with peaks at about 1 15 and 150" (Fig. 4a), while the Cb-C, separation exhibits a distribution qualitatively similar to that at 300K (Fig. 4b). The rms deviation is slightly broader than that at 300 K, but it is otherwise similar.

TABLE 4 Conformations ofjaspamide mimetic with a saturated nine-membered ring within 3 kcallmol from the lowest conformer as calculated with

M M 2 forcefields

Conformer Energy C;C:C,C, C&$ C&c,C, (kcal/mol) torsional distance RMS (A)

angle (deg) (A)

1 0.00 78.8 6.00 0.26 2 0.06 99.6 6.12 0.16 3 0.62 128.3 6.17 0.05 4 I .33 114.0 6.12 0.1 1 5 1.51 154.9 6.25 0.11 6 1.58 125.7 6.16 0.06 7 I .58 71.9 6.00 0.29 8 I .93 145.8 6.21 0.07 9 2.13 168.4 6.24 0.17

10 2.42 113.2 6.13 0.11 I I 2.48 51 .7 5.88 0.37

FlGURE 7 Stereo diagram with a superposition X-ray crystal structure of the nine-member double bond lactam from (12), against the energy-minimized jaspamide crystal structure. (-) Fragment of energy-minimized jaspamide crystal structure; (. . . .) crystal structure of double bond lactam. Superposition based on minimum rms difference of Ci, C:, C, and

\ C, atom positions

Previous IHNMR work has shown that 4 (with somewhat different substituents) exists as a mixture of interconverting conformational isomers at room temperature (13). This is consistent with 4 having three conformers with relative energies within about 0.6 kcal of the global minimum (Table 4), and with the molecular dynamics simulations which indicate significant conformational flexibility for 4 at both 300 and 400K.

Mimetic analysis. From the data in Tables 1 4 and Figs. 3-4, it appears that the saturated ring mimetic, 4, most closely mimics both the low energy crystal conformation of jaspamide and the flexibility of jaspamide in the positioning of the probable key pharmacophoric side chains. The double bond lactam, 3, is probably the next best mimic, but appears to exhibit somewhat less conformational flexibility than either mimetic 4 or jaspamide itself. The indolizidine mimetic, 2, appears to exhibit the greatest conformational rigidity, and the Ch-C, separation is significantly less than that of jaspamide. Our expectation thus would be that the probability of matching the biological activity of jaspamide would be in the order, 4 > 3 P 2 .

Synthesis. Based on this analysis, we embarked on the synthesis, shown schematically in Fig. 8, that was initially planned to generate the ring structures for all three mimetics, 2, 3, and 4. Boron triflouride assisted opening of cyclooctadiene monoepoxide with 4-lithio- anisole proceeded smoothly at - 78" (14). Subsequent Jones oxidation of the alcohol provides ketone 6. Carbomethoxylation of the kinetic enolate of 6 provides the requisite keto-ester for introduction of the indole moiety via a Mannich type condensation with

~

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Jaspamide mimetics

levels injected. A compound was considered effective if all meaningful movement of the larvae had stopped, although it might still be having uncoordinated spasms, for example. Compound 10 was found to have significant activity against both Sarcophaga Fulcuta and Heliothus virescens in an injection assay, with an LD,, N 50 pg for Heliothus virescens, and an LD,, < 5pg for Sarcophaga Fulcuta. Compounds 8 and 9 were essentially inactive. A contact assay, in which the compounds were dissolved in acetone, and spread on the backs of the larvae was also performed with Heliothus virescens. No activity for any of the compounds was found in this assay. In comparison, jaspamide has been reported to exhibit potent insecticidal activity against Heliothus virescens, with an LC,, of 4 ppm (1). The demonstration of insecticidal activity against two different species is encouraging, and suggests that the essential core of jaspamide has been correctly identified, although the lower activity suggests that additional structural elements may be essential for full activity.

n I I t I I c

-0Mc

7 8 I H

FIGURE 8 Synthesis scheme for preparation of mimetics.

gramine (1 5). However, Krapcho decarboxylation (16) provides an overall 12% yield of the undesired cis ketone 7 from cyclooctadiene epoxide. The cis relationship between the two side chain substituents was established via a difference NOE experiment. The thermodynamic decarboxylation conditions provide almost exclusively the cis ketone 7 . Unfortunately, at this juncture, all attempts to effect oxamination of ketone 7 were unsuccessful, either leading to the recovery of, or under more forcing conditions, the destruction of, the starting ketone. Believing that the olefin may be playing a role in the reluctance of ketone 7 to form an oxime, we removed the double bond by catalytic hydrogenation to provide ketone 8. However, at this stage we have erased the possibility of a facile transannular cyclization, and we thus decided to proceed on to a mimic of type 4, following the general approach outlined in Olson et al. (13). Gratifyingly, utilizing the protocol of Confalone (1 7) we were able not only to effect oxamination, but also fortuitously, we discovered we had epimerized the cis ketone and thus had generated the desired trans lactam 9 upon Beckman rearrangement. Subsequent bromination with NBS and silica gel (18) afforded the desired 10 in 34% yield from 8. Unfortunately all attempts to remove the methyl ether in the presence of the sensitive bromoindole were unsuccessful.

Biological evaluation. Compounds 8, 9 and 10 were assayed for insecticidal activity against both Sarco- phaga Falcuta (blowfly larvae) and Heliothus virescens (tobacco budworms) by Dr. Robert A. Houtchens (Dow Chemical Company). All compounds were dissolved in DMSO for use in injection assays. No toxic- ity of DMSO to either larvae was demonstrated at the

Summary In conclusion, we have designed a mimic, 4, of the b-turn region of jaspamide. Structural investigations by molecular modeling, NMR and crystallography from this study and previous work (1 3) suggest that the nine-membered ring lactam 4 is a reasonable model for the fl-turn region of jaspamide.

Preliminary biological investigations also suggest that lactam 10 (the 4-0-methyl ether of 4) exhibits a biological profile qualitatively similar to jaspamide.

EXPERIMENTAL PROCEDURES

General procedures NMR spectra were recorded on IBM WP2OOSY, Nicolet NT-360, Bruker AM-400 or GE GN-500 spectrometers. IR spectra were recorded on a Perkin- Elmer Model 137 infrared spectrophotometer. Low resolution mass spectra were recorded on an HP5985a system. All reactions were monitored by thin-layer chromatography carried out on 0.25mm E. Merck silica gel plates (60F-254) with UV light and 7% ethanolic phosphomolybdic acid and heat as developing agent. E. Merck silica gel 60 (particle size 0.040- 0.063mm) was used for flash chromatography. All reactions were carried out under an argon or nitrogen atmosphere with dry, freshly distilled solvents under anhydrous conditions unless otherwise noted. yields refer to chromatographically and spectroscopically (’ H NMR) homogeneous materials, unless otherwise noted.

5,6-Epoxycyclooctene To a solution of 1,5-cyclooctadiene (10.8 g, 100mmol) in 200mL methylene chloride, was added slowly 3-chloroperbenzoic acid (20 g, 120 mmol) at room

33 1

M. Kahn et al.

temperature. The mixture was stirred for 2h and quenched with saturated sodium bicarbonate (1 50 mL). The organic layer was dried over anhydrous mag- nesium sulfate and evaporated to afford 14g (100%) of 5,6-epoxycyclooctene as a clear liquid. 'H NMR (400MHz, CDCI,): 65.6 (m, 2H), 3.05 (m, 2H), 2.4 (m, 4 H), 2.1 (m, 4 H), ',CNMR (100 MHz, CDCI,): 6 128.83, 128.67, 56.71, 28.10, 23.67.

2-p-Anisoyl-5-cyclooctenol A stirred solution of 4-bromoanisole (7.59 mL, 60 mmol) in 400 mL of dry ether, was cooled to - 78". To this was added dropwise tert.-butyllithium (77.6mL of 1 . 7 ~ solution in pentane, 132mmol). After stirring for 60min at - 78", boron trifluoride etherate (8.86mL, 72 mmol) was added dropwise. After stirring for 10 min at - 78", 5,6-epoxycyclooctene (8.93g, 72mmol) was added dropwise. The reaction mixture was stirred an additional 15 min. at - 78", quenched with saturated sodium bicarbonate (200mL) and warmed to r.t. The aqueous layer was extracted with ether (3 x 150 mL) and the combined ether extracts were dried over sodium sulfate and evaporated in vacuo to yield crude product. Flash chromatography on 400g silica gel with 25% ethyl acetate in hexane as eluant, gave 6.27g (45%) of 2-p-anisoyl-5-cyclooctenol as a clear liquid. ' H NMR (400MH2, CDCI,): 6 7.2 (d, J = 8.81 Hz, 2 H), 6.9 (d, J = 8.8 Hz, 2 H), 5.6-5.8 (m, 2 H), 4.0 (m, 1 H), 3.8 (s, 3 H), 3.0 (m, J = 4.62, 7.18, 9.49, 3.85 Hz, 1 H), 2.1-2.7 (m, 4 H), 1.7-1.9 (m, 4 H). I3C NMR (100 MHz, CDCI,): 6 158.00, 131.46, 129.40, 126.85, 114.27, 74.32, 55.27, 48.53, 33.75, 33.62, 24.76, 24.54. MS (80eV) m/e (relative intensity) 232 (M', 18), 147 (13), 134 (IOO), 121 (41), 91 (31), 77 (23), 65 (15).

2-p-Anisoyl-5-cyclooctenone (6) A solution of 2-p-anisoyl-5-cyclooctenol (6.96 g, 30 mmol) in 100 mL ether was cooled to 0" and Jones reagent (26.728 of CrO, and 23mL of concentrated sulfuric acid diluted with water to a volume of 100 mL) was added dropwise until the formation of a stable red color was observed. The mixture was stirred for 30min, and aqueous sodium bisulfite added to quench the excess reagent. Water was added to the green mixture to completely dissolve the salts, and the mixture was extracted several times with ether. The ether extracts were dried over MgSO, and evaporated. Flash chromatography on 200g silica gel with 25% ethyl acetate in hexane as eluant afforded 5.52g (80%) of ketone 6 as a clear liquid. ' H NMR (400 MHz, CDC1,): 6 7.3 (d, 2 H), 6.85 (d, 2 H), 5.7-5.9 (m, 2 H), 3.92 (9, 1 H), 3.78 (s, 3 H), 2.65-2.80 (m, 2 H), 2.25- 2.4 (m, 2 H), 2.2 (m, 1 H), 1.9 (m, 1 H), 1.6 (m, 2 H). ',CNMR (lOOMHz, CDCI,): 6 188.00, 158.55, 132.17, 130.60, 129.68, 128.93, 113.71, 65.74, 55.11, 46.44, 31.76, 25.79, 22.24. IR (CHCI,): 1710cm-'.

332

MS (80eV) m/e (relative intensity) 230 (M', 27), 174 (17), 148 (14), 134 (loo), 121 (16), 119 (15), 91 (22).

2-p- Anisoyl-8-carbethoxy-5-cyclooctenone A stirred solution of lithium bis(trimethy1silyl)amide (40mL of 1 . 0 ~ solution in hexanes, Nmmol), in 200 mL of dry tetrahydrofuran, was cooled to - 78". To this was added dropwise ketone 6 (4.6 g, 20 mmol). After stirring for 30 min at - 78", methyl chlorofor- mate (3.1 mL, 40mmol) was added dropwise. The reaction mixture was warmed to r.t. and quenched with saturated ammonium chloride (100mL). The aqueous layer was extracted with ether (3 x 300mL), and the combined ether extracts were dried over anhydrous Na,SO, and evaporated in vacuo. Flash chromatography on 250g of silica gel with 25% ethyl acetate in hexane as eluant gave 4.15 g (72%) of 2-p- anisoyl-8-carbethoxy-5-cyclooctenone as a clear liquid. 'HNMR (400MH2, CDCI,): 6 7.25 (d, J = 8.47l+, 2 H), 6.80 (d, J = 8.47 Hz, 2 H), 5.80 (m, 2 H), 4.08 (dd, J = 2.82, 9.24Hz, 1 H), 3.80 (s, 3 H), 3.60 (s, 3 H), 3.44 (dd, J = 4.62,6.92, 1 H), 3.10 (9, J = 10.52, 13.08 Hz, 1 H), 2.40 (m, 2 H), 2.20 (m, 1 H), 1.93 (9, J = 12.31,12.05 Hz, 1 H), 1.70 (m, 1 H). 13CNMR (lOOMHz, CDCI,): 6 169.50, 158.53, 132.84, 132.25, 128.97, 127.61, 113.63, 62.11, 55.22, 53.20, 52.11, 34.10, 26.38, 35.15, IR (CHCI,): 1743, 1720cm-'. MS (80eV) m/e (relative intensity) 288 (M', lo), 174(9), 173(11), 134(100), 121 (29),91(22), 77 (1 2).

2-p-AnisoyI-8- (3'-methyIindoIe) -8-carbethoxy-5- cycloocrenone To a stirred solution of 2-p-anisoyl-8-carbethoxy-5- cyclooctenone (4.32 g, 15 mmol) in 50 mL of dry THF, was added dropwise lithium bis(trimethylsily1) amide (7.5mL of I.OM solution in hexane, 7.5mmol) at r.t. The reaction mixture was heated under reflux for 2-3 h, after which gramine (2.61 g, 15mmol) was added dropwise in THF (10mL). After stirring and refluxing overnight at 80-90" in an oil bath, the reaction mixture was cooled to r.t., and quenched with water. The aqueous layer was extracted with ether (3 x 50mL), and the combined ether extracts were dried over Na, SO,. Concentration followed by flash chromatography on 15Og silica gel with CH,Cl, as eluant afforded 2.5 g (40%) of 2-p-anisoyl-8-3'- methylindole)-8-carbethoxy-5-cyclooctenone as a white crystalline solid with m.p. 85-86". ' H NMR (400 MHz, CDCI,): 6 8.1 5 (d, 1 H), 7.69 (d, 1 H), 7.40 (d,2H),7.39(m,2H),7.2(m,2H),6.92(d, 1 H),6.85 (d, 2 H), 5.8-6.0 (m, 2 H), 4.35 (dd, 1 H), 3.80 (s, 3 H), 3.60(s, 3 H), 3.55 (q,2 H), 2.80(dd, 1 H), 2.63 (m, 1 H), 2.40 (m, 1 H), 2.25 (m, 1 H), 2.1 (9, 1 H), 1.80 (m, 1 H). I3C NMR (IOOMHz, CDCI,): 6 172.21, 158.70, 135.83, 132.09, 129.26, 129.06, 128.96, 128.05, 123.26, 122.11, 119.58, 118.84, 114.04, 111.23, 109.41, 71.04, 55.27, 52.56, 51.79, 34.60, 30.24, 28.52, 26.63. IR

Jaspamide mimetics

dine was reflwied for 24 h. The pyridine was evaporated, and the dark mixture was added to 20mL of saturated NaHCO, and extracted with chloroform (3 x 15mL). The organic layer was dried over Na2S04 and evaporated. The brown oil was chromatographed on silica gel using 2% methanol in CH2CI2 as eluant to yield 300mg (40%) of 2-p- anisoyl-8-(3'-methylindole)-cyclooctanone oxime as a white solid with m.p. 70-71". 'HNMR (400MHz, CDC1,): 8.75 (m, 1 H), 6.70-7.40 (m, 9 H), 3.80 (s, 3H), 3.60 (dd, 1 H), 3.20-3.40 (m, 2 H), 2.75 (m, 1 H), 1.40-2.30 (m, lOH). MS (80eV) m/e (relative intensity) 376 (M', l), 130 (loo), 121 (l l) , 113 (6), 77 (6).

(CHCI,): 1740, 1720cm-' . MS (80eV) m/e (relative intensity) 417 (M+, 4), 228 (2), 134 (9), 130 (loo), 121 (7), 117 (9), 77 (4). HRMS (EI) Calc. (M+) for C26H27N04: 417.487, found 417.1956, 100%.

2-p-Anisoyl-8-(3'-methylindole)-5-cyclooctenone (7) A mixture of lithium iodide (1.61 g, 12 mmol) in 15 mL of dry 2,4,6-collidine was heated to reflux. After all the lithium iodide had dissolved, 2-p-anisoyl-8-(3'-methylindole)-8-carbethoxy-5-cyclooctenone (2.5 g, 6 mmol) was dissolved in 5 mL of 2,4,6-collidine, and added to the refluxing solvent, generating a faintly yellow solution; with time, the solution turned darker in color and a precipitate formed. The mixture was heated under reflux for 3 h. It was cooled and poured onto a mixture of 15 mL of 6 N HCI, 15 mL of ether, and 7.5 g of ice. The residue in the flask was dissolved in a mixture of 6 N HCl and CH2C12, and this mixture was added to the ice. The aqueous layer was separated and extracted with two 10-mL portions of ether. The combined ethereal solution was washed once with 10 mL of 6 N HCI, once with 2 "a,CO, solution, twice with brine, and dried over anhydrous Na,SO,. The solvent was removed under reduced pressure, and the residue was purified by flash chromatography on silica gel with CH,CI, as eluant to afford 1.4 g (65%) of 2-p-anisoyl- 8-(3'-methylindole)-5-cyclooctenone as a white crystalline solid with m.p. 156-157'. 'HNMR (400 MHz, CDCI,): 6 8.0 (s, 1 H), 7.55 (d, 1 H), 7.30 (d, 1 H), 7.25 (d, 2 H), 7.15 (m, 2 H), 6.85 (d, 3 H), 5.75-5.90 (m, 2 H), 4.0 (dd, 1 H), 3.80 (s, 3 H), 3.1-3.25 (m, 2 H), 2.6-2.82 (m, 2 H), 2.25 (m, 3 H), 2.05 (m, 1 H), 1.60 (m, 1 H). I3CNMR (IOOMHz, CDCl,): 6 188.81, 158.54, 136.18, 131.99, 131.22, 128.97, 128.60, 127.55, 122.25, 121.91, 119.23, 116.71, 114.28, 113.81, 111.06, 56.19, 55.62, 55.24, 31.27. IR (CHCI,): 1705 cm-' . MS (80 eV) m/e (relative intensity) 361 (M+, 9), 156 (13), 130 (IOO), 121 (21). HRMS (EI) Calc. (M+) for C2,H2,N0,: 359.447, found 359.1878. 100%.

2-p-Anisoyl-8- (3'-methylindole) cyclooctanone (8) To a solution of 7 (1.78 g, 5 mmol) in 50 mL of methanol, was added a catalytic amount of 10% Pd/C and the mixture was stirred under one atmosphere of hydrogen for 24h. The solution was filtered and evaporated to yield 1.71 g (95%) of compound 8 as a white crystalline solid with m.p. 58-59'. ' H NMR (400MHz, CDCl,): 6 7.85 (s, 1 H), 7.55 (d, 1 H), 7.30 (d, 1 H), 7.20 (t, 1 H), 7.10 (t, 1 H), 7.00 (d, 2 H), 6.72 (m, 3 H), 3.75 (s, 3 H), 3,72 (d, 1 H), 3.15 (m, 2 H), 2.78 (m, 1 H), 2.20 (m, 1 H), 2.05 (m, 1 H), 1.80 (m, 4 H), 1.45 (m, 2 H), 1.20 (m, 1 H). IR (CHCI,): 1718cm-'. MS (80 eV) m/e 361.

2-p-Anisoyl-8- (3'-methylindole) cyclooctanone oxime A mixture of 8 (718 mg, 2 mmol) and hydroxylamine hydrochloride (690 mg, 10 mmol) in 10 mL of dry pyri-

3.9-Disubstituted nine-membered ring lactam (9) A solution of 2-p-anisoyl-8-(3'-methylindole)cyclooctanone oxime (36 mg, 0.1 mmol) in 2 mL of methylene chloride was cooled to 0'. Pyridine and catalytic amounts of 4-dimethylaminopyridine were added. After stirring for 10 min, methanesulfonyl chloride was added dropwise. The mixture was allowed to warm to r.t. and stirred an additional 30 min. Saturated aqueous sodium bicarbonate was added, and the mixture was stirred at r.t. for an additional 1 h. The, aqueous layer was extracted with methylene chloride (3 x 5 mL). The organic layer was dried over sodium sulfate and evaporated in vacuo. The brown oil was chromatographed on silica gel using 2.5% methanol, in methylene chloride as eluant to afford 12mg (33.3%) lactam 9 as an off-white solid with m.p.

7.25-7.4 (m, 3 H), 7.15 (t, J = 7.44, 7.69Hz, 1 H), 6.90 (t, J = 6.67, 8.46Hz, 1 H), 6.70 (d, J = 7.7Hz, 2 H), 6.45 (d, J = 8.0Hz, 2 H), 6.25 ( s , 1 H), 6.0 (9, J = 5.13, 5.38, 5.9Hz, 1 H), 3.75 ( s , 3 H), 3.26 (4, 1 H), 2.80 (m, 2 H), 2.40 (m, 1 H), 2.25 (m, 1 H), 1.50-1.80 (m, 8 H). I3CNMR (lOOMHz, CDCl,): 6 180.20, 159.39, 149.72, 136.09, 134.06, 129.41, 127.64, 126.53, 126.39, 123.00, 121.72, 119.30, 118.90, 113.98, 113.47, 110.81,55.20,45.10,32.00,31.33,28.85,28.00, 27.17. IR (CHCI,): 1665cm-'. MS (80eV) m/e (relative intensity) 374 (M+, 4), 162 (25), 149 (17), 134 (14), 130(100), 121 (13),77(19). HRMS(EI)Calc.(M+) for C,,H,,N2O2: 376.46, found 376.2045, 100%.

Bromoindole lactam (10) To a solution of (9) 7.4 mg (0.0 17 mmol) dissolved in 0.5mL of CH2C1, was added silica gel (l00mg) and 3.6 mg (0.02 mmol) of N-bromosuccinimide at r.t. in the dark. The reaction was stirred for 1 h. The Solids were removed by filtration and the solvent removed in vacuo. The residue was purified by preparative TLC (3% MeOH in CH,Cl,) to afford 4mg (52%) of an off-white solid that decomposed upon heating. ' H NMR (400 MHz, CDCl,): 6 8.07 (s, 1 H), 7.78 (d, J = 7.7Hz,2H),6.84-7.72(m,4H),6.92(d,J = 7.7, 2 H), 4.94 (m, 1 H), 1.3-3.8 (m, 13 H). IR (CJCI,): 1665cm-['. MS (80eV) m/e 453.

61-63'. 'HNMR (~OOMHZ, CDCl,): 6 8.05 ( s , 1 H),

333

M. Kahn et al.

ACKNOWLEDGMENTS

This research was supported in part by NIH grant GM 38260. Additionally, M.K. wishes to thank the Camille and Henry Dreyfus Foundation, the Searle Scholars Program/The Chicago Community Trust, the NSF (Presidential Young Investigators Award), Mon- santo, Procter and Gamble, Schering, Searle and Syntex for matching funds, and the American Cancer Society (Junior Faculty Fel- lowship for generous financial support. The molecular modelling facilities were provided, in part, by a BRSG shared instrumentation grant. We thank Dr. Robert Houtchens of Dow Chemical Co., Midland, MI, for testing our mimetics for insecticidal activity, Dr. Barbara Chen of Searle for the high resolution mass spectral analy- ses, and Professor Clark Still, Columbia University, for providing the Macro-Model and BATCHMIN programs for our use.

REFERENCES

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Address:

Dr. Michael E. Johnson Department of Medicinal Chemistry Pharmacognosy University of Illinois at Chicago P.O. Box 6998 (m/c 781) Chicago, IL 60680 USA

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design and synthesis of nonpeptide mimetics of jaspamide

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