Synthesis, chemical properties, and preliminary evaluation of substituted CBI analogs of CC-1065 and the duocarmycins incorporating the 7-cyano-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one alkylation subunit: Hammett quantitation of the magnitude of electronic effects on functional reactivity

4894

J . Org. Chem. 1996, 61, 4894-4912

Syn th esis, Ch em ica l P r op er ties, a n d P r elim in a r y Eva lu a tion of

Su bstitu ted CBI An a logs of CC-1065 a n d th e Du oca r m ycin s

In cor p or a tin g th e

7-Cya n o-1,2,9,9a -tetr a h yd r ocyclop r op a [c]ben z[e]in d ol-4-on e

Alk yla tion Su bu n it: Ha m m ett Qu a n tita tion of th e Ma gn itu d e of

Electr on ic Effects on F u n ction a l Rea ctivity

Dale L. Boger,* Nianhe Han, Christine M. Tarby, Christopher W. Boyce, Hui Cai,

Qing J in, and Paul A. Kitos

Department of Chemistry, The Scripps Research Institute, 10666 North Torrey Pines Road,

La J olla, California 92037, and Department of Biochemistry, University of Kansas,

Lawrence, Kansas 66045

Received March 19, 1996^X

The synthesis of 7-cyano-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (CCBI), a substituted

CBI derivative bearing a C7 cyano group, is described in efforts that establish the magnitude of

potential electronic effects on the functional reactivity of the agents. The CCBI alkylation subunit

was prepared by a modified Stobbe condensation/Friedel-Crafts acylation for generation of the

appropriately functionalized naphthalene precursors followed by 5-exo-trig aryl radical-alkene

cyclization for synthesis of the 1,2-dihydro-3H-benz[e]indole skeleton and final Ar-3′ alkylation for

introduction of the activated cyclopropane. The most concise approach provided the CCBI subunit

and its immediate precursor in 14-15 steps in superb overall conversions (15-20%). Resolution

of an immediate CCBI precursor and its incorporation into both enantiomers of 34-39, analogs of

CC-1065 and the duocarmycins, are detailed. A study of the solvolysis reactivity and regioselectivity

of N-BOC-CCBI (25) revealed that introduction of the C7 nitrile slowed the rate of solvolysis but

only to a surprisingly small extent. Classical Hammett quantitation of the effect provided a

remarkably small F (-0.3), indicating an exceptionally small C7 substituent electronic effect on

functional reactivity. Additional kinetic studies of acid-catalyzed nucleophilic addition proved

inconsistent with C4 carbonyl protonation as the slow and rate-determining step but consistent

with a mechanism in which protonation is rapid and reversible followed by slow and rate-

determining nucleophilic addition to the cyclopropane requiring both the presence and assistance

of a nucleophile (S_N2 mechanism). No doubt this contributes to the DNA alkylation selectivity of

this class of agents and suggests that the positioning of an accessible nucleophile (adenine N3)

and not C4 carbonyl protonation is the rate-determining step controlling the sequence selectivity

of the DNA alkylation reaction. This small electronic effect on the solvolysis rate had no impact

on the solvolysis regioselectivity, and stereoelectronically-controlled nucleophilic addition to the

least substituted carbon of the activated cyclopropane was observed exclusively. Consistent with

past studies, a direct relationship between solvolysis stability and cytotoxic potency was observed

with the CCBI-derived agents providing the most potent analogs in the CBI series, and these

observations were related to the predictable Hammett substituent effects. For the natural

enantiomers, this unusually small electronic effect on functional reactivity had no perceptible effect

on their DNA alkylation selectivity. Similar effects of the C7 cyano substituent on the unnatural

enantiomers were observed, and they proved to be 4-10× more effective than the corresponding

CBI-based unnatural enantiomers and 4-70× less potent than the CCBI natural enantiomers.

(+)-CC-1065 (1)¹and the duocarmycins 2-3^2-4consti-

tute the parent members of a class of potent antitumor

antibiotics that derive their biological effects through the

reversible, sequence-selective alkylation of DNA.^5-13

Since their disclosure, extensive efforts have been de-

voted to define and exploit their properties. In these

studies, the use of synthetic samples of the natural and

unnatural enantiomers of the natural products^14-16and

^XAbstract published in Advance ACS Abstracts, J une 15, 1996.

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Electronic Effects on Functional Reactivity

J . Org. Chem., Vol. 61, No. 15, 1996 4895

synthetic analogs bearing deep-seated structural

changes^17-40have proven especially valuable in the

definition and examination of such properties.⁴¹

oxy group para to the C4 carbonyl.⁴²Herein, we report

the complementary preparation and preliminary evalu-

ation of 7-cyano-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]-

indol-4-one (CCBI) bearing a cyano group para to the C4

carbonyl. The comparative evaluation of this key set of

agents incorporating the CBI, MCBI, and CCBI alkyla-

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In a preceding paper we described the preparation and

preliminary evaluation of MCBI, a substituted 1,2,9,9a-

tetrahydrocyclopropa[c]benz[e]indol-4-one bearing a meth-

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4896 J . Org. Chem., Vol. 61, No. 15, 1996

Boger et al.

Sch em e 1

Sch em e 2

tion subunits was designed to permit an accurate assess-

ment of the electronic influence of the C7 substituent on

the chemical and functional reactivity of CBI and the

fundamental role this may play in establishing the

biological properties of the agents.

material in lower conversions of 20-30%. A number of

alternatives on this direct approach were investigated^46-49

and failed to improve on the conversions or the isomer

ratio of 6 to 8. In part, this diminished conversion may

be attributed to the mixture of (E)- and (Z)-5 taken into

the Friedel-Crafts acylation reaction and the vigorous

reaction conditions and time required to affect the Z to

E isomerization for productive cyclization.

This was improved significantly by conducting the

Stobbe condensation in a more controlled manner. Con-

densation of 4 with the Wadsworth-Horner-Emmons

reagent 10⁵⁰(1.03 equiv, 1.1 equiv of NaH, THF, 0-25

°C, 12 h, 95-100%) provided 11 in which the required

E-isomer predominated (>11:1 E:Z), Scheme 2. Acid-

catalyzed deprotection of 11 (100%) followed by Friedel-

Crafts acylation of 5 affected by treatment with Ac₂O-

KOAc⁵⁰(reflux, 1 h, 30 min) provided 7 in 65% recrys-

tallized yield free of minor amounts of 9. In part, the

improved conversions to provide 7 may be attributed to

the facile closure of the correct E isomer of 5 and the

milder reaction conditions and shorter reaction times

employed since Z to E isomerization under the reaction

conditions was no longer required. Hydrolysis of the

O-acetate 7 by treatment with K₂CO₃-EtOH (reflux,

0.5-1 h, 81-100%) cleanly provided 6. This approach

dependably provided 6 in overall conversions of 45-50%

for the four steps and offered the additional advantages

of the clean generation of the required (E)-5 and the

ability to purify and characterize intermediates in route

to 6. Protection of the free phenol 6 as its benzyl ether

provided 12 (100%).

Syn th esis of CCBI (26) a n d N-BOC-CCBI (25). The

approach implemented for the synthesis of 25 and 26 and

their advanced analogs of CC-1065 and the duocarmycins

was developed concurrent with that of MCBI and con-

sequently relies on the strategy outlined in our preceding

work.⁴²However, recognizing that the incorporation of

a strong electron-withdrawing substituent into the fused

benzene ring would decelerate the Friedel-Crafts acyla-

tion reaction required of its construction, this was

conducted with the substrate 5 in which a halogen

occupies this para position. The expectation was that

conversion of the bromide to the nitrile could be ac-

complished at a later stage. Stobbe condensation⁴³of

3-bromobenzaldehyde (4) with diethyl succinate (1.5

equiv) affected by treatment with t-BuOK⁴⁴(1.1 equiv,

t-BuOH, reflux, 2 h, 75-100%) provided a 3.2:1 mixture

of the half esters 5 in excellent conversion (Scheme 1).

Subjection of this mixture of 5 to Friedel-Crafts acyla-

tion (1-1.1 equiv of NaOAc, Ac₂O, reflux, 6 h)⁴⁵provided

a mixture of 6, its O-acylation product 7, and significant

amounts of the isomeric products 8 and 9. The best

conversions were observed when the Friedel-Crafts

acylation was conducted under moderately dilute reaction

conditions (0.1 M versus 0.5 M). Subsequent ethanolysis

(3 M HCl-EtOH) of the resulting mixture served to

hydrolyze the O-acetates 7 and 9, providing a 7:1 mixture

of 6 and its isomer 8. Both 6 and 8 were readily

separated by chromatography or more conveniently by

simple recrystallization of the mixture. Although this

procedure has provided 6 in conversions as high as 53%

overall yield for the three steps, it generally provided the

At this stage, the C6 nitrile was introduced in a

remarkably clean and effective reaction by simply treat-

ing 12 with CuCN in refluxing DMF⁵¹to provide 13

(96%), Scheme 3. Hydrolysis of the ethyl ester (99-

100%) followed by Curtius rearrangement of 14 effected

by treatment of the carboxylic acid with the Shioiri-

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Electronic Effects on Functional Reactivity

J . Org. Chem., Vol. 61, No. 15, 1996 4897

Sch em e 3

Sch em e 4

eration of Ph₃P)CHOTHP⁵⁴in THF followed by reaction

with 18 in THF-HMPA⁵⁵over a sustained reaction

period. Treatment of 19 with Bu₃SnH (2 equiv, 0.2 equiv

of AIBN, C₆H₆, 80 °C, 2 h, 98-100%) provided the product

of clean 5-exo-trig aryl radical-alkene cyclization 20 in

excellent yield. Subsequent THP deprotection⁵⁶(100%),

conversion⁵⁷of the primary alcohol 21 to the chloride 22

(100%), and phenol deprotection⁵⁸(100%) provided 23.

Spirocyclization of 23 to provide N-BOC-CCBI (25) was

effected by treatment with NaH (3 equiv, 0 °C, 30 min,

95%), and acid-catalyzed deprotection of 23 (4 M HCl-

EtOAc, 25 °C, 30 min) followed by treatment of the crude

indoline hydrochloride salt 24 with 5% aqueous NaHCO₃-

THF (1:1, 25 °C, 1.5 h, 100%) cleanly provided CCBI (26).

Notably and because of the enhanced acidity of the

phenol, simple treatment of 23 with 5% aqueous NaH-

CO₃-THF (1:1, 25 °C, 9 h, 100%) led to clean spirocy-

clization to provide 25 without evidence of subsequent

hydrolysis of the labile BOC.

This approach to 25 and 26 was further shortened and

improved with implementation of the Tempo trap²⁶of the

5-exo-trig aryl radical-alkene cyclization of the substrate

28 containing an unactivated and unfunctionalized ac-

ceptor alkene. Selective, acid-catalyzed C4 iodination of

15 effected by low-temperature treatment with NIS

followed by alkylation of the sodium salt of 27 (1.5 equiv

of NaH, DMF, 25 °C, 2 h) with allyl bromide (5 equiv,

DMF, 25 °C, 2 h, 92%) provided 28. Treatment of 28 with

Bu₃SnH (5 equiv, 60 °C, 1.5-2 h) in benzene in the

presence of Tempo (5 equiv) cleanly provided 29 (Scheme

4). Reductive cleavage of 29 to provide 21 was effected

by treatment with Zn (80 equiv, 3:1:1 THF-HOAc-H₂O,

70 °C, 7 h, 73%).

Yamada reagent⁵²(1.2 equiv of DPPA, 1.2 equiv of Et₃N,

t-BuOH, reflux, 14 h, 87%) cleanly provided 15. In the

optimization of this latter reaction, it was determined

that use of rigorously dried t-BuOH, the use of dilute

(0.005 M) or moderately dilute reaction conditions (0.025

M), and the maintenance of anhydrous reaction condi-

tions through addition of 4 Å molecular sieves served to

significantly reduce the amount of competitive sym-

metrical urea generation.⁵³Preliminary efforts to reverse

the order of the steps in the conversion of 12 to 15 by

first converting the ethyl ester to the corresponding tert-

butyl carbamate followed by CuCN replacement of the

C6 bromide failed to provide 15 cleanly. Low-tempera-

ture, acid-catalyzed C4 bromination of 15 (1.2 equiv of

NBS, catalytic H₂SO₄, THF, -60 °C, 4 h, 87%) cleanly

provided 16, and alkylation of the sodium salt of 16 (1.3

equiv of NaH, DMF, 25 °C, 30 min) with 1-bromo-3-

methyl-2-butene (3 equiv, DMF, 0-25 °C, 12 h, 99-100%)

afforded 17. Low-temperature ozonolysis of 17 under

carefully controlled reaction conditions followed by im-

mediate reductive workup (Me₂S) of the crude ozonide

provided the aldehyde 18 (91%). The use of extended

reaction times or the failure to immediately quench the

excess O₃led to the rapid generation of a further

oxidation product. Introduction of the vinyl ether 19

(74%) proved most effective with low-temperature gen-

Resolu tion . The resolution of a late stage synthetic

intermediate was accomplished by the chromatographic

separation of the enantiomers of 22 on a Chiralcel-OD

semipreparative HPLC column (2 × 25 cm) using a 7%

i-PrOH-hexane eluent (7 mL/min), R ) 1.38.^35,42This

routinely provided the two enantiomers of 22 in greater

than 99.9% ee and with a 97% recovery. The intermedi-

ate 22 proved to be the only late-stage intermediate that

was effectively resolved on a Chiracel-OD column, and

similar efforts to separate 21 or 23 as well as N-BOC-

(53) For the symmetrical urea byproduct: ¹H NMR (250 MHz,

DMSO-d₆) δ 9.21 (s, 2H), 8.44 (s, 2H), 8.20 (d, 2H, J ) 8.3 Hz), 7.82 (s,

2H), 7.57 (m, 6H), 7.45 (m, 8H), 5.34 (s, 4H); IR (KBr) ν_max3400, 2240,

1650, 1590 cm^-1; FABMS (NBA) m/ z 575 (M + H⁺).

(54) Schlude, H. Tetrahedron 1975, 31, 89.

(56) Bongini, A.; Cardillo, G.; Orena, M.; Sandri, S. Synthesis 1979,

618.

(55) Corey, E. J .; Arai, Y.; Mioskowski, C. J . Am. Chem. Soc. 1979,

101, 6748.

(57) Hooz, J .; Gilani, S. S. H. Can. J . Chem. 1968, 46, 86.

(58) Beig, T.; Szeja, W. Synthesis 1985, 76.

4898 J . Org. Chem., Vol. 61, No. 15, 1996

Boger et al.

Ta ble 1. Ch r om a togr a p h ic Resolu tion ^a

Ta ble 2. Aqu eou s Solvolysis of N-BOC-CCBI a n d

Rela ted Agen ts

agent

solvent

R

N-BOC-MCBI

N-BOC-CBI

(41)

N-BOC-CCBI

(25)

21

22

23

25

3-7% i-PrOH/hexane gradient

5% i-PrOH/hexane

7% i-PrOH/hexane

2 or 3% i-PrOH/hexane

15, 25 or 30% i-PrOH/hexane

1.09

1.33

1.38

1.00

(40)

k, s^-1(pH 3)

1.75 × 10^-6

1.45 × 10^-6

0.99 × 10^-6

0.79 × 10^-5

24.2

t_1/2, h (pH 3) 110

133

213

k, s^-1(pH 2)

t_1/2, h (pH 2)

1.62 × 10^-5

1.53 × 10^-5

11.8

12.5

a

Analytical 4.6 × 250 mm, 10 µm Chiralcel-OD column, 1 mL/

min.

the absence of added base provided the agents 34, 36,

and 38, respectively. Interestingly, the agent 24 was

found to couple less effectively than prior agents, and the

slower coupling reactions in the series (e.g., CDPI₂) were

less successful. These slower coupling reactions, which

can be attributed principally to the insolubility of the

carboxylic acids even in DMF, suffered competitive ring

closure of 24 to CCBI (26). In part, this distinction of

24 may be attributed to the increased phenol acidity

leading to a more facile deprotonation and spirocycliza-

tion competitive with coupling. Subsequent treatment

of the coupled agents with NaH (3 equiv, 20% DMF-

THF, 0 °C, 30 min) provided CCBI-TMI (35, 99%) and

CCBI-indole₂(37, 66%) in good conversion. More re-

markable, simple exposure of 36 to 3% aqueous NaHCO₃-

THF (1:1, 25 °C, 3 h, 68%) or 38 to KHCO₃in DMF-

H₂O (5:2, 25 °C, 9-10 h, 69%) provided spirocyclization

to provide 37 and 39 without evidence of significant

subsequent hydrolysis of the labile amide.

Sch em e 5

Ch em ical Solvolysis: Reactivity. Ham m ett Qu an -

tita tion of th e Ma gn itu d e of th e C7 Su bstitu en t

Electr on ic Effect on F u n ction a l Rea ctivity. The

first important characteristic of the CCBI agents that was

examined was its relative reactivity toward solvolysis

which may be considered to reflect the relative functional

reactivity of the agents for acid or Lewis acid-catalyzed

DNA alkylation. Expectations were that the introduction

of the C7 cyano group would slow acid-catalyzed solvoly-

sis, which requires C4 carbonyl protonation, but the

magnitude of this electronic effect of the C7 substituent

was unknown. In preceding studies with the compari-

sons of the MCBI and CBI-based agents in which the

effect of a C7 electron-donating substituent (OCH₃) was

examined, the magnitude of this effect was found to be

exceptionally small.⁴²

CCBI (25) itself were not as successful, Table 1. Subjec-

tion of both enantiomers of 22 to the conditions of

catalytic hydrogenation for removal of the benzyl ether

provided the enantiomers of 23 which were incorporated

into the optically active agents 25, 26, and 34-39. The

assignment of the absolute configuration was tentatively

based on the optical rotations of the final agents 25, 26,

33, 37, and 39 for which the strong positive rotation was

assigned the natural configuration in analogy to all

closely related studies and unambiguously established

in the subsequent biological studies. Most notably, the

distinct DNA alkylation selectivities characteristic of the

natural and unnatural enantiomers for which prior

stereochemical assignments have been unambiguously

established were found to be in agreement with the initial

assignments.

CCBI-TMI (35), CCBI-In d ole₂(37), a n d CCBI-

CDP I₁(39). The CCBI alkylation subunit was incorpo-

rated into the CC-1065 and duocarmycin analogs 34-39

as detailed in Scheme 5. Acid-catalyzed deprotection of

23 (4 M HCl-EtOAc, 25 °C, 30 min, quantitative)

followed by coupling of the unstable indoline hydrochlo-

ride salt 24 with 5,6,7-trimethoxyindole-2-carboxylic acid

(30,¹⁴3 equiv of EDCI, DMF, 25 °C, 14 h, 85%), 31³⁹(3

equiv of EDCI, DMF, 25 °C, 16 h, 87%), and CDPI₁⁵⁹(32,

3 equiv of EDCI, DMF, 25 °C, 16 h, 81%) conducted in

Similar observations were made with the comparisons

now extended to N-BOC-CCBI (25) and CCBI (26).

Consistent with expectations, solvolysis at both pH 3 and

pH 2 revealed that N-BOC-CCBI (25) was the most stable

agent in the series while N-BOC-MCBI (40) was the most

reactive. However, the difference in reactivity was

remarkably modest and N-BOC-CCBI (25) was only

approximately two times more stable than N-BOC-MCBI

(40), which in turn was only slightly more reactive than

N-BOC-CBI (41) itself at both pH 2 and pH 3 (Table 2).

All three agents, including N-BOC-CCBI, were stable at

pH 7 (1:1 CH₃OH-H₂O), and no solvolysis was detected

over a 2 week monitoring period. Nearly identical

observations were made in the examination of CCBI (26)

in which it was found to be the most stable agent in the

series, but the differences in solvolysis rates at either pH

2 or 3 were surprisingly small: CCBI, k ) 1.5 × 10^-6

s^-1, t_1/2) 127 h (pH 2) and k ) 1.65 × 10^-7s^-1, t_1/2

)

1182 h (ph 3); CBI, k ) 2.07 × 10^-7s^-1, t_1/2, 930 h (pH 3);

MCBI, k ) 5.76 × 10^-7s^-1, t_1/2) 334 h (pH 3). Like the

observations disclosed in prior studies, CCBI was sub-

(59) Boger, D. L.; Coleman, R. S.; Invergo, B. J . J . Org. Chem. 1987,

52, 1521. Boger, D. L.; Coleman, R. S. J . Org. Chem. 1984, 49, 2240.

Electronic Effects on Functional Reactivity

J . Org. Chem., Vol. 61, No. 15, 1996 4899

F igu r e 1. Top: UV spectra of solvolysis study of N-BOC-CCBI

at pH 2 conducted in 50% CH₃OH buffer (4:1:20 1.0 M citric

acid, 0.2 M Na₂HPO₄, and H₂O). The spectra shown were

recorded at 0, 1, 2, 6, 11, 17, 37, and 63 h. Bottom: UV spectra

of solvolysis study of CCBI at pH 2 conducted in 50% CH₃OH

buffer. The spectra shown were recorded at 0, 20, 45, 70, 94,

142, 214, and 361 h.

F igu r e 2.

stantially more stable than N-BOC-CCBI presumably

because of preferential N versus O protonation.

consistent with nucleophilic addition and not C4 carbonyl

protonation as the rate-determining step.

Ch em ica l Solvolysis: Regioselectivity. Treatment

of N-BOC-CCBI (25) with 0.1 equiv of CF₃CO₂H in CH₃-

OH (0 °C, 24 h) resulted in the clean solvolysis to provide

a single product 42 (95%), eq 1. No BOC deprotection

The solvolysis was conducted in 1:1 CH₃OH-buffer (pH

3.1; buffer ) 4:1:20 v:v:v 0.1 M citric acid, 0.2 M Na₂-

HPO₄, H₂O; pH 2.05: buffer ) 4:1:20 v:v:v 1.0 M citric

acid, 0.2 M Na₂HPO₄, H₂O) and was followed spectro-

photometrically by UV with the disappearance of the

long-wavelength absorption of the CCBI chromophore

and with the appearance of a short-wavelength absorp-

tion attributable to the solvolysis products (Figure 1).

Although this was not examined in the present case, past

studies²⁴with N-BOC-CBI have demonstrated the pro-

duction of two solvolysis products derived from addition

of H₂O and CH₃OH to the least-substituted carbon of the

cyclopropane. Consistent with these observations, sol-

volysis in CH₃OH produced a single product as detailed

below.

Hammett plots revealed unusually small F values of

-0.30 (pH 3) or -0.34 (pH 2) for the acid-catalyzed

solvolysis (Figure 2). This exceptionally small F value

of -0.3 indicates little differential positive charge buildup

in the reaction transition state consistent with a strict

S_N2 versus S_N1 mechanism requiring the presence and

assistance of the nucleophile for ring opening of the

cyclopropane. Moreover, the observation of the remark-

ably small 2× solvolysis rate difference in going from a

strong C7 electron-donating substituent to a strong

electron-withdrawing substituent (OMe versus CN) is

or olefin was observed, and the methanolysis proceeded

without alteration of the stereoelectronically-controlled

regioselectivity. Clean cleavage of the C8b-C9 bond with

addition of CH₃OH to the least substituted C9 cyclopro-

pane carbon was observed to provide 42, and no cleavage

of the C8b-C9a bond with ring expansion and addition

4900 J . Org. Chem., Vol. 61, No. 15, 1996

Ta ble 3. CH₃OH-THF Solvolysis of 25^a

Boger et al.

suggests that positioning of an accessible nucleophile^5,6,9,11

(adenine N3) and not C4 carbonyl protonation^8,12,60is the

rate-determining step controlling the DNA alkylation

selectivity.

In Vitr o Cytotoxic Activity. The results of a study

of the comparative cytotoxic properties of the CCBI-based

agents alongside the corresponding CBI and MCBI

agents are detailed in Table 4. In preliminary studies,

the natural enantiomers of the CCBI-based agents have

been found to exhibit the most potent cytotoxic activity

in CBI series (Table 4).

Consistent with past observations, the agents were

found to follow a direct relationship between chemical

stability (-log k) and in vitro cytotoxic potency (L1210,

log 1/IC₅₀) over the narrow range of reactivity examined

by the series. This is illustrated in Figure 3 with the

N-BOC derivatives. Presumably, this may be attributed

to the more effective delivery of the more stable agents

to their intracellular target, and the solvolysis rates may

be taken to accurately represent the relative functional

reactivity/stability of the agents.

Less obvious, but more fundamental, the observations

were found to follow a predictable direct relationship

between in vitro cytotoxic activity and the Hammett σ_F

constant of the C7 substituent with CCBI providing the

most potent agent (Figure 4). This fundamental rela-

tionship should prove useful in the design of new analogs

possessing further enhanced properties.

equiv of H⁺

equiv of CH₃OH

k (s^-1

)

t_1/2(h)

0.25

0.1

100

500

20

7.4 × 10^-4

2.1 × 10^-4

5.6 × 10^-4

0.2 × 10^-4

0.26

0.92

0.34

9.2

0.1

a

Catalytic CF₃SO₃H, 0.3 mM in THF, 25 °C.

of CH₃OH to C9a to provide 43 was detected (>20:1). This

is in sharp contrast to solvolysis studies of the more

reactive alkylation subunits of CC-1065,⁶⁰duocarmycin

A,⁷and CBQ²⁰where significant or substantial amounts

of the abnormal ring expansion solvolysis products have

been detected. Nonetheless, the observations are con-

sistent with prior studies with N-BOC-CBI (41) and

N-BOC-MCBI (40) where no (>20:1) ring expansion

solvolysis product was detected.^24,42Thus, within the

reactivity range represented by the agents 25, 40, and

41 and their C7 substituents, no effect of the substituents

on the regioselectivity of acid-catalyzed nucleophilic

addition was observed. Given the potential range rep-

resented by the C7 substituents examined, it is unlikely

that any substituent will effect a perceptible change in

this inherent regioselectivity with the CBI-based agents.

To date, the abnormal ring expansion solvolysis prod-

ucts have only been detected with the chemically more

reactive agents and are only especially prevalent in the

CBQ system where the stereoelectronic alignment of both

cyclopropane bonds are equivalent.²⁰However, this

stereoelectronically-controlled acid-catalyzed nucleophilic

addition to the CBI-based agents including the CCBI-

based agents that proceed with >20:1 regioselectivity

provides an additional advantage of the agents over the

CPI-based analogs of CC-1065 that have been found to

exhibit a more modest 4:1 selectivity.⁶⁰

While the latter two correlations are limited to the

comparisons made on the three presently available

agents, they also follow trends established in the exami-

nation of 44-49 (Figure 5) and their related full structure

analogs (Figure 6), further supporting their potential

generality. Notably, 25 is the most stable and one of the

most synthetically accessible alkylation subunits dis-

closed to date.

Ch em ica l Solvolysis: Mech a n ism . As illustrated

by the relative aqueous solvolysis rates measured at pH

3 versus pH 2, the 10-fold rate increase at pH 2 for all

three agents is indicative of a solvolysis first-order

dependence on the acid concentration. In additional

studies conducted with N-BOC-CCBI (25), the solvolysis

not only exhibited this first-order rate dependence on acid

concentration but also exhibited a first-order rate depen-

dence on the concentration of the nucleophile when the

acid-catalyzed addition of CH₃OH was conducted in THF

(0.1-0.25 equiv of CF₃SO₃H, 20-500 equiv of CH₃OH,

25 °C), Table 3.⁶¹Together, these studies illustrate that

the slow step for acid-catalyzed nucleophilic addition to

the activated cyclopropane under the conditions exam-

ined is not C4 carbonyl protonation, but rather nucleo-

philic addition to the activated cyclopropane following

rapid and reversible C4 carbonyl protonation (eq 2).

Analogous to prior observations, the corresponding seco

precursors 23, 34, 36, and 38 exhibited cytotoxic activity

indistinguishable from the cyclopropane-containing agents.

DNA Alk yla tion Selectivity a n d Efficien cy. The

DNA alkylation properties of the agents were examined

within w794 duplex DNA,¹⁰a 144 base-pair segment of

duplex DNA for which comparative results are available

for related agents. The alkylation site identification and

the assessment of the relative selectivity among the

available sites were obtained by thermally-induced strand

cleavage of the singly 5′ end-labeled duplex DNA after

exposure to the agents. Following treatment of the end-

labeled duplex DNA with a range of agent concentrations,

the unbound agent was removed by EtOH precipitation

of the DNA. Redissolution of the DNA in aqueous buffer,

thermolysis (100 °C, 30 min) to induce strand cleavage

at the sites of DNA alkylation, denaturing high resolution

polyacrylamide gel electrophoresis (PAGE) adjacent to

Sanger dideoxynucleotide sequencing standards, and

autoradiography led to identification of the DNA cleavage

and alkylation sites. The full details of this procedure

have been disclosed and discussed elsewhere.¹⁰The DNA

alkylation reaction selectivities observed under the in-

cubation conditions for the agents detailed herein have

proven identical to the alkylation selectivities observed

with shorter or extended reaction periods or when the

Although these latter studies were conducted under

conditions different than that employed for DNA alkyla-

tion, the results have significant implications on the

origin of the DNA alkylation selectivity of the agents and

(60) Warpehoski, M. A.; Harper, D. E. J . Am. Chem. Soc. 1994, 116,

7573. Warpehoski, M. A.; Harper, D. E. J . Am. Chem. Soc. 1995, 117,

2951.

(61) Boger, D. L.; McKie, J . A.; Han, N.; Tarby, C. M.; Riggs, H. W.;

Kitos, P. A. Bioorg. Med. Chem. Lett. 1996, 6, 659.

Electronic Effects on Functional Reactivity

J . Org. Chem., Vol. 61, No. 15, 1996 4901

Ta ble 4. Cytotoxic Activity

agent

IC₅₀(L1210)

agent

IC₅₀(L1210)

agent

(+)-MCBI

(+)-N-BOC-MCBI

(+)-N-MCBI-TMI

(+)-MCBI-indole₂

(+)-MCBI-CDPI₁

(+)-MCBI-CDPI₂

IC₅₀(L1210)

Natural Enantiomers

25, (+)-CCBI

2 mM

20 nM

7 pM

6 pM

nd

(+)-CBI

(+)-N-BOC-CBI

(+)-CBI-TMI

(+)-CBI-indole₂

(+)-CBI-CDPI₁

(+)-CBI-CDPI₂

nd

5 mM

90 nM

8 pM

10 pM

6 pM

26, (+)-N-BOC-CCBI

35, (+)-CCBI-TMI

37, (+)-CCBI-indole₂

39, (+)-CCBI-CDPI₁

(+)-CCBI-CDPI₂

80 nM

30 nM

10 pM

5 pM

6 pM

Unnatural Enantiomers

25, (-)-CCBI

3 mM

80 nM

450 pM

400 pM

80 pM

nd

(-)-CBI

11 mM

900 nM

2000 pM

4000 pM

380 pM

40 pM

(-)-MCBI

30 mM

200 nM

400 pM

30 pM

10 pM

26, (-)-N-BOC-CCBI

35, (-)-CCBI-TMI

37, (-)-CCBI-indole₂

39, (-)-CCBI-CDPI₁

(-)-CCBI-CDPI₂

(-)-N-BOC-CBI

(-)-CBI-TMI

(-)-N-BOC-MCBI

(-)-MCBI-TMI

(-)-MCBI-indole₂

(-)-MCBI-CDPI₁

(-)-MCBI-CDPI₂

(-)-CBI-indole₂

(-)-CBI-CDPI₁

(-)-CBI-CDPI₂

F igu r e 4.

N-BOC-MCBI (40) were detected. Both natural enanti-

omers exhibited comparable efficiences of DNA alkylation

detectable at 10^-3M (37 °C, 72 h) and prominent at 10^-2

M, and both were only slightly more efficient than the

corresponding unnatural enantiomers (1-2×). This ef-

ficiency of DNA alkylation proved analogous to that of

(+)-N-BOC-CBI (41) but distinct from the unnatural

enantiomer of N-BOC-CBI: (+)-N-BOC-CBI/ent-(-)-N-

BOC-CBI (5-10×)³³versus (+)-N-BOC-CCBI/ent-(-)-N-

BOC-CCBI and (+)-N-BOC-MCBI/ent-(-)-N-BOC-MCBI

(1-2×). This distinction between the enantiomers of 41

which was not observed with 40 or 25 was also accurately

reflected in the relative cytotoxic potencies of the agents

where the unnatural enantiomers of both N-BOC-CCBI

(4×) and N-BOC-MCBI (2×) more closely approach that

of the corresponding natural enantiomers than that of

N-BOC-CBI (11×). Like the preceding BOC derivatives

examined,^18,20,33,42the two enantiomers of 25 alkylated

DNA much less efficiently than 34-39 (10⁴×), providing

detectable alkylation at 10^-2-10^-3M (37 °C, 24-72 h)

much less selectively than 34-39 exhibiting a two base-

pair AT-rich alkylation selectivity (5′-AA > 5′-TA), and

did so with alkylation of the same sites. This unusual

behavior of the two enantiomers alkylating the same sites

is analogous to past observations.^11,12It is a natural

consequence of the reversed binding orientations of the

two enantiomers and the diastereomeric relationship of

the two adducts that result in the two enantiomers

covering the exact same binding site surrounding the

alkylated adenine. This has been discussed in detail and

F igu r e 3.

reactions were conducted at different temperatures (37

or 4 °C, 0.5-7 d). As discussed below, the rates and

efficiencies but not final relative efficiencies of DNA

alkylation were altered by changing the reaction tem-

peratures.

DNA Alk yla tion P r op er ties of (+)- a n d en t-(-)-N-

BOC-CCBI. A representative comparison of the DNA

alkylation properties of both enantiomers of N-BOC-

CCBI (25) alongside both enantiomers of N-BOC-MCBI

(40) within w794 DNA is illustrated in Figure 7. No

substantial distinctions between N-BOC-CCBI (25) and

4902 J . Org. Chem., Vol. 61, No. 15, 1996

Boger et al.

F igu r e 5.

illustrated elsewhere,^6,9,11,35and N-BOC-CCBI (25) con-

forms nicely to these past observations and models.

DNA Alk yla tion P r op er ties of th e Na tu r a l En a n -

tiom er s of CCBI-TMI (35), CCBI-In d ole₂(37), a n d

CCBI-CDP I₁(39). A comparison of the DNA alkylation

by the natural enantiomers of 35, 37, and 39 alongside

(+)-duocarmycin SA (2) and (+)-CC-1065 (1) within w794

DNA is illustrated in Figure 8 and is representative of

comparisons that have been made with the agents. (+)-

Duocarmycin SA (2) and (+)-CCBI-TMI (35) were in-

distinguishable, and the two agents exhibited the same

selectivity and efficiency of DNA alkylation. This is

illustrated nicely in Figure 8 where the two agents

detectably alkylate the same high affinity site of 5′-

AATTA at 10^-6-10^-7M (25 °C, 24 h). This is analogous

to the observations made in our prior comparisons of

duocarmycin SA (2) and CBI-TMI³⁵or MCBI-TMI.⁴²

The alkylation selectivities have been documented and

described in detail elsewhere^5,6,9,34,36and summarized in

reviews,¹¹and each alkylation site detected was adenine

followed by two 5′ A or T bases in a three base-pair site

that follows the following preference: 5′-AAA > 5′-TTA

> 5′-TAA > 5′-ATA. For the shorter agents CCBI-TMI

and CCBI-CDPI₁, there was also a strong preference but

not absolute requirement for the fourth 5′ base to be A

or T versus G or C, and this preference distinguished

many of the high versus low affinity sites (e.g., 5′-AAAA).

For the longer agent, CCBI-indole₂, not only was there

a stronger preference for the fourth base to be A or T

but that preference extended to include a fifth 5′ A or T

base (e.g., 5′-AAAAA). Thus, like the preceding agents,

the CCBI-based agents exhibited AT-rich adenine N3

F igu r e 6.

alkylation selectivities that start at the 3′ adenine N3

alkylation site with agent binding in the minor groove

in the 3′ f 5′ direction covering 3.5 or 5 base pairs.

DNA Alk yla tion P r op er ties of th e Un n a tu r a l

En a n tiom er s of CCBI-TMI (35), CCBI-In d ole₂(37),

a n d CCBI-CDP I₁(39). A representative comparison

of the DNA alkylation by the unnatural enantiomers of

the CCBI-based agents alongside the unnatural enanti-

omer of duocarmycin SA (1) and the natural enantiomer

of CC-1065 (1) in w794 of DNA is illustrated in Figure

9. Several important findings analogous to those made

in our prior studies with the CBI-based agents are also

observed with the CCBI-based agents. First, the un-

natural enantiomer DNA alkylation is considerably

slower, and the results shown in Figure 9 for the

unnatural enantiomers were obtained only with incuba-

tion at 25 °C (72 h) versus incubation at 25 °C (24 h,

Figure 8) for the natural enantiomers. Even with the

more vigorous reaction conditions (37 °C) or the more

extended reaction periods, the extent of alkylation by the

unnatural enantiomers is lower, requiring higher agent

concentrations to detect. This distinguishing difference

in the rate and efficiency of DNA alkylation was most

prominent with the smaller agents CCBI-TMI (35) and

duocarmycin SA (2) and readily perceptible but less

prominent with the intermediate-sized agents CCBI-

CDPI₁(39) and CCBI-indole₂(37). This trend is similar

to that observed in the relative cytotoxic potency of the

enantiomeric pairs.

Electronic Effects on Functional Reactivity

J . Org. Chem., Vol. 61, No. 15, 1996 4903

F igu r e 7. Thermally-induced strand cleavage of double-stranded DNA (SV40 DNA fragment, 144 bp, nucleotide no. 5238-138,

clone w794) after 72 h incubation of agent with DNA at 37 °C followed by removal of unbound agent, and 30 min incubation at

100 °C, 8% denaturing PAGE, and autoradiography: lane 1, (+)-N-BOC-MCBI (1 × 10^-2M); lane 2, ent-(-)-N-BOC-MCBI (1 ×

10^-2M); lane 3, control DNA; lane 4, (+)-CC-1065 (1 × 10^-6M); lanes 5-8, Sanger G, C, A, and T reactions; lanes 9-11 (+)-N-

BOC-CCBI (1 × 10^-2to 1 × 10^-4M); lanes 12-14, ent-(-)-N-BOC-CCBI (1 × 10^-2to 1 × 10^-4M).

The DNA alkylation selectivity and efficiency observed

with ent-(-)-CCBI-TMI (35) and ent-(-)-duocarmycin SA

(2) were nearly indistinguishable with the latter agent

being slightly more effective. This observation is analo-

gous to that made in our prior comparisons with (-)-

MCBI-TMI but different from that made with (-)-CBI-

TMI where the distinction was even larger (10×).³⁵The

larger agents were more effective at alkylating DNA, (-)-

CCBI-indole₂> (-)-CCBI-CDPI₁> (-)-CCBI-TMI,

and even with incubation at 25 °C for 72 h the more

effective agents did not achieve the efficiency observed

with the natural enantiomers. This is illustrated nicely

in Figure 9 with the comparison of the unreacted DNA

observed at 10^-6M for (+)-CC-1065 (1) versus the full

set of CCBI unnatural enantiomers. Again, no distinc-

tions in the DNA alkylation selectivity of the unnatural

enantiomers of the CCBI-based agents and the agents

described previously were perceptible. Each of the alky-

lation sites proved to be adenine, which was flanked on

both sides nearly always by an A or T base and the

preference for this three-base AT-rich site was 5′-AAA

> 5′-TAA > 5′-AAT > 5′-TAT. For the shorter agents,

there was a strong preference for the second 3′ base to

be A or T (e.g., 5′-AAAA), which for the larger agents

extended to the third 3′ base as well (e.g., 5′-AAAAA).

Thus, each alkylation site for the unnatural enantiomers

proved consistent with adenine N3 alkylation with agent

binding in the minor groove in the reverse 5′ f 3′

direction across a 3.5 or 5 base-pair AT-rich site sur-

rounding the alkylation site. This is analogous to the

natural enantiomer alkylation selectivity except that it

extends in the reverse 5′ f 3′ direction in the minor

groove and, because of the diastereomeric nature of the

adducts, is offset by one base pair relative to the natural

enantiomers.

En a n tiom er Distin ction s. Prior studies have sug-

gested an attractive explanation for the confusing be-

havior of enantiomeric pairs of agents that we have

proposed may be attributed to a single structural

featuresthe degree of steric bulk surrounding the CPI/

DSA C7 or CBI/MCBI C8 center in the alkylation subunit

for which the unnatural enantiomers are especially

sensitive.^6,35The enantiomer differences have proven

distinguishable with simple derivatives of the alkylation

subunits themselves (i.e., N-BOC-CCBI), are most promi-

nent with the dimer-based agents (i.e., CCBI-TMI), and

are less prominent or not readily distinguishable with

the larger trimer- or tetramer-based agents (i.e., CCBI-

CDPI₂). In general, less distinction in the biological

potency and relative DNA alkylation efficiency was

observed with the CI and duocarmycin SA enantiomeric

pairs, both of which lack substituents or steric bulk at

this position. Moreover, the distinctions among the

enantiomeric CI-based agents that lack the pyrrole ring

4904 J . Org. Chem., Vol. 61, No. 15, 1996

Boger et al.

F igu r e 8. Thermally-induced strand cleavage of double-stranded DNA (SV40 DNA fragment, 144 bp, nucleotide no. 5238-138,

clone w794) after 24 h incubation of agent-DNA at 25 °C followed by removal of unbound agent, 30 min incubation at 100 °C, 8%

denaturing PAGE, and autoradiography: lane 1, control DNA; lanes 2-4, (+)-duocarmycin SA (2, 1 × 10^-5to 1 × 10^-7M); lanes

5-7, (+)-CCBI-TMI (1 × 10^-5to 1 × 10^-7M); lanes 8-11, Sanger G, C, A, and T reactions; lanes 12-14, (+)-CC-1065 (1, 1 ×

10^-5to 1 × 10^-7M); lanes 15-17, (+)-CCBI-indole₂(1 × 10^-5to 1 × 10^-7M); lanes 18-19, (+)-CCBI-CDPI₁(1 × 10^-6and 1 ×

10^-7M).

altogether are small and less pronounced than those

observed with the DSA-based agents (DSA > CI). In

contrast, the CPI-, CBI-, and DA-based agents exhibit

more pronounced distinctions (CPI > DA > CBI), follow-

ing an order that reflects the relative steric differences.

Consistent with these observations, the CCBI-TMI

enantiomers were found to exhibit analogous distinctions

(Table 5). As detailed and illustrated elsewhere,^6,35this

distinguishing behavior of the unnatural enantiomers is

derived from a pronounced steric interaction of the CPI/

DSA C7 or CBI/CCBI C8 center with the 5′ base adjacent

to the adenine N3 alkylation site present in the un-

natural enantiomer 5′ f 3′ binding model.

(1.9×) > CBI-TMI (1.0) > duocarmycin SA (0.9×)

(Figure 10). The relative rates of DNA alkylation by

CCBI-indole₂(2.5×), MCBI-indole₂(1.4×), and CBI-

indole₂(1.0) at 25 °C were determined to exhibit nearly

identical trends. Although the studies are limited to a

very narrow reactivity range difficult to distinguish, the

rate of DNA alkylation did not correlate with the relative

reactivity of the agents toward acid-catalyzed solvolysis

suggesting that other or additional factors may contribute

to the rate or are responsible for the catalysis of the DNA

alkylation reaction.²⁰In prior studies, we have high-

lighted similar observations with agents that span a

much larger range of relative reactivities.³²

Ra te of DNA Alk yla tion . Analogous to two prior

studies,^35,42the relative rates of DNA alkylation for the

natural enantiomers of CCBI-TMI (35) and CCBI-

indole₂(37) versus those of the corresponding CBI and

MCBI agents were measured at the single high affinity

site in w794, 5′-AATTA. In these prior studies, the

relative rates of DNA alkylation for MCBI-TMI, CBI-

TMI, and duocarmycin SA were determined to be quite

similar at 4 °C, 1.8:1.0:0.9, respectively. The same

relative rates were observed in the present study now

extended to include CCBI-TMI (35), and this latter

agent proved to alkylate DNA with the fastest relative

rate (10^-6M, 25 °C): CCBI-TMI (2.5×) > MCBI-TMI

More interestingly, the final relative efficiency of DNA

alkylation observed under these reaction conditions did

more closely follow the trends of the relative reactivities

of the agents. The chemically more stable CCBI-based

agents 35 and 37 alkylated DNA 1.5-2× more efficiently

than the MCBI- or CBI-based agents which in turn were

essentially indistinguishable. Nearly identical trends

were observed in the relative stability of the agents with

CCBI being 1.6-2.1× more stable than CBI or MCBI

which in turn were very close in stability (1.06-1.2×).

Such observations are consistent with our prior studies

that suggest it is not the rate^12,60of DNA alkylation that

may be related to the cytotoxic potency of the agents, but

Electronic Effects on Functional Reactivity

J . Org. Chem., Vol. 61, No. 15, 1996 4905

F igu r e 9. Thermally-induced strand cleavage of duplex DNA (SV40 DNA segment, 144 bp, nucleotide no. 5238-138, clone w794)

after 72 h incubation at 25 °C followed by removal of unbound agent, 30 min incubation at 100 °C, 8% denaturing PAGE, and

autoradiography: lane 1, control DNA; lane 2, (+)-CC-1065 (1 × 10^-6M); lanes 3-6, Sanger G, C, A, and T sequencing reactions;

lane 7, ent-(-)-duocarmycin SA (1 × 10^-6M); lanes 8-9, ent-(-)-CCBI-TMI (1 × 10^-6and 10^-7M); lanes 10-11, ent-(-)-CCBI-

indole₂(1 × 10^-6and 10^-7M); lanes 12-13, ent-(-)-CCBI-CDPI₁(1 × 10^-6and 10^-7M).

Ta ble 5. En a n tiom er Distin ction s

but the effect was very small. Classical Hammett quan-

titation of the effect provided a remarkably small F

(-0.3), indicating an exceptionally small C7 substituent

electronic effect on functional reactivity. Additional

kinetic studies demonstrated that protonation of the C4

carbonyl is not the rate-determining step of solvolysis or

acid-catalyzed nucleophilic addition, but rather that it

is rapid and reversible followed by slow and rate-

determining nucleophilic addition to the cyclopropane

requiring the presence and assistance of a nucleophile

(S_N2 mechanism). No doubt this contributes to the DNA

alkylation selectivity and suggests that the positioning

of an accessible nucleophile (adenine N3) and not C4

carbonyl protonation or Lewis acid complexation is the

rate-determining step controlling the sequence selectivity

of DNA alkylation. This exceptionally small electronic

effect on the solvolysis rate had no impact on the

solvolysis regioselectivity, and stereoelectronically-con-

trolled nucleophilic addition to the least substituted

carbon of the activated cyclopropane was observed ex-

clusively. Consistent with past studies, a direct relation-

ship between solvolysis stability and cytotoxic potency

was demonstrated and related to the predictable Ham-

mett substituent effects. For the natural enantiomers,

this very small electronic effect on functional reactivity

had no perceptible effect on their DNA alkylation selec-

tivity. Although the range of reactivity spanned by the

agents is quite small and the effects are difficult to

distinguish, the efficiencies but not the rates of DNA

alkylation were found to correlate with the relative

reactivities of the agents with the most stable agent in

the series providing the most efficient DNA alkylation

as well as most potent cytotoxic activity. Similar effects

rel IC₅₀

rel DNA

agent

CI-TMI

duocarmycin SA

MCBI-TMI

CCBI-TMI

(L1210)^a

alkylation intensity^b

1

10

50

65

70

0.5-2.0

10

50

50-100

100

CBI-TMI

duocarmycin A

>100

a

b

IC₅₀(unnatural)/(natural) enantiomer. Concentrations of

unnatural/natural enantiomer required to detect DNA alkylation.

rather the ultimate efficiency of DNA alkylation that may

be more relevant to the expression of their biological

properties.^{6,9,11,13,29,31-35,42,61}J ust as importantly, they also

suggest that other factors are contributing to the DNA

alkylation reaction and that other or additional features²⁰

beyond C4 carbonyl protonation⁶⁰or Lewis acid complex-

ation may be responsible for catalysis. Most prevalent

among these possibilities is activation by ground-state

destabilization derived through a DNA binding induced

conformational change that activates the agent for nu-

cleophilic addition.²⁰Studies which address such issues

are in progress and will be reported in due course.

Con clu sion s. A short and efficient synthesis of CCBI

and its immediate precursors is detailed. Its evaluation

permitted an accurate assessment of the electronic effect

of substituents on the chemical and functional reactivity

of the agents and the impact this may have on their

biological properties. A study of the solvolysis reactivity

of N-BOC-CCBI and its comparison with related agents

revealed that the introduction of a strong electron-

withdrawing C7 cyano group slowed the rate of solvolysis

4906 J . Org. Chem., Vol. 61, No. 15, 1996

Boger et al.

Meth od B. NaH (2.99 g, 74.6 mmol, 1.05 equiv, 60%

dispersion in mineral oil) was washed with Et₂O (3 × 20 mL)

and suspended in anhydrous THF (100 mL) under Ar. The

suspension was cooled to 0 °C, 10⁵⁰(24.77 g, 72.3 mmol, 1.03

equiv) was added dropwise under Ar, and the reaction mixture

was stirred at 0 °C for 2 h. The solution was then transferred

by cannula to a solution of m-bromobenzaldehyde (4, 13.15 g,

71.1 mmol, 1 equiv) in 80 mL of THF at 0 °C. The resulting

reaction mixture was stirred at 0 °C for 1 h before being

allowed to warm to 25 °C, and the mixture was stirred

overnight. The solvent was removed under vacuum, and the

residue was partitioned between H₂O and CH₂Cl₂. The

aqueous phase was extracted with CH₂Cl₂(3 × 200 mL), and

the combined organic layers were dried (MgSO₄) and concen-

trated. The diester was purified by passage through a plug

of SiO₂(10% EtOAc-hexane) to provide 11 (26.3 g, 26.3 g

theoretical, 100%) as a clear oil: ¹H NMR (CDCl₃, 250 MHz)

δ 7.73 (s, 1H), 7.48 (s, 1H), 7.45 (m, 1H), 7.26 (d, 1H, J ) 5.8

Hz), 7.25 (d, 1H, J ) 6.8 Hz), 4.25 (q, 2H, J ) 7.1 Hz), 3.38 (s,

2H), 1.44 (s, 9H), 1.30 (t, 3H, J ) 7.1 Hz); ¹³C NMR (CDCl₃,

62.5 MHz) δ 169.6, 166.7, 139.2, 137.0, 131.4, 131.3, 129.8,

127.9, 127.3, 122.3, 80.9, 60.9, 34.5, 27.7, 14.0; IR (film) ν_max

2978, 2930, 1728, 1641, 1560, 1474, 1368, 1329, 1279, 1198,

1154, 1097, 786, 682 cm^-1; FABHRMS (NBA-CsI) m/z 500.9664

(M⁺+ Cs, C₁₇H₂₁BrO₄requires 500.9678).

A solution of 11 (16.6 g, 46.5 mmol) in 90% aqueous CF₃-

CO₂H (75 mL) was stirred for 30 min at 25 °C. The solvent

was removed under reduced pressure, and the residue was

azeotroped two times with benzene. The half-ester 5 was

purified by dissolution in saturated aqueous NaHCO₃solution

(50 mL). Acidification of the aqueous phase with 10% aqueous

HCl (pH ) 1) and extraction with EtOAc (3 × 50 mL) followed

by drying the combined organic phase (Na₂SO₄) and concen-

tration afforded pure 5 (14.6 g, 14.6 g theoretical, 100%) as a

pale yellow oil that crystallized under vacuum: mp 78-80 °C;

¹H NMR (CDCl₃, 400 MHz) δ 7.83 (s, 1H), 7.50 (m, 2H), 7.29

(m, 2H), 4.30 (q, 2H, J ) 7.1 Hz), 3.52 (s, 2H), 1.33 (t, 3H, J )

7.1 Hz); ¹³C NMR (CDCl₃, 100 MHz) δ 175.6, 167.1, 140.4,

136.8, 131.9, 131.8, 130.3, 127.3, 126.8, 122.8, 61.5, 33.5, 13.6;

IR (neat) ν_max2984, 1709, 1639, 1556, 1472, 1370, 1278, 1194,

1092, 1018, 782 cm^-1; FABHRMS (NBA-NaI) m/z 334.9883

(M⁺+ Na, C₁₃H₁₃BrO₄requires 334.9895).

The half-ester 5 (14.56 g, 46.5 mmol) was dissolved in Ac₂O

(310 mL, 0.15 M) under Ar. KOAc (5.93 g, 60.5 mmol, 1.3

equiv) was added, and the reaction mixture was warmed at

reflux for 30 min. The hot solution was poured into H₂O (400

mL), and the resulting mixture was stirred and allowed to cool

to 25 °C. The product was collected by filtration and recrystal-

lized from CH₃OH to yield pure 7 (10.06 g, 15.68 g theoretical,

65%) free of contaminant 9. For 7: mp 113-114 °C (CH₃OH);

¹H NMR (CDCl₃, 250 MHz) δ 8.34 (s, 1H, C1-H), 8.08 (d, 1H,

J ) 1.6 Hz, C8-H), 7.77-7.59 (m, 3H), 4.38 (q, 2H, J ) 7.1

Hz), 2.45 (s, 3H), 1.37 (t, 3H, J ) 7.1 Hz); IR (neat) ν_max2986,

1770, 1717, 1591, 1369, 1273, 1238, 1190, 1155, 1099, 1068,

1057, 1020, 907, 815 cm^-1; FABHRMS (NBA) m/z 358.9882

(M⁺, C₁₅H₁₃BrO₄requires 358.9895).

F igu r e 10. Plot of percent integrated optical density (IOD%)

versus time established through autoradiography of 5′-³²P end-

labeled DNA and used to monitor the relative rate of w794

alkylation at the 5′-AATTA high affinity site for 35, 37, (+)-

CBI-TMI, (+)-MCBI-TMI, (+)-CBI-indole₂, and (+)-MCBI-

indole₂.

of the C7 cyano group on the unnatural enantiomers were

detected and they proved to be 4-10× more effective than

the corresponding CBI-based unnatural enantiomer and

4-70× less potent than the corresponding CCBI natural

enantiomer.

Exp er im en ta l Section

Eth yl 7-Br om o-4-h yd r oxy-2-n a p h th a len eca r boxyla te

(6). Meth od A. A solution of t-BuOK (7.19 g, 64.1 mmol) in

t-BuOH (100 mL) at 45 °C was treated with a mixture of

m-bromobenzaldehyde (4, 10.78 g, 58.3 mmol) and diethyl

succinate (15.23 g, 87.4 mmol) dropwise. The reaction mixture

was warmed at reflux for 2 h. After cooling to 25 °C, the

mixture was neutralized with the addition of 10% aqueous HCl

(pH ) 1), and the t-BuOH was removed from the organic layer

under reduced pressure. The residue was extracted with

EtOAc (3 × 30 mL). The half ester was extracted from the

organic layer with 5% aqueous NaHCO₃(5 × 30 mL). The

combined basic aqueous layers were reacidified with aqueous

3 M HCl and extracted with EtOAc (3 × 30 mL). The

combined organic layers were washed with saturated aqueous

NaCl and dried (MgSO₄). Solvent removal yielded a mixture

of the two isomeric half-esters 5 (13.7 g, 18.2 g theoretical,

75%) as an amber oil.

The half-esters 5 (13.66 g) were dissolved in 300 mL of Ac₂O.

Anhydrous NaOAc (3.93 g, 48.0 mmol) was added, and the

reaction mixture was warmed at reflux for 6 h. The Ac₂O was

removed under reduced pressure, and the residue was sus-

pended in 10% aqueous Na₂CO₃(100 mL) and extracted with

EtOAc (3 × 100 mL). The combined organic layers were dried

(Na₂SO₄) and concentrated. The residue was dissolved in 150

mL of 3 M HCl-EtOH at 0 °C, and the solution was allowed

to warm to 25 °C and stir for 16 h. The solvent was removed

under reduced pressure. Chromatography (SiO₂, 5 × 20 cm,

20% EtOAc-hexane) and subsequent recrystallization from

toluene afforded pure 6 as a white solid free of contaminant

8.

The acetate 7 (15.7 g, 46.6 mmol) was dissolved in EtOH

(200 mL), and K₂CO₃(32.2 g, 233 mmol, 5 equiv) was added.

The reaction mixture was warmed at reflux for 1 h, cooled to

25 °C, and poured into H₂

O (200 mL). The mixture was

acidified with the addition of 10% aqueous HCl (pH ) 1), and

the desired product was extracted into EtOAc (3 × 250 mL).

The combined organic layers were washed with saturated

aqueous NaCl and dried (MgSO₄), and the solvent was

removed under reduced pressure. The resulting solid was

recrystallized from toluene to yield 6 (14.62 g, 14.62 g

theoretical, 100%): mp 180 °C (needles, toluene); ¹H NMR

(acetone-d₆, 250 MHz) δ 9.53 (br s), 8.23 (d, 1H, J ) 1.8 Hz),

8.18 (d, 1H, J ) 9.0 Hz), 8.09 (s, 1H), 7.68 (dd, 1H, J ) 2.0,

8.9 Hz), 7.50 (d, 1H, J ) 1.4 Hz), 4.37 (q, 2H, J ) 7.1 Hz),

1.38 (t, 3H, J ) 7.1 Hz); ¹³C NMR (acetone-d₆, 100 MHz) δ

166.5, 154.3, 136.0, 131.6, 130.9, 130.5, 126.3, 125.2, 121.7,

121.6, 108.4, 61.7, 14.5; IR (KBr) ν_max3379, 2989, 1701, 1588,

1476, 1420, 1399, 1386, 1360, 1284, 1252, 1080, 1026, 966, 893,

816, 768 cm^-1; FABHRMS (NBA) m/z 293.9888 (M⁺, C₁₃H₁₁

-

BrO₃requires 293.9892).

Electronic Effects on Functional Reactivity

J . Org. Chem., Vol. 61, No. 15, 1996 4907

Anal. Calcd for C₁₃H₁₁BrO₃: C, 52.91; H, 3.76. Found: C,

53.17; H, 3.46.

mmol, 1.2 equiv) was added, and the reaction mixture was

warmed at reflux for 14 h. The mixture was cooled to 25 °C,

and the solvent was removed under vacuum. The residue was

dissolved in EtOAc, and the organic phase was washed with

10% aqueous HCl, dried (Na₂SO₄), and concentrated in vacuo.

Chromatography (SiO₂, 3 × 20 cm, 20% EtOAc-hexane)

afforded 15 (534 mg, 618 mg theoretical, 87%) as a white

crystalline solid: mp 145 °C (white needles, 10% EtOAc-

hexane); ¹H NMR (CDCl₃, 250 MHz) δ 8.26 (d, 1H, J ) 8.7

Hz), 8.01 (s, 1H), 7.45 (m, 7H), 7.23 (s, 1H), 6.84 (s, 1H), 5.21

(s, 2H), 1.57 (s, 9H); ¹³C NMR (CDCl₃, 62.5 MHz) δ 154.9,

152.5, 138.1, 136.1, 133.6, 132.6, 128.6, 128.2, 127.4, 124.1,

123.6, 123.5, 119.3, 110.5, 106.5, 101.7, 81.1, 70.4, 28.3; IR

(KBr) ν_max3327, 2980, 2223, 1701, 1586, 1545, 1420, 1369,

Eth yl 4-(Ben zyloxy)-7-br om o-2-n a p h th a len eca r boxy-

la te (12). A solution of 6 (8.04 g, 2.2 mmol) in anhydrous DMF

(150 mL) under Ar was treated with K₂CO₃(5.65 g, 40.9 mmol),

benzyl bromide (5.59 g, 32.7 mmol, 1.2 equiv), and Bu₄NI (402

mg, 1.1 mmol, 0.04 equiv). After being stirred for 11 h at 25

°C, the reaction mixture was poured into H₂O (200 mL) and

extracted with EtOAc (3 × 200 mL). The combined organic

layers were washed with saturated aqueous NaCl, dried

(MgSO₄), and concentrated. The resulting solid was recrystal-

lized from 5% EtOAc-hexane to afford 12 (8.36 g, 10.49 g

theoretical, 80%) as white needles. An additional 2.13 g (20%)

of 12 was obtained by chromatography (SiO₂, 4 × 20 cm, 10%

EtOAc-hexane) of the crystallization mother liquors: mp 105

°C (needles, hexane); ¹H NMR (CDCl₃, 250 MHz) δ 8.19 (d,

1H, J ) 8.8 Hz), 8.10 (s, 1H), 8.05 (d, 1H, J ) 1.8 Hz), 7.61

(dd, 1H, J ) 1.9, 8.9 Hz), 7.50 (m, 6H), 5.28 (s, 2H), 4.45 (q,

2H, J ) 7.1 Hz), 1.46 (t, 3H, J ) 7.1 Hz); ¹³C NMR (CDCl₃,

100 MHz) δ 166.5, 154.7, 136.4, 134.7, 130.8, 130.7, 129.1,

128.7, 128.3, 127.6, 126.3, 124.2, 122.5, 121.5, 104.6, 70.4, 61.4,

14.4; IR (KBr) ν_max2986, 1716, 1589, 1578, 1406, 1369, 1331,

1341, 1251, 1160, 1107, 1064, 1002, 910, 820, 744, 694 cm^-1

;

FABHRMS (NBA) m/z 374.1641 (M⁺, C₂₃H₂₂N₂O₃requires

374.1630).

Anal. Calcd for

C₂₃H₂₂N₂O₃: C, 73.78; 5.92; N, 7.48.

Found: C, 73.40; H, 5.84; N, 7.23.

N-(ter t-Bu t yloxyca r b on yl)-4-(b en zyloxy)-1-b r om o-7-

cya n o-2-n a p h th yla m in e (16). A solution of 15 (137 mg,

0.366 mmol) in freshly distilled THF (7.3 mL) and cooled to

-78 °C under Ar was treated with 10 µL of a 1 µL/mL solution

of H₂SO₄in THF, and the solution was stirred for 20 min before

the addition of NBS (78 mg, 439 mmol, 1.2 equiv). The

reaction mixture was allowed to warm to -60 °C and was

stirred for 4 h at which time the reaction was complete by TLC.

Et₂O (7.3 mL) was added, and the resulting organic phase was

washed with 5% aqueous NaHCO₃(1 × 10 mL) and saturated

aqueous NaCl (1 × 10 mL), dried (MgSO₄), and concentrated

in vacuo. Chromatography (SiO₂, 2 × 20 cm, 10% EtOAc-

hexane) afforded 16 (145 mg, 166 mg theoretical, 87%) as a

white crystalline solid: mp 179 °C dec (white needles, 10%

EtOAc-hexane); ¹H NMR (CDCl₃, 250 MHz) δ 8.40 (s, 1H),

8.26 (d, 1H, J ) 8.5 Hz), 8.23 (s, 1H), 7.5-7.3 (m, 6H), 5.23 (s,

2H), 1.58 (s, 9H); ¹³C NMR (CDCl₃, 75 MHz) δ 154.4, 152.4,

137.0, 136.0, 132.2, 132.0, 128.7, 128.6, 128.4, 128.0, 124.9,

124.8, 124.2, 119.1, 111.9, 102.0, 81.9, 70.8, 28.3; IR (KBr) ν_max

3416, 2978, 2230, 1738, 1623, 1603, 1570, 1498, 1405, 1364,

1279, 1238, 1098, 1025, 962, 892, 816, 767, 725, 691 cm^-1

;

FABHRMS (NBA) m/z 384.0370 (M⁺, C₂₀H₁₇BrO₃requires

384.0361).

Anal. Calcd for C₂₀H₁₇BrO₃: C, 62.35; H, 4.45. Found: C,

62.23; H, 4.61.

Eth yl 4-(Ben zyloxy)-7-cya n o-2-n a p h th a len eca r boxy-

la te (13). A solution of 12 (9.46 g, 27.2 mmol) in anhydrous

DMF (12.6 mL) under Ar was treated with CuCN (2.92 g, 32.6

mmol, 1.2 equiv), and the mixture was warmed at reflux for

20 h. The reaction mixture was cooled to 25 °C and poured

into 250 mL of H₂O to which FeCl₃(5.29 g, 32.6 mmol, 1.2

equiv) was added with swirling. The solution was extracted

with EtOAc (3 × 200 mL), and the combined organic layers

were washed with saturated aqueous NaCl, dried (MgSO₄),

and concentrated. Recrystallization from 20% EtOAc-hexane

provided 13 (6.70 g, 9.00 g theoretical, 74%) as a white solid.

Chromatography (SiO₂, 4 × 20 cm, 10% EtOAc-hexane) of the

crystallization mother liquors afforded additional 13 (2.00 g,

22%, 96% combined) as white solid: mp 125 °C (needles,

EtOH); ¹H NMR (CDCl₃, 250 MHz) δ 8.43 (d, 1H, J ) 8.7 Hz),

8.29 (s, 1H), 8.25 (s, 1H), 7.69 (dd, 1H, J ) 1.6, 8.7 Hz), 7.66

(s, 1H), 7.56-7.39 (m, 5H), 5.32 (s, 2H), 4.46 (q, 2H, J ) 7.2

Hz), 1.46 (t, 3H, J ) 7.1 Hz); ¹³C NMR (CDCl₃, 62.5 MHz) δ

165.9, 154.3, 135.9, 134.6, 132.3, 129.8, 128.6, 128.3, 128.2,

127.8, 127.5, 123.8, 123.2, 118.6, 110.7, 106.9, 70.5, 61.5, 14.3;

IR (KBr) ν_max2994, 2226, 1711, 1577, 1502, 1284, 1252, 1093,

1335, 1229, 1158, 990, 970, 878, 850, 823, 758, 696 cm^-1

;

FABHRMS (NBA-CsI) m/z 584.9793 (M⁺+ Cs, C₂₃H₂₁BrN₂O₃

requires 584.9790).

Anal. Calcd for C₂₃H₂₁BrN₂O₃: C, 60.94; H, 4.67; N, 6.18.

Found: C, 61.12; H, 4.75; N, 6.04.

N-(ter t-Bu t yloxyca r b on yl)-N-(3-m et h yl-2-bu t en -1-yl)-

4-(ben zyloxy)-1-br om o-7-cya n o-2-n a p h th yla m in e (17). A

solution of 16 (1.77 g, 3.90 mmol) in anhydrous DMF (20 mL)

under Ar was treated with NaH (206 mg, 5.1 mmol, 1.3 equiv,

60% oil dispersion), and the reaction mixture was stirred for

30 min. The mixture was cooled to 0 °C, and 4-bromo-2-

methyl-2-butene (1.35 mL, 11.7 mmol, 3 equiv) was added

dropwise by cannula. The solution was stirred at 0 °C for 1 h

before being allowed to warm to 25 °C and stirred overnight.

Water (20 mL) was added, and the aqueous phase was

extracted with EtOAc (3 × 15 mL). The combined organic

phases were washed with saturated aqueous NaCl (1 × 30 mL)

and dried (Na₂SO₄), and the solvent was removed under

vacuum. Chromatography (SiO₂, 4 × 20 cm, 10% EtOAc-

hexane) afforded 17 (2.05 g, 2.03 g theoretical, >99%) as an

amber oil: ¹H NMR (CDCl₃, 400 MHz) δ 8.66 (d, 1H, J ) 0.5

Hz), 8.40 (d, 1H, J ) 8.6 Hz), 7.63 (d, 1H, J ) 8.6 Hz), 7.51-

7.34 (m, 5H), 6.85 (s, 1H), 5.24 (d, 1H, J ) 9.5 Hz), 5.20 (d,

1H, J ) 11.7 Hz), 5.17 (d, 1H, J ) 11.7 Hz), 4.40 (dd, 1H, J )

6.1, 14.5 Hz), 4.01 (dd, 1H, J ) 7.7, 14.9 Hz), 1.61 (s, 3H),

1.57 (s, 3H), 1.37 and 1.30 (two s, 9H); ¹³C NMR (CDCl₃, 100

MHz) δ 153.7, 140.9, 136.3, 135.8, 133.5, 128.8, 128.5, 127.3,

126.7, 124.1, 119.8, 119.5, 118.8, 111.7, 110.9, 81.0, 80.5, 70.8,

47.7, 46.3, 28.5, 28.2, 25.7; IR (film) ν_max3467 (br), 2928, 2229,

1701, 1676, 1596, 1503, 1438, 1387, 1335, 1255, 1164, 1090,

1029, 916, 827 cm^-1; FABHRMS (NBA) m/z 332.1276 (M⁺

H, C₂₁H₁₇NO₃requires 332.1287).

+

Anal. Calcd for C₂₁H₁₇NO₃: C, 76.11; H, 5.17; N, 4.23.

Found: C, 75.96; H, 5.42; N, 4.31.

4-(Ben zyloxy)-7-cya n o-2-n a p h th a len eca r boxylic Acid

(14). A solution of 13 (8.67 g, 26.2 mmol) in 260 mL of THF-

CH₃OH-H₂O (3:1:1) was treated with LiOH-H₂O (5.49 g, 131

mmol, 5 equiv), and the mixture was stirred at 25 °C for 25 h.

The solution was acidified with the addition of 10% aqueous

HCl (pH < 1) and the product partially precipitated. The

product was collected by filtration, and the remaining aqueous

phase was extracted with EtOAc (3 × 200 mL). The combined

organic layers were dried (MgSO₄) and concentrated in vacuo

to afford a combined 14 (7.94 g, 7.94 g theoretical, 100%): mp

235 °C (white powder, EtOH); ¹H NMR (DMSO-d₆, 400 MHz)

δ 8.73 (d, 1H, J ) 1.1 Hz), 8.34 (d, 1H, J ) 8.9 Hz), 8.32 (s,

1H), 7.88 (dd, 1H, J ) 1.5, 8.9 Hz), 7.63 (s, 1H), 7.57-7.34 (m,

5H), 5.38 (s, 2H); ¹³C NMR (CDCl₃, 100 MHz) δ 167.3, 153.8,

136.6, 135.5, 132.3, 130.3, 128.6, 128.3, 128.1, 127.6, 126.4,

123.4, 121.9, 118.8, 110.0, 107.5, 70.0; IR (KBr) ν_max3000 (br),

2227, 1687, 1577, 1499, 1419, 1343, 1283, 1250, 1105, 996, 915,

836, 740 cm^-1; FABHRMS (NBA) m/z 304.0964 (M⁺+ H,

C₁₉H₁₃NO₃requires 304.0974).

863, 738 cm^-1

.

Anal. Calcd for C₂₈H₂₉BrN₂O₃: C, 64.60; H, 5.62; N, 5.38.

Found: C, 64.51; H, 5.74; N, 5.18.

N-(ter t-Bu t yloxyca r b on yl)-N-(for m ylm et h yl)-4-(b en -

zyloxy)-1-br om o-7-cya n o-2-n a p h th yla m in e (18). A solu-

tion of 17 (180 mg, 0.345 mmol) in 22 mL (0.016 M) of 20%

CH₃OH-CH₂Cl₂was cooled to -78 °C. A stream of 3% O₃/O₂

N-(ter t-Bu t yloxyca r b on yl)-4-(b en zyloxy)-7-cya n o-2-

n a p h th yla m in e (15). A solution of 14 (500 mg, 1.65 mmol)

in freshly distilled t-BuOH (165 mL) was treated with Et₃N

(0.276 mL, 1.98 mmol, 1.2 equiv) and 5 g of activated 4 Å

molecular sieves. Diphenyl phosphorazidate (0.426 mL, 1.98

4908 J . Org. Chem., Vol. 61, No. 15, 1996

Boger et al.

(160 L/min) was bubbled through the solution for 72 s. The

reaction was immediately quenched with the addition of 0.81

mL of dimethyl sulfide, and the mixture was allowed to stir

at -78 °C for 5 min before being allowed to warm to 25 °C

and stirred for 5 h. The solvent was removed in vacuo.

Chromatography (SiO₂, 2 × 20 cm, 30% EtOAc-hexane)

afforded 18 (155 mg, 171 mg theoretical, 91%) as a white foamy

solid: ¹H NMR (CDCl₃, 400 MHz) δ 9.76 and 9.73 (two s), 8.63

and 8.61 (two s, 1H), 8.41 and 8.36 (two d, 1H, J ) 8.7 Hz),

7.64 and 7.60 (two dd, 1H, J ) 1.4, 8.7 Hz), 7.50-7.34 (m,

5H), 7.17 and 7.14 (two s, 1H), 5.26, 5.29 and 5.17 (one s and

two d, 2H, J ) 11.4 Hz), 4.76 and 4.65 (two d, 1H, J ) 18.8

Hz), 4.01 and 3.95 (two d, 1H, J ) 18.8 Hz), 1.31 and 1.52

(two s, 9H); ¹³C NMR (CDCl₃, 100 MHz, major rotamer) δ

197.3, 154.1, 140.8, 135.6, 133.4, 132.0, 128.8, 128.7, 128.5,

127.9, 127.6, 127.1, 124.2, 118.7, 113.8, 112.0, 110.5, 81.8, 70.9,

58.9, 28.1; IR (film) ν_max2976, 2229, 1706, 1596, 1415, 1369,

1335, 1259, 1225, 1152, 1096, 848 cm^-1; FABHRMS (NBA) m/z

495.0922 (M⁺+ H, C₂₅H₂₃BrN₂O₄requires 495.0919).

(SiO₂, 1 × 20 cm, 20% EtOAc-hexane) afforded 21 (29.7 mg,

29.7 mg theoretical, 100%) as an off-white solid: ¹H NMR

(CDCl₃, 400 MHz) δ 8.32 (d, 1H, J ) 8.7 Hz, C6-H), 8.09 (s,

1H, C9-H), 8.02 (br s, 1H, C4-H), 7.51 (d, 1H, J ) 8.7 Hz, C7-

H), 7.43 (m, 5H), 5.25 (s, 2H, CH₂Ph), 4.21 (dd, 1H, J ) 11.5,

2.3 Hz, C2-H), 4.14 (dd, 1H, J ) 11.5, 8.9 Hz, C2-H), 3.92 (dd,

1H, J ) 4.0, 10.3 Hz, CHHOH), 3.80 (m, 1H, C1-H), 3.75 (dd,

1H, J ) 7.2, 10.3 Hz, CHHOH), 1.59 (s, 9H); ¹³C NMR (CDCl₃,

100 MHz) δ 155.5, 153.0, 136.2, 129.7, 128.7, 128.5, 128.2,

127.7, 124.9, 123.5, 123.2, 119.4, 115.0, 110.7, 99.1, 80.5, 70.6,

64.8, 52.5, 47.0, 41.1, 28.4; IR (KBr) ν_max3450 (br), 2926, 2226,

1702, 1592, 1458, 1410, 1368, 1328, 1143, 1032, 735, 696 cm^-1

;

FABHRMS (NBA) m/z 430.1896 (M⁺, C₂₆H₂₃N₂O₄requires

430.1893).

F r om 29. A solution of 29 (341 mg, 0.599 mmol) in THF-

HOAc-H₂O (3:1:1, 20 mL) was treated with Zn powder (3.13

g, 80 equiv), and the mixture was warmed at 70 °C for 6 h.

The Zn powder was removed by filtration through Celite, and

the mixture was concentrated under vacuum. Chromatogra-

phy (SiO₂, 1.3 × 13 cm, 0-25% EtOAc-hexane) afforded 21

(188 mg, 258 mg theoretical, 73%) as an off-white solid

identical in all respects to that described above.

Anal. Calcd for C₂₅H₂₃BrN₂O₄: C, 60.62; H, 4.68; N, 5.65.

Found: C, 60.39; H, 4.61; N, 5.69.

2-[N-(ter t-Bu tyloxyca r bon yl)-N-[3-(tetr a h yd r op yr a n y-

loxy)-2-p r op en -1-yl]a m in o]-4-(ben zyloxy)-1-br om o-7-cy-

a n on a p h th a len e (19). A suspension of triphenyl[(2-tetrahy-

dropyranyloxy)methyl]phosphonium chloride⁵⁴(635 mg, 1.51

mmol, 3 equiv) in THF (5 mL) at -78 °C was treated dropwise

with n-BuLi (1.44 mmol, 0.58 mL, 2.5 M in hexane, 2.86 equiv).

The reaction mixture was stirred at -78 °C for 10 min and

allowed to warm to 0 °C over 20 min. The mixture was

recooled to -78 °C, and HMPA (2.11 mL, 12.1 mmol, 24 equiv)

was added followed immediately by the addition of 18 (250

mg, 0.505 mmol) in 2.5 mL of THF. The reaction was stirred

at -78 °C for 1.5 h and 24 h at 25 °C before being quenched

with the addition of 20 mL of phosphate buffer (pH 7.0). The

mixture was extracted with EtOAc (3 × 50 mL), and the

combined organic phase was dried (Na₂SO₄) and concentrated

in vacuo. Chromatography (SiO₂, 2 × 30 cm, 10% EtOAc-

hexane with 2% Et₃N) afforded 19 (221 mg, 300 mg theoretical,

74%) as an oil and as a mixture of E- and Z-isomers: ¹H NMR

(CDCl₃, 400 MHz) δ 8.65 (s, 1H), 8.38 (m, 1H), 7.62 (d, 1H, J

) 8.1 Hz), 7.49-7.36 (m, 6H), 6.96-6.79 (m, 1H), 6.22-6.07

(m, 1H), 5.28-5.15 (m, 2H), 4.83-4.29 (m, 3H), 3.85-2.77 (m,

2H), 1.98-1.22 (m, 15H); IR (film) ν_max2940, 2229, 1704, 1596,

1415, 1367, 1332, 1258, 1225, 1163, 1021, 965, 902, 849, 739,

5-(Be n zyloxy)-3-(t er t -b u t yloxyca r b on yl)-1-(ch lor o-

m eth yl)-8-cya n o-1,2-d ih yd r o-3H-ben z[e]in d ole (22).

A

solution of 21 (47 mg, 0.10 mmol) in freshly distilled anhydrous

CH₂Cl₂(0.35 mL) under Ar was treated sequentially with Ph₃P

(82 mg, 0.31 mmol, 3 equiv) and CCl₄(91 µL, 0.94 mmol, 9

equiv). The reaction mixture was stirred at 25 °C for 2 h. The

solvent was evaporated under a stream of N₂. Chromatogra-

phy (SiO₂, 0.8 × 10 cm, 10% EtOAc-hexane) afforded 22 (49

mg, 49 mg theoretical, 100%) as a white solid: mp 212-214

°C; ¹H NMR (CDCl₃, 400 MHz) δ 8.33 (d, 1H, J ) 8.7 Hz, C6-

H), 7.98 (s, 1H, C9-H), 7.70 (br s, 1H, C4-H), 7.50-7.24 (m,

6H), 5.24 (s, 2H), 4.23 (d, 1H, J ) 11.0 Hz, C2-H), 4.14 (dd,

1H, J ) 11.0, 8.9 Hz, C2-H), 3.95 (dddd, 1H, J ) 2.6, 9.2, 11.1,

12.1 Hz, C1-H), 3.83 (dd, 1H, J ) 3.1, 11.1 Hz, CHHCl), 3.47

(dd, 1H, J ) 9.9, 11.0 Hz, CHHCl), 1.54 (s, 9H); ¹³C NMR

(CDCl₃, 100 MHz) δ 155.9, 144.3, 136.1, 134.3, 133.8, 133.6,

130.4, 128.7, 128.5, 128.4, 128.3, 127.7, 125.1, 123.5, 119.2,

111.1, 99.0, 70.6, 53.1, 46.3, 41.3, 28.3; IR (KBr) ν_max2978,

2231, 1692, 1594, 1478, 1409, 1374, 1337, 1166, 1146, 1084,

973, 960, 859, 756, 697 cm^-1; FABHRMS (NBA) m/z 448.1570

(M⁺, C₂₆H₂₅ClN₂O₃requires 448.1554).

Resolu tion of 22. Samples of racemic 22 were resolved

by preparative HPLC chromatography on a Diacel Chiralcel-

OD column (10 µm, 2 × 25 cm) using 7% i-PrOH-hexane

eluant (7 mL/min). The enantiomers eluted with retention

times of 20.70 min (unnatural enantiomer) and 28.54 min

(natural enantiomer), R ) 1.38.

697 cm^-1

.

5-(Ben zyloxy)-3-(ter t-bu tyloxycar bon yl)-8-cyan o-1-[(tet-

r a h yd r op yr a n yloxy)m et h yl]-1,2-d ih yd r o-3H -b en z[e]in -

d ole (20). A solution of 19 (41 mg, 69 µmol) in freshly distilled

benzene (3.5 mL) under Ar was treated with AIBN (2 mg, 0.2

equiv) followed by Bu₃SnH (40 mg, 0.014 mmol, 2 equiv). The

reaction mixture was warmed at reflux for 2 h and cooled to

25 °C, and the solvent was removed under a stream of N₂. The

residue was azeotroped with THF (1 × 2 mL). Chromatogra-

phy (SiO₂, 1 × 20 cm, 10% EtOAc-hexane) afforded 20 (35

mg, 35.5 mg theoretical, 99%) as a clear oil: ¹H NMR (CDCl₃,

400 MHz) δ 8.29 (d, 1H, J ) 8.7 Hz), 8.24 and 8.19 (s and d,

1H, J ) 1.3 Hz), 8.01 (br s, 1H), 7.50 (br d, 1H, J ) 7.1 Hz),

7.44-7.34 (m, 5H), 5.24 (s, 2H), 4.58 and 4.55 (two br m, 1H),

4.12 (br s, 1H), 4.10 (br s, 1H), 3.96 (dd, 1H, J ) 5.8, 9.6 Hz),

3.87 (m, 1H), 3.78 (dd, 1H, J ) 8.9, 9.6 Hz), 3.64 and 3.58 (ddd

and dd, 1H, J ) 3.1, 8.1, 11.3 Hz and 6.2, 9.4 Hz), 3.50-3.38

(m, 1H), 1.73-1.55 (m, 15H); ¹³C NMR (CDCl₃, 100 MHz) δ

155.3, 153.0, 136.3, 129.8, 129.5, 128.6, 128.2, 127.7, 124.5,

123.4, 123.1, 119.5, 110.3, 99.7, 99.1, 80.5, 70.5, 70.1, 62.6, 62.4,

52.8, 38.9, 30.5, 28.4, 28.2, 25.3, 19.5; IR (film) ν_max2943, 2226,

1703, 1622, 1592, 1454, 1367, 1328, 1258, 1141, 1033, 967, 854,

(1S)-22: [R]²⁵-9.5 (c 0.5, CHCl₃).

D

en t-(1R)-22: [R]²⁵+9.5 (c 0.3, CHCl₃).

D

3-(ter t-Bu tyloxyca r bon yl)-1-(ch lor om eth yl)-8-cya n o-5-

h yd r oxy-1,2-d ih yd r o-3H-ben z[e]in d ole (23). A solution of

22 (71.5 mg, 0.159 mmol) and 10% Pd-C (40 mg) in anhydrous

EtOAc (5 mL) was degassed with a stream of N₂

for 30 s. The

resulting mixture was placed under an atmosphere of H₂

and

stirred at 25 °C for 2.5 h. The mixture was diluted with THF

(1 mL) and filtered through Celite (EtOAc wash). The solvent

was removed in vacuo. Chromatography (SiO₂, 1.5 × 6 cm,

20% EtOAc-hexane) afforded 23 (57.1 mg, 57.1 mg theoretical,

100%) as a white solid: ¹H NMR (CDCl₃, 400 MHz) δ 8.25 (d,

1H, J ) 8.7 Hz, C6-H), 7.97 (s, 1H, C9-H), 7.87 (br s, 1H, C4-

H), 7.42 (dd, 1H, J ) 1.5, 8.7 Hz, C7-H), 6.67 (br s, 1H, OH),

4.23 (d, 1H, J ) 11.4 Hz, C2-H), 4.14 (dd, 1H, J ) 8.8, 11.8

Hz, C2-H), 3.95 (m, 1H, C1-H), 3.82 (dd, 1H, J ) 3.2, 11.3 Hz,

CHHCl), 3.46 (dd, 1H, J ) 9.8, 11.2 Hz, CHHCl), 1.59 (s, 9H);

¹³C NMR (CDCl₃, 100 MHz) δ 154.2, 153.1, 142.9, 129.4, 127.8,

125.2, 123.2, 122.8, 119.3, 114.7, 111.0, 101.7, 82.5, 53.3, 46.3,

41.3, 28.4; IR (film) ν_max3315 (br), 2964, 2923, 2227, 1676,

1585, 1421, 1369, 1331, 1235, 1141, 729 cm^-1; FABHRMS

(NBA) m/z 358.1076 (M⁺+ H, C₁₉H₁₉ClN₂O₃requires 358.1084).

736, 697 cm^-1; FABHRMS (NBA-CsI) m/z 647.1549 (M⁺

Cs, C₃₁H₃₄N₂O₅requires 647.1552).

+

5-(Ben zyloxy)-3-(ter t-bu tyloxyca r bon yl)-8-cya n o-1-(h y-

d r oxym eth yl)-1,2-d ih yd r o-3H-ben z[e]in d ole (21). F r om

20. A solution of 20 (35 mg, 69 µmol) in freshly distilled CH₃-

OH (1 mL) was treated with 0.5 mg of Amberlyst-15 ion

exchange resin, and the reaction mixture was stirred at 45 °C

for 5 h. The resin was removed by filtration and washed with

CH₃OH (1 × 2 mL). The filtrates were combined, and the

solvent was removed under a stream of N₂. Chromatography

(1S)-23: [R]²³-15 (c 0.08, CHCl₃).

D

en t-(1R)-23: [R]²³+16 (c 0.09, CHCl₃).

D

N-(ter t-Bu t yloxyca r b on yl)-7-cya n o-1,2,9,9a -t et r a h y-

d r ocyclop r op a [c]ben z[e]in d ol-4-on e (25, N-BOC-CCBI).

Meth od A. A solution of 23 (1.4 mg, 3.91 µmol) in 2:1 DMF-

Electronic Effects on Functional Reactivity

J . Org. Chem., Vol. 61, No. 15, 1996 4909

THF (112 µL) at 0 °C was treated with NaH (1.6 mg, 39 µmol,

60% oil dispersion), and the mixture was stirred for 30 min.

The solvent was removed under a stream of N₂and vacuum.

PTLC (SiO₂, 0.25 mm × 10 × 15 cm, 30% EtOAc-hexane)

afforded 25 (1.20 mg, 1.26 mg theoretical, 95%) as a white

solid: ¹H NMR (CDCl₃, 400 MHz) δ 8.29 (d, 1H, J ) 8.1 Hz,

C5-H), 7.64 (dd, 1H, J ) 1.5, 8.1 Hz, C6-H), 7.14 (d, 1H, J )

1.1 Hz, C8-H), 6.87 (br s, 1H, C3-H), 4.03 (m, 2H, C1-H₂), 2.81

(dt, 1H, J ) 4.9, 7.5 Hz, C9a-H), 1.65 (dd, 1H, J ) 4.7, 7.9 Hz,

C9-H), 1.55 (s, 10H, C9-H and C(CH₃)₃); ¹³C NMR (CDCl₃, 100

MHz) δ 180.4, 159.8, 151.4, 140.9, 135.8, 129.6, 127.7, 125.3,

118.2, 115.0, 108.8, 83.9, 52.9, 33.2, 29.4, 28.1, 23.8; IR (film)

ν_max2977, 2230, 1727, 1634, 1608, 1396, 1369, 1295, 1277,

1250, 1159, 1131, 843 cm^-1; UV (THF) λ_max259 (ꢀ ) 22 200),

267 (ꢀ ) 24 000), 300 nm (ꢀ ) 12 000); UV (CH₃OH) λ_max260

(ꢀ ) 24 700), 266 (ꢀ ) 25 200), 317 nm (ꢀ ) 13 400); FABHRMS

(NBA) m/z 323.1383 (M⁺+ H, C₁₉H₁₈N₂O₃requires 323.1396).

N-(ter t-Bu tyloxycar bon yl)-N-(2-pr open yl)-4-(ben zyloxy)-

7-cya n o-1-iod o-2-n a p h th yla m in e (28). A solution of 27 (160

mg, 0.31 mmol) in anhydrous DMF (5 mL) under Ar was

treated with NaH (19 mg, 0.47 mmol, 1.5 equiv, 60% oil

dispersion), and the reaction mixture was stirred for 30 min

at 0 °C. Allyl bromide (194 mg, 139 µL, 1.55 mmol, 5 equiv)

was added dropwise over 5 min, and the solution was allowed

to warm to 25 °C and stirred for 2 h. Saturated aqueous

NaHCO₃(10 mL) was added, and the aqueous phase was

extracted with EtOAc (4 × 10 mL), dried (Na₂SO₄), and

concentrated. Chromatography (SiO₂, 2 × 15 cm, 10% EtOAc-

hexane) afforded 28 (155 mg, 168 mg theoretical, 92%) as a

clear oil that crystallized under vacuum: ¹H NMR (CDCl₃, 400

MHz) δ 8.60 (s, 1H), 8.38 (d, 1H, J ) 8.6 Hz), 7.63 (d, 1H, J )

8.4 Hz), 7.47-7.34 (m, 5H), 6.90 and 6.79 (two s, 1H), 5.94-

5.84 (m, 1H), 5.27 and 5.22 (two d, 2H, J ) 12.3 Hz), 5.00 (m,

2H), 4.50 (dd, 1H, J ) 5.3, 14.4 Hz), 3.81 (dd, J ) 7.0, 14.4

Hz), 1.57 and 1.29 (two s, 9H); ¹³C NMR (CDCl₃, 100 MHz) δ

154.8, 144.9, 138.7, 135.7, 134.8, 133.2, 128.8, 128.3, 127.3,

126.9, 126.8, 124.2, 118.8, 118.4, 117.2, 112.2, 110.7, 80.8, 70.7,

53.3, 52.0, 28.4; IR (film) ν_max2977, 2228, 1703, 1593, 1503,

1410, 1368, 1324, 1250, 1223, 1150, 1108, 931, 830, 735, 696

(+)-N-BOC-CCBI (25): [R]²³+124 (c 0.04, THF).

D

en t-(-)-N-BOC-CCBI (25): [R]²³-121 (c 0.03, THF).

D

Meth od B. A solution of 23 (8.6 mg, 24.0 µmol) was

dissolved in 1:1 THF-5% aqueous NaHCO₃(2 mL), and the

mixture was stirred at 25 °C for 9 h. The THF was removed

by evaporation, and the product was extracted with EtOAc (4

× 1 mL). Chromatography (SiO₂, 0.8 × 5 cm, 0-25% EtOAc-

hexane gradient) afforded 25 (7.7 mg, 7.7 mg theoretical, 100%)

as a white solid identical to that described above.

cm^-1; FABHRMS (NBA-CsI) m/z 672.9948 (M⁺+ Cs, C₂₆H₂₅

-

IN₂O₃, requires 672.9964).

Anal. Calcd for C₂₆H₂₅IN₂O₃: C, 57.99; H, 4.66; N, 5.18.

Found: C, 57.60; H, 4.41; N, 5.27.

5-(Be n zyloxy)-3-(t er t -b u t yloxyca r b on yl)-8-cya n o-1-

[(2′,2′,6′,6′- tetr a m eth ylp ip er id in o)oxy]m eth yl-1,2-d ih y-

d r o-3H-ben z[e]in d ole (29). A solution of 28 (200 mg, 0.37

mmol) in freshly distilled benzene (12 mL) was treated

sequentially with TEMPO (3.0 equiv) and Bu₃SnH (1.0 equiv).

The reaction mixture was warmed to 60 °C. After 20 min, an

additional 1 equiv of Bu₃SnH was added. After 30 min,

additional TEMPO (2 equiv) and Bu₃SnH (1.0 equiv) were

added. After 20 min, 2 equiv of TEMPO and Bu₃SnH in two

separate portions at 15 min intervals were added. After 45

min at 60 °C, the solvent was removed by evaporation. PCTLC

(4 mm SiO₂, 0-25% EtOAc-hexane gradient) afforded 29 (144

mg, 212 mg theoretical, 68%) as a semisolid: ¹H NMR (CDCl₃,

400 MHz) δ 8.31 (d, 1H, J ) 8.7 Hz), 8.19 (s, 1H), 8.01 (s, 1H),

7.53 (d, 2H, J ) 7.0 Hz), 7.44 (t, 2H, J ) 7.0 Hz), 7.40 (m,

2H), 5.26 (s, 2H), 4.13 (m, 2H), 3.96 (m, 1H), 3.88 (t, 1H, J )

7.0 Hz), 3.80 (m, 1H), 1.59 (s, 9H), 1.39 (m, 4H), 1.10 (s, 3H),

1.03 (s, 6H), 0.96 (s, 3H), 0.92 (t, 2H, J ) 7.3 Hz); ¹³C NMR

(CDCl₃, 100 MHz) δ 155.2, 152.6, 143.0, 136.4, 129.8, 129.6,

128.7, 128.2, 127.7, 124.5, 123.3, 123.1, 119.5, 117.1, 110.3,

99.1, 81.1, 78.7, 70.5, 59.9, 52.8, 39.6, 39.5, 38.1, 33.0, 28.5,

26.6, 20.4, 20.2, 17.1, 13.6; IR (film) ν_max2968, 2926, 1701,

7-Cyan o-1,2,9,9a-tetr ah ydr ocyclopr opa[c]ben z[e]in dol-

4-on e (26, CCBI). A solution of 23 (2.5 mg, 7.0 µmol) in 4 M

HCl-EtOAc (400 µL) was stirred at 25 °C under Ar for 30 min.

The solvent was removed under a stream of N₂. After being

dried in vacuo, the residue 30¹⁴was dissolved in THF (200

µL) and treated with 200 µL of 5% aqueous NaHCO₃. The

reaction mixture was stirred at 25 °C for 5 h before the solvent

was removed in vacuo. PTLC (SiO₂, 0.25 mm × 20 × 20 cm,

70% THF-hexane) afforded 26 (1.6 mg, 1.6 mg theoretical,

100%) as a cream colored solid: ¹H NMR (CDCl₃, 250 MHz) δ

8.30 (d, 1H, J ) 8.1 Hz, C5-H), 7.61 (dd, 1H, J ) 1.5, 8.1 Hz,

C6-H), 7.11 (d, 1H, J ) 1.5 Hz, C8-H), 5.77 (s, 1H, C3-H), 4.88

(br s, 1H, NH) 3.87 (dd, 1H, J ) 5.2, 10.3 Hz, C1-H), 3.69 (d,

1H, J ) 10.3 Hz, C1-H), 2.92 (dt, J ) 3.6, 6.7 Hz, 1H, C9a-H),

1.63 (dd, 1H, J ) 4.3, 7.9 Hz, C9-H), 1.49 (t, 1H, J ) 4.7 Hz,

C9-H); IR (film) ν_max3097, 2851, 2227, 1622, 1583, 1519, 1328,

1241, 1092, 809 cm^-1; UV (THF) λ_max337 (ꢀ ) 4400), 312 (ꢀ )

4000), 266 (ꢀ ) 6300), 246 (ꢀ ) 13 500), 222 nm (ꢀ ) 15 000);

UV (CH₃OH) λ_max348 (ꢀ ) 5500), 317 (ꢀ ) 4300), 264 (ꢀ )

6500), 248 (ꢀ ) 16 300), 221 nm (ꢀ ) 12 800); FABHRMS (NBA)

m/z 223.0865 (M⁺+ H, C₁₄H₁₀N₂O requires 223.0871).

(+)-CCBI (26): [R]²³+64 (c 0.05, THF).

1623, 1586, 1450, 1407, 1352, 1326, 1143, 1041, 855 cm^-1

;

D

en t-(-)-CCBI (26): [R]²³-67 (c 0.05, THF).

FABHRMS (NBA) m/z 570.3330 (M⁺+ H, C₃₅H₄₃N₃O₄requires

570.3332).

D

N-(ter t-Bu tyloxycar bon yl)-4-(ben zyloxy)-7-cyan o-1-iodo-

2-n a p h th yla m in e (27). A solution of 23 (250 mg, 0.67 mmol)

in 10 mL of THF-CH₃OH (1:1) at -40 °C was treated with a

catalytic amount of TsOH-H₂O (20 mg) and NIS (180 mg, 0.80

mmol, 1.2 equiv) in 2 mL of THF. The reaction mixture was

stirred under Ar at -40 °C for 1 h and then warmed to 0 °C.

Additional TsOH-H₂O (10 mg) and NIS (75 mg, 0.5 equiv)

were added. After the reaction mixture was stirred for 1 h at

25 °C, it was quenched with the addition of 5 mL of saturated

aqueous NaHCO₃and extracted with Et₂O (4 × 15 mL). The

combined organic layer was washed with saturated aqueous

NaCl and dried (Na₂SO₄). PCTLC (2 mm SiO₂, 0-10%

EtOAc-hexane gradient) afforded 27 (230 mg, 338 mg theo-

retical, 68%) as a solid: mp 177 °C dec (needles, EtOAc-

hexane); ¹H NMR (CDCl₃, 400 MHz) δ 8.38 (d, 1H, J ) 1.5

Hz), 8.26 (d, 1H, J ) 8.6 Hz), 8.21 (s, 1H), 7.52 (dd, 2H, J )

1.0, 7.8 Hz), 7.47 (dd, 1H, J ) 1.5, 8.6 Hz), 7.45-7.35 (m, 3H),

7.33 (br s, 1H), 5.26 (s, 2H), 1.59 (s, 9H); ¹³C NMR (CDCl₃,

100 MHz) δ 155.4, 152.5, 140.2, 137.0, 135.9, 134.2, 128.7,

128.4, 127.9, 125.1, 125.0, 124.3, 119.0, 112.2, 102.2, 81.8, 78.8,

70.7, 28.3; IR (film) ν_max3383, 2978, 2228, 1732, 1600, 1563,

seco-CCBI-TMI (34). A solution of 23 (1.5 mg, 4.2 µmol)

in 150 µL of 4 M HCl-EtOAc was stirred at 25 °C for 20 min.

The solvent was removed under a stream of N₂. After being

dried in vacuo, the residue, 30¹⁴(1.0 mg, 4.2 µmol, 1 equiv),

and EDCI (2.9 mg, 12.6 µmol, 3 equiv) were dissolved in

anhydrous DMF, and the reaction mixture was stirred at 25

°C for 16 h. The solvent was removed under vacuum, and the

residue was dissolved in THF and loaded directly onto a silica

gel column. Chromatography (SiO₂, 0.5 × 6 cm, 50% EtOAc-

hexane) afforded 34 (1.7 mg, 2.0 mg theoretical, 85%) as a

mustard-colored solid: ¹H NMR (DMSO-d₆, 250 MHz) δ11.50

(br s, 1H, NH), 10.86 (s, 1H, OH), 8.53 (s, 1H, C9-H), 8.22 (d,

1H, J ) 8.7 Hz, C6-H), 8.04 (s, 1H, C4-H), 7.59 (dd, 1H, J )

1.5, 8.7 Hz, C7-H), 7.07 (d, 1H, J ) 1.7 Hz, C4′-H), 6.95 (s,

1H, C3′-H), 4.75 (t, 1H, J ) 10.0 Hz, C2-H), 4.46 (d, 1H, J )

10.5 Hz, C2-H), 4.26 (m, C1-H), 4.04 (dd, 1H, J ) 2.8, 11.3

Hz, CHHCl), 3.92 (s, 3H), 3.88 (dd, 1H, J ) 3.9, 11.3, Hz), 3.81

(s, 3H), 3.79 (s, 3H); IR (film) ν_max3422, 3122, 2938, 2225, 1587,

1525, 1493, 1454, 1389, 1312, 1235, 1110, 1050, 997, 824, 794

cm^-1; FABHRMS (NBA) m/z 492.1347 (M⁺+ H, C₂₆H₂₂ClN₃O₅

requires 492.1326).

1495, 1397, 1366, 1331, 1230, 1154, 987, 879, 735 cm^-1

;

FABHRMS (NBA-CsI) m/z 632.9674 (M⁺+ Cs, C₂₃H₂₁IN₂O₃

requires 632.9651).

Anal. Calcd for C₂₃H₂₁IN₂O₃: C, 55.21; H, 4.23; N, 5.60.

Found: C, 55.28; H, 4.03; N, 5.40.

(1S)-34: [R]²⁵-19 (c 0.12, CHCl₃).

D

en t-(1R)-34: [R]²⁵+19 (c 0.12, CHCl₃).

D

CCBI-TMI (35). A solution of 34 (2.2 mg, 4.4 µmol) in

20% DMF-THF (0.25 mL, 0.018 M) under Ar was cooled to 0

4910 J . Org. Chem., Vol. 61, No. 15, 1996

Boger et al.

°C, and NaH (0.5 mg, 3 equiv) was added. The reaction

mixture was stirred at 0 °C for 30 min before the solvent was

removed under a stream of N₂with care to maintain the 0 °C

temperature. PTLC (SiO₂, 0.25 mm × 10 cm × 15 cm, 50%

EtOAc-hexane) afforded 35 (2.1 mg, 2.1 mg theoretical, 99%)

as a pale yellow solid: ¹H NMR (DMSO-d₆, 400 MHz) δ 11.66

(br s, 1H, NH), 8.10 (d, 1H, J ) 8.3 Hz, C5-H), 7.87 (s, 1H,

C8-H), 7.85 (dd, 1H, J ) 1.5, 8.3 Hz, C6-H), 7.12 (d, 1H, J )

2.2 Hz, C4′-H), 6.92 (s, 1H, C3′-H), 6.74 (s, 1H, C3-H), 4.54

(dd, 1H, J ) 5.6, 10.5 Hz, C1-H), 4.37 (d, 1H, J ) 10.5 Hz,

C1-H), 3.89 (s, 3H), 3.81 (s, 3H), 3.80 (s, 3H), 3.4 (1H, C9a-H

masked by H₂O), 1.93 (dd, 1H, J ) 4.0, 7.6 Hz, C9-H), 1.80 (t,

1H, J ) 5.0 Hz, C9-H); IR (film) ν_max3440, 2920, 2850, 2226,

1734, 1653, 1457, 1389, 1307, 1233, 1108 cm^-1; FABHRMS

(NBA) m/z 456.1546 (M⁺+ H, C₂₆H₂₁N₃O₅requires 456.1559).

µmol, 3 equiv). The reaction mixture was stirred at 25 °C for

14 h. The crude reaction mixture was concentrated and loaded

directly onto a preparative TLC plate. Chromatography (SiO₂,

0.25 mm × 20 × 15 cm, 20% DMF-toluene) afforded 38 (4.4

mg, 5.4 mg theoretical, 81%) as a pale yellow solid: ¹H NMR

(DMSO-d₆, 400 MHz) δ11.68 (s, 1H, NH), 10.88 (br s, 1H, OH),

8.55 (s, 1H, C9-H), 8.22 (d, 1H, J ) 8.7 Hz, C6-H), 8.13 (s, 1H,

C4-H), 8.00 (d, 1H, J ) 8.9 Hz, C4′-H), 7.59 (d, 1H, J ) 8.7

Hz, C7-H), 7.23 (d, 1H, J ) 8.9 Hz, C5′-H), 7.04 (s, 1H, C8′-

H), 6.10 (s, 2H, NH₂), 4.82 (t, 1H, J ) 10.1 Hz, C2-H), 4.55

(dd, 1H, J ) 2.0, 11.0 Hz, C2-H), 4.33 (m, 1H, C1-H), 4.05 (dd,

1H, J ) 3.1, 11.1 Hz, CHHCl), 3.99 (m, 3H, CHHCl and C2′-

H₂), 3.26 (m, 2H, C1′-H₂obscured by H₂O); IR (film) ν_max3350,

2921, 2260, 1723, 1658, 1620, 1590, 1503, 1452, 1414, 1342,

1283, 1252, 1024, 799 cm^-1; FABHRMS (NBA) m/z 486.1340

(M⁺+ H, C₂₆H₂₀ClN₅O₃requires 486.1333).

(+)-CCBI-TMI (35): [R]²³+144 (c 0.05, acetone).

D

en t-(-)-CCBI-TMI (35): [R]²³-135 (c 0.04, acetone).

(1S)-38: [R]²⁵+37 (c 0.15, DMF).

D

seco-CCBI-In d ole₂(36). A solution of 23 (2.8 mg, 7.8

µmol) in 250 µL of 4 M HCl-EtOAc under Ar was stirred at

25 °C for 20 min. The solvent was removed under a stream

of N₂, and the crude hydrochloride salt was dried under

vacuum. A solution of 24, 31³⁹(2.5 mg, 7.8 µmol, 1.0 equiv),

and EDCI (4.5 mg, 23.5 µmol, 3 equiv) was added, and the

mixture was slurried in 142 µL (0.55 M) of anhydrous DMF.

The reaction mixture was stirred at 25 °C for 16 h before the

solvent was removed under vacuum. PTLC (SiO₂, 0.25 mm ×

15 × 20 cm, 30% DMF-toluene) afforded 36 (3.8 mg, 87%) as

a tan solid: ¹H NMR (DMSO-d₆, 400 MHz) δ 11.76 (s, 1H, NH),

11.71 (s, 1H, NH), 10.85 (s, 1H, OH), 10.16 (s, 1H, NH), 8.55

(s, 1H, C9-H), 8.23 (d, 1H, J ) 8.6 Hz, C6-H), 8.22 (s, 1H, C4′-

H), 8.14 (s, 1H, C4-H), 7.67 (d, 1H, J ) 7.8 Hz, C4′′-H), 7.59

(m, 2H), 7.48 (apparent t, 2H, J ) 8.8 Hz, C7′- and C7′′-H),

7.41 (s, 1H, C3′′-H), 7.25 (s, 1H, C3′-H), 7.22 (t, 1H, J ) 8.1

Hz, C6′′-H), 7.06 (t, 1H, J ) 7.3 Hz, C5′′-H), 4.85 (t, 1H, J )

10.0 Hz, C2-H), 4.60 (d, 1H, J ) 11.2 Hz, C2-H), 4.34 (m, 1H,

C1-H), 4.06 (d, 1H, J ) 10.9 Hz, CHHCl), 3.92 (dd, 1H, J )

6.9, 11.4 Hz, CHHCl); IR (film) ν_max3284, 2921, 2225, 1651,

1589, 1557, 1516, 1411, 1391, 1316, 1230, 1137, 1059, 805, 743

cm^-1; FABHRMS (NBA) m/z 560.1470 (M⁺+ H, C₃₂H₂₂ClN₅O₃

requires 560.1489).

en t-(1R)-38: [R]²⁵-36 (c 0.22, DMF).

D

CCBI-CDP I₁(39). A solution of 38 (3.6 mg, 7.4 µmol) in

DMF-H₂O (5:2, 600 µL + 240 µL) was treated with KHCO₃

(20 equiv). The reaction mixture was stirred at 25 °C for 9 h.

After removal of solvent, PTLC (SiO₂, 0.25 mm × 20 × 20 cm,

20% DMF-toluene) afforded 39 (2.3 mg, 3.3 mg theoretical,

69%) as a tan solid: ¹H NMR (DMSO-d₆, 400 MHz) δ 11.74 (s,

1H, NH), 8.12 (d, 1H, J ) 8.1 Hz, C5-H), 8.02 (d, 1H, J ) 8.9

Hz, C4′-H), 7.87 (s, 1H, C8-H), 7.85 (d, 1H, J ) 8.1 Hz, C6-H),

7.21 (d, 1H, J ) 8.9 Hz, C5′-H), 7.10 (s, 1H, C3-H), 7.01 (s,

1H, C8′-H), 6.13 (s, 2H, NH₂), 4.63 (dd, 1H, J ) 5.1, 10.3 Hz,

C1-H), 4.51 (d, 1H, J ) 10.3 Hz, C1-H), 3.97 (t, 2H, J ) 8.8

Hz, C2′-H), 3.42 (m, 1H, C9a-H), 3.28 (t, 2H, J ) 8.8 Hz, C1′-

H), 1.91 (dd, 1H, J ) 4.1, 7.7 Hz, C9-H), 1.76 (t, 1H, J ) 4.6

Hz, C9-H); IR (neat) ν_max3364, 2911, 1652, 1598, 1499, 1455,

1386, 1263, 1130 cm^-1; FABHRMS (NBA) m/z 450.1578 (M +

H⁺, C₂₆H₁₉N₅O₃requires 450.1566).

(+)-CCBI-CDP I₁(39): [R]²³+124 (c 0.10, DMF).

D

en t-(-)-CCBI-CDP I₁(39): [R]²³-117 (c 0.07, DMF).

D

Aqu eou s Solvolysis Rea ctivity. p H 3. N-BOC-CCBI

(25, 100 µg) and CCBI (26, 50 µg) were dissolved in CH₃OH

(1.5 mL) and mixed with pH 3 aqueous buffer (1.5 mL). The

buffer contained 4:1:20 (v:v:v) 0.1 M citric acid, 0.2 M Na₂-

HPO₄, and H₂O, respectively. The solvolysis solution was

sealed and kept at 25 °C protected from light. The UV

spectrum was measured at regular intervals every 2 h during

the first day, every 12 h for another week, and every 24 h for

an additional week. For 26, the measurements continued

every 5 days for 6 months. For 25, the decrease in the long-

wavelength absorption at 322 nm and the increase in the short-

wavelength absorption at 268 nm were monitored, Figure 1.

The solvolysis rate constant (k ) 9.90 × 10^-7s^-1) and half-life

(t_1/2) 213 h) were calculated from data recorded at the short

wavelength from the least-squares treatment (r ) 0.999) of

the slope of the plot of time versus 1n[(A_f- A_i)/(A_f- A)], Figure

11. For 26, the decrease in the long-wavelength absorption

(1S)-36: [R]²⁵+49 (c 0.17, DMF).

D

en t-(1R)-36: [R]²⁵-44 (c 0.21, DMF).

D

CCBI-In d ole₂(37). Meth od A. A solution of 36 (3.4 mg,

6.10 µmol) in 20% DMF-THF (0.34 mL) under Ar was cooled

to 0 °C and treated with NaH (0.8 mg, 18.3 µmol, 60% in oil,

3 equiv). The reaction mixture was stirred for 30 min at 0 °C

before the solvent was removed under a stream of N₂with care

to maintain the 0 °C temperature. PTLC (SiO₂, 0.25 mm ×

15 × 20 cm, 15% DMF-toluene) afforded 37 (2.1 mg, 3.2 mg

theoretical, 66%) as a tan solid: ¹H NMR (DMSO-d₆, 400 MHz)

δ 11.87 (s, 1H, NH), 11.72 (s, 1H, NH), 10.18 (s, 1H, NH), 8.23

(s, 1H, C4′-H), 8.12 (d, 1H, J ) 8.2 Hz, C5-H), 7.88 (s, 1H,

C8-H), 7.86 (d, 1H, J ) 8.2 Hz, C6-H), 7.66 (d, 1H, J ) 7.9 Hz,

C4′′-H), 7.61 (dd, 1H, J ) 8.9, 2.0, Hz, C6′-H), 7.49 (d, 1H, J

) 8.2, Hz, C7′′-H), 7.50 (d, 1H, J ) 8.9 Hz, C7′-H), 7.41 (s,

1H, C3′′-H), 7.31 (s, 1H, C3′-H), 7.20 (t, 1H, J ) 7.2 Hz, C6′′-

H), 7.06 (t, 1H, J ) 7.2 Hz, C5′′-H), 7.05 (s, 1H, C3-H), 4.66

(dd, J ) 4.8, 10.2 Hz, C1-H), 4.53 (d, 1H, J ) 10.2 Hz, C1-H),

3.37 (m, 1H, C9a-H), 1.91 (dd, 1H, J ) 4.4, 8.4 Hz, C9-H), 1.78

(t, 1H, J ) 4.8 Hz, C9-H); IR (neat) ν_max3274, 1644, 1601, 1549,

at 353 nm was monitored providing k ) 1.65 × 10^-7s^-1(t_1/2

)

1182 h, r ) 0.99) by the same treatment.

p H 2. Samples of 25 (100 µg) and 26 (50 µg) were dissolved

in CH₃OH (1.5 mL), and the solutions were mixed with

aqueous buffer (pH 2.05, 1.5 mL). The buffer contained 4:1:

20 (v:v:v) 1.0 M citric acid, 0.2 M Na₂HPO₄, and H₂O,

respectively. Immediately after mixing, the UV spectra of the

solutions were measured against a reference solution contain-

ing CH₃OH (1.5 mL) and the aqueous buffer (1.5 mL), and

these readings were used for the initial absorbance values. The

solutions were stoppered, protected from the light, and allowed

to stand at 25 °C. UV spectra were recorded at regular

intervals until constant values were obtained for the long and

short wavelength absorbances. The solvolysis rate constants

were determined from the slope of the lines obtained from

linear least-squares treatment of plots of 1n[(A_f- A_i)/(A_f-

A)] versus time using the short wavelength measurements for

25 and long wavelength measurements for 26 (Figure 11). The

first-order rate constant determined under these conditions

was 7.94 × 10^-6s^-1(t_1/2) 24.2 h, r ) 0.999) for N-BOC-CCBI

(25) and 1.5 × 10^-6s^-1(t_1/2) 127 h, r ) 0.99) for CCBI (26).

Solvolysis of N-BOC-CCBI in THF -CH₃OH. The sol-

volysis of N-BOC-CCBI (25) was carried out in THF with 20-

1516, 1454, 1388, 1308, 1265, 1237, 1133, 806, 745 cm^-1

;

FABHRMS (NBA) m/z 524.1737 (M⁺+ H, C₃₂H₂₁N₅O₃requires

524.1723).

(+)-CCBI-In d ole₂(37): [R]²⁵+80 (c 0.04, THF).

D

en t-(-)-CCBI-In d ole₂(37): [R]²⁵-81 (c 0.09, THF).

D

Meth od B. A solution of 36 (2.7 mg, 4.8 µmol) in 1:1 THF-

3% aqueous NaHCO₃(700 µL) was stirred at 25 °C for 10 h.

The solvent was removed under a stream of N₂. PTLC (SiO₂,

0.25 mm × 20 × 20 cm, 10% DMF-toluene) afforded 37 (1.7

mg, 2.5 mg theoretical, 68%) as a tan solid.

seco-CCBI-CDP I₁(38). A solution of 23 (4.0 mg, 11.1

µmol) in 500 µL of 4 M HCl-EtOAc was stirred at 0 °C for 30

min. The solvent was removed under a stream of N₂, and the

crude hydrochloride salt was dried under vacuum. The salt

and 32⁵⁹(3.0 mg, 12.3 µmol, 1.1 equiv) were dissolved in 300

µL of anhydrous DMF and treated with EDCI (6.4 mg, 33.3

Electronic Effects on Functional Reactivity

J . Org. Chem., Vol. 61, No. 15, 1996 4911

25 in THF with 20 equiv of CH₃OH in the presence of CF₃-

SO₃H (0.1 equiv). A stock solution was prepared by addition

of CF₃SO₃H (5.6 µL) and anhydrous CH₃OH (502 µL) to

anhydrous THF (99.49 mL). A sample of 25 (100 µg) in a UV

cell was dissolved in THF (1950 µL), and the THF stock

solution (50 µL) that contained CF₃SO₃H (0.1 equiv) and CH₃-

OH (20 equiv) was added. The solvolysis solution was sealed,

and the UV spectrum was measured with an automated cycle

program at regular intervals (10 min/cycle). The decrease in

the long wavelength absorption at 313 nm was monitored, and

the solvolysis was complete after 80 h. The solvolysis rate (k

) 0.2 × 10^-4s^-1) and half-life (t_1/2) 9.2 h) were calculated

from data recorded at 313 nm from the least-squares treatment

(r ) 0.984) of the slope of the plot of time versus ln[(A_f- A_i)/

(A_f- A)].

Solvolysis Regioselectivity: 3-(ter t-Bu tyloxycar bon yl)-

8-cya n o-5-h yd r oxy-1-(m et h oxym et h yl)-1,2-d ih yd r o-3H -

ben z[e]in d ole (42). A solution of 25 (2.5 mg, 7.8 µmol) in

CH₃OH (1 mL) containing 0.12 equiv of CF₃CO₂H was stirred

at 0 °C for 24 h. NaHCO₃(2.5 mg) was added, and the reaction

mixture was stirred at 0 °C, warmed to 25 °C, filtered through

a plug of Celite, and concentrated. PTLC (SiO₂, 0.25 mm ×

20 × 20 cm, 20% EtOAc-hexane) provided 42 as a semisolid

(2.3 mg, 2.7 mg theoretical, 85%, 95% based on conversion)

and recovered 25 (0.3 mg, 12%). For 42: ¹H NMR (CDCl₃,

400 MHz) δ 8.23 (d, 1H, J ) 8.7 Hz), 8.08 (s, 1H), 7.88 (br s,

1H), 7.40 (dd, 1H, J ) 1.4, 8.7 Hz), 6.79 (br s, 1H), 4.10

(apparent d, 2H, J ) 7.9 Hz), 3.85 (m, 1H), 3.65 (dd, 1H, J )

4.6, 9.4 Hz), 3.39 (s, 3H), 3.35 (t, 1H, J ) 9.2 Hz), 1.59 (s, 9H);

IR (film) ν_max3339, 2974, 2925, 1704, 1674, 1620, 1586, 1452,

1418, 1369, 1329, 1250, 1220, 1137 cm^-1; FABHRMS (NBA-

NaI) m/z 377.1488 (M + Na⁺, C₂₀H₂₂N₂O₄requires 377.1477).

DNA Alk yla tion Stu d ies: Selectivity a n d Efficien cy.

General procedures, the preparation of singly ³²P 5′ end-labeled

double-stranded DNA, the agent binding studies, gel electro-

phoresis, and autoradiography were conducted according to

procedures described in full detail elsewhere.¹⁰Eppendorf

tubes containing the 5′ end-labeled DNA (9 µL) in TE buffer

(10 mM Tris, 1 mM EDTA, pH 7.5) were treated with the agent

in DMSO (1 µL at the specified concentration). The solution

was mixed by vortexing and brief centrifugation and subse-

quently incubated at 4 or 25 °C for 24 h (natural enantiomers)

and 25 or 37 °C (unnatural enantiomers) for 72 h. The

covalently modified DNA was separated from unbound agent

by EtOH precipitation and resuspended in TE buffer (10 µL).

The solution of DNA in an Eppendorf tube sealed with

parafilm was warmed at 100 °C for 30 min to induce cleavage

at the alkylation sites, allowed to cool to 25 °C, and centrifuged.

Formamide dye (0.03% xylene cyanol FF, 0.03% bromophenol

blue, 8.7% Na₂EDTA 250 mM) was added (5 µL) to the

supernatant. Prior to electrophoresis, the sample was dena-

tured by warming at 100 °C for 5 min, placed in an ice bath,

and centrifuged, and the supernatant (3 µL) was loaded

directly onto the gel. Sanger dideoxynucleotide sequencing

reactions were run as standards adjacent to the reaction

samples. Polyacrylamide gel electrophoresis (PAGE) was run

on an 8% sequencing gel under denaturing conditions (8 M

urea) in TBE buffer (100 mM Tris, 100 mM boric acid, 0.2 mM

Na₂EDTA) followed by autoradiography.

DNA Alk yla tion Rela tive Ra te of (+)-CCBI-TMI (35),

(+)-CBI-TMI, a n d (+)-MCBI-TMI. Following the proce-

dure detailed above, Eppendorf tubes containing 5′ end-labeled

w794 DNA (9µL) in TE buffer (pH 7.5) were treated with (+)-

CCBI-TMI (35), (+)-CBI-TMI, or (+)-MCBI-TMI (1 µL, 10^-6

M in DMSO). The solutions were mixed and incubated at 25

°C for 2, 4, 6, 9, 12, 15, and 24 h, respectively. Subsequent

isolation of the alkylated DNA by EtOH precipitation, resus-

pension in TE buffer (10 µL, pH 7.5), thermolysis (30 min, 100

°C), PAGE, and autoradiography were conducted as detailed

above. Relative rates for alkylation at the w794 high-affinity

5′-AATTA site were derived from the slopes of the plots of

percent integrated optical density (IOD) of the high-affinity

alkylation cleavage bands versus time.

F igu r e 11.

500 equiv of CH₃OH in the presence of 0.1-0.25 equiv of

CF₃SO₃H. The following is the procedure for the solvolysis of

DNA Alk yla tion Rela tive Ra te of (+)-CCBI-In d ole₂

(37), (+)-CBI-In d ole₂, a n d (+)-MCBI-In d ole₂. Following

4912 J . Org. Chem., Vol. 61, No. 15, 1996

Boger et al.

the procedure detailed above, Eppendorf tubes containing 5′

end-labeled w794 DNA (9 µL) in TE buffer (pH 7.5) were

treated with (+)-CCBI-indole₂(37), (+)-CBI-indole₂, and (+)-

MCBI-indole₂(1 µL, 10^-6M in DMSO). The solutions were

mixed and incubated at 25 °C for 2, 4, 6, 9, 12, 15, and 24 h,

respectively. Subsequent isolation of the alkylated DNA by

EtOH precipitation, resuspension in TE buffer (10 µL, pH 7.5),

thermolysis (30 min, 100 °C), PAGE, and autoradiography

were conducted as detailed above. Relative rates for alkylation

at the w794 high-affinity 5′-AATTA site were derived from the

slopes of the plots of percent integrated optical density (IOD)

of the high-affinity alkylation cleavage bands versus time.

Ack n ow led gm en t. We gratefully acknowledge the

financial support of the National Institutes of Health

(CA 55276).

Su p p or tin g In for m a tion Ava ila ble: Summary tables of

the in vitro cytotoxic activity (L1210) employed for Figure 6

and ¹H NMR spectra of 5, 7, 11, 14, 19-23, 25, 26, 29, 34-

39, and 42 (22 pages). This material is contained in libraries

on microfiche, immediately follows this article in the microfilm

version of the journal, and can be ordered from the ACS; see

any current masthead page for ordering information.

J O9605298

Article Doi

Synthesis, chemical properties, and preliminary evaluation of substituted CBI analogs of CC-1065 and the duocarmycins incorporating the 7-cyano-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one alkylation subunit: Hammett quantitation of the magnitude of electronic effects on functional reactivity

DOI: 10.1021/jo9605298

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Article abstract of DOI:10.1021/jo9605298

Full text of DOI:10.1021/jo9605298

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