Synthesis and DNA-cleaving activity of lactenediynes conjugated with DNA-complexing moieties

Available online at www.sciencedirect.com

Bioorganic & Medicinal Chemistry 16 (2008) 3501–3518

Synthesis and DNA-cleaving activity of lactenediynes

conjugated with DNA-complexing moieties

Luca Banﬁ,^a,*Andrea Basso,^aElisabetta Bevilacqua,^aValentina Gandolfo,^a

Giuseppe Giannini,^bGiuseppe Guanti,^a,*Loana Musso,^c

Monica Paravidino^aand Renata Riva^a

^aDipartimento di Chimica e Chimica Industriale, via Dodecaneso 31, I-16146 Genova, Italy

^bSigma-Tau, Via Pontina km 30,400 I-00040 Pomezia (RM), Italy

^cDipartimento di Scienze Molecolari Agroalimentari, via Celoria 2, 20133 Milano, Italy

Received 30 October 2007; accepted 8 February 2008

Available online 13 February 2008

Abstract—Lactenediynes are compounds characterized by the fusion of a b-lactam with a cyclodeca-3-ene-1,5-diyne. In this work

the most promising members of this family have been activated by attaching a carbalkoxy or a carbamoyl group to the azetidinone

nitrogen, and conjugated to various DNA-complexing moieties, either acting by intercalation or through groove binding. These con-

jugated artiﬁcial enediynes have been demonstrated to possess in vitro ability to produce single and double strand cleavage of plas-

mid DNA. As potency and capacity to induce double cut, they rank among the best simple enediyne analogues ever prepared. A

thorough investigation was carried out in order to develop the best suited linkers for assembling these conjugates.

O

1. Introduction

S

HO

NHCOOMe

S

The natural enediyne antibiotics are among the most po-

tent anti-cancer chemotherapeutic agents known to

date.^1,2They act through an unique mechanism, involv-

ing direct radical attack on cellular DNA. Particularly

noteworthy is the ability to induce not only single but

also simultaneous double strand DNA cleavage, leading

to apoptosis. The high potency of the natural com-

pounds (e.g., Calicheamicin, Fig. 1)³is counterbalanced

by their poor selectivity, making them unsuitable for

clinical use as such. In order to solve this problem, Cal-

icheamicin has been conjugated to a selective antibody.

The resulting conjugate (Gemtuzumab, Mylotarg^TM) has

shown very promising activity towards some types of

previously intractable tumours.⁴

Me

H

O

Me

S

O

N

O

I

HO

OH

O

H

O

OMe

EtN

OMe

MeO

Me

HO

O

I

Calicheamicin γ₁

MeO

OH

Figure 1.

simpler analogues operating through a similar mode of

action.⁵Towards this goal some years ago we designed

an original class of simpliﬁed ‘artiﬁcial’ enediynes, called

lactenediynes, and characterized by the fusion of a 10-

membered enediyne ring with a b-lactam. Three diﬀerent

classes of lactenediynes have been prepared⁶or ap-

proached⁷so far. Among them, the most useful seems

to be the one characterized by the trans fusion of a b-lac-

tam with atoms 8,9 of a 10-membered cyclodeca-3-ene-

1,5-diyne, depicted in Scheme 1 as general formula 1.

First of all these compounds are very stable in the dry

state, contrary to what often happens with simple ened-

iyne compounds, including other type of lactenediynes.

In these compounds, the b-lactam ring behaves as a

However, the high structural complexity of natural

enediynes makes highly desirable the development of

Keywords: Enediynes; Antitumor; DNA cleavage; b-Lactams; DNA

intercalating agents; DNA groove binders; Camptothecin.

*

Corresponding authors. Tel.: +39 010 3536105; fax: +39 010

3536105; e-mail addresses: banﬁ@chimica.unige.it; guanti@chimica.

unige.it

doi:10.1016/j.bmc.2008.02.022

3502

L. Banﬁ et al. / Bioorg. Med. Chem. 16 (2008) 3501–3518

a: R = benzyl

b: R = -naphthyl

c: R = -naphthyl

R²

d: R = CH₂-( -naphthyl)

e: R = CH₂-( -naphthyl)

f: R = CH₂CH₂-( -naphthyl)

g: R = CH₂CH₂-( -naphthyl)

R³

R²

R¹

R²

H₂N R¹

OAc

R³

O

R³

H₂N R¹

H

N

O

N

O

R

1

2

3

8a-g

O

R¹= H or SiMe₂tBu or CH₂CH₂R⁴

R²= H or OH

d or e

R³= H or OR⁵

Scheme 1.

b

c

OAc

O

N

a

safety-catch, completely suppressing cycloaromatization

of the 10-membered enediyne moiety, whereas opening

of the b-lactam ring to 2 unlashes the typical reactivity of

monocyclic cyclodeca-3-ene-1,5-diynes, leading to the

formation of diradical 3.

N

H

N

O

SiMe₂tBu

O

tBu

5

7

6

O

During the past 10 years, we have prepared several com-

pounds of general formula 1^8–13and validated the chem-

ical principle that lays behind their design. An important

diﬀerence among the various substances prepared so far

is represented by the number of ‘handles’, that is the

attachment points that can be used for joining appropri-

ate substituents or substructures. These handles can be

used to append ‘activating’ substituents, ‘triggering’ de-

vices, or DNA-complexing structures (‘delivery units’).

We have prepared compounds provided with 1–3 han-

dles, represented by substituents R¹, R²and R³.

f

g,h

OH

O

R

O

R

N

OtBu

O

SiMe₂tBu

O

SiMe₂tBu

O

4

9a-c

10a-c

O

a: R = CH₂-( -naphthyl)

b: R = CH₂CH₂-( -naphthyl)

c: R = CH₂CH₂-( -naphthyl)

Scheme 2. Reagents: (a) Ac₂O, pyridine, DMAP, 96%; (b) 40% aq HF/

MeCN, 94%; (c) Boc₂O, DMAP, MeCN, 91%; (d) R–N@C@O, Et₃N,

DMAP, CH₂Cl₂. Yields: 8a: 90%, 8b: 45%, 8d: 99%, 8e: 71%, 8f: 69%,

8g: 36%; (e) N-(p-nitrophenyloxycarbonyl) b-naphthyl-amine, DMAP,

CH₃CN. Yield: 8c: 30%; (f) RCOCl, pyridine. Yields: 9a: 84%, 9b:

97%, 9c: 100%; (g) 40% aq HF/MeCN 1:40. Yields: from 9a: 75%, from

9b: 100%, from 9c: 98%; (h) Boc₂O, DMAP, MeCN. Yields: 10a: 93%,

10b: 79%, 10c: 100%.

The b-lactam is normally too stable to undergo sponta-

neous opening under physiological conditions. In order

to circumvent this limitation we are following two alter-

native routes.

oxamazins.¹⁵Preliminary studies on simple monocyclic

b-lactams have, however, shown that this type of activa-

tion was not strong enough for our purposes. We there-

fore turned our attention to carbonyl derivatives, such

as urethane-type compounds (see 7 in Scheme 2) or

urea-type compounds (see 8 in Scheme 2). The former

activation has been widely employed in the use of b-lac-

tams as acylating agents, for example, in the synthesis of

Taxol derivatives or ACE inhibitors.¹⁶On the other

hand, urea-type compounds were previously employed

in medicinal chemistry in the ﬁeld of protease inhibi-

tors.^17,18These previous reports have shown that urea-

type compounds, such as 8, should be hydrolytically less

reactive than urethane-type adducts like 7.¹⁸This was

conﬁrmed by us, working on model systems.

In the ﬁrst one we take advantage of an intramolecular

opening of the b-lactam by a suitable nucleophile (an

amino group) attached to one of the handles. In partic-

ular, compounds 1 with R¹= CH₂CH₂NH₂were dem-

onstrated to be eﬀective DNA-cleaving agents, whereas

protection of the amino group completely suppresses

this activity. Although the potency was not impressive,

those results were promising in view of selective prodrug

activation.^11,13

In this paper, we report instead the results of a second,

alternative, approach, that involves the attachment of

appropriate activating substituents to the b-lactam

nitrogen. Moreover, the cleaving activity has been im-

proved by joining also DNA-complexing moieties to

the basic scaﬀold. This study has led to very eﬃcient

enediyne prodrugs, capable to induce both single and

double strand DNA cleavage at concentrations as low

as 10^ꢀ7M.

Therefore, we prepared two simple members of the two

classes, namely compounds 7 and 8a. The synthesis

(Scheme 2)¹⁹started from the previously described race-

mic lactenediyne 4.⁹Since the last step of the total syn-

thesis aﬀords an 86:14 ratio of the two diastereoisomers,

we did our ﬁrst exploration work on the major isomer,

which has the hydroxy group in a pseudo-axial position,

and that was available in higher amount. The prepara-

tion of 7 and 8a involved a three step sequence: acetyla-

tion of the hydroxy group, removal of the silylated

protection, and activation, by reaction with di-tert-butyl

dicarbonate or with benzyl isocyanate. The overall se-

2. Results and discussion

In principle, activation of a b-lactam towards hydrolysis

can be achieved by placing on the lactam nitrogen an

electron withdrawing group. This activation has been

exploited, for example, in the development of monocy-

clic b-lactam antibiotics, such as monobactams¹⁴or

L. Banﬁ et al. / Bioorg. Med. Chem. 16 (2008) 3501–3518

3503

Table 1. Results of cleavage of plasmid DNA with compounds 7, 8,

10, 14, 15^a

These promising results led us to test the cytotoxicity of

7 (Table 2). The most interesting results are evidenced in

bold. As can be seen, on some tumour cell lines, GI₅₀

values in the nanomolar range were obtained. For two

cell lines (SF-539 and CAKI-1) even a 50% decrease of

cell count was observed at concentrations between 10

and 100 lM. Reduction of tumour mass was also dem-

onstrated in vivo on murine models (see Section 4.35).

b

c

d

Compound

CS₅₀

CS_min

CD_min

D/S^e

7

8a

40

70

5

25

40

1:15

1:10

No D^f

1:8

8

8b

8c

25

1

8

200

25

10

1

>200

5

8d

8e

70

40

n.d.

5

n.d.

8

1:20

1:10

No D^f

1:20

1:12

1:8

8f

8g

These simple activated lactenediynes proved to be supe-

rior to other simple artiﬁcial enediynes, but deﬁnitely

less potent than natural compounds, and in particular

Calicheamicin, which is able to cleave 50% of plasmid

DNA at concentrations in the low nanomolar range.

Most importantly, Calicheamicin induces a higher per-

centage of double strand break (2:1 compared to single

break!). However, it has been demonstrated that this

high level of potency is mainly due to the oligosaccha-

ride part, which acts as the ‘delivery system’ of this po-

tent warhead. The aglycon itself is indeed 1000 times less

potent, aﬀording only a small percentage of double cut

(1:30 single/double),²²and being therefore not any better

than 7 or 8a.

150

80

10

5

>100

100

10

10a

10b

10c

14

80

150

30

15

25

1

5

^aFor conditions see Section 4.

^bCS₅₀, concentration of lactenediyne (lM) producing a form ratio of

form I/(form II + form III) = 50:50.

^cCS_min, minimum concentration of lactenediyne producing detectable

quantity of form II.

^dCD_min, minimum concentration of lactenediyne producing detectable

quantity of form III.

^eD/S, ratio of form III/form II at 100 lM.

^fForm III was not visible at 100 lM.

This fact led us to reason that we might be able to in-

crease the potency of our activated lactenediynes and

to improve the percentage of double cut, by conjugating

them with suitable ‘delivery systems’, that is groups able

to complex tightly (and maybe selectively) with DNA

through intercalation or groove binding. These moieties

should be however structurally simpler than the highly

complex oligosaccharide unit of Calicheamicin! This

strategy was previously proved to be successful for other

simple artiﬁcial enediynes^20,21,23or for other type of rad-

ical generating species.²⁴

quences proceeded in very high yield, conﬁrming once

again the stability of this class of enediynes.

Compounds 7 and 8a, despite the presence of activating

groups, were found to be quite stable. They could be

conveniently chromatographed and even stored in free-

zer as DMSO solution for several months. Moreover,

they were demonstrated to be stable in plasma. In order

to assess their ability to cleave DNA, we incubated

them, at various concentrations, with supercoiled plas-

mid pBR 322 for 24 h at 37 ꢁC. After gel electrophoresis

(see Table 1) we were able to determine the relative ratio

of forms I (native, supercoiled), II (relaxed cyclic) and

III (linear). Both compounds were found to give single

and double strand breaks at concentrations much lower

than those required by simple 10-membered monocyclic

enediynes.²⁰For example, 7 gave 50% of cleavage at

40 lM (Table 1), while typically monocyclic cyclodeca-

3-ene-1,5-diynes display the same eﬀect at 500 lM.^20,21

Most importantly, while simple monocyclic enediynes

do not provoke the formation of form III (deriving from

double cut), 7 aﬀorded also detectable amounts of form

III, although the main mechanism involved single cleav-

age, with a single/double ratio of about 15:1.

We ﬁrst studied the incorporation of naphthyl groups,

as very simple intercalating moieties. Since lactenediyne

4 possesses two ‘handles’, we attached this group alter-

natively on both, varying also the spacer. For the prep-

aration of compounds 8a–g, various carboxylic acids

containing a naphthyl group have been converted into

the corresponding isocyanates, which were in turn used

for the preparation of the urea-type adducts. Only in the

case of b-naphthyl urea 8c this approach, which started

from b-naphthoic acid, was not successful. We started

therefore from b-naphthylamine and prepared the corre-

sponding p-nitrophenyl carbamate as the acylating

agent. In the case of esters 10a–c we used the second

handle instead, placing the naphthyl containing group

on the hydroxy group to give 9a–c. After removal of

the silyl protection, the nitrogen was ﬁnally activated

as the t-butyl urethane to give 10a–c.

These results ﬁrst indicated that the presence of the b-

lactam is beneﬁcial in improving the ability of these

compounds to bind to DNA. We strongly believe that

DNA cleavage takes place only after opening of the aze-

tidinone. In fact, analogous incubation with an unacti-

vated compound 1 (R¹= b OMe, R²= Me, R³= H)⁸

gave no cleavage at all even at 1 mM concentration.

Urea-type compound 8a was about 50% less potent than

7, probably because of slower hydrolysis of the azetidi-

none. By incubating 7 and 8ab for 24, 48 and 72 h, we

found indeed a slight increase of form II with time for

8a but not for 7.

All these compounds were incubated with plasmid pBR

322 and compared with 7. The results are reported in

Table 1. As can be seen, the cleaving activity was heavily

dependent on: (a) the type of handle used for attaching

the intercalating moiety; (b) the type of attachment of

the naphthyl group (a or b). On the other hand, the

length of the spacer was less important. The best results

were achieved with compound 8d which was about 1.5–2

times more active as overall cleaving agent than 7. Most

3504

L. Banﬁ et al. / Bioorg. Med. Chem. 16 (2008) 3501–3518

Table 2. Cytotoxicity of compound 7 on various tumour cell lines^a

importantly, however, the amount of double cut in-

creased remarkably, and double cut was still visible at

1 lM! We guess that stronger complexation with DNA

leads to increased probability of simultaneous double

cut.

Cellular line

GI₅₀

TGI

LC₅₀

Leukemias

MOLT-4

RPMI-8226

ꢀ6.39

>ꢀ4.00

ꢀ4.75

Lung tumours

A549/ATCC

EKVX

In order to check the eﬀect of the relative stereochemis-

try, we prepared also 14 and 15, that is the epimers of 7

and 8d, starting from the minor pseudo-equatorial iso-

mer 11 (Scheme 3). The last two lines of Table 1 clearly

show that relative stereochemistry has a negligible eﬀect.

ꢀ4.55

>4.00

>ꢀ4.00

ꢀ4.42

>ꢀ4.00

HOP-62

NCI-H226

NCI-H23

ꢀ5.30

ꢀ4.40

ꢀ4.77

>ꢀ4.00

ꢀ7.03

>ꢀ4.00

ꢀ4.15

NCI-H322M

NCI-H460

>ꢀ4.00

For further improving the activity of these conjugated

lactenediynes, we decided to attach to them also minor

groove binders, in particular polypyrroles of the netrop-

sin–distamycin family.^21,25In order to do that we needed

to develop appropriate linkers. The methodology used

for attaching the naphthyl groups has indeed some lim-

itations, since, in order to vary the length of the spacer, a

diﬀerent substrate must be prepared each time. While

this was not a big problem for the synthesis of 8 and

10 (we just needed to prepare naphthylacetic or naph-

thylpropionic acids) this was anticipated to be problem-

atic for more complex substructures, especially when the

function to be attached is a primary amine.

Colon tumors

COLO 205

HCC-2998

HCT-116

HCT-15

ꢀ4.40

ꢀ4.46

>ꢀ4.00

ꢀ4.59

ꢀ4.33

HT 29

KM12

SW-620

>ꢀ4.00

ꢀ4.87

CNS tumours

SF-295

SF-539

ꢀ4.51

ꢀ6.14

ꢀ4.58

ꢀ4.42

ꢀ4.79

>ꢀ4.00

ꢀ5.27

>ꢀ4.00

ꢀ4.59

SNB-19

SNB-75

U251

>ꢀ4.00

A ﬁrst logical way to build a general ﬂexible linker is to

use the hydroxy group at C-9 as handle. Towards this

goal we treated lactenediyne 4 with succinic anhydride

and the resulting monosuccinate was coupled with a

model amine (Scheme 4). Deprotection of the silyl group

aﬀorded 16 in acceptable overall yields. However, our

attempt to introduce the activating substituent at nitro-

gen to give 17 failed. Despite the mildness of the basic

catalyst used (DMAP), complete intramolecular acyl

substitution to release succinimide 18 occurred.

Melanomas

M14

ꢀ4.40

ꢀ4.32

ꢀ4.06

ꢀ4.74

ꢀ4.67

>ꢀ4.00

SK-MEL-2

SK-MEL-28

UACC-257

UACC-62

Ovarian tumours

IGROV1

ꢀ4.71

ꢀ4.21

>ꢀ4.00

4.05

>ꢀ4.00

OVCAR-3

OVCAR-4

OVCAR-5

OVCAR-8

SK-OV-3

>ꢀ4.00

ꢀ4.84

In order to spare the precious compound 4, this ap-

proach was thoroughly studied using the simpliﬁed

model compound 20, obtained from 19⁹in three steps

(Scheme 4). However we never succeeded to avoid the

unwanted intramolecular process leading to the succini-

mide, either by changing the acylating reagent (using

ꢀ4.06

>ꢀ4.00

Kidney tumours

786–0

A498

ꢀ4.94

ꢀ4.77

ꢀ5.58

ꢀ6.45

ꢀ4.69

ꢀ4.45

ꢀ5.23

ꢀ4.16

ꢀ4.01

>ꢀ4.00

ꢀ4.22

ACHN

ꢀ4.59

CAKI-1

RXF 393

TK-10

ꢀ5.19

ꢀ4.16

>ꢀ4.00

UO-31

a

b

OH

OAc

Prostate tumours

DU-145

ꢀ4.79

ꢀ4.25

>ꢀ4.00

N

81%

N

78%

O

SiMe₂tBu

O

SiMe₂tBu

O

H

Breast tumours

MCF7

11

12

13

ꢀ5.96

ꢀ4.54

>ꢀ4.00

ꢀ4.27

>ꢀ4.00

ꢀ4.06

ꢀ4.61

ꢀ4.00

>ꢀ4.00

d

93%

c

NCI/ADR-RES

MDA-MB-231/ATCC

HS 578T

90%

MDA-MB-435

MDA-N

BT-549

OAc

H

N

O

t

N

T-47D

O

Bu

O

14

^aValues are log[concn] in M units. If PG (% growth) is the percentage

of cell growth compared to the control experiment, GI₅₀: concn of 7

at which PG = 50; TGI: concn at which PG = 0; LC₅₀: concn at

which PG = ꢀ50.

15

O

Scheme 3. Reagents: (a) Ac₂O, pyridine, DMAP; (b) 40% aq HF/

MeCN; (c) Boc₂O, DMAP, MeCN; (d) R–N@C@O, Et₃N, DMAP,

CH₂Cl₂.

L. Banﬁ et al. / Bioorg. Med. Chem. 16 (2008) 3501–3518

3505

O

a, b, c, d

(for 22)

O

NH

R

N

R

NCO

a,b 47%

HN

OMe

e,f

22 R = H

OH

O

(for 23)

23 R = Me

c

93%

R = H or Me

O

N

NH

16

O

SiMe₂tBu

O

g

MeO

Ph

MeO

O

Ph

4

67%

d

N

O

H

O

N

25

23

24

22

OMe

Bn

O

OMe

i

63%

MeO

h

O

HN

O

OMe

Ph

H

O

N

18

N

Boc

26

O

Bn

N

H

O

17

SiMe₃

27

O

SiMe₃

Scheme 5. Reagents and condition: (a) Z-b-alanine, EDC, HOBT,

CH₂Cl₂–DMF, 90%; (b) H₂, Pd–C, MeOH; (c) HCl; (d) COCl₂,

NaHCO₃, toluene, 33%; (e) succinic anhydride, pyridine, CH₂Cl₂; (f)

1—Ph₂PON₃; 2—D; (g) CAN, CH₃CN, CH₂Cl₂, H₂O; (h) DMAP,

CH₂Cl₂; (i) DMAP, DBU, CH₂Cl₂.

a, 92%

e, 95%

c, 51%

O

H

nBu

OH

N

NH

O

N

O

N

O

SiMe₂tBu

O

H

O

19

20

SiMe₃

This time we used, as a model azetidinone, known com-

pound 25,²⁶which was conveniently obtained by us

through deprotection of 24 (Scheme 5).¹²We converted

both a model primary amine (benzylamine) and a model

secondary amine (N-methylbenzylamine) into the corre-

sponding isocyanates 22 and 23. In the second case we

used a shorter route, proceeding through Curtius rear-

rangement of a monosuccinic amide. In the ﬁrst case,

however, this protocol was not successful, because of

the formation of diketopiperazine 26 during Curtius

rearrangement. Therefore we employed a longer route,

which involved acylation with a protected b-alanine, fol-

lowed by deprotection and reaction of the resulting

amine with phosgene. The use of the conditions reported

by Nowick, was essential, in order to avoid dihydropyri-

midinedione formation.²⁷When we attempted to join

22–25 as usual, once again we could not isolate the ex-

pected azetidinyl urea, because of the formation of dihy-

dropyrimidinedione 26. While one can argue that this

product may be formed directly from isocyanate 22, it

should be noted that, when the same crude isocyanate

was reacted with N-methylaniline, formation of the urea

took place smoothly, with negligible formation of dihy-

dropyrimidinedione. Probably the isocyanate attacks

regularly the azetidinone, but then, under the reaction

conditions, intramolecular displacement by the second-

ary amide occurs. This process is obviously not possible

when the amide is tertiary. Therefore isocyanate 23 gives

the expected conjugated azetidinone 27 without prob-

lems. Interestingly, in this case the reaction was slow un-

der the usual conditions (DMAP as catalyst), but was

strongly accelerated in the presence of DBU.

f, 91%

g, 100%

e, 69%

c, 55%

H

nBu

N

NH

O

N

H

N

O

N

O

H

21

Scheme 4. Reagents: (a) succinic anhydride, DMAP, CH₂Cl₂; (b) p-

anisidine, DCC; (c) HF, CH₃CN–H₂O; (d) Boc₂O, DMAP, CH₂Cl₂;

(e) N-methyl 4-amino-1-methyl-pyrrolocarboxyamide, Py-BOP,

CH₂Cl₂, N-methyl morpholine; (f) ethyl isocyanatoacetate, Et₃N,

DMAP, CH₂Cl₂; (g) Candida antarctica lipase.

Boc₂O, Boc-ON, benzyl isocyanate) or by using other

bases instead of DMAP (Et₃N, pyridine). We also pre-

pared 21, the carbamate analogue of 20, reasoning that

an urethane moiety should be more resistant to nucleo-

philic acylic substitution reactions. However, also in this

case it was not possible to introduce the Boc group on

the b-lactam nitrogen: under the usual conditions

(Boc₂O, DMAP) the only observed product was indeed

an imidazolidinedione deriving from intramolecular at-

tack by the secondary amide nitrogen onto the urethane

carbonyl.

A possible alternative solution that avoids this problem

would be to join a linker to the hydroxy group at C-9

through an ether bond. However, all our attempts to

perform a Williamson reaction on 4 failed, because of

the already observed easy silyl transfer from nitrogen

to oxygen under basic conditions.¹³

Therefore we decided to use the other handle and to ap-

pend our linker to the azetidinone nitrogen through an

activating urea-type function, like that present in com-

pounds 8. There are two possibilities: (a) to attach the

linker to the DNA-complexing moiety ﬁrst and then join

it to the lactenediyne; (b) to attach the linker to the aze-

tidinone ﬁrst and then join the DNA-complexing struc-

ture. We initially thought that the ﬁrst strategy had

more chances to be successful, because of the anticipated

reactivity of the N-carbamoyl azetidinone towards intra-

molecular nucleophilic acyl substitution.

Although successful, this last method requires the use of

a secondary amine, and we were not very happy with

that, because of the lack of generality, and the antici-

pated diﬃculties in the coupling of secondary aromatic

amines. Thus we explored, with some concern, also the

second strategy, involving attachment of the linker to

the azetidinone ﬁrst (Scheme 6). For this purpose we

prepared, in two high yielding steps, isocyanate 28, by

opening of succinic anhydride with tert-butanol,²⁸fol-

3506

L. Banﬁ et al. / Bioorg. Med. Chem. 16 (2008) 3501–3518

We developed also a linker useful for binding struc-

O

tures containing a carboxy group (Scheme 7). This

time we started from N-methyl caprolactam, which

was converted, through the known protected amino-

acid 32,²⁹into isocyanate 33. The isocyanate could

be coupled in good yields with the model azetidi-

none 29 to give the azetidinyl urea 34. Now acid

catalysed deblocking was followed by one pot cou-

pling with carboxylic acid 37, corresponding to a

truncated netropsin analogue. The required acid

37³⁰was prepared by us in three steps from deriva-

tive 36.^31–34Reduction of the nitro group was car-

ried out through hydrogenation^31,33to give the

corresponding amine,³⁵which was directly acetylated

and saponiﬁed to aﬀord 37.

a,b

OCN

c

O

t

Bu

O

28

O

MeO

O

MeO

Ph

N

96%

O

H

25

29

28

d, 95%

Ph

MeO

O

MeO

O

e,f

58%

H

N

H

N

CO₂tBu

Bn

30

O

31

O

t

Scheme 6. Reagents and condition: (a) BuOH, NIS, DMAP, Et₃N,

toluene; (b) 1—Ph₂PON₃; 2—D; (c) H₂, Pd–C, EtOH; (d) DMAP,

DBU, CH₂Cl₂; (e) CF₃CO₂H, CH₂Cl₂; (f) BnNH₂, DCC, CH₂Cl₂.

Having successfully demonstrated, working on model

systems, the eﬀectiveness of these two linkers, we

implemented their use on the lactenediyne structure

itself. Scheme 8 shows all the conjugated compounds

that we have prepared and tested. Using the linker

suited for primary amines we obtained conjugated

compounds 40 and 41 containing again a truncated

netropsin analogue and a camptothecin derivative.

In order to prepare these two adducts we utilized

amines 45 and 46. Compound 46 was prepared at

the Dipartimento di Scienze Molecolari Agroalimen-

tari as previously reported,³⁶whereas amine 45 was

prepared following essentially the literature proce-

dures.³⁷We also synthesized the derivative of benzyl-

lowed by Curtius rearrangement. This isocyanate was

coupled with model azetidinone 25 in high yield. How-

ever, acidic deblocking of the tert-butyl ester brought

about opening of the azetidinone as well. This opening

appears to involve breaking of N-C4 bond, probably

facilitated by the styryl double bond. Actually, when

we used the saturated analogue 29 instead, tert-butyl es-

ter cleavage with CF₃CO₂H took place uneventfully,

and the b-lactam resisted. The resulting carboxylic acid

was coupled with a model primary amine (benzylamine)

aﬀording in good (unoptimized) yields the desired ad-

duct. Interestingly, and somehow surprisingly, this time

dihydropyrimidinedione 26 was not formed, despite the

basic conditions used for the coupling (Et₃N). Thus, this

linker appears to be very good for joining primary

amines to our lactenediynes.

amine 39, which served as

a

control, since

benzylamine was not anticipated to have a particular

aﬃnity to DNA. We also prepared conjugated ad-

ducts 43 and 44, containing a truncated netropsin

analogue and a naphthylacetic acid, using compound

42, endowed with the linker appropriate for joining

carboxylic acids.

The results of incubation with plasmid pBR 322 are

shown in Table 3. As a comparison, the results with

7 and 8d are also included. All compounds were

found to be more active than the parent derivative

7. It is interesting to note that the benzylamine deriv-

ative 39 had an activity identical to the naphthyl

derivative 8d, which had been selected as the best

one by the results of Table 1. This means that the lin-

ker itself has a positive eﬀect, because 39 is consider-

ably more active than 8a, which also contains a

benzylamine, but without any linker.

O

HO₂C

OCN

N

a,b

Boc

N

32

c

69%

N

Boc

O

29

d

33

Ph

92%

MeO

N

Ph

H

N

MeO

O

N

O

Boc

H

N

NH

N

e,f

O

N

44%

35

O

However the most striking results were found with

compounds 40 and 41, conjugated with a netropsin

analogue and with camptothecin. Not only they were

2–3 times more active than 8d, but also the amount

of double cleavage increased signiﬁcantly. It is very

diﬃcult to achieve this level of double cut, Calicheami-

cin being a real exception. Most other natural anti-

cancer agents acting through radical attack to DNA

aﬀord a lower percentage of double strand cleavage.

For example, with Bleomycin the ratio is only

1:9.^22,38Figure 2 shows the electrophoresis gel after

incubation with compounds 40 and 41 at 15 lM

concentration.

34

NHAc

O

N

MeO

HO

g,h,i

78%

NH

O

36

37

N

NO₂

NHAc

Scheme 7. Reagents and conditions: (a) H₂O, H₂SO₄; (b) Boc₂O,

K₂CO₃, dioxane–H₂O; (c) 1—Ph₂PON₃; 2—D; (d) DMAP, DBU,

CH₂Cl₂; (e) CF₃CO₂H, CH₂Cl₂; (f) 37, PyBOP, Et₃N, CH₂Cl₂; (g) H₂,

Pd–C, DMF; (h) Ac₂O, pyridine; (i) NaOH, MeOH–H₂O, 50 ꢁC.

L. Banﬁ et al. / Bioorg. Med. Chem. 16 (2008) 3501–3518

3507

b,c

a

f

OAc

80%

50%

96%

H

N

OtBu

N

O

H

O

6

38

39

b,h

O

42

O

b,e

90%

Boc

b,d

80%

b,g

N

68%

OAc

H

N

H

N

O

N

O

44

O

43

O

N

O

41

40

N

NH

O

N

O

H₂N

O

N

O

N

HN

O

N

H

N

O

NH

O

N

AcHN

N

OH O

N

45

O

46

O

N

OH O

H

Scheme 8. Reagents: (a) 28, DMAP, DBU, CH₂Cl₂; (b) CF₃CO₂H, CH₂Cl₂; (c) benzylamine, Py-BOP, Et₃N, CH₂Cl₂; (d) 45, Py-BOP, Et₃N,

CH₂Cl₂; (e) 46, Py-BOP, Et₃N, CH₂Cl₂; (f) 33, DMAP, DBU, CH₂Cl₂; (g) 37, Py-BOP, Et₃N, CH₂Cl₂; (h) 1-naphthylacetic acid, Py-BOP, Et₃N,

CH₂Cl₂.

3. Conclusions

Table 3. Results of cleavage of plasmid DNA with conjugate

compounds 7, 8d, 38–40, 42, 43^a

In conclusion, this study has allowed to develop an eﬃ-

cient activation strategy able to transform highly stable

lactenediyne derivatives into reactive prodrugs, which

have shown a remarkable in vitro activity as DNA-

cleaving agents, as well as cytotoxicity in the micromolar

(or high nanomolar range) against some tumour cell

lines.

Compound

CS₅₀

CS_min

CD_min

D/S

7

40

25

15

10

35

25

5

25

5

1:15

1:8

8d

39

40

41

43

44

1

5

1:8

1:6

0.3

5

1

1:5

15

10

1:10

1

A thorough study on model compounds has allowed the

development of suitable linkers for attaching DNA-

complexing moieties, such as aromatic intercalators or

groove binders. Analysis of a small collection of conju-

gated compounds has permitted the discovery of potent

DNA-cleaving agents, which have in particular demon-

strated a remarkable ability to induce simultaneous dou-

ble strand break. These results rank them among the

most eﬀective simpliﬁed artiﬁcial enediynes synthesized

so far.¹Further in vitro and in vivo tests on these con-

jugated lactenediynes are in progress.

^aFor the meanings of the column headers, see Table 1.

4. Experimental

4.1. General

NMR spectra were taken in CDCl₃at 200 MHz (¹H),

and 50 MHz (¹³C), or, when stated, at 300 MHz (¹H)

and 75 MHz (¹³C), using TMS as internal standard for

¹H NMR and the central peak of CDCl₃(at

Figure 2. Result of incubation of compounds 40 and 41 (15 lM) with

pBR 322 plasmid (67.5 lM/bp) for 24 in, pH 7.5, buﬀer at 37 ꢁC.

3508

L. Banﬁ et al. / Bioorg. Med. Chem. 16 (2008) 3501–3518

77.02 ppm) for ¹³C NMR. Chemical shifts are reported

in ppm (d scale), coupling constants are reported in

hertz. Peak assignment in ¹³C spectra was made with

the aid of DEPT experiments. In ABX systems, proton

A is the one upﬁeld. GC–MS were carried out on a

HP-5971A instrument, using an HP-1 column (12 m

long, 0.2 mm wide), electron impact at 70 eV, and a

mass temperature of about 170 ꢁC. Only m/z > 33 were

detected. All analyses were performed with a constant

He ﬂow of 0.9 mL/min with initial temperature of

100 ꢁC, init. time 2 min, rate 20 ꢁC/min, ﬁnal tempera-

ture 280 ꢁC, ﬁnal time 4 min, inj. temperature 250 ꢁC,

det. temperature 280 ꢁC. t_Rare in minutes. IR spectra

have been measured as CHCl₃solutions. Melting points

solution. Extraction with AcOEt (three times), drying

(Na₂SO₄), evaporation and chromatography (PE/

AcOEt 3:7 + 1% MeOH) gave the pure product 6 as a

white solid (66 mg, 94%). R_f0.35 (PE/AcOEt 1:1).

Found: C, 68.0; H, 4.95, N, 6.05. C₁₃H₁₁NO₃requires

C, 68.11; H, 4.84; N, 6.11%. GC–MS: t_R8.23 min. m/

z: 229 (M⁺, 30.9); 187 (23.9); 186 (38.1); 170 (7.5); 159

(14.5); 158 (33.9); 144 (16.1) 142 (12.4); 141 (14.2); 140

(9.4); 130 (71.1); 115 (57.9); 114 (18.7); 112 (55.1); 103

(14.6) 91 (11.8); 89 (15.5); 84 (15.8); 77 (17.1); 63

1

(23.9); 51 (14.1); 43 (100). H NMR: d 6.05–5.80 [4H,

m, CH@CH, CH–OAc, NH]; 3.98 [1H, t, CH–N,

J = 2.4]; 3.76 [1H, ddd, CH–C@O, J = 2.6, 3.9, 12.5];

2.93 [1H, ddd, CHH–C„C, J = 1.8, 4.0, 18.0]; 2.67

[1H, dd, CHH–C„C, J = 12.7, 18.0]. 2.15 [3H, s,

CH₃C@O]. ¹³C NMR (50 MHz): d 170.3 and 167.4

[C@O]; 127.0, 121.9 [CH@CH]; 100.4, 93.2, 88.2, 84.0

[C„C]; 61.6, 58.1; 53.5 [CH–OAc, CH–N, CH–C@O];

20.7 [CH₃C@O]; 19.3 [CH₂–C„C].

were measured on a Buchi 535 apparatus and are uncor-

¨

rected. TLC analyses were carried out on silica gel plates

and developed at UV (when not otherwise stated) or

with ‘molibdic’ reagent (21 g (NH₄)MoO₄Æ4H₂O, 1 g

Ce(SO₄)₂, 469 mL H₂O, 31 mL H₂SO₄) or with ninhy-

drin. R_fwere measured after an elution of 7–9 cm. Chro-

matographies were carried out on 220–400 mesh silica

gel using the ‘ﬂash’ methodology. Petroleum ether (40–

60 ꢁC) is abbreviated as PE. All reactions employing

dry solvents were carried out under a nitrogen atmo-

sphere. Abbreviations used are listed in a note.¹⁹

4.4. (1R^*,9R^*,10S^*)(Z)-9-Acetoxy-11-(tert-butoxycarbonyl)-

11-azabicyclo[8.2.0]dodec-5-ene-3,7-diyn-12-one (7)

A solution of compound 6 (101.5 mg, 0.443 mmol) in

dry CH₃CN (5 mL) was treated sequentially with 4-

dimethylaminopyridine (32.7 mg, 0.268 mmol) and di-

tert-butyl dicarbonate (190 mg, 0.871 mmol). After

30 min the mixture was poured into saturated aqueous

NH₄Cl and extracted with ethyl acetate. After drying

(Na₂SO₄) and evaporation, chromatography (PE/Et₂O

70:30) gave pure 7 as a white solid (133 mg, 91%).

R_f: 0.60 (PE/Et₂O 1:1). Found: C, 65.8; H, 5.8, N,

4.2. C₁₈H₁₉NO₅requires C, 65.64; H, 5.81; N, 4.25%.

GC–MS: t_R9.35 min. m/z: 329 [M⁺, 6.1%]; 231 [11.4];

187 [11.1]; 186 [15.0]; 171 [5.9]; 159 [6.5]; 158 [11.9];

144 [9.2]; 130 [18.6]; 116 [7.0]; 115 [19.1]; 114 [6.5];

112 [7.0]; 63 [6.3]; 57 [100]; 56 [8.0]; 44 [10.6]; 43

[71.9]; 41.[23.3] ¹H NMR: d 6.08 [1H, t, CH–OAc,

J = 1.9]; 6.01 and 5.92 [2H, AB syst., CH@CH,

J_AB= 9.6]; 4.23 [1H, t, CH–N, J = 2.6]; 3.80 [1H, dt,

CH–C@O, J_d= 12.5, J_t= 3.6]; 2.96 [1H, ddd, CHH–

C„C, J = 1.3, 4.0, 17.9]; 2.71 [1H, dd, CHH–C„C,

J = 12.5, 17.9]; 2.12 [3H, s, CH₃–C@O]; 1.51 [9H, s,

(CH₃)₃C].

4.2. (1R^*,9R^*,10S^*)(Z)-9-Acetoxy-11(tert-butyldimethyl-

silyl)-11-azabicyclo[8.2.0]dodec-5-ene-3,7-diyn-12-one (5)

A solution of lactenediyne 4⁹(100 mg, 0.332 mmol) in

dry CH₂Cl₂(1.2 mL) was sequentially treated with pyr-

idine (1.2 mL), 4-dimethylaminopyridine (DMAP)

(6 mg, 0.053 mmol) and acetic anhydride (157 lL,

1.66 mmol). After stirring for 75 min at rt, the mixture

was evaporated to dryness, taken up with n-heptane

and evaporated again. This procedure was repeated

once again and ﬁnally the crude product was chromato-

graphed (PE/AcOEt 8:2 ! 6:4) to give pure 5 as a white

solid (96%). R_f0.48 (PE/Et₂O 1:1). Found: C, 66.6; H,

7.4, N, 4.0. C₁₉H₂₅NO₃Si requires C, 66.44; H, 7.34;

N, 4.08%. GC–MS: t_R9.57 min. m/z: 286 (Mꢀ57, 7.4);

144 (10.2); 126 (4.7); 117 (100); 115 (17.0); 100 (5.5);

75 (29.3); 73 (17.6); 57 (6.9); 43 (21.7). IR: m_max3060,

2995, 2970, 2940, 2870, 2305, 1745, 1605 (w), 1425,

1370, 1325, 1265, 1230, 1200, 1155, 1090, 1010,

920 cm^ꢀ1

.

¹H NMR: d 6.00, 5.90 [2H, AB syst.,

4.5. General procedure for the preparation of N-carbam-

oyl derivatives 8a,b,d–g: (1R^*,9R^*,10S^*)(Z)-9-acetoxy-11-

[(1-naphthylmethyl)carbamoyl]-11-azabicyclo[8.2.0]dodec-

5-ene-3,7-diyn-12-one 8d

CH@CH, J = 9.7]; 5.61 [1H, t, CH–OAc, J = 1.9];

3.90–3.73 [2H, m, CH–N and CH–C@O]; 2.93 [1H,

ddd, CHH–C„C, J = 1.8, 3.9, 17.9]; 2.61 [1H, dd,

CHH–C„C, J = 12.5, 17.9]; 2.12 [3H, s, CH₃C@O];

0.95 [9H, s, (CH₃)₃C]; 0.25 and 0.20 [2· 3H, 2 s,

(CH₃)₂Si].

A

solution of (1-naphthyl)acetic acid (372 mg,

2.00 mmol) in dry toluene (35 mL) was treated with

Et₃N (1.00 mL, 6.8 mmol) and diphenyl phosphoryl

azide (1.20 mL, 6.0 mmol). After stirring at rt for

30 min, TLC showed complete conversion of the acid

into the corresponding acyl azide. The solution was then

heated at reﬂux, monitoring the Curtius rearrangement

through IR. When the azide band at 2180 cm^ꢀ1disap-

peared (about 30 min) being replaced by an isocyanate

band at 2250 cm^ꢀ1, the solution was cooled, diluted with

a, pH 7, buﬀer solution (phosphate) and extracted three

times with Et₂O. After drying (Na₂SO₄) and evapora-

tion, the resulting yellow oil was taken up in dry CH₂Cl₂

4.3. (1R^*,9R^*,10S^*)(Z)-9-Acetoxy-11-azabicyclo[8.2.0]dodec-

5-ene-3,7-diyn-12-one (6)

A solution of 5 (105.2 mg, 0.306 mmol) in CH₃CN

(4.75 mL) was cooled to ꢀ20 ꢁC, and treated with a

40% aqueous HF solution (0.250 mL, 5.74 mmol). After

20 min, the temperature was allowed to reach 0 ꢁC and

the solution stirred at this temperature for 2 h. After fur-

ther 1.5 h at rt, the reaction was complete and the solu-

tion was poured into a saturated NaHCO₃aqueous

L. Banﬁ et al. / Bioorg. Med. Chem. 16 (2008) 3501–3518

3509

(8 mL). This solution was approximately 0.25 M in the

isocyanate.

H, 4.9, N, 6.75. C₂₅H₂₀N₂O₄requires C, 72.80; H, 4.89;

N, 6.79%. ¹H NMR: d 7.85–7.74 [3H, m]; 7.50–7.31 [3H,

m]; 7.24–7.19 [1H, m]; 6.82 [1H, t, NH–CH₂, J = 5.9];

6.12 [1H, t, CH–OCO, J = 2.0]; 5.99 and 5.93 [2H, AB

syst., CH@CH, J_AB= 9.7]; 4.62 [2H, d, CH₂–NH,

J = 6.0]; 4.36 [1H, t, CH–N, J = 2.7]; 3.86 [1H, ddd,

J = 4.0, 2.9, 12.3]; 2.93 and 2.72 [2H, AB part of an

ABXY system, CH₂–C„C, J_AB= 17.8, J_AX= 12.8,

J_BX= 3.9, J_AY= 0, J_BY= 1.5]; d 2.07 [3H, s, CH₃–CO].

Compound 7 (40 mg, 0.174 lmol) was dissolved in

dry CH₂Cl₂(0.5 mL) and treated with Et₃N (121 lL,

0.872 mmol),

4-dimethylaminopyridine

(3 mg,

0.024 mmol) and 2.8 mL of the above described isocya-

nate solution (supposed 0.70 mmol). After stirring over-

night at rt, the mixture was treated with 5% aqueous

NH₄H₂PO₄and extracted three times with CH₂Cl₂.

Drying (Na₂SO₄), evaporation and chromatography

(PE/AcOEt 8:2) gave pure 8d as a white solid (71 mg,

98%). Mp: 167–168 ꢁC (dec). R_f0.43 (PE/AcOEt 7:3).

Found: C, 72.75; H, 4.9, N, 6.75. C₂₅H₂₀N₂O₄requires

C, 72.80; H, 4.89; N, 6.79%. IR: m_max3372, 3041,

2994, 2956, 2212 (weak), 1769, 1700, 1599, 1511, 1365,

4.5.4. (1R^*,9R^*,10S^*)(Z)-9-Acetoxy-11-[(2-(1-naphthyl)-

ethyl)carbamoyl]-11-azabicyclo[8.2.0]dodec-5-ene-3,7-

diyn-12-one (8f). It was prepared from 3-(a-naph-

thyl)propanoic acid^39,40following the general procedure

described above for 8d. Yield: 69%. R_f0.32 (ETP/Et₂O

1:1). Found: C, 73.15; H, 5.3, N, 6.45. C₂₆H₂₂N₂O₄re-

1334, 1295, 1191, 1141, 1102, 1009, 958 cm^ꢀ1

.

¹H

quires C, 73.23; H, 5.20; N, 6.57%. H NMR: d 8.08

1

NMR: d 8.02 [1H, d, J = 8.4]; 7.91–7.80 [2H, m]; 7.60–

7.51 [2H, m]; 7.48–7.42 [2H, m]; 6.70 [1H, t, NH–CH₂,

J = 5.7]; 6.15 [1H, t, CH–OCO, J = 2.0]; 5.98 and 5.92

[2H, AB syst., CH@CH, J_AB= 9.5]; 5.00 and 4.85 [2H,

AB part of an ABX system, CH₂–NH, J_AB= 14.5,

J_AX= 5.8, J_BX= 6.1]; 4.35 [1H, t, CH–N, J = 2.7]; 3.83

[1H, ddd, J = 4.0, 2.9, 12.4]; 2.90 and 2.69 [2H, AB part

of an ABXY system, CH₂–C„C, J_AB= 17.8,

J_AX= 12.8, J_BX= 3.8, J_AY= 0, J_BY= 1.6]; d 2.02 [3H,

s, CH₃–CO].

[1H, d, J = 8.6]; 7.87 [1H, dd, J = 2.1, 7.3]; 7.77 [1H,

d, J = 7.8]; d 7.61–7.31 [4H, m]; d 6.68 [1H, t, NH–

CH₂, J = 4.7]; 6.08 [1H, t, CH–OCO, J = 1.9]; 5.99

and 5.92 [2H, AB system, CH@CH, J_AB= 9.7]; 4.31

[1H, t, CH–N, J = 2.7]; 3.83 [1H, ddd, CH–CO,

J = 3.1, 4.1, 12.5]; 3.72–3.59 [2H, m, CH₂–NH]; 3.33

[2H, t, CH₂–CH₂, J = 7.0]; 2.68 and 2.91 [2H, AB part

of an ABXY system, CH₂–C„C, J_AB= 17.8,

J_AX= 12.8, J_BX= 3.9, J_AY= 0, J_BY= 1.5]; 2.09 [3H, s,

CH₃–CO].

4.5.1. (1R^*,9R^*,10S^*)(Z)-9-Acetoxy-11-(benzylcarbamoyl)-

11-azabicyclo[8.2.0]dodec-5-ene-3,7-diyn-12-one (8a). It

was prepared from commercially available benzyl isocy-

anate following the general procedure described above

for 8d. Yield: 90%. R_f0.48 (PE/AcOEt 6:4). Found: C,

69.65; H, 5.0, N, 7.65. C₂₁H₁₈N₂O₄requires C, C,

4.5.5. (1R^*,9R^*,10S^*)(Z)-9-Acetoxy-11-[(2-(2-naphthyl)-

ethyl)carbamoyl]-11-azabicyclo[8.2.0]dodec-5-ene-3,7-diyn-

12-one (8g). It was prepared from 3-(b-naphthyl)propa-

noic acid^40,41following the general procedure described

above for 8d. Yield: 36%. R_f0.23 (ETP/Et₂O 1:1).

Found: C, 73.1; H, 5.4, N, 6.4. C₂₆H₂₂N₂O₄requires

C, 73.23; H, 5.20; N, 6.57%. ¹H NMR: d 7.86–7.75

[3H, m]; 7.65 [1H, s]; 7.53–7.40 [2H, m]; 7.34 [1H, dd,

J = 8.5, 1.0]; 6.47 [1H, t, NH, J = 5.7]; 6.05 [1H, t,

CH–OCO, J = 1.8]; 5.99 and 5.91 [2H, AB system,

CH@CH, J_AB= 9.7]; 4.29 [1H, t, CH–N, J = 2.5]; 3.83

[1H, dt, CH–CO, J_t= 3.1, J_d= 12.5]; 3.62 [2H, q,

CH₂–NH, J = 6.8]; 3.02 [2H, t, CH₂–CH₂, J = 7.2];

2.65 and 2.91 [2H, AB part of an ABX system, CH₂–

C„C, J_AB= 17.8, J_AX= 12.5, J_BX= 4.0]; 2.04 [3H, s,

CH₃–CO].

1

69.60; H, 5.01; N, 7.73%. H NMR: d 7.40–7.26 [5H,

m, aromatics]; 6.74 [1H, t, NH, J = 5.6]; 6.10 [1H, t,

CH–OAc, J = 2.0]; 5.99 and 5.93 [AB system, CH@CH,

J = 9.7]; 4.46 [2H, d, CH₂NH, J = 6.0]; 4.34 [1H, t, CH–

N–C@O, J = 2.6]; 3.86 [1H, ddd, CH–C@O, J = 12.4,

4.1, 3.0]; 2.93 and 2.71 [AB part of ABXY system,

CH₂C„C, J_AB= 17.8, J_AX= 12.4, J_BX= 3.9, J_AY= 0,

J_BY= 1.6]; 2.09 [3H, s, CH₃].

4.5.2. (1R^*,9R^*,10S^*)(Z)-9-Acetoxy-11-(1-naphthylcar-

bamoyl)-11-azabicyclo[8.2.0]dodec-5-ene-3,7-diyn-12-one

(8b). It was prepared from a-naphthoic acid following

the general procedure described above for 8d. Yield:

45%. R_f0.60 (PE/Et₂O 1:1). Found: C, 72.1; H, 4.65,

N, 6.95. C₂₄H₁₈N₂O₄requires C, 72.35; H, 4.55; N,

7.03%. ¹H NMR: d 8.98 [1H, s, NH]; 8.08 [1H, d,

J = 7.6]; 7.98–7.84 [2H, m]; 7.69 [1H, d, J = 8.2]; 7.62–

7.44 [3H, m]; 6.18 [1H, t, CH–OAc, J = 2.0]; 6.03 and

5.96 [AB system, CH@CH, J = 9.7]; 4.50 [1H, t, CH–

N–C@O, J = 2.7]; 4.03 [1H, ddd, CH–C@O, J = 12.4,

3.0, 4.2]; 3.05 and 2.83 [AB part of ABXY system,

CH₂C„C, J_AB= 17.8, J_AX= 12.4, J_BX= 4.2, J_AY= 0,

J_BY= 1.5]; 2.14 [3 H, s, CH₃].

4.6. (1R^*,9R^*,10S^*)(Z)-9-Acetoxy-11-(2-naphthylcarba-

moyl)-11-azabicyclo[8.2.0]dodec-5-ene-3,7-diyn-12-one (8c)

b-Naphthylamine (415 mg, 2.90 mmol) was dissolved in

CHCl₃(5 mL) and treated with a solution of p-nitro-

phenyl chloroformate (300 mg, 1.49 mmol) in CHCl₃

(1.5 mL). A purple precipitate forms at once. After

15 min the reaction is complete. It was ﬁltered and the

mother liquors evaporated to dryness. Crystallization

from CHCl₃/pentane aﬀorded N-(2-naphthyl) p-nitro-

phenyl carbamate (218 mg), as a purple solid (mp

174.8–175.8 ꢁC. R_f0.60 in PE/AcOEt 7:3), which was

used immediately for the next reaction. A solution of

compound 7 (37.2 mg, 162 lmol) in dry CH₃CN

(2 mL) was treated with DMAP (10.8 mg, 98 lmol)

and with the nitrophenyl carbamate (100 mg, 324 lmol).

The solution was stirred overnight. Water (200 lL) was

added and the mixture stirred for 20 min. The solution

4.5.3. (1R^*,9R^*,10S^*)(Z)-9-Acetoxy-11-[(2-naphthyl-

methyl)carbamoyl]-11-azabicyclo[8.2.0]dodec-5-ene-3,7-

diyn-12-one (8e). It was prepared from b-naphthylacetic

acid following the general procedure described above for

8d. Yield: 71%. R_f0.48 (PE/Et₂O 4:6). Found: C, 72.75;

3510

L. Banﬁ et al. / Bioorg. Med. Chem. 16 (2008) 3501–3518

becomes lemon yellow. Extraction (three times) with

ACOEt, washing with saturated aqueous NaCl, drying

(Na₂SO₄), evaporation and chromatography (PE/

CH₂Cl₂/AcOEt 7:2:1) gave pure 8c (19 mg, 30%). R_f

0.65 (PE/AcOEt 65:35). Found: C, 72.0; H, 4.45, N,

6.8. C₂₄H₁₈N₂O₄requires C, 72.35; H, 4.55; N, 7.03%.

¹H NMR: d 8.49 [1H, s, NH]; 8.10 [1H, d, J = 1.9];

8.0–7.83 [3H, m]; 7.51–7.37 [3H, m]; 6.16 [1H, t, CH–

OAc, J = 1.9]; 6.02 and 5.95 [AB system, CH@CH,

J = 9.7]; 4.46 [1H, t, CH–N–C@O, J = 2.6]; 3.99 [1H,

ddd, CH–C@O, J = 12.5, 3.0, 4.2]; 3.02 and 2.79 [AB

part of ABXY system, CH₂C„C, J_AB= 17.8,

J_AX= 12.8, J_BX= 4.0, J_AY= 0, J_BY= 1.5]; 2.13 [3H, s,

CH₃].

CH₂CH₂CO, J = 8.1]; 2.92 and 2.60 [2H, AB part of

an ABXY system, CH₂–C„C, J_AB= 17.9, J_AX= 12.9,

J_BX= 3.7, J_AY= 0, J_BY= 1.7]; 2.88–2.76 [2H, m,

CH₂CO]; 0.92 [9H, s, (CH₃)₃–C]; 0.20 and 0.161 [2·

3H, s, (CH₃)₂Si].

4.9. (1R^*,9R^*,10S^*)(Z)-11(tert-butyldimethylsilyl)-9-[3-

(2-naphthyl)propanoyloxy]-11-azabicyclo[8.2.0]dodec-5-

ene-3,7-diyn-12-one (9c)

It was prepared in 100% yield from 2-naphthylpropa-

noic acid and lactenediyne 4 following the same proce-

dure described above for 9a. R_f: 0.64 (Et₂O/PE 6:4).

Found: C, 74.25; H, 6.95, N, 2.8. C₃₀H₃₃NO₃Si

requires C, 74.50; H, 6.88; N, 2.90%. ¹H NMR: d

7.82–7.75 [3H, m]; 7.64 [1H, s]; 7.50–7.38 [2H, m]; d

7.32 [1H, dd, J = 1.6, 8.4]; d 5.97 and 5.86 [2H, AB

system, CH₂@CH₂, J_AB= 9.6]; 5.60 [1H, t, CH–OCO,

J = 1.9]; 3.84 [1H, t, CH–N, J = 2.5]; 3.70 [1H, ddd,

CH–CH₂, J = 12.6, 3.8, 2.7]; 3.13 [2H, t, CH₂CH₂CO,

J = 7.6]; 2.88 and 2.58 [2H, AB part of an ABXY

system, CH₂–C„C, J_AB= 17.9, J_AX= 12.8, J_BX= 3.7,

J_AY= 0, J_BY= 1.6]; 2.83–2.75 [2H, m, CH₂C@O]; 0.91

[9H, s, (CH₃)₃–C]; 0.17 and 0.11 [2· 3H, s, (CH₃)₂Si].

4.7. (1R^*,9R^*,10S^*)(Z)-11(tert-butyldimethylsilyl)-9-(1-

naphthylacetoxy)-11-azabicyclo[8.2.0]dodec-5-ene-3,7-diyn-

12-one (9a)

A crude solution (approximately 1.34 M) of 1-naphthy-

lacetyl chloride was prepared by treating 1-naphthylace-

tic acid (500 mg, 2.69 mmol) in dry CH₂Cl₂(10 mL)

with oxalyl chloride (2 mL of a 2.06 M solution,

4.04 mmol). After stirring for 1 h at rt, the solution

was evaporated to dryness, taken up with CH₂Cl₂and

re-evaporated twice. The resulting yellow solid was ta-

ken up with 2 mL of CH₂Cl₂. A solution of lactenediyne

4⁹(30 mg, 99 lmol) in dry CH₂Cl₂(1.0 mL) was treated

with pyridine (600 lL) and with the above cited 1.34 M

solution of 1-naphthylacetyl chloride in CH₂Cl₂

(380 lL, 509 lmol). The solution was stirred overnight

and then quenched with 5% aqueous NH₄H₂PO₄.

Extraction for three times with CH₂Cl₂(resulting pH

5–6), washing with saturated aqueous NaCl, Drying

(Na₂SO₄), evaporation and chromatography (Et₂O/PE

2:8 ! 4:6) gave pure 9a as a white solid (84%). R_f:

0.71 (Et₂O/PE 1:1). Found: C, 74.1; H, 6.75, N, 2.95.

4.10. (1R^*,9R^*,10S^*)(Z)-11-(tert-butoxycarbonyl)-9-(1-

naphthylacetoxy)-11-azabicyclo[8.2.0]dodec-5-ene-3,7-

diyn-12-one (10a)

Substrate 9a (22.2 mg, 47.3 lmol) was dissolved in

CH₃CN (2 mL) and cooled to ꢀ20 ꢁC. The solution was

treated with 40% aqueous HF (40 lL). After 20 min, the

solution was brought to 0 ꢁC, and, after 2 h, to rt. After

stirring for 3.5 h the mixture was quenched with saturated

aq NaHCO₃and extracted three times with AcOEt. after

washing with saturated aq NaCl, drying (Na₂SO₄) evapo-

ration, and chromatography (PE/AcOEt 6:4), pure

(1R^*,9R^*,10S^*)(Z)-9-(1-naphthylacetoxy)-11-azabicy-

clo[8.2.0]dodec-5-ene-3,7-diyn-12-one was obtained

(12.6 mg, 75%). R_f: 0.11 (Et₂O/PE 1:1). ¹H NMR: d

7.93–7.79 [3H, m]; 7.62–7.39 [4H, m]; 6.01 and 5.91 [2H,

AB system, CH@CH, J_AB= 9.8]; 5.83 [1H, t, CH–OCO,

J = 1.93]; 5.64 [1H, s, NH–CO]; 4.15 [2H, s, CH₂–CO];

3.86 [1H, t, CH–N, J = 2.3]; 3.63 [1H, ddd, CH–C@O,

J = 2.6, 3.8, 12.6]; 2.88 and 2.62 [2H, AB part of an ABXY

system, CH₂–C„C, J_AB= 18.0, J_AX= 12.8, J_BX= 3.7,

J_A= 0, J_BY= 1.8].

1

C₂₉H₃₁NO₃Si requires C, 74.16; H, 6.65; N, 2.98%. H

NMR: d 7.98 [1H, dd, J = 9.5, 0.6]; 7.86 [1H, dd,

J = 7.3, 2.1]; 7.73 [1H, dd, J = 7.2, 2.0]; 7.58–7.49 [2H,

m]; d 7.46–7.44 [2H, m]; d 6.00 and 5.89 [2H, AB system,

CH₂@CH₂, J_AB= 9.5]; 5.60 [1H, t, CH–OCO, J = 1.8];

4.15 [2H, s, CH₂–CO]; 3.83 [1H, t, CH–N, J = 2.3];

3.69 [1H, ddd, CH–CH₂, J = 12.6, 3.9, 2.7]; 2.98 and

2.51 [2H, AB part of an ABXY system, CH₂–C„C,

J_AB= 17.9, J_AX= 12.6, J_BX= 4.0, J_AY= 0, J_BY= 1.6];

0.85 [9H, s, (CH₃)₃–C]; 0.01 and ꢀ0.01 [2· 3H, s,

(CH₃)₂Si].

This compound (12.6 mg, 35.4 mmol) was dissolved in

dry CH₃CN (2 mL) and treated with 4-dimethylamino-

pyridine (2.6 mg, 21 lmol) and di-tert-butyl dicarbonate

(15.2 mg, 71 lmol). After 40 min the reaction was

complete. It was treated with saturated aqueous NH₄Cl

and extracted three times with AcOEt. After drying

(Na₂SO₄), evaporation and chromatography (PE/Et₂O

65:25), pure 9a was obtained (7.5 mg, 93%). R_f: 0.58

4.8. (1R^*,9R^*,10S^*)(Z)-11(tert-butyldimethylsilyl)-9-[3-

(1-naphthyl)propanoyloxy]-11-azabicyclo[8.2.0]dodec-5-

ene-3,7-diyn-12-one (9b)

It was prepared in 97% yield from 1-naphthylpropanoic

acid and lactenediyne 4 following the same procedure

described above for 9a. R_f: 0.74 (Et₂O/PE 6:4). Found:

C, 74.3; H, 6.9, N, 2.9. C₃₀H₃₃NO₃Si requires C,

74.50; H, 6.88; N, 2.90%. ¹H NMR: d 7.99 [1H, d,

J = 8.3]; 7.86 [1H, dd, J = 7.3, 2.1]; 7.73 [1H, dd,

J = 7.3, 2.1]; 7.59–7.33 [4H, m]; d 6.00 and 5.91 [2H,

AB system, CH₂@CH₂, J_AB= 9.7]; 5.64 [1H, t, CH–

OCO, J = 1.9]; 3.87 [1H, t, CH–N, J = 2.4]; 3.73 [1H,

ddd, CH–CH₂, J = 12.6, 3.8, 2.7]; 3.44 [2H, t,

1

(Et₂O/ETP 1:1). H NMR: d 7.94–7.79 [3H, m]; 7.56–

7.40 [4H, m]; 6.09 [1H, t, CH–OCO, J = 1.9]; 6.00 and

5.91 [2H, AB system, CH@CH, J_AB= 9.7]; 4.17–4.13

[3H, m, CH₂–CO and CH–N]; 3.60 [1H, dt, CH–CH₂,

J_d= 12.3, J_t= 3.6]; 2.88 and 2.65 [2H, AB part of an

ABXY system, CH₂–C„C, J_AB= 17.9, J_AX= 12.8,

J_BX= 3.8, J_AY= 0, J_BY= 1.4]; 1.32 [9H, s, (CH₃)₃C].

L. Banﬁ et al. / Bioorg. Med. Chem. 16 (2008) 3501–3518

3511

4.11. (1R^*,9R^*,10S^*)(Z)-11-(tert-butoxycarbonyl)-9-[(1-

naphthyl)propanoyloxy-11-azabicyclo[8.2.0]dodec-5-ene-

3,7-diyn-12-one (10b)

in this case the reaction is much slower requiring 10 h

at rt to reach completion. R_f0.31 (PE/AcOEt 1:1).

Found: C, 68.35; H, 4.95, N, 6.0. C₁₃H₁₁NO₃requires

1

C, 68.11; H, 4.84; N, 6.11%. H NMR: d 6.21 [1H, br

It was prepared in 79% yield (100% and 79% for the two

steps) from lactenediyne 9b following the same procedure

described above for 10a. R_f: 0.64 (Et₂O/ETP 1:1). Found:

C, 74.0; H, 5.85, N, 2.75. C₂₉H₂₇NO₅requires C, 74.18; H,

s, NH–CO]; 5.91 [2H, s, CH@CH]; 5.41 [1H, d, CH–

OCO, J = 9.3]; 3.52 [1H, ddd, CH–CH₂, J = 12.2, 4.1,

2.6]; 3.41 [1H, dd, CH–NH, J = 9.4, 2.6]; 2.93 and 2.75

[2H, AB part of ABXY syst., CH₂–C„C, J_AB= 17.8,

J_AX= 12.6, J_BX= 3.6, J_AY= 0, J_BY= 1.0]; 2.14 [3H, s,

CH₃–CO].

1

5.80; N, 2.98%. H NMR: d 7.98 [1H, d, J = 8.05]; 7.86

[1H, dd, J = 7.3, 2.0]; 7.73 [1H, d, 7.4]; 7.59–7.29, [4H,

m] 6.12 [1H, t, CH–OCO, J = 2.0]; 6.01 and 5.93 [2H,

AB system, CH@CH, J_AB= 10.0]; 4.22 [1H, t, CHNCO,

J = 2.0]; 3.69 [1H, dt, CH–CH₂, J_d= 12.3, J_t= 3.9]; 3.42

[2H, t, CH₂CH₂CO, J = 8.1]; 2.93 and 2.69 [2H, AB part

of an ABXY system, CH₂–C„C, J_AB= 17.9, J_AX= 12.5,

J_BX= 3.9, J_AY= 0, J_BY= 1.6]; 2.87–2.77 [2H, m,

CH₂CO]; 1.49 [9H, s, (CH₃)₃C].

4.15. (1R^*,9S^*,10S^*)(Z)-9-Acetoxy-11-(tert-butoxycar-

bonyl)-11-azabicyclo[8.2.0]dodec-5-ene-3,7-diyn-12-one

(14)

It has been prepared in 90% yield starting from 13, fol-

lowing the same procedure described for 7. R_f: 0.60 (PE/

Et₂O 1:1). Found: C, 65.75; H, 5.9, N, 4.3. C₁₈H₁₉NO₅

1

4.12. (1R^*,9R^*,10S^*)(Z)-11-(tert-butoxycarbonyl)-9-[(2-

naphthyl)propanoyloxy-11-azabicyclo[8.2.0]dodec-5-ene-

3,7-diyn-12-one (10c)

requires C, 65.64; H, 5.81; N, 4.25% H NMR d 5.94

[2H, s, CH@CH]; 5.84 [1H, d, CH–OCO, J = 9.1]; 4.33

[1H, dd, CH–N, J = 9.16, 3.21]; 3.45 [1H, dt, CH–

CH₂, J_d= 12.0, J_t= 3.7]; 2.95 and 2.75 [2H, AB part

of an ABX system, CH₂–C„C, J_AB= 17.8,

J_AX= 12.5, J_BX= 3.6]; 2.11 [3H, s, CH₃–CO]; 1.51

[9H, s, (CH₃)₃CO].

It was prepared in 98% yield (100% and 98% for the two

steps) from lactenediyne 9c following the same proce-

dure described above for 10a. R_f: 0.62 (Et₂O/ETP 1:1).

Found: C, 74.15; H, 5.8, N, 2.7. C₂₉H₂₇NO₅requires

C, 74.18; H, 5.80; N, 2.98%. ¹H NMR: d 7.82–7.75

[3H, m]; 7.63 [1H, s]; 7.49–7.39 [2H, m]; 7.32 [1H, dd,

J = 1.7, 8.4]; 6.07 [1H, t, CH–OCO, J = 2.1]; 5.98 and

5.88 [2H, AB system, CH@CH, J_AB= 9.7]; 4.20 [1H, t,

CHN, J = 2.6]; 3.65 [1H, dt, CH–CH₂, J_d= 12.4,

J_t= 3.9]; 3.12 [2H, t, CH₂CH₂CO]; 2.88 and 2.66 [2H,

AB part of an ABXY system, CH₂–C„C, J_AB= 18.0,

J_AX= 12.8, J_BX= 3.9, J_AY= 0, J_BY= 1.6]; 2.79 [2 H, t,

CH₂CO, J = 7.8]; 1.45 [9H, s, (CH₃)₃C].

4.16. (1R^*,9S^*,10S^*)(Z)-9-Acetoxy-11-[(1-naphthylmethyl)-

carbamoyl]-11-azabicyclo[8.2.0]dodec-5-ene-3,7-diyn-12-

one (15)

It was prepared in 67% yield starting from 13, following

the same procedure described for 8d. R_f0.57 (PE/AcOEt

7:3). Found: C, 72.6; H, 4.95, N, 6.65. C₂₅H₂₀N₂O₄re-

1

quires C, 72.80; H, 4.89; N, 6.79%. H NMR: d 7.90–

7.78 [2H, m]; 7.62–7.42 [4H, m]; 6.81 [1H, t, NH–CH₂,

J = 5.5]; 5.93 [1H, d, CH–OCO, J = 9.0]; 5.93 [2H, s,

CH@CH]; 4.97 and 4.83 [2H, AB part of an ABX sys-

tem, CH₂–NH, J_AB= 14.9, J_AX= 5.5, J_BX= 6.0]; 4.46

[1H, dd, CH–N, J = 9.2, 2.9]; 3.52 [1H, dt, CH–CH₂,

J = 12.0, 3.6]; 2.92 and 2.74 [2H, AB part of an ABX

system, CH₂–C„C, J_AB= 17.9, J_AX= 12.4, J_BX= 3.4];

2.21 [3H, s, CH₃–CO].

4.13. (1R^*,9S^*,10S^*)(Z)-9-Acetoxy-11(tert-butyldimeth-

ylsilyl)-11-azabicyclo[8.2.0]dodec-5-ene-3,7-diyn-12-one (12)

It has been prepared in 81% starting from 11, following

the same procedure described for 5. R_f0.54 (PE/Et₂O

1:1). Found: C, 66.7; H, 7.45, N, 4.0. C₁₉H₂₅NO₃Si re-

quires C, 66.44; H, 7.34; N, 4.08%. GC–MS: t_R

9.54 min; m/z 286 (Mꢀ57, 1.7); 144 (3.8); 118 (9.9);

100 (4.2); 75 (28.8); 59 (2.7); 43 (19.5). IR: m_max3006,

2929, 2857, 2398, 1745, 1602, 1364, 1278, 2214, 1163,

4.17. N-Benzyl 3-isocyanatopropanamide (22)

N-(Benzyloxycarbonyl) b-alanine (824 mg, 3.70 mmol)

was dissolved in dry CH₂Cl₂(6 mL) and DMF

(6 mL). The solution was cooled to 0 ꢁC and treated

with N-hydroxybenzotriazole (HOBT) (500 mg,

3.70 mmol), benzylamine (445 lL, 4.07 mmol) and

EDC¹⁹(852 mg, 4.44 mmol). After 10 min the cooling

bath was removed and the mixture stirred for 4 h at

rt. The reaction was quenched with water, and ex-

tracted with AcOEt. The organic extracts were washed

with 1 M NaOH and 5% NH₄H₂PO₄. Drying

(Na₂SO₄), evaporation and chromatography (CH₂Cl₂/

AcOEt 45:55 to 30:70) gave pure N-benzyl 3-(benzyl-

oxycarbonylamino)propanamide⁴²as a solid (1.044 g,

90%). R_f0.67 (CH₂Cl₂/AcOEt 3:7, ninhydrin). ¹H

NMR: d 7.65–7.22 [10H, m, aromatics]; 6.00 [1H, s,

NH]; 5.49 [1H, s, NH]; 5.06 [2H, s, CH₂O]; 4.41 [2H,

d, NHCH₂Ph, J = 5.6]; 3.49 [2H, q, NHCH₂CH₂,

J = 5.8]; 2.43 [2H, t, CH₂CH₂CO, J = 5.8].

1098, 1007, 952 cm^ꢀ1

.

¹H NMR: d 5.90 [2H, s,

CH@CH]; 5.22 [1H, d, CH–OCO, J = 9.3]; 3.47 [1H,

dt, CH–CH₂, J_d= 12.2, J_t= 3.5]; 2.95 and 2.66 [2H,

AB part of an ABX syst., CH₂–C„C, J_AB= 17.7,

J_AX= 12.3, J_BX= 3.3]; 2.15 [3H, s, CH₃–CO]; 0.94

[9H, s, (CH₃)₃C]; 0.28 and 0.27 [2· 3H, s, (CH₃)₂Si].

¹³C NMR: d 172.50 [COO]; 169.54 [CO–N]; 125.12

and 122.35 [CH@CH]; 100.44, 93.83, 87.15, 84.32

[C„C]; 69.19 [CH–OCO]; 59.71 and 56.97 [CH–N and

CH–CO]; 26.15 [(CH₃)₃C]; d 20.31 [C(CH₃)₃]; d ꢀ5.03

and ꢀ5.32 [(CH₃)₂Si].

4.14. (1R^*,9S^*,10S^*)(Z)-9-Acetoxy-11-azabicyclo[8.2.0]-

dodec-5-ene-3,7-diyn-12-one (13)

It has been prepared in 78% starting from 12, following

the same procedure described for 6. Note however that

3512

L. Banﬁ et al. / Bioorg. Med. Chem. 16 (2008) 3501–3518

This compound (502 m, 1.61 mmol) was dissolved in

MeOH (12 mL) and hydrogenated over 10% Pd/C

(54 mg) at room temp. and pressure for 80 min. After

ﬁltration of the catalyst, the solvent was evaporated.

the resulting amine was taken up in dry CH₂Cl₂

(3 mL) and treated with 1 M HCl in diethyl ether

(3 mL). A yellowish solid separated. Filtration aﬀor-

ded 264 mg of the solid hydrochloride (76%). This so-

lid (1.23 mmol) was suspended in CH₂Cl₂(12 mL) and

treated with saturated aqueous NaHCO₃(12 mL).

After vigorously stirring for 1 min, the mixture was

cooled to 0 ꢁC and stirring stopped, allowing the

phases to separate. Then a 1.93 M toluene solution

of phosgene (1.275 mL, 2.46 mmol) was added, via

syringe, directly to the organic phase. Then stirring

was resumed for 15 min at 0 ꢁC. The phases were sep-

arated, and the aqueous one re-extracted three times

with CH₂Cl₂. Drying (Na₂SO₄) and evaporation aﬀor-

ded crude isocyanate 22 (108 mg, 33% from N-benzyl

3-(benzyloxycarbonylamino)propanamide) as a yellow

oil. This compound was used as such, without further

TLC showed complete conversion of the acid into the

corresponding acyl azide. The solution was then heated

at 80 ꢁC, monitoring the Curtius rearrangement through

TLC. When the reaction was complete (140 min), the

solution was cooled, diluted with 5% aqueous NaHCO₃

and extracted three times with AcOEt. After drying

(Na₂SO₄) and evaporation, the resulting yellow oil was

taken up in dry CH₂Cl₂(7 mL). This solution was

approximately 0.135 M in N-Benzyl-N-methyl-3-isocy-

anatopropanamide 23 and was used as such for the fol-

lowing reaction.

Azetidinone 25 (24.9 mg, 122 lmol) was dissolved in

CH₂Cl₂(300 lL) and treated with the above prepared

isocyanate solution (1.35 mL, 183 lmol), DMAP¹⁹

(7.5 mg, 61 lmol), and DBU¹⁹(18 lL, 121 lmol). The

solution was reﬂuxed for 4 h. The mixture was diluted

with AcOEt and 5% NH₄H₂PO₄. Extraction with

AcOEt (three times), followed by washing with satu-

rated NaCl, drying (Na₂SO₄) and evaporation to dry-

ness gave a crude product that was chromatographed

(PE/AcOEt 2:8) to give pure 27 as an oil (32.0 mg,

63%). R_f0.36 (PE/AcOEt 3:7). Found: C, 68.45; H,

6.5, N, 9.9. C₂₄H₂₇N₃O₄requires C, 68.39; H, 6.46;

1

puriﬁcations. However, H NMR showed that it was

essentially pure. IR: m_max2247, 1665 cm^ꢀ1

.

¹H

NMR: d 7.39–7.19 [5H, m]; 5.94 [1H, s, NH]; 4.47

[2H, d, CH₂Ph, J = 5.4]; 3.66 [2H, t, NCH₂CH₂,

J = 5.8]; 2.46 [2H, t, CH₂CO, J = 6.2].

1

N, 9.97%. H NMR (2 conformers are present: A (ma-

jor) and B (minor) in ratio 58:42): d 7.45–7.07 [11H, m,

aromatics and NH]; 6.83 (conf. A) [1H, d, CH@CHPh,

J = 15.8]; 6.81 (conf. B) [1H, d, CH@CHPh, J = 15.8];

6.23 (conf. A) [1H, dd, CH@CHPh, J = 8.4, 15.8]; 6.22

(conf. B) [1H, dd, CH@CHPh, J = 8.4, 15.8]; 4.81

(conf. A) [1H, dd, H-4, J = 5.0, 8.4]; 4.79 (conf B)

[1H, dd, H-4, J = 5.0, 8.4]; 4.69 (conf. A) [1H, d, H-

3, J = 5.0]; 4.68 (conf. B) [1H, d, H-3, J = 5.0]; 4.59

(conf. A) [2H, s, CH₂Ph]; 4.49 (conf. B) [2H, s,

CH₂Ph]; 3.70–3.54 [2H, m, CH₂CH₂CON(Me)Bn];

3.49 [3H, s, OCH₃]; 2.96 (conf. B) [3H, s, NCH₃];

2.86 (conf. A) [3H, s, NCH₃]; 2.66–2.56 [2H, m,

CH₂CH₂CON(Me)Bn].

4.18. (3R^*,4S^*)(E)-3-Methoxy-4-styryl-2-azetidinone (25)

To a solution of (3R^*,4S^*)(E)-3-methoxy-1-(p-methoxy-

phenyl)-4-styryl-2-azetidinone 24 (608.2 mg, 1.96 mmol)

in CH₂Cl₂(6.5 mL) and CH₃CN (14 mL), cooled to

ꢀ18 ꢁC, a solution of CAN¹⁹(2.699 g, 4.92 mmol) in

H₂O (6 mL) was dropped during 5 min. After further

stirring for 15 min, the mixture was dropped into a solu-

tion of Na₂S₂O₅(644 mg) in H₂O (9 mL). The resulting

pH was 1. Extraction with Et₂O (three times), followed

by washing of the united organic layers with 5% aqueous

NaHCO₃and with saturated NaCl, and ﬁnally drying

(Na₂SO₄) and evaporation, gave a crude product that

was chromatographed with PE/AcOEt 7:3 to 4:6 to give

compound 25 as a yellowish solid (266 mg, 67%). R_f0.20

(PE/AcOEt 1:1). The analytical data were identical with

those reported.²⁶

4.20. (3R^*,4S^*)(E)-3-Methoxy-4-(2-phenylethyl)-2-aze-

tidinone (29)

A solution of azetidinone 25 (302.9 mg, 1.49 mmol) in

96% EtOH (6 mL) was hydrogenated over 10% Pd–C

(97 mg) for 5 h. The reaction was followed by GC–

MS. t_Rof 25: 7.45 min; t_Rof 29: 6.97 min. MS of 29:

m/z: 162 (M⁺ꢀ43 (–HN@C@O), 6.0); 117 (1.6); 91

(12.8); 71 (100.0); 65 (5.0); 43 (2.7); 41 (12.7); 39 (3.0).

Filtration of the catalyst and evaporation aﬀorded com-

pound 29 (294.5 mg, 96%), which was not further purif-

4.19. (3R^*,4S^*)(E)-1-[2-[(N-benzyl-N-methyl)carbamoyl]-

ethylcarbamoyl]-3-methoxy-4-styryl-2-azetidinone (27)

N-Methylbenzylamine (301 mg, 2.41 mmol) was dis-

solved in dry CH₂Cl₂(2 mL). The solution was cooled

in cold water and treated with pyridine (0.50 mL) and

succinic anhydride (372 mg, 3.72 mmol). After 5 min

the cooling bath was removed and the solution allowed

to stir for 70 min at rt. The mixture was diluted with sat-

urated aqueous NH₄Cl, acidiﬁcated with HCl to pH 3,

and extracted with CH₂Cl₂. The organic extracts were

washed with saturated aqueous NaCl, dried (Na₂SO₄)

and evaporated to dryness. Chromatography

(AcOEt + 1% AcOH) aﬀorded a white solid (510 mg).

Part of this solid (209.2 mg, supposed 0.946 mmol) was

taken up in dry toluene (4 mL) and treated with Et₃N

(160 lL, 1.12 mmol) and diphenyl phosphoryl azide

(250 lL, 1.12 mmol). After stirring at rt for 135 min,

1

icated, but used as such for further reactions. H NMR

(300 MHz.): d 7.40–7.12 [5H, m, aromatics]; 5.97 [1H, br

s, NH]; 4.50 [1H, dd, H-3, J = 2.7, 5.1]; 3.75 [1H, dt, H-

4, J_d= 8.1, J_t= 5.1]; 3.55 [3H, s, OCH₃]; 2.80–2.60 [2H,

m, CH₂Ph]; 2.08–1.82 [2H, m, CH₂CH₂Ph].

4.21. (3R^*,4S^*)1-[(2-(tert-Butoxycarbonyl)ethyl)carbam-

oyl]-3-methoxy-4-(2-phenylethyl)-2-azetidinone (30)

Succinic acid mono-tert-butyl ester was prepared as pre-

viously described²⁸: succinic anhydride (1.521 g,

15.20 mmol) was dissolved in dry toluene (10 mL) and

treated with NIS¹⁹(532.5 mg, 4.6 mmol), DMAP¹⁹

L. Banﬁ et al. / Bioorg. Med. Chem. 16 (2008) 3501–3518

3513

(189.3 mg,

1.5 mmol),

triethylamine

(635 lL,

4.23. (3R^*,4S^*)1-[(5-(N-(tert-butoxycarbonyl)-N-methyl-

amino)pentyl)carbamoyl]-3-methoxy-4-(2-phenylethyl)-2-

azetidinone (34)

4.56 mmol) and dry tert-butyl alcohol (4.3 mL). The

solution was reﬂuxed for 24 h and then cooled, diluted

with AcOEt and washed with 0.5 M aqueous citric acid

(pH 2). The aqueous phase was re-extracted with AcOEt

and the united organic layers were washed with satu-

rated aqueous NaCl, dried (Na₂SO₄) and evaporated

to give crude succinic acid mono-tert-butyl ester as a

brown solid (2.409 g). A part of this solid (201 mg,

1.15 mmol) was dissolved in toluene (4.5 mL) and trea-

ted with Et₃N (196 lL, 1.41 mmol) and diphenyl phos-

phoryl azide (295 lL, 1.37 mmol). After stirring for 5 h

at rt, the mixture was reﬂuxed for 3.5 h. After cooling

and evaporating to dryness, the residue was taken up

in AcOEt and washed with 5% aqueous NaHCO₃. Dry-

ing (Na₂SO₄) and evaporation gave crude tert-butyl 3-

isocyanatopropionate 28. It was taken up in dry CH₂Cl₂

(2.56 mL) in order to have a theoretical 0.45 M solution.

A 0.45 M solution of crude isocyanate 33 was prepared

from know acid 32²⁹following the same procedure de-

scribed above for the synthesis of isocyanate 28 (see

the preparation of 30).

Azetidinone 29 (92.0 mg, 448 lmol) was dissolved in

CH₂Cl₂(5 mL) and treated with the solution of 28

(2.0 mL, 900 lmol), DMAP¹⁹(27.4 mg, 224 lmol) and

DBU¹⁹(67 lL, 448 lmol). The solution was stirred for

16 h at rt, diluted with AcOEt, and washed with 5%

aqueous NH₄H₂PO₄and saturated aqueous NaCl. Dry-

ing (Na₂SO₄), evaporation, and chromatography (PE/

AcOEt 6:4) gave pure 34 as an oil (184.3 mg, 92%). R_f

0.47 (PE/AcOEt 1:1). Found: C, 64.4; H, 8.3, N, 9.35.

1

C₂₄H₃₇N₃O₅requires C, 64.41; H, 8.33; N, 9.39%. H

Compound 29 (50.9 mg, 248 lmol) was dissolved in dry

CH₂Cl₂(700 lL) and treated with the above prepared

solution of isocyanate 28 (1.10 mL, 495 lmol), DMAP

(15.2 mg, 124 lmol), and DBU (37 lL, 248 lmol). The

solution was stirred for 16 h at rt, diluted with AcOEt,

and washed with 5% aqueous NH₄H₂PO₄and saturated

aqueous NaCl. Drying (Na₂SO₄), evaporation and chro-

matography (PE/AcOEt 7:3) gave pure 30 as an oil

(89.3 mg, 95%). R_f0.41 (PE/AcOEt 6:4). Found: C,

64.1; H, 7.65, N, 7.5. C₂₀H₂₈N₂O₅requires C, 63.81;

NMR d 7.34–7.15 [5H, m, aromatics]; 6.58 [1H, br t,

NH, J = 5.7]; 4.55 [1H, d, H-3, J = 5.4]; 4.21 [1H, dt,

H-4, J_d= 7.2 J_t= 5.2]; 3.59 [3H, s, OCH₃]; 3.29 [2H, t,

CH₂N, J = 6.6]; 3.19 [2H, t, CH₂N, J = 6.8]; 2.82 [3H,

s, NCH₃]; 2.78 [2H, t, CH₂Ph, J = 8.2]; 2.35–2.10 [2H,

m, CH₂CH₂Ph]; 1.63–1.45 [4H, m, CH₂]; 1.44 [9H, s,

C(CH₃)₃]; 1.20–1.10 [2H, m, CH₂].

4.24. 4-(4-Acetamido-1-methyl-1H-pyrrole-2-carboxa-

mido)-1-methyl-1H-pyrrole-2-carboxylic acid (37)

1

H, 7.50; N, 7.44%. H NMR d 7.40–7.12 [5H, m, aro-

matics]; 6.95 [1H, br t, NH, J = 5.9]; 4.54 [1H, d, H-3,

J = 5.6]; 4.21 [1H, dt, H-4, J_d= 7.8, J_t= 5.6]; 3.59 [3H,

s, OCH₃]; 3.52 [2H, q, NHCH₂, J = 6.2]; 2.77 [2H, t,

CH₂Ph, J = 7.8]; 2.48 [2H, t, CH₂CO₂Bu, J = 6.2];

2.35–2.13 [2H, m, CH₂CH₂Ph]; 1.46 [9H, s, C(CH₃)₃].

A solution of known compound 36^31,32,34(111 mg,

362 lmol) in dry DMF (4 mL) was hydrogenated over

10% Pd–C (65 mg)³⁴for 6 h at rt. The catalyst was rap-

idly ﬁltered through cotton, washing with a total of

7 mL of DMF. The ﬁltrate was immediately treated

with acetic anhydride (61 lL, 651 lmol), and pyridine

(52 lL, 651 lmol). After stirring for 100 min at rt the

acetylation is complete. Most DMF was evaporated

at 1 mbar. The residue was taken up in AcOEt and ﬁl-

tered through a paper ﬁlter to remove traces of the

hydrogenation catalyst. The clear orange-yellow solu-

tion was washed with 5% aqueous NH₄H₂PO₄and sat-

urated NaCl, dried (Na₂SO₄) and evaporated to

dryness. The crude product was chromatographed with

CH₂Cl₂/acetone 8:2 to CH₂Cl₂/acetone 7:3 + 1%

MeOH to give pure methyl 4-(4-acetamido-1-methyl-

1H-pyrrole-2-carboxamido)-1-methyl-1H-pyrrole-2-car-

boxylate (89.4 mg, 78%). It is worth noting that this

product is not very soluble in most organic solvents.

Therefore, introduction to the chromatographic col-

umn has been made by dissolving it in CH₂Cl₂/MeOH

8:2, adding 500 mg of 40–60 mesh silica, evaporating

the solvents and introducing the resulting powder at

the head of the column. R_f0.45 (CH₂Cl₂/acetone

70:30 + 1% MeOH). ¹H NMR (d₆-DMSO): d 9.89

and 9.83 [2H, 2 s, NH]; 7.46 [1H, d, J = 2.0]; 7.15

[1H, d, J = 1.4]; 6.90 [1H, d, J = 1.8]; 6.85 [1H, d,

J = 1.8]; 3.84, 3.82, 3.74 [3· 3H, 3 s, CH₃O and

CH₃N]; 1.98 [3H, CH₃C@O]. ¹³C NMR (d₆-DMSO,

75 MHz.): d 166.4, 160.7, 158.3 [C@O]; 122.8, 122.4,

122.1, 118.4 [aromatic quat.]; 120.7, 118.1, 108.3,

103.8 [aromatic CH]; 50.9 [CH₃O]; 36.1 and 36.0

[CH₃N]; 23.0 [CH₃C@O].

t

4.22. (3R^*,4S^*)1-[(2-(Benzylcarbamoyl)ethyl)carbamoyl]-

3-methoxy-4-(2-phenylethyl)-2-azetidinone (31)

A solution of azetidinyl urea 30 (37.3 mg, 99 lmol) in

dry CH₂Cl₂(500 lL) was treated with triﬂuoroacetic

acid (250 lL). After stirring for 30 min at rt, the solvent

was rapidly evaporated, taken up with CH₂Cl₂and

evaporated again (this process was repeated three

times). It was ﬁnally taken up in CH₂Cl₂(500 lL), trea-

ted with benzylamine (14 lL, 129 lmol) and with

DCC¹⁹(25.3 mg, 123 lmol). After stirring for 45 min

at rt, the reaction was complete. Dilution with AcOEt,

washing with 5% aqueous NH₄H₂PO₄and with satu-

rated aqueous NaCl, drying (Na₂SO₄) and evaporation

aﬀorded a crude product that was chromatographed

(PE/AcOEt 3:7 to 2:8) to give pure 31 as a white solid

(23.4 mg, 58% from 30). R_f0.17 (PE/AcOEt 1:1 + 2%

AcOH). R_fof intermediate acid (same eluent): 0.32.

Found: C, 67.6; H, 6.8, N, 10.0. C₂₃H₂₇N₃O₄requires

C, 67.46; H, 6.65; N, 10.26%. ¹H NMR d 7.37–7.12

[10H, m, aromatics]; 7.06 [1H, br t, NH, J = 5.8]; 6.05

[1H, br t, NHBn, J = not measurable]; 4.51 [1H, d, H-

3, J = 5.6]; 4.43 [2H, d, CH₂Ph, J = 6.0]; 4.15 [1H, dt,

H-4, J_d= 7.6 J_t= 5.6]; 3.59 [3H, s, OCH₃]; 3.59 [2H,

q, NHCH₂, J = 6.2]; 2.75 [2H, t, CH₂Ph, J = 8.1]; 2.47

[2H, t, CH₂CONH, J = 6.2]; 2.35–2.10 [2H, m,

CH₂CH₂Ph].

3514

L. Banﬁ et al. / Bioorg. Med. Chem. 16 (2008) 3501–3518

1

This ester (78.8 mg, 248 lmol) was dissolved (by warm-

ing) in MeOH (2 mL) and treated with a 3 M solution of

NaOH in H₂O (1 mL, 3.0 mmol). The solution was stir-

red overnight. Most MeOH was evaporated, and the

residue taken up in AcOEt. The organic phase was

washed with 5% aqueous NH₄H₂PO₄(further acidiﬁed

with HCl so that pH 3). The aqueous phase was ex-

tracted ﬁve times with AcOEt. Evaporation aﬀorded

the crude acid 37 (64.3 mg) that was used as such for

the subsequent reactions.

H, 6.04; N, 7.00%. H NMR (300 MHz) d 6.80 [1H, t,

NH, J = 6.1]; 6.04 [1H, t, CHOAc, J = 2.0]; 5.98 and

5.92 [2H, AB syst. (with small long range couplings),

CH@CH, J = 9.8]; 4.30 [1H, t, CHNC@O, J = 2.5];

3.85 [1H, ddd, CHC@O, J = 3.0, 3.9, 12.6]; 3.57–3.46

[2H, m, CH₂N]; 2.93 and 2.70 [2H, AB part of an ABXY

system, CH₂–C„C, J_AB= 17.8, J_AX= 12.6, J_BX= 3.9,

t

J_AY= 0, J_BY= 1.8]; 2.48 [2H, t, CH₂CO₂Bu, J = 6.1];

2.10 [3H, s, CH₃CO]; 1.46 [9H, s, C(CH₃)₃]. ¹³C NMR

(75 MHz): d 170.9, 169.1, 166.5, 149.9 [C@O]; 126.5,

122.6 [CH@CH]; 98.7, 93.7, 87.6, 84.4 [C„C]; 81.4

[C(CH₃)₃]; 60.9, 59.5, 52.0 [CHOAc, CHN, CHCO];

35.6, 35.5 [NCH₂CH₂CO₂tBu]; 28.1 [C(CH₃)₃]; 20.7

[CH₃C@O]; 19.1 [CH₂C„]. IR: m_max3384, 2963, 1773,

1712, 1506, 1368, 1335, 1297, 1258, 1192, 1151,

4.25. Compound 35

A solution of azetidinyl urea 34 (55.0 mg, 123 lmol) in

dry CH₂Cl₂(500 lL) was treated with triﬂuoroacetic

acid (500 lL). After stirring for 90 min at rt, the solvent

was rapidly evaporated, taken up with CH₂Cl₂/n-hep-

tane and evaporated again (this process was repeated

three times). It was ﬁnally taken up in CH₂Cl₂

(1.0 mL), and treated with Py-BOP¹⁹(46 mg, 104 lmol),

crude carboxylic acid 37 (24.0 mg, 80 lmol), and Et₃N

(40 lL, 287 lmol). The initial suspension (due to poor

solubility of 37) soon became a yellowish solution. This

solution was stirred for 24 h at rt. Then it was diluted

with AcOEt and washed with 5% aqueous NaHCO₃

and with saturated aqueous NaCl. Drying (Na₂SO₄),

evaporation and chromatography (Et₂O/acetone 4:6)

aﬀorded pure 35 (21.8 mg, 44%). R_f0.41 (Et₂O/acetone

1103 cm^ꢀ1

.

4.27. (1R^*,9R^*,10S^*)(Z)-9-Acetoxy-11-[(2-(benzylcarba-

moyl)ethyl)carbamoyl]-11-azabicyclo[8.2.0]dodec-5-ene-

3,7-diyn-12-one (39)

A solution of lactenediyne 38 (35.3 mg, 88 lmol) in dry

CH₂Cl₂(500 lL) was treated with triﬂuoroacetic acid

(250 lL). After stirring for 30 min at rt, the solvent

was rapidly evaporated, taken up with CH₂Cl₂and

evaporated again (this process was repeated three

times). It was ﬁnally taken up in CH₂Cl₂(500 lL), trea-

ted with benzylamine (12 lL, 110 lmol) and with

DCC¹⁹(22.3 mg, 108 lmol). After stirring for 100 min

at rt, the reaction was complete. Dilution with CH₂Cl₂,

washing with 5% aqueous NH₄H₂PO₄and with satu-

rated aqueous NaCl, drying (Na₂SO₄) and evaporation

aﬀorded a crude product that was chromatographed

(ﬁrst with Et₂O/AcOEt 1:2 and then with CH₂Cl₂/

AcOEt 1:1) to give pure 39 as a white solid (19.2 mg,

50% from 38). R_f0.28 (CH₂Cl₂/AcOEt 1:1). Found: C,

66.25; H, 5.4; N, 9.5. C₂₄H₂₃N₃O₅requires C, 66.50;

1

4:6). H NMR (d₆-DMSO, 300 MHz): d 9.84 and 9.82

[2H, 2 s, NH]; 7.32–7.11 [7H, m, aromatics]; 7.00 [1H,

t, NH urea, J = 5.7]; 6.85 [1H, s, CH pyrrole]; 6.39

[1H, s, CH pyrrole]; 4.73 [1H, d, H-3, J = 5.7]; 4.14

[1H, dt, H-4, J_d= 7.2, J_t= 5.6]; 3.82, 3.61 [2· 3H, 2 s,

CH₃N pyrrole]; 3.50–3.38 [2H, m, CH₂N]; 3.47 [3H, s,

OCH₃]; 3.21–3.06 [2H, m, CH₂N]; 3.01 [3H, br s,

CH₃N tertiary amide]; 2.67 [2 H, t, CH₂Ph, J = 7.9];

2.06–1.80 [2H, m, CH₂CH₂Ph]; 1.97 [3H, CH₃C@O];

1.62–1.40 [4H, m, CH₂]; 1.31–1.15 [2H, m, CH₂]. ¹³C

NMR (d₆-DMSO, 75 MHz): d 166.6, 166.4, 162.8,

158.2, 150.0 [C@O]; 141.4 [quat. of Ph]; 128.3 (·2),

128.1 (·2), 125.8 [CH of Ph]; 122.7, 122.6, 122.1, 121.9

[quat. of pyrrole]; 117.9, 115.9, 103.6 (·2) [CH of pyr-

role]; 82.4 [C-3]; 59.1 [OCH₃]; 56.2 [C-4]; 48.0 (very

broad) [CH₂N]; 39.04 [CH₂N]; 36.0, 34.9 [CH₃N of pyr-

roles]; 30.3 and 29.5 [CH₃N of tertiary amide (2 con-

formers)]; 31.4, 29.8 [CH₂CH₂Ph]; 28.9, 26.7 (broad),

23.3 [CH₂CH₂CH₂]; 23.0 [CH₃C@O].

1

H, 5.35; N, 9.69%. H NMR (300 MHz): d 7.38–7.21

[5H, m, aromatics]; 6.96 [1H, t, NH, J = 6.0]; 6.05 [1H,

t, CHOAc, J = 1.9]; 6.07–6.04 [1H, br s, NHBn]; 5.99

and 5.91 [2H, AB system, CH@CH, J = 10.0]; 4.45

[2H, d, CH₂Ph, J = 5.7]; 4.27 [1H, t, CHNC@O,

J = 2.7]; 3.84 [1H, ddd, CHC@O, J = 3.0, 3.9, 12.6];

3.59 [2H, q, NHCH₂CH₂, J = 6.0]; 2.93 and 2.69 [2H,

AB part of an ABXY system, CH₂–C„C, J_AB= 17.7,

J_AX= 12.9, J_BX= 3.9, J_AY= 0, J_BY= 1.8]; 2.57–2.40 [2

H, m, CH₂CONH]; 2.03 [3H, s, CH₃CO]. IR: m_max

:

3438, 3382, 3003, 2928, 2854, 1776, 1755, 1702, 1663,

4.26. (1R^*,9R^*,10S^*)(Z)-9-Acetoxy-11-[(2-(tert-butoxy-

carbonyl)ethyl)carbamoyl]-11-azabicyclo[8.2.0]dodec-5-

ene-3,7-diyn-12-one (38)

1509, 1369, 1336, 1298, 1191, 1141, 1102 cm^ꢀ1

.

4.28. Conjugated lactenediyne (40)

A solution of azetidinone 6 (50.2 mg, 219 lmol) in dry

CH₂Cl₂(1.0 mL) was treated with a 0.45 M solution of

isocyanate 2, prepared as above described (see the syn-

thesis of 30) (980 lL, 441 mol), DMAP¹⁹(16.4 mg,

134 lmol) and DBU¹⁹(32 lL, 214 lmol). After stirring

for 16 h at rt the mixture was diluted with AcOEt and

washed with 5% aqueous NH₄H₂PO₄and aqueous satu-

rated NaCl. Drying (Na₂SO₄), evaporation and chroma-

tography (PE/Et₂O 50:50) gave pure 38 as a colourless

oil (70.6 mg, 80%). R_f0.26 (PE/Et₂O 40:60). Found: C,

63.05; H, 6.1, N, 6.95. C₂₁H₂₄N₂O₆requires C, 62.99;

Amine 45 was prepared according to the literature.³⁷

However the last step (hydrogenation of the correspond-

ing nitro derivative) was carried out in DMF. After ﬁl-

tration of the catalyst, the DMF solution of 45 was

directly used for the next coupling. Starting from

60.0 mg of nitro derivative, we obtained a solution of

crude 45 (theoric 172 lmol) in 8 mL of DMF. Mean-

while, a solution of lactenediyne 38 (30.8 mg, 77 lmol)

in dry CH₂Cl₂(500 lL) was treated with triﬂuoroacetic

acid (500 lL). After stirring for 45 min at rt, the solvent

was rapidly evaporated, taken up with CH₂Cl₂and

L. Banﬁ et al. / Bioorg. Med. Chem. 16 (2008) 3501–3518

3515

evaporated again (this process was repeated three times).

It was ﬁnally taken up in CH₂Cl₂(500 lL), and added to

the DMF solution of 45. Et₃N (40 lL, 272 lmol) and

Py-BOP¹⁹(45 mg, 102 lmol) were added. The resulting

solution was stirred for 16 h at rt. After evaporation

of DMF at 1 mbar, the residue was taken up in CH₂Cl₂,

ﬁltered on paper in order to remove traces of hydroge-

nation catalyst, washed with 5% aqueous NH₄H₂PO₄

and with saturated aqueous NaCl. Drying (Na₂SO₄)

and evaporation aﬀorded a crude product that was chro-

matographed (AcOEt/acetone 9:1 + 1% MeOH) to give

pure 40 as a solid (39.8 mg, 80% from 38). R_f0.46

(AcOEt/acetone 9:1). Found: C, 61.5; H, 5.85; N,

14.95. C₃₃H₃₇N₇O₇requires C, 61.58; H, 5.79; N,

contrary, due to the distance between the stereogenic

centres, the signals of the two 1:1 diastereoisomers

are nearly always superimposed. The signals that can

1

be seen clearly distinct at H NMR are: H-10⁰of the

Z oxime, and CH₃C@O of both geometric isomers.

¹H NMR (300 MHz.) (the main numbers are used for

camptothecin, the primed number for the lac-

tenediyne): d 9.05 [0.62H, s, CH@N (E)]; 8.23–8.17

[1.62H, m, H-9 (E) + H-12 (E + Z)]; 8.10 [0.38H, s,

CH@N (Z)]; 7.95 [0.38H, d, J = 8.1, H-9 (Z)]; 7.83

[1H, t, J = 8.0, H-10]; 7.73–7.67 [1H, m, H-11]; 7.68

[0.38H, s, H-14 (Z)]; 7.59 [0.62H, s, H-14 (E)]; 6.95

[0.62H, t, NH (E), J = 6.0]; 6.95 [0.38H, t, NH (Z),

J = 6.0]; 6.61 [0.38H, br t, NH (Z)]; 6.47 [0.62H, br

s, NH (E)]; 6.20–5.85 [3H, m, H-5⁰, H-6⁰, H-9⁰]; 5.72

[1H, d, H-17, J = 16.5]; 5.37 [1.24H, s, H-5 (E)]; 5.32

[0.76H, s, [H-5 (Z)]; 5.28–5.19 [1H, m, H-17]; 4.48

[1.24H, t, OCH₂CH₂N (E), J = 5.4]; 4.40 [0.76H, t,

OCH₂CH₂N (Z), J = 4.5]; 4.27 [0.62H, t, H-10⁰(E),

J = 2.7]; 4.23 [0.17H, t, H-10⁰(Z) (1 diast.), J = 2.7];

4.22 [0.17H, t, H-10⁰(Z) (1 diast.), J = 2.7]; 4.00

[0.38H, br s, OH (Z)]; 3.94 [0.62H, br s, OH (E)];

3.88–3.53 [4H, m, CH₂N]; 3.52–3.42 [1H, m, H-1⁰];

2.96–2.82 [1H, m, H-2⁰]; 2.74–2.58 [1H, m, H-2⁰]; 2.53

[1.24H, t, CH₂CH₂CON (E), J = 6.1]; 2.43 [0.76H, t,

CH₂CH₂CON (Z), J = 6.1]; 2.10 and 2.09 [1.86H, 2 s,

CH₃CO (E) (2 diast.)]; 2.05 and 2.03 [1.14H, 2 s,

CH₃CO (Z) (2 diast.)]; 1.98–1.80 [2H, m, H-19]; 1.03

[3H, t, H-18, J = 7.5].

1

15.23%. H NMR (300 MHz): d 8.58 [1H, s, NH]; 7.78

[1H, s, NH]; 7.25 [1H, d, CH pyrrole, J = 1.5]; 7.16

[1H, t, urea NHCH₂, J = 5.9]; 6.88 [1H, d, CH pyrrole,

J = 1.2]; 6.70 [1H, d, CH pyrrole, J = 1.5]; 6.67 [1H, d,

CH pyrrole, J = 1.8]; 6.48 [1H, br t, NHBu]; 6.22 [1H,

t, CHOAc, J = 1.9]; 5.98 and 5.88 [2H, ABX system,

CH@CH, J_AB= 9.8, J_AX= 1.5, J_BX= 0]; 4.34 [1H, t,

CHNC@O, J = 2.7]; 3.90 [6H, s, CH₃N]; 3.85 [1H,

ddd, CHC@O, J = 3.0, 5.1, 11.7]; 3.60–3.43 [2H, m,

CH₂NH]; 3.36 [2H, q, NHCH₂CH₂, J = 6.7]; 2.95–2.75

[2H, m, CH₂–C„C]; 2.62–2.40 [2H, m, CH₂CONH];

2.08 [3H, s, CH₃CO]; 1.63–1.50 [2H, m, CH₂]; 1.39

[2H, hexuplet, CH₃CH₂, J = 7.5]; 0.94 [3H, t, CH₃CH₂,

J = 7.2]. ¹³C (75 MHz): d 169.9, 168.1, 166.8, 162.0,

159.0, 151.0 [C@O]; 126.8, 122.2 [CH@CH]; 123.7,

123.5, 121.7, 121.5 [quat. of pyrroles]; 118.5, 118.4,

103.8, 103.1 [CH of pyrroles]; 99.0, 93.3, 88.0, 84.4

[C„C]; 60.8, 59.7, 52.0 [CHOAc, CHN, CHCO]; 39.1,

¹³C NMR (75 MHz.): d 173.8 [(E)]; 173.7 [(Z)]; 171.4

(Z), 171.1 (E), 169.23 (E), 169.19 (Z), 166.4 (E), 166.3

(Z) [4 C@O]; 157.5 (Z), 157.4 (E) [C@O pyridone];

152.3 (E), 152.2 (Z), 150.3 (Z), 150.2 (E), 150.03 (E),

149.99 (Z), 149.5 (E), 148.8 (Z), 146.0 (E + Z) [1 C@O

+4 quat. aromatics]; 144.5 [CH@N (E)]; 141.7 [CH@N

(Z)]; 131.7 (Z), 130.9 (E), 127.4 (Z), 125.9 (E), 125.3

(E), 125.1 (Z), 119. 1 (Z), 118.8 (E) [4 quat. aromatics];

130.7 (E + Z), 130.5 (E + Z), 128.7 (Z), 128.6 (E), 124.2

(Z), 122.8 (E), 98.3 (Z), 98.0 (E) [5 aromatic CH]; 126.5

and 122.4 [C-5⁰and C-6⁰(E + Z)]; 98.7, 93.5, 87.7, 84.4

[C-3⁰, C-4⁰, C-7⁰, C-8⁰]; 74.7 (Z), 74.2 (E) [CH₂ON]; 72.7

(E + Z) [C-20]; 66.3 (E), 66.2 (Z) [C-17]; 60.8, 59.5 [C-9⁰,

C-10⁰]; 52.40 (E), 51.39 (Z) [CH₂N]; 51.9 (E + Z) [C-1⁰];

39.2 (Z), 38.8 (E), 36.1 (E + Z), 35.8 (E), 35.4 (Z) [2

CH₂N+CH₂CON]; 31.6 [C-19]; 20.8 [CH₃CO]; 19.0 [C-

2⁰]; 7.8 [C-18].

t

36.1, 35.8 [NCH₂and CH₂CO₂Bu]; 36.6, 36.5 [CH₃N];

31.9, 20.2 [other CH₂]; 20.8 [C₃C@O]; 18.8 [CH₂C„];

13.8 [CH₃CH₂]. IR: m_max: 3438, 3358, 2956, 2930, 1778,

1754, 1654, 1581, 1516, 1464, 1432, 1403, 1337, 1298,

1249, 1191, 1141, 1100 cm^ꢀ1

.

4.29. Conjugated lactenediyne (41)

A solution of lactenediyne 38 (34.8 mg, 87 lmol) in dry

CH₂Cl₂(500 lL) was treated with triﬂuoroacetic acid

(250 lL). After stirring for 80 min at rt, the solvent

was rapidly evaporated, taken up with CH₂Cl₂and

evaporated again (this process was repeated three

times). It was ﬁnally taken up in dry CH₂Cl₂(3.5 mL),

and treated with compound 46 (20 mg, 46 lmol), Et₃N

(43 lL, 308 lmol) and Py-BOP¹⁹(50 mg, 113 lmol)

were added. The resulting solution was stirred for 20 h

at rt. After dilution with CH₂Cl₂, the solution was

washed with H₂O. Drying (Na₂SO₄) and evaporation

aﬀorded a crude product that was chromatographed

(CH₂Cl₂/acetone 1:1 + 1% MeOH) to give pure 41 as a

yellow ﬂuorescent solid (31.1 mg, 90% from 46). R_f

0.57 (CHCl₃/MeOH 9:1). Found: C, 65.15; H, 5.1; N,

10.95. C₄₁H₃₈N₆O₉requires C, 64.90; H, 5.05; N,

11.08%.

4.30. (1R^*,9R^*,10S^*)(Z)-9-Acetoxy-11-[(5-(N-(tert-butox-

ycarbonyl)-N-methylamino)pentyl)carbamoyl]-11-azabi-

cyclo[8.2.0]dodec-5-ene-3,7-diyn-12-one (42)

It was prepared in 96% yield from 6 and 33 using the

same procedure used for synthesizing 34. R_f0.12 (PE/

Et₂O 6:4). Found: C, 63.7; H, 7.1; N, 8.8. C₂₅H₃₃N₃O₆

1

requires C, 63.68; H, 7.05; N, 8.91%. H NMR: d 6.38

[1H, t, NH, J = 5.7]; 6.05 [1H, t, CHOAc, J = 2.0];

5.99 and 5.93 [2H, AB syst. (with small long range cou-

plings), CH@CH, J = 10.0]; 4.31 [1H, t, CHNC@O,

J = 2.6]; 3.85 [1H, ddd, CHC@O, J = 3.0, 4.0, 12.4];

3.37–3.10 [4H, m, CH₂N]; 2.94 and 2.72 [2H, AB part

Since we started from enantiomerically pure 46 and

racemic 38, this product is obviously a 1:1 diastereoiso-

meric mixture. Moreover, NMR shows the presence of

two geometric isomers in a 62:38 E:Z ratio. In this lat-

ter case the signals are often distinct at NMR. On the

of an ABXY system, CH₂–C„C, J_AB= 17.9, J_AX

12.7, J_BX= 3.8, J_AY0, J_BY= 1.6]; 2.83 [3H, s, NCH₃];

=

3516

L. Banﬁ et al. / Bioorg. Med. Chem. 16 (2008) 3501–3518

2.11 [3H, s, CH₃CO]; 1.67–1.45 [4H, m, CH₂]; 1.45 [9H,

s, C(CH₃)₃]; 1.38–1.23 [2H, m, CH₂].

4.33. Incubation with plasmid DNA and analysis via gel

electrophoresis

4.31. Conjugated lactenediyne (43)

Working buﬀer: it was prepared dissolving 4.84 g of

TRIS, 584 mg of EDTA and 1.142 mL of acetic acid

in 1 L of distilled water. Loading buﬀer: it was prepared

from 75 mg of Ficoll, 500 lL of working buﬀer and

500 lL of a solution containing 0.25% bromophenol

blue and 0.25% xylene cyanol in, pH 8.3, TRIS–bo-

rate–EDTA buﬀer.

It was prepared in 68% yield from 42 and carboxylic

acid 37 following the same methodology described

above for 35. R_f0.23 (CH₂Cl₂/acetone 1:1). Found: C,

62.1; H, 6.0; N, 14.7. C₃₄H₃₉N₇O₇requires C, 62.09;

1

H, 5.98; N, 14.91%. H NMR (300 MHz): d 8.18 [2H,

br s, NH of pyrrole]; 7.27 [1H, s, CH pyrrole (covered

by CHCl₃)]; 7.15 [1H, d, CH pyrrole, J = 1.8]; 6.48–

6.40 [2H, m, NH and CH pyrrole]; 6.28 [1H, s, CH pyr-

role]; 6.05 [1H, t, CHOAc, J = 2.1]; 5.99 and 5.90 [2H,

AB syst. (with small long range couplings), CH@CH,

J = 9.6]; 4.24 [1H, t, CHNC@O, J = 2.6]; 3.89 [3H, s,

NCH₃]; 3.82 [1H, ddd, CHC@O, J = 3.0, 3.9, 12.3];

3.69 [3H, s, NCH₃]; 3.60–3.38 [2H, m, CH₂N]; 3.38–

3.18 [2H, m, CH₂N]; 3.07 [3H, s, NCH₃]; 2.88 and

2.71 [2H, AB part of an ABXY system, CH₂–C„C,

J_AB= 17.8, J_AX= 12.7, J_BX= 3.8, J_AY= 0, J_BY= 1.8];

2.11 and 2.10 [2· 3H, 2 s, CH₃CO]; 1.72–1.50 [4H, m,

CH₂]; 1.40–1.27 [2H, m, CH₂]. ¹³C NMR (75 MHz): d

169.3, 167.6, 166.9, 164.0, 159.0, 150.3 [C@O]; 126.6,

122.3 [CH@CH]; 123.2, 123.1, 121.6, 121.4 [aromatic

quat.]; 119.4, 117.1, 103.9 (broad), 103.3 [aromatic

CH]; 98.8, 93.4, 87.8, 84.4 [C„C]; 60.8, 59.6 [CHN

and CHOAc]; 52.0 [CHC@O]; 49.5 (very broad)

[CH₂N]; 39.3 [CH₂N]; 36.7, 35.6 [pyrrole CH₃N]; 30.9

[CH₃N]; 29.1, 27.1, 23.4 [other CH₂]; 23.5, 20.7

[CH₃C@O]; 18.9 [CH₂C„C]. IR: m_max3439, 3375,

2989, 2938, 1770, 1752, 1697, 1658, 1609, 1528, 1436,

The enediyne derivatives were dissolved in DMSO in or-

der to have a 10 mM solution. These parent solutions

were diluted with DMSO to the desired concentrations

just before incubation.

Plasmid pBR 322 (Fermentas, 90% in form I, 500 lg/

mL) was diluted 1:10 with a, pH 7.5, Tris (40 mM)/

EDTA (4 mM) buﬀer (prepared using molecular biol-

ogy water) in order to have a 50 lg/mL, 75 lM/bp

concentration. Eighteen microliters of this solution

was treated with 2 lL of the appropriately diluted

DMSO lactenediyne solution. For example, in order

to have

a

ﬁnal lactenediyne concentration of

100 lM, 2 lL of a 1 mM solution was added. The

resulting mixtures were incubated at 37 ꢁC for 24 h.

At the end of this period, the solutions were treated

with 15 lL of loading buﬀer and analysed on agarose

gel (prepared from 300 mg agarose, 32 mL working

buﬀer and 1.5 lg ethidium bromide), with the submar-

ine methodology. The gel was immersed in 325 mL of

working buﬀer containing 165 lg of ethidium bromide

and eluted at 80 mV. After elution, the gel was ob-

served at 302 nm (transilluminator), and photo-

graphed. The intensity ratio between form I and

II + III was determined, without corrections (apart

from substraction of form II already present in the

control), by densitometry. The II/III ratio was deter-

mined after incubation with a 100 lM concentration

of the enediyne.

1399, 1336, 1295, 1253, 1098 cm^ꢀ1

.

4.32. Conjugated lactenediyne (44)

It was prepared in 90% yield from 42 and 1-naphthylace-

tic acid following the same methodology described

above for 35. R_f0.15 (PE/AcOEt 4:6). Found: C,

70.95; H, 6.15; N, 7.65. C₃₂H₃₃N₃O₅requires C, C,

1

71.22; H, 6.16; N, 7.79%. H NMR: d 8.02–7.91 [1H,

m]; 7.90–7.73 [2H, m]; 7.60–7.28 [4H, m]; 6.43–6.28

[1H, m, NH]; 6.05 [1H, t, CHOAc, J = 1.8]; 5.99 and

5.92 [2H, AB syst., CH@CH, J = 9.9]; 4.30 [1H, t,

CHNC@O, J = 2.4]; 4.18–4.05 [2H, m, CH₂-naphthyl];

3.85 [1H, dt, CHC@O, J_d= 12.4, J_t= 3.3]; 3.48–3.35

[2H, m, CH₂N]; 3.33–3.10 [2H, m, CH₂N]; 2.99 and

2.97 [3H, 2 s, NCH₃(2 conformers)]; 2.92 and 2.70

[2H, AB part of an ABXY system, CH₂–C„C,

J_AB= 17.9, J_AX= 12.7, J_BX= 3.8, J_AY= 0, J_BY= 1.6];

2.10 and 2.07 [3H, 2 s, CH₃CO (2 conformers)]; 1.67–

1.10 [6H, m, CH₂]. ¹³C NMR (several signals are splitted

due to conformers at the tertiary amide): d 171.0, 166.85,

166.76, 150.1 [4 C@O]; 133.8, 132.1, 131.4 [aromatic

quat.]; 128.8, 127.6, 126.4, 126.3, 125.8, 125.5, 123.5

[aromatic CH]; 126.5, 122.5 [CH@CH]; 98.7, 96.6,

93.8, 93.7, 87.7, 87.6, 84.4 [C„C]; 60.9, 59.5 [CHOAc

and CHN]; 52.0 [CHC@O]; 50.3, 47.8, 39.6, 39.5, 39.0,

38.4 [CH₂]; 35.9, 33.6 [CH₃N (2 conf.)]; 29.41, 29.38,

28.0, 26.7, 23.9, 23.8 [other CH₂]; 20.7 [CH₃C@O];

19.04 [CH₂C„C]. IR: m_max3380, 2997, 2931, 2859,

1771, 1705, 1633, 1518, 1397, 1370, 1335, 1294, 1262,

4.34. Cytotoxicity tests on lactenediyne (7)

These tests were carried out by NCI, Bethesda, Maryland

(USA). The cell lines listed in Table 2 were treated for 48 h

with various concentrations of 7. The control tests have

been carried out under the same conditions, but without

addition of 7. The cell growth has been estimated through

the Sulforhodamine B test⁴³followed by colorimetric

determination. The percent growth (PG) has been then

calculated through the following equations: (a)

PG = 100 · (OD_testꢀ OD_zero)/(OD_controlꢀ OD_zero

)

if

ꢀ

(OD_testꢀ OD_zero) P 0.

(b)

PG = 100 · (OD_test

OD_zero)/(OD_zero) if (OD_testꢀ OD_zero) < 0, where OD_test

is the absorbance measured after 48 h in the presence of

7, OD_zerois the absorbance measured at the start of the

experiment and OD_controlis the absorbance measured

on the cells after 48 h in the absence of 7. Plotting the

log₁₀of the molar concentrations of 7 versus PG, the fol-

lowing values can be determined: GI₅₀: concentration at

which PG = 50. TGI: concentration at which PG = 0.

LC₅₀: concentration at which PG = ꢀ50.

1141, 1102 cm^ꢀ1

.

L. Banﬁ et al. / Bioorg. Med. Chem. 16 (2008) 3501–3518

3517

9. Banﬁ, L.; Basso, A.; Guanti, G. Tetrahedron 1997, 53,

3249.

4.35. In vivo anti-cancer activity tests on lactenediyne (7)

These tests were carried out in a ‘hollow ﬁbre assay’⁴⁴by

NCI, Bethesda, Maryland (USA). Twelve diﬀerent cell

lines related to human tumours (NCI-H23, NCI-H522,

MDA-MB-231, MDA-MB-435, SW-620, COLO 205,

LOX IMVI, UACC-62, OVCAR-3, OVCAR-5, U251,

SF-295) have been implanted (both intraperitoneally

and subcutaneously) in aythmic nude mice. After 3–4

days, the mice have been then subjected to four intra-

peritoneal treatments (once a day for 4 days) with com-

pound 7. After collection of the ﬁbres, they have been

examined by the MTT test for evaluation of the cellular

mass, which has been compared to that of analogues

tests where the mice had been treated with the solvent

only. In 3 instances out of 12 (intraperitoneal implant)

and in 1 instance out of 12 (subcutaneous implant) the

test has shown more than 50% reduction of the cellular

mass compared to the control test.

10. Banﬁ, L.; Basso, A.; Guanti, G. Eur. J. Org. Chem. 2000,

939.

11. Banﬁ, L.; Guanti, G. Tetrahedron Lett. 2002, 43, 7427.

12. Banﬁ, L.; Guanti, G. Eur. J. Org. Chem. 2002, 3745.

13. Banﬁ, L.; Guanti, G.; Rasparini, M. Eur. J. Org. Chem.

2003, 1319.

14. Floyd, D. M.; Fritz, A. W.; Cimarusti, C. M. J. Org.

Chem. 1982, 47, 176.

15. Woulfe, S. R.; Miller, M. J. J. Med. Chem. 1985, 28, 1447.

16. Farina, V.; Hauck, S. I.; Walker, D. G. Synlett 1992, 761;

Baldwin, J. E.; Adlington, R. M.; Gollins, D. W.;

Godfrey, C. R. A. Tetrahedron 1995, 51, 5169; Ojima, I.;

Sun, C. M.; Zucco, M.; Park, Y. H.; Duclos, O.; Kuduk,

S. Tetrahedron Lett. 1993, 34, 4149.

´

17. Ogilvie, W. W.; Yoakim, C.; Do, F.; Hache, B.; Lagace,

L.; Naud, J.; O’Meara, J. A.; Deziel, R. Bioorg. Med.

´

Chem. 1999, 7, 1521; Finke, P. E.; Shah, S. K.; Fletcher,

D. S.; Ashe, B. M.; Brause, K. A.; Chandler, G. O.;

Dellea, P. S.; Hand, K. M.; Maycock, A. L.; Osinga, D.;

Underwood, D. J.; Weston, H.; Davies, P.; Doherty, J. B.

J. Med. Chem. 1995, 38, 2449; Hagmann, W. K.;

Kissinger, A. L.; Shah, S. K.; Finke, P. E.; Dorn, C. P.

J.; Brause, K. A.; Ashe, B. M.; Weston, H.; Maycock, A.

L.; Knight, W. B.; Dellea, P. S.; Fletcher, D. S.; Hand, K.

M.; Osinga, D.; Davies, P.; Doherty, J. B. J. Med. Chem.

1993, 36, 771.

Acknowledgments

We thank Miss Roberta Miroglio for her very valuable

collaboration to this work, Prof. Lucio Merlini (Univer-

sity of Milano) and Prof. Sergio Penco (Sigma-Tau) for

their helpful advices, Dr. Claudio Pisano (Sigma-Tau)

and Prof. Franco Zunino (Istituto Nazionale per lo Stu-

dio e la Cura dei Tumori, Milano) for further (unpub-

lished) biological tests on some of the here reported

compounds. This work has been ﬁnancially assisted by

C.N.R., MUR (PRIN 2000), Sigma-Tau, and Fonda-

zione San Paolo.

18. Shah, S. K.; Dorn, C. P. J.; Finke, P. E.; Hale, J. J.;

Hagmann, W. K.; Brause, K. A.; Chandler, G. O.;

Kissinger, A. L.; Ashe, B. M.; Weston, H.; Knight, W.

B.; Maycock, A. L.; Dellea, P. S.; Fletcher, D. S.; Hand,

K. M.; Mumford, R. A.; Underwood, D. J.; Doherty, J. B.

J. Med. Chem. 1992, 35, 3745.

19. Abbreviations used in the text and in the schemes: Boc-ON:

2-(tert-butoxycarbonyloxyimino)-2-phenyl-acetonitrile.

CAN: cerium ammonium nitrate. DBU: 1,8-diazabicy-

clo[5.4.0]undec-7-ene. DCC: dicyclohexyl carbodiimide.

DMAP: 4-dimethylaminopyridine. EDC: N-ethyl-N⁰-

(dimethylaminopropyl)carbodiimide. EDTA: ethylenedia-

mino-tetracetic acid. HOBT: N-hydroxybenzotriazole.

NIS: N-iodosuccinimide. Py-BOP: (benzotriazolyloxy) (tri-

pyrrolidino)-phosphonium hexaﬂuorophosphate. TRIS:

tris(hydroxymethyl)aminomethane.

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Synthesis and DNA-cleaving activity of lactenediynes conjugated with DNA-complexing moieties

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