Clarification of the binding mode of teleocidin and benzolactams to the Cys2 domain of protein kinase Cδ by synthesis of hydrophobically modified, teleocidin-mimicking benzolactams and computational docking simulation

1476

J . Med. Chem. 1998, 41, 1476-1496

Cla r ifica tion of th e Bin d in g Mod e of Teleocid in a n d Ben zola cta m s to th e Cys2

Dom a in of P r otein Kin a se Cδ by Syn th esis of Hyd r op h obica lly Mod ified ,

Teleocid in -Mim ick in g Ben zola cta m s a n d Com p u ta tion a l Dock in g Sim u la tion

Yasuyuki Endo,*^,†Shunji Takehana,^†Michihiro Ohno,^†Paul E. Driedger,^‡Silvia Stabel,^§Miho Y. Mizutani,^|

Nobuo Tomioka,^|Akiko Itai,^|,∇and Koichi Shudo^†

Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113, J apan, Procyon

Pharmaceuticals, Inc., 165 New Boston Street, Woburn, Massachusetts 01801, Max-Delbu¨ck Laboratorium, Carl-von-Linne´-Weg

10, D-5000 Ko¨ln 30, Germany, and Institute of Medicinal Molecular Design, 5-24-5, Hongo, Bunkyo-ku, Tokyo 113, J apan

Received October 16, 1997

Phorbol esters (12-O-tetradecanoylphorbol 13-acetate; TPA) and teleocidins are known to be

potent tumor promoters and to activate protein kinase C (PKC) by binding competitively to

the enzyme. The relationship between the chemical structures and the activities of these

compounds has attracted much attention because of the marked structural dissimilarities. The

benzolactam 5, with an eight-membered lactam ring and benzene ring instead of the nine-

membered lactam ring and indole ring of teleocidins, reproduces the active ring conformation

and biological activities of teleocidins. Herein we describe the synthesis of benzolactams with

hydrophobic substituents at various positions. Structure-activity data indicate that the

existence of a hydrophobic region between C-2 and C-9 and the steric factor at C-8 play critical

roles in the appearance of biological activities. We also computationally simulated the docking

of teleocidin and the modified benzolactam molecules to the Cys2 domain structure observed

in the crystalline complex of PKCδ with phorbol 13-acetate. Teleocidin and benzolactams fitted

well into the same cavity as phorbol 13-acetate. Of the three functional groups hydrogen-

bonding to the protein, two hydrogen-bonded with protein atoms in common with phorbol 13-

acetate, but the third one hydrogen-bonded with a different protein atom from that in the case

of phorbol 13-acetate. The model explains well the remarkable difference in activity between

5 and its analogue having a bulky substituent at C-8.

In tr od u ction

IL-V acetate was ∆G^q) 19.2 kcal/mol at -10 °C) and

the short half-life of interconversion (estimated to be

1.2 s at 37 °C for (-)-IL-V acetate) made it difficult to

interpret the mode of interaction of these promoters

with common macromolecular targets (i.e., protein ki-

nase C (PKC)). It is particularly important to determine

the active ring conformation of teleocidins in order to

explain the relationships between the structures and

activities of several classes of TPA-type tumor promoters

with various skeletal structures. Several molecular

modeling studies based upon ligand structures and the

structure-activity relationships of TPA-type tumor

promoters with different skeletons have failed to provide

conclusive evidence to identify the common structural

requirements.⁷

After previous synthetic efforts⁸and computational

prediction⁹of the conformational status of the indola-

ctams, we had designed and synthesized benzolactam-

Vs, in which the indole ring of indolactams is replaced

with a benzene ring, in an attempt to reproduce

separately the two conformations of teleocidins. Among

these benzolactams, eight-membered lactams (benzolac-

tam-V8, (-)-BL-V8 (4)) can only exist in the twist form

and nine-membered lactams (benzolactam-V9) exist

exclusively in the sofa form in solution.¹⁰Eight-

membered derivatives having a sufficiently hydrophobic

moiety, (e.g., (-)-benzolactam-V8-310, (-)-BL-V8-310,

5) exhibited biological activities similar to those of

teleocidin B-4, i.e., differentiation-inducing activity

The teleocidins (e.g., teleocidin B-4, 1) (Figure 1) are

a family of TPA-type tumor promoters¹which include

diterpene esters represented by 12-O-tetradecanoylphor-

bol 13-acetate (TPA, 2) and aplysiatoxins. (-)-Indolac-

tam-V ((-)-IL-V, 3), which lacks the hydrophobic group

at the 6,7-position of the indole ring of teleocidins, was

synthesized by us²and was later isolated from Strep-

toverticillium blastmyceticum.³(-)-IL-V is considered

to be the minimum-sized structure exhibiting tumor-

promoter activity,⁴and the synthesis of (-)-IL-V has

become of interest in recent years.⁵All teleocidins

including (-)-IL-V exist in an equilibrium of two con-

formational states, twist and sofa forms,⁶as shown in

Figure 2. The twist form is characterized by the cis-

amide bond, whereas the sofa form is characterized by

the trans-amide bond. The twist-sofa equilibrium is

considered to be due to cis-trans isomerization of the

amide bond and the steric effects of substituents on the

nine-membered lactam ring. However, the low-energy

barrier between the two conformers found in kinetic

studies^6b(the observed free energy of activation of (-)-

* To whom correspondence should be addressed, at the following:

fax, +81-3-5802-3347; e-mail, yendo@mol.f.u-tokyo.ac.jp.

^†University of Tokyo.

^‡Procyon Pharmaceuticals, Inc.

^§Max-Delbu¨ck Laboratorium.

^|Institute of Medicinal Molecular Design.

^∇Address correspondence pertaining to computational aspects of this

work to this author: fax, +81-3-5689-4053; e-mail, itai@immd.co.jp.

Published on Web 04/03/1998

Clarification of the Binding Mode of Benzolactams

J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9 1477

membered derivatives, such as benzolactam-V9-310

((()-BL-V9-310, 6), were inactive in these biological

assays. These results clearly indicated that the twist

form is close to the biologically active conformation of

teleocidins. Irie et al. recently established the impor-

tance of the twist form of teleocidin by a different

approach based on the synthesis of conformationally

restricted analogues of (-)-IL-V.¹²

Recently, a crystallographic study revealed the direct

interaction of phorbol 13-acetate with the PKCδ Cys2

domain.¹³The binding site thus identified can only bind

with the twist form, but not the sofa form, of teleocidins

in computer modeling analyses.¹⁴This is in good

agreement with our conclusion based on synthesis and

biological activity assay of benzolactams. Derivatization

of (-)-BL-V8-310, a PKC modulator with a new skeletal

systems, would provide further opportunities for exami-

nation of the interaction of ligands with PKC.

We report herein the synthesis of benzolactams with

hydrophobic substituents at various positions for inves-

tigation of specific hydrophobic contacts in the PKC-

ligand interaction.¹⁵We also report a computational

simulation of the docking of these molecules and teleo-

cidin B-4 to the Cys2 domain of PKCδ.¹⁴This modeling

study explains well the remarkable differences in

biological activity for benzolactams with hydrophobic

substituents at specified positions.

Ch em istr y

The relative position of the hydrogen-bonding func-

tional groups of teleocidins required for the appearance

of biological activity has been established by the syn-

thesis and biological evaluation of two benzolactams

((-)-BL-V8-310 (5) and (()-BL-V9-310 (6)) reproducing

respectively the two conformational states of teleoci-

dins.¹⁰Although hydrogen-bonding and other polar

interactions are important factors for recognition of a

biologically active molecule at a receptor, hydrophobic

interaction is also important for stability of binding to

many receptors. In fact, the hydrophobic moiety on the

indole ring of teleocidins plays a critical role in increas-

ing the biological potency. The activity of teleocidin B-4

(1) in terms of the binding affinity to PKC and growth

inhibition of HL-60 cells is 100-500 times higher than

that of (-)-IL-V (3), which lacks the hydrophobic

moiety.¹⁶Various structure-activity studies have re-

ported that teleocidins B-1-B-4, which are stereochem-

ical isomers of the 19- and 22-positions of the terpenoid

side chain, and their open-chain analogues, teleocidins

A-1 and A-2, exhibit similar biological activities.¹⁷The

terpenoid substituents of teleocidins can be replaced by

a simple linear alkyl group without loss of activity.¹⁸

The active PKC-ligand complex is thought to contain

from 3 to as many as 12 molecules of phospholipid bound

to PKC.¹⁹At present, studies on the role of the

hydrophobic moieties of TPA-type tumor promoters are

focused on distinguishing between the direct interaction

of their hydrophobic regions with PKC versus external

hydrophobic interaction with phospholipid. The ben-

zolactams are useful compounds for analyzing these

points. In the case of benzolactams, the activity of (-)-

BL-V8 (4), which lacks the hydrophobic moiety, is 1000

times weaker than that of (-)-BL-V8-310 (5). Recently,

we have reported the synthesis and biological activity

F igu r e 1. Structures of TPA-type tumor promoters and

related compounds.

F igu r e 2. Equilibrium between the twist (left) and the sofa

(right) forms in (-)-IL-V. Top: Views from the face of the

indole ring. Bottom: Views from the side of the indole ring.

toward human promyelocytic leukemia (HL-60) cells

and binding activity to PKC.¹⁰(-)-BL-V8-310 showed

tumor-promoting activity in two-stage carcinogenesis

experiments on mouse skin.¹¹On the other hand, nine-

1478 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9

Endo et al.

tosylation (63%). Condensation of 21 with diethyl

acetamidomalonate gave 22 (77%). Deprotection and

decarboxylation gave the amino acid, which was pro-

tected as the ethyl ester for the carboxylic acid group

and with Boc for the amino group, and the ester group

was reduced with LiBH₄to afford 23 (49%). The nitro

alcohol 23 was converted into the methylamino alcohol

24 (99%) by catalytic hydrogenation and formylation,

followed by reduction with BH₃. Reaction of 24 with

the triflate of (R)-benzyl R-hydroxyisovalerate^5dgave

diastereomeric esters 25 (88%). After hydrogenolysis

of the benzyl ester, condensation with N-hydroxysuc-

cinimide using DCC gave the activated esters (96%).

After removal of the Boc group using CF₃COOH, cy-

clization was carried out under dilute conditions to give

9 (48%) and the epimer 26 (43%), which were isolated

at this stage.

Synthesis of (-)-BL-V8-23TM (10) was performed

from 1,2,3,4-tetrahydro-1,1,4,4,7-pentamethylnaphtha-

lene (27), prepared by Friedel-Crafts alkylation of

toluene with 2,5-dichloro-2,5-dimethylhexane. Nitra-

tion of 27 followed by benzylic bromination gave the

nitro bromide 28 (51%). Condensation of 28 with

diethyl acetamidomalonate gave 29 (90%). Deprotection

and decarboxylation gave the amino acid, which was

protected as the ethyl ester for the carboxylic acid group

and with Boc for the amino group, and the ester group

was reduced with LiBH₄to afford 30 (94%). The nitro

alcohol 30 was converted into the methylamino alcohol

31, (83%) by catalytic hydrogenation and formylation,

followed by reduction with BH₃. Reaction of 31 with

the triflate of (R)-benzyl R-hydroxyisovalerate gave

diastereomeric esters 32 (88%). After hydrogenolysis

of the benzyl ester, condensation with N-hydroxysuc-

cinimide using DCC gave the activated esters (96%).

After removal of the Boc group using CF₃COOH, cy-

clization was carried out under dilute conditions to give

10 (48%) and the epimer 33 (43%).

F igu r e 3. Structures of designed benzolactams ((-)-BL-8-C10

(8), (-)-BL-VA-210 (9), and (-)-BL-V8-23TM (10)).

of 9-alkylated (-)-BL-V8s.²⁰Among the (-)-BL-V8s,

substitution of a C10-C14 linear alkyl chain at the

9-position of the aromatic nucleus is optimum for

maximal biological activity, though substitution of a

C8-C16 cyclic alkyl group (cyclooctyl, cylcododecyl,

cyclohexadecyl) or even a bulky adamantanemethyl

group at the 9-position retains almost the same activity.

This suggests that the hydrophobic alkyl group on (-)-

BL-V8s is folded when the molecule binds to a receptor.

Diterpene ester tumor promoters, such as TPA (2) and

3-tetradecanoylingenol (3-TI, 7), which are essentially

identical pharmacologically, have different skeletons

with hydrophobic esters at different positions on the

molecules. Thus, it seems likely that a large, ap-

propriately oriented hydrophobic region on the molecule

plays a critical role in the appearance of biological

activities.

For the purpose of characterizing the hydrophobic

region, (-)-BL-8-C10 (8) with a decyl group at the

2-position and (-)-BL-V8-210 (9) with a decyl group at

the 8-position of the (-)-BL-V8 skeleton were designed.

We supposed that location of the hydrophobic group in

the former corresponds to that in 3TI, and the latter to

that in TPA, in analogy to (-)-BL-V8-310 (5). In

addition to these (-)-BL-V8s with a linear alkyl sub-

stituent, (-)-BL-V8-23TM (10) with an 8,9-cyclized alkyl

group, which corresponds to that in teleocidin Bs, was

designed (Figure 3).

Syntheses of the benzolactams (BLs) were carried out

in a manner similar to that used for (-)-BL-V8-310 (5)^10c

(Scheme 1). In the synthesis of (-)-BL-8-C10 (8), the

n-decylglycine unit was introduced as the triflate of (R)-

benzyl R-hydroxy-n-dodecanoate (11), which was pre-

pared as follows. Racemic n-decylglycine (12) was

optically resolved by stereoselective hydrolysis of the

N-chloroacetate 13 using aminoacylase (yield: 35%)

according to Mori and Otsuka.²¹After hydrolysis of (R)-

13, conversion of the amino group of (R)-12 to a hydroxy

group via the diazotization followed by esterification

with benzyl alcohol-SOCl₂gave (R)-benzyl R-hydroxy-

n-dodecanoate (14; 53%). Treatment of 14 with triflic

anhydride gave the triflate 11 (74%). Substitution of

N-Boc-2-(methylamino)phenylalaninol (15)^10cwith the

triflate 11 gave 16 (80%). Deprotection of the benzyl

ester, formation of an activated ester, deprotection of

the N-Boc group, and cyclization afforded 8 (27%) and

its epimer 17 (24%).

The benzolactams 8-10 were confirmed to take the

twist conformational form, which has been established

1

in the case of 4, on the basis of the similarity of the H

NMR spectral data and nuclear Overhauser effect

(NOE) experiments. Optical purity of the target com-

pounds was determined by ¹H NMR measurements with

(-)-R-methoxy-R-(trifluoromethyl)phenylacetic

((-)-MTPA) to be >99.8% ee.

acid

Biologica l Eva lu a tion

The biological activities of the benzolactams 8-10

were examined by means of two bioassays related to in

vivo tumor promotion. One of the most sensitive and

specific biological activities of the TPA-type tumor

promoters is induction of growth inhibition, cell adhe-

sion, and differentiation to monocytes of HL-60 cells.²²

The effective concentration (EC₅₀) of teleocidin B-4 for

growth inhibition is 5 × 10^-9M, whereas that of (-)-

IL-V is 5 × 10^-7M. Results are presented in Figure 4

along with comparative data for (-)-BL-V8-310 (5) and

(-)-BL-V8 itself (4), which lacks a hydrophobic group

at C-9.

(-)-BL-V8-210 (9) was prepared starting from 3-car-

bomethoxy-4-nitrobenzoic acid (18). Reduction of 18

with borane-dimethyl sulfide complex followed by

oxidation with PCC gave 3-carbomethoxy-4-nitroben-

zaldehyde (19) (86%). Reaction of the aldehyde with

nonylphosphoniumylide gave 20 (84%), which was con-

verted to the tosylate 21 by hydride reduction and

Despite the switching of its decyl group from C-9 to

C-2, (-)-BL-8-C10 (8) showed distinct, though de-

creased, activity. Its activity is 50 times stronger than

that of (-)-BL-V8 (4). This suggests that the hydro-

Clarification of the Binding Mode of Benzolactams

J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9 1479

Sch em e 1. Synthesis of Benzolactams 7-9^a

a

Key: (a) ClCH₂COCl, NaOH/H₂O; (b) Aspergillus aminoacylase, CoCl₂, pH 7.26; (c) HCl/H₂O; (d) NaNO₂, H₂SO₄/H₂O; (e) C₆H₅CH₂OH,

SOCl₂; (f) Tf₂O, 2,6-lutidine/CH₂Cl₂; (g) (S)-2-(methylamino)phenylalaninol (15), 2,6-lutidine/CH₂ClCH₂Cl; (h) H₂, Pd-C/EtOH; (i)

N-hydroxysuccinimide, DCC/CH₃CN; (j) CF₃COOH/CH₂Cl₂then aq NaHCO₃/CH₃COOEt; (k) BH₃-S(CH₃)₂/THF; (l) PCC/CH₂Cl₂; (m)

C₉H₁₉P⁺Br^-, n-BuLi/THF; (n) LiBH₄/THF; (o) NaH, TsCl/toluene; (p) CH₃CONHCH(COOC₂H₅)₂, NaH/DMF; (q) HCl, AcOH; (r) SOCl₂/

EtOH; (s) Boc₂O/CH₂Cl₂; (t) HCOOH, Ac₂O; (u) BH₃/THF; (v) triflate of benzyl (R)-R-hydroxyisovalerate, 2,6-lutidine/CH₂ClCH₂Cl; (w)

HNO₃, H₂SO₄; (x) NBS/CCl₄, hν.

phobic region at C-2 and C-9 has a common function in

determining biological activity. Unexpectedly, switch-

ing of the decyl group from C-9 to the neighboring C-8

also caused a distinct decrease of the activity; i.e., the

activity of (-)-BL-V8-210 (9) is 30 times weaker than

that of 5. Further introduction of a bulky substituent

at C-8 caused a significant decrease in activity; i.e., the

activity of (-)-BL-V8-23TM (10) is 1000 times weaker

than that of 5 and almost the same as that of 4.

Binding affinity of the BLs to PKCδ is directly related

to ligand-PKC interaction and is useful for interpreting

modeling studies. Assay of the inhibition of [³H]PDBu

binding (K_d) 2.9 nM) to recombinant PKCδ and the

determination of inhibition constants from binding

curve IC₅₀values were done as previously described.²³

The difference in PKCδ binding potency between 5 (K_i

) 5.9 ( 1.7 nM) and 10 (K_i) 5100 ( 730 nM) is

particularly striking. These BLs (4, 5, 8-10) and

teleocidins exist in a similar conformational state, which

means that the hydrophilic functional groups are ar-

ranged in appropriate positions for binding to the

receptor. Therefore, the significant difference in the

activity caused by the substituents indicates that a

steric factor that disfavors binding to the receptor exists

around the 8-position of BLs. This is apparently the

first observation of an effect on PKC binding attribut-

1480 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9

Endo et al.

F igu r e 4. Biological activities of benzolactams 8-10. Left: Growth inhibition of HL-60 cells. Right: Inhibition of specific [³H]-

PDBu binding with PKCδ. Legend: [, BL-V8-310 (5); 2, BL-8-C10 (8); O, BL-V8-210 (9); b, BL-V8-23TM (10); 4, BL-V8 (4).

groups (Figure 5, 35-38) were designed and synthe-

sized. The syntheses were carried out in a manner

similar to that used for (-)-BL-V8-23TM (10) as shown

in Schemes 2 and 3. (-)-BL-V8-DM1 (35), which has

two methyl groups on the C-8 side, was prepared

starting from 4,4-dimethyl-γ-butyrolactone. Friedel-

Crafts reaction of benzene with the lactone followed by

bromination on the aromatic nucleus gave 7-bromo-4,4-

dimethyl-1,2,3,4-tetrahydronaphthalen-1-one (39) (47%).

Reduction of the ketone to a methylene group with

triethylsilane gave 6-bromo-1,1-dimethyl-1,2,3,4-tet-

rahydronaphthalene (40) (89%). Conversion of the

bromide 40 to an aldehyde via lithiation/DMF followed

by hydride reduction gave the alcohol 41 (95%). This

was converted to the benzyl bromide 42 (82%), which

was nitrated to give the 7-nitro bromide 43 (32%) and

its 5-nitro isomer. Condensation of 43 with diethyl

acetamidomalonate gave 44 (97%). Deprotection and

decarboxylation gave the amino acid, which was pro-

tected as the ethyl ester for the carboxylic acid group

and with Boc for the amino group, and the ester group

was reduced with LiBH₄to afford 45 (71%). The nitro

alcohol 45 was converted into the methylamino alcohol

46 (75%) by catalytic hydrogenation and formylation,

followed by reduction with BH₃. Reaction of 46 with

the triflate of (R)-benzyl R-hydroxyisovalerate gave

diastereomeric esters 47 (82%). After hydrogenolysis

of the benzyl ester, condensation with N-hydroxysuc-

cinimide using DCC gave the activated esters. After

removal of the Boc group using CF₃COOH, cyclization

was carried out under dilute conditions to give 35 (33%)

and the epimer 48 (30%).

F igu r e 5. Structures of 6,7-cyclic (-)-IL-V 34 and 8,9-cyclic

(-)-BL-Vs 35-38.

able to essentially steric changes in the hydrophobic

alkyl portion of a PKC ligand. Also, the difference

between the K_ivalue of 3.0 ( 0.45 nM of 8 and its EC₅₀

of 200 nM for HL-60 differentiation suggests that 8 will

be useful as a probe for examination of the mechanism

of the biological effects of binding of TPA-type tumor

promoters to PKC.

F u r th er Eva lu a tion of Sp ecific Hyd r op h obic

Con ta cts in th e P KC-Liga n d In ter a ction . The

activity of the BLs is markedly affected by the nature

of the substituent at the 8-position. However, the

tetramethyltetramethylene analogue of (-)-IL-V (Figure

5, 34) has been shown to possess activity comparable

to that of TPA in the PKC binding assay.²⁴Superposi-

tion of the active twist form of teleocidin B-4 (1) and

(-)-BL-V8-23TM (10) as determined by MM-2 calcula-

tions is illustrated in Figure 6. The significant differ-

ence in the activity seems to be caused by an extremely

specific steric effect of the substituent on the 8-position

of the BL molecule, which would hinder fitting into the

cavity through van der Waals contact with PKCδ. For

the purpose of investigation of the steric hindrance and

the appropriate direction of the hydrophobic moiety for

strong binding, four (-)-BL-V8s with 8,9-cyclized alkyl

(-)-BL-V8-DM3 (36), which has two methyl groups

on the C-9 side, was prepared starting from 4-bro-

mobenzaldehyde (Scheme 2). Aldol condensation of the

aldehyde with 3-methylbutan-2-one afforded the R,â-

unsaturated ketone 49 (45%). Hydride reduction fol-

lowed by intramolecular Friedel-Crafts reaction em-

ploying polyphosphoric acid gave 7-bromo-1,1-dimethyl-

1,2,3,4-tetrahydronaphthalene (50) (64%). Conversion

of the bromide 50 to an aldehyde via lithiation/DMF

followed by hydride reduction gave the alcohol 51 (93%),

which was converted to the benzyl bromide 52 (63%).

Nitration, followed by condensation with diethyl aceta-

midomalonate, gave 53 (22%). Deprotection and decar-

boxylation gave the amino acid, which was protected as

the ethyl ester for the carboxylic acid group and with

Clarification of the Binding Mode of Benzolactams

J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9 1481

F igu r e 6. Molecular superposition of teleocidin B-4 (1) (dotted line) and (-)-BL-V8-23TM (10) (solid line). The two molecules

were superposed on the basis of root-mean-square fit of the pairs of atoms marked with asterisks.

Sch em e 2. Synthesis of Benzolactams 35 and 36^a

a

Key: (a) benzene, AlCl₃; (b) Br₂, AlCl₃/CH₂Cl₂; (c) (C₂H₅)₃SiH, CF₃COOH; (d) n-BuLi/Et₂O then DMF; (e) NaBH₄/MeOH; (f) CHBr₃,

Ph₃P/CH₂Cl₂; (g) fuming HNO₃/Ac₂O; (h) CH₃CONHCH(COOC₂H₅)₂, NaH/DMF; (i) HCl, AcOH; (j) SOCl₂/EtOH; (k) Boc₂O/CH₂Cl₂; (l)

LiBH₄/THF; (m) H₂, Pd-Cl/EtOH; (n) HCOOH, Ac₂O; (o) BH₃/THF; (p) triflate of benzyl (R)-R-hydroxyisovalerate, 2,6-lutidine/CH₂ClCH₂Cl;

(q) N-hydroxysuccinimide, DCC/CH₃CN; (r) CF₃COOH/CH₂Cl₂then aq NaHCO₃/CH₃COOEt; (s) 3-methylbutan-2-one, aq NaOH/EtOH;

(t) LiAlH₄/Et₂O; (u) polyphosphoric acid.

Boc for the amino group, and the ester group was

reduced with LiBH₄to afford 54 (89%). The nitro

alcohol 54 was converted into the methylamino alcohol

55 (75%) by catalytic hydrogenation and formylation,

followed by reduction with BH₃. Reaction of 55 with

the triflate of (R)-benzyl R-hydroxyisovalerate gave

diastereomeric esters 56 (84%). After hydrogenolysis

of the benzyl ester, condensation with N-hydroxysuc-

cinimide using DCC gave the activated esters. After

removal of the Boc group using CF₃COOH, cyclization

was carried out under dilute conditions to give 36 (38%)

and the epimer 57 (38%).

(-)-BL-V8-SP1 (37), which has a spiro[cyclohexane]

group on the C-9 side as a hydrophobic moiety, was

formed as follows (Scheme 3). Reaction of cyclohex-

anone with (3-phenylpropyl)magnesium bromide gave

1482 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9

Sch em e 3. Synthesis of Benzolactams 37 and 38^a

Endo et al.

a

Key: (a) (3-phenylpropyl)magnesium bromide/EtO; (b) 90% H₂SO₄; (c) CrO₃/AcOH; (d) Br₂, AlCl₃/CH₂Cl₂; (e) (C₂H₅)₃SiH, CF₃COOH;

(f) n-BuLi/Et₂O then DMF; (g) NaBH₄/MeOH; (h) CHBr₃, Ph₃P/CH₂Cl₂; (i) fuming HNO₃/Ac₂O; (j) CH₃CONHCH(COOC₂H₅)₂, NaH/DMF;

(k) HCl, AcOH; (l) SOCl₂/EtOH; (m) Boc₂O/CH₂Cl₂; (n) LiBH₄/THF; (o) H₂, Pd-C/EtOH; (p) HCOOH, Ac₂O; (q) BH₃/THF; (r) triflate of

benzyl (R)-R-hydroxyisovalerate, 2,6-lutidine/CH₂ClCH₂Cl; (s) N-hydroxysuccinimide, DCC/CH₃CN; (t) CF₃COOH/CH₂Cl₂then aq NaHCO₃/

CH₃COOEt.

1-(3-phenylpropyl)cyclohexanol (58) (70%). Intramo-

lecular Friedel-Crafts reaction of 58 afforded 3′,4′-

dihydrospiro[cyclohexane-1,1′(2′H)-naphthalene] (59)

(88%). After benzylic oxidation with CrO₃, introduction

of a bromo group at the 6′-position followed by reduction

with triethylsilane gave the bromide 60 (35%). This was

converted to the benzyl bromide 61 in a manner similar

to that used for 43 (69%). Nitration of 61 afforded the

nitro bromide 62 (40%) and its isomer. Condensation

of 62 with diethyl acetamidomalonate gave 63 (94%).

Deprotection and decarboxylation gave the amino acid,

which was protected as the ethyl ester for the carboxylic

acid group and with Boc for the amino group, and the

ester group was reduced with LiBH₄to afford 64 (86%).

The nitro alcohol 64 was converted into the methyl-

amino alcohol 65 (75%) by catalytic hydrogenation and

formylation, followed by reduction with BH₃. Reaction

of 65 with the triflate of (R)-benzyl R-hydroxyisovalerate

gave diastereomeric esters 66 (64%). After hydro-

genolysis of the benzyl ester, condensation with N-

hydroxysuccinimide using DCC gave the activated

esters. After removal of the Boc group using CF₃COOH,

cyclization was carried out under dilute conditions to

give 37 (38%) and the epimer 67 (28%).

The biological activities of the benzolactams 35-38

were examined by assay of growth inhibition of HL-60

cells and inhibition of specific binding of [³H]PDBu to

recombinant PKCδ. The results are presented in Figure

7 along with comparative data for (-)-BL-V8-310 (5) and

(-)-BL-V8-23TM (10). As is apparent, (-)-BL-V8-DM3

(36) with two methyl groups on the C-8 side showed

weak activity (EC₅₀of 7 µM for HL-60 growth inhibition

and K_i) 1700 ( 220 nM for PKC affinity). On the other

hand, (-)-BL-V8-DM1 (35), with two methyl groups on

the C-9 side, showed about 30 times stronger activity

than 36. The difference in PKCδ binding potency

between 36 and 35 (K_i) 65 ( 0.6 nM) clearly indicates

that a steric factor that disfavors binding to the receptor

exists around the 8-position of BLs. The relatively weak

activities of 35 compared to 5 can be explained by a lack

of some favorably directed hydrophobic group. Intro-

duction of the hydrophobic spiro[cyclohexane] group on

the C-9 side enhanced the biological activity. (-)-BL-

V8-SP1 (37) and (-)-BL-V8-SP3 (38) potently inhibited

the growth of HL-60 cells (EC₅₀) 70 and 20 nM,

respectively) and bound strongly to PKCδ (K_i) 19 (

1.1 and 14 ( 0.94 nM, respectively). The difference in

PKCδ binding potency between 38 and 9, with a linear

decyl group at C-8 (K_i) 32 ( 8.7 nM) indicates that

the hydrophobic region of 38 is in a favorable location

for biological activity.

BL-V8-SP3 (38), which has a dimethylspiro[cyclohex-

ane] group on the C-9 side as a hydrophobic moiety, was

prepared in the same manner as that used for 37,

starting from 4,4-dimethylcyclohexanone. The inter-

mediates and yields are described in the Experimental

Section. Optical purity of the target compounds (35-

38) was determined by ¹H NMR measurements with

(-)-MTPA to be >99.8% ee.

Dock in g Stu d y. In 1995, the crystal structure of the

PKCδ Cys2 domain was elucidated in the complex with

phorbol 13-acetate.¹³Docking simulations to this struc-

ture would be useful to clarify the relationship between

the three-dimensional structures and activities of other

Clarification of the Binding Mode of Benzolactams

J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9 1483

F igu r e 7. Biological activities of hydrophobically modified benzolactams 35-38. Left: Growth inhibition of HL-60 cells. Right:

Inhibition of specific [³H]PDBu binding with PKCδ. Legend: [, BL-V8-310 (5); 4, BL-V8-DM1 (35); 2, BL-V8-DM3 (36); 9, BL-

V8-SP1 (37); 0, BL-V8-SP3 (38); b, BL-V8-23TM (10).

PKC activators. Such simulation would also be useful

to distinguish direct hydrophobic interaction of the

hydrophobic moiety of teleocidins and indolactams with

PKC from external hydrophobic interaction of the

moiety with external phospholipids. Thus, we per-

formed a docking study to obtain stable docking mod-

els¹⁴for the twist form of (-)-IL-V (3), (-)-BL-V8 (4),

teleocidin B-4 (1), and two (-)-BL-V8s with 8,9-cyclic

alkyl groups (10 and 35) bound to the PKCδ Cys2

domain, based upon the coordinates of the X-ray struc-

ture.¹³

In advance of the ADAM docking, the allowed binding

region for the compounds to be docked was roughly

indicated on the protein structure interactively by the

program GREEN,²⁶so that the region fully included the

phorbol 13-acetate molecule complexed with the enzyme

in the crystal. A three-dimensional grid is generated

inside the region, and various potentials are calculated

in order to estimate intermolecular interaction energy

very rapidly. van der Waals potentials for several probe

atoms and electrostatic potentials at each grid point are

calculated, using all protein atoms. Then, several

dummy atoms, used as probes to cover likely binding

modes and ligand conformations at the initial step of

ADAM, were located at the estimated positions of

partner atoms of hydrogen-bonding groups of the en-

zyme inside the allowed region.

Up to several dozen stable docking models initially

output from the ADAM program for each molecule were

reranked after energy minimization using the SANDER

program of the AMBER package²⁷in order to take into

account conformational changes in the protein structure

upon ligand binding. In this study, the most stable

docking model after minimization was taken as a

candidate structure for each compound and used as the

basis for later discussion.

First, the redocking of phorbol 13-acetate was at-

tempted in order to confirm the reliability of the docking

method and the validity of all assumptions concerning

the protein structure, using the modeled structure of

phorbol 13-acetate itself. The most stable docking

model selected by the docking method closely resembled

the structure of the complex in the crystal, fully

reproducing the ligand conformation and relative posi-

tion and orientation, as shown in Figure 8, top. The

hydrogen-bond network found in the crystal was also

reproduced accurately in the predicted model. Three

functional groups hydrogen-bonded to main-chain amide

groups at the bottom of the cavity: the C3-carbonyl to

the NH of Gly253, C-4 OH to the carbonyl of Gly253,

and C-20 OH to both the carbonyl of Leu251 and the

NH of Thr242, as found in the crystal (Table 1). Then,

the twist forms of (-)-IL-V (3) and (-)-BL-V8 (4) were

docked into the cavity by the same procedure. In the

most stable docking structure for 3, the molecule was

well-fitted to the cavity, to the same extent as phorbol

Docking simulations in this study were performed

using an automatic docking program (ADAM) developed

by us previously.²⁵The most stable docking model

between a given protein and a small molecule is

obtained by this method without presumptions, covering

all possible docking combinations of ligand conforma-

tions and binding modes. All the flexibilities in rotat-

able bonds of the ligand molecule are automatically

considered in the docking procedure, whereas the

protein structure is treated as fixed in the step of

obtaining initial docking models. As regards ring

conformations, it is necessary to for possible conformers

to be input in advance. Force-field energy values are

used to select energetically favorable conformations

together with favorable binding modes at the same time

throughout the docking process. A noteworthy feature

of the method is that torsion angles in the ligand are

repeatedly optimized in continuous space together with

relative positions and orientations of the two molecules

during the process of docking. The efficiency of the

method was confirmed by the fact that crystal structures

of more than 10 enzyme-inhibitor complexes were well-

reproduced by the program ADAM. In every case, not

only was the docking mode in the crystal reproduced

by the most stable docking model output from ADAM,

but also the predicted structure coincided with the

crystal structure very accurately with rms values of less

than 2 Å. If torsion angles were not optimized during

the docking process, the correct model would not be

predicted or reproduced by the highest ranked model

(most stable docking model) with such accuracy, even

if conformational flexibilities were considered by sys-

tematic rotation of rotatable bonds in increments of 30°

or 60°.

1484 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9

Endo et al.

F igu r e 8. Stereoviews of the most stable docking models for phorbol 13-acetate (top), (-)-IL-V (3) (middle), and (-)-BL-V8 (4)

(bottom). Bound ligands are shown with broad lines, and intermolecular hydrogen bonds (distances less than 3.2 Å) are shown

with yellow lines.

13-acetate, forming four hydrogen bonds with the

enzyme, as shown in Figure 8, middle. Three functional

groups, the amide carbonyl, amide NH, and C-14 OH,

were involved in the hydrogen-bond network. The

amide carbonyl group hydrogen-bonded to the NH of

Gly253 and the C-14 OH to both the carbonyl of Leu251

and the NH of Thr242, corresponding to the C3-carbonyl

and C-20 OH groups in phorbol 13-acetate, respectively.

Clarification of the Binding Mode of Benzolactams

J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9 1485

Ta ble 1. Hydrogen-Bond Distances (Heteroatom-Heteroatom Distance in Å) in the Most Stable Docking Models and Those in the

Crystal Structure

ligand

T242 N‚‚‚HO^a

L251 O‚‚‚HO^a

L251 O‚‚‚HN^b

G253 NH‚‚‚OdC^c

G253 O‚‚‚HO^d

phorbol 13-acetate (crystal)

phorbol 13-acetate (docked)

(-)-IL-V (3)

2.95

2.94

3.00

3.03

3.01

3.07

3.04

2.81

2.80

2.78

2.88

2.78

none

2.96

2.97

2.93

3.10

2.99

3.03

2.97

3.09

2.98

2.64

2.77

none

(-)-BL-V8 (4)

teleocidin B-4 (1)

(-)-BL-V8-23TM (10)

(-)-Bl-V8-DM1 (35)

a

b

Phorbol 20-OH; (-)-IL-V and teleocidin B-4 14-OH; (-)-BL-Vs 5-OH. (-)-IL-V and teleocidin B-4 10-NH; (-)-BL-Vs 4-NH. ^cPhorbol

d

3-CdO; (-)-IL-V and teleocidin B-4 11-CdO; (-)-BL-Vs 3-CdO. Phorbol 4-OH.

Most notably, however, the partner of the amide NH in

3 was the carbonyl of Leu251, different from the partner

of the third hydrogen-bonding group (C-4 OH) in phorbol

13-acetate, i.e.; the carbonyl of Gly253. The (-)-BL-V8

(4) molecule also formed a stable complex with the same

hydrogen-bond network as IL-V, as expected from the

superposition study of (-)-BL-V8 skeleton and teleocidin

B-4 (1) (see Figure 6). The amide carbonyl hydrogen-

bonded to the NH of Gly253, the C-5 CH₂OH hydrogen-

bonded to both the carbonyl of Leu251 and the NH of

Thr242, and the amide NH hydrogen-bonded to the

carbonyl of Leu251, as shown in Figure 8, bottom.

Then, docking of teleocidin B-4 (1) was attempted in

order to investigate the influence of the large hydro-

phobic group. The molecule 1 fitted well into the

enzyme cavity without any unfavorable contact with

protein atoms, as shown in Figure 9, top. The hydro-

phobic moiety of 1 points toward the outside of the

protein, which is supposed to be important for the

interaction with the lipid membrane. The docking of

the two congeners of (-)-BL-V8, (-)-BL-V8-23TM (10)

and (-)-BL-V8-DM1 (35), was also attempted in order

to assess the effect of hydrophobic substituent groups

on binding affinity. In the initial docking models, one

of the methyl groups near C-8 of 10 clashed slightly with

protein atoms at the entrance of the cavity, whereas 35

fitted well in the cavity. The clash was removed by

energy minimization without causing marked changes

in the hydrogen-bond networks with the proteins, as

shown in Figure 9, middle and bottom.

difference between the hydrophobic interaction of the

hydrophobic proline ring with the indole ring versus the

benzene ring. The indole ring of 3 seems to have a more

effective hydrophobic interaction compared to the ben-

zene ring of 4 as judged from the present docking of 3

to the PKCδ Cys2 domain. The difference of electro-

static interaction between the 1-NH of 3 and an amide

group of the peptide backbone may be also affected.

1-NH of 3 is close to the carbonyl of Met239 (N‚‚‚O

distance: 2.8 Å), but relative orientation of the two

groups is not ideal for forming a stable hydrogen bond.

Nevertheless, some electrostatic interaction is expected

between them.²⁹A more important difference is the

difference in substitution effects between 3 and 4. The

6,7-tetramethyltetramethylene analogue of (-)-IL-V

(34) has a greater PKC binding activity than (-)-IL-V

(3); its potency is comparable to that of TPA.²⁴On the

other hand, (-)-BL-V8-23TM (10), which has an 8,9-

tetramethyltetramethylene group, showed very low

activity. When we designed (-)-BL-V8-310,¹⁰which is

still the most potent benzolactam known, such a subtle

substituent effect was not expected. The present dock-

ing model explains well the great difference in activity.

Teleocidin B-4 formed a stable complex with the same

hydrogen-bonding pattern as the twist structure of 3

(Figure 9, top). Substituent groups at the 7-position of

teleocidin B-4 do not hinder the fitting. Methyl and

vinyl groups at the 22-position of teleocidin B-4 also

satisfactorily evade van der Waals contacts with the

protein. In contrast, bulky substituent groups at the

8-position of 10 would hinder the fitting of the molecule

into the cavity through van der Waals contacts with

Ser240 and Met239 (Figure 9, middle). Compound 35,

which lacks the bulky dimethyl group at the 8-position,

formed a complex with the same hydrogen-bonding

pattern as the twist structure of 3 (Figure 9, bottom).

Discu ssion

Recently, molecular modeling of the (-)-BL-V8 (4)

molecule complexed with the PKCδ Cys2 domain using

another calculation method was reported by Kozikowski

et al.²⁸The hydrogen-bonding pattern that they ob-

tained was the same as the present result. They also

described the importance of the hydrophobic interaction

between the hydrophobic moiety of 4 and hydrophobic

residues in the protein; the phenyl ring resides on top

of Pro241, parallel to the proline ring, and the N-1

methyl group and the isopropyl group of 4 all interact

with the hydrophobic side chains of Leu251 and Leu

254, respectively.

Although the hydrogen-bonding patterns of (-)-IL-V

(3) and (-)-BL-V8 (4) are very similar, the structure-

activity results for these two types of compounds are

somewhat different. For example, there is a difference

in PKCδ binding potency between 3 (80 nM) and 4 (1700

nM) and a difference in HL-60 growth-inhibitory activity

between 3 (EC₅₀: 10^-7M) and 4 (EC₅₀> 10^-6M). The

difference in activity may be explained in terms of the

To interpret the structure-activity relations among

these compounds, force-field energies of all the most

stable docking models were calculated by using the

ANAL program of the AMBER package. Intramolecu-

lar, intermolecular, and total interaction energy values

are summarized in Table 2. Total energy is defined as

the sum of difference-intramolecular energy and inter-

molecular interaction energy. Here, the difference-

intramolecular energy is defined as the difference

between the conventional intramolecular energy for the

ligand structure in the most stable docking model and

that for the energy-minimized structure of the ligand

alone extracted from the complex. For considering

intramolecular relative stabilities among compounds of

different structural classes, the raw energy values

obtained by force-field calculation are less reliable than

1486 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9

Endo et al.

F igu r e 9. Stereoviews of the most stable docking models for teleocidin B-4 (1) (top), (-)-BL-V8-23TM (10) (middle), and (-)-

BL-V8-DM1 (35) (bottom). The colored cage represents the allowed region for carbon atoms in ligands, calculated from the protein

structure. Colors represent the electrostatic potential levels (positive, red; neutral, green; negative, blue).

Ta ble 2. Energy Decomposition Analysis of the Most Stable Docking Models of (-)-IL-V and (-)-BLs^a

ligand

E_ligand,bound

E_ligand,free

E_ligand,∆

E_inter

E_total

K_ifor PKCδ (nM)

(-)-IL-V (3)

22.15

24.31

32.58

31.53

27.65

21.43

23.54

31.56

29.91

26.87

0.72

0.77

1.02

1.62

0.78

-30.31

-27.06

-31.25

-27.97

-29.16

-29.59

-26.29

-30.23

-26.35

-28.38

80 ( 29

1700 ( 30

0.32 ( 0.043

5100 ( 730

65 ( 0.6

(-)-BL-V8 (4)

teleocidin B-4 (1)

(-)-BL-V8-23TM (10)

(-)-BL-V8-DM1 (35)

a

Energies are in kcal/mol. E_ligand,bound: energy of the ligand in the complexed structure. E_ligand,free: energy of the ligand minimized

alone. E_ligand,∆: E_ligand,bound- E_ligand,free. E_inter: interaction energy between protein and ligand. E_total: E_ligand,∆+ E_inter

.

the difference energies used here, because of the high

dependency on the force-field parameters.

The order of stability, based on the total energy for

related teleocidin-type benzolactams: teleocidin B-4 (1)

(-30.23), (-)-BL-V8-DM1 (35) (-28.38), (-)-BL-V8-

23TM (10) (-26.35), and (-)-BL-V8 (4) (-26.29), can be

well-correlated with the order of PKCδ binding potency,

as seen from Table 2. The reason why docking models

Clarification of the Binding Mode of Benzolactams

J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9 1487

added a solution of chloroacetyl chloride (62 g, 548.6 mmol) in

2 N NaOH (200 mL) dropwise over 2 h with stirring. After

the addition, the solution was acidified to pH 2 with concen-

trated HCl. The precipitate was collected and dissolved in

AcOEt. The solution was washed with brine, dried (Na₂SO₄),

and concentrated in vacuo to give 13 as a solid. Recrystalli-

zation from acetone-n-hexane gave 20.5 g (63%) of pure 13.

Mp: 91 °C. Anal. (C₁₄H₂₆NO₃Cl) C, H, N.

of 10 and 4 exhibit almost the same stability seems to

be as follows: the lower stability of 10 compared to 35,

by 2.03 kcal/mol, can be ascribed to the steric hindrance

of the additional dimethyl groups, whereas the higher

stability of 35 compared to 4, by 2.09 kcal/mol, can be

ascribed to favorable van der Waals interaction of the

additional dimethylcyclohexyl group with protein atoms.

The hydrophobic regions of ligands bound to PKC are

known to interact with the hydrophobic regions of

phospholipid molecules presumably specifically associ-

ated with PKC.³⁰Moreover, increasing the size of the

hydrophobic regions of PKC ligands does increase their

affinity for PKC. Thus it might be expected that the

greater potency of more hydrophobic ligands for PKC

in comparison to less hydrophobic ligands due to hy-

drophobic interactions with phospholipid would not be

fully reflected in calculations of binding interactions of

PKC ligand with PKC. However, this calculation of

hydrophobically modified benzolactams reveals that the

direct hydrophobic interaction of the ligand with PKC

plays an important role in the affinity.

(R)-2-Am in od od eca n oic Acid ((R)-12). A solution of 13

(22.56 g, 97.4 mmol) in NaOH(aq) (3.44 g in 800 mL of distilled

water) was warmed to 37 °C, adjusted to pH 7.27 with 3 N

HCl, and diluted with 2 L of distilled water. To this were

added Aspergillus aminoacylase (Tokyo Kasei Co.; 3.58 g) and

CoCl₂(15 mg), and the mixture was left to stand for 19 h at

37 °C. The separated (S)-12 was collected, washed with water,

methanol, and Et₂O, and dried to give 8.22 g of (S)-12 (49%,

mp 220 °C). The aqueous filtrate was left to stand for 22 h at

37 °C, and the precipitate was removed again by filtration.

The filtrate was acidified with 3 N HCl, and the separated

(R)-13 was collected. It was dissolved in AcOEt. The solution

was washed with 2 N HCl, water, and brine, dried over MgSO₄,

and concentrated in vacuo. The residue was crystallized from

acetone-n-hexane to give 7.92 g (35%) of (R)-13. Mp: 79-

80.5 °C. (R)-13 (5.60 g, 24.2 mmol) and 3 N HCl (100 mL) were

stirred and heated under reflux for 4.5 h. After cooling, the

mixture was neutralized with 15% aqueous NH₃. The pre-

cipitate was collected, washed with water, methanol, and

AcOEt, and dried in vacuo to give 4.74 g (99%) of (R)-12. Mp:

Con clu sion

Synthesis and testing of benzolactams as conforma-

tionally restricted analogues of the biologically active

conformation of teleocidins have clarified the conforma-

tional flexibility problem in structure-activity studies

of teleocidins. However, the roles of the hydrophobic

moieties of teleocidins and benzolactams and the flexible

hydrophobic ester chains of phorbol esters are still not

clear. Benzolactams are useful medicinal leads for

elucidation of the role of PKC and the mechanism of

tumor promotion, especially in conjunction with the

structural determination of the PKCδ Cys2 domain-

phorbol 13-acetate complex.¹³The present synthesis

and biological evaluation of hydrophobically modified

benzolactams have demonstrated the importance of a

continuous substituent in hydrophobic region between

C2 and C9 of BLs for binding to PKCδ Cys2 domain. It

is noteworthy that the significant difference in the

activity between (-)-BL-V8-310 (5) and (-)-BL-V8-

23TM (10), as well as the difference between 5 and (-)-

BL-V8-210 (8), can be accounted for by steric hindrance

in binding of the ligand with PKCδ: docking simulation

of teleocidin, (-)-IL-V, and benzolactams to the PKCδ

Cys2 domain based upon the coordinates of the X-ray

structure of the complex with phorbol 13-acetate, using

an advanced docking program, explains well the above

experimental results. These findings should be helpful

in the design of compounds as biological tools for

analyzing the mechanism of tumor promotion, or in

designing potent antagonists.

205 °C dec. [R]²²-20.5° (c ) 1.02, AcOH). Anal. (C₁₂H₂₅

-

D

NO₂) C, H, N.

(R)-Ben zyl 2-Hyd r oxyd eca n oa te (14). A solution of

sodium nitrite (2.0 g, 29.0 mmol) in water (23 mL) was added

dropwise to a stirred solution of (R)-12 (2.69 g, 12.5 mmol) in

2 N sulfuric acid over 1 h at 95 °C. After stirring for 2.5 h at

95 °C and cooling, the mixture was extracted with AcOEt,

washed with brine, and dried over MgSO₄. Concentration gave

the crude (R)-2-hydroxydecanoic acid as a colorless solid, which

was used without purification. The acid was dissolved in dry

benzene (50 mL); then benzyl alcohol (8.5 mL, 81.8 mmol) and

thionyl chloride (1.0 mL, 13.7 mmol) were added. The solution

was heated at reflux with a Dean-Stark trap for 10 h. An

additional portion of thionyl chloride (1.0 mL) was added, and

the reaction was continued for 16 h. After cooling, the solution

was diluted with saturated NaHCO₃(aq) and extracted with

AcOEt. The organic phase was washed with water, 10% citric

acid aqueous solution, water, and brine. After drying (MgSO₄)

and concentration in vacuo, the resultant residue was chro-

matographed on silica gel (hexane/AcOEt, 5:2) to give (R)-

benzyl 2-hydroxydecanoate (14) as a viscous liquid (2.04 g,

53%). [R]²²+10.46° (c ) 1.09, CHCl₃).

D

(R)-Ben zyl 2-[[(Tr iflu or om eth yl)su lfon yl]oxy]decan oate

(11). A solution of 14 (1.17 g, 4.12 mmol) in dry dichlo-

romethane (20 mL) was cooled to -40 °C, and 2,6-lutidine (0.95

g, 8.88 mmol) was added, followed by triflic anhydride (1.92

g, 6.8 mmol). After 1 h, the mixture was warmed to room

temperature, and ice-water was added. The organic phase

was separated, washed with water and brine, dried (MgSO₄),

and concentrated to give an oil. Purification by column

chromatography on silica gel (hexane/AcOEt, 5:1) gave (R)-

benzyl 2-[[(trifluoromethyl)sulfonyl]oxy]decanoate (11) as a

colorless oil. [R]²²_D+27.5° (c ) 1.16, CHCl₃). ¹H NMR (CDCl₃):

0.88 (t, 3H, J ) 7 Hz, (CH₂)₈CH₃), 1.24 (br, 12H, -(CH₂)₆CH₃),

1.38 (br, 2H, -CH₂(CH₂)₆CH₃), 1.48 (br, 2H, -CH₂(CH₂)₇CH₃),

1.65 (m, 1H, CHCH₂-), 1.78 (m, 1H, CHCH₂-), 5.14 (t, J ) 6

Hz, 1H, CHOTf), 5.26 (m, 2H, CH₂C₆H₅), 7.38 (m, 5H, C₆H₅).

Dia ster eom er ic Ester s 16. A mixture of 640 mg (2.29

mmol) of (()-2-(methylamino)-N-Boc-phenylalaninol (15),^10c

the (R)-triflate 11 (978 mg, 2.30 mmol), and 2,6-lutidine (294

mg, 2.75 mmol) in 10 mL of CH₂ClCH₂Cl was refluxed for 2

days, and then the solvent was removed by evaporation. The

residue was chromatographed on silica gel to give 1.01 g of a

diastereomeric mixture of 16 as a colorless oil (80%). ¹H NMR

(CDCl₃): 0.88 (t, 3H, J ) 7 Hz, (CH₂)₈CH₃), 1.24 (br, 16H,

-(CH₂)₈CH₃), 1.44 (s, 9H, Boc-CH₃), 1.49 (m, 0.5H, -CH₂(CH₂)₈-

Exp er im en ta l Section

Gen er a l Rem a r k s. Melting points were obtained on a

Yanagimoto micro-hot-stage apparatus without correction.

Spectra were recorded with the following instruments: ¹H

NMR spectra, J EOL J MN-GX-400 (400 MHz); mass spectra,

J EOL J MN-DX 303; IR spectra, J ASCO DS-402G; optical

rotational spectra, J ASCO DIP-181. NMR spectra were

recorded with tetramethylsilane as an internal standard, and

the chemical shifts are given as δ values from TMS. Column

chromatography was performed on silica gel (Merck 9385).

(()-2-[(Ch lor oa cetyl)a m in o]d od eca n oic Acid (13). A

solution of n-decylglycine ((()-12; 51.1 g, 237.6 mmol) in 2 N

NaOH (120 mL) was stirred and cooled at 0 °C. To this was

1488 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9

Endo et al.

CH₃), 1.66 (m, 0.5H, -CH₂(CH₂)₈CH₃), 1.90 (m, 1H, -CH₂(CH₂)₈-

CH₃), 2.72 (s, 1.5H, N-CH₃), 2.77 (s, 1.5H, N-CH₃), 2.7-3.0 (m,

1H, ArCH₂CH-), 3.27 (m, 0.5H, CH₂OH), 3.38 (m, 1H, CH₂-

OH), 3.61 (m, 1H, CH₃-N-CH-CO), 3.70 (m, 0.5H, CH₂OH), 4.00

(m, 1H, -CHCH₂OH), 5.06 (s, 1H, CH₂C₆H₅), 5.14 (d, 0.5H, J

) 12 Hz, CH₂C₆H₅), 5.18 (d, 0.5H, J ) 12 Hz, CH₂C₆H₅), 5.42

(bs, 0.5H, NHBoc), 5.49 (d, 0.5H, J ) 8 Hz, NHBoc), 7.05-

7.25 (m, 4H, ArH), 7.3-7.4 (m, 5H, CH₂C₆H₅).

was stirred for 40 min under an Ar atmosphere. To the ylide

solution was added a solution of the aldehyde 19 (8.83 g, 42.2

mmol) in 100 mL of THF at 0 °C. The mixture was stirred for

3.5 h at 0 °C. The reaction was quenched by the addition of 5

mL of 2 N HCl, and then the solvent was evaporated. The

residue was poured into 2 N HCl and extracted with CH₂Cl₂.

The extract was washed with water and brine and dried over

MgSO₄. Concentration and purification by column chroma-

tography on silica gel with CH₂Cl₂/n-hexane ) 1:1 afforded

11.3 g of 20 as a yellow viscous liquid (84%), E,Z-mixture of

the olefin. ¹H NMR (CDCl₃): 0.87 (m, 3H, CH₂(CH₂)₅CH₃), 1.27

(br, 10H, CH₂(CH₂)₅CH₃), 1.46 (m, 2H, CHdCHCH₂CH₂(CH₂)₅-

CH₃), 2.23-2.33 (m, 2H, CHdCHCH₂CH₂(CH₂)₅CH₃), 3.93 (s,

3H, COOCH₃), 5.90 (m, 0.6H, ArCHdCH(CH₂)₇CH₃), 6.44 (m,

1.4H, ArCHdCH(CH₂)₇CH₃), 7.48 (m, 1H, ArH), 7.54-7.59 (m,

1H, ArH), 7.93 (m, 1H, ArH).

(-)-BL-8-C10 (8) a n d (-)-Ep i-BL-8-C10 (17). A mixture

of the diastereomeric esters 16 (1.01 g, 1.78 mmol) and 150

mg of 10% Pd-C in 120 mL of ethanol was vigorously stirred

under 1 atm of H₂at room temperature for 2 h and then

filtered. The filtrate was concentrated to afford the crude

carboxylic acid. The acid and 818 mg (7.1 mmol) of N-

hydroxysuccinimide were dissolved in 18 mL of CH₃CN, and

then a solution of 733 mg (3.6 mmol) of DCC in 2 mL of CH₃-

CN was added at 0 °C with stirring. Stirring was continued

for 2 h at room temperature; then the solvent was removed

under reduced pressure. The residue was suspended in a

small amount of AcOEt, and insoluble urea was filtered off.

The filtrate was concentrated and purified by column chro-

matography on silica gel to give an activated ester as a

colorless liquid. This product was unstable and was used

without further purification. Trifluoroacetic acid (5 mL) was

added to a solution of the activated ester in 5 mL of CH₂Cl₂at

0 °C with stirring. The mixture was stirred for 4 h at room

temperature; then the solvent was removed under reduced

pressure at below 30 °C. The residue was dissolved in 700

mL of AcOEt, and 50 mL of saturated NaHCO₃(aq) was added.

The mixture was refluxed for 6 h with vigorous stirring. The

organic phase was separated, and the aqueous layer was

extracted with AcOEt. The combined extract was washed with

brine, dried over MgSO₄, and concentrated. The crude product

was separated by column chromatography to give 171 mg

(27%) of (-)-BL-8-C10 (8) and 151 mg (24%) (-)-epi-BL-8-C10

(17).

Nitr o Tosyla te 21. To a solution of 2.7 g (126.0 mmol) of

LiBH₄in 500 mL of THF was added 11.2 g (35.1 mmol) of 20

at 0 °C with stirring. The solution was stirred for 1 h at 0 °C

and then for 16 h at room temperature and concentrated. The

residue was poured carefully into ice-water and extracted

with AcOEt. The extract was washed with 10% citric acid-

(aq), water, and brine and dried over MgSO₄. Evaporation and

purification by column chromatography on silica gel (CH₂Cl₂/

AcOEt, 10:1) afforded 7.64 g of the benzyl alcohol (82%) as a

pale-yellow oil. Sodium hydride (60% in oil; 1.20 g, 30 mmol)

was washed with n-hexane, dried under reduced pressure, and

suspended in 40 mL of toluene under an Ar atmosphere. To

this suspension was added a solution of the above benzyl

alcohol (6.4 g, 22.0 mmol) in toluene (80 mL) followed by

p-toluenesulfonyl chloride (5.75 g, 30.2 mmol). After stirring

for 2 h at room temperature, the mixture was poured into 10%

citric acid(aq) and extracted with AcOEt. The organic phase

was washed with water and brine, dried over MgSO₄, and

concentrated. The residue was chromatographed on silica gel

(hexane/CH₂Cl₂, 1:1) to give the tosylate 21 as a pale-red,

viscous oil, E,Z-mixture of the olefin. ¹H NMR (CDCl₃): 0.87

(m, 3H, CH₂(CH₂)₅CH₃), 1.27 (br, 10H, CH₂(CH₂)₅CH₃), 1.46

(m, 2H, CHdCHCH₂CH₂(CH₂)₅CH₃), 2.30 (m, 2H, CHdCHCH₂-

CH₂(CH₂)₅CH₃), 2.46 (s, 3H, ArCH₃), 5.48 (m, 2H, ArCH₂OH),

5.90 (m, 0.6H, ArCHdCH(CH₂)₇CH₃), 6.43 (m, 1.4H, ArCHd

CH(CH₂)₇CH₃), 7.36 (m, 3H, Ts, ArH), 7.61 (s, 0.7H, ArH), 7.63

(s, 0.3H, ArH), 7.61 (s, 0.3H, ArH), 7.84 (d, 2H, J ) 9 Hz, Ts),

8.07 (d, 0.3H, J ) 8 Hz, ArH), 8.10 (d, 0.7H, J ) 8 Hz, ArH).

The optical purity of the BL-8-C10s was 80% ee, because

the hydroxydediazoniation in the preparation of (R)-benzyl

2-hydroxydecanoate (14) required high temperature. Final

optical purification was conducted as follows: the optically

crude 8 was converted to N-tosylvaline ester,^6bwhich was

purified by silica gel column chromatography and recrystal-

lization. Hydrolysis of the purified ester afforded optically

pure 8, colorless viscous oil. [R]²²-237.9° (c ) 0.66, CHCl₃).

D

¹H NMR (CDCl₃): 0.88 (t, 3H, J ) 7 Hz, (CH₂)₈CH₃), 1.22 (m,

16H, -(CH₂)₈CH₃), 1.90 (m, 2H, -CH₂(CH₂)₈CH₃), 2.77 (s, 3H,

N-CH₃), 2.93 (m, 2H, ArCH₂CH-), 3.57 (m, 1H, CH₂OH), 3.67

(t, 1H, CH₃-N-CH-CO), 3.75 (m, 1H, CH₂OH), 4.31 (m, 2H,

-CHCH₂OH), 6.28 (bs, 1H, -NHCO-), 6.93 (m, 1H, ArH), 7.05

(m, 2H, ArH), 7.20 (m, 1H, ArH). HRMS: calcd for C₂₂H₃₆N₂O₂,

360.2777; found, 360.2834.

Diester 22. Sodium hydride (60% in oil; 1.15 g, 28.8 mmol)

was washed with n-hexane, dried under reduced pressure, and

suspended in 50 mL of DMF under an Ar atmosphere.

A

solution of 6.67 g (30.9 mmol) of diethyl acetamidomalonate

in 10 mL of DMF was added to it, followed by a solution of

tosylate 21 (10.77 g, 24.2 mol) in 50 mL of DMF, and the

reaction mixture was stirred for 16 h at room temperature.

The solvent was removed under reduced pressure. The residue

was poured into 2 N HCl and the mixture was extracted with

AcOEt. The organic phase was washed with water and brine,

dried over MgSO₄, and concentrated. Purification by column

chromatography on silica gel (CH₂Cl₂/AcOEt, 10:1) afforded

9.12 g (77%) of the diester 22 as pale-yellow columns. Mp:

58-61 °C. ¹H NMR (CDCl₃): 0.87 (t, 3H, J ) 7 Hz, CH₂-

(CH₂)₅CH₃), 1.27 (m, 16H, CH₂(CH₂)₅CH₃, 2 × -OCH₂CH₃),

1.45 (m, 2H, CHdCHCH₂CH₂(CH₂)₅CH₃), 1.95 (s, 3H, NH-

COCH₃), 2.28 (m, 2H, CHdCHCH₂CH₂(CH₂)₅CH₃), 4.08 (m,

2H, ArCH₂CH), 4.25 (m, 4H, 2 × -OCH₂CH₃), 5.84 (m, 0.6H,

ArCHdCH(CH₂)₇CH₃), 6.34 (m, 1.4H, ArCHdCH(CH₂)₇CH₃),

6.46 (bs, 1H, -NHCO-), 7.10 (d, 0.7H, J ) 1 Hz, ArH), 7.17

(d, 0.3H, J ) 1 Hz, ArH), 7.29 (m, 1H, ArH), 7.83 (d, 0.3H, J

) 8 Hz, ArH), 7.84 (d, 0.7H, J ) 8 Hz, ArH). Anal.

(C₂₆H₃₈N₂O₇) C, H, N.

3-Ca r bom eth oxy-4-n itr oben za ld eh yd e (19). A solution

of 3-carbomethoxy-4-nitrobenzoic acid (18; 22.6 g, 100.4 mmol)

in THF (450 mL) was cooled to 0 °C, and BH₃-methyl sulfide

complex (16 mL, 168.8 mmol) was added. The mixture was

stirred for 30 min at room temperature, refluxed for 5 h, and

then cooled. The reaction was quenched by the addition of

methanol, and the whole was concentrated. The residue was

diluted with saturated NaHCO₃(aq) and extracted with AcOEt.

The organic phase was washed with water and brine, dried

(MgSO₄), and concentrated in vacuo. The crude product was

crystallized from hexane-AcOEt to give 3-carbomethoxy-4-

nitrobenzyl alcohol (19.2 g, 91%). A solution of the benzyl

alcohol (15.5 g, 73.5 mmol) in CH₂Cl₂(100 mL) was added to

a suspension of pyridinium chlorochromate (PCC; 16.8 g, 78

mmol) and neutral activity I Al₂O₃(21.1 g; Merck 70-230

mesh) in CH₂Cl₂(150 mL) under Ar at 0 °C with vigorous

stirring. The stirring was continued for 18 h; then the mixture

was concentrated, charged on a silica gel column, and chro-

matographed to give 3-carbomethoxy-4-nitrobenzaldehyde (19)

(14.4 g, 94%). Mp: 74.5-75.0 °C. Anal. (C₉H₇NO₅) C, H, N.

Nitr o Alcoh ol 23. A mixture of 9.12 g (18.6 mmol) of 22,

20 mL of acetic acid, and 50 mL of concentrated HCl was

refluxed gently for 24 h. The mixture was cooled to room

temperature and added to 200 mL of ice water. The aqueous

layer was washed twice with 50 mL of CH₂Cl₂and then

concentrated to dryness. The residue was dried under reduced

pressure over P₂O₅to give the amino acid as a white solid. A

Nitr o Ester 20. A solution of nonyltriphenylphosphonium

bromide (45.8 g, 97.7 mmol) in 800 mL of THF was treated

with 30 mL of 1.6 M n-BuLi in hexane at 0 °C, and the mixture

Clarification of the Binding Mode of Benzolactams

J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9 1489

solution of the crude amino acid in dry ethanol (40 mL) was

added to a solution of 20.86 g of SOCl₂in 100 mL of dry ethanol

(prepared at -10 °C) with stirring at -40 °C. The mixture

was stirred for 1 h at -40 °C and for 30 h at room temperature

and then concentrated under reduced pressure to remove most

of the ethanol. The residue was poured into saturated

NaHCO₃(aq) and extracted with CH₂Cl₂. The organic phase

was washed with saturated NaHCO₃(aq), water, and brine.

The solution was dried over MgSO₄and then concentrated,

and the residue was redissolved in 200 mL of CH₂Cl₂. To this

solution was added 5.04 g (23.1 mmol) of Boc₂O at 0 °C. The

mixture was stirred for 18 h at room temperature and

concentrated. Purification of the residue by column chroma-

tography on silica gel (CH₂Cl₂) afforded 8.10 g of the N-Boc-

amino acid ethyl ester (91%) as a pale-yellow oil. A solution

of the N-Boc-amino acid ethyl ester (8.10 g, 17.0 mmol) in THF

(50 mL) was added to a stirred solution of 1.28 g (59.7 mmol)

of LiBH₄in THF (150 mL). The solution was stirred for 1 h

at 0 °C and for 17.5 h at room temperature and then

concentrated. The residue was poured carefully into ice water

and extracted with CH₂Cl₂. The extract was washed with

water and brine and dried over MgSO₄. Evaporation and

purification by column chromatography on silica gel afforded

3.93 g of the nitro alcohol 23 (49%) as colorless needles. Mp:

91-93 °C. ¹H NMR (CDCl₃): 0.88 (m, 3H, CH₂(CH₂)₅CH₃), 1.27

(m, 10H, CH₂(CH₂)₅CH₃), 1.37 (s, 9H, Boc-CH₃), 1.46 (m, 2H,

CHdCHCH₂CH₂(CH₂)₅CH₃), 2.24 (dd, 0.6H, J ) 7, 13 Hz,

CHdCHCH₂CH₂(CH₂)₅CH₃), 2.31 (m, 1.4H, CHdCHCH₂-

CH₂(CH₂)₅CH₃), 2.41 (bs, 1H, OH), 3.06 (m, 2H, ArCH₂CH),

3.70 (m, 2H, CH₂OH), 4.00 (m, 1H, CHCH₂OH), 5.04 (bs, 1H,

-NHCO-), 5.85 (m, 0.6H, ArCHdCH(CH₂)₇CH₃), 6.39 (m,

1.4H, ArCHdCH(CH₂)₇CH₃), 7.31 (m, 2H, ArH), 7.95 (m, 1H,

ArH). Anal. (C₂₄H₃₈N₂O₅) C, H, N.

4.77 mmol) in 15 mL of CH₂ClCH₂Cl was refluxed for 24 h,

and then the solvent was removed by evaporation. The residue

was chromatographed on silica gel to give 1.095 g of a

diastereomeric mixture of 25 as a colorless oil (86%). ¹H NMR

(CDCl₃): 0.88 (m, 6H, CH₂(CH₂)₅CH₃, CH(CH₃)₂), 1.16 (m, 3H,

CH(CH₃)₂), 1.26 (m, 14H, CH₂(CH₂)₇CH₃), 1.37 (s, 4.5H, Boc-

CH₃), 1.45 (s, 4.5H, Boc-CH₃), 1.55 (m, 2H, ArCH₂CH₂(CH₂)₇-

CH₃), 2.26 (m, 1H, CH(CH₃)₂), 2.50 (m, 2H, ArCH₂CH₂(CH₂)₇-

CH₃), 2.82 (s, 1.5H, N-CH₃), 2.84 (s, 1.5H, N-CH₃), 2.95 (m,

2H, ArCH₂CH), 3.20 (d, 0.5H, J ) 10 Hz, CH₃NCHCO-), 3.26

(d, 0.5H, J ) 10 Hz, CH₃NCHCO-), 3.41 (m, 0.5H, CH₂OH),

3.60 (m, 1H, CH₂OH), 3.73 (m, 1H, CH₂OH, CHCH₂OH), 3.95

(m, 0.5H, CHCH₂OH), 4.87 (d, 0.5H, J ) 11 Hz, COOCH₂C₆H₅),

4.91 (d, 0.5H, J ) 11 Hz, COOCH₂C₆H₅), 4.94 (m, 1H,

COOCH₂C₆H₅), 5.41 (bd, 0.5H, J ) 5 Hz, -NHCO-), 6.06 (bd,

0.5H, J ) 2 Hz, -NHCO-), 6.85-7.07 (m, 5H, ArH), 7.26 (m,

3H, COOCH₂C₆H₅).

(-)-BL-V8-210 (9) a n d (-)-Ep i-BL-V8-210 (26). A mix-

ture of diastereomeric esters 25 (1.09 g, 1.78 mmol) and 150

mg of 10% Pd-C in 120 mL of ethanol was vigorously stirred

under 1 atm of H₂at room temperature for 2 h and then

filtered. The filtrate was concentrated to afford the crude

carboxylic acid. The acid and 818 mg (7.1 mmol) of N-

hydroxysuccinimide were dissolved in 18 mL of CH₃CN, and

then a solution of 733 mg (3.6 mmol) of DCC in 2 mL of CH₃-

CN was added at 0 °C with stirring. Stirring was continued

for 2 h at room temperature; then the solvent was removed

under reduced pressure. The residue was suspended in a

small amount of AcOEt, and insoluble urea was filtered off.

The filtrate was concentrated and purified by column chro-

matography on silica gel to give an activated ester as a

colorless liquid. This product was unstable and was used

without further purification. Trifluoroacetic acid (5 mL) was

added to a solution of the activated ester in 5 mL of CH₂Cl₂at

0 °C with stirring. The mixture was stirred for 4 h at room

temperature; then the solvent was removed under reduced

pressure at below 30 °C. The residue was dissolved in 1.2 L

of AcOEt, and 100 mL of saturated NaHCO₃(aq) was added.

The mixture was refluxed for 6 h with vigorous stirring. The

organic phase was separated, and the aqueous layer was

extracted with AcOEt. The combined extract was washed with

brine, dried over MgSO₄, and concentrated. The crude product

was separated by column chromatography to give 240 mg

(40%) of (-)-BL-V8-210 (9) and 151 mg (38%) of (-)-epi-BL-

V8-210 (26). 9: colorless viscous oil. [R]²²_D-231.9° (c ) 1.16,

CHCl₃). ¹H NMR (CDCl₃): 0.88 (t, 3H, J ) 7 Hz, (CH₂)₂CH₃),

0.93 (d, 3H, J ) 7 Hz, CH(CH₃)₂), 1.07 (d, 3H, J ) 7 Hz, CH-

(CH₃)₂), 1.26 (m, 14H, -CH₂(CH₂)₇CH₃), 1.58 (m, 2H,

ArCH₂CH₂(CH₂)₇CH₃), 2.48 (m, 1H, CH(CH₃)₂), 2.49 (t, 2H, J

) 8 Hz, ArCH₂CH₂(CH₂)₇CH₃), 2.78 (s, 3H, N-CH₃), 2.85 (d,

1H, J ) 17 Hz, ArCH₂CH-), 3.00 (dd, 1H, J ) 8, 17 Hz, ArCH₂-

CH-), 3.42 (d, 1H, J ) 8 Hz, CH₃-N-CH-CO), 3.55 (dd, 1H, J

) 8, 11 Hz, CH₂OH), 3.74 (dd, 1H, J ) 4, 11 Hz, CH₂OH),

4.27 (m, 1H, -CHCH₂OH), 6.21 (bd, 1H, J ) 2 Hz, -NHCO-

), 6.85 (s, 1H, ArH), 6.96 (d, 1H, J ) 8 Hz, ArH), 7.00 (dd, 1H,

J ) 1, 8 Hz, ArH). HRMS: calcd for C₂₅H₄₂N₂O₂, 402.3248;

Meth yla m in o Alcoh ol 24. A mixture of 2.44 g (5.62 mmol)

of 23 and 240 mg of 10% Pd-C in 200 mL of ethanol was

vigorously stirred under 1 atm of H₂at room temperature for

10 h and then filtered. The filtrate was concentrated, and the

residue was crystallized from benzene to afford 2.09 g of the

amine with a saturated alkyl substituent as colorless needles

(92%). Mp: 115 °C. Anal. (C₂₄H₄₂N₂O₃) C, H, N. A solution

of the amine (1.99 g, 4.90 mmol) in 30 mL of THF was added

at 0 °C to a mixed anhydride solution (prepared by addition

of 1.22 g of formic acid to 2.66 g of acetic anhydride and THF

(5 mL) at 0 °C, followed by heating for 2 h at 60-70 °C). After

having been stirred for 2 h at room temperature, the mixture

was concentrated under reduced pressure. The residue was

poured into saturated NaHCO₃(aq), and the whole was ex-

tracted with AcOEt. The extract was washed with water and

brine and dried over MgSO₄. Concentration and purification

by column chromatography on silica gel with AcOEt/n-hexane

) 1:1 afforded 1.97 g of the N-formate as a pale-yellow oil

(93%). Mp: 93 °C. Anal. (C₂₅H₄₂N₂O₄) C, H, N. To a solution

of the formate (1.89 g, 4.35 mmol) in 300 mL of THF was added

dropwise 19 mL of 1.0 M BH₃in THF solution at 0 °C, and

the mixture was stirred for 2.5 h at 0 °C. The reaction was

quenched by the addition of 10 mL of 10% citric acid, and the

whole was extracted with AcOEt. After concentration, the

residue was poured into saturated NaHCO₃(aq) and extracted

with AcOEt. The organic layer was washed with water and

brine and dried over MgSO₄. Concentration and purification

by column chromatography on silica gel with AcOEt/n-hexane

) 1:1 gave 1.82 g of the methylamino alcohol 24 as colorless

prisms (99%). Mp: 53-55 °C. ¹H NMR (CDCl₃): 0.88 (t, 3H,

J ) 7 Hz, CH₂(CH₂)₅CH₃), 1.26 (m, 14H, CH₂(CH₂)₇CH₃), 1.47

(s, 9H, Boc-CH₃), 1.54 (m, 2H, ArCH₂CH₂(CH₂)₇CH₃), 2.48 (t,

2H, J ) 8 Hz, ArCH₂CH₂(CH₂)₇CH₃), 2.83 (m, 2H, ArCH₂CH),

2.85 (s, 3H, N-CH₃), 3.50 (dd, 1H, J ) 2, 8 Hz, CH₂OH), 3.56

(d, 1H, J ) 8 Hz, CH₂OH), 3.70 (m, 1H, CHCH₂OH), 5.12 (bd,

1H, -NHCO-), 6.61 (d, 1H, J ) 8 Hz, ArH), 6.85 (d, 1H, J )

2 Hz, ArH), 6.99 (dd, 1H, J ) 2, 8 Hz, ArH). Anal.

(C₂₅H₄₄N₂O₃) C, H, N.

found, 402.3237. 26: colorless viscous oil. [R]²²-145.9° (c

D

) 0.92, CHCl₃). HRMS: calcd for C₂₅H₄₂N₂O₂, 402.3248; found,

402.3219.

7-Nitr o-6-(br om om eth yl)-1,2,3,4-tetr a h yd r o-1,1,4,4-tet-

r a m eth yln a p h th a len e (28). To a solution of 1,1,4,4,6-

pentamethyl-1,2,3,4-tetrahydronaphthalene (27; 16.6 g, 82.2

mmol) in 65 mL of AcOH was added dropwise a mixture of

concentrated HNO₃(8.2 mL) and concentrated H₂SO₄(18 mL)

at below 20 °C (required ice bath) with vigorous stirring. After

stirring for 4 h, the mixture was poured into ice-water (200

mL), and the precipitate was collected. The precipitate was

dissolved in CH₂Cl₂and washed with saturated NaHCO₃(aq)

and water. Drying (MgSO₄) and concentration gave a pale-

yellow solid. The crude product was recrystallized from

ethanol to give 12.6 g (62.1%) of 7-nitro-1,1,4,4,6-pentamethyl-

1,2,3,4-tetrahydronaphthalene. Mp: 152-153 °C. Anal.

(C₁₅H₂₁NO₂) C, H, N. To a solution of the nitrotoluene (11.3

g, 45.7 mmol) and N-bromosuccinimide (7.65 g, 45.7 mmol) in

Dia ster eom er ic Ester 25. A mixture of 870 mg (2.07

mmol) of 24, (R)-benzyl 2-[[(trifluoromethyl)sulfonyl]oxy]-

isovalerate^5d(808 mg, 2.38 mmol), and 2,6-lutidine (510 mg,

1490 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9

Endo et al.

CCl₄(100 mL) was added AIBN (150 mg, 0.91 mmol), followed

by a catalytic amount of benzyltrimethylammonium tribromide

at room temperature with stirring. The mixture was heated

to reflux for 3 h. After removal of the precipitate by filtration,

the filtrate was concentrated and chromatographed on silica

gel (hexane/AcOEt, 10:1) to afford 7.37 g (49%) of the benzyl

bromide 28. Mp: 87.5-89 °C (n-hexane). ¹H NMR (CDCl₃):

1.31 (m, 12H, CH₃), 1.71 (s, 4H, -(CH₂)₂-), 4.83 (s, 2H, ArCH₂-

Br), 7.43 (s, 1H, ArH), 8.02 (s, 1H, ArH). Anal. (C₁₅H₂₀NO₂-

Br) C, H, N.

solution of the formate (2.90 g, 7.18 mmol) in 80 mL of THF

was added dropwise 21 mL of 1.0 M BH₃in THF solution at 0

°C, and the mixture was stirred for 3 h at 0 °C. The reaction

was quenched by the addition of 10 mL of 10% citric acid, and

the whole was extracted with AcOEt. After concentration, the

residue was poured into saturated NaHCO₃(aq) and extracted

with AcOEt. The organic layer was washed with water and

brine and dried over MgSO₄. Concentration and purification

by column chromatography on silica gel (hexane/AcOEt, 2:1)

gave 2.72 g of the methylamino alcohol 31 as a colorless

amorphous gum (97%). ¹H NMR (CDCl₃): 1.25 (m, 12H, CH₃),

1.46 (s, 9H, Boc-CH₃), 1.65 (s, 4H, -(CH₂)₂-), 2.67 (m, 1H,

ArCH₂CH), 2.83 (m, 1H, ArCH₂CH), 2.87 (s, 3H, N-CH₃), 3.52

(m, 2H, CH₂OH), 3.79 (m, 1H, CHCH₂OH), 5.13 (bs, 1H,

-NHCO-), 6.70 (s, 1H, ArH), 6.97 (s, 1H, ArH). HRMS: calcd

for C₂₃H₃₈N₂O₃, 390.2883; found, 390.2896.

Diester 29. The nitrobenzyl bromide 28 (7.37 g, 22.6 mmol)

was converted into the diester 29 by the same method as that

used for the preparation of 22 employing 1.08 g (27.0 mmol)

of NaH, 4.90 g (22.6 mmol) of diethyl acetamidomalonate, and

50 mL of DMF for 2.5 h at room temperature under an Ar

atmosphere. Purification by column chromatography on silica

gel (hexane/AcOEt, 4:1) gave 9.44 g (90%) of the diester 29 as

pale-yellow prisms. Mp: 131-132 °C (hexane/AcOEt). ¹H

NMR (CDCl₃): 1.28 (m, 18H, CH₃, 2 × -OCH₂CH₃), 1.69 (s,

4H, -(CH₂)₂-), 1.95 (s, 3H, NHCOCH₃), 4.03 (s, 2H, ArCH₂-

CH), 4.2 (m, 4H, 2 × -OCH₂CH₃), 6.46 (bs, 1H, -NHCO-),

7.13 (s, 1H, ArH), 7.79 (s, 1H, ArH). Anal. (C₂₄H₃₄N₂O₇) C,

H, N.

Dia ster eom er ic Ester 32. A mixture of 2.0 g (5.12 mmol)

of 31, (R)-benzyl 2-[[(trifluoromethyl)sulfonyl]oxy]isovalerate^5d

(4.36 g, 12.8 mmol), and 2,6-lutidine (1.65 g, 15.4 mmol) in 80

mL of CH₂ClCH₂Cl was refluxed for 48 h, and then the solvent

was removed by evaporation. The residue was chromato-

graphed on silica gel to give 2.42 g of a diastereomeric mixture

of 32 as a colorless amorphous gum (82%).

Nitr o Alcoh ol 30. A mixture of 9.24 g (20.0 mmol) of 29,

40 mL of acetic acid, and 20 mL of concentrated HCl was

refluxed gently for 14 h. The mixture was concentrated, and

water was removed azeotropically by the addition of ethanol

three times. The residue was dissolved in dry ethanol (40 mL),

and the solution was added to a solution of 20.0 g of SOCl₂in

60 mL of dry ethanol (prepared at -10 °C) with stirring at

-40 °C. The mixture was stirred for 1 h at -40 °C and then

for 20 h at room temperature and then concentrated under

reduced pressure to remove most of the ethanol. The residue

was poured into saturated NaHCO₃(aq) and extracted with

AcOEt. The organic phase was washed with saturated

NaHCO₃(aq), water, and brine. The solution was dried over

MgSO₄and then concentrated, and the residue was redissolved

in 20 mL of CH₂Cl₂. To this solution was added 5.45 g (25.0

mmol) of Boc₂O at 0 °C. The mixture was stirred for 18 h at

room temperature and concentrated. Purification by column

chromatography on silica gel (hexane/AcOEt, 8:1) afforded 8.41

g of the N-Boc-amino acid ethyl ester (94%) as colorless plates.

Mp: 134-135.5 °C (hexane). Anal. (C₂₄H₃₆N₂O₆) C, H, N. A

solution of the N-Boc-amino acid ethyl ester (8.41 g, 18.8 mmol)

in THF (80 mL) was added to a stirred solution of 1.23 g (56.4

mmol) of LiBH₄in THF (80 mL). The solution was stirred for

1 h at 0 °C and for 18 h at room temperature and concentrated.

The residue was poured carefully into ice water and extracted

with AcOEt. The extract was washed with water and brine

and dried over MgSO₄. Evaporation afforded 7.66 g of the nitro

alcohol 30 (100%) as a pale-yellow gum. ¹H NMR (CDCl₃):

1.30 (m, 12H, CH₃), 1.36 (s, 9H, Boc-CH₃), 1.70 (s, 4H,

-(CH₂)₂-), 2.96 (m, 1H, ArCH₂CH), 3.20 (m, 1H, ArCH₂CH),

3.78 (m, 2H, CH₂OH), 3.98 (m, 1H, CHCH₂OH), 4.95 (bd, 1H,

J ) 8 Hz, -NHCO-), 7.24 (s, 1H, ArH), 7.89 (s, 1H, ArH).

HRMS: calcd for C₂₂H₃₄N₂O₅, 406.2468; found, 406.2467.

Meth yla m in o Alcoh ol 31. A mixture of 5.66 g (13.9 mmol)

of 30 and 500 mg of 10% Pd-C in 500 mL of ethanol was

vigorously stirred under 1 atm of H₂at room temperature for

6 h and then filtered. The filtrate was concentrated, and the

residue was crystallized from n-hexane to afford 4.84 g of the

amine as a colorless powder (92%). Mp: 114-115 °C. Anal.

(C₂₂H₃₆N₂O₃) C, H, N. A solution of the amine (3.0 g, 7.98

mmol) in 30 mL of THF was added at 0 °C to a mixed

anhydride solution (prepared by addition of 2.0 g of formic acid

to 4.0 g of acetic anhydride and THF (5 mL) at 0 °C, followed

by heating at 60 °C for 2 h at 60-70 °C). After having been

stirred for 2.5 h at room temperature, the mixture was

concentrated under reduced pressure. The residue was poured

into saturated NaHCO₃(aq), and the whole was extracted with

AcOEt. The extract was washed with water and brine and

dried over MgSO₄. Concentration and purification by column

chromatography on silica gel (hexane/AcOEt, 5:6) afforded 2.90

g of the N-formate as a colorless amorphous gum (90%). To a

(-)-BL-V8-23TM (10) a n d (-)-Ep i-BL-V8-23TM (33). A

mixture of diastereomeric esters 32 (2.40 g, 4.14 mmol) and

300 mg of 10% Pd-C in 200 mL of ethanol was vigorously

stirred under 1 atm of H₂at room temperature for 2 h and

then filtered. The filtrate was concentrated to afford the crude

carboxylic acid. The acid and 952 mg (8.28 mmol) of N-

hydroxysuccinimide were dissolved in 18 mL of CH₃CN, and

then a solution of 1.27 g (6.17 mmol) of DCC in 10 mL of CH₃-

CN was added at 0 °C with stirring. Stirring was continued

for 2 h at room temperature; then the solvent was removed

under reduced pressure. The residue was suspended in a

small amount of AcOEt, and insoluble urea was filtered off.

The filtrate was concentrated and purified by column chro-

matography on silica gel (hexane/AcOEt, 5:3) to give an

activated ester as a colorless amorphous gum. This product

was unstable and was used without further purification.

Trifluoroacetic acid (20 mL) was added to a solution of the

activated ester in 20 mL of CH₂Cl₂at 0 °C with stirring. The

mixture was stirred for 2 h at room temperature; then the

solvent was removed under reduced pressure at below 30 °C.

The residue was dissolved in 800 mL of AcOEt, and 60 mL of

saturated NaHCO₃(aq) was added. The mixture was refluxed

for 4 h with vigorous stirring. The organic phase was

separated, and the aqueous layer was extracted with AcOEt.

The combined extract was washed with brine, dried over

MgSO₄, and concentrated. The crude product was separated

by column chromatography to give 532 mg (37%) of (-)-BL-

V8-23TM (10) and 485 mg (33%) of (-)-epi-BL-V8-23TM (33).

10: colorless needles. Mp: 210-212 °C (CH₂Cl₂/hexane). [R]²²

D

-287.0° (c ) 0.46, CHCl₃). ¹H NMR (CDCl₃): 0.93 (d, 3H, J )

7 Hz, CH(CH₃)₂), 1.07 (d, 3H, J ) 7 Hz, CH(CH₃)₂), 1.24 (m,

12H, CH₃), 1.65 (s, 4H, -(CH₂)₂-), 2.42 (m, 1H, CH(CH₃)₂),

2.78 (m, 1H, ArCH₂CH-), 2.79 (s, 3H, N-CH₃), 3.01 (m, 1H,

ArCH₂CH-), 3.50-3.78 (m, 3H, CH₂OH, CH₃-N-CH-CO), 4.10

(m, 1H, -CHCH₂OH), 6.21 (bs, 1H, -NHCO-), 6.90 (s, 1H,

ArH), 6.98 (s, 1H, ArH). Anal. (C₂₃H₃₆N₂O₂) C, H, N. 33:

colorless needles. Mp: 209.5-211 °C (CH₂Cl₂/hexane). [R]²²

D

-153.5° (c ) 0.34, CHCl₃). HRMS: calcd for C₂₂H₃₆N₂O₂,

372.2787; found, 372.2763.

7-Br om o-3,4-d ih yd r o-4,4-d im e t h yl-1(2H )-n a p h t h a -

len on e (39). A solution of 4,4-dimethyl-γ-butyrolactone (20.45

g, 0.179 mol) in dry benzene (30 mL) was added over 45 min

to a stirred mixture of AlCl₃(71.5 g, 0.536 mol) and dry

benzene (120 mL) held at 5 °C. The mixture was warmed

slowly to 90-100 °C and held at that temperature for 2.5 h.

Decomposition with ice and HCl gave a benzene layer, which

was washed successively with 2 N HCl, water, and saturated

NaHCO₃(aq), then dried (MgSO₄), and concentrated. Distil-

lation of the residue through a Vigreux column gave 22.32 g

(72%) of 3,4-dihydro-4,4-dimethyl-1(2H)-naphthalenone³¹as a

colorless liquid. Bp: 120 °C (7 mmHg). This ketone (22.32 g)

Clarification of the Binding Mode of Benzolactams

J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9 1491

was added dropwise to a stirred mixture of AlCl₃(42.47 g,

0.318 mol) and CH₂Cl₂(10 mL), followed by similar addition

of bromine (8 mL).³²The mixture was vigorously stirred for

30 min at 80 °C. After cooling, the mixture was acidified with

6 N HCl at 0 °C and extracted with ether. The organic phase

was washed with water and saturated NaHCO₃(aq), dried

(Na₂SO₄), and concentrated. Distillation afforded 21.17 g

(65%) of 7-bromo-3,4-dihydro-4,4-dimethyl-1(2H)-naphthale-

none (39) as colorless cubes. Bp: 110 °C (0.3 mmHg). Mp:

56-57 °C (n-hexane). ¹H NMR (CDCl₃): 1.38 (s, 6H, CH₃), 2.01

(t, 2H, J ) 8.2 Hz, -COCH₂CH₂-), 2.73 (t, 2H, J ) 8.2 Hz,

-COCH₂CH₂-), 7.31 (d, 1H, J ) 8.4 Hz, ArH), 7.62 (dd, 1H,

J ) 2.2, 8.4 Hz, ArH), 8.13 (dd, 1H, J ) 1.5, 2.2 Hz, ArH).

Anal. (C₁₂H₁₃OBr) C, H, N.

6-Br om o-1,1-d im et h yl-1,2,3,4-t et a h yd r on a p h t h a len e

(40). A solution of 39 (11.52 g, 45.5 mmol) in CF₃COOH (50

mL) was added to triethylsilane (12.77 g, 0.11 mol) at 0 °C

with stirring.³³The mixture was stirred for 2 h at 0 °C and

then for 6 h at room temperature. It was poured into

saturated NaHCO₃(aq) and extracted with hexane. The

organic phase was washed with water and brine, dried

(MgSO₄), and concentrated. Distillation afforded 21.17 g (65%)

of 6-bromo-1,1-dimethyl-1,2,3,4-tetrahydronaphthalene (40) as

a colorless liquid. Bp: 88-89 °C (1 mmHg). ¹H NMR (CDCl₃):

1.38 (s, 6H, CH₃), 1.64 (m, 2H, -CH₂CH₂C(CH₃)₂), 1.78 (m,

2H, ArCH₂CH₂CH₂), 2.73 (t, 2H, J ) 6.4 Hz, ArCH₂CH₂CH₂),

7.18 (m, 2H, ArH), 7.24 (dd, 1H, J ) 2.2, 8.4 Hz, ArH). HRMS:

calcd for C₁₂H₁₅⁷⁹Br, 238.0358; found, 238.0347.

1,1-Dim et h yl-6-(h yd r oxym et h yl)-1,2,3,4-t et r a h yd r o-

n a p h th a len e (41). A stirred solution of 40 (7.98 g, 33.4 mmol)

in dry ether (80 mL) was treated with 1.6 M n-BuLi in hexanes

(25 mL, 40 mmol) at room temperature. The mixture was

stirred for 20 min at room temperature; then 2.6 mL of

dimethylformamide was added at -20 °C. Stirring was

continued at 0 °C for 20 min; then the reaction was quenched

by the addition of a small amount of water, and the mixture

was poured into 2 N HCl. The mixture was extracted with

ether and washed with water and brine. After drying (MgSO₄),

the solution was concentrated to give 1,1-dimethyl-1,2,3,4-

tetrahydro-6-naphthalenaldehyde. The aldehyde was dis-

solved in methanol (180 mL), and NaBH₄(2.28 g) was added

at 0 °C, with stirring. After stirring for 30 min at 0 °C, the

mixture was extracted with ether. The organic phase was

washed with brine and dried (MgSO₄). Concentration and

purification by column chromatography on silica gel (hexane/

CH₂Cl₂, 1:0 then 1:3) afforded 6.03 g (95%) of 1,1-dimethyl-6-

(hydroxymethyl)-1,2,3,4-tetrahydronaphthalene (41) as a col-

orless oil. ¹H NMR (CDCl₃): 1.28 (s, 6H, CH₃), 1.66 (m, 2H,

-CH₂CH₂C(CH₃)₂), 1.81 (m, 2H, ArCH₂CH₂CH₂), 2.77 (t, 2H,

J ) 6.4 Hz, ArCH₂CH₂CH₂), 4.62 (s, 2H, ArCH₂OH), 7.06 (s,

1H, ArH), 7.15 (d, 1H, J ) 8.1 Hz, ArH), 7.33 (d, 1H, J ) 8.1

Hz, ArH). HRMS: calcd for C₁₃H₁₈O, 190.1358; found, 190.1360.

1,1-Dim eth yl-6-(br om om eth yl)-1,2,3,4-tetr a h yd r on a p h -

th a len e (42). Triphenylphosphine (13.22 g, 50.4 mmol) was

added as a bolus to a stirred solution of 41 (6.40 g, 33.6 mmol)

and CCl₄(13.95 g, 42.1 mmol) in CH₂Cl₂(50 mL) at 0 °C. After

stirring for 15 min at 0 °C, the mixture was concentrated, and

ether was added. The precipitate was filtered off, and the

filtrate was chromatographed on silica gel (n-hexane) to give

1,1-dimethyl-6-(bromomethyl)-1,2,3,4-tetrahydronaphtha-

lene (42) as a pale-yellow oil. HRMS: calcd for C₁₃H₁₇⁷⁹Br,

252.0513; found, 252.0517.

(CDCl₃): 1.31 (s, 6H, CH₃), 1.69 (m, 2H, -CH₂CH₂C(CH₃)₂),

1.84 (m, 2H, ArCH₂CH₂CH₂), 2.81 (t, 2H, J ) 6.4 Hz, ArCH₂-

CH₂CH₂), 4.80 (s, 2H, ArCH₂Br), 7.20 (s, 1H, ArH), 8.05 (s,

1H, ArH). HRMS: calcd for C₁₃H₁₆NO₂⁷⁹Br, 299.0345; found,

299.0335.

Diester 44. The nitrobenzyl bromide 43 (2.86 g, 9.59 mmol)

was converted into the diester 44 by the same method as that

used for the preparation of 29, employing 387 mg (9.68 mmol)

of NaH, 2.09 g (9.62 mmol) of diethyl acetamidomalonate, and

30 mL of DMF for 2.5 h at room temperature under an Ar

atmosphere. Purification by column chromatography on silica

gel (hexane/AcOEt, 4:1) gave 4.04 g of the diester 44 as a

colorless powder (97%). Mp: 131-132 °C (hexane/AcOEt). ¹H

NMR (CDCl₃): 1.28 (m, 12H, CH₃, 2 × -OCH₂CH₃), 1.66 (m,

2H, -CH₂CH₂C(CH₃)₂), 1.81 (m, 2H, ArCH₂CH₂CH₂), 1.97 (s,

3H, NHCOCH₃), 2.72 (t, 2H, J ) 6.4 Hz, ArCH₂CH₂CH₂), 3.99

(s, 2H, ArCH₂CH), 4.24 (m, 4H, 2 × -OCH₂CH₃), 6.45 (bs, 1H,

-NHCO-), 6.86 (s, 1H, ArH), 7.81 (s, 1H, ArH). Anal.

(C₂₂H₃₀N₂O₇) C, H, N.

Nitr o Alcoh ol 45. The diester 44 (3.97 g, 9.14 mmol) was

converted into the nitro alcohol 45 by the same method as that

used for the preparation of 30 from 29. Purification of the

intermediate N-Boc nitro monoester by column chromatogra-

phy on silica gel (n-hexane/AcOEt, 2:1) and recrystallization

of the nitro alcohol afforded 2.76 g (71%) of 45 as colorless

prisms. Mp: 138.5-140 °C (hexane/AcOEt). ¹H NMR (CDCl₃):

1.27 (s, 6H, CH₃), 1.37 (s, 9H, Boc-CH₃), 1.68 (m, 2H,

-CH₂CH₂C(CH₃)₂), 1.82 (m, 2H, ArCH₂CH₂CH₂), 2.78 (t, 2H,

J ) 6.4 Hz, ArCH₂CH₂CH₂), 3.00 (m, 1H, ArCH₂CH), 3.15 (m,

1H, ArCH₂CH), 3.70 (m, 2H, CH₂OH), 3.95 (m, 1H, CHCH₂-

OH), 5.03 (bs, 1H, -NHCO-), 7.08 (s, 1H, ArH), 7.93 (s, 1H,

ArH). Anal. (C₂₀H₃₀N₂O₅) C, H, N.

Meth yla m in o Alcoh ol 46. The procedure was the same

as that used for the preparation of 31, employing 2.76 g (7.30

mmol) of 45. Purification by column chromatography on silica

gel (n-hexane/AcOEt, 3:1) afforded 1.97 g (75%) of the methyl-

amino alcohol 46 as a colorless amorphous gum. ¹H NMR

(CDCl₃): 1.28 (s, 3H, CH₃), 1.29 (s, 3H, CH₃), 1.46 (s, 9H, Boc-

CH₃), 1.63 (m, 2H, -CH₂CH₂C(CH₃)₂), 1.76 (m, 2H, ArCH₂CH₂-

CH₂), 2.64 (m, 3H, ArCH₂CH₂CH₂, ArCH₂CH), 2.79 (m, 1H,

ArCH₂CH), 2.87 (s, 3H, NCH₃), 3.53 (m, 2H, CH₂OH), 3.71 (m,

1H, CHCH₂OH), 5.16 (bs, 1H, -NHCO-), 6.71 (s, 1H, ArH),

6.75 (s, 1H, ArH). HRMS: calcd for C₂₁H₃₄N₂O₃, 362.2569;

found, 362.2581.

Dia ster eom er ic Ester 47. The methylamino alcohol 46

(1.61 g, 4.44 mmol) was converted into the diastereomeric ester

47 by the same method as that used for the preparation of 32,

employing 6.64 g (19.5 mmol) of (R)-benzyl 2-[[(trifluorometh-

yl)sulfonyl]oxy]isovalerate, 1.62 g (15.1 mmol) of 2,6-lutidine,

and 50 mL of CH₂ClCH₂Cl for 40 h at refluxing temperature.

Purification by column chromatography on silica gel (hexane/

AcOEt, 20:1) gave 2.01 g (82%) of the diastereomeric ester 47

as a colorless amorphous gum.

(-)-BL-V8-DM1 (35) a n d (-)-Ep i-BL-V8-DM1 (48). The

procedure was the same as that used for the preparation of

10 and 33 employing 1.68 g (3.33 mmol) of 47 via the activated

ester. Purification of the activated ester by column chroma-

tography on silica gel (n-hexane/AcOEt, 7:1) followed by

condensation and separation of 35 and 48 by column chroma-

tography on silica gel (n-hexane/AcOEt, 1:2) afforded 343 mg

(33%) of (-)-BL-V8-DM1 (35) and 310 mg (30%) of (-)-epi-

BL-V8-DM1 (48). 35: colorless needles. Mp: 179-181 °C (CH₂-

1,1-Dim eth yl-6-(br om om eth yl)-7-n itr o-1,2,3,4-tetr a h y-

d r on a p h th a len e (43). To a solution of 42 (5.6 g, 22.2 mmol)

in 12 mL of Ac₂O was added dropwise a mixture of fuming

HNO₃(2 mL), AcOH (2 mL), and Ac₂O (2 mL) at 0 °C with

vigorous stirring. After stirring for 20 min at 0 °C, the mixture

was poured into ice-water (100 mL) and extracted with

AcOEt. The organic phase was washed with saturated water

and brine. Drying (Na₂SO₄) and concentration gave a pale-

yellow solid. The crude product was chromatographed on silica

gel (hexane/CH₂Cl₂, 1:0 then 1:8) to give 2.09 g (32%) of 1,1-

dimethyl-6-(bromomethyl)-7-nitro-1,2,3,4-tetrahydronaphtha-

lene (43) and the 5-nitro isomer (1.31 g, 19%). ¹H NMR

Cl₂/hexane). [R]²²-294.0° (c ) 0.94, CHCl₃). ¹H NMR

D

(CDCl₃): 0.94 (d, 3H, J ) 6.6 Hz, CH(CH₃)₂), 1.06 (d, 3H, J )

6.6 Hz, CH(CH₃)₂), 1.25 (s, 6H, CH₃), 1.62 (m, 2H, -CH₂CH₂C-

(CH₃)₂), 1.76 (m, 2H, ArCH₂CH₂CH₂), 2.42 (m, 1H, CH(CH₃)₂),

2.64 (t, 2H, J ) 6.3 Hz, ArCH₂CH₂CH₂), 2.79 (s, 3H, N-CH₃),

2.80 (m, 1H, ArCH₂CH-), 2.94 (dd, 1H, J ) 8.4, 16.4 Hz,

ArCH₂CH-), 3.42 (d, 1H, CH₃-N-CH-CO), 3.52 (dd, 1H, J )

8.0, 10.6 Hz, -CHCH₂OH), 3.70 (dd, 1H, J ) 4.0, 10.6 Hz,

-CHCH₂OH), 4.26 (m, 1H, -CHCH₂OH), 6.33 (bs, 1H, -NH-

CO-), 6.72 (s, 1H, ArH), 7.00 (s, 1H, ArH). Anal. (C₂₁H₃₂N₂O₂)

1492 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9

Endo et al.

C, H, N. 48: colorless needles. Mp: 215.5-217 °C (CH₂Cl₂/

hexane). [R]²²_D-154.4° (c ) 1.08, CHCl₃). Anal. (C₂₁H₃₂N₂O₂)

C, H, N.

NHCOCH₃), 2.77 (t, 2H, J ) 6.4 Hz, ArCH₂CH₂CH₂), 4.04 (s,

2H, ArCH₂CH), 4.24 (m, 4H, 2 × -OCH₂CH₃), 6.44 (bs, 1H,

-NHCO-), 7.15 (s, 1H, ArH), 7.55 (s, 1H, ArH). HRMS: calcd

for C₂₂H₃₀N₂O₇, 434.2053; found, 434.2069.

1-(4′-Br om op h en yl)-4-m eth yl-1-p en ten -3-on e (49). To

a stirred solution of 4-bromobenzaldehyde (29.45 g, 0.16 mol)

and 3-methylbutan-2-one (13.71 g, 0.16 mol) in ethanol was

added 10% NaOH(aq) (6 mL). The mixture was stirred at

room temperature for 3 h; then water was added, and the

whole was extracted with benzene. The organic phase was

washed with water and brine, dried (MgSO₄), and concen-

trated. Distillation gave 1-(4′-bromophenyl)-4-methyl-1-penten-

3-one (49) as a yellow oil. Bp: 120 °C (1.5 mmHg). HRMS:

calcd for C₁₂H₁₃O⁷⁹Br, 252.0150; found, 252.0134.

7-B r o m o -1,1-d im e t h y l-1,2,3,4-t e t r a h y d r o n a p h t h a -

len e (50). A solution of 49 (15.99 g, 63.1 mmol) in dry ether

(100 mL) was added to a stirred suspension of LiAlH₄(1.55 g)

in dry ether (60 mL) at 0 °C under an Ar atmosphere. The

mixture was stirred at room temperature for 15 min; then

the reaction was quenched by the addition of a small amount

of AcOEt, and the mixture was poured into 2 N HCl. The

mixture was extracted with AcOEt, and the extract was

washed with water and saturated NaHCO₃(aq). Drying

(MgSO₄) and concentration gave the crude alcohol. To the

alcohol was added polyphosphoric acid (96 g), and the mixture

was heated at 150 °C for 15 min with stirring and then cooled.

Water was added, and the whole was extracted with ether.

The organic phase was washed with water and brine, dried

over MgSO₄, and concentrated. Distillation gave 7-bromo-1,1-

dimethyl-1,2,3,4-tetrahydronaphthalene (50) as a colorless oil.

Bp: 105 °C (2 mmHg). ¹H NMR (CDCl₃): 1.26 (s, 6H, CH₃),

1.64 (m, 2H, -CH₂CH₂C(CH₃)₂), 1.80 (m, 2H, ArCH₂CH₂CH₂),

2.69 (t, 2H, J ) 6.4 Hz, ArCH₂CH₂CH₂), 6.90 (d, 1H, J ) 8.1

Hz, ArH), 7.16 (dd, 1H, J ) 2.2, 8.1 Hz, ArH), 7.42 (d, 1H, J

) 8.1 Hz, ArH). HRMS: calcd for C₁₂H₁₅⁷⁹Br, 238.0358; found,

238.0368.

1,1-Dim et h yl-7-(h yd r oxym et h yl)-1,2,3,4-t et r a h yd r o-

n a p h th a len e (51). The procedure was the same as that used

for the preparation of 41, employing 9.35 g (39.1 mmol) of 50.

Purification by column chromatography on silica gel (n-hexane/

CH₂HCl₂, 1:0 then 1:3) afforded 6.90 g (93%) of 1,1-dimethyl-

7-(hydroxymethyl)-1,2,3,4-tetrahydronaphthalene (51) as a

colorless oil. ¹H NMR (CDCl₃): 1.29 (s, 6H, CH₃), 1.67 (m, 2H,

-CH₂CH₂C(CH₃)₂), 1.80 (m, 2H, ArCH₂CH₂CH₂), 2.76 (t, 2H,

J ) 6.4 Hz, ArCH₂CH₂CH₂), 4.64 (s, 2H, ArCH₂OH), 7.04 (d,

1H, J ) 7.7 Hz, ArH), 7.07 (dd, 1H, J ) 1.8, 7.7 Hz, ArH),

7.32 (s, 1H, ArH). HRMS: calcd for C₁₃H₁₈O, 190.1358; found,

190.1356.

1,1-Dim eth yl-7-(br om om eth yl)-1,2,3,4-tetr a h yd r on a p h -

th a len e (52). The procedure was the same as that used for

the preparation of 42, employing 6.85 g (36.0 mmol) of 51.

Purification by column chromatography on silica gel (n-hexane)

afforded 5.76 g (63%) of 1,1-dimethyl-7-(bromomethyl)-1,2,3,4-

tetrahydronaphthalene (52) as a colorless oil. HRMS: calcd

for C₁₃H₁₇⁷⁹Br, 252.0513; found, 252.0507.

Nitr o Alcoh ol 54. The diester 53 (2.19 g, 5.04 mmol) was

converted into the nitro alcohol 54 by the same method as that

used for the preparation of 30 from 29. Purification of the

intermediate N-Boc nitro monoester by column chromatogra-

phy on silica gel (n-hexane/AcOEt, 2:1) and column chroma-

tography of the nitro alcohol on silica gel (n-hexane/AcOEt,

2:1) afforded 1.12 g (61%) of 54 as a yellow amorphous gum.

¹H NMR (CDCl₃): 1.29 (s, 6H, CH₃), 1.38 (s, 9H, Boc-CH₃), 1.68

(m, 2H, -CH₂CH₂C(CH₃)₂), 1.82 (m, 2H, ArCH₂CH₂CH₂), 2.79

(t, 2H, J ) 6.2 Hz, ArCH₂CH₂CH₂), 3.01 (m, 1H, ArCH₂CH),

3.18 (m, 1H, ArCH₂CH), 3.70 (m, 2H, CH₂OH), 3.96 (m, 1H,

CHCH₂OH), 4.97 (bs, 1H, -NHCO-), 7.30 (s, 1H, ArH), 7.67

(s, 1H, ArH). HRMS: calcd for C₂₀H₃₀N₂O₅, 378.2155; found,

378.2142.

Meth yla m in o Alcoh ol 55. The procedure was the same

as that used for the preparation of 31, employing 1.10 g (2.89

mmol) of 54. Purification by column chromatography on silica

gel (n-hexane/AcOEt, 2:1) afforded 621 mg (63%) of the

methylamino alcohol 55 as a colorless amorphous gum. ¹H

NMR (CDCl₃): 1.26 (s, 3H, CH₃), 1.28 (s, 3H, CH₃), 1.46 (s,

9H, Boc-CH₃), 1.63 (m, 2H, -CH₂CH₂C(CH₃)₂), 1.76 (m, 2H,

ArCH₂CH₂CH₂), 2.64 (m, 3H,ArCH₂CH₂CH₂, ArCH₂CH), 2.72

(t, 2H, J ) 6.2 Hz, ArCH₂CH), 2.82 (m, 1H, ArCH₂CH), 2.83

(s, 3H, NCH₃), 3.53 (m, 2H, CH₂OH), 3.71 (m, 1H, CHCH₂-

OH), 5.18 (bs, 1H, -NHCO-), 6.36 (s, 1H, ArH), 6.96 (s, 1H,

ArH). HRMS: calcd for C₂₁H₃₄N₂O₃, 362.2569; found, 362.2567.

Dia ster eom er ic Ester 56. The methylamino alcohol 55

(511 mg, 1.41 mmol) was converted into the diastereomeric

ester 56 by the same method as that used for the preparation

of 32, employing 1.08 g (3.18 mmol) of (R)-benzyl 2-[[(trifluo-

romethyl)sulfonyl]oxy]isovalerate, 509 mg (4.75 mmol) of 2,6-

lutidine, and 15 mL of CH₂ClCH₂Cl for 40 h at refluxing

temperature. Purification by column chromatography on silica

gel (hexane/AcOEt, 20:1) gave 654 mg (84%) of the diastere-

omeric ester 56 as a colorless amorphous gum.

(-)-BL-V8-DM3 (36) a n d (-)-Ep i-BL-V8-DM3 (57). The

procedure was the same as that used for the preparation of

10 and 33 employing 612 mg (1.11 mmol) of 56 via the

activated ester. Purification of the activated ester by column

chromatography on silica gel (n-hexane/AcOEt, 7:1), followed

by condensation and separation of 36 and 57 by column

chromatography on silica gel (n-hexane/AcOEt, 1:2), afforded

148 mg (38%) of (-)-BL-V8-DM3 (36) and 148 mg (38%) of (-)-

epi-BL-V8-DM3 (57). 36: colorless needles. Mp: 186-187.5

°C (CH₂Cl₂/hexane). [R]²²-299.7° (c ) 0.86, CHCl₃). ¹H

D

NMR (CDCl₃): 0.93 (d, 3H, J ) 6.6 Hz, CH(CH₃)₂), 1.07 (d,

3H, J ) 6.6 Hz, CH(CH₃)₂), 1.23 (s, 6H, CH₃), 1.62 (m, 2H,

-CH₂CH₂C(CH₃)₂), 1.76 (m, 2H, ArCH₂CH₂CH₂), 2.43 (m, 1H,

CH(CH₃)₂), 2.68 (t, 2H, J ) 6.3 Hz, ArCH₂CH₂CH₂), 2.76 (s,

3H, N-CH₃) 2.84 (m, 1H, ArCH₂CH-), 2.99 (dd, 1H, J ) 8.4,

16.5 Hz, ArCH₂CH-), 3.43 (d, 1H, CH₃-N-CH-CO), 3.55 (dd,

1H, J ) 8.0, 10.6 Hz, -CHCH₂OH), 3.74 (dd, 1H, J ) 4.0, 10.6

Hz, -CHCH₂OH), 4.27 (m, 1H, -CHCH₂OH), 6.24 (bs, 1H,

-NHCO-), 6.69 (s, 1H, ArH), 6.94 (s, 1H, ArH). Anal. Calcd

for C₂₁H₃₂N₂O₂: C, 73.22; H, 9.36; N, 8.13. Found: C, 72.94;

H, 9.41; N, 8.04. 57: colorless amorphous gum. [R]²²_D-142.4°

(c ) 1.08, CHCl₃). HRMS: calcd for C₂₁H₃₂N₂O₂, 344.2464;

found, 344.2468.

Diester 53. A mixture of fuming HNO₃(2 mL), AcOH (2

mL) and Ac₂O (2 mL) was added dropwise to a solution of 52

(5.75 g, 22.7 mmol) in 12 mL of Ac₂O at 0 °C with vigorous

stirring. The mixture was stirred for 20 min at 0 °C, poured

into ice-water (100 mL), and extracted with AcOEt. The

organic phase was washed with saturated water and brine.

Drying (Na₂SO₄) and concentration gave a pale-yellow solid.

The crude product was chromatographed on silica gel (hexane/

CH₂Cl₂, 1:30) to give a mixture of 1,1-dimethyl-7-(bromo-

methyl)-6-nitro-1,2,3,4-tetrahydronaphthalene and the 5-nitro

isomer (4.97 g, 73%). The mixture of nitrobenzyl bromides

(4.96 g, 16.6 mmol) was converted into the diester 53 by the

same method as that used for the preparation of 44, employing

669 mg (16.75 mmol) of NaH, 3.61 g (16.6 mmol) of diethyl

acetamidomalonate, and 30 mL of DMF for 2.5 h at room

temperature under an Ar atmosphere. Purification by column

chromatography on silica gel (hexane/AcOEt, 4:1) gave 2.19 g

of the diester 53 as a yellow amorphous gum (30%). ¹H NMR

(CDCl₃): 1.29 (m, 12H, CH₃, 2 × -OCH₂CH₃), 1.67 (m, 2H,

-CH₂CH₂C(CH₃)₂), 1.82 (m, 2H, ArCH₂CH₂CH₂), 1.96 (s, 3H,

1-(3-P h en ylpr opyl)cycloh exan ol (58). A solution of 1-bro-

mo-3-phenylpropane (19.9 g, 99.9 mml) in dry ether (30 mL)

was added to a stirred mixture of Mg turnings (2.39 g) and

dry ether (5 mL) under gentle reflux, under an Ar atmosphere.

The mixture was cooled to 0 °C, and a solution of cyclohex-

anone (9.80 g, 132.4 mmol) in dry ether (20 mL) was added

dropwise with stirring. Stirring was continued at room

temperature for 1 h; then the mixture was poured into 2 N

HCl and extracted with ether. The organic phase was washed

with brine, dried (MgSO₄), and concentrated. Distillation gave

15.17 g (70%) of 1-(3-phenylpropyl)cyclohexanol (58) as a

colorless oil. Bp: 152 °C (3 mmHg). ¹H NMR (CDCl₃): 1.22-

Clarification of the Binding Mode of Benzolactams

J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9 1493

1.60 (m, 12H, aliphatic), 1.69 (m, 2H, ArCH₂CH₂CH₂), 2.61

(m, 2H, ArCH₂CH₂CH₂), 7.15-7.19 (m, 3H, ArH), 7.25-7.29

(m, 2H, ArH). HRMS: calcd for C₁₅H₂₂O-H₂O, 200.1565; found,

200.1589.

lonate, and 30 mL of DMF for 2.5 h at room temperature

under an Ar atmosphere. Purification by column chromatog-

raphy on silica gel (hexane/AcOEt, 4:1) gave 5.18 g of the

diester 63 as colorless prisms (94%). Mp: 169-170 °C (hexane/

AcOEt). ¹H NMR (CDCl₃): 1.25-1.83 (m, 20H, aliphatic, 2 ×

-OCH₂CH₃), 1.97 (s, 3H, NHCOCH₃), 2.70 (t, 2H, J ) 6.4 Hz,

ArCH₂CH₂CH₂), 3.99 (s, 2H, ArCH₂CH), 4.23 (m, 4H, 2 ×

-OCH₂CH₃), 6.45 (bs, 1H, -NHCO-), 6.86 (s, 1H, ArH), 7.91

(s, 1H, ArH). Anal. (C₂₅H₃₄N₂O₇) C, H, N.

Nitr o Alcoh ol 64. The diester 63 (5.05 g, 10.64 mmol) was

converted into the nitro alcohol 64 by the same method as that

used for the preparation of 30 from 29. Purification of the

intermediate N-Boc nitro monoester by column chromatogra-

phy on silica gel (n-hexane/AcOEt, 8:1) and column chroma-

tography of the nitro alcohol on silica gel (n-hexane/AcOEt,

2:1) afforded 3.75 g (86%) of 64 as pale-yellow plates. Mp:

152-153 °C (hexane/AcOEt). ¹H NMR (CDCl₃): 1.24-1.84 (m,

23H, aliphatic, Boc-CH₃), 2.76 (t, 2H, J ) 6.4 Hz, ArCH₂CH₂-

CH₂), 3.00 (m, 1H, ArCH₂CH), 3.15 (m, 1H, ArCH₂CH), 3.69

(m, 2H, CH₂OH), 3.95 (m, 1H, CHCH₂OH), 5.03 (bs, 1H,

-NHCO-), 7.07 (s, 1H, ArH), 8.03 (s, 1H, ArH). Anal.

(C₂₃H₃₄N₂O₅) C, H, N.

Meth yla m in o Alcoh ol 65. The procedure was the same

as that used for the preparation of 31, employing 2.67 g (6.38

mmol) of 64. Purification of the methylamino alcohol by

column chromatography on silica gel (n-hexane/AcOEt, 3:1)

afforded 1.77 g (64%) of the methylamino alcohol 65 as a

colorless amorphous gum. ¹H NMR (CDCl₃): 1.24-1.77 (m,

23H, aliphatic, Boc-CH₃), 2.62 (m, 3H, ArCH₂CH₂CH₂, ArCH₂-

CH), 2.78 (m, 1H, ArCH₂CH), 2.88 (s, 3H, NCH₃), 3.53 (m, 2H,

CH₂OH), 3.71 (m, 1H, CHCH₂OH), 5.13 (bs, 1H, -NHCO-),

6.37 (s, 1H, ArH), 6.74 (s, 1H, ArH). HRMS: calcd for

3′,4′-Dih yd r osp ir o [c yc lo h e x a n e -1,1′(2H )-n a p h t h a -

len e] (59). The alcohol 58 (15.0 g, 68.8 mmol) was added to

50 mL of 85% H₂SO₄at 0 °C over a period of 20 min with

stirring.³⁴After stirring at room temperature for 30 min, the

mixture was poured into ice-water and extracted with hexane.

The organic phase was washed with brine, dried (MgSO₄), and

concentrated. Distillation gave 9.36 g (68%) of the spiro-

[cyclohexane-1,1′-tetralin] 59 as a colorless oil. Bp: 111 °C (2

mmHg). ¹H NMR (CDCl₃): 1.22-1.76 (m, 12H, aliphatic), 1.82

(m, 2H, ArCH₂CH₂CH₂), 2.75 (t, 2H, J ) 6.2 Hz, ArCH₂CH₂-

CH₂), 7.05 (m, 2H, ArH), 7.16 (dt, 1H, J ) 2.2, 6.2 Hz, ArH),

7.41 (d, 1H, J ) 8.1 Hz, ArH). HRMS: calcd for C₁₅H₂₀

200.1565; found, 200.1561.

,

6′-Br om o-3′,4′-dih ydr ospir o[cycloh exan e-1,1′(2H)-n aph -

th a len e] (60). A solution of CrO₃(39.59 g) in AcOH-H₂O

(220 mL, 10:1) was added to a stirred solution of 59 (21.5 g,

107.3 mol) in AcOH (1000 mL) over a period of 1.5 h.³⁵The

mixture was stirred for 1.5 h, 2-propanol (2 mL) was added,

and the whole was concentrated in vacuo. The residue was

taken up in water and extracted with hexane. The organic

phase was washed with water and brine, dried (MgSO₄), and

concentrated. Purification by column chromatography (hex-

ane/CH₂Cl₂, 2:1) gave 14.3 g (62%) of the 4′-ketone. The ketone

(19.14 g, 89.3 mmol) was added dropwise to a mixture of AlCl₃

(29.42 g, 220.6 mmol) and CH₂Cl₂(10 mL) at room temperature

with vigorous stirring. Then 5.5 mL of bromine was added.

The mixture was stirred for 1 h, poured into 6 N HCl, and

extracted with ether. The organic phase was washed with

water and saturated NaHCO₃(aq), dried (Na₂SO₄), and con-

centrated. Purification by column chromatography (hexane/

CH₂Cl₂, 3:1) gave 14.95 g (57%) of the 6′-bromo 4′-ketone as

colorless needles. Mp: 115.5-116.5 °C (n-hexane) Anal.

(C₁₅H₁₇OBr) C, H. A solution of the bromo ketone (14.68 g,

50.1 mmol) in CF₃COOH (50 mL) was added to triethylsilane

(17.64 g, 0.152 mol) at 0 °C with stirring.³¹The mixture was

stirred for 1 h at 0 °C and for 18 h at room temperature, then

poured into saturated NaHCO₃(aq) and extracted with hexane.

The organic phase was washed with water and brine, dried

(MgSO₄) and concentrated. Purification by column chroma-

tography (n-hexane) gave 13.92 g (100%) of 6′-bromo-3′,4′-

dihydrospiro[cyclohexane-1,1′(2H)-naphthalene] (60) as a col-

orless oil. ¹H NMR (CDCl₃): 1.26-1.82 (m, 14H, aliphatic),

2.72 (t, 2H, J ) 6.8 Hz, ArCH₂CH₂), 7.18-7.29 (m, 3H, ArH).

HRMS: calcd for C₁₅H₁₉⁷⁹Br, 278.0671, found, 278.0673.

6′-(Br om om eth yl)-3′,4′-d ih yd r osp ir o[cycloh exa n e-1,1′-

(2H)-n a p h th a len e] (61). The procedure was the same as

that used for the preparation of 42 from 40, employing 11.82

g (42.3 mmol) of 60. Purification by column chromatography

on silica gel (n-hexane) afforded 8.70 g (70%) of 6′-(bromo-

methyl)-3′,4′-dihydrospiro[cyclohexane-1,1′(2H)-naphtha-

lene] (61) as colorless prisms. Mp: 55-57 °C (methanol). ¹H

NMR (CDCl₃): 1.27-1.82 (m, 14H, aliphatic), 2.73 (t, 2H, J )

6.2 Hz, ArCH₂CH₂), 4.45 (s, 2H, ArCH₂Br), 7.07 (s, 1H, ArH),

7.18 (dd, 1H, J ) 2.0, 8.2 Hz, ArH), 7.38 (d, 1H, J ) 8.2 Hz,

ArH). Anal. (C₁₆H₂₁Br) C, H.

6′-(Br om om eth yl)-3′,4′-d ih yd r o-7′-n itr osp ir o[cycloh ex-

a n e-1,1′(2H)-n a p h th a len e] (62). The procedure was the

same as that used for the preparation of 43 from 42, employing

8.61 g (29.4 mmol) of 61. Purification by column chromatog-

raphy on silica gel (n-hexane) afforded 4.02 g (40%) of

6′-(bromomethyl)-3′,4′-dihydro-7′-nitrospiro[cyclohexane-1,1′-

(2H)-naphthalene] (62) as pale-yellow prisms and its 5′-nitro

isomer (3.08 g, 31%). Mp: 90.5-91.5 °C (n-hexane). ¹H NMR

(CDCl₃): 1.26-1.85 (m, 14H, aliphatic), 2.79 (t, 2H, J ) 6.2

Hz, ArCH₂CH₂), 4.80 (s, 2H, ArCH₂Br), 7.20 (s, 1H, ArH), 8.15

(s, 1H, ArH). Anal. (C₁₆H₂₀NO₂Br) C, H, N.

C

₂₄H₃₈N₂O₃, 402.2883; found, 402.2883.

Dia ster eom er ic Ester 66. The methylamino alcohol 65

(1.66 g, 4.12 mmol) was converted into the diastereomeric ester

66 by the same method as that used for the preparation of 32,

employing 3.76 g (11.1 mmol) of (R)-benzyl 2-[[(trifluorometh-

yl)sulfonyl]oxy]isovalerate, 1.54 g (14.4 mmol) of 2,6-lutidine,

and 50 mL of CH₂ClCH₂Cl for 40 h at refluxing temperature.

Purification by column chromatography on silica gel (hexane/

AcOEt, 20:1) gave 2.02 g (83%) of the diastereomeric ester 66

as a colorless amorphous gum.

(-)-BL-V8-SP 1 (37) a n d (-)-Ep i-BL-V8-SP 1 (67). The

procedure was the same as that used for the preparation of

10 and 33, employing 1.94 g (3.28 mmol) of 66 via the activated

ester. Purification of the activated ester by column chroma-

tography on silica gel (n-hexane/AcOEt, 7:1), followed by

condensation and separation of 37 and 67 by column chroma-

tography on silica gel (n-hexane/AcOEt, 1:2), afforded 477 mg

(38%) of (-)-BL-V8-SP1 (37) and 357 mg (28%) of (-)-epi-BL-

V8-SP1 (67). 37: colorless needles. Mp: 198-199 °C (CH₂-

Cl₂/hexane). [R]²²-297.9° (c ) 0.98, CHCl₃). ¹H NMR

D

(CDCl₃): 0.95 (d, 3H, J ) 6.6 Hz, CH(CH₃)₂), 1.07 (d, 3H, J )

6.6 Hz, CH(CH₃)₂), 1.26-1.80 (m, 14H, aliphatic), 2.44 (m, 1H,

CH(CH₃)₂), 2.64 (t, 2H, J ) 6.2 Hz, ArCH₂CH₂CH₂), 2.84 (s,

3H, N-CH₃), 2.84 (m, 1H, ArCH₂CH-), 2.93 (dd, 1H, J ) 8.4,

16.5 Hz, ArCH₂CH-), 3.43 (d, 1H, J ) 8.1 Hz, CH₃-N-CH-

CO), 3.51 (dd, 1H, J ) 7.9, 10.8 Hz, -CHCH₂OH), 3.70 (dd,

1H, J ) 3.9, 10.8 Hz, -CHCH₂OH), 4.29 (m, 1H, -CHCH₂-

OH), 6.42 (bs, 1H, -NHCO-), 6.72 (s, 1H, ArH), 7.09 (s, 1H,

ArH). Anal. (C₂₄H₃₆N₂O₂) C, H, N. 67: colorless needles.

[R]²²-133.7° (c ) 1.43, CHCl₃). Anal. (C₂₄H₃₆N₂O₂) C, H,

D

N.

1-(3-P h en ylpr opyl)-4,4-dim eth ylcycloh exan ol (68). The

procedure was the same as that used for the preparation of

58, employing 9.80 g (77.7 mmol) of 4,4-dimethylcyclohex-

anone. Purification by distillation gave 34.44 g (66%) of 1-(3-

phenylpropyl)-4,4-dimethylcyclohexanol (68) as a colorless oil.

Bp: 146 °C (1 mmHg). HRMS: calcd for

228.1878; found, 228.1876.

C₁₇H₂₆O‚H₂O,

Diester 63. The nitrobenzyl bromide 62 (3.94 g, 11.65

mmol) was converted into the diester 63 by the same method

as that used for the preparation of 29, employing 466 mg (11.65

mmol) of NaH, 2.54 g (11.69 mmol) of diethyl acetamidoma-

3′,4′-Dih yd r o-4,4-d im eth ylsp ir o[cycloh exa n e-1,1′(2H)-

n a p h th a len e] (69). The procedure was the same as that used

for the preparation of 59, employing 34.43 g (0.14 mol) of 68.

Purification by distillation gave 23.78 g (74%) of the spiro-

1494 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9

Endo et al.

[cyclohexane-1,1′-tetralin] 69 as a colorless oil. Bp: 148 °C (4

mmHg). HRMS: calcd for C₁₇H₂₄, 228.1878; found, 228.1875.

6′-Br om o-3′,4′-d ih yd r o-4,4-d im eth ylsp ir o[cycloh exa n e-

1,1′(2H)-n a p h th a len e] (70). The spiro[cyclohexane-1,1′-te-

tralin] 69 (23.75 g, 0.104 mol) was converted into the bromide

70 by the same method as that used for the preparation of 60

from 59. Purification of the final product by column chroma-

tography (hexane) gave 17.84 g (57%) of 6′-bromo-3′,4′-dihydro-

4,4-dimethylspiro[cyclohexane-1,1′(2H)-naphthalene] (70) as

-CH₃), 1.07 (d, 3H, J ) 6.6 Hz, CH(CH₃)₂), 1.05 (s, 3H, -CH₃),

1.15-1.93 (m, 12H, aliphatic), 2.41 (m, 1H, CH(CH₃)₂), 2.63

(t, 2H, J ) 6.0 Hz, ArCH₂CH₂CH₂), 2.79 (m, 1H, ArCH₂CH-),

2.80 (s, 3H, N-CH₃), 2.92 (dd, 1H, J ) 8.4, 16.5 Hz, ArCH₂-

CH-), 3.42 (d, 1H, J ) 8.1 Hz, CH₃-N-CH-CO), 3.51 (dd, 1H,

J ) 7.9, 10.8 Hz, -CHCH₂OH), 3.69 (dd, 1H, J ) 3.7, 10.8

Hz, -CHCH₂OH), 4.31 (m, 1H, -CHCH₂OH), 6.63 (bs, 1H,

-NHCO-), 6.72 (s, 1H, ArH), 7.11 (s, 1H, ArH). Anal. Calcd

for C₂₆H₄₀N₂O₂: C, 75.68; H, 9.77; N, 6.79. Found: C, 75.66;

H, 9.81; N, 6.89. 77: colorless amorphous gum. [R]²²_D-113.7°

(c ) 1.11, CHCl₃). HRMS: calcd for C₂₆H₄₀N₂O₂, 412.3090;

found, 412.3087.

colorless prisms. Mp: 47-48 °C (n-hexane). Anal. (C₁₇H₂₃

Br) C, H.

-

6′-(Br om om et h yl)-3′,4′-d ih yd r o-4,4-d im et h ylsp ir o[cy-

cloh exa n e-1,1′(2H)-n a p h th a len e] (71). The procedure was

the same as that used for the preparation of 42 from 40,

employing 12.15 g (39.5 mmol) of 70. Purification by column

chromatography on silica gel (n-hexane) afforded 8.90 g (70%)

of 6′-(bromomethyl)-3′,4′-dihydro-4,4-dimethylspiro[cyclohex-

ane-1,1′(2H)-naphthalene] (71) as colorless needles. Mp: 68-

69 °C (methanol). Anal. Calcd for C₁₈H₂₅Br: C, 67.29; H, 7.84.

Found: C, 67.05; H, 7.81.

6′-(Br om om et h yl)-3′,4′-d ih yd r o-4,4-d im et h yl-7′-n it r o-

sp ir o[cycloh exa n e-1,1′(2H)-n a p h th a len e] (72). The pro-

cedure was the same as that used for the preparation of 43

from 42, employing 8.67 g (27.0 mmol) of 71. Purification by

column chromatography on silica gel (n-hexane/CH₂Cl₂) af-

forded 3.61 g (37%) of 6′-(bromomethyl)-3′,4′-dihydro-4,4-

dimethyl-7′-nitrospiro[cyclohexane-1,1′(2H)-naphthalene] (72)

as pale-yellow prisms and its 5′-nitro isomer (2.68 g, 27%). Mp:

82-83 °C (n-hexane). Anal. (C₁₈H₂₄NO₂Br) C, H, N.

Diester 73. The nitrobenzyl bromide 72 (3.38 g, 9.23 mmol)

was converted into the diester 73 by the same method as that

used for the preparation of 29, employing 371 mg (9.27 mmol)

of NaH, 2.0 g (9.23 mmol) of diethyl acetamidomalonate, and

25 mL of DMF for 2 h at room temperature under an Ar

atmosphere. Purification by column chromatography on silica

gel (hexane/AcOEt, 4:1) gave 4.66 g of the diester 73 as

colorless prisms (100%). Mp: 131-132 °C (hexane/AcOEt).

Anal. (C₂₇H₃₈N₂O₇) C, H, N.

HL-60 Gr ow th -In h ibitor y Test. The HL-60 cells were

provided by Prof. F. Takaku (Faculty of Medicine, University

of Tokyo) and were maintained in continuous suspension

culture. The cells were cultured in plastic flasks in RPMI1640

medium, supplemented with 5% fetal bovine serum (FBS, not

delipidized) and antibiotics (penicillin G and streptomycin),

in a humidified atmosphere of 5% CO₂in air at 37 °C. Test

compounds were dissolved in ethanol at 2 mM and added to

the cells, which were seeded at about 8 × 10⁴cells/mL. The

final ethanol concentration was kept below 0.5%. The con-

centration of ethanol did not affect the HL-60 cell growth by

comparison with control (ethanol-free) experiment. Cell growth

was determined from a count of the cell number after 4 days.

In h ibition of Sp ecific [³H]P DBu Bin d in g to P KCδ.

Assay of the inhibition of [³H]PDBu binding (K_d) 2.9 nM) to

recombinant PKCδ and the determination of inhibition con-

stants from binding curve IC₅₀values were done as previously

described.²³K_ivalues of the testing compounds are described

below. The number of repetitions for each experiment is noted

in parentheses: 1, 0.32 ( 0.043 nM (2); 3, 80 ( 29 nM (2); 4,

1700 ( 30 nM (2); 5, 5.9 ( 1.7 nM (4); 8, 3.0 ( 0.45 nM (3); 9,

32 ( 8.7 nM (2); 10, 5100 ( 730 nM (3); 35, 65 ( 0.6 nM (2);

36, 1700 ( 220 nM (2); 37, 19 ( 1.1 nM (2); 38, 14 ( 0.94 nM

(3).

Mod elin g a n d Dock in g Sim u la tion . The most stable

docking models for all compounds were estimated by using the

automatic docking program ADAM version 3.4. Energy mini-

mization of the docking models was done by the SANDER

module of the AMBER 4.1 program package. Energy decom-

position analyses of the final docking models were done by the

ANAL module of AMBER 4.1. The structural data used for

docking were prepared as follows. The atomic coordinates of

phorbol 13-acetate were constructed by adding an acetyl group

to 13-OH of the crystal structure of phorbol.³⁶The structure

of the twist form of teleocidin B-4 (1) was taken from that in

the crystal.³⁷The structure of the twist form of (-)-IL-V (3)

was modeled by removing the alkyl moieties on the indole ring

of the teleocidin B-4 structure. The structure of (-)-BL-V8

(4) was obtained computationally, because neither the molecule

nor its congeners could be crystallized. Starting from a

structure constructed by conventional modeling procedures,

stable structures were searched by high-temperature molec-

ular dynamics calculation using the AMBER program.²⁷The

stable conformers were selected from 2000 snapshot structures

sampled from a 100-ps MD calculations (dielectric constant:

ꢀ ) 4r) at 3000 K, followed by energy minimization. The force-

field parameters used for both MD and energy-minimization

calculations were those which we previously employed to

reproduce the conformational equilibrium between twist and

sofa forms observed in (-)-IL-V.^9aAs the most stable con-

former was very similar to the twist form of (-)-IL-V having

the cis-amide structure, this was used for docking as the

putative active conformation. The structures of (-)-BL-V8-

23TM (10) and BL-V8-DM1 (35) were modeled by adding the

corresponding alkyl moieties to the (-)-BL-V8 structure. In

all compounds, hydrogen atoms were computationally located

or relocated at appropriate positions. The atomic charges on

atoms of all molecules were calculated by the Mulliken

analysis method based on molecular orbital calculation using

the MNDO method in the MOPAC program.³⁸The structure

of the PKCδ Cys2 domain was obtained from the complexed

crystal structure of the PKCδ Cys2 domain and phorbol 13-

Nitr o Alcoh ol 74. The diester 73 (4.05 g, 8.07 mmol) was

converted into the nitro alcohol 74 by the same method as that

used for the preparation of 30 from 29. Purification of the

intermediate N-Boc nitro monoester by column chromatogra-

phy on silica gel (n-hexane/AcOEt, 8:1) and column chroma-

tography of the nitro alcohol on silica gel (n-hexane/AcOEt,

2:1) afforded 2.82 g (84%) of the nitro alcohol 74 as a pale-

yellow powder. Mp: 116-117 °C (n-hexane). HRMS: calcd for

C

₂₅H₃₈N₂O₅, 446.2781; found, 446.2768.

Meth yla m in o Alcoh ol 75. The procedure was the same

as that used for the preparation of 31, employing 1.97 g (4.39

mmol) of 74. Purification of the methylamino alcohol by

column chromatography on silica gel (n-hexane/AcOEt, 3:1)

afforded 980 mg (55%) of the methylamino alcohol 75 as a

colorless amorphous gum. HRMS: calcd for

430.3195; found, 430.3198.

C₂₆H₄₂N₂O₃,

Dia ster eom er ic Ester 76. The methylamino alcohol 75

(920 mg, 2.14 mmol) was converted into the diastereomeric

ester 76 by the same method as that used for the preparation

of 32, employing 1.94 g (5.70 mmol) of (R)-benzyl 2-[[(trifluo-

romethyl)sulfonyl]oxy]isovalerate, 798 mg (7.46 mmol) of 2,6-

lutidine, and 30 mL of CH₂ClCH₂Cl for 40 h at refluxing

temperature. Purification by column chromatography on silica

gel (hexane/AcOEt, 20:1) gave 826 mg (62%) of the diastere-

omeric ester 76 as a colorless amorphous gum.

BL-V8-SP 3 (38) a n d Ep i-BL-V8-SP 3 (77). The procedure

was the same as that used for the preparation of 10 and 33,

employing 797 mg (1.28 mmol) of 76 via the activated ester.

Purification of the activated ester by column chromatography

on silica gel (n-hexane/AcOEt, 7:1), followed by condensation

and separation of 38 and 77 by column chromatography on

silica gel (n-hexane/AcOEt, 1:2), afforded 211 mg (40%) of (-)-

BL-V8-SP3 (38) and 145 mg (27%) of (-)-epi-BL-V8-SP3 (77).

38: colorless needles. Mp: 207-209 °C (CH₂Cl₂/hexane).

[R]²²-252.9° (c ) 1.13, CHCl₃). ¹H NMR (CDCl₃):: 0.95 (d,

D

3H, J ) 6.6 Hz, CH(CH₃)₂), 0.96 (s, 3H, -CH₃), 1.05 (s, 3H,

Clarification of the Binding Mode of Benzolactams

J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9 1495

acetate (Protein Data Bank code 1PTR)¹³by removing the

phorbol 13-acetate molecule. All hydrogen atoms that are

missing in the crystal structure but should exist in the protein

structure were computationally located at appropriate posi-

tions and optimized further by the AMBER program.

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conformation of a tumor promoter, teleocidin. A molecular

dynamic study. J . Med. Chem. 1992, 25, 2248-2253. (b) Kawai,

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indolactams. Crystal and solution structures. J . Org. Chem.

1992, 57, 6150-6155.

(10) (a) Ohno, M.; Endo, Y.; Hirano, M.; Itai, A.; Shudo, K. Designed

molecules reproducing the two conformations of teleocidins.

Tetrahedron Lett. 1993, 34, 8119-8122. (b) Endo, Y.; Ohno, M.;

Hirano, M.; Takeda, M.; Itai, A.; Shudo, K. Chiral requirement

for the tumor promoters. Conformation and activity study of

benzolactams. BioMed. Chem. Lett. 1994, 4, 491-494. (c) Endo,

Y.; Ohno, M.; Hirano, M.; Itai, A.; Shudo, K. Synthesis, confor-

mation, and biological activity of teleocidin mimics, benzolac-

tams. A clarification of the conformational flexibility problem

in structure-activity studies of teleocidins. J . Am. Chem. Soc.

1996, 118, 1841-1855.

(11) Fujiki, H. Personal communication. The same amounts (5.5

nmol/application) of (-)-BL-V8-310 and teleocidin B-4 induced

tumors on mouse skin initiated with 7,12-dimethylbenz[a]-

anthracene (DMBA) in 13.3% and 86.7%, respectively, of tumor-

bearing mice in week 20.

(12) (a) Okuno, S.; Irie, K.; Suzuki, Y.; Koshimizu, K.; Nishino, H.;

Iwashima, A. Synthesis and biological activities of fluorine-

substituted (-)-indolactam-V, the core structure of tumor promter

teleocidins. BioMed. Chem. Lett. 1994, 4, 431-434. (b) Irie, K.;

Isaka, T.; Iwata, Y.; Yanai, Y.; Nakamura, Y.; Koizumi, F.;

Ohigashi, H.; Wender, P. A.; Satomi, Y.; Nishino, H. Synthesis

and biological activities of new conformationally restricted

analogues of (-)-indolactam-V: elucidation of the biologically

active conformation of the tumor-promoting teleocidins. J . Am.

Chem. Soc. 1996, 118, 10733-10743.

(13) Zhang, G.; Kazanietz, M. G.; Blumberg, P. M.; Hurley, J . H.

Crystal structure of the Cys2 activator-binding domain of protein

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924.

(14) A part of this work has been reported in preliminary form: Itai,

A.; Matsuo, A.; Mizutani, M.; Tomioka, N.; Sitaka, M.; Endo,

Y.; Shudo, K. Advanced computational docking of two teleocidin

congeners to Cys2 domain of protein kinase Cδ. Chem. Pharm.

Bull. 1997, 45, 573-575.

(15) A part of this work has been reported in a preliminary form.

Endo, Y.; Ohno, M.; Takehana, S.; Driedger, P. E.; Stabel, S.;

Shudo, K. Role of hydrophobic moiety of tumor promoters.

Synthesis and activity of benzolactams with alkyl substituents

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J M970704S

Article Doi

Clarification of the binding mode of teleocidin and benzolactams to the Cys2 domain of protein kinase Cδ by synthesis of hydrophobically modified, teleocidin-mimicking benzolactams and computational docking simulation

DOI: 10.1021/jm970704s

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