Synthesis and biological evaluation of B-ring analogues of (-)-rhazinilam

Bioorganic & Medicinal Chemistry 14 (2006) 2314–2332

Synthesis and biological evaluation of B-ring analogues of

(ꢀ)-rhazinilam

´

´ `

`

Anne Decor, Barbara Monse, Marie-Therese Martin, Angele Chiaroni, Sylviane Thoret,

*

´

Daniel Guenard, Franc¸oise Gueritte and Olivier Baudoin

Institut de Chimie des Substances Naturelles, CNRS, avenue de la Terrasse, 91198 Gif-sur-Yvette cedex, France

Received 27 May 2005; revised 3 November 2005; accepted 8 November 2005

Available online 28 November 2005

Abstract—Three macrocyclic analogues of rhazinilam 1 having a 11- or 12-membered B-ring with an endocyclic carbamate group or

an amino-acid residue were synthesized from the natural product. These analogues 3 and 4 displayed a very low activity on tubulin.

Thirty N-1 and C-16 substituted analogues of rhazinilam were also synthesized regioselectively from rhazinilam. Stereochemical

analyses showed that N-1 and C-16a analogues have the same conformation as rhazinilam, whereas C-16b analogues adopt a dif-

ferent conformation for rings B and D. All N-1 and C-16 analogues were less active than rhazinilam on tubulin, though analogues

5a, 6aa, 6ba, and 6f having the less bulky substituents retained close aﬃnities. A few analogues either active (like 6f) or inactive (like

5o) on tubulin showed signiﬁcant inhibition of the growth of KB cancer cells.

16

1. Introduction

O

Et

O

H

O

20

D

¹N

B

H

N

In the past decades, the mitotic spindle has been an

attractive target for the development of anticancer

agents. Small molecules disturbing the assembly or

disassembly of microtubules, in particular natural com-

pounds, have shown a remarkable ability to impair cell

proliferation and some of them, as, for instance vinca

alkaloids and taxoids, are successfully used in cancer

therapy.¹The search for antimitotic compounds having

a novel mode of tubulin binding is still highly relevant,

in order to solve issues of selectivity and drug resistance.

(ꢀ)-Rhazinilam 1 is a natural compound isolated from

various Apocynaceae, whose tetracyclic structure pos-

sesses an axially chiral phenyl–pyrrole subunit bridged

by a nine-membered lactam ring.²It was found to have

unique antimitotic properties, with in vitro inhibition of

both microtubule assembly and disassembly, and the

formation of abnormal tubulin spirals.³As a conse-

quence of these tubulin-binding properties, rhazinilam

showed in vitro cytotoxicity toward various cancer cell

lines in the low micromolar range, however no activity

was found in vivo.

N

B

C

A

C

1

2

carbamate,

amino-acid

R²

O

H

R¹

D

N

B

C

A

5-6

3-4

Driven by the original in vitro properties of rhazinilam,

several groups have undertaken structure–activity rela-

tionships (SAR) aimed at increasing the in vitro activity

and solving the problem of in vivo inactivity. In the ab-

sence of structural data on the rhazinilam binding site in

tubulin, a number of observations were made through

the testing of analogues obtained by semi-synthesis^4,5

or total synthesis.^6–10In particular, it was shown that

the introduction of substituents on rings A, C, and D

has a negative eﬀect on the activity, suggesting a narrow

tubulin binding pocket. In addition, these data suggest

that the conservation of the boat–chair conformation

of B-ring, imposed by the cis-amide group and the qua-

ternary C-20 carbon, is important for preserving the

Keywords: Rhazinilam; Tubulin; Cytotoxicity; Structure–activity

relationships.

*

Corresponding author. Tel.: +33 169823038; fax: +33 169077247;

e-mail: baudoin@icsn.cnrs-gif.fr

doi:10.1016/j.bmc.2005.11.011

´

A. Decor et al. / Bioorg. Med. Chem. 14 (2006) 2314–2332

2315

18

aﬃnity for tubulin. Indeed, analogues with a secondary

N

16

19

15

14

or tertiary C-20 as well as analogues with a smaller B-

ring were less active than rhazinilam. Biphenyl-carba-

mate 2 was found to be the most active analogue of

rhazinilam so far, with a 2-fold activity on tubulin com-

pared to rhazinilam, however its in vitro cytotoxicity is

still in the low micromolar range, very close to that of

the natural compound.^7b,10In three dimensions, com-

pound 2 is almost superimposable to rhazinilam, and

its improved activity originates mainly in the replace-

ment of the amide group by a carbamate. These results

prompted us to perform further modiﬁcations of B-ring

by semi- and total syntheses.

ref. (5)

O

2

¹⁷₁N

3

H

21

N

H

8

5

CO₂Me

10

6

1

(+)-vincadifformine 7

a

O

R

O

( )₃

HN

N

H₂N

c

N

3

8 R=CO₂H

9 R=CH₂OH

b

In this paper, we describe the synthesis, structural anal-

ysis and biological activity of three analogues 3 and 4

with an enlarged (11- and 12-membered) B-ring as well

as thirty rhazinilam analogues 5 and 6 substituted in

the N-1 and C-16 positions of B-ring. The ﬁrst series

of compounds 3 and 4 were obtained by opening of

the lactam ring of (ꢀ)-rhazinilam, functionalization

and ring re-closure. These ﬁrst analogues of rhazinilam

with a larger B-ring, containing either an additional

C–O bond (compound 3) or an amino-acid residue

(compounds 4a and 4b) compared to rhazinilam, were

ﬁrst designed to provide further information on the role

of B-ring conformation and ﬂexibility. In addition, the

presence of the additional hydrogen-bond donor/accep-

tor amide group in compounds 4 was thought to be of

potential beneﬁt for the interaction with tubulin. The

second series of analogues 5 and 6 were synthesized by

regioselective direct functionalization of (ꢀ)-rhazinilam

1 and were designed to analyze further the inﬂuence of

the substitution of the amide group nitrogen and

a-carbon on the biological activity.

Scheme 1. Reagents and conditions: (a) KOH (42 equiv), EtOH/H₂O

1:1, microwave heating (120 ꢁC, sealed tube), 30 min, 85%; (b) LiAlH₄

(20 equiv), THF, reﬂux, 22 h, 69%; (c) NaHMDS (2 equiv),

(Cl₃CO)₂C@O (1 equiv), THF, 0 ꢁC, 10 min, 21%. NaHMDS, sodium

bis(trimethylsilyl)amide.

by deprotonation of 9 with 2 equiv of NaHMDS,

followed by reaction with diluted triphosgene in THF

at 0 ꢁC.¹¹

1

By contrast with intermediates 8 and 9 the H NMR

spectrum (CDCl₃) of compound 3 showed the presence

of only one conformer, as the cyclization is much more

sterically favored with the phenyl–pyrrole axis of 9 in

the same (aR) conﬁguration as rhazinilam. Molecular

modeling experiments (Monte-Carlo random search)

indicated that the amide group of 3 adopts the more sta-

ble trans conformation (Fig. 1), whereas that of rhazini-

lam is ﬁxed in the cis conformation by the more rigid

nine-membered ring (see Fig. 3A). The plane angle be-

tween the phenyl and pyrrole rings is similar in 3 and

in rhazinilam, with respective values of 87ꢁ and 89ꢁ,

indicating that the enlargement of B-ring does not

signiﬁcantly aﬀect the ACD ring system.

2. Results and discussion

2.1. Analogues 3 and 4 with a macrocyclic B-ring

The synthesis of macrocycles 4a and 4b incorporating a

glycine or ^L-valine in their B-ring was performed as fol-

lows (Scheme 2). Initially, the carboxylic acid group of 8

was converted to the methyl ester and the resulting com-

pound coupled with Fmoc-glycine. However, the hydro-

lysis of the ester group and subsequent removal of the

Fmoc group were low yielding. A more straightforward

approach consisted of reacting directly amino-acid 8

(ꢀ)-Rhazinilam 1 was synthesized in two steps from the

alkaloid (+)-vincadiﬀormine 7 by a recently optimized

procedure (Scheme 1).^5bMacrocycle 3 was obtained in

three steps from rhazinilam as follows. The hydrolysis

of the lactam group of rhazinilam was ﬁrst performed

in 82% yield under relatively harsh conditions, using ex-

cess KOH in reﬂuxing ethanol and water for 23 h. Alter-

natively, the reaction time could be substantially

shortened under microwave heating (120 ꢁC, 100 W,

sealed tube), giving amino-acid 8 in 85% yield in only

30 min. Reduction of the carboxylic acid with excess

LiAlH₄in reﬂuxing THF provided amino-alcohol 9 in

1

69% yield. The H NMR spectra of compounds 8 and

9 showed that these compounds occur as a mixture of

two conformers (87/13 for 8 and 80/20 for 9 in CDCl₃)

arising from probable atropisomerization around the

phenyl–pyrrole bond. These two conformers intercon-

vert rapidly as shown by the presence of exchange corre-

lations in NOESY spectra recorded at 295 K. A number

of reagents and reaction conditions were tested for the

macrocyclization of amino-alcohol 9 to give 11-mem-

bered carbamate 3. The best yield (21%) was obtained

Figure 1. Lowest energy conformer of macrocycle 3 obtained by

molecular modeling (all hydrogen atoms, except for H-1, were omitted

for clarity).

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A. Decor et al. / Bioorg. Med. Chem. 14 (2006) 2314–2332

2316

HO₂C

cycle 4b energetically favors one conformer compared to

R

NHX

macrocycle 4a. Molecular modeling calculations

(Monte-Carlo random search) were performed with

compounds 4a and 4b. For each compound, four con-

formers of similar energies were obtained, in which the

N(1)–C(22) amide is always trans, whereas the N(24)–

C(2) amide is either trans or cis (with two diﬀerent ori-

entations in each case). Amongst these four conformers,

only one could be ﬁtted with the NMR dataset of one of

the conformers of 4a, 4ax, as well as of the major con-

former of 4b, 4bx (Fig. 2).

HN

O

a

N

8

R

11a R=H, X=Fmoc

11b R=iPr, X=Fmoc

Cl

NHFmoc

b

c

O

12a R=H, X=H

12b R=iPr, X=H

10a R=H

10b R=iPr

O

NH

2

24

R

22

HN₁

O

N

The second conformers 4ay and 4by could not be as-

signed unambiguously. The NMR data arguing for the

proposed structure 4ax (Fig. 2A), where both amide

groups have trans conformations, are the following: (1)

observed NOESY correlations (Fig. 2A); (2) deshielding

of proton H-12, resoning at 8.41 ppm (compared to

7.20 ppm for rhazinilam), due to the proximity of the

anisotropy cone of the C-22 carbonyl group; (3) torsion

angles of 26ꢁ for H(23a)–C(23)–N(24)–H(24) and 142ꢁ

for H(23b)–C(23)–N(24)–H(24), calculated from the

4a R=H

4b R=iPr

Scheme 2. Reagents and conditions: (a) CH₂Cl₂/10% aq NaHCO₃1/1,

10a or 10b (1 equiv), 20 ꢁC, 90 min, 46% (11a) or 66% (11b); (b) THF/

piperidine 9:1, 20 ꢁC, 88% (R=H) or 92% (R=iPr); (c) TBTU (3 equiv),

HOBT (3 equiv), EtN(iPr)₂(4 equiv), DMF (c = 1 mM), 20 ꢁC, 24 h,

30% (4a) or 10% (4b). Fmoc, 9-ﬂuorenylmethoxycarbonyl; TBTU, O-

benzotriazol-1-yl-N,N,N⁰,N⁰-tetramethyluronium tetraﬂuoroborate;

HOBT, 1-hydroxybenzotriazole.

3

coupling constants J(H-23a–H-24) = 5.8 Hz and J(H-

23b–H-24) = 7.0 Hz.¹⁵Similarly, the NMR data arguing

for the proposed structure 4bx (Fig. 2B), where the

N(1)–C(22) amide is trans and the N(24)–C(2) amide is

cis, are the following: (1) observed NOESY correlations

(Fig. 2B); (2) deshielding of proton H-12, resoning at

8.14 ppm, less pronounced than for 4ax probably be-

cause H-12 is farther away from the C-22 carbonyl; (3)

torsion angle of 153ꢁ for H(23)–C(23)–N(24)–H(24), cal-

culated from the coupling constant ³J(H-23–H-

24) = 8.3 Hz. In this structure, the C-23 isopropyl group

lies in a pseudo-equatorial position which probably sta-

bilizes this particular conformation and may explain the

preponderance of conformer 4bx, whereas the glycine

conformers 4ax and 4ay are equally abundant. The

plane angle between phenyl and pyrrole rings is 91ꢁ

for both conformers 4ax and 4bx, and is again very sim-

ilar to that of rhazinilam (89ꢁ), indicating the conserva-

tion of the three-dimensional structure of the ACD ring

system.

with the acid chloride 10a, obtained in high yield from

Fmoc-glycine and thionyl chloride.¹²This method aﬀor-

ded the coupled product 11a in 46% yield. After Fmoc

group removal with piperidine (88% yield of amino-acid

12a), the macrocyclization occurred in the presence of

TBTU and HOBT as coupling reagents to give macrocy-

cle 4a in 30% yield.^13,14The compound was puriﬁed

twice, by reverse-phase column chromatography fol-

lowed by HPLC in order to remove traces of the peptide

coupling reagents, which in part is responsible for the

low yield of isolated compound. Other peptide coupling

reagents such as BOP (benzotriazol-1-yloxytris(dimeth-

ylamino)phosphonium hexa-ﬂuorophosphate) did not

aﬀord improved yields.

The same strategy was applied for the synthesis of mac-

rocycle 4b containing a ^L-valine (Scheme 2). This other

amino-acid was chosen in order to study the inﬂuence

of a bulky Ca-substituent on the conformation and bio-

logical activity of the macrocyclic rhazinilam analogue.

Thus, compound 4b was obtained in three steps from

amino-acid 8 and Fmoc-valine acid chloride 10b. A dif-

ﬁcult puriﬁcation again explains in part the low macro-

cyclization yield. Similar to acyclic intermediates 8 and

9, NMR experiments indicated that intermediates

11a,b and 12a,b exist as a mixture of conformers inter-

converting at room temperature. However, by contrast

to macrocycle 3 which showed only one conformer,

macrocycles 4a and 4b both exist as a mixture of two

conformers (named 4ax/4ay and 4bx/4by) in acetone-d₆

at 295 K, in a ratio of, respectively, 1/1 and 85/15. These

conformers interconvert at 295 K, as shown by correla-

tions observed on the NOESY spectrum recorded at this

temperature. These correlations disappeared on

NOESY spectrum recorded at 233 K, which shows that

the interconversion is blocked at this temperature. The

two conformers most probably arise from classical cis–

trans isomerization of one or both amide groups. It

seems that the presence of the isopropyl group in macro-

In summary, the three-dimensional structures proposed

for macrocycle 3, for one conformer of macrocycle 4a,

A

B

6

17

16

12

17

16

1

12

22

2

1

23

2

22

24

23

Figure 2. Molecular modeling structure of macrocycles 4a–b with

selected NOESY correlations. (A) Conformer 4ax. (B) Major con-

former 4bx (some hydrogen atoms were omitted for clarity).

´

A. Decor et al. / Bioorg. Med. Chem. 14 (2006) 2314–2332

2317

and the major conformer of macrocycle 4b show a very

O

diﬀerent B-ring conformation compared to rhazinilam,

with a trans N(1)–C(22) amide, whereas the amide group

of rhazinilam is cis. In addition, the presence of two

interconverting conformers for 4a and 4b shows that

the 12-membered B-ring of these compounds is ﬂexible,

by contrast to the rigid nine-membered B-ring of

rhazinilam.

R¹

N

a

N

16

5a-s

O

H

¹N

R²

N

O

H

1

N

The 11- and 12-membered macrocycles 3, 4a, and 4b

showed a very weak activity on the assembly and disas-

sembly of microtubules.¹⁶In addition, these compounds

showed no cytotoxicity toward KB cancer cells. Acyclic

intermediates 8, 9 and 11a,11b were also evaluated and

showed no activity on tubulin and on KB cells. The

low activity of macrocycles 3 and 4 might be imputed

to the excessive ﬂexibility of B-ring compared to that

of rhazinilam, which enables the N-1 amide group of

the analogues to adopt the more stable trans conforma-

tion. The importance of the cis amide conformation for

the binding to tubulin could suggest that H-1 amide pro-

ton is engaged in a hydrogen-bond with the protein and

has to remain intact and in the right orientation in order

to preserve the activity.

b

6a-e (α :β )

Scheme 3. Reagents and conditions: (a) nBuLi (1.05 equiv), THF,

0 ꢁC, 30 min, then electrophile (1.0 equiv); (b) nBuLi (2.5 equiv), THF,

0 ꢁC, 30 min, then electrophile (1.0 equiv).

1

ratio was measured from H NMR and HPLC analy-

sis of the mixture, then the two diastereoisomers were

separated by preparative HPLC for further stereo-

chemical analysis and biological testing. The perﬂuori-

nated N-tBoc analogue 5p (entry 16) was synthesized

by a diﬀerent procedure, by reacting (ꢀ)-rhazinilam

with perﬂuoro-tert-butyl alcohol, triphosgene, and

pyridine in CH₂Cl₂at 0 ꢁC.¹¹Finally, 16-hydroxyrhaz-

inilam 6f (entry 25) was obtained as a by-product dur-

ing the large-scale conversion of (+)-vincadiﬀormine

into (ꢀ)-rhazinilam.⁵It was converted to its

acetate 6g (entry 26) using Ac₂O/Et₃N/DMAP in

dichloromethane.

2.2. N-1 and C-16 analogues 5 and 6

In consequence of the above results showing a loss of

tubulin-binding activity upon enlargement of rhazinilam

B-ring, it was decided to maintain the nine-membered

rigid structure of rhazinilam and to perform direct mod-

iﬁcations around the amide group on the N1 or C16 po-

sition. The improved activity of carbamate 2 compared

to that of the corresponding amide also encouraged us

in this way.^7bThe introduction of three substituents

(Me, CH₂CO₂Ph, and tBoc) on the N-1 amide nitrogen

of rhazinilam as well as the biological properties of these

analogues were brieﬂy described in a previous report.^5a

N-1-methylrhazinilam was the most active N-1 analogue

reported, with a four times lower activity compared to

that of rhazinilam on the inhibition of microtubule dis-

assembly. In continuation to this work, we describe the

introduction of a larger number of chemically diverse

substituents on the amide group at N-1 and C-16.

The three-dimensional structure of (ꢀ)-rhazinilam 1 was

solved in 1972 by X-ray analysis.¹⁸We have again

solved this structure with a better resolution for the pur-

pose of the present study (Fig. 3A). The nine-membered

lactam B-ring of rhazinilam adopts a boat–chair confor-

mation. In addition, the C-14 atom on piperidine D-ring

points down, toward B-ring. This conformation is also

preferred in solution (CDCl₃), as shown by NOESY

experiments (Fig. 3A). The X-ray crystal structure of

N-tBoc-rhazinilam 5o (Fig. 3B) shows that the introduc-

tion of the large tBoc group does not modify the confor-

mation of rings B and D.

The absolute conﬁguration of the major and minor C-16

diastereoisomers of analogues 6a–g was determined by

X-ray analysis as well as NMR experiments. First, the

X-ray crystal structure of the major diastereoisomer of

16-ethylrhazinilam 6b was solved (Fig. 3C), showing

that this diastereoisomer has the a conﬁguration at C-

16. Rings B and D of this diastereoisomer 6ba adopt

the same conformation as rhazinilam. By extension,

the minor diastereoisomer of compound 6b has the b

conﬁguration at C-16.

Deprotonation of the amide proton of (ꢀ)-rhazinilam 1

by 1 equiv n-butyllithium at 0 ꢁC followed by quenching

with an electrophile aﬀorded N-substituted analogues

5a–s regioselectively and in moderate to good yield

(Scheme 3 and Table 1, entries 1–19). On the other hand,

using 2 equiv nBuLi generated the amide dianion which

reacted regioselectively on C-16 with electrophiles to

give analogues 6a–e (Scheme 3 and Table 1, entries

20–24). In case of C-16 ethyl (6b, entry 21) and benzoyl

(6d, entry 23) analogues, the corresponding N-substitut-

ed compounds 5a and 5k were also isolated in 33% and

25% yield, respectively. In both N- and C-substitutions,

unreacted rhazinilam was recovered in substantial

amounts. Among the strong bases screened for this

functionalization, nBuLi gave the highest yields.

The conﬁguration of other C-16 analogues was deter-

mined indirectly, as no other X-ray structure could be

obtained. It was noticed that all major diastereoisomers

of C-16 analogues 6a–e have similar chemical shifts of

¹H and ¹³C atoms located on D-ring and to a lesser ex-

tent on B-ring (Table 2, entries 2–6). These chemical

shifts are also similar to those of rhazinilam (entry 1).

This suggests that all major diastereoisomers have the

a conﬁguration at C-16 and adopt the same conforma-

The C-substitution products 6a–e were obtained as

mixtures of two diastereoisomers a and b.¹⁷The a:b

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A. Decor et al. / Bioorg. Med. Chem. 14 (2006) 2314–2332

2318

Table 1. Synthesis, cytotoxicity, and antitubulin activity of N-1 and C-16 analogues of (ꢀ)-rhazinilam produced via Scheme 3

Entry

Compound

R¹or R^2a

Electrophile

Yield^b(%)

Ratio a:b

Inhibition of

microtubule

assembly

Inhibition of

microtubule

disassembly

IC₅₀(1)/ IC₅₀

Cytotoxicity

KB cell line

IC₅₀(1)/ IC₅₀

d

c

IC₅₀(1)/ IC₅₀

1

2

5a

5b

Et

nPr

EtI

n-PrI

66

32

46

81

76

62

71

53

50

66

59

62

53

50

85

12

61

46

67

43

1/11

1/22

In

1/2

In

1/33

In

3

5c

5d

Allyl

(Allyl)Cl

BrCH₂CN

MOMBr

MEMCl

SEMCl

BnBr

In

4

CH₂CN

MOM

MEM

1/22

In

1/65

In

5

5e

In

6

5f

5g

In

1/23

In

7

SEM

Bn

1/9

In

8

5h

5i

In

9

Ac

Piv

AcBr

PivCl

BzCl

1/13

1/58

1/4

1/33

1/8

In

10

11

12

13

14

15

16

17

18

19

20

5j

In

5k

5l

Bz

1/6

1/9

1/22

In

(pOMe)Bz

(pCF₃)Bz

C(@O)OEt

tBoc

(pOMe)BzCl

(pCF₃)BzCl

ClC(@O)OEt

Boc₂O

In

5m

5n

In

5o

5p

1/2

1/5

In

e

C(@O)OC(CF₃)₃

C(@O)OPh

Ms

—

In

5q

5r

ClC(@O)OPh

MsCl

TsCl

1/18

In

5s

Ts

In

2

1/11

1/7

In

6aa

6ab

6ba

6bb

6ca

6cb

6da

6db

6ea

6f

a-Me (S)

b-Me (R)

a-Et (S)

b-Et (R)

a-Bn (S)

b-Bn (R)

a-Bz (R)

b-Bz (S)

a-tBoc (S)

a-OH (S)

a-OAc (S)

MeI

80:20

79:21

62:38

82:18

98:2

1/2

In

1/5

1/14

1/3

1/11

In

21

22

23

EtI

56

52

30

10

1/3

1/15

In

1/7

In

BnBr

BzCl

Boc₂O

1/5

In

1/8

1/14

1/22

1/4

In

24

25

26

1/25

1/2

In

f

—

1/7

In

g

6g

In = inactive (or IC₅₀not measurable).

^aAbbreviations: MOM, methoxymethyl; MEM, 2-methoxyethoxymethyl; SEM, 2-(trimethylsilyl)ethoxymethyl; Piv, pivaloyl; Bz, benzoyl; Ms,

methanesulfonyl; Ts, 4-toluenesulfonyl; for compounds 6a–e, the absolute [(R) or (S)] conﬁguration at C-16 is indicated.

^bIsolated yields (for compounds 6a–e, combined yields after separation of the diastereoisomers by HPLC).

^cIC₅₀is the concentration of compound required to inhibit 50% of the rate of microtubule assembly or disassembly, average of three experiments;

IC₅₀(1) = 7 lM for assembly and 3 lM for disassembly.

^dIC₅₀is the concentration of compound corresponding to 50% growth inhibition after 72 h incubation, average of three experiments; IC₅₀

(1) = 0.5 lM.

^eObtained from (CF₃)₃COH, (Cl₃CO)₂C@O, pyridine, CH₂Cl₂, 0 ꢁC.

^fObtained from 7 by semi-synthesis.

^gObtained from 6f.

tion as rhazinilam. In addition, large coupling constants

observed for H-16 and H-17 indicate that H-16 is in the

axial position in these molecules (see Fig. 3C). For in-

stance in compounds 6da and 6ea (which have a more

simple coupling pattern for H-16 compared to other

analogues), H-16 appears as a doublet with J = 11 Hz.

The same observations apply to the minor diastereoiso-

to the axial–axial and axial–equatorial couplings with

H-17 (J = 13 and 5 Hz, respectively), H-16 shows a

1.7 Hz coupling constant corresponding to a W-cou-

pling with the amide proton.

A more precise picture of the conformation of the b dia-

stereoisomers was obtained by NOESY experiments

performed on both diastereoisomers of the benzoyl ana-

logue 6d (Fig. 4). Whereas the NOE correlations for the

major 6da diastereoisomer (Fig. 4A) were identical to

those obtained with rhazinilam (Fig. 3A), the correla-

tions observed for the minor 6db diastereoisomer are

consistent with ring B adopting a boat–boat conforma-

tion, whereas C-14 on ring D now points up, opposite to

ring B (Fig. 4B). In both diastereoisomers, the H-16 pro-

ton is axial and gives the large coupling constant with

the axial H-17, whereas in 6db the H–N(1)–C(2)–

C(16)–H bond system is planar and gives the W-cou-

1

mers, which have similar H and ¹³C chemical shifts for

B and D rings (Table 2, entries 9–12), whereas these sig-

nals diﬀer markedly in each couple of diastereoisomers

(see values under brackets). This suggests that all minor

diastereoisomers have the b conﬁguration at C-16, and

that B and D-rings of these diastereoisomers adopt a dif-

ferent conformation than rhazinilam and the a diastere-

oisomers. In this case, one H-17 proton also shows large

coupling constants pointing at an axial position of H-16

in the b diastereoisomers. By contrast, the coupling pat-

tern of H-16 is more complex than in the a diastereoiso-

mers. For instance, in benzoyl analogue 6db, in addition

1

pling on the H spectrum.

´

A. Decor et al. / Bioorg. Med. Chem. 14 (2006) 2314–2332

2319

Figure 4. Selected NOESY correlations observed in CDCl₃with (A)

diastereoisomer 6da and (B) diastereoisomer 6db of 16-benzoylrhaz-

inilam. The 3D structures were obtained from molecular modeling.

less unfavorable steric interactions between the C-16

substituent and the rest of the molecule. The stereo-

chemical outcome of the substitution of rhazinilam at

C-16 could be explained by the attack of the electrophil-

ic reagent by the amide dianion formed from rhazinilam

preferentially from the a position, as the b position is

shielded both by the phenyl A-ring and the C-20 ethyl

group. With this explanation larger electrophiles would

tend to give a higher diastereoisomeric ratio, which is

particularly obvious in the case of the tBoc group (Table

1, entry 24), for which almost only the a diastereoisomer

was observed. However, an accurate relationship be-

tween the size of the substituent and the diastereoiso-

meric ratio is not clearly demonstrated by the present

data (entries 20–24) and a number of other stereo-elec-

tronic eﬀects could be operating (such as p-stacking

interactions between aromatic electrophiles and rhazini-

lam A-ring). The major a diastereoisomer stays in the

stable BC-D conformation of rhazinilam, with the C-

16 substituent in the equatorial position pointing away

from other residues (Fig. 3C). By contrast, the minor

b diastereoisomer interconverts to the more stable BB-

U conformation where the C-16 substituent is again

equatorial and pointing away from other residues

(Fig. 3D).

Figure 3. (A) X-ray crystal structure of (ꢀ)-rhazinilam 1 and selected

NOESY correlations observed in CDCl₃(white arrows). (B) X-ray

crystal structure of N-tBoc-rhazinilam 5o. (C) X-ray crystal structure

of 16 a-ethylrhazinilam 6ba. (D) Molecular modeling structure of 16

b-ethylrhazinilam 6bb.

In order to study these conformational changes into

more detail, molecular modeling experiments were con-

ducted, ﬁrst with rhazinilam. The steric energies of the

two possible conformers of ring B [boat–chair (BC)

and boat–boat (BB)] and ring D [C-14 down (D) or up

(U)] were computed (MMFF94 force ﬁeld). The four

conformers could be classiﬁed as follows by decreasing

stability: BC-D > BB-U > BC-U > BB-D. The most sta-

ble BC-D conformation is identical to that of rhazinilam

in solution and in the crystal. It also corresponds to the

conformation of the major a C-16 diastereoisomers. The

BB-U conformation, which comes next in order of sta-

bility, corresponds to the minor b C-16 diastereoisomer.

Complementary conclusions were drawn from modeling

the two 16-ethyl diastereoisomers 6ba and 6bb: the low-

est energy conformer of diastereoisomer 6ba has the BC-

D conformation and is superimposable to the X-ray

structure of the molecule (Fig. 3C). The lowest energy

conformer of diastereoisomer 6bb has the BB-U confor-

mation and is identical to the related structure of com-

pound 6db deduced by NOESY experiments (Fig. 3D

and Fig. 4B). The BC-D and the BB-U conformations

adopted by the two diastereoisomers are those giving

Finally, the conﬁguration of the 16-hydroxy and 16-

acetoxy analogues 6f and 6g, synthesized by a diﬀerent

route than the other C-16 analogues, was determined

by similar NMR experiments. First, the chemical shifts

Table 2. Selected ¹H and ¹³C chemical shifts for a and b C-16 analogues^a

Entry Compound Dd(H-3)

d(C-3)

Dd(H-14) d(C-14)

Dd(H-15)

d(C-15) d(C-16)

Dd(H-17) d(C-17) d(C-19)

1

2

1

ꢀ0.23

ꢀ0.22

ꢀ0.23

ꢀ0.20

ꢀ0.22

ꢀ0.16

ꢀ0.20

46.1

46.2

45.9

46.1

0.39

0.41

0.40

0.46

0.39

0.19

0.32

19.4

19.5

0.15

0.17

0.14

0.09

0.10

0.00

0.09

33.1

33.3

33.5

33.4

33.6

33.5

33.0

33.9

28.1

31.9

39.2

40.1

47.1

44.9

65.8

67.7

1.00

1.31

1.26

1.22

0.91

0.68

0.77

1.09

36.6

45.8

44.7

43.3

40.7

39.0

45.9

43.0

30.1

30.6

30.5

30.4

31.6

30.8

6aa

6ba

6ca

6da

6ea

6f

3

4

5

6

7

8

6g

9

6ab

6bb

6cb

6db

ꢀ0.08 (ꢀ0.15) 45.7 (0.4) 0.23 (0.18) 20.0 (ꢀ0.6) 0.38 (ꢀ0.21) 28.8 (4.5) 42.3 (ꢀ10.4) 0.28 (1.03) 41.7 (4.1) 36.0 (ꢀ5.4)

ꢀ0.09 (ꢀ0.13) 45.7 (0.4) 0.22 (0.18) 20.0 (ꢀ0.6) 0.51 (ꢀ0.34) 28.9 (4.6) 49.1 (ꢀ9.9) 0.21 (1.05) 40.4 (4.3) 36.1 (ꢀ5.5)

ꢀ0.09 (ꢀ0.14) 45.6 (0.5) 0.21 (0.19) 20.0 (ꢀ0.6) 0.53 (ꢀ0.39) 28.8 (4.6) 49.0 (ꢀ8.9) 0.26 (0.96) 39.4 (3.9) 36.1 (ꢀ5.6)

ꢀ0.08 (ꢀ0.12) 45.7 (0.4) 0.22 (0.24) 20.0 (ꢀ0.6) 0.50 (ꢀ0.41) 28.8 (4.8) 55.7 (ꢀ8.6) 0.66 (0.25) 36.2 (4.5) 36.2 (ꢀ5.8)

10

11

12

^aDd = d(axial proton) ꢀ d(equatorial proton). Values under brackets refer to d(a diastereoisomer) ꢀ d(b diastereoisomer). All values in ppm.

Attributions were made on the basis of usual 2D NMR experiments.

´

A. Decor et al. / Bioorg. Med. Chem. 14 (2006) 2314–2332

2320

of B and D-rings atoms of 6f and 6g have close values to

those of the 6a–e a-diastereoisomers (Table 2, entries 7

and 8), although this eﬀect is more pronounced with

6g than with 6f. In addition, NOESY correlations

similar to those observed with rhazinilam (Fig. 3A)

and 16 a-benzoylrhazinilam (Fig. 4A) were observed

with compound 6f. These experiments show that 6f

and 6g have the a conﬁguration at C-16 and also adopt

the BC-D conformation.

pound 6aa was twice more active than rhazinilam on

microtubule assembly. This inﬂuence of steric interac-

tions was observed in all previous SAR studies and fur-

ther indicates that the compounds bind to a narrow

tubulin binding pocket.²In a number of cases, the ana-

logues which had little or no activity on tubulin were sig-

niﬁcantly cytotoxic on KB cells, which could arise from

a number of phenomena, including the possibility

that tubulin is not the sole cellular target of these

compounds.

The antitubulin activity and the cytotoxicity of ana-

logues 5 and 6 were evaluated and compared to those

of rhazinilam 1 (Table 1). All N-1 analogues (entries

1–19) were less active than rhazinilam on the inhibition

of both microtubule assembly and disassembly. The

most active analogue was the N-ethyl analogue 5a (entry

1), which was twice less active than 1 on the inhibition of

microtubule disassembly. However, there was a rather

poor correlation between the tubulin-binding eﬀect

and the cytotoxicity. For instance, compound 5a was

inactive on KB cells. Conversely, several cytotoxic N-1

analogues and in particular carbamates 5o–q (entries

15–17) had almost no eﬀect on tubulin.¹⁹Flow cytome-

try experiments conducted with compound 5o indicated

that this compound is active during the G2/M phase of

the cell cycle, like rhazinilam, however it was not possi-

ble to determine if tubulin was the actual intracellular

target of this compound. Control experiments indicated

that the deprotection of the tBoc group to release free

rhazinilam did not occur in the cell culture medium.

Available NMR and X-ray diﬀraction data for N-1 ana-

logues (see Fig. 3B for 5o) show that they adopt the

same overall conformation as rhazinilam. In conse-

quence, the decrease in the binding to tubulin observed

for these compounds seems to reﬂect mostly steric repul-

sions between the N-1 substituent and the protein. The

diﬀerence of polarity and lipophilicity of the substituent

may explain the discrepancies observed among the dif-

ferent analogues. A second explanation could put for-

ward the possibility that the H-1 proton of rhazinilam

is engaged in an hydrogen-bond with tubulin, in what

case the presence of any substituent at N-1 would lower

the aﬃnity.

3. Summary and conclusions

This paper describes the synthesis of 11- and 12-mem-

bered macrocyclic analogues of rhazinilam 3 and 4, as

well as nine-membered B-ring analogues 5 and 6

substituted in the N-1 or C-16 position. Stereochem-

ical analyses showed that N-1 and C-16a analogues

have the same conformation as rhazinilam, whereas

C-16b analogues adopt a diﬀerent conformation for

rings B and D. All analogues were less active than

rhazinilam on tubulin, though analogues 5a, 6aa,

6ba, and 6f having the less bulky substituents retained

interesting aﬃnities for the protein, comparable to

those of the most active analogues of rhazinilam pre-

viously described. This poor tolerance to bulky sub-

stituents indicates that rhazinilam binds to a very

narrow pocket of the protein. The correlation between

the aﬃnity for tubulin and the cytotoxicity toward KB

cancer cells was not clear for the N-1 analogues (e.g.,

5a and 5o), whereas it was better for C-16 analogues.

The importance of the conservation of the rigid boat–

chair conformation of rhazinilam B-ring, with a cis

N(1)–C(2) amide conformation, was clearly established

by this study. In particular, macrocycles 3 and 4 hav-

ing a ﬂexible B-ring with a trans N(1)–C(22) amide

group have almost no aﬃnity for tubulin. In conse-

quence, future design of other analogues of rhazinilam

should conserve the space occupation and structural

integrity of the natural product B-ring. The replace-

ment of C-ring by other aromatic groups, which was

initiated by the synthesis of biphenyl and phenyl–pyr-

idine analogues,^7,9,10could be a privileged option. A

metabolization study of rhazinilam by cytochromes

P450, mimicking its in vivo transformation and sug-

gesting other research directions, will be reported in

due course.

C-16 analogues were in general less active than rhazini-

lam both on tubulin and KB cells (entries 20–26), but to

a lesser extent than N-1 analogues. In this case, there

was also a better correlation between the tubulin-bind-

ing eﬀect and the cytotoxicity. Of the two a and b diaste-

reoisomers, the a diastereoisomer was generally the

most active, except in the case of benzyl analogue 6c

where both diastereoisomers had, in ﬁrst approxima-

tion, the same level of activity. As we have demonstrated

before that a diastereoisomers have the same conforma-

tion as rhazinilam, whereas that of b diastereoisomers

diﬀers signiﬁcantly, these data conﬁrm that the conser-

vation of B-ring conformation is an essential feature

for the binding to tubulin, in accordance with previous

SAR studies. For both N-1 and C-16 analogues, the de-

crease in the aﬃnity for tubulin seems to arise mainly

from the bulkiness of the substituent. Indeed the most

active compounds (5a, 6aa, 6ba, and 6f) were the ones

bearing the less bulky substituents. For instance, com-

4. Experimental

Reagents were commercially available and used without

further puriﬁcation unless otherwise stated. THF was

distilled from sodium/benzophenone and methylene

chloride from di-phosphorus pentoxide (P₂O₅) immedi-

ately before use. Yields refer to chromatographically

and spectroscopically homogeneous materials. Reac-

tions were monitored by thin-layer chromatography

carried out on 0.25 mm SDS silica gel coated glass

plates (60F254) using UV light as visualizing agent

and ethanolic sulfuric molybdate or cerium ammonium

sulfate and heat as staining agents. Merck silica gel 60

´

A. Decor et al. / Bioorg. Med. Chem. 14 (2006) 2314–2332

2321

(particle size 40–63 lM) or Econosil Prep C18 HL (par-

4.2. Amino-acid (8)

ticle size 40–63 lM) was used for ﬂash chromatography.

1 and 2 mm SDS silica gel-coated glass plates (60F254)

were used for preparative TLC using UV light as visu-

alizing agent. Microwave heating was performed with

a CEM Discover focused reactor in adapted sealed

tubes. NMR spectra were recorded on Bruker AMX-

300, AMX-400 or AMX-500 instruments at 295 K and

calibrated using tetramethylsilane as an internal refer-

ence. Attributions were made on the basis of 2D exper-

iments. The following abbreviations were used to

designate the multiplicities: s, singlet; d, doublet; t, trip-

let, q, quartet; m, multiplet; br, broad; ar, aromatic ring;

eq, equatorial; ax, axial; and Cq, quaternary carbon.

Atom numbering refers to rhazinilam (Scheme 1). IR

spectra were recorded on a Perkin Elmer BX FT-IR

spectrometer. Mass spectra (MS) and high resolution

mass spectra (HRMS) were recorded under Electro-

spray Ionization (ESI) or Matrix-Assisted Laser

Desorption Ionization-Time-of-Flight (MALDI-TOF)

In a sealed tube, (ꢀ)-rhazinilam 1 (426 mg, 1.45 mmol)

was dissolved in water/ethanol 1:1 (3 mL). Powdered

KOH (3.44 g, 61.3 mmol) was added, the tube was

sealed, and the reaction mixture was heated under

microwave irradiation at 120 ꢁC for 30 min. After cool-

ing, the reaction mixture was transferred into an Erlen-

meyer, cooled to 0 ꢁC, and treated with a 1 N HCl

aqueous solution until pH 4–5. The aqueous solution

was extracted with dichloromethane and the organic

layer was dried over MgSO₄. After evaporation, the

crude mixture was puriﬁed by ﬂash chromatography

(dichloromethane/ethyl acetate 4:1) to give compound

8 (384 mg, 85%) as a white powder; mp 74 ꢁC; ¹H

NMR (300 MHz, CDCl₃) 2 conformers in a 87/13 ratio,

major conformer: d 7.20 (m, 2H, H_ar), 6.92 (m, 2H, H_ar),

6.61 (d, J = 2.7 Hz, 1H, H-5), 5.99 (d, J = 2.7 Hz, 1H,

H-6), 3.90 (dd, J = 5.4, 5.4 Hz, 2H, H-3), 2.36 (m, 1H,

H-15a), 2.04 (m, 1H, H-15b), 1.97 (m, 1H, H-14a),

1.77 (m, 2H, H-14b and H-16a), 1.73 (m, 1H, H-19a),

1.63 (m, 1H, H-17a), 1.60 (m, 1H, H-16b), 1.57 (m,

1H, H-17b), 1.48 (m, 1H, H-19b), 0.80 (t, 3H,

J = 7.2 Hz, 3H, H-18) ppm; minor conformer (character-

istic signals): d 7.42 (dd, J = 7.5, 2.1 Hz, 1H, H_ar), 7.33

(ddd, J = 7.5, 7.5, 2.1 Hz, 1H, H_ar), 7.09 (m, 1H, H_ar),

6.69 (m, 1H, H_ar), 6.51 (d, J = 2.7 Hz, 1H, H-5), 5.75

(d, J = 2.4 Hz, 1H, H-6), 0.71 (t, J = 7.2 Hz, 3H, H-18)

ppm; ¹³C NMR (75.5 MHz, CDCl₃) major conformer:

d 175.9 (C-2), 142.7 (C-13), 132.0 (CH_ar), 130.8 (C-21),

128.3 (CH_ar), 126.6 (C-8), 120.4 (CH_ar), 119.3 (C-5),

116.8 (CH_ar), 115.2 (C-7), 109.3 (C-6), 46.2 (C-3), 39.6

(C-20), 36.9 (C-19), 35.6 (C-15), 33.2 (C-17), 30.1 (C-

16), 21.8 (C-14), 9.1 (C-18) ppm, minor conformer (char-

acteristic signals): d 179.0, 145.4, 131.5, 127.9, 118.9,

´

conditions at the Laboratoire de Spectrometrie de Mas-

se (ICSN). Melting points (mp) are uncorrected and

were recorded on a Buchi B-540 capillary melting point

¨

apparatus. Optical rotations were recorded on a Jasco

P-1010 polarimeter. HPLC analyses and puriﬁcations

were performed on Waters systems equipped with a

photodiode array detector (monitoring at 200–

400 nm), using a Hypercarb column (10 cm · 0.46 cm

for analytic, 15 cm · 1 cm for preparative), by Thermo

Electron, Inc. or

a

C18 Symmetry column

(25 cm · 0.46 cm for analytic, 25 cm · 1.0 cm for semi-

preparative), by Waters, Inc. Molecular modeling stud-

ies were conducted using Sybyl 6.9.1 software (linux).

Conformational searches were performed with the

Monte-Carlo random search procedure using MMFF94

force ﬁeld parameters.

117.7, 115.5, 114.7, 110.2, 46.1, 39.2, 30.3, 29.8, 21.3,

24

D

~

9.3 ppm; ½aꢁ ꢀ9 (c 1.0, CHCl₃); IR (ﬁlm) m 3470,

4.1. (ꢀ)-Rhazinilam (1)^5,20,21

3370, 2960, 2873, 1730 cm^ꢀ1; HRMS (MALDI-TOF)

calcd for C₁₉H₂₅N₂O₂[(M+H)⁺]: 313.1916, found:

313.1909.

1

Beige crystals; mp 217 ꢁC (lit. 214–216 ꢁC)²¹; H NMR

(300 MHz, CDCl₃) d 7.43 (dd, J = 6.6, 2.4 Hz, 1H, H-

9), 7.34 (ddd, J = 7.5, 7.5, 2.4 Hz, 1H, H-11), 7.29

(ddd, J = 7.5, 7.5, 1.8 Hz, 1H, H-10), 7.19 (dd,

J = 7.5, 1.8 Hz, 1H, H-12), 6.63 (br s, 1H, H-1), 6.50

(d, J = 2.7 Hz, 1H, H-5), 5.75 (d, J = 3.0 Hz, 1H, H-

6), 4.00 (dd, J = 12.0, 4.8 Hz, 1H, H-3eq), 3.77 (ddd,

J = 12.0, 12.0, 4.8 Hz, 1H, H-3ax), 2.46 (dd, J = 12.3,

12.3 Hz, 1H, H-17ax), 2.37 (dd, J = 12.6, 12.6 Hz,

1H, H-16ax), 2.26 (ddddd, J = 12.3, 12.3, 12.3, 5.7,

2.7 Hz, 1H, H-14ax), 1.96 (dd, J = 12.6, 7.8 Hz, 1H,

H-16eq), 1.87 (m, 1H, H-14eq), 1.71 (ddd, J = 13.2,

13.2, 3.0 Hz, 1H, H-15ax), 1.56 (m, 1H, H-15eq), 1.44

(m, 1H, H-17eq), 1.40 (m, 1H, H-19a), 1.26 (m, 1H, H-

19b), 0.71 (t, J = 7.2 Hz, 3H, H-18) ppm; ¹³C NMR

(75.5 MHz, CDCl₃) d 177.3 (C-2), 140.4 (C-8), 138.1

(C-13), 131.4 (C-9), 130.6 (C-21), 128.0 (C-11), 127.2

(C-10), 126.8 (C-12), 119.1 (C-5), 117.3 (C-7), 109.6

(C-6), 46.1 (C-3), 38.9 (C-20), 36.6 (C-17), 33.1 (C-

4.3. Amino-alcohol (9)

To a suspension of LiAlH₄(2.06 g, 54.3 mmol) in THF

(20 mL) at 0 ꢁC under argon was added dropwise a solu-

tion of compound 8 (848 mg, 2.71 mmol) in THF

(10 mL). After stirring for 5 min at 0 ꢁC, the mixture

was reﬂuxed for 22 h. After cooling to 0 ꢁC, the mixture

was treated sequentially with water (2 mL), a 15%

NaOH aqueous solution (2 mL), water (6 mL), and ﬁl-

tered through Celite. The solution was dried over

MgSO₄and evaporated under vacuum. The residue

was puriﬁed by ﬂash chromatography (heptane/ethyl

acetate 4:1), to give compound 9 as an oil (562 mg,

1

69%); H NMR (300 MHz, CDCl₃) 2 conformers in a

4/1 ratio, major conformer: d 7.14 (m, 2H, H_ar), 6.76

(m, 2H, H_ar), 6.58 (d, J = 1.2 Hz, 1H, H-5), 5.99 (d,

J = 1.2 Hz, 1H, H-6), 3.91 (dd, J = 5.5, 5.5 Hz, 2H, H-

3), 3.61 (m, 3H, H-1 and H-2a), 3.30 (m, 1H, H-2b),

1.92 (m, 1H, H-14a), 1.80 (m, 1H, H-19a), 1.73 (m,

1H, H-14b), 1.71 (m, 2H, H-15), 1.56 (m, 1H, H-17a),

1.54 (m, 1H, H-16a), 1.49 (m, 1H, H-19b), 1.28 (m,

1H, H-16b), 1.23 (m, 1H, H-17b), 0.84 (t, 3H,

15), 30.1 (C-19), 28.1 (C-16), 19.4 (C-14), 8.2 (C-

24

D

18) ppm; ½aꢁ ꢀ432 (c 1.0, CHCl₃) (lit. ꢀ421, c 1.0,

CHCl₃) ; IR (ﬁlm) m 3225, 1672 cm^ꢀ1; HRMS (ESI)

21

~

calcd for C₁₉H₂₂N₂NaO [(M+Na)⁺]: 317.1630, found:

317.1644.

´

A. Decor et al. / Bioorg. Med. Chem. 14 (2006) 2314–2332

2322

J = 6.9 Hz, 3H, H-18) ppm, minor conformer (character-

istic signal): d 0.74 (t, J = 6.9 Hz, 3H, H-18) ppm; ¹³C

NMR (75.5 MHz, CDCl₃) major conformer: d 144.9

((Cq)_ar), 132.4 (C-21), 132.2 (CH_ar), 128.4 (CH_ar),

126.0 ((Cq)_ar), 119.1 and 119.0 (CH_arand C-5), 115.9

(CH_ar), 115.8 (C-7), 109.7 (C-6), 63.3 (C-2), 46.6 (C-3),

39.9 (C-20), 37.8 (C-17), 36.2 (C-19), 31.0 (C-15), 29.0

(C-16), 22.3 (C-14), 9.6 (C-18) ppm, minor conformer

(characteristic signals): d 146.0, 132.0, 128.2, 125.1,

formers in a 3/1 ratio, major conformer: d 8.47 (d, 1H,

J = 7.5 Hz, H_ar), 8.11 (br s, 1H, H-1), 7.78 (m, 2H,

H_ar), 7.61 (m, 1H, H_ar), 7.39–7.20 (m, 7H, H_ar), 7.07

(m, 1H, H_ar), 6.44 (m, 1H, H-5), 6.28 (br s, 1H,

NHFmoc), 5.96 (m, 1H, H-6), 4.42 (m, 1H,

C(@O)OCH₂), 4.23 (m, 1H, C(@O)OCH₂CH), 4.17

(m, 1H, C(@O)OCH₂), 4.03 (m, 1H, C(@O)CH₂NH),

3.89 (m, 1H, C(@O)CH₂NH), 3.81 (m, 1H, H-3a), 3.63

(m, 1H, H-3b), 2.25 (m, 2H), 2.05–1.50 (m, 7H), 1.42

(m, 1H, H-19), 0.80 (t, J = 7.5 Hz, 3H, H-18) ppm, min-

117.6, 115.0, 110.4, 63.7, 46.6, 39.7, 37.7, 33.9, 30.4,

23

D

28.9, 21.9, 9.8 ppm; ½aꢁ ꢀ7 (c 0.70, CHCl₃); IR (ﬁlm)

or conformer (characteristic signals): d 8.38 (d,

J = 7.5 Hz, 1H, H_ar), 5.91 (m, 1H, H-6), 0.65 (t,

m 3436, 2962 cm^ꢀ1; HRMS (MALDI-TOF) calcd for

~

C₁₉H₂₇N₂O [(M+H)⁺]: 299.2123, found: 299.2122.

J = 7.5 Hz, 3H, H-18) ppm; ¹³C NMR (75.5 MHz,

CDCl₃) major conformer:

d 177.7 (C@O), 168.1

4.4. Macrocycle (3)

(C@O), 156.7 (C(@O)OCH₂), 143.8 ((Cq)_ar), 144.0

((Cq)_ar), 141.4 (2(Cq)_ar), 136.2 ((Cq)_ar), 131.9 (CH_ar),

131.4 (CH_ar), 128.2 (CH_ar), 127.9 (CH_ar), 127.2 (CH_ar),

125.3 (CH_ar), 123.4 (CH_ar), 120.1 (CH_ar), 119.7 (CH_ar),

113.8 ((Cq)_ar), 110.0 (C-6), 67.5 (C(@O)OCH₂), 47.2

(C(@O)OCH₂CH), 46.2 (C-3), 45.6 (C(@O)CH₂NH),

39.4 (C-20), 35.4 (CH₂), 34.0 (CH₂), 30.1 (CH₂), 29.5

(CH₂), 21.1 (CH₂), 9.4 (C-18) ppm, minor conformer

(characteristic signals): d 179.0 (C@O), 166.4 (C@O),

156.4 (C@O), 109.8 (C-6), 68.5 (C(@O)OCH₂), 46.5

(C(@O)OCH₂CH), 46.0 (CH₂), 45.3 (CH₂), 36.1

To a solution of compound 9 (42 mg, 0.14 mmol) in

THF (9.5 mL) at 20 ꢁC under argon was added dropwise

NaHMDS (2 M in THF, 141 lL, 0.14 mmol). After

cooling to 0 ꢁC, a solution of triphosgene (42 mg,

0.14 mmol) in THF (9.5 mL) was added dropwise and

the mixture was stirred at 0 ꢁC for 10 min. A saturated

aqueous solution of NaHCO₃was added and the aque-

ous layer was extracted with dichloromethane. After

drying over Na₂SO₄, the solvents were removed under

vacuum and the residue was puriﬁed by preparative

TLC (heptane/ethyl acetate 2:3) to give compound 3 as

(CH₂), 33.4 (CH₂), 30.4 (CH₂), 30.2 (CH₂), 21.4 (CH₂)

24

D

~

ppm; ½aꢁ ꢀ27 (c 0.43, CHCl₃); IR (ﬁlm) m 3349, 2963,

1

a beige powder (9.5 mg, 21%); mp 190 ꢁC; H NMR

1719 cm^ꢀ1; HRMS (ESI) calcd for C₃₇H₃₉N₃NaO₅

(300 MHz, CDCl₃) d 7.34 (m, 1H, H_ar), 7.26 (m, 3H,

H_ar), 6.54 (d, J = 2.7 Hz, 1H, H-5), 6.03 (br s, 1H, H-

1), 5.91 (d, J = 2.7 Hz, 1H, H-6), 4.17 (m, 1H, H-2a),

3.89 (t, J = 5.6 Hz, 1H, H-3), 3.44 (td, J = 10.5, 2.1 Hz

1H, H-2b), 2.00–1.90 (m, 2H, H-14eq and H-19a), 1.83

(m, 1H, H-14ax), 1.75 (m, 1H, H-15ax), 1.67 (m, 1H,

H-19b), 1.62 (m, 1H, H-15eq), 1.52 (m, 1H, H-16a),

1.46 (m, 1H, H-17a), 1.34 (m, 1H, H-17b), 1.28 (m,

1H, H-16b), 0.89 (t, J = 7.3 Hz, 3H, H-18) ppm; ¹³C

NMR (75.5 MHz, CDCl₃) d 156.5 (C@O), 138.6

((Cq)_ar), 136.2 ((Cq)_ar), 132.3 (C-12), 130.6 (C-21),

128.8 (CH_ar), 127.3 (CH_ar), 126.8 (CH_ar), 118.9 (C-5),

116.4 (C-7), 110.4 (C-6), 64.2 (C-2), 46.4 (C-3), 40.0

[(M+Na)⁺]: 614.2631, found: 614.2638.

4.6. Compound (11b)

Compound 11b was synthesized in the same manner as

for compound 11a from compound

8 (404 mg,

1.29 mmol) and Fmoc-^L-valine acid chloride 10b¹²

(510 mg, 1.29 mmol). The crude mixture was puriﬁed

by preparative TLC (dichloromethane/ethyl acetate

7:3), to give compound 11b as a white powder

(545 mg, 66%); mp 78 ꢁC; ¹H NMR (300 MHz, ace-

tone-d₆) 2 conformers in a 1/1 ratio: d 8.55 (m, 2H,

H_arand H⁰_ar), 8.36 (m, 2H, H_arand H_a⁰_r), 7.96 (d,

J = 7.2 Hz, 2H, H_arand H⁰_ar), 7.83 (d, J = 7.2Hz, 2H,

H_arand H⁰_ar), 7.50 (m, 2H, H_arand H_a⁰_r), 7.44 (m, 2H,

H_arand H_ar), 7.32 (m, 2H, H_arand H⁰_ar), 7.14 (m, 2H,

H_arand H⁰_ar), 6.73 (m, 2H, H-5 and H-5⁰), 5.96 (br s,

2H, NHFmoc and NHFmoc⁰), 5.96 (d, J = 2.1 Hz, 2H,

H-6 and H-6⁰), 4.65 (m, 4H, C(@O)OCH₂and

C(@O)OCH⁰₂), 4.32 (m, 1H, CHiPr), 4.22 (m, 1H,

CHiPr⁰), 3.91 (m, 4H, H-3 and H-3⁰), 2.46 (m, 2H,

CH(CH₃)₂and CH(CH₃)⁰₂), 2.38 (m, 4H), 2.27 (m,

(C-20), 37.3 (C-19), 35.4 (C-17), 30.7 (C-15), 25.2 (C-

24

D

16), 21.5 (C-14), 9.5 (C-18) ppm; ½aꢁ ꢀ90 (c 1.0,

CHCl₃); IR (ﬁlm) m 3265, 2957, 1721 cm^ꢀ1; HRMS

~

(ESI) calcd for C₂₀H₂₄N₂NaO₂[(M+Na)⁺]: 347.1735,

found: 347.1723.

4.5. Compound (11a)

To a vigorously stirred solution of compound 8 (564 mg,

1.81 mmol) in dichloromethane (12 mL) and 10% aque-

ous NaHCO₃(12 mL) at 20 ꢁC was added dropwise a

solution of Fmoc-glycine acid chloride 10a¹²(569 mg,

1.81 mmol) in dichloromethane (14 mL). After stirring

for 1.5 h, the mixture was diluted with water and dichlo-

romethane, and the two layers were decanted. The aque-

ous layer was acidiﬁed to pH 5 by addition of a 1 N HCl

aqueous solution and extracted with dichloromethane.

The organic layers were gathered, dried over MgSO₄,

and evaporated under vacuum. The residue was puriﬁed

by preparative TLC (dichloromethane/ethyl acetate 7:3),

to give compound 11a as a pale yellow powder (490 mg,

4H),

2.15

(m,

2H,

C(@O)OCH₂CH

and

C(@O)OCH₂CH⁰), 1.93 (m, 4H), 1.78 (m, 1H, H-19a),

1.70 (m, 4H), 1.60 (m, 1H, H-19a⁰), 1.55 (m, H-19b),

1.31 (m, 1H, H-19b⁰), 1.04 (m, 12H, CH(CH₃)₂and

CH(CH₃)⁰₂), 0.90 (t, J = 6.9 Hz, 3H, H-18), 0.76 (t,

J = 6.9 Hz, 3H, H-18⁰) ppm; ¹³C NMR (75.5 MHz, ace-

tone-d₆) d 175.3 (C@O), 174.8 (C@O), 169.4 (C@O),

169.3 (C@O), 157.0 (2C@O) ; 144.5 ((Cq)_ar), 141.7

((Cq)_ar), 137.6 ((Cq)_ar), 137.4 ((Cq)_ar), 132.0 ((Cq)_ar),

131.8 ((Cq)_ar), 131.5 (CH_ar), 129.2 ((Cq)_ar), 128.9

((Cq)_ar), 128.4 (CH_ar), 127.9 (CH_ar), 127.7 (CH_ar),

126.0 (CH_ar), 125.7 (CH_ar), 123.2 (CH_ar), 123.0 (CH_ar),

120.9 (CH_ar), 120.3 (CH_ar), 119.6 (CH_ar), 114.2 ((Cq)_ar),

1

46%); mp 146 ꢁC; H NMR (300 MHz, CDCl₃) 2 con-

´

A. Decor et al. / Bioorg. Med. Chem. 14 (2006) 2314–2332

2323

110.1 and 109.9 (C-6 and C-6⁰), 67.1 (C(@O)OCH₂

and C(@O)OCH⁰₂), 61.8 (CHiPr and CHiPr⁰), 46.5

and 46.4 (C-3 and C-3⁰), 39.8 (C(@O)OCH₂CH and

C(@O)OCH₂CH⁰), 39.6 (CH₂), 36.6 (CH₂), 36.0

(CH₂), 34.2 (CH₂), 33.3 (CH₂), 30.8 (CH₂), 30.6

(CH(CH₃)₂and CH(CH₃)⁰₂), 30.3 (CH₂), 30.2 (CH₂),

29.0 (C-20 and C-20⁰), 21.5 (CH₂), 21.3 (CH₂), 17.5

and 17.3 (CH(CH₃)₂and CH(CH₃)⁰₂), 9.33 and 8.96

(C-18 and C-18⁰) ppm; HRMS (ESI) calcd for

C₃₉H₄₃N₃NaO₅[(M+Na)⁺]: 656.3100, found: 656.3137.

(CH(CH₃)₂), 17.0 (CH(CH₃)₂), 7.89 (C-18) ppm; minor

conformer (characteristic signals): d 8.0 (C-18) ppm; IR

(ﬁlm) m 3363, 2964, 2360, 1710 cm^ꢀ1; HRMS (ESI) calcd

~

for C₂₄H₃₄N₃O₃[(M+Na)⁺]: 412.2600, found: 412.2605.

4.9. Macrocycle (4a)

To a solution of compound 12a (31 mg, 0.084 mmol)

in dry DMF (83 mL) under argon at 20 ꢁC were add-

ed TBTU (80 mg, 0.25 mmol), HOBT (34 mg,

0.25 mmol), and di isopropylethylamine (58 lL,

0.34 mmol). After stirring for 24 h, water was added

and the aqueous layer was extracted with ethyl ace-

tate. The organic layer was dried over Na₂SO₄and

concentrated to dryness. The crude mixture was puri-

ﬁed by ﬂash chromatography (C18, ethanol/water

60:40, then 65:35) followed by HPLC (semi-prepara-

4.7. Compound (12a)

To a solution of compound 11a (412 mg, 0.70 mmol) in

THF (23 mL) at 20 ꢁC was added piperidine (2.5 mL).

The mixture was stirred for 2.5 h and evaporated under

vacuum. The residue was puriﬁed by ﬂash chromatogra-

phy (C18, ethanol/water 9:1), to give compound 12a as a

white powder (227 mg, 88%); mp 178 ꢁC; ¹H NMR (300

MHz, CDCl₃) 2 conformers in a 82/18 ratio, major con-

former: d 9.27 (m, 1H, H-1), 8.45 (d, J = 7.5 Hz, 1H,

H_ar), 7.27 (m, 2H, H_ar), 7.03 (m, 1H, H_ar), 6.59 (m,

1H, H-5), 6.09 (m, 1H, H-6), 4.06 (m, 2H, H-3), 3.35

(m, 2H, C(@O)CH₂NH₂), 2.93 (br s, 2H, NH₂), 2.15

(m, 2H), 1.93–1.59 (m, 6H), 1.41 (m, 1H, H-19a), 0.81

(m, 1H, H-19b), 0.81 (t, J = 6.3 Hz, 3H, H-18) ppm,

minor conformer (characteristic signals): d 9.17 (m,

H-1), 0.69 (t, J = 6.6 Hz, 3H, H-18) ppm; ¹³C NMR

(75.5 MHz, CDCl₃) major conformer: d 179.9 (C-2),

171.3 (NHC(@O)), 137.1 (CH_ar), 132.1 ((Cq)_ar), 131.7

(CH_ar), 128.9 ((Cq)_ar), 127.7 (CH_ar), 126.8 (CH_ar),

119.7 (C-5), 114.3 ((Cq)_ar), 110.1 (C-6), 46.4 (C-3 and

C(@O)CH₂NH₂), 39.6 (C-20), 38.1 (CH₂), 30.1 (CH₂),

tive C18 Symmetry, H₂O/CH₃CN 3:2, 4.7 mL min^ꢀ1

)

to give compound 4a as a white ﬁlm (9 mg, 30%);

HPLC analysis (analytic C18 Symmetry, H₂O/CH₃CN

3:2, 1 mL min^ꢀ1) t_R11.1 min; ¹H NMR (500 MHz,

acetone-d₆) 2 conformers in a 1/1 ratio, conformer

4ax: d 8.41 (d, J = 8.0 Hz, 1H, H-12), 8.34 (dd,

J = 7.0, 7.0 Hz, 1H, CH₂NHC(@O)), 8.22 (s, 1H, H-

1), 7.26 (dd, J = 7.5, 7.5 Hz, 1H, H-11), 7.10 (m,

1H, H-9), 6.99 (dd, J = 7.5, 7.5 Hz, 1H, H-10), 6.77

(d, J = 2.5 Hz, 1H, H-5), 5.75 (d, J = 2.5 Hz, 1H, H-

6), 4.09 (m, 1H, H-3a), 3.89 (m, 1H, H-3b), 3.86

(dd, J = 16.0, 7.0 Hz, 1H, C(@O)CH₂NH), 3.56 (dd,

J = 16.0, 5.8 Hz, 1H, C(@O)CH₂NH), 2.42 (m, 1H,

H-16a), 2.04 (m, 1H, H-16b), 1.96 (m, 1H, H-14a),

1.89 (m, 1H, H-17a), 1.83 (m, 1H, H-15a), 1.80 (m,

1H, H-14b), 1.69 (m, 1H, H-19a), 1.60 (m, 1H, H-

15b), 1.27 (m, 1H, H-19b), 0.80 (t, J = 7.5 Hz, 3H,

H-18) ppm, conformer 4ay: d 8.57 (s, 1H, H-1), 8.34

(dd, J = 7.0, 7.0 Hz, 1H, CH₂NHC(@O)), 7.92 (m,

1H, H-12), 7.30 (dd, J = 7.5, 7.5 Hz, 1H, H-11), 7.11

(m, 1H, H-10), 7.10 (m, 1H, H-9), 6.72 (d,

J = 2.3 Hz, 1H, H-5), 5.97 (d, J = 2.3 Hz, 1H, H-6),

4.09 (m, 1H, H-3a), 3.89 (m, 1H, H-3b), 3.86 (dd,

J = 16.0, 7.0 Hz, 1H, C(@O)CH₂NH), 3.56 (dd,

J = 16.0, 5.8 Hz, 1H, C(@O)CH₂NH), 2.42 (m, 1H,

H-16a), 2.04 (m, 1H, H-16b), 1.96 (m, 1H, H-14a),

1.89 (m, 1H, H-17a), 1.83 (m, 1H, H-15a), 1.80 (m,

1H, H-14b), 1.60 (m, 1H, H-15b), 1.36 (m, 1H, H-

17b), 1.25 (m, 1H, H-19a), 1.05 (m, 1H, H-19b),

0.60 (t, J = 7.5 Hz, 3H, H-18) ppm; ¹³C NMR

(125.8 MHz, acetone-d₆) conformer 4ax: d 177.3 (C-

2), 169.5 (C(@O)CH₂), 138.1 (C-13), 132.0 (C-9),

130.8 (C-21), 128.4 (C-11), 128.2 (C-8), 122.6 (C-10),

120.8 (C-5), 116.7 (C-12), 113.5 (C-7), 111.3 (C-6),

48.0 (C(@O)CH₂NH), 46.5 (C-3), 41.2 (C-20), 39.9

(C-17), 36.9 (C-19), 32.0 (C-15), 29.8 (C-16), 23.2

(C-14), 9.9 (C-18) ppm, conformer 4ay: d 177.3 (C-

2), 169.5 (C(@O)CH₂), 136.9 (C-13), 131.8 (C-9),

129.1 (C-21), 128.2 (C-8), 128.0 (C-11), 124.3 (C-10),

119.7 (C-5), 116.7 (C-12), 114.9 (C-7), 111.3 (C-6),

48.0 (C(@O)CH₂NH), 46.5 (C-3), 39.9 (C-17), 39.8

26.4 (CH₂), 22.9 (CH₂), 21.8 (CH₂), 9.55 (C-18) ppm,

24

~

m

½aꢁ ꢀ23 (c 0.40, CHCl₃); IR (ﬁlm)

3361,

D

2962, 2360, 1706 cm^ꢀ1; HRMS (MALDI-TOF) calcd

for C₂₁H₂₇N₃NaO₅[(M+Na)⁺]: 392.1950, found:

392.1947.

4.8. Compound (12b)

Compound 12b was synthesized in the same manner

as for compound 12a from compound 11b (491 mg,

0.77 mmol). Yellow powder (294 mg, 92%); mp

1

100 ꢁC; H NMR (300 MHz, DMSO-d₆) 2 conformers

in a 4/1 ratio, major conformer: d 8.89 (d, J = 8.1 Hz,

1H, H_ar), 7.70 (dd, J = 8.1, 8.1 Hz, 1H, H_ar), 7.64 (m,

1H, H_ar), 7.41 (m, 1H, H_ar), 7.22 (d, J = 2.1 Hz, 1H,

H-5), 6.32 (d, J = 2.1 Hz, 1H, H-6), 4.36 (m, 2H, H-

3), 4.17 (d, J = 3.3 Hz, 1H, CHiPr), 3.44 (m, 2H),

2.64 (m, 1H), 2.44 (m, 1H, CH(CH₃)₂), 2.19–1.94

(m, 7H), 1.68 (m, 1H, H-19a), 1.53 (m, 1H, H-19b),

1.34 (d, J = 6.6 Hz, 3H, CH(CH₃)₂), 1.24 (d,

J = 6.6 Hz, 3H, CH(CH₃)₂), 0.94 (t, J = 7.2 Hz, 3H,

H-18) ppm, minor conformer (characteristic signals):

d 7.13 (d, J = 2.1 Hz, 1H, H-5), 6.26 (d, J = 2.1 Hz,

1H, H-6) ppm; ¹³C NMR (75.5 MHz, DMSO-d₆)

major conformer d 174.3 (C-2), 169.3 (NHC(@O)),

136.5 ((Cq)_ar), 130.4 (CH_ar), 130.0 ((Cq)_ar), 127.7

((Cq)_ar), 126.8 (CH_ar), 121.5 (CH_ar), 118.0 (C-5),

117.8 (CH_ar), 113.0 ((Cq)_ar), 108.8 (C-6), 68.1 (CHiPr),

45.1 (C-3), 36.3 (CH₂), 32.6 (CH₂), 32.3 (CH₂), 29.8

(CH₂), 29.1 (CH₂), 29 (C-20), 20.5 (CH₂), 18.3

(C-20), 35.9 (C-19), 32.0 (C-15), 29.8 (C-16), 23.2

23

D

(C-14), 9.4 (C-18) ppm; ½aꢁ ꢀ10 (c 0.58, CHCl₃);

IR (ﬁlm) m 3353, 2963, 1682 cm^ꢀ1; HRMS (ESI) calcd

~

for C₂₁H₂₅N₃NaO₂[(M+Na)⁺]: 374.1844, found:

374.1836.

´

A. Decor et al. / Bioorg. Med. Chem. 14 (2006) 2314–2332

2324

4.10. Macrocycle (4b)

(dd, J = 12.7, 12.7 Hz, 1H, H-17ax), 2.37 (dd, J = 13.2,

13.2 Hz, 1H, H-16ax), 2.24 (ddddd, J = 12.8, 12.8,

12.8, 5.5, 2.7 Hz, 1H, H-14ax), 1.98 (dd, J = 13.2,

7.5 Hz, 1H, H-16eq), 1.84 (m, 1H, H-14eq), 1.70 (ddd,

J = 13.0, 13.0, 2.8Hz, 1H, H-15ax), 1.54 (m, 1H, H-

15eq), 1.43 (m, 1H, H-17eq), 1.38 (m, 1H, H-19a), 1.18

(m, 1H, H-19b), 1.03 (m, J = 7.0 Hz, 3H, NCH₂CH₃),

0.68 (t, J = 7.2 Hz, 3H, H-18) ppm; ¹³C NMR

(75.5 MHz, CDCl₃) d 174.6 (C-2), 143.6 (C-13), 139.4

((Cq)_ar), 131.8 (CH_ar), 130.7 (C-21), 128.0 (CH_ar),

126.6 (CH_ar), 126.5 (CH_ar), 118.2 (C-5), 116.5 ((Cq)_ar),

110.3 (C-6), 46.2 (C-3), 43.8 (NCH₂), 38.9 (C-20), 36.6

(C-17), 33.3 (C-15), 30.2 (C-16 or C-19), 29.7 (C-19 or

C-16), 19.5 (C-14), 13.7 (NCH₂CH₃), 8.2 (C-18) ppm;

Compound 4b was synthesized in the same manner as

for compound 4a from compound 12b (61 mg,

0.15 mmol). White ﬁlm (6 mg, 10%); ¹H NMR

(500 MHz, acetone-d₆) 2 conformers in a 85/15 ratio,

major conformer: d 8.14 (dd, J = 8.2, 1.3 Hz, 2H, H-12

and H-1), 7.30 (ddd, J = 7.7, 7.7, 1.6 Hz, 1H, H_ar),

7.17 (dd, J = 7.7, 1.4 Hz, 1H, H-9), 7.09 (ddd, J = 7.5,

7.5, 1.4 Hz, 1H, H_ar), 6.70 (d, J = 2.7 Hz, 1H, H-5),

6.47 (d, J = 8.3 Hz, 1H, CHNHC(@O)), 5.86 (d,

J = 2.7 Hz, 1H, H-6), 3.93 (dd, J = 5.5, 5.5 Hz, 2H, H-

3), 3.82 (dd, J = 9.8, 8.0 Hz, 1H, CHiPr), 2.48 (m, 1H,

H-16a), 2.16 (m, 1H, H-16b), 2.00 (m, 1H, H-14a),

1.87 (m, 1H, H-14b), 1.79 (m, 2H, H-15), 1.58 (m, 1H,

CH(CH₃)₂), 1.52 (m, 2H, H-17), 1.34 (m, 1H, H-19a),

1.13 (m, 1H, H-19b), 1.01 (d, J = 7.4 Hz, 3H,

CH(CH₃)₂), 0.95 (d, J = 7.4 Hz, 3H, CH(CH₃)₂), 0.65

(t, J = 7.4 Hz, 3H, H-18) ppm, minor conformer (charac-

teristic signals): d 7.47 (ddd, J = 7.5, 7.5, 2.0 Hz, 1H, H-

12), 7.24 (ddd, J = 7.5, 7.5, 2.0 Hz, 1H, H_ar), 6.96 (dd,

J = 7.5, 7.5 Hz, 1H, H_ar), 6.66 (d, J = 2.5 Hz, 1H, H-

5), 5.67 (d, J = 2.5 Hz, 1H, H-6), 0.79 (t, J = 7.3 Hz,

3H, H-18) ppm; ¹³C NMR (125.8 MHz, acetone-d₆)

major conformer (characteristic signals): d 132.0 (CH_ar),

128.1 (CH_ar), 124.0 (CH_ar), 122.1 (CH_ar), 120.2 (C-5),

114.4 (C-6), 64.6 (CHiPr), 46.4 (C-3), 41.5 (C-17), 36.3

(C-19), 32.1 (C-15), 30.7 (C-16), 30.3 (CH(CH₃)₂), 23.9

(C-14), 19.3 (CH(CH₃)₂), 9.3 (C-18) ppm; HRMS

(ESI) calcd for C₂₄H₃₁N₃NaO₂[(M+Na)⁺]: 416.2314,

found: 416.2300.

25

ꢀ1

~

½aꢁ ꢀ338 (c 1.0, CHCl₃); IR (ﬁlm) m 2962, 1651 cm

;

D

HRMS (ESI) calcd for C₂₁H₂₆N₂NaO [(M+Na)⁺]:

345.1943, found: 345.1942.

4.13. 1-Propylrhazinilam (5b)

White powder (26 mg, 32% from 71 mg rhazinilam); mp

165 ꢁC; ¹H NMR (300 MHz, CDCl₃) d 7.45 (dd, J = 6.9,

1.8Hz, 1H, H_ar), 7.38 (ddd, J = 7.5, 7.5, 1.8 Hz, 1H,

H_ar), 7.29 (ddd, J = 7.5, 7.5, 1.8 Hz, 1H, H_ar), 7.21

(dd, J = 7.5, 1.2Hz, 1H, H_ar), 6.47 (d, J = 2.7 Hz, 1H,

H-5), 5.83 (d, J = 2.7 Hz, 1H, H-6), 4.03 (dd, J = 12.0,

5.1Hz, 1H, H-3eq), 3.81 (ddd, J = 12.0, 12.0, 4.5Hz,

1H, H-3ax), 3.68 (ddd, J = 13.5, 9.3, 6.6Hz, 1H,

NCH₂), 2.93 (ddd, J = 13.2, 9.6, 6.2 Hz, 1H, NCH₂),

2.50 (dd, J = 12.3, 12.3, 1H, H-17ax), 2.38 (dd,

J = 12.9, 12.9 Hz, 1H, H-16ax), 2.22 (ddddd, J = 12.3,

12.3, 12.3, 5.4, 3.0 Hz, 1H, H-14ax), 1.99 (dd, J = 13.2,

7.8 Hz, 1H, H-16eq), 1.84 (m, 1H, H-14eq), 1.72 (ddd,

J = 13.2, 13.2, 3.0 Hz, 1H, H-15ax), 1.52 (m, 4H, H-

15eq and NCH₂CH₂), 1.42 (m, 1H, H-17eq), 1.37 (m,

1H, H-19a), 1.16 (m, 1H, H-19b), 0.84 (t, J = 7.5 Hz,

3H, N(CH₂)₂CH₃), 0.70 (t, J = 7.4 Hz, 3H, H-18) ppm;

¹³C NMR (75.5 MHz, CDCl₃) d 174.8 (C-2), 144.2 (C-

13), 139.3 (C-8), 131.8 (CH_ar), 130.7 (C-21), 128.0

(CH_ar), 126.6 (CH_ar), 126.5 (CH_ar), 118.3 (C-5), 116.5

(C-7), 110.2 (C-6), 51.1 (NCH₂), 46.2 (C-3), 38.9 (C-

20), 36.6 (C-17), 33.3 (C-15), 30.2 (C-19), 29.7 (C-16),

4.11. General procedure for the N-1 and C-16 substitution

of (ꢀ)-rhazinilam (1)

To a solution of (ꢀ)-rhazinilam 1 (100 mg, 0.34 mmol)

in THF (2.5 mL) at 0 ꢁC under argon was added drop-

wise n-butyllithium (1.6 M in hexane, 212 lL,

0.34 mmol for N-1 substitutions or 424 lL, 0.68 mmol

for C-16 substitutions). After stirring the mixture for

30 min at 0 ꢁC, the electrophilic reagent (see Table 1,

0.34 mmol) was added dropwise and the mixture was al-

lowed to warm to 20 ꢁC. The evolution of the reaction

was monitored by TLC. When no more evolution was

observed, the reaction was quenched with a saturated

aq solution of NH₄Cl and the aqueous layer was

extracted with dichloromethane. After drying over

MgSO₄, the solution was evaporated under reduced

pressure and the residue was puriﬁed by ﬂash or thin

layer chromatography. For compounds 6a–d, the

diastereoisomeric mixture obtained after puriﬁcation

on silica gel was separated by preparative HPLC.

21.5 (NCH₂CH₂), 19.5 (C-14), 11.5 (N(CH₂)₂CH₃), 8.2

~

23

(C-18) ppm; ½aꢁ ꢀ332 (c 1.0, CHCl₃); IR (ﬁlm) m

D

2963, 1648 cm^ꢀ1; HRMS (ESI) calcd for C₂₂H₂₈N₂NaO

[(M+Na)⁺]: 359.2099, found: 359.2105.

4.14. 1-Allylrhazinilam (5c)

White powder (58 mg, 46% from 112 mg rhazinilam);

1

mp 158 ꢁC; H NMR (300 MHz, CDCl₃) d 7.40 (dd,

J = 7.2, 1.8 Hz, 1H, H_ar), 7.31 (ddd, J = 7.7, 7.7,

1.8 Hz, 1H, H_ar), 7.25 (ddd, J = 7.5, 7.5, 1.8 Hz, 1H,

H_ar), 7.18 (dd, J = 7.5, 1.5Hz, 1H, H_ar), 6.44 (d,

J = 2.4 Hz, 1H, H-5), 5.77 (d, J = 3.0 Hz, 1H, H-6),

5.74 (m, 1H, NCH₂CH), 5.03 (m, 2H, NCH₂CHCH₂),

4.41 (dd, J = 14.7, 6.0H z, 1H, NCH₂), 4.00 (dd,

J = 12.0, 5.1 Hz, 1H, H-3eq), 3.77 (ddd, J = 12.0, 12.0,

4.5 Hz, 1H, H-3ax), 3.49 (dd, J = 14.7, 6.9 Hz, 1H,

NCH₂), 2.47 (dd, J = 12.6, 12.6 Hz, 1H, H-17ax), 2.38

(dd, J = 12.8, 12.8 Hz, 1H, H-16ax), 2.22 (ddddd,

J = 13.0, 13.0, 13.0, 5.4, 2.7 Hz, 1H, H-14ax), 1.98 (dd,

J = 13.2, 7.8 Hz, 1H, H-16eq), 1.84 (m, 1H, H-14eq),

4.12. 1-Ethylrhazinilam (5a)

White powder (65 mg, 66% from 89 mg rhazinilam); mp

169 ꢁC; ¹H NMR (300 MHz, CDCl₃) d 7.42 (dd,

J = 7.3Hz, J = 1.1Hz, 1H, H_ar), 7.34 (m, 2H, H_ar), 7.22

(dd, J = 8.5Hz, J = 1.5Hz, 1H, H_ar), 6.45 (d,

J = 2.8Hz, 1H, H-5), 5.81 (d, J = 3.2 Hz, 1H, H-6),

4.02 (dd, J = 12.0, 5.2Hz, 1H, H-3eq), 3.79 (ddd,

J = 12.0, 12.0, 4.7 Hz, 1H, H-3ax), 3.69 (m, 1H,

NCH₂), 3.14 (dq, J = 13.5, 7.2Hz, 1H, NCH₂), 2.49

´

A. Decor et al. / Bioorg. Med. Chem. 14 (2006) 2314–2332

2325

1.69 (ddd, J = 13.5, 13.5, 3.0 Hz, 1H, H-15ax), 1.51 (m,

4.17. 1-(2-Methoxy)ethoxymethylrhazinilam (5f)

1H, H-15eq), 1.44 (m, 1H, H-17eq), 1.38 (m, 1H, H-

19a), 1.17 (m, 1H, H-19b), 0.66 (t, J = 7.4Hz, 3H, H-

18) ppm; ¹³C NMR (75.5 MHz, CDCl₃) d 174.7 (C-2),

143.8 (C-13), 139.4 (C-8), 134.5 (NCH₂CH), 131.8

(CH_ar), 130.7 (C-21), 128.0 (CH_ar), 126.9 (CH_ar), 126.8

(CH_ar), 118.5 (C-5), 117.2 (NCH₂CHCH₂), 116.6 (C-

7), 110.3 (C-6), 52.1 (NCH₂), 46.3 (C-3), 39.0 (C-20),

1

Oil (243 mg, 62% from 300 mg rhazinilam); H NMR

(300 MHz, CDCl₃) d 7.40 (d, J = 7.5 Hz, 1H, H_ar),

7.34 (m, 3H, H_ar), 6.45 (d, J = 2.7 Hz, 1H, H-5), 5.75

(d, J = 2.4 Hz, 1H, H-6), 5.39 (d, J = 10.2 Hz, 1H,

NCH₂), 4.28 (d, J = 10.5 Hz, 1H, NCH₂), 4.00 (dd,

J = 12.1, 5.3 Hz, 1H, H-3eq), 3.79 (m, 1H, H-3ax),

3.74 (m, 1H, CH₂OCH₂), 3.58–3.49 (m, 3H,

NCH₂OCH₂and CH₂OCH₃), 3.38 (s, 3H, OCH₃),

2.46 (m, 1H, H-17a), 2.42 (m, 1H, H-16a), 2.18 (ddddd,

J = 13.2, 13.2, 13.2, 5.4, 2.7 Hz, 1H, H-14ax), 1.96 (m,

1H, H-16b), 1.83 (m, 1H, H-14eq), 1.70 (ddd, J = 13.2,

13.2, 3.0 Hz, 1H, H-15ax), 1.52 (m, 1H, H-15eq), 1.47

(m, 1H, H-17b), 1.40 (m, 1H, H-19a), 1.20 (dq,

J = 14.5, 7.3 Hz, 1H, H-19b), 0.70 (t, J = 7.3 Hz, 3H,

H-18) ppm; ¹³C NMR (75.5 MHz, CDCl₃) d 176.0 (C-

2), 143.1 (C-13), 138.9 (C-8), 131.4 (CH_ar), 130.4 (C-

21), 128.2 (CH_ar), 127.1 (CH_ar), 126.9 (CH_ar), 118.7

(C-5), 116.8 (C-7), 109.8 (C-6), 77.7 (NCH₂), 71.7

(CH₂OCH₃), 67.9 (NCH₂OCH₂), 59.0 (OCH₃), 46.1

36.7 (C-17), 33.3 (C-15), 30.3 (C-19), 29.7 (C-16), 19.6

24

D

(C-14), 8.3 (C-18) ppm; ½aꢁ ꢀ302 (c 1.0, CHCl₃);

IR (ﬁlm) m 2964, 1651 cm^ꢀ1; HRMS (ESI) calcd

~

for C₂₂H₂₆N₂NaO [(M+Na)⁺]: 357.1943, found:

357.1953.

4.15. 1-Cyanomethylrhazinilam (5d)

White powder (70 mg, 81% from 76 mg rhazinilam); mp

1

138 ꢁC; H NMR (300 MHz, CDCl₃) d 7.47–7.34 (m,

4H, H_ar), 6.50 (d, J = 3.0 Hz, 1H, H-5), 5.82 (d,

J = 2.1Hz, 1H, H-6), 4.95 (d, J = 17.4 Hz, 1H, NCH₂),

4.02 (dd, J = 11.4, 5.1Hz, 1H, H-3eq), 3.77 (ddd,

J = 12.0, 12.0, 4.2 Hz, 1H, H-3ax), 3.62 (d,

J = 17.4 Hz, 1H, NCH₂), 2.44 (m, 1H, H-17a), 2.42

(m, 1H, H-16a), 2.18 (ddddd, J = 13.2, 13.2, 13.2, 5.4,

2.7 Hz, 1H, H-14ax), 2.04 (m, 1H, H-16b), 1.88 (m,

1H, H-14eq), 1.71 (ddd, J = 13.2, 13.2, 3.0 Hz, 1H, H-

15ax), 1.54 (m, 1H, H-15eq), 1.50 (m, 1H, H-17b),

1.37 (m, 1H, H-19a), 1.13 (m, 1H, H-19b), 0.69 (t,

J = 7.5 Hz, 3H, H-18) ppm; ¹³C NMR (75.5 MHz,

CDCl₃) d 175.1 (C-2), 141.9 (C-13), 139.4 ((Cq)_ar),

132.1 (CH_ar), 130.7 (C-21), 128.9 (CH_ar), 128.4 (CH_ar),

127.0 (CH_ar), 119.2 ((Cq)_ar), 116.1 (C-5), 115.7

(NCH₂CN), 109.7 (C-6), 46.2 (C-3), 39.0 (C-20), 36.7

(C-3), 38.9 (C-20), 36.7 (C-17), 33.1 (C-15), 30.2 (C-

19), 29.5 (C-16), 19.5 (C-14), 8.2 (C-18) ppm; ½aꢁ

25

D

;

ꢀ1

~

ꢀ312 (c 1.0, CHCl₃); IR (ﬁlm) m 2941, 1667 cm

HRMS (ESI) calcd for C₂₃H₃₀N₂NaO₃[(M+Na)⁺]:

405.2154, found: 405.2156.

4.18. 1-(2-Trimethylsilyl)ethoxymethylrhazinilam (5g)

1

Oil (399 mg, 71% from 399 mg rhazinilam); H NMR

(300 MHz, CDCl₃) d 7.40 (d, J = 7.5 Hz, 1H, H_ar),

7.34 (dd,J = 6.3, 1.2 Hz, 1H, H_ar), 7.29 (m, 2H, H_ar),

6.46 (d, J = 2.7 Hz, 1H, H-5), 5.76 (d, J = 2.7 Hz, 1H,

H-6), 5.37 (d, J = 10.1 Hz, 1H, NCH₂), 4.14 (d,

J = 10.1 Hz, 1H, NCH₂), 3.98 (dd, J = 12.0, 5.1 Hz,

1H, H-3eq), 3.78 (ddd, J = 12.0, 12.0, 4.8 Hz, 1H, H-

3ax), 3.62 (td, J = 9.9, 6.4 Hz, 1H, NCH₂OCH₂), 3.47

(td, J = 9.9, 6.4 Hz, 1H, NCH₂OCH₂), 2.47 (m, 1H,

H-17a), 2.41 (m, 1H, H-16a), 2.19 (ddddd, J = 12.9,

12.9, 12.9, 5.6, 2.7 Hz, 1H, H-14ax), 2.01 (m, 1H, H-

16b), 1.84 (m, 1H, H-14eq), 1.71 (ddd, J = 12.9, 12.9,

3.0 Hz, 1H, H-15ax), 1.53 (m, 1H, H-15eq), 1.47 (m,

1H, H-19a), 1.43 (m, 1H, H-17b), 1.20 (dq, J = 14.4,

7.4 Hz, 1H, H-19b), 0.96 (m, 1H, CH₂Si), 0.92 (m, 1H,

CH₂Si), 0.70 (t,J = 7.4 Hz, 3H, H-18), 0.00 (s, 9H,

Si(CH₃)₃) ppm; ¹³C NMR (75.5 MHz, CDCl₃) d 177.1

(C-2), 144.6 (C-13), 140.4 (C-8), 132.8 (CH_ar), 131.8

(C-21), 129.5 (CH_ar), 128.4 (CH_ar), 128.3 (CH_ar), 120.1

(C-5), 118.2 (C-7), 111.1 (C-6), 78.3 (NCH₂), 67.2

(NCH₂OCH₂), 47.5 (C-3), 40.3 (C-20), 38.1 (C-17),

(NCH₂), 36.5 (C-17), 33.0 (C-15), 30.2 (C-19), 29.0 (C-

24

16), 19.5 (C-14), 8.3 (C-18) ppm; ½aꢁ ꢀ321 (c 1.0,

D

CHCl₃); IR (ﬁlm) ~m 2943, 2250, 1669 cm^ꢀ1; HRMS

(ESI) calcd for C₂₂H₂₆N₂NaO [(M+Na)⁺]: 356.1739,

found: 356.1719.

4.16. 1-Methoxymethylrhazinilam (5e)

White powder (53 mg, 76% from 60 mg rhazinilam); mp

1

131 ꢁC; H NMR (300 MHz, CDCl₃) d 7.43 (m, 1H,

H_ar), 7.36 (m, 1H, H_ar), 7.31 (m, 2H, H_ar), 6.48 (d,

J = 2.7 Hz, 1H, H-5), 5.78 (d, J = 2.7 Hz, 1H, H-6),

5.30 (d, J = 10.2 Hz, 1H, NCH₂), 4.18 (d, J = 10.2 Hz,

1H, NCH₂), 4.02 (dd, J = 12.0, 5.1 Hz, 1H, H-3eq),

3.79 (ddd, J = 12.0, 12.0, 4.5 Hz, 1H, H-3ax), 3.33 (s,

1H, OCH₃), 2.48 (m, 1H, H-17a), 2.44 (m, 1H, H-

16a), 2.22 (ddddd, J = 13.2, 13.2, 13.2, 5.1, 2.7 Hz, 1H,

H-14ax), 2.04 (m, 1H, H-16b), 1.86 (m, 1H, H-14eq),

1.73 (ddd, J = 13.5, 13.5, 3.0 Hz, 1H, H-15ax), 1.54

(m, 1H, H-15eq), 1.47 (m, 1H, H-17b), 1.38 (m, 1H,

H-19a), 1.21 (m, 1H, H-19b), 0.71 (t, J = 7.4 Hz, 3H,

H-18) ppm; ¹³C NMR (75.5 MHz, CDCl₃) d 176.0 (C-

2), 143.1 (C-13), 139.0 (C-8), 131.5 (CH_ar), 130.4

((Cq)_ar), 128.2 (CH_ar), 127.1 (CH_ar), 126.8 (CH_ar),

118.7 (C-5), 116.7 ((Cq)_ar), 109.7 (C-6), 79.0 (NCH₂),

56.4 (OCH₃), 46.1 (C-3), 38.9 (C-20), 36.7 (C-17), 33.1

34.5 (C-15), 31.6 (C-19), 30.9 (C-16), 20.9 (C-14), 19.5

22

D

(CH₂Si), 9.6 (C-18), 0.0 (Si(CH₃)₃) ppm; ½aꢁ ꢀ183 (c

1.0, CHCl₃); IR (ﬁlm) m 2950, 1668 cm^ꢀ1; HRMS

~

(ESI) calcd for C₂₅H₃₆N₂NaO₂Si [(M+Na)⁺]: 447.2444,

found: 447.2418.

4.19. 1-Benzylrhazinilam (5h)

Oil (41 mg, 53% from 60 mg rhazinilam); ¹H NMR

(300 MHz, CDCl₃) d 7.44 (dd, J = 7.5, 1.5 Hz, 1H,

H_ar), 7.30–7.15 (m, 7H, H_ar), 6.81 (dd, J = 7.5, 1.5 Hz,

1H, H_ar), 6.48 (d, J = 2.7 Hz, 1H, H-5) ; 5.77 (d,

J = 2.7 Hz, 1H, H-6), 5.33 (d, J = 14.6 Hz, 1H,

(C-15), 30.2 (C-19), 29.5 (C-16), 19.5 (C-14), 8.2 (C-18)

~

22

ppm; ½aꢁ ꢀ317 (c 1.0, CHCl₃); IR (ﬁlm) m 2937, 1669

D

HRMS (ESI) calcd for C₂₁H₂₆N₂NaO₂

cm^ꢀ1

;

[(M+Na)⁺]: 361.1892, found: 361.1869.

´

A. Decor et al. / Bioorg. Med. Chem. 14 (2006) 2314–2332

2326

NCH₂), 4.05 (dd, J = 11.7, 5.1 Hz 1H, H-3eq), 3.81

(ddd, J = 12.0, 12.0, 4.5 Hz, 1H, H-3ax), 3.70 (d,

J = 14.6 Hz, 1H, NCH₂), 2.54 (m, 1H, H-17a), 2.43

(m, 1H, H-16a), 2.30 (ddddd, J = 12.9, 12.9, 12.9, 5.4,

2.7 Hz, 1H, H-14ax), 2.05 (m, 1H, H-16b), 1.86 (m,

1H, H-14eq), 1.73 (ddd, J = 13.2, 13.2, 3.0 Hz, 1H, H-

15ax), 1.55 (m, 1H, H-15eq), 1.48 (m, 1H, H-17b),

1.41 (m, 1H, H-19a), 1.19 (m, 1H, H-19b), 0.70 (t,

J = 7.2 Hz, 3H, H-18) ppm; ¹³C NMR (75.5 MHz,

CDCl₃) d 174.9 (C-2), 144.0 ((Cq)_ar), 139.2 ((Cq)_ar),

138.3 ((Cq)_ar), 131.7 (CH_ar), 130.5 (C-21), 129.0 (CH_ar),

128.3 (CH_ar), 127.8 (CH_ar), 127.1 (CH_ar), 126.8 (CH_ar),

126.7 (CH_ar), 118.5 (C-5), 116.5 ((Cq)_ar), 109.8 (C-6),

52.7 (NCH₂), 46.2 (C-3), 39.0 (C-20), 36.6 (C-17), 33.2

(CH_ar), 127.2 (CH_ar), 126.2 (CH_ar), 119.0 (C-5), 117.1

(C-7), 110.8 (C-6), 46.1 (C-3), 44.0 (NCOC(CH₃)₃),

39.1 (C-20), 36.4 (C-17), 32.7 (C-15), 31.1 (C-16), 30.0

(C-19), 28.4 (2 NCOC(CH₃)₃), 27.2 (NCOC(CH₃)₃),

24

D

19.7 (C-14), 8.4 (C-18) ppm; ½aꢁ ꢀ491 (c 1.0, CHCl₃);

IR (ﬁlm) m 2961, 1693 cm^ꢀ1; HRMS (ESI) calcd

~

for C₂₄H₃₀N₂NaO₂[(M+Na)⁺]: 402.2239, found:

402.2274.

4.22. 1-Benzoylrhazinilam (5k)

Pale yellow solid (66 mg, 59% from 82 mg rhazinilam);

mp 90 ꢁC; ¹H NMR (300 MHz, CDCl₃) d 8.04 (d,

J = 6.3 Hz, 1H, H_ar), 7.52 (m, 2H, H_ar), 7.46–7.24

(m, 5H, H_ar), 7.16 (d, J = 7.2 Hz, 1H, H_ar), 6.40 (d,

J = 2.7 Hz, 1H, H-5), 5.87 (d, J = 3.0 Hz, 1H, H-6),

3.86 (dd, J = 12.3, 5.1 Hz 1H, H-3eq), 3.66 (ddd,

J = 12.3, 12.3, 4.6 Hz, 1H, H-3ax), 2.58 (m, 1H, H-

16a), 2.36 (ddd, J = 12.9, 12.9, 1.5 Hz, 1H, H-17ax),

2.11 (m, 1H, H-16b), 2.02 (m, 1H, H-14a), 1.86 (m,

1H, H-14b), 1.70 (m, 1H, H-15a), 1.47 (m, 1H, H-

19a), 1.40 (m, 1H, H-15b), 1.32 (m, 1H, H-17eq),

1.17 (m, 1H, H-19b), 0.71 (t, J = 7.2 Hz, 3H, H-18)

ppm; ¹³C NMR (75.5 MHz, CDCl₃) d 176.6 (C-2),

173.6 (NCO), 141.0 ((Cq)_ar), 139.4 ((Cq)_ar), 136.1

((Cq)_ar), 133.8 (CH_ar), 131.7 (CH_ar), 131.6 (CH_ar),

130.2 (CH_ar), 130.1 (C-21), 128.6 (CH_ar), 128.5 (CH_ar),

128.2 (CH_ar), 127.6 (CH_ar), 126.7 (CH_ar), 119.6 (C-5),

116.9 ((Cq)_ar), 110.1 (C-6), 46.0 (C-3), 39.1 (C-20),

(C-15), 30.2 (C-19), 29.6 (C-16), 19.5 (C-14), 8.2 (C-18)

~

25

ppm; ½aꢁ ꢀ293 (c 1.0, CHCl₃); IR (ﬁlm) m 2962, 1652

D

HRMS (ESI) calcd for C₂₆H₂₈N₂NaO

cm^ꢀ1

;

[(M+Na)⁺]: 407.2058, found: 407.2099.

4.20. 1-Acetylrhazinilam (5i)

White powder (32 mg, 50% from 56 mg rhazinilam); mp

118 ꢁC; ¹H NMR (300 MHz, CDCl₃) d 7.40 (dd, J = 7.6,

1.8 Hz, 1H, H_ar), 7.35 (ddd, J = 7.8, 7.8, 1.2 Hz, 1H,

H_ar), 7.28 (ddd, J = 7.2, 7.2, 1.5 Hz, 1H, H_ar), 7.13

(dd, J = 7.5, 1.5 Hz, 1H, H_ar), 6.43 (d, J = 2.1 Hz, 1H,

H-5), 5.71 (d, J = 2.7 Hz, 1H, H-6), 3.94 (dd, J = 11.4,

5.4 Hz 1H, H-3eq), 3.70 (ddd, J = 12.0, 12.0, 4.5 Hz,

1H, H-3ax), 2.48 (m, 1H, H-16a), 2.41 (m, 1H, H-17a),

2.34 (s, 3H, NCOCH₃), 2.17 (ddddd, J = 12.9, 12.9,

12.9, 5.1, 2.7 Hz, 1H, H-14ax), 1.98 (m, 1H, H-16b),

1.82 (m, 1H, H-14eq), 1.70 (ddd, J = 13.2, 13.2,

2.7 Hz, 1H, H-15ax), 1.45 (m, 1H, H-15eq), 1.41 (m,

1H, H-19a), 1.35 (m, 1H, H-17b), 1.12 (m, 1H, H-

19b), 0.70 (t, J = 7.5 Hz, 3H, H-18) ppm; ¹³C NMR

(75.5 MHz, CDCl₃) d 176.4 (C-2), 173.8 (NCO), 140.4

((Cq)_ar), 138.5 ((Cq)_ar), 131.6 (CH_ar), 130.1 (C-21),

128.3 (CH_ar), 127.6 (CH_ar), 127.5 (CH_ar), 119.7 (C-5),

117.0 (C-7), 109.4 (C-6), 46.2 (C-3), 39.1 (C-20), 37.5

36.6 (C-17), 32.4 (C-15), 31.5 (C-16), 30.1 (C-19),

23

D

19.5 (C-14), 8.5 (C-18) ppm; ½aꢁ ꢀ381 (c 1.0, CHCl₃);

IR (ﬁlm) m 2956, 1689 cm^ꢀ1; HRMS (ESI) calcd

~

for C₂₆H₂₆N₂NaO₂[(M+Na)⁺]: 421.1892, found:

421.1898.

4.23. 1-(p-Methoxybenzoyl)rhazinilam (5l)

White powder (81 mg, 62% from 89 mg rhazinilam);

mp 169 ꢁC; ¹H NMR (300 MHz, CDCl₃) d 7.54 (d,

J = 8.7 Hz, 2H, H_ar), 7.43 (dd, J = 7.5, 1.8 Hz, 1H,

H_ar), 7.33 (ddd, J = 7.5, 7.5, 2.1 Hz, 1H, H_ar), 7.27

(ddd, J = 7.5, 7.5, 1.5 Hz, 1H, H_ar), 7.13 (dd,

J = 7.5, 1.5 Hz, 1H, H_ar), 6.79 (d, J = 9.0 Hz, 2H,

H_ar), 6.40 (d, J = 3.0 Hz, 1H, H-5), 5.88 (d,

J = 3.0 Hz, 1H, H-6), 3.85 (dd, J = 12.0, 5.1 Hz, 1H,

H-3eq), 3.73 (s, 3H, OCH₃), 3.66 (ddd, J = 12.0,

12.0, 4.2 Hz, 1H, H-3ax), 2.58 (dd, J = 13.5, 13.5 Hz,

1H, H-16ax), 2.39 (ddd, J = 13.5, 13.5, 1.5 Hz, 1H,

H-17ax), 2.12 (m, 1H, H-16eq), 2.04 (m, 1H, H-14a),

1.72 (m, 1H, H-14b), 1.69 (m, 1H, H-15a), 1.48 (m,

1H, H-19a), 1.44 (m, 1H, H-15b), 1.30 (m, 1H, H-

17eq), 1.18 (m, 1H, H-19b), 0.72 (t, J = 7.5 Hz, 3H,

H-18) ppm; ¹³C NMR (75.5 MHz, CDCl₃) d 176.6

(C-2), 172.6 (NCO), 162.5 ((Car)OCH₃), 141.3

((Cq)_ar), 139.5 (C-8), 131.6 (CH_ar), 130.7 (CH_ar),

130.1 (C-21), 128.5 (CH_ar), 128.1 ((Cq)_ar), 127.3

(CH_ar), 126.5 (CH_ar), 119.4 (C-5), 116.9 (C-7), 113.5

(CH_ar), 110.2 (C-6), 55.4 (OCH₃), 46.0 (C-3), 39.1

(C-17), 32.5 (C-15), 32.0 (C-16), 30.3 (C-19), 26.8

24

D

(NCOCH₃), 19.7 (C-14), 8.5 (C-18) ppm; ½aꢁ ꢀ345 (c

1.0, CHCl₃); IR (ﬁlm) m 2941, 1705 cm^ꢀ1; HRMS

~

(ESI) calcd for C₂₁H₂₄N₂NaO₂[(M+Na)⁺]: 359.1735,

found: 359.1722.

4.21. 1-Pivaloylrhazinilam (5j)

White powder (52 mg, 66% from 61 mg rhazinilam); mp

1

90 ꢁC; H NMR (300 MHz, CDCl₃) d 7.36 (dd, J = 7.5,

1.8 Hz, 1H, H_ar), 7.27 (ddd, J = 7.7, 7.7, 1.8 Hz, 1H,

H_ar), 7.20 (ddd, J = 7.5, 7.5, 1.5 Hz, 1H, H_ar), 6.90

(dd, J = 7.8, 1.5 Hz, 1H, H_ar), 6.39 (d, J = 3.0 Hz, 1H,

H-5), 5.84 (d, J = 2.7 Hz, 1H, H-6), 3.93 (dd, J = 12.0,

5.0 Hz, 1H, H-3eq), 3.68 (ddd, J = 12.3, 12.3, 4.8 Hz,

1H, H-3ax), 2.44 (m, 1H, H-16a), 2.39 (m, 1H, H-17a),

2.18 (ddddd, J = 13.1, 13.1, 13.1, 5.4, 2.7 Hz, 1H, H-

14ax), 1.99 (m, 1H, H-16b), 1.77 (m, 1H, H-14eq),

1.69 (ddd, J = 13.4, 13.4, 3.0 Hz, 1H, H-15ax), 1.46

(m, 1H, H-15eq), 1.38 (m, 1H, H-19a), 1.30 (m, 1H,

H-17b), 1.21 (s, 9H, NCOC(CH₃)₃), 1.09 (m, 1H, H-

19b), 0.68 (t, J = 7.4 Hz, 3H, H-18) ppm; ¹³C NMR

(75.5 MHz, CDCl₃) d 188.2 (NCO), 176.5 (C-2), 142.0

(C-13), 139.9 (C-8), 131.9 (CH_ar), 130.4 (C-21), 128.5

(C-20), 36.4 (C-17), 32.4 (C-15), 31.5 (C-16), 30.1

24

D

(C-19), 19.5 (C-14), 8.4 (C-18) ppm; ½aꢁ ꢀ413 (c

1.0, CHCl₃); IR (ﬁlm) m 2961, 1686 cm^ꢀ1; HRMS

~

(ESI) calcd for C₂₇H₂₈N₂NaO₃[(M+Na)⁺]: 452.2031,

found: 452.2051.

´

A. Decor et al. / Bioorg. Med. Chem. 14 (2006) 2314–2332

2327

4.24. 1-(p-Triﬂuoromethylbenzoyl)rhazinilam (5m)

7.14 (dd, J = 7.9, 1.6 Hz, 1H, H_ar), 6.46 (d, J = 2.4 Hz,

1H, H-5), 5.78 (d, J = 1.8 Hz, 1H, H-6), 3.95 (dd,

J = 12.3, 4.8 Hz, 1H, H-3eq), 3.73 (ddd, J = 12.3, 12.3,

4.2 Hz, 1H, H-3ax), 2.48 (dd, J = 13.8, 13.8 Hz, 1H,

H-17ax), 2.37 (dd, J = 13.8, 13.8 Hz, 1H, H-16ax), 2.15

(ddddd, J = 13.2, 13.2, 13.2, 5.1, 3.0 Hz, 1H, H-14ax),

1.99 (dd, J = 14.4, 6.3 Hz, 1H, H-16eq), 1.82 (m, 1H,

H-14eq), 1.71 (ddd, J = 13.2, 13.2, 2.7 Hz, 1H, H-

15ax), 1.48 (m, 1H, H-15eq), 1.43 (m, 1H, H-19a),

1.35 (m, 1H, H-17eq), 1.35 (s, 9H, C(CH₃)₃), 1.14 (m,

1H, H-19b) ; 0.71 (t, J = 7.2 Hz, 3H, H-18) ppm; ¹³C

NMR (75.5 MHz, CDCl₃) d 174.2 (C-2), 151.6 (NCO),

140.8 (C-13), 138.5 (C-8), 131.3 (CH_ar), 130.0 (C-21),

128.0 (CH_ar), 127.0 (CH_ar), 126.9 (CH_ar), 119.4 (C-5),

116.7 (C-7), 109.2 (C-6), 82.2 (C(CH₃)₃), 46.0 (C-3),

39.0 (C-20), 37.0 (C-17), 32.5 (C-15), 31.4 (C-16), 30.2

White powder (80 mg, 53% from 94 mg rhazinilam); mp

86 ꢁC; ¹H NMR (300 MHz, CDCl₃) d 7.63 (m, 3H, H_ar),

7.55 (dd, J = 7.5, 2.0 Hz, 1H, H_ar), 7.46 (ddd, J = 7.5,

7.5, 1.8 Hz, 1H, H_ar), 7.39 (ddd, J = 7.5, 7.5Hz,

1.8 Hz, 1H, H_ar), 7.24 (m, 2H, H_ar), 6.49 (d,

J = 2.4 Hz, 1H, H-5), 5.91 (d, J = 2.7 Hz, 1H, H-6),

3.96 (dd, J = 12.0, 5.1 Hz 1H, H-3eq), 3.73 (ddd,

J = 12.0, 12.0, 4.8 Hz 1H, H-3ax), 2.65 (ddJ = 13.9,

13.9 Hz, 1H, H-16ax), 2.42 (ddd, J = 13.4, 13.4,

1.8 Hz, 1H, H-17ax), 2.16 (m,1H, H-16eq), 2.08 (m,

1H, H-14a), 1.84 (m, 1H, H-14b), 1.77 (ddd, J = 13.5,

13.5, 3.3 Hz, 1H, H-15ax), 1.50 (m, 1H, H-19a), 1.45

(m, 1H, H-15eq), 1.40 (m, 1H, H-17eq), 1.24 (m, 1H,

H-19b), 0.80 (t, J = 7.2 Hz, 3H, H-18) ppm; ¹³C NMR

(75.5 MHz, CDCl₃) d 176.5 (C-2), 172.3 (NCO), 140.3

((Cq)_ar), 139.7 ((Cq)_ar), 139.3 ((Cq)_ar), 133.3 (q,

²J_C,F= 32 Hz, (Car)CF₃), 131.8 (CH_ar), 130.2 (C-21),

(C-19), 27.9 (C(CH₃)₃), 19.5 (C-14), 8.4 (C-18) ppm;

~

24

½aꢁ ꢀ385 (c 1.0, CHCl₃); IR (ﬁlm) m 2967, 1760, 1687

D

cm^ꢀ1

;

HRMS (ESI) calcd for C₂₄H₃₀N₂NaO₃

1

129.2 (q, J_C,F= 272 Hz, CF₃), 128.8 (CH_ar), 128.1

[(M+Na)⁺]: 417.2154, found: 417.2152.

(CH_ar), 127.9 (CH_ar), 126.7 (CH_ar), 125.3 (m, CH_ar(C-

q)_arCF₃), 119.7 (C-5), 116.8 (C-7), 109.9 (C-6), 46.1

(C-3), 39.1 (C-20), 36.8 (C-17), 32.3 (C-15), 31.5 (C-

4.27. 1-Phenoxycarbonylrhazinilam (5q)

16), 30.1 (C-19), 19.5 (C-14), 8.5 (C-18) ppm; ¹⁹F

Oil (51 mg, 61% from 60 mg rhazinilam); ¹H NMR

(300 MHz, CDCl₃) d 7.53 (m, 1H, H_ar), 7.42–7.27 (m,

5H, H_ar), 7.20 (dd, J = 7.2, 7.2 Hz, 1H, H_ar), 7.11 (d,

J = 8.1 Hz, 2H, H_ar), 6.59 (d, J = 2.7 Hz, 1H, H-5),

5.93 (d, J = 2.7 Hz, 1H, H-6), 4.06 (dd, J = 12.0,

4.8 Hz, 1H, H-3eq), 3.82 (ddd, J = 12.0, 12.0, 4.8 Hz,

1H, H-3ax), 2.58 (dd, J = 12.6, 12.6 Hz, 1H, H-17ax),

2.50 (dd, J = 12.6, 12.6 Hz, 1H, H-16ax), 2.26 (ddddd,

J = 12.9, 12.9, 12.9, 5.1, 2.7 Hz, 1H, H-14ax), 2.13 (m,

1H, H-16eq), 1.88 (m, 1H, H-14eq), 1.79 (ddd,

J = 13.5, 13.5, 3.0 Hz, 1H, H-15ax), 1.53 (m, 2H, H-

15eq and H-19a), 1.44 (m, 1H, H-17b), 1.27 (m, 1H,

H-19b), 0.79 (t, J = 7.2 Hz, 3H, H-18) ppm; ¹³C NMR

(75.5 MHz, CDCl₃) d 174.3 (C-2), 151.4, 150.8, 140.2

(NCO, CO₂Car and C-13), 138.7 (C-8), 131.6 (CH_ar),

130.3 (C-21), 129.3 (CH_ar), 128.5 (CH_ar), 127.8 (CH_ar),

127.1 (CH_ar), 125.9 (CH_ar), 121.4 (CH_ar), 120.0 (C-5),

116.6 (C-7), 109.3 (C-6), 46.2 (C-3), 39.2 (C-20), 37.3

24

D

NMR (282 MHz, CDCl₃) d ꢀ63.0 ppm; ½aꢁ ꢀ390_ꢀ(₁c

~

1.0, CHCl₃); IR (ﬁlm) m 2946, 1694, 1682, 1327 cm

;

HRMS (ESI) calcd for C₂₇H₂₅F₃N₂NaO₂[(M+Na)⁺]:

489.1766, found: 489.1763.

4.25. 1-Ethoxycarbonylrhazinilam (5n)

White crystals (36 mg, 50% from 61 mg rhazinilam); mp

137 ꢁC; ¹H NMR (300 MHz, CDCl₃) d 7.43 (dd, J = 7.5,

1.4 Hz, 1H, H_ar), 7.34 (ddd, J = 7.5, 7.5, 1.5 Hz, 1H,

H_ar), 7.28 (ddd, J = 7.5, 7.5, 1.8 Hz, 1H, H_ar), 7.18

(dd,J = 7.4, 1.6 Hz, 1H, H_ar), 6.45 (d, J = 3.0 Hz, 1H,

H-5), 5.76 (d, J = 2.1 Hz, 1H, H-6), 4.14 (m, 2H,

OCH₂), 3.95 (dd, J = 12.0, 4.8 Hz, 1H, H-3eq), 3.71

(ddd, J = 12.0, 12.0, 4.2 Hz, 1H, H-3ax), 2.48 (dd,

J = 13.5, 13.5 Hz, 1H, H-17ax), 2.37 (dd, J = 13.8,

13.8 Hz, 1H, H-16ax), 2.17 (ddddd, J = 12.9, 12.9,

12.9, 5.1, 3.0 Hz, 1H, H-14ax), 2.00 (dd, J = 13.8,

6.3 Hz, 1H, H-16eq), 1.82 (m, 1H, H-14eq), 1.70 (ddd,

J = 13.2, 13.2, 3.0 Hz, 1H, H-15ax), 1.43 (m, 1H, H-

15eq), 1.39 (m, 1H, H-19a), 1.31 (m, 1H, H-17eq), 1.17

(t, J = 7.5 Hz, 3H, OCH₂CH₃), 1.12 (m, 1H, H-19b),

0.71 (t, J = 7.2 Hz, 3H, H-18) ppm; ¹³C NMR

(75.5 MHz, CDCl₃) d 174.3 (C-2), 153.2 (NCO), 140.4

(C-13), 138.6 (C-8), 131.4 (CH_ar), 130.1 (C-21), 128.2

(CH_ar), 127.3 (CH_ar), 126.9 (CH_ar), 119.6 (C-5), 116.6

(C-7), 109.2 (C-6), 62.7 (OCH₂), 46.0 (C-3), 39.0 (C-

(C-17), 32.5 (C-15), 31.4 (C-16), 30.4 (C-19), 19.6 (C-

24

D

14), 8.5 (C-18) ppm; ½aꢁ ꢀ459 (c 1.0, CHCl₃); IR (ﬁlm)

m 2943, 1784, 1725 cm^ꢀ1; HRMS (ESI) calcd for

~

C₂₆H₂₆N₂NaO₃[(M+Na)⁺]: 437.1841, found: 437.1852.

4.28. 1-Methanesulfonylrhazinilam (5r)

White powder (36 mg, 46% from 61 mg rhazinilam); mp

1

171 ꢁC; H NMR (300 MHz, CDCl₃) d 7.45–7.36 (m,

4H, H_ar), 6.52 (d, J = 2.7 Hz, 1H, H-5), 5.96 (d,

J = 2.7 Hz, 1H, H-6), 4.04 (dd, J = 12.0, 4.8 Hz, 1H,

H-3eq), 3.82 (ddd, J = 12.0, 12.0, 4.8 Hz, 1H, H-3ax),

3.14 (s, 3H, SO₂CH₃), 2.49 (m, 1H, H-17a), 2.45 (m,

1H, H-16a), 2.20 (m, 2H, H-14a and H-16b), 1.91 (m,

1H, H-14b), 1.73 (ddd, J = 13.2, 13.2, 3.0 Hz, 1H, H-

15ax), 1.59 (m, 1H, H-15eq), 1.50 (m, 1H, H-17b),

1.38 (m, 1H, H-19a), 1.16 (m, 1H, H-19b), 0.68 (t,

J = 7.5 Hz, 3H, H-18) ppm; ¹³C NMR (75.5 MHz,

CDCl₃) d 175.3 (C-2), 138.9 (C-8), 136.7 (C-13), 131.8

(CH_ar), 130.8 (C-21), 130.0 (CH_ar), 128.7 (CH_ar), 128.2

(CH_ar), 119.0 (C-5), 116.0 (C-7), 110.9 (C-6), 46.3

(C-3), 42.3 (SO₂CH₃), 39.1 (C-20), 37.1 (C-17), 32.8

20), 37.0 (C-17), 32.4 (C-15), 31.3 (C-16), 30.2 (C-19),

19.5 (C-14), 14.1 (OCH₂CH₃), 8.4 (C-18) ppm; ½aꢁ

24

D

~

ꢀ427 (c 1.0, CHCl₃); IR (ﬁlm) m 2963, 1771, 1724

cm^ꢀ1

;

HRMS (ESI) calcd for C₂₂H₂₆N₂NaO₃

[(M+Na)⁺]: 389.1841, found: 389.1818.

4.26. 1-tert-Butoxycarbonylrhazinilam (5o)

White crystals (250 mg, 85% from 219 mg rhazinilam);

1

mp 197 ꢁC; H NMR (300 MHz, CDCl₃) d 7.40 (dd,

J = 7.4, 1.7 Hz, 1H, H_ar), 7.33 (ddd, J = 7.2, 7.2,

1.2 Hz, 1H, H_ar), 7.25 (dd, J = 7.2, 7.2 Hz, 1H, H_ar),

´

A. Decor et al. / Bioorg. Med. Chem. 14 (2006) 2314–2332

2328

(C-15), 31.4 (C-16, 30.4 (C-19), 19.4 (C-14), 8.2 (C-18)

~

H_ar), 7.34 (m, 2H, H_ar), 7.29 (m, 1H, H_ar), 6.54 (d,

J = 2.7 Hz, 1H, H-5), 6.28 (br s, 1H, H-1), 5.86 (d,

J = 2.7 Hz, 1H, H-6), 3.96 (m, 1H, H-3a), 3.88 (m, 1H,

H-3b), 2.80 (m, 1H, H-16), 2.13 (m, 1H, H-14a), 2.06

(ddd, J = 12.3, 12.3, 2.4 Hz, 1H, H-15ax), 1.90 (m, 1H,

H-14b), 1.65 (m, 1H, H-15eq), 1.56 (dd, J = 14.1,

14.1 Hz, 1H, H-17ax), 1.53 (m, 1H, H-19a), 1.35 (m,

1H, H-19b), 1.28 (m, 1H, H-17eq), 1.20 (d, 3H,

J = 6.9 Hz, CH₃), 0.64 (t, J = 7.5 Hz, 3H, H-18) ppm;

¹³C NMR (75.5 MHz, CDCl₃) d 178.7 (C-2), 140.7 (C-

8), 136.8 (C-13), 132.7 (C-21), 131.2 (CH_ar), 128.3

(CH_ar), 128.0 (CH_ar), 127.8 (CH_ar), 119.6 (C-5), 116.9

(C-7), 108.5 (C-6), 45.7 (C-3), 42.3 (C-17), 41.7 (C-16),

24

ppm; ½aꢁ ꢀ255 (c 1.0, CHCl₃); IR (ﬁlm) m 2962, 1685,

D

1348 cm^ꢀ1; HRMS (ESI) calcd for C₂₀H₂₄N₂NaO₃S

[(M+Na)⁺]: 395.1405, found: 395.1397.

4.29. 1-(p-Toluenesulfonyl)rhazinilam (5s)

White powder (62 mg, 67% from 61 mg rhazinilam); mp

196 ꢁC; ¹H NMR (300 MHz, CDCl₃) d 7.86 (d,

J = 9.0 Hz, 2H, H_ar), 7.60 (dd, J = 7.8, 0.9 Hz, 1H,

H_ar), 7.48–7.35 (m, 3H, H_ar), 7.26 (d, J = 9.0 Hz, 2H,

H_ar), 6.25 (d, J = 2.6 Hz, 1H, H-5), 5.45 (d, J = 2.6 Hz,

1H, H-6), 3.94 (dd, J = 12.0, 5.1 Hz, 1H, H-3eq), 3.71

(ddd, J = 12.0, 12.0, 4.5 Hz, 1H, H-3ax), 2.41 (s, 3H,

(Car)CH₃), 2.36 (m, 2H, H-16a and H-17a), 2.15 (m,

1H, H-14a), 2.05 (m, 2H, H-16b), 1.82 (m, 1H, H-

14b), 1.67 (ddd, J = 13.2, 13.2, 3.0 Hz, 1H, H-15ax),

1.47 (m, 1H, H-15eq), 1.40 (m, 1H, H-17b), 1.29 (m,

1H, H-19a), 1.09 (m, 1H, H-19b), 0.66 (t, J = 7.2 Hz,

3H, H-18) ppm; ¹³C NMR (75.5 MHz, CDCl₃) d 173.9

(C-2), 144.3 (Cq(ar)), 139.9 (C-8), 136.9 (C-13), 136.1

(Cq(ar)), 132.0 (CH_ar), 130.2 (C-21), 129.5 (2 CH_ar),

129.0 (CH_ar), 128.2 (CH_ar), 128.1 (CH_ar), 118.5 (C-5),

115.7 (C-7), 110.8 (C-6), 46.1 (C-3), 38.9 (C-20), 36.7

38.6 (C-20), 36.0 (C-19), 28.8 (C-15), 20.2 (CH₃), 20.0

23

D

(C-14), 8.6 (C-18) ppm; ½aꢁ ꢀ24 (c 0.50, CHCl₃); IR

(ﬁlm) m 3195, 2852, 1664 cm^ꢀ1; HRMS (ESI) calcd for

~

C₂₀H₂₄N₂NaO [(M+Na)⁺]: 331.1786, found: 331.1783.

4.32. 16(S)-Ethylrhazinilam (6ba)

White powder (46 mg, 45% from 94 mg rhazinilam);

HPLC (preparative Hypercarb column, MeOH,

3.0 mL/min) t_R14.0 min; mp 142 ꢁC; ¹H NMR

(300 MHz, CDCl₃) d 7.44 (dd, J = 7.1, 2.0 Hz, 1H, H-

12), 7.35 (ddd, J = 7.5, 7.5, 1.8 Hz, 1H, H_ar), 7.30

(ddd, J = 7.5, 7.5, 1.5 Hz, 1H, H_ar), 7.18 (dd, J = 7.2,

1.8 Hz, 1H, H-9), 6.59 (br s, 1H, H-1), 6.49 (d,

J = 2.7 Hz, 1H, H-5), 5.76 (d, J = 2.4 Hz, 1H, H-6),

3.99 (dd, J = 11.7, 5.4 Hz, 1H, H-3eq), 3.77 (ddd,

J = 12.3, 12.3, 4.8 Hz,1H, H-3ax), 2.46 (dd, J = 11.7,

11.7 Hz, 1H, H-17ax), 2.41 (m, 1H, H-16), 2.24 (ddddd,

J = 12.7, 12.7, 12.7, 5.4, 2.9 Hz, 1H, H-14ax), 1.84 (m,

1H, H-14eq), 1.69 (m, 2H, H-15a and CH₂CH₃), 1.52

(m, 1H, H-15b), 1.41 (m, 1H, H-19a), 1.28 (m, 2H,

CH₂CH₃and H-19b), 1.20 (d, J = 11.7 Hz, 1H, H-

17eq), 0.86 (t, J = 7.4 Hz, 3H, CH₂CH₃), 0.70 (t,

J = 7.2 Hz, 3H, H-18) ppm; ¹³C NMR (75.5 MHz,

CDCl₃) d 179.7 (C-2), 140.7 (C-8), 137.9 (C-13), 131.4

(C-12), 130.9 (C-21), 128.1 (CH_ar), 127.5 (CH_ar), 127.2

(CH_ar), 118.9 (C-5), 117.2 (C-7), 109.5 (C-6), 46.1 (C-

3), 44.7 (C-17), 39.2 (C-16), 38.7 (C-20), 33.5 (C-15),

(C-17), 32.8 (C-15), 31.5 (C-16), 30.1 (C-19), 21.7

24

D

((Car)CH₃), 19.4 (C-14), 8.3 (C-18) ppm; ½aꢁ ꢀ259 (c

1.0, CHCl₃); IR (ﬁlm) m 2963, 1709, 1360 cm^ꢀ1; HRMS

~

(ESI) calcd for C₂₆H₂₈N₂NaO₃S [(M+Na)⁺]: 471.1718,

found: 471.1715.

4.30. 16(S)-methylrhazinilam (6aa)

White powder (43 mg, 34% from 120 mg rhazinilam);

HPLC separation (preparative Hypercarb column,

MeOH/CH₂Cl₂4:1, 3.0 mL/min) t_R17.6 min; mp

238 ꢁC; ¹H NMR (300 MHz, CDCl₃) d 7.42 (dd,

J = 6.9, 1.8 Hz, 1H, H-12), 7.37–7.26 (m, 2H, H_ar),

7.21 (dd, J = 7.4, 1.7 Hz, 1H, H_ar), 6.68 (br s, 1H, H-

1), 6.49 (d, J = 2.7 Hz, 1H, H-5), 5.75 (d, J = 2.7 Hz,

1H, H-6), 3.99 (dd, J = 12, 5.6 Hz, 1H, H-3eq), 3.76

(ddd, J = 12.3, 12.3, 5.0 Hz, 1H, H-3ax), 2.56 (m, 1H,

H-16), 2.47 (dd, J = 13.3, 13.3 Hz, 1H, H-17ax), 2.24

(ddddd, J = 13.2, 13.2, 13.2, 5.7, 3.0 Hz, 1H, H-14ax),

1.83 (m, 1H, H-14eq), 1.68 (ddd, J = 13.5, 13.5, 3.0

Hz, 1H, H-15ax), 1.51 (m, 1H, H-15eq), 1.41 (m, 1H,

H-19a), 1.28 (m, 1H, H-19b), 1.16 (d, J = 13.3 Hz, 1H,

H-17eq), 1.06 (d, J = 6.3 Hz, 3H, CH₃), 0.70 (t,

J = 7.3 Hz, 3H, H-18) ppm; ¹³C NMR (75.5 MHz,

CDCl₃) d 181.1 (C-2), 140.4 (C-8), 137.9 (C-13), 131.4

(C-12), 130.7 (C-21), 128.1 (CH_ar), 127.3 (CH_ar), 127.0

(CH_ar), 119.1 (C-5), 117.2 (C-7), 109.5 (C-6), 46.1 (C-

3), 45.8 (C-17), 38.8 (C-20), 33.3 (C-15), 31.9 (C-16),

30.6 (C-19), 29.1 (CH₂CH₃), 19.4 (C-14), 11.9

23

D

(CH₂CH₃), 8.2. (C-18) ppm; ½aꢁ ꢀ254 (c 1.0, CHCl₃);

IR (ﬁlm) m 3190, 2961, 1666 cm^ꢀ1; HRMS (ESI) calcd

~

for C₂₁H₂₆N₂NaO [(M+Na)⁺]: 345.1943, found:

345.1935.

4.33. 16(R)-Ethylrhazinilam (6bb)

White powder (12 mg, 12% from 94 mg rhazinilam);

HPLC (preparative Hypercarb column, MeOH,

3.0 mL/min) t_R18.5 min; mp 171 ꢁC; ¹H NMR

(300 MHz, CDCl₃) d 7.36 (m, 1H, H_ar), 7.30 (m, 2H,

H_ar), 7.28 (m, 1H, H_ar), 6.54 (d, J = 2.7 Hz, 1H, H-5),

6.27 (br s, 1H, H-1), 5.85 (d, J = 2.4 Hz, 1H, H-6),

3.96 (m, 1H, H-3a), 3.87 (m, 1H, H-3b), 2.54 (m, 1H,

H-16), 2.10 (m, 2H, H-14a and H-15a), 1.88 (m, 1H,

H-14b), 1.64 (m, 1H, CH₂CH₃), 1.59 (m, 1H, H-15b),

1.49 (m, 2H, H-17a and H-19a), 1.36 (m, 1H, H-19b),

1.28 (m, 2H, H-17b and CH₂CH₃), 1.00 (t, J = 7.3 Hz,

3H, CH₂CH₃), 0.64 (t, J = 7.3 Hz, 3H, H-18) ppm; ¹³C

NMR (75.5 MHz, CDCl₃) d 177.5 (C-2), 140.7 (C-8),

136.8 (C-13), 132.8 (C-21), 131.0 (CH_ar), 128.2 (CH_ar),

30.6 (C-19), 21.0 (CH₃), 19.4 (C-14), 8.2 (C-18) ppm;

~

23

½aꢁ ꢀ203 (c 1.0, CHCl₃); IR (ﬁlm) m 3194, 2962,

D

1669 cm^ꢀ1; HRMS (ESI) calcd for C₂₀H₂₄N₂NaO

[(M+Na)⁺]: 331.1786, found: 331.1780.

4.31. 16(R)-methylrhazinilam (6ab)

White powder (11 mg, 9% from 120 mg rhazinilam);

HPLC separation (preparative Hypercarb column,

MeOH/CH₂Cl₂4:1, 3.0 mL/min) t_R26.2 min; mp

1

195 ꢁC; H NMR (300 MHz, CDCl₃) d 7.40 (m, 1H,

´

A. Decor et al. / Bioorg. Med. Chem. 14 (2006) 2314–2332

2329

128.0 (CH_ar), 127.7 (CH_ar), 119.6 (C-5), 116.9 (C-7),

108.6 (C-6), 49.1 (C-16), 45.7 (C-3), 40.4 (C-17), 38.5

4.36. 16(R)-Benzoylrhazinilam (6da)

(C-20), 36.1 (C-19), 28.9 (C-15), 27.2 (CH₂CH₃), 20.0

White powder (30 mg, 25% from 91 mg rhazinilam);

HPLC (preparative Hypercarb column, MeOH/CH₂Cl₂

3:2, 3.0 mL/min) t_R10.0 min; mp 186 ꢁC; ¹H NMR

(300 MHz, CDCl₃) d 7.71 (m, 2H, H_ar), 7.58–7.47 (m,

3H, H_ar), 7.42 (m, 3H, H_ar), 7.27 (m, 1H, H_ar), 6.60

(br s, 1H, H-1), 6.49 (d, J = 2.7 Hz, 1H, H-5), 5.73 (d,

J = 2.7 Hz, 1H, H-6), 4.43 (d, J = 10.7 Hz, 1H, H-16),

4.00 (dd, J = 12.0, 5.3 Hz, 1H, H-3eq), 3.80 (ddd,

J = 12.3, 12.3, 4.5 Hz, 1H, H-3ax), 2.79 (dd, J = 14.4,

10.7 Hz, 1H, H-17ax), 2.34 (ddddd, J = 13.2, 13.2,

13.2, 5.4, 3.3 Hz, 1H, H-14ax), 1.88 (m, 2H, H-14eq

and H-17eq), 1.76 (ddd, J = 13.5, 13.5, 3.3 Hz, 1H, H-

15ax), 1.67 (m, 1H, H-15eq), 1.52 (m, 1H, H-19a),

1.33 (m, 1H, H-19b), 0.70 (t, J = 7.4 Hz, 3H, H-18)

ppm; ¹³C NMR (75.5 MHz, CDCl₃) d 196.0 (COPh),

172.1 (C-2), 141.1 (C-8), 136.8 ((Cq)_ar), 136.5 ((Cq)_ar),

133.1 (CH_ar), 132.0 (CH_ar), 130.5 (C-21), 128.5 (CH_ar),

128.4 (CH_ar), 128.1 (CH_ar), 128.0 (CH_ar), 127.4 (CH_ar),

119.3 (C-5), 116.8 (C-7), 109.7 (C-6), 47.1 (C-16), 46.1

23

D

(C-14), 12.1 (CH₂CH₃), 8.6 (C-18) ppm; ½aꢁ ꢀ59 (c

1.0, CHCl₃); IR (ﬁlm) m 3188, 2960, 1653 cm^ꢀ1; HRMS

~

(ESI) calcd for C₂₁H₂₆N₂NaO [(M+Na)⁺]: 345.1943,

found: 345.1935.

4.34. 16(S)-Benzylrhazinilam (6ca)

Beige powder (69 mg, 32% from 163 mg rhazinilam);

HPLC (preparative Hypercarb column, MeOH/CH₂Cl₂

3:2, 3.0 mL/min) t_R9.8 min; mp 177 ꢁC; ¹H NMR

(300 MHz, CDCl₃) d 7.39 (dd, J = 7.1, 2.0 Hz, 1H,

H_ar), 7.30–7.18 (m, 5H, H_ar), 7.03 (m, 2H, H_ar), 6.56

(dd, J = 8.3, 2.0 Hz, 1H, H_ar), 6.49 (d, J = 2.4 Hz,

1H, H-5), 6.39 (br s, 1H, H-1), 5.73 (d, J = 2.7 Hz,

1H, H-6), 4.00 (dd, J = 12.0, 5.4 Hz, 1H, H-3eq), 3.77

(ddd, J = 12.3, 12.3, 4.8 Hz, 1H, H-3ax), 2.91 (dd,

J = 12.6, 8.7 Hz, 1H, CH₂Ph), 2.74 (ddd, J = 10.2,

6.0, 6.0 Hz, 1H, H-16), 2.55 (m, 1H, CH₂Ph), 2.51

(dd, J = 10.8, 10.8 Hz, 1H, H-17ax), 2.24 (ddddd,

J = 12.9, 12.9, 12.9, 5.4, 2.7 Hz, 1H, H-14ax), 1.84

(m, 1H, H-14eq), 1.66 (ddd, J = 13.2, 13.2, 3.0 Hz,

1H, H-15ax), 1.52 (m, 1H, H-15eq), 1.38 (m, 1H, H-

19a), 1.29 (d, J = 10.8 Hz, 1H, H-17eq), 1.18 (m, 1H,

H-19b), 0.57 (t, J = 7.4 Hz, 3H, H-18) ppm; ¹³C

NMR (75.5 MHz, CDCl₃) d 178.7 (C-2), 140.4 (C-8),

139.4 (C-23), 137.5 (C-13), 131.2 (CH_ar), 130.8 (C-

21), 129.2 (CH_ar), 128.2 (CH_ar), 127.8 (CH_ar), 127.3

(CH_ar), 127.2 (CH_ar), 126.2 (CH_ar), 119.0 (C-5), 117.3

(C-7), 109.5 (C-6), 46.1 (C-3), 43.3 (C-17), 41.5

(C-3), 40.7 (C-17), 38.2 (C-20), 33.6 (C-15), 30.4 (C-

24

D

19), 19.4 (C-14), 8.2. (C-18) ppm; ½aꢁ ꢀ108 (c 1.0,

ꢀ1

~

CHCl₃); IR (ﬁlm) m 3307, 2961, 1699, 1651 cm

;

HRMS (ESI) calcd for C₂₆H₂₆N₂NaO₂[(M+Na)⁺]:

421.1892, found: 421.1874.

4.37. (S)-Benzoylrhazinilam (6db)

White powder (7 mg, 5% from 91 mg rhazinilam);

HPLC (preparative Hypercarb column, MeOH/CH₂Cl₂

3:2, 3.0 mL/min) t_R22.3 min; mp 186 ꢁC; ¹H NMR

(300 MHz, CDCl₃) d 8.09 (dd, J = 8.3, 1.2 Hz, 2H,

CO(o-H_ar)), 7.55 (dd, J = 7.4, 7.4 Hz, 1H, CO(p-H_ar)),

7.45 (dd, J = 7.7, 7.7 Hz, 2H, CO(m-H_ar)), 7.37 (m,

2H, H_ar), 7.31 (ddd, J = 7.5, 7.5, 1.9 Hz, 1H, H_ar),

7.07 (d, J = 7.8 Hz, 1H, H_ar), 6.56 (d, J = 2.7 Hz, 1H,

H-5), 6.29 (br s, 1H, H-1), 5.84 (d, J = 2.7 Hz, 1H, H-

6), 4.57 (ddd, J = 13.1, 4.8, 1.7 Hz, 1H, H-16), 4.00

(ddd, J = 13.7, 2.9, 2.9 Hz, 1H, H-3eq), 3.92 (ddd,

J = 11.9, 11.9, 5.0 Hz, 1H, H-3ax), 2.18 (m, 1H, H-

17ax), 2.16 (m, 2H, H-14a and H-15a), 1.94 (m, 1H,

H-14b), 1.66 (dd, J = 9.9, 5.4 Hz, 1H, H-15b), 1.58 (m,

1H, H-19a), 1.52 (dd, J = 14.2, 4.5 Hz, 1H, H-17eq),

1.47 (m, 1H, H-19b), 0.67 (t,J = 7.5 Hz, 3H, H-18)

ppm; ¹³C NMR (75.5 MHz, CDCl₃) d 195.5 (COPh),

170.9 (C-2), 140.1 (C-8), 136.5 ((Cq)_ar), 136.1 ((Cq)_ar),

133.4 (CH_ar), 132.3 (C-21), 130.7 (CH_ar), 129.0 (CH_ar),

128.8 (CH_ar), 128.6 (CH_ar), 128.5 (CH_ar), 127.9 (CH_ar),

119.9 (C-5), 117.1 (C-7), 108.7 (C-6), 55.7 (C-16), 45.7

(CH₂Ph), 40.1 (C-16), 38.7 (C-20), 33.4 (C-15), 30.5

24

D

(C-19), 19.4 (C-14), 7.9 (C-18) ppm; ½aꢁ ꢀ423 (c 1.0,

CHCl₃); IR (ﬁlm) m 3201, 2963, 1673 cm^ꢀ1; HRMS

~

(ESI) calcd for C₂₆H₂₈N₂NaO [(M+Na)⁺]: 407.2099,

found: 407.2077.

4.35. 16(R)-Benzylrhazinilam (6cb)

Beige powder (42 mg, 20% from 163 mg rhazinilam);

HPLC (preparative Hypercarb column, MeOH/CH₂Cl₂

3:2, 3.0 mL/min) t_R13.5 min; mp 178 ꢁC; ¹H NMR

(300 MHz, CDCl₃) d 7.39 (m, 4H, H_ar), 7.24 (m, 2H,

H_ar), 7.17 (m, 3H, H_ar), 6.54 (d, J = 3.0 Hz, 1H, H-5),

6.33 (br s, 1H, H-1), 5.86 (d, J = 2.4 Hz, 1H, H-6), 3.94

(m, 1H, H-3a), 3.85 (m, 1H, H-3b), 3.09 (dd, J = 13.2,

6.3 Hz, 1H, CH₂Ph), 2.97 (m, 1H, H-16), 2.60 (dd,

J = 13.2, 9.0 Hz, 1H, CH₂Ph), 2.04 (m, 1H, H-14a), 1.95

(ddd, J = 12.9, 12.9, 2.6 Hz, 1H, H-15ax), 1.83 (m, 1H,

H-14b), 1.52 (dd, J = 13.5, 13.5 Hz, 1H, H-17ax), 1.42

(m, 2H, H-15eq and H-19a), 1.33 (m, 1H, H-19b), 1.26

(dd, J = 12.0, 4.1 Hz, 1H, H-17eq), 0.55 (t, J = 7.5 Hz,

3H, H-18) ppm; ¹³C NMR (75.5 MHz, CDCl₃) d 177.2

(C-2), 140.7 (C-8), 139.3 (CH₂(Cq)_ar), 136.9 (C-13),

132.6 (C-21), 131.2 (CH_ar), 129.0 (CH_ar), 128.5 (CH_ar),

128.2 (CH_ar), 128.1 (CH_ar), 127.8 (CH_ar), 126.4 (CH_ar),

119.6 (C-5), 116.8 (C-7), 108.5 (C-6), 49.0 (C-16), 45.6

(C-3), 38.2 (C-20), 36.2 (C-17 and C-19), 28.8 (C-15),

24

D

20.0 (C-14), 8.6 (C-18) ppm; ½aꢁ ꢀ99 (c 1.0, CHCl₃);

IR (ﬁlm) m 3241, 2925, 1651 cm^ꢀ1; HRMS (ESI) calcd

~

for C₂₆H₂₆N₂NaO₂[(M+Na)⁺]: 421.1892, found:

421.1892.

4.38. 16(S)-tert-Butoxycarbonylrhazinilam (6ea)

(C-3), 40.1 (CH₂Ph), 39.4 (C-17), 38.4 (C-20), 36.1 (C-

Oil (45 mg, 10% from 319 mg rhazinilam); ¹H NMR

(300 MHz, CDCl₃) d 7.42 (m, 1H, H_ar), 7.38–7.28 (m,

2H, H_ar), 7.23 (m, 1H, H_ar), 6.66 (br s, 1H, H-1), 6.48

(d, J = 2.7 Hz, 1H, H-5), 5.75 (d, J = 2.4 Hz, 1H, H-

6), 3.99 (dd, J = 11.7, 5.1 Hz, 1H, H-3eq), 3.77 (ddd,

24

D

19), 28.8 (C-15), 20.0 (C-14), 8.6 (C-18) ppm; ½aꢁ ꢀ18

(c 1.0, CHCl₃); IR (ﬁlm) m 3436, 2961, 1651 cm^ꢀ1; HRMS

~

(ESI) calcd for C₂₆H₂₈N₂NaO [(M+Na)⁺]: 407.2099,

found: 407.2084.

´

A. Decor et al. / Bioorg. Med. Chem. 14 (2006) 2314–2332

2330

J = 12.0, 12.0, 4.8 Hz, 1H, H-3ax), 3.39 (d, J = 11.1 Hz,

1H, H16), 2.53 (dd, J = 14.4, 11.1 Hz, 1H, H-17ax), 2.24

(ddddd, J = 13.0, 13.0, 13.0, 5.4, 3.3 Hz, 1H, H-14ax),

1.85 (m, 2H, H-14eq and H-17eq), 1.71 (ddd, J = 13.2,

13.2, 3.0 Hz, 1H, H-15ax), 1.61 (m, 1H, H-15eq), 1.42

(s, 9H, C(CH₃)₃), 1.33 (m, 1H, H-19a), 1.25 (m, 1H,

H-19b), 0.69 (t, J = 7.4 Hz, 3H, H-18) ppm; ¹³C NMR

(75.5 MHz, CDCl₃) d 173.5 (C-2), 169.0 (CO₂C(CH₃)₃),

140.7 (C-8), 137.5 (C-13), 131.5 (CH_ar), 130.4 (C-21),

128.2 (CH_ar), 127.7 (CH_ar), 127.4 (CH_ar), 119.1 (C-5),

116.9 (C-7), 109.6 (C-6), 81.3 (C(CH₃)₃), 46.2 (C-3),

44.9 (C-16), 39.0 (C-17), 38.2 (C-20), 33.5 (C-15), 30.4

1H, H_ar), 7.08 (br s, 1H, H-1), 6.48 (d, J = 2.7 Hz,

1H, H-5), 5.82 (d, J = 2.7 Hz, 1H, H-6), 4.13 (m, 1H,

H-16), 3.96 (ddd, J = 12.0, 4.2, 4.2 Hz, 1H, H-3eq),

3.83 (ddd, J = 12.0, 12.0, 4.8 Hz, 1H, H-3ax), 3.30 (d,

J = 7.5 Hz, OH), 2.39 (dd, J = 11.4, 11.4 Hz, 1H, H-

17ax), 2.09 (m, 1H, H-14ax), 1.62 (dd, J = 11.4,

2.4 Hz, 1H, H-17eq), 1.90 (m, 1H, H-14eq), 1.71 (m,

2H, H-15ax and H-15eq), 1.41 (m, 1H, H-19a), 1.32

(m, 1H, H-19b), 0.70 (t, J = 7.2 Hz, 3H, H-18) ppm;

¹³C NMR (75.5 MHz, CDCl₃) d 177.5 (C-2), 140.2

(C-8), 135.4 (C-13), 131.3 (CH_ar), 130.8 (C-21), 128.3

(CH_ar), 128.2 (CH_ar), 127.7 (CH_ar), 119.2 (C-5), 116.8

(C-7), 109.1 (C-6), 65.7 (C-16), 45.9 (C-3 and C-17),

(C-19), 27.8 (C(CH₃)₃), 19.4 (C-14), 8.1 (C-18) ppm;

~

24

D

½aꢁ

ꢀ148 (c 0.35, CHCl₃); IR (ﬁlm)

m

3315,

37.6 (C-20), 33.0 (C-15), 31.6 (C-19), 19.4 (C-14), 8.2

23

D

2965, 1732, 1681 cm^ꢀ1; HRMS (ESI) calcd for

C₂₄H₃₀N₂NaO₃[(M+Na)⁺]: 417.2154, found: 417.2126.

(C-18) ppm; ½aꢁ ꢀ168 (c 1.0, CHCl₃); IR (ﬁlm) m

~

3386, 3062, 2934, 2873, 1680 cm^ꢀ1; HRMS (ESI) calcd

for C₁₉H₂₂N₂NaO₂[(M+Na)⁺]: 333.1579, found:

333.1585.

4.39. 1-Perﬂuoro-tert-butoxycarbonylrhazinilam (5p)

To a solution of perﬂuoro- tert-butyl alcohol (47 lL,

0.34 mmol) in dichloromethane (1 mL) under argon at

0 ꢁC were added a solution of triphosgene (50 mg,

0.17 mmol) in dichloromethane (2 mL) and dry pyridine

(136 lL, 1.70 mmol). The mixture was stirred for 30 min

at 0 ꢁC and for 4.5 h at 20 ꢁC, then a solution of (ꢀ)-

rhazinilam 1 (100 mg, 0.34 mmol) in dichloromethane

(2 mL) was added dropwise. After stirring for 45 min

at 20 ꢁC, a saturated aq solution of NaHCO₃was added

and the aqueous layer was extracted with dichlorometh-

ane. The organic layer was washed with a 1 M aq solu-

tion of CuSO₄, dried over MgSO₄, and the solvent was

evaporated under vacuum. The residue was puriﬁed by

preparative TLC (heptane/ethyl acetate 1:1) to give com-

4.41. 16(S)-Acetoxyrhazinilam (6g)

To a solution of compound 6f (57 mg, 0.18 mmol) in dry

pyridine (2.2 mL) under argon at 20 ꢁC was added acetic

anhydride (1.1 mL, 11.8 mmol). After stirring for 24 h,

dichloromethane and water were added and the aqueous

layer was extracted with dichloromethane. The organic

layer was washed with a 1 M aq solution of CuSO₄,

dried over MgSO₄, and the solvent was evaporated un-

der vacuum. The crude mixture was puriﬁed by prepara-

tive TLC (heptane/ethyl acetate 1:4), to give compound

6g (42 mg, 58%) as a beige solid, and 1-acetyl-16(S)-

1

acetoxyrhazinilam (29 mg, 32%); mp 230 ꢁC; H NMR

(300 MHz, CDCl₃) d 7.45–7.33 (m, 4H, H_ar), 6.68 (br

s, 1H, H-1), 6.50 (d, J = 3.0 Hz, 1H, H-5), 5.80 (d,

J = 2.7 Hz, 1H, H-6), 4.96 (dd, J = 11.1, 1.5 Hz, 1H,

H-16), 4.00 (dd, J = 12.0, 4.8 Hz, 1H, H-3eq), 3.80

(ddd, J = 12.0, 12.0, 4.8 Hz, 1H, H-3ax), 2.72 (dd,

J = 13.8, 11.1 Hz, 1H, H-17ax), 2.18 (ddddd, J = 13.2,

13.2, 13.2, 5.4, 3.0 Hz, 1H, H-14ax), 2.06 (s, 3H,

COCH₃), 1.86 (m, 1H, H-14eq), 1.72 (ddd, J = 13.2,

13.2, 3.0 Hz, 1H, H-15ax), 1.63 (m, 2H, H-15eq and

H-17eq), 1.46 (m, 1H, H-19a), 1.33 (m, 1H, H-19b),

0.70 (t, J = 7.4 Hz, 3H, H-18) ppm; ¹³C NMR

(75.5 MHz, CDCl₃) d 172.8, 170.3 (C-2 and COCH₃),

140.1, 135.6 (C-8 and C-13), 131.1 (CH_ar), 129.8 (C-

21), 128.3 (3 CH_ar), 119.3 (C-5), 117.4 (C-7), 109.6 (C-

6), 67.7 (C-16), 46.1 (C-3), 43.0 (C-17), 37.3 (C-20),

1

pound 5p as an oil (23 mg, 12%); H NMR (300 MHz,

CDCl₃) d 7.50 (dd, J = 6.9, 2.1 Hz, 1H, H_ar), 7.42

(ddd, J = 7.5, 7.5, 2.1 Hz, 1H, H_ar), 7.37 (ddd, J = 7.2,

7.2, 1.8 Hz, 1H, H_ar), 7.21 (dd, J = 7.2, 2.0 Hz, 1H,

H_ar), 6.53 (d, J = 3.0 Hz, 1H, H-5), 5.75 (d, J = 2.7 Hz,

1H, H-6), 4.01 (dd, J = 12.0, 4.8 Hz, 1H, H-3eq), 3.77

(ddd, J = 12.0, 12.0, 4.8 Hz, 1H, H-3ax), 2.49 (dd,

J = 12.1, 12.1 Hz, 1H, H-17ax), 2.41 (dd, J = 12.6,

12.6 Hz, 1H, H-16ax), 2.22 (ddddd, J = 13.5, 13.5,

13.5, 5.4, 3.0 Hz, 1H, H-14ax), 2.07 (dd, J = 12.6,

6.6 Hz, 1H, H-16eq), 1.88 (m, 1H, H-14eq), 1.75 (ddd,

J = 13.2, 13.2, 3.0 Hz, 1H, H-15ax), 1.46 (m, 1H, H-

17eq), 1.40 (m, 1H, H-19a), 1.16 (m, 1H, H-19b), 0.75

(t, J = 7.2 Hz, 3H, H-18) ppm; ¹³C NMR (75.5 MHz,

CDCl₃) d 173.4 (C-2), 152.3 ((Cq)_ar), 138.5 ((Cq)_ar),

131.7 (CH_ar), 130.2 ((Cq)_ar), 128.7 (CH_ar), 128.4 (CH_ar),

126.6 (CH_ar), 120.3 (C-5), 118.0 ((Cq)_ar), 108.9 (C-6),

46.2 (C-3), 39.1 (C-20), 37.5 (C-17), 32.4 (C-15), 32.2

33.9 (C-15), 30.8 (C-19), 20.9 (COCH₃), 19.5 (C-14),

24

D

8.2 (C-18) ppm; ½aꢁ ꢀ288 (c 1.0, CHCl₃); IR (ﬁlm)

m 3471, 2962, 1741, 1681 cm^ꢀ1; HRMS (ESI) calcd for

~

C₂₁H₂₄N₂NaO₃[(M+Na)⁺]: 375.1685, found: 375.1683.

(C-16), 30.4 (C-19), 19.5 (C-14), 8.5 (C-18) ppm; ¹⁹F

24

D

NMR (282 MHz, CDCl₃) d ꢀ69.5 ppm; ½aꢁ ꢀ382 (c

4.42. X-ray diﬀraction analysis for compounds 1, 5o, and

6ba

0.64, CHCl₃); IR (ﬁlm) m 2921, 1822, 1687 cm^ꢀ1; HRMS

~

(ESI) calcd for C₂₄H₂₁F₉N₂NaO₃[(M+Na)⁺]: 579.1306,

found: 579.1312.

For all structures, data were collected with a Nonius

Kappa-CCD diﬀractometer (Mo Ka, graphite mono-

chromator).^22aThe absolute conﬁguration of (ꢀ)-rhazil-

inam 1 was assigned to each compound. The structures

have been solved by direct methods using program

SHELXS86^22band reﬁned by program SHELXL93^22c

using full matrix least-squares based on F². All details

are listed in the experimental section. Reﬁnement was

4.40. 16(S)-Hydroxyrhazinilam (6f)

This compound was obtained as a side-product during

the conversion of (+)-vincadiﬀormine 7 into (ꢀ)-rhaz-

inilam 1;⁵beige powder; mp 228 ꢁC; ¹H NMR

(300 MHz, CDCl₃) d 7.43–7.32 (m, 3H, H_ar), 7.22 (m,

´

A. Decor et al. / Bioorg. Med. Chem. 14 (2006) 2314–2332

2331

disturbed only in compound 6ba by the presence of one

References and notes

desordered solvent molecule (ethanol) in the unit cell.

CCDC253697 (compound 1), CCDC253698 (compound

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These data can be obtained free of charge at

www.ccdc.cam.ac.uk/conts/retrieving.html [or from the

Cambridge Crystallographic Data Centre, 12 Union

Road, Cambridge CB 1EZ, UK; fax/(+44)1223/336-

033; E-mail: deposit@ccdc.cam.ac.uk].

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¨

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20. For comparative purposes, the complete physical data of

(ꢀ)-rhazinilam including ¹H and ¹³C NMR attributions

were again determined carefully.

Acknowledgments

We thank Dr. C. Thal for his initial contribution, C.

Richez for rhazinilam substitutions, M.-T. Adeline

and B. Dumontet for assistance with HPLC separa-

tions, C. Gaspard and G. Aubert for cytotoxicity

assays, and Dr J. Bignon for ﬂow cytometry experi-

ments. This work was ﬁnancially supported by the

Centre National de la Recherche Scientiﬁque

(France).

´

A. Decor et al. / Bioorg. Med. Chem. 14 (2006) 2314–2332

2332

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Article Doi

Synthesis and biological evaluation of B-ring analogues of (-)-rhazinilam

DOI: 10.1016/j.bmc.2005.11.011

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Article abstract of DOI:10.1016/j.bmc.2005.11.011

Full text of DOI:10.1016/j.bmc.2005.11.011

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