New antitubulin derivatives in the combretastatin A4 series: Synthesis and biological evaluation

Bioorganic & Medicinal Chemistry 13 (2005) 3853–3864

New antitubulin derivatives in the combretastatin

A4 series: synthesis and biological evaluation

a

Christine Borrel, Sylviane Thoret, Xavier Cachet, Daniel Guenard, Franc¸ois Tillequin,

b

a

b

a

´

Michel Koch^aand Sylvie Michel^a,*

a

´

Laboratoire de Pharmacognosie, Universite Rene Descartes (Paris 5), CNRS UMR 8638, Faculte des Sciences Pharmaceutiques

et Biologiques, 4 avenue de l’Observatoire, 75270 Paris cedex 06, France

^bCNRS—ICSN, UPR 2301, 91190 Gif-sur-Yvette, France

Received 12 October 2004; accepted 15 February 2005

Available online 9 April 2005

Abstract—Two series of combretastatin A4 derivatives (acrylamide = carboxamide and carbamate) were synthesized in order to

improve the water solubility and stabilize the cis-conﬁguration of the double bond. Their cytotoxic eﬀects were evaluated against

MCF-7, KB-3-1 and IGROV human cancer cell lines, as well as their inhibitory activity on tubulin polymerization. Results were

compared to those of carboxamide 1, chosen as reference. Potent inhibitions were observed on both tests in the carboxamide series,

particularly for compound 4d bearing a ﬂuorine group in replacement of the 3-hydroxyl of CA4. In contrast, most of the carbamates

were either inactive or displayed only moderate cytotoxicities. Interestingly, a submicromolar IC₅₀was measured on MCF-7 cells for

6g, although this compound was totally devoid of antitubulin activity.

1. Introduction

(i.e., hypoxia, lack of nutrients and waste products accu-

mulation) lead to tumour cells death. Tubulin binding

agents that display such an activity, target tubulin of

the cytoskeleton on which they act as assembling inhib-

itors. Rapid endothelial cell shape changes are induced,

leading to the aforementioned chain of fatal events for

the tumour.^5,6

Tubulin is a heterodimeric protein that polymerizes into

microtubules and both forms coexist in a dynamic equi-

librium. These ﬁlaments are involved in many kinetic

processes in eukaryotic cells, for example, intracellular

organelle transport, mitosis, cell motion or cell shape.¹

It constitutes a major target in the ﬁeld of anticancer

drugs research. Indeed, agents that inhibit the polymer-

ization of tubulin and those that stabilize the resultant

microtubules are widely used in current cancer chemo-

therapy, but have undesirable side eﬀects and are subject

to multidrug resistance.^2,3Apart from these antimitotic

properties, some antitubulin agents were recently shown

to exhibit antivascular activities at dose levels far from

the MTD (maximal tolerated dose).⁴These agents, like

other vascular targeting agents (VTAs), disrupt the asso-

ciated vasculature of tumour and lead to vascular shut-

down and haemorrhagic necrosis, leaving normal

vasculature intact. Consequences of blood supply arrest

In this context, two compounds, combretastatin A4

(CA4) and N-acetylcolchinol (ZD 6126), appear particu-

larly promising due to potency and selectivity of their

antivascular activity.^5–11The former one is a natural stil-

bene extracted from the bark of Combretum caﬀrum

Kuntze, whereas the last one is a semi-synthetic ana-

logue of the natural antimitotic agent colchicine. Both

corresponding CA4 and ZD 6126 phosphate prodrugs,

are currently under clinical trials.^4,12

MeO

NHCOCH₃

OMe

OH

Keywords: Tubulin; Combretastatin A4; Carboxamides; Carbamates.

*

OH

OMe

combretastatin A4 (CA4)

Corresponding author. Tel.: +33 153 739812; fax: +33 140 469658;

e-mail: sylvie.michel@univ-paris5.fr

N-acetylcolchinol

doi:10.1016/j.bmc.2005.02.039

3854

C. Borrel et al. / Bioorg. Med. Chem. 13 (2005) 3853–3864

Nevertheless, the antitumour activity of these com-

pounds, in terms of tumour mass reduction, is rather

limited. Indeed, a rim of viable tumour cells survive at

the periphery of the tumour, perfused by unaﬀected ves-

sels in the surrounding normal tissues and it proliferates

after drug removal. The search for compounds that dis-

play both antivascular and direct antimitotic activities is

now well-established, particularly since AVE 8062A and

Oxi 4503, two combretastatin analogues, were proved to

possess such a dual activity correlated with increased

antitumour eﬀect.^13,14

and various substituents were introduced on the 3⁰-posi-

tion (R₁= NH₂, H, OH, F).

CONH₂

X

MeO

carboxamides

X = CONH₂

carbamates

1"

A

1'

X = NHCOOR

B

OMe

NH₂

R₁

4'

OMe

R₂

1

General Formula of CA4 analogues

MeO

In order to complete this study, the synthesis of several

isosteric carbamates (general formula: X = NHCOOR)

was undertaken. Both series were prepared from com-

mon acrylic acid precursors (general formula:

X = COOH). Biological evaluation of these intermedi-

ates is also presented.

OPO₃Na₂

OMe

NHSer, HCl

OMe

AVE 8062A

OMe

Oxi 4503 (CA1-P)

From the literature review, it appeared that numerous

CA4 analogues were previously prepared.^15,16In the

course of structure–activity relationship studies, many

modiﬁcations, relative to chemical features of the mole-

cule (i.e., double bond geometry, substitution pattern of

both rings) were evaluated in order to make their inﬂu-

ence on biological activity more precise. Following these

works, diﬀerent series of analogues where the double

bond of CA4 was replaced by a mimicking structure,

were synthesized. Excellent results obtained with ﬁve-

membered rings have to be underlined. In such restricted

analogues, the essential cis-geometry between both A/B-

rings is similar to that of CA4.

2. Chemistry

Acrylic acids 3a–c were synthesized by means of a Per-

kin reaction, as previously reported.^18,20,21This method

was also applied to the preparation of new acrylic acids

3d–f (Scheme 1). Reaction of 4-hydroxy-3-methoxy-

benzaldehyde (2e) with 3,4,5-trimethoxyphenylacetic

acid in the presence of acetic anhydride aﬀorded the ace-

tate of corresponding acrylic acid 3e. In contrast, the

regioisomeric isovanillin led to the phenol 3b when trea-

ted under similar conditions.²¹Deprotection of 3e in

mild basic conditions aﬀorded 3f. Pure cis-(E) isomers

were obtained in moderate yields (31–44%) after puriﬁ-

cation of the crude precipitate by fractional crystalliza-

tion. For each compound, the cis conﬁguration was

In contrast, only a few results concerned acyclic ana-

logues where the double bond was maintained and sta-

bilized by an electron-withdrawing group.¹⁷A series of

(Z)-acrylonitriles was prepared by Ohsumi and co-work-

ers.^18,19The authors showed that the insertion of a

nitrile group (CN) onto the oleﬁn site adjacent to the

A-ring, did not aﬀect the activity of CA4 and in terms

of steric hindrance, CN was about the maximum tolera-

ble size. Apart from these studies, a number of N-alkyl

acrylamides were studied by Cushman et al. N-Alkyl-

ation in this position leads to a dramatic decrease of bio-

logical activities.²⁰

1

unambiguously determined by H NMR, on the basis

of typical chemical shifts observed for the ethylenic pro-

ton H₃signal.^18,21As a general rule, the isomeric trans-

(Z) isomers, known to be biologically inactive in the

combretastatin series, were not separated from the crude

reaction mixture.^20,22Only trans-(Z) compound 3a was

isolated for spectroscopic comparison with the corre-

sponding cis-(E) isomer. There is an upﬁeld chemical

shift displacement of 0.6 ppm in going from (E)-3a to

(Z)-3a.

These results have encouraged the preparation of CA4

analogues with the goal of both a stabilized double bond

and an increased hydrosolubility. Two series of com-

pounds bearing a substituent, either a carboxamide or

a carbamate group, on the 1 position of the oleﬁn bridge

adjacent to the A-ring were prepared.

COOH

OMe

CHO

R₂

MeO

a

+

31-44%

R₁MeO

OMe

R₁

OMe

To our knowledge, only one unsubstituted carboxamide

was described.¹⁸Compound 1 was less cytotoxic than

CA4, but displayed increased antitubulin activity. In

order of studying the structure–activity relationships in

this series, carboxamide 1 was chosen as a lead com-

pound for further chemical modulations on the B-ring

(general formula: X = CONH₂). The trimethoxy A-ring

and the 4⁰-OCH₃on the B-ring of CA4, which was re-

ported necessary for potent activity were conserved

R₂

3a : R

₁= -NO₂; R₂= -OCH₃

2a :

R₁= -NO₂; R₂= -OCH₃

R¹₁= -H ; R₂= -OCH₃

3b

R

:

₁= -OH ; R₂= -OCH₃

₁= -H ; R = -OCH₃

: R = -OH ; R₂= -OCH₃

2b

3c : R

:

2c :

3d R₁= -F ; R₂²= -OCH₃

2d : R₁= -F; R₂= -OCH₃

3e :R₁= -OCH , R₂= -OAc

2e

: R₁= -OCH₃; R₂= -OH

3f :R₁= -OCH³, R₂= -OH

b

70%

3

Scheme 1. Syntheses of acrylic acids 3a–f. Reagents and conditions: (a)

(CH₃CO)₂O, Et₃N, 140 °C; (b) K₂CO₃, MeOH/H₂O 3/1, rt.

C. Borrel et al. / Bioorg. Med. Chem. 13 (2005) 3853–3864

3855

OMe

NO₂

methyl carbamates (6a,c,d,g,h) starting from the corre-

sponding acrylic acids.²³Thus, activation of the carbox-

ylic function by treatment with ethylchloroformate led

to a mixed anhydride, which reacted with sodium azide

to aﬀord a carbonyl azide derivative. Thermal rear-

rangement in toluene at 80 °C gave the corresponding

isocyanate. Finally, alcoholysis by either trimethylsilyl-

ethan-2-ol or methyl alcohol aﬀorded the corresponding

carbamates in weak to good yield, depending on the

starting acrylic acid and alcohol. Indeed, when the

B-ring of acrylic acid bears either an electronegative or

a mesomeric electron-withdrawing group such as –F or

–NO₂, high yields were obtained (78–96%). In contrast,

synthesis of carbamates bearing either a hydroxy or an

amino group at R₁failed. All other attempts toward

the reduction of nitro carbamates 5a and 6a were unsuc-

cessful, only leading to degradation products. Degrada-

tion was also observed when trying to deprotect the

carbonate group of carbamates 5h and 6h. In order

to circumvent the drawback of alkaline deprotection,

O-(tert-butyldimethylsilanyloxy) acrylic acid 3g was syn-

thesized and subsequently converted into the corre-

sponding carbamates 5g and 6g. Nevertheless,

attempts to cleave the protecting group also failed in this

case. The chemical stability of 5g and 6g strongly sug-

gests that instability in the carbamate series mainly

depends upon the presence of a proton donor group at

R₁. Finally, carbonates 5h and 6h as well as silyl ethers

5g and 6g were submitted to biological tests.

COOH

H₃

MeO

OMe

(Z)-3a

Reference compound 1 was prepared as previously de-

scribed.¹⁸Treatment of acrylic acid 3a with SOCl₂fol-

lowed by aminolysis of the corresponding acylchloride

with aqueous NH₃aﬀorded the nitro compound 4a

(Scheme 2). Finally, zinc reduction in acetic acid aﬀor-

ded 1 in quantitative yield. Similarly, activation with

SOCl₂was applied to the synthesis of the new acryl-

amides 4c and 4d from acrylic acids 3c and 3d, respec-

tively. When ester 3e was used as starting material,

phenol 4f was directly obtained since deprotection

occurred during aminolysis step. For the synthesis of

the acrylamide analogous to CA4 (4b), the protection

of the phenol group and the activation of the carboxylic

function were both ensured by treatment with ethylchlo-

roformate. Thereby, reaction with ammonium chloride

aﬀorded an ethylcarbonate intermediate, which was

directly deprotected by LiOH.

As outlined in Scheme 3, Curtius conditions gave an ac-

cess to trimethylsilylethyl carbamates (5a,c,d,g,h) and

COOH

CONH₂

MeO

a,b (4a, 4c-d, 4f) 50-95 %*

or

MeO

c-e (4b) 66 %*

MeO

* from two steps

OMe

R₁

R₂

4a : R₁= -NO , R₂= -OCH₃

1 : R₁= -NH², R₂= -OCH₃

f 100 %

3a : R

₁= -NO₂; R₂= -OCH₃

₁= -OH ; R₂= -OCH₃

4b : R₁= -OH²,R₂= OCH₃

:

3b

R

3c : R₁= -H ; R = -OCH₃

3d: R

3e : R₁= -OCH₃, R₂= -OAc

₁= -F ; R₂²= -OCH₃

:

4c R₁= -H ;R₂= -OCH₃

:

4d R₁= -F; R₂= -OCH₃

:

4f R₁= -OCH₃, R₂= -OH

Scheme 2. Syntheses of acrylamides 4a–d,f and 1. Reagents and conditions: (a) SOCl₂, Et₃N, CH₂Cl₂, rt; (b) NH₄OH, CH₂Cl₂, rt; (c) Et₃N,

ClCOOEt, DMF, ꢀ10 °C; (d) NH₄Cl, Et₃N, DMF, ꢀ10 °C; (e) LiOH, MeOH/H₂O 6/1, rt; (f) Zn, AcOH, rt.

COOH

NHCOOR

a-d (5 or 6 c-d, g-h) 26-71 / 31-86 %*

MeO

or

a,e, c-d (5a, 6a) 78 / 96 %*

* overall yield

OMe

R₁

R₂

3a : R

₁= -NO , R₂= -OCH₃

5a, c-d, g-h : R = -(CH₂)₂-TMS

6a, c-d, g-h : R = -CH₃

3b : R₁= -OH², R₂= -OCH₃

f 92%

3g : R

₁= -OTBDMS, R₂= -OCH₃

3c : R₁= -H, R₂= -OCH₃

5a-6a :R₁= -NO₂, R₂= -OCH₃

5c-6c :R₁= -H, R₂= -OCH₃

:

3d R₁= -F, R₂= -OCH₃

5d-6d :

R₁= -F, R₂= -OCH₃

5g-6g : R₁= -OTBDMS,R₂= -OCH₃

5h-6h :R₁= -OCOOEt, R₂= -OCH₃

Scheme 3. Syntheses of carbamates 5a,c,d,g,h and 6a,c,d,g,h. Reagents and conditions: (a) Et₃N, ClCOOEt, anhyd acetone, ꢀ5 °C; (b) NaN₃, H₂O,

ꢀ5 °C; (c) anhyd toluene, 80 °C; (d) trimethylsilylethan-2-ol (5a,c,d,g,h) or MeOH (6a,c,d,g,h), 50 °C; (e) NaN₃, THF, ꢀ5 °C; (f) TBDMSCl,

imidazole, DMF, rt.

3856

C. Borrel et al. / Bioorg. Med. Chem. 13 (2005) 3853–3864

3. Biological activity

fect on the interaction between the inhibitor and tubulin.

Indeed, with the exception of nitro derivative 4a and 4f,

which derive from the aforementioned acrylic acid 3f, all

acrylamides 4b–d displayed moderate to strong ITP

activities. Surprisingly, compound 4b, which bears the

substitution pattern of CA4, showed about eightfold

lower antitubulin activity than 1. Only a slight decrease

(threefold) is observed for 4c (R₁= H) and 4d (R₁= –F).

Therefore, in the carboxamide series as well as in the

CA4 series, the nature of the substituent on the 3⁰-

position of the B-ring dramatically inﬂuences ITP

activity.^18,20,22

3.1. Inhibition of tubulin polymerization

Inhibition of tubulin polymerization (ITP) was deter-

mined for acrylic acids 3a–d,f and 3g; acrylamides 4a–

d,f and carbamates 5a/6a, 5c–d/6c–d and 5g–h/6g–h in

comparison with the parent compound 1 and colchicine

used as standard. Results expressed in terms of the rela-

tive IC₅₀/IC_50colchare summarized in Table 1. The po-

tent activity of 1 was conﬁrmed in our hands where

this compound displayed an ITP value of 0.5, in good

agreement with the literature data.¹⁸

All the newly synthesized carbamates exhibited weak

ITP activities. Particularly, nitro compounds 5a and 6a

(R₁= –NO₂) and carbonates 5h and 6h (R₁=

–OCOOEt) were totally devoid of eﬀect. Surprisingly,

the N-methylcarbamate 5g (R₁= –OTBDMS) displayed

a ITP value of 20, whereas, in the silylated series, 6g was

devoid of activity. In comparison to the corresponding

acrylamides 4c and 4d, which possess potent inhibitory

activities, unsubstituted carbamates 5c and 6c (R₁= H)

as well as those bearing a ﬂuorine on the 3⁰-position

of the B-ring (5d and 6d) showed a 10- to 60-fold de-

creased antitubulin eﬀects. The results also clearly

underlined the inﬂuence of the bulkiness of the substitut-

ing group on the oleﬁn site adjacent to the A-ring on

ITP properties. Indeed, the N-methylcarbamates 6c

and 6d were slightly more active than their bulky N-

(trimethylsilyl)ethyl homologues 5c and 5d.

ITP activity of acrylic acid 3c had been previously deter-

mined by Cushman et al. and it was concluded that

introduction of carboxylic group on the oleﬁn site

resulted in a lack of activity in comparison to that of

CA4.²⁰In good agreement with this statement, all the

newly synthesized acrylic acids 3b,d,f and 3g, as well

as 3a were found inactive, except 3f bearing a –OCH₃

group on the 3⁰-position of the B-ring, which displayed

a weak activity with a ITP value of 21. Results obtained

in carboxylic series dramatically contrasted with those

observed in carboxamide series and an acidic function

on this position of the oleﬁn has likely a deleterious ef-

Table 1. IC₅₀values of ITP^a

Compound

IC₅₀/IC_50colch

Acrylic acids

3.2. Cytotoxicity

3a

3b

3c

3d

3f

Inactive

21

Compounds were evaluated for their cytotoxic activities

against three human cancer cell lines (KB-3-1, MCF-7

and IGROV) using an MTT assay. IC₅₀values are sum-

marized in Tables 2,3 and represent the concentration

inducing a 50% decrease of cell growth after 3 days incu-

bation. Values superior to 10 lM are not reported.

3g

Inactive

Acrylamides

4a

4b

4c

4d

4f

1

Inactive

3.7

1.7

All the new acrylic acids were devoid of cytotoxic ef-

fects, even 3f, which displayed a weak ITP activity. In

the acrylamide series (Table 2), the cytotoxicity results

were well-correlated with those of ITP tests. Indeed,

ineﬀective compounds against tubulin polymerization

assay (4a and 4f) were also not cytotoxic, except a slight

eﬀect observed for the nitro acrylamide 4a against

MCF-7 cell line. In our hands, 1 displayed moderate

cytotoxic activities against MCF-7 and KB-3-1 cells,

and a strong one against IGROV cells. Interestingly,

replacement of amino group by a hydroxy leads to a

decreased cytotoxicity, except in the case of KB-3-1 cell

lines. Thus, compared with 1, acrylamide 4b displayed 6-

fold and 30-fold lower cytotoxic activity against IGROV

and MCF-7 cells, respectively, but a 1.5-fold increased

potency against KB-3-1 cells. In comparison with CA4

values, introduction of a carboxamide group on the ole-

ﬁn site near the trimethoxyphenyl ring severely weak-

ened the cell growth inhibition potency. This decrease

1.5

Inactive

0.5^b

Methylcarbamates

6a

6c

6d

6g

6h

Inactive

27

15

Inactive

Silylcarbamates

5a

5c

5d

5g

5h

Inactive

91

71

20

Inactive

Reference

Colchicine

1.0^c

^aInhibition of tubulin polymerization. ITP was determined as descri-

bed in Section 5.

^bLit. IC₅₀= 3 lM.¹⁸

^cIC_50colchvaries from 1.8 to 6.7 lM for the diﬀerent experiments

according to the tubulin concentration.

According to Ref. 20, 3c gave an IC₅₀of 12.8 lM on MCF-7 cells.

C. Borrel et al. / Bioorg. Med. Chem. 13 (2005) 3853–3864

3857

Table 2. IC₅₀values (lM, mean of 8 determinations SD) for

cytotoxicity of carboxamides, colchicine and CA4

tively, submicromolar and micromolar IC₅₀, but only

against one cancer cell line (KB-3-1). According to their

cytotoxic eﬀects, three categories of N-methylcarba-

mates might be considered. Firstly, compound 6a was

devoid of any activity for both ITP and cytotoxicity

tests. Secondly, 6c (R₁= –H) and 6d (R₁= –F), which

displayed moderate ITP activities, were shown to exhibit

micromolar IC₅₀against the three cell lines. Thirdly, 6h

(R₁= –OCOOEt) and 6g (R₁= –OTBDMS) were signif-

icantly cytotoxic, particularly 6g, which displayed a IC₅₀

of 0.84 lM against MCF-7, but neither 6h nor 6g exhib-

ited antitubulin properties. Another mechanism might

be evoked in order to explain observed cytotoxicities

of these N-methylcarbamates.

Compound

KB-3-1

IGROV

MCF-7

4a

4b

>10

8.00 1.55

1.13 0.17

0.46 0.12

0.14 0.13

>10

0.15 0.01

0.051 0.006

0.016 0.02

>10

0.12 0.02

0.18 0.02

0.10 0.03

>10

4c

4d

4f

1^a

0.20 0.01

0.003 0.0002

0.005 0.0006

0.028 0.012

0.021 0.002

0.014 0.001

0.44 0.13

0.032 0.013

0.069 0.013

Colchicine

CA4^b

^aLit. IC₅₀= 0.20 lM on Colon 26.¹⁸

^bFor an eﬃcient synthesis of CA4, see Ref. 21.

Table 3. IC₅₀values (lM, mean of 8 determinations SD) for

cytotoxicity of carbamates, colchicine and CA4

4. Conclusion

Compound KB-3-1

IGROV

MCF-7

In conclusion, two series of analogues of CA4 were syn-

thesized and evaluated for their ITP and cytotoxic activ-

ities. Results for carbamates were rather disappointing

likely due to the bulkiness of the alkoxy groups. Fur-

thermore, the instability of the synthesis intermediates

could not allow the access to the expected molecules

bearing a hydroxy or an amino group on the 3⁰-position

of the B-ring. In contrast, acrylamide derivatives dis-

played promising antitumour and cytotoxic properties.

As far as SARare concerned, introduction of polar car-

boxamide group on oleﬁn site near the A-ring seemed to

induce deleterious eﬀects on both cytotoxic and ITP

activities. Substitution on the 3⁰-position of the B-ring

with a polar amino group increased ITP eﬀect but re-

duced cytotoxicity.¹⁸A more hydrophobic ﬂuorine

group could restore cytotoxic activity against several

cancer cell lines and in this case slightly weakened ITP

properties. These results could be advantageously

exploited for the development of new drugs with poten-

tial antivascular and correlated direct cytotoxicity. Fluo-

rine analogue 4d whose biological activity is within the

same range as those of reference compound 1 is cur-

rently selected for further biological studies.

Methylcarbamates

6a

6c

6d

6g

6h

>10

1.21 0.12

0.91 0.30

2.87 0.27

1.41 0.22

2.34 0.22

1.27 0.08

6.96 1.60

>10

2.02 0.43

1.47 0.14

0.84 0.40

2.83 1.15

Silylcarbamates

5a

5c

5d

5h

5g

>10

0.80 0.33

2.43 0.60

>10

References

Colchicine

CA4

0.003 0.0002 0.021 0.002 0.032 0.013

0.005 0.0006 0.014 0.001 0.069 0.013

is correlated with a ITP decrease observed for com-

pound 4b. Compared to 1, 4c (R₁= –H) and 4d

(R₁= –F) were signiﬁcantly more potent in terms of

cytotoxicity against KB-3-1 cell line (i.e., respectively,

4-fold and 13-fold) and also, but to a lower extent,

against MCF-7 (respectively, by a 1.2- and 4-fold). In

contrast, both compounds were less potent than 1

against the IGROV cell line, but the decrease was lim-

ited to a 3.6-factor for ﬂuoro compound 4d. Results ob-

tained for 4d are remarkable since cytotoxicity against

KB-3-1 was comparable to that of CA4. This observa-

tion^àdid not comply with Hsieh groupÕs hypothesis

involving that presence of an available donor group in

the B-ring was important for cytotoxic activity in CA4

analogues.²⁵

5. Experimental

Most reagents were commercially available reagent-

grade chemicals and used without further puriﬁcation.

Reactions were monitored by thin-layer chromatogra-

phy using silica gel plates (Merck Si gel 60F₂₅₄). Where

appropriate, crude products were separated by column

chromatography, silica gel (Merck, particle size 0.035–

0.070 mm or 0.025–0.045 mm) with an overpressure of

300 mbars. All melting points were determined on a

Leica micromelting point apparatus and are uncorrected.

As previously noted for ITP activity, carbamates dis-

played on the whole, signiﬁcantly lower potency than

the corresponding isosteric carboxamide (Table 3).

Bulky silylcarbamates 5a,c and 5g were considered inac-

tive. In contrast, compounds 5d and 5h, showed, respec-

1

NMRspectra were recorded at 300 or 400 MHz ( H

NMR) and at 75 or 100 MHz (¹³C NMR) using a

Bruker AC 300 or a AVANCE 400 spectrophotometer;

J values are given in hertz (Hz) and d in ppm, relative to

solvent peaks as internal standards (d: CDCl₃:

7.27 ppm (¹H), 77 ppm (¹³C); Me₂CO-d₆: 2.10 ppm

(¹H), 29.8 ppm (¹³C); Me₂SO-d₆: 2.50 ppm (¹H), 40.6

ppm (¹³C)); ¹H and ¹³C signals were unambiguously

attributed by 2D NMRexperiments. Mass spectra

^àA synthesis of 3-ﬂuoro-3-deoxy CA4 was recently published.²⁴

Cytotoxicity against K562 human chronic myelogenous leukaemia

cell line was reported and was similar to that of CA4. ITP activity was

not evaluated.

3858

C. Borrel et al. / Bioorg. Med. Chem. 13 (2005) 3853–3864

(MS) were measured on a Nermag R10-10C (DIC) or a

ZQ 2000 Waters (ES) spectrophotometer. The IRspec-

tra were obtained using a Nicolet 510 FT-IRspectro-

photometer. The UV spectra were recorded on a

Beckman DU 640 apparatus. Elemental analysis was

determined by the microanalyses service of Pierre

et Marie Curie University.

818, 772. Anal. Calcd (C₁₉H₁₉FO₆) C = 62.98%,

H = 5.29%. Found C = 60.33%, H = 5.36%.

5.1.3. (E)-3-(4⁰-Acetoxy-3⁰-methoxyphenyl)-2-(3⁰⁰,4⁰⁰,5⁰⁰-

trimethoxyphenyl)acrylic acid (3e). The general proce-

dure was performed with vanilline 2e (670 mg,

4.4 mmol). The reaction mixture was heated for 3 h

30 min and the precipitate was crystallized from ethanol

to give 479 mg (30%) of 3e as pale yellow crystals. Mp

5.1. General procedure for the preparation of acrylic acids

3b,d–e

1

205–207 °C; H NMR(DMSO- d₆): d 7.70 (s, 1H, H3),

6.99 (d, 1H, J = 8 Hz, H5⁰), 6.86 (dd, 1H, J = 2, 8 Hz,

H5⁰), 6.70 (d, 1H, J = 2 Hz, H2⁰), 6.45 (s, 2H,

H2⁰⁰,H6⁰⁰), 3.68 (s, 6H, 3⁰⁰,5⁰⁰-OCH₃), 3.66 (s, 3H, 4⁰⁰-

OCH₃), 3.40 (s, 3H, 4⁰-OCH₃), 2.20 (s, 3H, –COCH₃);

¹³C NMR(DMSO- d₆): d 169.7 (–COCH₃), 169.5 (C1),

154.5 (C3⁰⁰,C5⁰⁰), 151.4 (C3⁰), 141.1 (C4⁰), 139.4 (C3),

138.3 (C4⁰⁰), 134.6 (C2), 134.4 (C1⁰), 133.1 (C1⁰⁰), 125.1

(C6⁰), 124.0 (C5⁰), 114.7 (C2⁰), 108.0 (C2⁰⁰,C6⁰⁰), 61.3

(4⁰⁰-OCH₃), 57.3 (3⁰⁰,5⁰⁰-OCH₃), 56.3 (3⁰-OCH₃), 21.5

(–COCH₃); MS(ES) m/z = 425 [M+Na]⁺; UV (MeOH)

A mixture of 3,4,5-trimethoxyphenylacetic acid (2 g,

8.84 mmol), substituted benzaldehyde (4.4 mmol) and

triethylamine (2 mL) in Ac₂O (4 mL) was heated at

140 °C until the benzaldehyde had disappeared. After

cooling, the reaction mixture was acidiﬁed with 35%

aqueous HCl (6 mL) and kept at room temperature

overnight. The precipitate was collected by ﬁltration

and recrystallized from absolute EtOH to give pure

acids.

k

(nm) 205 (loge = 4.69), 271 (loge = 4.15), 301

5.1.1. (E)-3-(3⁰-Hydroxy-4⁰-methoxyphenyl)-2-(3⁰⁰,4⁰⁰,5⁰⁰-

trimethoxyphenyl)acrylic acid (3b).²⁰The general proce-

dure was performed with isovanilline 2b (670 mg,

4.4 mmol). The reaction mixture was heated for 3 h

and the precipitate was recrystallized from ethanol to

give 500 mg (31%) of 3b as pale yellow crystals. Mp

(loge = 4.05); IR(KBr) m⁰(cm^ꢀ1) 3068, 2942, 1762,

1676, 1584, 1510, 1411, 1257, 1238, 1222, 1126, 1035,

1009, 913, 854, 837, 772, 714. Anal. Calcd (C₂₁H₂₂O₈)

C = 62.68%, H = 5.51%. Found C = 62.31%, H = 5.61%.

5.2. General procedure for the preparation of acrylic acids

3a and 3c

1

184–186 °C; H NMR(DMSO- d₆): d 8.95 (s, 1H, OH),

7.56 (s, 1H, H3), 6.80 (d, 1H, J = 8.5 Hz, H5⁰), 6.59

(dd, 1H, J = 2, 8.5 Hz, H6⁰), 6.52 (d, 1H, J = 2 Hz,

H2⁰), 6.43 (s, 2H, H2⁰⁰, H6⁰⁰), 3.72 (s, 3H, 4⁰-OCH₃),

3.70 (s, 3H, 4⁰⁰-OCH₃), 3.68 (s, 6H, 3⁰⁰,5⁰⁰-OCH₃); ¹³C

NMR(DMSO- d₆): 169.2 (C1), 153.6 (C3⁰⁰,C5⁰⁰), 149.5

(C4⁰), 146.4 (C3⁰), 139.7 (C3), 137.5 (C4⁰⁰), 132.7 (C1⁰⁰),

130.9 (C2), 127.6 (C1⁰), 123.5 (C6⁰), 117.8 (C2⁰), 112.1

(C5⁰), 107.3 (C2⁰⁰,C6⁰⁰), 60.7 (4⁰⁰-OCH₃), 56.5 (3⁰⁰,5⁰⁰-

OCH₃), 56.0 (4⁰-OCH₃); MS (DIC/NH₃) m/z = 378

[M+NH₄]⁺, 361 [M+H]⁺; UV (MeOH) k (nm) 203

A mixture of 3,4,5-trimethoxyphenylacetic acid (1 g,

4.4 mmol), substituted benzaldehyde (4.4 mmol) and tri-

ethylamine (1 mL) in Ac₂O (10 mL) was heated at

140 °C. After cooling, the mixture was acidiﬁed with

35% aqueous HCl dropwise added (6 mL). The mixture

was left overnight and then the precipitated product was

ﬁltered. The precipitate was recrystallized from absolute

EtOH to give pure products.

(loge = 4.56), 284 (loge = 3.98); IR(KBr) m⁰(cm^ꢀ1

)

5.2.1. (E)-3-(4⁰-Methoxy-3⁰-nitrophenyl)-2-(3⁰⁰,4⁰⁰,5⁰⁰-tri-

methoxyphenyl)acrylic acid (E-3a) and (Z)-3-(4⁰-meth-

oxy-3⁰-nitrophenyl)-2-(3⁰⁰,4⁰⁰,5⁰⁰-trimethoxyphenyl)acrylic

acid (Z-3a).¹⁸The general procedure was performed

with 4-methoxy-3-nitrobenzaldehyde 2a (850 mg,

4.7 mmol). The reaction mixture was stirred for 20 h.

After acidiﬁcation, E-3a precipitated and was collected

by ﬁltration. The aqueous ﬁltrate was allowed to stand

overnight at rt leading to precipitation of Z-3a, which

was collected by ﬁltration as an amorphous yellow pow-

der (50 mg, <3%). The former precipitate was recrystal-

lized from ethanol to give 800 mg (44%) of pure E-3a as

3328, 2912, 1665, 1583, 1502, 1443, 1266, 1124, 1017.

Anal. Calcd (C₁₉H₂₀O₇) C = 63.33%, H = 5.59%. Found

C = 63.30%, H = 5.61%.

5.1.2. (E)-3-(3⁰-Fluoro-4⁰-methoxyphenyl)-2-(3⁰⁰,4⁰⁰,5⁰⁰-tri-

methoxyphenyl)acrylic acid (3d). The general procedure

was performed with 3-ﬂuoro-4-methoxybenzaldehyde

2d (494 mg, 3.2 mmol). The reaction mixture was heated

for 18 h and the precipitate was recrystallized from eth-

anol to give 484 mg (42%) of 3d as white crystals. Mp

1

203–205 °C; H NMR(DMSO- d₆): d 7.64 (s, 1H, H3),

1

7.05 (t, 1H, J = 9 Hz, H5⁰), 6.98 (dd, 1H, J = 1.5,

9 Hz, H6⁰), 6.81 (dd, 1H, J = 13, 1.5 Hz, H2⁰), 6.45 (s,

2H, H2⁰⁰,H6⁰⁰), 3.79 (s, 3H, 4⁰-OCH₃), 3.70 (s, 3H, 4⁰⁰-

OCH₃), 3.68 (s, 6H, 3⁰⁰,5⁰⁰-OCH₃); ¹³C NMR(DMSO-

d₆): d 169.3 (C1), 154.4 (C3⁰⁰,C5⁰⁰), 151.82 (J = 244 Hz,

C3⁰), 149.1 (J = 11 Hz, C4⁰), 138.7 (C3), 138.3 (C4⁰⁰),

133.1 (C1⁰⁰), 132.9 (C2), 128.9 (C6⁰), 128.3 (C1⁰),

118.03 (J = 19 Hz, C2⁰), 114.6 (C5⁰), 107.7 (C2⁰⁰,C6⁰⁰),

61.3 (4⁰⁰-OCH₃), 57.1 (4⁰,3⁰⁰,5⁰⁰-OCH₃); MS(DIC/NH₃)

yellow crystals. Compound E-3a: mp 223 °C; H NMR

(DMSO-d₆): d 7.70 (s, 1H, H3), 7.53 (d, 1H, J = 1.5 Hz,

H2⁰), 7.40 (dd, 1H, J = 1.5, 9 Hz, H6⁰), 7.26 (d, 1H,

J = 9 Hz, H5⁰), 6.48 (s, 2H, H2⁰⁰,H6⁰⁰), 3.88 (s, 3H, 4⁰-

OCH₃), 3.70 (s, 3H, 4⁰⁰-OCH₃), 3.68 (s, 6H, 3⁰⁰,5⁰⁰-

OCH₃); ¹³C NMR(DMSO- d₆): d 169.0 (C1), 154.5

(C3⁰⁰,C5⁰⁰), 153.3 (C4⁰), 139.8 (C3⁰), 138.6 (C4⁰⁰), 137.3

(C3), 137.2 (C6⁰), 134.5 (C2), 132.3 (C1⁰⁰), 127.9 (C1Õ),

127.5 (C2⁰), 115.3 (C5⁰), 107.7 (C2⁰⁰,C6⁰⁰), 61.2 (4⁰⁰-

OCH₃), 57.9 (4⁰-OCH₃), 57.1 (3⁰⁰,5⁰⁰-OCH₃); MS (ES)

m/z = 363 [M+H]⁺; UV (MeOH)

k

(nm) 205

(loge = 4.60), 282 (loge = 4.17), 297 (loge = 4.16); IR

(KBr) m⁰(cm^ꢀ1) 3448, 2998, 2942, 2626, 1667, 1616,

1516, 1506, 1414, 1278, 1257, 1129, 1024, 1003, 924,

m/z = 412 [M+Na]⁺; UV (MeOH)

(loge = 4.63), 297 (loge = 3.95); IR(KBr) m⁰(cm^ꢀ1

3448, 2990, 2941, 2626, 1666, 1612, 1583, 1536, 1413,

k

(nm) 204

)

C. Borrel et al. / Bioorg. Med. Chem. 13 (2005) 3853–3864

3859

1291, 1266, 1128, 1088, 1015, 996, 930, 830; Anal. Calcd

(C₁₉H₁₉NO₈) C = 58.61%, H = 4.92%, N = 3.60%.

Found C = 58.63%, H = 4.88%, N = 3.76%.Compound

(4⁰⁰-OCH₃), 57.1 (3⁰⁰,5⁰⁰-OCH₃), 55.7 (3⁰-OCH₃); MS(ES)

m/z = 383 [M+Na]⁺; UV (MeOH)

(nm) 207

(loge = 4.59), 292 (loge = 4.06), 319 (loge = 4.16); IR

(NaCl ﬁlm) m⁰(cm^ꢀ1) 3316, 2937, 1767, 1672, 1588,

1503, 1460, 1413, 1245, 1169, 1126, 1025, 991. Anal.

Calcd (C₂₁H₂₂O₈) C = 63.33%, H = 5.59%. Found

C = 62.75%, H = 5.60%.

k

1

Z-3a: H NMR(DMSO- d₆): d 8.03 (d, 1H, J = 2Hz,

H2⁰), 7.78 (dd, 1H, J = 9, 2 Hz, H6⁰), 7.42 (d, 1H,

J = 9 Hz, H5⁰), 7.10 (s, 1H, H3), 6.78 (s, 2H,

H2⁰⁰,H6⁰⁰), 3.95 (s, 3H, 4⁰⁰-OCH₃), 3.81 (s, 6H, 3⁰⁰,5⁰⁰-

OCH₃), 3.69 (s, 3H, 4⁰-OCH₃); ¹³C NMR(DMSO- d₆):

d 171.4 (C1), 154.0 (C3⁰⁰,C5⁰⁰), 152.7 (C4⁰), 140.0 (C3⁰),

139.1 (C4⁰⁰), 137.0 (C2), 134.8 (C6⁰), 132.8 (C1⁰⁰), 129.2

(C1Õ), 125.9 (C3), 125.6 (C2⁰), 115.7 (C5⁰), 104.7

(C2⁰⁰,C6⁰⁰), 61.2 (4⁰⁰-OCH₃), 57.9 (4⁰-OCH₃), 57.1

(3⁰⁰,5⁰⁰-OCH₃); MS (DIC/NH₃) m/z = 407 [M+NH₄]⁺,

390 [M+H]⁺; UV (MeOH) k (nm) 202 (loge = 4.55),

207 (loge = 4.50), 237 (loge = 4.23), 306 (loge = 4.42);

IR(KBr) m⁰(cm^ꢀ1) 3440, 2941, 1691, 1618, 1582, 1529,

1508, 1465, 1413, 1340, 1283, 1270, 1242, 1128, 1009,

815. Anal. Calcd (C₁₉H₁₉NO₈) C = 58.61%, H =

4.92%, N = 3.60%. Found C = 58.20%, H = 4.92%,

N = 3.92%.

5.2.4.

(E)-3-(4⁰-Methoxy-3⁰-[(tert-butyldimethylsilyl)-

oxy]phenyl)-2-(3⁰⁰,4⁰⁰,5⁰⁰-trimethoxyphenyl)acrylic acid (3g).

TBDMSCl (440 mg, 2.8 mmol) was added to a solution

of 3b (200 mg, 0.56 mmol) and imidazole (161 mg,

2.44 mmol) in DMF (2 mL). The mixture was stirred

at rt for 3 h, then diluted with water (5 mL) and

extracted with CH₂Cl₂. The combined extracts were

dried over anhydrous MgSO₄and evaporated to dryness.

The crude residue was dissolved in a mixture of THF

(2 mL) and 1 mL of a saturated aqueous NaHCO₃solu-

tion. The solution was stirred for 2 h at rt, then water

was added and the reaction mixture was extracted with

AcOEt. Combined organic layers were washed with

water, brine and dried over anhydrous MgSO₄and

evaporated to dryness to give 245 mg (92%) of 3g as a

white powder. Mp (CH₂Cl₂/n-hexane) 170 °C; ¹H

NMR(CDCl ₃): d 7.84 (s, 1H, H3), 6.85 (dd, 1H,

J = 2, 8.5 Hz, H6⁰), 6.73 (d, 1H, J = 8.5 Hz, H5⁰), 6.62

(d, 1H, J = 2 Hz, H2⁰), 6.46 (s, 2H, H2⁰⁰, H6⁰⁰), 3.89 (s,

3H, 4⁰⁰-OCH₃), 3.79 (s, 9H, 3⁰⁰,5⁰⁰,4⁰-OCH₃), 0.90 (s,

6H, C–CH₃in TBDMS), 0.01 (s, 9H, Si–CH₃); ¹³C

NMR(CDCl ₃): d 170.1 (C1), 153.8 (C3⁰⁰,C5⁰⁰), 152.9

(C4⁰), 144.6 (C3⁰), 142.5 (C3), 137.7 (C4⁰⁰), 131.3 (C1⁰⁰),

128.8 (C2), 127.1 (C1⁰), 126.8 (C6⁰), 122.5 (C2⁰), 111.3

(C5⁰), 106.5 (C2⁰⁰,C6⁰⁰), 61.0 (4⁰⁰-OCH₃), 56.1 (3⁰⁰,5⁰⁰-

OCH₃), 55.5 (4⁰-OCH₃), 25.6 (C–CH₃in TBDMS),

18.4 (Si–C), ꢀ4.8 (Si–CH₃); MS(ES) m/z = 497

5.2.2. (E)-3-(4⁰-Methoxyphenyl)-2-(3⁰⁰,4⁰⁰,5⁰⁰-trimethoxy-

phenyl)acrylic acid (3c).²¹The general procedure was

performed with 4-methoxybenzaldehyde 2c (536 mL,

4.4 mmol). The reaction mixture was heated during

20 h and the precipitate was recrystallized from ethanol

to give 617 mg (41%) of 3c as yellow crystals. mp 212 °C;

¹H NMR(DMSO- d₆): d 7.67 (s, 1H, H3), 7.05 (d, 2H,

J = 8.5 Hz, H2⁰,H6⁰), 6.80 (d, 2H, J = 8.5 Hz,

H3⁰,H5⁰), 6.44 (s, 2H, H2⁰⁰,H6⁰⁰), 3.70 (s, 6H, 4⁰,4⁰⁰-

OCH₃), 3.67 (s, 6H, 3⁰⁰,5⁰⁰-OCH₃); ¹³C NMR(DMSO-

d₆): d 169.1 (C1), 160.6 (C4⁰), 153.8 (C3⁰⁰,C5⁰⁰), 139.3

(C3), 137.5 (C4⁰⁰), 132.8 (C1⁰⁰), 132.6 (C2⁰,C6⁰), 131.1

(C2), 127.3 (C1Õ), 114.4 (C3⁰,C5⁰), 107.1 (C2⁰⁰,C6⁰⁰),

60.7 (4⁰⁰-OCH₃), 56.5 (3⁰⁰,5⁰⁰-OCH₃), 55.7 (4⁰-OCH₃);

MS (DIC/NH₃) m/z = 362 [M+NH₄]⁺, 345 [M+H]⁺;

UV (EtOH) k (nm) 301 (loge = 4.17); IR(KBr) m⁰

(cm^ꢀ1) 2994, 2942, 2630, 1664, 1604, 1582, 1508, 1464,

1414, 1253, 1242, 1180, 1127, 1029, 1008, 830. Anal.

Calcd (C₁₉H₂₀O₆) C = 66.27%, H = 5.85%. Found

C = 65.76%, H = 5.85%.

[M+Na]⁺, 433, 301; UV (EtOH)

k

(nm) 206

(loge = 4.64), 285 (loge = 4.15), 313 (loge = 4.17); IR

(KBr) m⁰(cm^ꢀ1) 3001, 2929, 1665, 1598, 1583, 1506,

1272, 1127, 1028, 992, 862, 836, 778. Anal. Calcd

(C₂₅H₃₄O₇Si) C = 63.27%, H = 7.22%. Found C =

62.36%, H = 7.27%.

5.2.3. (E)-3-(4⁰-Hydroxy-3⁰-methoxyphenyl)-2-(3⁰⁰,4⁰⁰,5⁰⁰-

5.3. General procedure for the preparation of amides

(4a,c–d and 4f)

trimethoxyphenyl)acrylic acid (3f).

A solution of

K₂CO₃(21 mg, 0.15 mmol) in water (0.5 mL) was added

to a solution of 3e (20 mg, 0.05 mmol) in methanol

(1.5 mL). The reaction mixture was stirred overnight

at rt and then neutralized with 1 N aqueous HCl. After

concentration, the resulting solution was extracted with

AcOEt. The combined organic layers were washed with

water, brine and dried over anhydrous MgSO₄, ﬁltered

and evaporated to dryness. Puriﬁcation of the residue

by ﬂash chromatography (eluent: cyclohexane/AcOEt,

60/40 (v/v), 1% AcOH) aﬀorded 12 mg (70%) of 3f as

Step 1: To a solution of carboxylic acid (0.28 mmol) in

freshly distilled CH₂Cl₂(1 mL), triethylamine (167 lL,

1.21 mmol) and thionyl chloride (38 lL, 0.52 mmol)

were added dropwise. The reaction mixture was stirred

at rt for 2 h and concentrated to dryness under reduce

pressure. Step 2: To 4 mL of an aqueous 28% NH₄OH

solution a solution of the crude residue dissolved in

CH₂Cl₂(4 mL) was added. The reaction mixture was

vigorously stirred at room temperature overnight, then

diluted with water and extracted with CH₂Cl₂. The com-

bined organic extracts were dried over anhydrous

MgSO₄, ﬁltered and evaporated to dryness under

reduce pressure. The residue was puriﬁed by ﬂash

chromatography.

1

an amorphous white powder. H NMR(DMSO- d₆): d

9.50 (s, 1H, –OH), 7.61 (s, 1H, H3), 6.75 (dd, 1H,

J = 1.5, 8 Hz, H6⁰), 6.66 (d, 1H, J = 8 Hz, H5⁰), 6.48

(d, 1H, J = 1.5 Hz, H2⁰), 6.47 (s, 2H, H2⁰⁰,H6⁰⁰), 3.70

(s, 6H, 3⁰⁰,5⁰⁰-OCH₃), 3.67 (s, 3H, 4⁰⁰-OCH₃), 3.35 (s,

3H, 3⁰-OCH₃); ¹³C NMR(DMSO- d₆): d 169.6 (C1),

154.4 (C3⁰⁰,C5⁰⁰), 149.3 (C4⁰), 148.0 (C3⁰), 140.6 (C3),

137.9 (C4⁰⁰), 134.6 (C1⁰⁰), 130.6 (C2), 126.7 (C1⁰), 126.6

(C6⁰), 116.3 (C5⁰), 113.9 (C2⁰), 107.9 (C2⁰⁰,C6⁰⁰), 61.0

5.3.1. (E)-3-(4⁰-Methoxy-3⁰-nitrophenyl)-2-(3⁰⁰,4⁰⁰,5⁰⁰-tri-

methoxyphenyl)acrylamide (4a).¹⁸The general proce-

dure was performed with E-3a (108 mg, 0.28 mmol)

3860

C. Borrel et al. / Bioorg. Med. Chem. 13 (2005) 3853–3864

and the residue was puriﬁed by ﬂash column chromato-

graphy (CH₂Cl₂/MeOH 99/1 (v/v)) to give 82 mg (81%)

of 4a as a pale yellow powder. Mp (CH₂Cl₂/n-hexane)

187–189 °C; ¹H NMR(CDCl ₃): d 7.57 (s, 1H, H3),

7.52 (d, 1H, J = 2 Hz, H2⁰), 7.27 (d, 1H, J = 9 Hz,

H5⁰), 6.92 (dd, 1H, J = 2, 9 Hz, H6⁰), 6.49 (s, 2H,

H2⁰⁰,H6⁰⁰), 5.62 (s, 2H, –CONH₂), 3.93 (s, 6H, 4⁰,4⁰⁰-

5.3.4. (E)-3-(3⁰-Fluoro-4⁰-methoxyphenyl)-2-(3⁰⁰,4⁰⁰,5⁰⁰-tri-

methoxyphenyl)acrylamide (4d). The general procedure

was performed with 3d (100 mg, 0.28 mmol) and the res-

idue was puriﬁed by ﬂash column chromatography

(CH₂Cl₂/MeOH 99/1 (v/v)) to give 65 mg (64%) of 4d

1

as a brown powder. Mp (CH₂Cl₂/n-hexane) 165 °C; H

NMR(CDCl ): d 7.72 (s, 1H, H3), 6.89 (dd, 1H, J = 2,

3

OCH₃), 3.82 (s, 6H, 3⁰⁰,5⁰⁰-OCH₃); ¹³C NMR(CDCl ):

8.5 Hz, H6⁰), 6.79 (dd, 1H, J = 9, 8.5 Hz, H5⁰), 6.71

(dd, 1H, J = 13, 2 Hz, H2⁰), 6.49 (s, 2H, H2⁰⁰,H6⁰⁰), 5.76

(bs, 1H, –CONH), 5.58 (bs, 1H, –CONH), 3.90 (s, 3H,

4⁰⁰-OCH₃), 3.85 (s, 3H, 4⁰-OCH₃), 3.80 (s, 6H, 3⁰⁰,5⁰⁰-

OCH₃); ¹³C NMR(CDCl ₃): d 168.9 (C1), 154.4

(C3⁰⁰,C5⁰⁰), 151.6 (C3⁰, J = 244 Hz), 148.3 (C4⁰,

J = 11 Hz), 138.3 (C4⁰⁰), 136.4 (C3), 134.4 (C2), 131.4

(C1⁰⁰), 127.6 (C1⁰,C6⁰), 117.3 (C2⁰, J = 19 Hz), 112.7

(C5⁰), 106.2 (C2⁰⁰,C6⁰⁰), 61.0 (4⁰⁰-OCH₃), 56.2 (3⁰⁰,5⁰⁰-

OCH₃), 56.0 (4⁰-OCH₃); MS (DIC/NH₃) m/z = 362

[M+H]⁺; UV (MeOH) k (nm) 207 (loge = 4.56), 28_ꢀ2.₁5

3

d 168.3 (C1), 154.7 (C3⁰⁰,C5⁰⁰), 153.0 (C4⁰), 139.3 (C3⁰),

138.8 (C4⁰⁰), 136.1 (C6⁰), 134.9 (C3), 134.2 (C2), 130.7

(C1⁰⁰), 127.3 (C1⁰,C2⁰), 113.2 (C5⁰), 106.2 (C2⁰⁰,C6⁰⁰),

61.2 (4⁰⁰-OCH₃), 56.6 (4⁰-OCH₃), 56.4 (3⁰⁰,5⁰⁰-OCH₃);

MS(ES) m/z = 411 [M+Na]⁺; UV (EtOH) k (nm) 209

(loge = 4.7), 287 (loge = 4.3); IR(NaCl ﬁlm) m⁰(cm^ꢀ1

)

3418, 3173, 2912, 1679, 1528, 1351, 1279, 1126. Anal.

Calcd (C₁₉H₂₀N₂O₇) C = 58.76%, H = 5.19%,

N = 7.21%. Found C = 58.70%, H = 5.14%, N = 7.30%.

5.3.2. (E)-3-(3⁰-Amino-4⁰-methoxyphenyl)-2-(3⁰⁰,4⁰⁰,5⁰⁰-tri-

methoxyphenyl)acrylamide (1).¹⁸To a solution of 4a

(100 mg, 0.26 mmol) in AcOH (5 mL) zinc powder

(1.0 g) was added at rt. The suspension was vigorously

stirred for 1 h and then, was ﬁltered over Celite. The ﬁl-

trate was evaporated to dryness to give 1 (90 mg, quan-

titative yield) as a yellow powder in a pure form (1 was

analyzed without further puriﬁcation). Mp (CH₂Cl₂/

n-hexane) 189 °C; ¹H NMR(CDCl ₃): d 7.73 (s, 1H,

H3), 6.62 (d, 1H, J = 8.5 Hz, H5⁰), 6.54 (dd, 1H, J = 2,

8.5 Hz, H6⁰), 6.51 (s, 2H, H2⁰⁰,H6⁰⁰), 6.40 (d, 1H,

J = 2 Hz, H2⁰), 5.86 (s, 1H, –CONH), 5.55 (bs, 1H,

–CONH), 3.92 (s, 3H, 4⁰⁰-OCH₃), 3.81 (s, 9H, 4⁰,3⁰⁰,5⁰⁰-

OCH₃); ¹³C NMR(CDCl ₃): d 169.3 (C1), 154.2

(C3⁰⁰,C5⁰⁰), 148.3 (C4⁰), 138.5 (C3), 138.1 (C4⁰⁰), 135.7

(C3⁰), 132.3 (C1⁰⁰), 130.5 (C2), 127.4 (C1⁰), 122.5 (C6⁰),

116.6 (C2⁰), 109.9 (C5⁰), 106.6 (C2⁰⁰,C6⁰⁰), 61.1 (4⁰⁰-

OCH₃), 56.3 (3⁰⁰,5⁰⁰-OCH₃), 55.4 (4⁰-OCH₃); MS(ES)

(loge = 4.12), 303.5 (loge = 4.16); IR(KBr) m⁰(cm

)

3421–3157, 2912, 1681, 1585, 1508, 1278, 1237, 1129.

Anal. Calcd (C₁₉H₂₀FNO₅) C = 63.15%, H = 5.19%,

N = 7.21%. Found C = 63.63%, H = 5.47%, N = 4.27%.

5.3.5. (E)-3-(4⁰-Hydroxy-3⁰-methoxyphenyl)-2-(3⁰⁰,4⁰⁰,5⁰⁰-

trimethoxyphenyl)acrylamide (4f). The general procedure

was performed with 3e (112 mg, 0.28 mmol) and the res-

idue was puriﬁed by ﬂash column chromatography

(cyclohexane/AcOEt 20/80 (v/v)) to give 75 mg (77%)

of 4g as an amorphous brown powder. ¹H NMR

(CDCl₃): d 7.77 (s, 1H, H3), 6.79 (s, 2H, H5⁰,H6⁰),

6.54 (s, 2H, H2⁰⁰,H6⁰⁰), 6.46 (bs, 1H, H2⁰), 5.86 (bs,

1H, OH), 5.71 (bs, 1H, –CONH), 5.57 (bs, 1H, –

CONH), 3.89 (s, 3H, 4⁰⁰-OCH₃), 3.83 (s, 6H, 3⁰⁰,5⁰⁰-

OCH₃), 3.53 (s, 3H, 3⁰-OCH₃); ¹³C NMR(CDCl ): d

3

169.0 (C1), 154.5 (C3⁰⁰,C5⁰⁰), 146.9 (C4⁰), 146.0 (C3⁰),

138.1 (C3, C4⁰⁰), 132.4 (C1⁰⁰), 130.7 (C2), 126.9 (C1⁰),

126.1 (C5⁰), 114.3 (C6⁰), 111.8 (C2⁰), 106.5 (C2⁰⁰,C6⁰⁰),

60.9 (4⁰⁰-OCH₃), 56.3 (3⁰⁰,5⁰⁰-OCH₃), 55.3 (3⁰-OCH₃);

MS(ES) m/z = 382 [M+Na]⁺; UV (MeOH) k (nm) 202

(loge = 4.64), 206 (loge = 4.64), 234 (loge = 4.24), 281

m/z = 381 [M+Na]⁺; UV (MeOH)

k

(nm) 204

(loge = 4.6), 251 (loge = 4.2), 297 (loge = 4.0), 336

(loge = 3.9); IR(NaCl ﬁlm) m⁰(cm^ꢀ1) 3421, 2900,

1653, 1581, 1236, 1124.

(loge = 4.02), 321 (loge = 4.16); IR(NaCl ﬁlm)

m⁰

5.3.3. (E)-3-(4⁰-Methoxyphenyl)-2-(3⁰⁰,4⁰⁰,5⁰⁰-trimethoxy-

phenyl)acrylamide (4c). The general procedure was per-

formed with 3c (96 mg, 0.28 mmol) and the residue

was puriﬁed by ﬂash column chromatography using an

eluting gradient of AcOEt in cyclohexane to give

90 mg (94%) of 4c as a brown powder. Mp (CH₂Cl₂/

n-hexane)172–174 °C, ¹H NMR(CDCl ₃): d 7.79 (s,

1H, H3), 7.02 (d, 2H, J = 8 Hz, H2⁰,H6⁰), 6.72 (d, 2H,

J = 8 Hz, H3⁰,H5⁰), 6.50 (s, 2H, H2⁰⁰,H6⁰⁰), 5.75 (bs,

1H, –CONH), 5.57 (bs, 1H, –CONH), 3.93 (s, 3H, 4⁰⁰-

(cm^ꢀ1) 3471, 3347, 3179, 2935, 1663, 1582, 1514, 1463,

1412, 1280, 1237, 1126, 1030, 1104, 895, 818, 734. Anal.

Calcd

(C₁₉H₂₁NO₆)

C = 63.50%,

H = 5.89%,

N = 3.90%. Found C = 63.21%, H = 6.39%, N = 3.77%.

5.3.6. (E)-3-(3⁰-Hydroxy-4⁰-methoxyphenyl)-2-(3⁰⁰,4⁰⁰,5⁰⁰-

trimethoxyphenyl)acrylamide (4b). A solution of 3b

(0.055 mmol) in DMF (1 mL) was cooled at ꢀ10 °C

and stirred under argon. Triethylamine (31 lL,

0.22 mmol) and ethylchloroformate (16 lL, 0.17 mmol)

were added dropwise. After 30 min, a solution of NH₄Cl

(5 mg, 0.11 mmol) and triethylamine (15 lL, 0.11 mmol)

in DMF (1 mL) was added. The reaction mixture was

stirred for 1 h and evaporated to dryness under reduced

pressure. The residue was taken up with AcOEt and

washed with water and brine. The combined organic

layers were dried over anhydrous MgSO₄, ﬁltered and

evaporated to dryness. To a solution of the residue in

methanol (3 mL), a solution (0.5 mL) of LiOH (5 mg,

0.11 mmol) in water was added. After 5 h, the reaction

mixture was neutralized with 1 N aqueous HCl and

evaporated to dryness under vacuum. The residue was

OCH₃), 3.81 (s, 6H, 3⁰⁰,5⁰⁰-OCH₃), 3.77 (s, 3H, 4⁰-

13

OCH₃); C NMR(CDCl ): d 169.3 (C1), 160.3 (C4⁰),

3

154.4 (C3⁰⁰,C5⁰⁰), 138.1 (C4⁰⁰), 137.8 (C3), 132.4

(C2⁰,C6⁰), 132.3 (C1⁰⁰), 131.0 (C2), 127.2 (C1⁰), 113.9

(C3⁰,C5⁰), 106.4 (C2⁰⁰,C6⁰⁰), 61.2 (4⁰⁰-OCH₃), 56.3

(3⁰⁰,5⁰⁰-OCH₃), 55.3 (4⁰-OCH₃); MS(ES) m/z = 366

[M+Na]⁺; UV (MeOH) k (nm) 205 (loge = 4.57), 228

(loge = 4.31), 282.5 (loge = 4.16); IR(KBr) m⁰(cm^ꢀ1

)

3429, 3162, 2937, 1677, 1582, 1508, 1236, 1128, 1029,

995, 823, 691. Anal. Calcd (C₁₉H₂₁NO₅) C = 66.46%,

H = 6.16%, N = 4.08%. Found C = 65.66%, H =

6.19%, N = 4.16%.

C. Borrel et al. / Bioorg. Med. Chem. 13 (2005) 3853–3864

3861

treated with AcOEt. The resulting solution was washed

¹³C NMR(CDCl ₃):

d

153.9 (C3⁰⁰,C5⁰⁰), 153.6

successively with water and brine, dried over anhydrous

MgSO₄, ﬁltered and then evaporated to dryness. The

crude residue was puriﬁed by recrystallization in

(–NHCOO), 150.7 (C4⁰), 139.4 (C3⁰), 139.3 (C4⁰⁰),

136.0 (C1⁰⁰), 134.2 (C6⁰), 131.3 (C1), 129.7 (C1⁰), 125.5

(C2⁰), 112.9 (C5⁰), 111.2 (C2), 106.4 (C2⁰⁰,C6⁰⁰), 63.8

(-CH₂O), 61.0 (4⁰⁰-OCH₃), 56.5 (3⁰⁰,5⁰⁰-OCH₃), 56.4 (4⁰-

OCH₃), 17.7 (–CH₂–TMS), ꢀ1.4 (Si–CH₃); MS(ES) m/

z = 527 [M+Na]⁺; UV (MeOH) k (nm) 204 (loge = 4.60),

CH₂Cl₂/n-hexane to give 13 mg (66%) of 4b as yellow

1

crystals. Mp 163–165 °C; H NMR(CDCl ): d 7.75 (s,

3

1H, H3), 6.69 (d, 1H, J = 8 Hz, H5⁰), 6.65 (dd, 1H,

J = 2, 8 Hz, H6⁰), 6.64 (bs, 1H, H2⁰), 6.51 (s, 2H,

H2⁰⁰,H6⁰⁰), 5.54 (bs, 2H, –CONH₂), 5.50 (s, 1H, OH),

3.93 (s, 3H, 4⁰⁰-OCH₃), 3.86 (s, 3H, 4⁰-OCH₃), 3.82 (s,

6H, 3⁰⁰,5⁰⁰-OCH₃); ¹³C NMR(CDCl ₃): d 169.1 (C1),

154.3 (C3⁰⁰,C5⁰⁰), 145.2 (C4⁰), 144.7 (C3), 137.8

(C4⁰⁰,C3⁰), 131.9 (C1⁰⁰), 131.5 (C2), 129.0 (C1⁰), 123.8

(C6⁰), 116.3 (C2⁰), 110.2 (C5⁰), 106.5 (C2⁰⁰,C6⁰⁰), 61.1

(4⁰⁰-OCH₃), 56.3 (3⁰⁰,5⁰⁰-OCH₃), 55.9 (4⁰-OCH₃); MS(ES)

240 (loge = 4.23), 304 (loge = 4.13); IR(KBr) m⁰(cm^ꢀ1

)

3332, 2920, 1588, 1456, 1406, 1126, 1045, 839. Anal.

Calcd (C₂₄H₃₂N₂O₅Si) C = 57.13%, H = 6.39%,

N = 5.55%. Found C = 57.35%, H = 6.50%, N = 5.52%.

5.4.2.

(3⁰⁰,4⁰⁰,5⁰⁰-trimethoxyphenyl)ethen-1-yl]carbamate

(E-Methyl-N-[2-(4⁰-methoxy-3⁰-nitrophenyl)-1-

(6a).

The general procedure was performed with E-3a

(56 mg, 0.14 mmol). The reaction mixture was heated

for 4 h 30 min and the residue was puriﬁed by ﬂash col-

umn chromatography (eluent: cyclohexane/AcOEt 70/30

(v/v)) to give 52 mg (96%) of 6a as an amorphous yellow

m/z = 382 [M+Na]⁺; UV (MeOH)

(loge = 4.53), 207 (loge = 4.53), 242 (loge = 4.10), 296

k

(nm) 205

(loge = 3.94), 320 (loge = 3.99); IR(NaCl ﬁlm)

m⁰

(cm^ꢀ1) 3460, 3346, 3186, 2934, 1661, 1581, 1508, 1459,

1410, 1275, 1237, 1125, 1024. Anal. Calcd

(C₁₉H₂₁NO₆) C = 63.50%, H = 5.89%, N = 3.90%.

Found C = 56.28%, H = 5.70%, N = 3.45%.

1

powder. H NMR(CDCl ₃): d 7.44 (d, 1H, J = 2 Hz,

H2⁰), 7.08 (m, 2H, H2, H6⁰), 6.82 (d, 1H, J = 9 Hz,

H5⁰), 6.51 (s, 2H, H2⁰⁰,H6⁰⁰), 6.16 (bs, 1H, NH), 3.89

(s, 6H, 4⁰,4⁰⁰-OCH₃), 3.79 (s, 3H, –COOCH₃), 3.75 (s,

3H, 3⁰⁰,5⁰⁰-OCH₃); ¹³C NMR(CDCl ): d 153.9 (–NH-

5.4. General procedure for the preparation of carbamates

5a and 6a

3

COOCH₃, C3⁰⁰,C5⁰⁰), 150.8 (C4⁰), 139.3 (C3⁰), 139.2

(C4⁰⁰), 135.9 (C1), 134.2 (C6⁰), 131.2 (C1⁰⁰), 129.6 (C1⁰),

125.6 (C2⁰), 113.0 (C5⁰), 111.5 (C2), 106.3 (C2⁰⁰,C6⁰⁰),

61.1 (4⁰⁰-OCH₃), 56.4 (4⁰-OCH₃), 56.3 (3⁰⁰,5⁰⁰-OCH₃),

52.5 (–COOCH₃); MS(ES) m/z = 441 [M+Na]⁺; UV

(MeOH) k (nm) 202 (loge = 4.59), 203 (loge = 4.57),

220 (loge = 4.44), 245 (loge = 4.10), 282 (loge = 4.04);

IR(KBr) m⁰(cm^ꢀ1) 3300, 2943, 1585, 1518, 1460, 1414,

1282, 1127, 1021, 1006, 764. Anal. Calcd

(C₂₀H₂₂N₂O₈) C = 57.41%, H = 5.30%, N = 6.70%.

Found C = 56.36%, H = 5.57%, N = 6.18%.

Step 1: To a solution of E-3a (0.14 mmol) in anhydrous

acetone (3 mL), cooled at ꢀ5 °C and stirred under

argon, triethylamine (39 lL, 0.28 mmol) and ethylchlo-

roformate (19 lL, 0.18 mmol) were added dropwise.

After 30 min, ice (3 mL) was added and the solution

was extracted with CH₂Cl₂(4 mL). The combined

organic layers were washed with water, dried over anhy-

drous MgSO₄, ﬁltered and evaporated to dryness. Step

2: The residue was taken up in 3 mL of THF and the

solution was cooled at 0 °C. A solution of NaN₃

(19 mg, 0.28 mmol) in water (1 mL) was added. The

solution was extracted with ether (4 mL). The combined

organic layers were washed with water, dried over anhy-

drous MgSO₄, ﬁltered and evaporated to dryness under

reduced pressure. Step 3: The residue was taken up with

3 mL of anhydrous toluene and heated under argon at

80 °C for 1 h. Step 4: The reaction mixture was cooled

at 50 °C and 2-trimethylsilylethanol or methanol

(1.4 mmol) was added. The solution was stirred under

argon at 50 °C, then allowed to cool at rt and evapo-

rated to dryness. The crude residue was puriﬁed by ﬂash

column chromatography.

5.5. General procedure for the preparation of carbamates

(5c,d,g,h, 6c,d,g–h)

Step 1: A solution of acrylic acid (0.14 mmol) in freshly

distilled anhydrous acetone (3 mL) was cooled at ꢀ5 °C

and stirred under argon. Triethylamine (39 lL,

0.28 mmol) and ethyl chloroformate (19 lL, 0.18 mmol)

were added dropwise. Step 2: After 30 min, a solution of

NaN₃(19 mg, 0.28 mmol) in water (1 mL) was added

and the reaction was quenched after 1 h by addition of

ice (3 mL). Further steps are identical to those previ-

ously described for the preparation of carbamates 5a

and 6a.

5.4.1. (E)-[2-(Trimethylsilyl)ethyl]-N-[2-(4⁰-methoxy-3⁰-

nitrophenyl)-1-(3⁰⁰,4⁰⁰,5⁰⁰-trimethoxyphenyl)ethen-1-yl] carb-

amate (5a). The general procedure was performed with

E-3a (56 mg, 0.14 mmol). The reaction mixture was

heated for 18 h and the residue was puriﬁed by ﬂash col-

umn chromatography (eluent: cyclohexane/AcOEt 75/25

5.5.1. (E)-[2-(Trimethylsilyl)ethyl]-N-[2-(4⁰-methoxyphen-

yl)-1-(3⁰⁰,4⁰⁰,5⁰⁰-trimethoxyphenyl)ethen-1-yl]carbamate (5c).

The general procedure was performed with 3c (48 mg,

0.14 mmol). The reaction mixture was heated for 5 h

and the residue was puriﬁed by ﬂash column chromato-

graphy (eluent: cyclohexane/AcOEt 90/10 (v/v)) to give

(v/v)) to give 55 mg (78%) of 5a as yellow powder. Mp

1

(CH₂Cl₂/n-hexane) = 141–142 °C; H NMR(CDCl ): d

20 mg (31%) of 5c as an amorphous white powder. H

3

7.42 (d, 1H, J = 2Hz, H2⁰), 7.08 (dd, 2H, J = 2,

8.5 Hz, H2, H6⁰), 6.71 (d, 1H, J = 8.5 Hz, H5⁰), 6.50

(s, 2H, H2⁰⁰,H6⁰⁰), 6.11 (bs, 1H, NH), 4.27 (t, 2H,

OCH₂), 3.88 (s, 6H, 4⁰,4⁰⁰-OCH₃), 3.72 (s, 3H, 3⁰⁰,5⁰⁰-

OCH₃), 1.50 (t, 2H, –CH₂–TMS), 0.7 (s, 9H, Si–CH₃);

NMR(CDCl ₃): d 7.00 (s, 1H, H2), 6.91 (bd, 2H,

J = 8.8 Hz, H2⁰,H6⁰), 6.67 (bd, 2H, J = 8.8 Hz,

H3⁰,H5⁰), 6.54 (s, 2H, H2⁰⁰,H6⁰⁰), 6.02 (bs, 1H, –CONH),

4.24 (t, 2H, –OCH₂), 3.87 (s, 3H, 4⁰⁰-OCH₃), 3.74 (s, 3H,

4⁰-OCH₃), 3.71 (s, 6H, 3⁰⁰,5⁰⁰-OCH₃), 1.03 (t, 2H,

3862

C. Borrel et al. / Bioorg. Med. Chem. 13 (2005) 3853–3864

–CH₂–TMS), 0.06 (9H, s, Si–CH₃); ¹³C NMR(CDCl ):

(MeOH) k (nm) 201 (loge = 4.66), 204 (loge = 4.66),

3

d 157.9 (C4⁰), 154.2 (–NHCO), 153.5 (C3⁰⁰, C5⁰⁰), 138.5

(C4⁰⁰), 133.3 (C1), 132.4 (C1⁰⁰), 130.1 (C2⁰,C6⁰), 129.2

(C1⁰), 115.5 (C2), 113.5 (C3⁰,C5⁰), 106.4 (C2⁰⁰,C6⁰⁰), 63.6

(–OCH₂), 61.1 (4⁰⁰-OCH₃), 56.2 (3⁰⁰,5⁰⁰-OCH₃), 55.3 (4⁰-

OCH₃), 17.8 (–CH₂–TMS), ꢀ1.4 (Si–CH₃); MS(ES) m/

242 (loge = 4.24), 290 (loge = 4.13); IR(KBr)

m⁰

(cm^ꢀ1) 3305, 2951, 2899, 2858, 1710, 1585, 1544, 1508,

1288, 1244, 1228, 1127, 866, 838. Anal. Calcd

(C₃₀H₄₇NO₇Si₂) C = 61.09%, H = 8.03%, N = 2.37%.

Found C = 60.69%, H = 8.11%, N = 1.98%.

z = 482 [M+Na]⁺; UV (MeOH)

k

(nm) 206

(loge = 4.63), 244 (loge = 4.26), 290 (loge = 4.17); IR

(NaCl ﬁlm) m⁰(cm^ꢀ1) 3379, 2954, 1705, 1584, 1510,

1421, 1249, 1129, 1037, 836. Anal. Calcd (C₂₄H₃₃NO₆Si)

C = 62.72%, H = 7.24%, N = 3.05%. Found C = 62.16%,

H = 7.12%, N = 3.27%.

5.5.4. (E)-Ethyl [5⁰-{2-[2-(trimethylsilyl)ethylcarbamoyl]-

2-(3⁰⁰,4⁰⁰,5⁰⁰-trimethoxyphenyl)ethen-1-yl}]-2⁰-methoxyphen-

yl carbonate (5h). The general procedure was performed

with 3b (50 mg, 0.14 mmol). The reaction mixture was

heated for 4 h 30 min and the residue was puriﬁed by

ﬂash column chromatography (eluent: cyclohexane/

AcOEt 80/20 (v/v)) to give 38 mg (50%) of 3b as an

5.5.2.

(E)-[2-(Trimethylsilyl)ethyl]-N-[2-(3⁰-ﬂuoro-4⁰-

1

methoxyphenyl)-1-(3⁰⁰,4⁰⁰,5⁰⁰-trimethoxyphenyl)ethen-1-yl]-

carbamate (5d). The general procedure was monitored

with 3d (50 mg, 0.14 mmol). The reaction mixture was

heated for 5 h and the residue was puriﬁed by ﬂash col-

umn chromatography (eluent: cyclohexane/AcOEt 80/20

(v/v)) to give 48 mg (71%) of 5d as a white powder. Mp

(CH₂Cl₂/n-hexane) 126 °C; ¹H NMR(DMSO- d₆): d 8.88

(bs, 1H, –CONH), 6.94 (t, 1H, J = 9 Hz, H5⁰), 6.70 (s,

1H, H2), 6.69 (dd, 1H, J = 2, 9 Hz, H6⁰), 6.60 (dd, 1H,

J = 13, 2 Hz, H2⁰), 6.49 (s, 2H, H2⁰⁰,H6⁰⁰), 4.10 (t, 2H,

–OCH₂), 3.74 (s, 3H, 4⁰-OCH₃), 3.66 (s, 3H, 4⁰⁰-

OCH₃), 3.62 (s, 6H, 3⁰⁰,5⁰⁰-OCH₃), 0.94 (t, 2H, –CH₂–

TMS), 0.03 (9H, s, Si–CH₃); ¹³C NMR(DMSO- d₆): d

155.0 (–NHCO), 153.8 (C3⁰⁰,C5⁰⁰), 151.83 (C3⁰,

J = 252 Hz), 145.5 (C4⁰), 138.8 (C4⁰⁰), 136.8 (C1), 132.5

(C1⁰⁰), 130.95 (C1⁰, J = 8 Hz), 125.9 (C6⁰), 116.35 (C2⁰,

J = 17 Hz), 114.8 (C2), 114.4 (C5⁰), 107.8 (C2⁰⁰,C6⁰⁰),

63.0 (–OCH₂), 61.2 (4⁰⁰-OCH₃), 56.9 (4⁰,3⁰⁰,5⁰⁰-OCH₃),

18.3 (–CH₂–TMS), ꢀ0.4 (Si–CH₃); MS(ES) : m/z = 500

[M+Na]⁺; UV (MeOH) k (nm) 202 (loge = 4.60), 204

(loge = 4.57), 243 (loge = 4.31), 290 (loge = 4.17); IR

(KBr) m⁰(cm^ꢀ1) 3371, 2928, 1724, 1635, 1518, 1437,

1127, 1052, 862. Anal. Calcd (C₂₄H₃₂FNO₆Si)

C = 60.36%, H = 6.75%, N = 2.93%. Found C =

58.78%, H = 6.72%, N = 2.77%.

amorphous white powder. H NMR(CDCl ₃): d 6.98

(s, 1H, H1), 6.83 (dd, 1H, J = 1.5, 8.5 Hz, H4⁰), 6.73

(d, 1H, J = 8.5 Hz, H3⁰), 6.71 (d, 1H, J = 1.5 Hz, H6⁰),

6.52 (s, 2H, H2⁰⁰,H6⁰⁰), 6.07 (bs, 1H, –CONH), 4.25

(4H, m, 2-OCH₂), 3.85 (s, 3H, 4⁰⁰-OCH₃), 3.75 (s, 3H,

2⁰-OCH₃), 3.72 (s, 6H, 3⁰⁰,5⁰⁰-OCH₃), 1.33 (t, 3H,

–CH₂CH₃), 1.03 (t, 2H, –CH₂–TMS), 0.02 (s, 9H, Si–

CH₃); ¹³C NMR(CDCl ₃): d 153.9 (–NHCO), 153.5

(C3⁰⁰,C5⁰⁰), 153.1 (–COOEt), 149.4 (C2⁰), 139.6 (C1⁰),

138.5 (C4⁰⁰), 134.3 (C2), 131.8 (C1⁰⁰), 129.7 (C5⁰), 127.6

(C4⁰), 122.6 (C6⁰), 113.9 (C1⁰), 112.0 (C3⁰), 106.4

(C2⁰⁰,C6⁰⁰), 64.7 (–OCH₂–CH₃), 63.6 (–OCH₂–CH₂),

60.9 (4⁰⁰-OCH₃), 56.2 (3⁰⁰,5⁰⁰-OCH₃), 55.9 (2⁰-OCH₃),

17.7 (–CH₂–TMS), 14.1 (–OCH₂CH₃), ꢀ1.4 (Si–CH₃);

MS(ES) m/z = 570 [M+Na]⁺; UV (EtOH) k (nm) 201

(loge = 4.69), 203 (loge = 4.66), 244 (loge = 4.24), 289

(loge = 4.14); IR(KBr) m⁰(cm^ꢀ1) 3329, 2955, 1762,

1732, 1588, 1544, 1504, 1268, 1234, 1125, 1050, 862,

834. Anal. Calcd (C₂₇H₃₇NO₉Si) C = 59.21%, H =

6.81%, N = 2.56%. Found C = 57.91%, H = 6.98%,

N = 2.60%.

5.5.5. (E)-Methyl-N-[-2-(4⁰-methoxyphenyl)-1-(3⁰⁰,4⁰⁰,5⁰⁰-

trimethoxyphenyl)ethen-1-yl]carbamate (6c). The general

procedure was performed with 3c (48 mg, 0.14 mmol).

The reaction mixture was heated for 4 h 10 min and

the residue was puriﬁed by ﬂash column chromatogra-

phy (eluent: cyclohexane/AcOEt 80/20 (v/v)) to give

20 mg (38%) of 6c as an amorphous pale yellow powder.

¹H NMR(CDCl ₃): d 7.00 (s, 1H, H2), 6.91 (d, 2H,

J = 8.5 Hz, H2⁰,H6⁰), 6.67 (d, 2H, J = 8.5 Hz,

H3⁰,H5⁰), 6.53 (s, 2H, H2⁰⁰,H6⁰⁰), 6.11 (bs, 1H, –CONH),

3.86 (s, 3H, 4⁰⁰-OCH₃), 3.76 (s, 3H, 4⁰-OCH₃), 3.74 (s,

3H, –COOCH₃), 3.71 (s, 6H, 3⁰⁰,5⁰⁰-OCH₃); ¹³C NMR

(CDCl₃): d 158.0 (–NHCOOCH₃), 154.5 (C4⁰), 153.5

(C3⁰⁰, C5⁰⁰), 138.3 (C4⁰⁰), 133.1 (C1), 132.3 (C1⁰⁰), 130.2

(C2⁰,C6⁰), 129.1 (C1⁰), 115.6 (C2), 113.5 (C3⁰,C5⁰),

106.5 (C2⁰⁰,C6⁰⁰), 61.1 (4⁰⁰-OCH₃), 56.2 (3⁰⁰,5⁰⁰-OCH₃),

55.3 (–COOCH₃), 52.4 (4⁰-OCH₃); MS (DIC/NH₃) m/

z = 374 [M+H]⁺; UV (MeOH) k (nm) 202 (loge = 4.50),

205 (loge = 4.48), 242 (loge = 4.16), 292 (loge = 4.08);

IR(NaCl ﬁlm) m⁰(cm^ꢀ1) 3328, 3002, 2940, 1730, 1583,

1509, 1245, 1127, 1031, 1004, 826. Anal. Calcd

(C₂₀H₂₃NO₆) C = 64.33%, H = 6.21%, N = 3.75%.

Found C = 62.98%, H = 6.26%, N = 3.48%.

5.5.3. (E)-[2-(Trimethylsilyl)ethyl]-N-[2-(4⁰-methoxy-3⁰-

[(tert-butyldimethylsilyl)oxy]phenyl)-1-(3⁰⁰,4⁰⁰,5⁰⁰-trimeth-

oxyphenyl)ethen-1-yl]carbamate (5g). The general proce-

dure was performed with 3g (63 mg, 0.14 mmol). The

reaction mixture was heated for 4 h 30 min and the res-

idue was puriﬁed by ﬂash column chromatography (elu-

ent: cyclohexane/AcOEt 90/10 (v/v)) to give 31 mg (26%)

of 5g as an amorphous white powder. ¹H NMR

(CDCl₃): d 6.98 (s, 1H, H2), 6.65 (d, 1H, J = 8.5 Hz,

H5⁰), 6.61 (dd, 1H, J = 2, 8.5 Hz, H6⁰), 6.54 (s, 2H,

H2⁰⁰,H6⁰⁰), 6.48 (d, 1H, J = 2 Hz, H2⁰), 5.96 (bs, 1H,

NH), 4.24 (t, 2H, –OCH₂), 3.85 (s, 3H, 4⁰⁰-OCH₃),

3.73 (s, 3H, 4⁰-OCH₃), 3.72 (s, 3H, 3⁰⁰,5⁰⁰-OCH₃), 1.02

(t, 2H, –CH₂–TMS), 0.89 (s, 6H, C–CH₃in TBDMS),

0.05 (s, 9H, Si–CH₃), 0.00 (s, 9H, Si–CH₃); ¹³C NMR

(CDCl₃): d 153.9 (–NHCO), 153.5 (C3⁰⁰,C5⁰⁰), 149.5

(C4⁰), 144.4 (C3⁰), 138.3 (C4⁰⁰), 133.3 (C1), 132.6 (C1⁰⁰),

129.6 (C1⁰), 122.9 (C6⁰), 121.2 (C2⁰), 115.2 (C2), 111.7

(C5⁰), 106.5 (C2⁰⁰,C6⁰⁰), 63.5 (CH₂O), 60.9 (4⁰⁰-OCH₃),

56.1 (3⁰⁰,5⁰⁰-OCH₃), 55.5 (4⁰-OCH₃), 25.6 (C–CH₃in

TBDMS), 18.2 (Si–C in TBDMS), 17.7 (–CH₂-TMS),

ꢀ1.5 (Si–CH₃), ꢀ5.0 (Si–CH₃in TBDMS); MS(DIC/

NH₃) m/z = 590 [M+H]⁺, 562, 446, 196, 90, 73; UV

5.5.6. (E)-Methyl-N-[2-(3⁰-ﬂuoro-4⁰-methoxyphenyl)-1-

(3⁰⁰,4⁰⁰,5⁰⁰-trimethoxyphenyl)ethen-1-yl]carbamate

(6d).

The general procedure was performed with 3d (50 mg,

C. Borrel et al. / Bioorg. Med. Chem. 13 (2005) 3853–3864

3863

0.14 mmol). The reaction mixture was heated for 4 h

and the residue was puriﬁed by ﬂash column chromato-

graphy (eluent: cyclohexane/AcOEt 70/30 (v/v)) to give

H6⁰), 6.52 (s, 2H, H2⁰⁰,H6⁰⁰), 6.10 (bs, 1H, –CONH),

4.25 (q, 2H, –OCH₂), 3.86 (s, 3H, 4⁰⁰-OCH₃), 3.79 (s,

3H, 2⁰-OCH₃), 3.76 (s, 3H, –NHCOOCH₃), 3.73 (s,

6H, 3⁰⁰,5⁰⁰-OCH₃), 1.35 (t, 3H, –CH₂CH₃); ¹³C NMR

(CDCl₃): d 154.3 (–NHCOOCH₃), 153.6 (C3⁰⁰, C5⁰⁰),

153.3 (–COOEt), 149.5 (C2⁰), 139.6 (C1⁰), 138.5 (C4⁰⁰),

134.5 (C2), 131.8 (C1⁰⁰), 129.6 (C5⁰), 127.7 (C4⁰), 122.7

(C6⁰), 114.1 (C1), 112.0 (C3⁰), 106.3 (C2⁰⁰,C6⁰⁰), 64.9

(–OCH₂), 61.0 (4⁰⁰-OCH₃), 56.2 (3⁰⁰,5⁰⁰-OCH₃), 56.0 (2⁰-

OCH₃), 52.5 (–COOCH₃), 14.2 (–CH₂–CH₃); MS(ES)

1

48 mg (86%) of 6d as an amorphous white powder. H

NMR(CDCl ₃): d 7.00 (s, 1H, H2), 6.72 (dd, 1H,

J = 8, 9 Hz, H5⁰), 6.70 (dd, 1H, J = 1.5, 8 Hz, H6⁰),

6.68 (dd, 1H, J = 12, 1.5 Hz, H2⁰), 6.52 (s, 2H,

H2⁰⁰,H6⁰⁰), 6.08 (bs, 1H, –CONH), 3.87 (s, 3H, 4⁰⁰-

OCH₃), 3.82 (s, 3H, 4⁰-OCH₃), 3.77 (s, 3H, –COOCH₃),

3.74 (s, 6H, 3⁰⁰,5⁰⁰-OCH₃); ¹³C NMR(CDCl ): d 154.1

3

(–NHCOOCH₃), 153.6 (C3⁰⁰,C5⁰⁰), 151.8 (C3⁰,

J = 243 Hz), 145.77 (C4⁰, J = 12 Hz), 138.5 (C4⁰⁰),

134.3 (C1), 131.7 (C1⁰⁰), 129.95 (C1⁰, J = 7.5 Hz), 124.9

(C6⁰), 116.173 (C2⁰, J = 18 Hz), 113.8 (C2), 112.9

(C5⁰), 106.4 (C2⁰⁰,C6⁰⁰), 61.0 (4⁰⁰-OCH₃), 56.4 (4⁰,3⁰⁰,5⁰⁰-

OCH₃), 52.4 (–COOCH₃); MS(ES) m/z = 414

[M+Na]⁺; UV (MeOH) k (nm) 202 (loge = 4.63), 204

(loge = 4.58), 242 (loge = 4.20), 274 (loge = 3.95), 281

m/z = 484 [M+Na]⁺; UV (MeOH)

k

(nm) 202

(loge = 4.46), 216 (loge = 4.33), 243 (loge = 4.06), 293

(loge = 4.04); IR(NaCl ﬁlm) m⁰(cm^ꢀ1) 3313, 2940,

1763, 1732, 1584, 1505, 1236, 1127, 1055, 1003, 917,

770, 731. Anal. Calcd (C₂₃H₂₇NO₉) C = 59.86%,

H = 5.90%, N = 3.04%. Found C = 59.42%, H =

6.01%, N = 3.05%.

(loge = 3.94), 295 (loge = 3.84); IR(NaCl ﬁlm)

m⁰

(cm^ꢀ1) 3336, 2936, 1584, 1518, 1441, 1416, 1281, 1129,

1023. Anal. Calcd (C₂₀H₂₂FNO₆) C = 61.38%,

H = 5.67%, N = 3.58%. Found C = 61.35%, H =

5.80%, N = 3.59%.

5.6. Tubulin binding assay

Calf brain tubulin was puriﬁed according to the method

of Shelanski, by three cycles of assembly–disassembly

and then dissolved in the assembly buﬀer containing

0.1 M MES, 0.5 mM MgCl₂, 2 mM EGTA and 1 mM

GTP pH = 6.6 (the concentration of tubulin was about

2–3 mg/mL).²⁶Tubulin assembly was monitored and

recorded continuously by turbidimetry at 400 nm in a

UV spectrophotometer, equipped with a thermostated

cell at 37 °C.²⁷The IC₅₀, deﬁned as the concentration

value of inhibitor, which decreased by 50% the maxi-

mum assembly rate of tubulin without drug, was deter-

mined. The IC₅₀for all compounds were compared to

the IC₅₀of colchicine, measured the same day under

the same conditions.

5.5.7. (E)-Methyl-N-[2-(4⁰-methoxy-3⁰-[(tert-butyldimeth-

ylsilyl)oxy]phenyl)-1-(3⁰⁰,4⁰⁰,5⁰⁰-trimethoxyphenyl)ethen-1-

yl]carbamate (6g). The general procedure was performed

with 3g (63 mg, 0.14 mmol). The reaction mixture was

heated for 4 h 30 min and the residue was puriﬁed by

ﬂash column chromatography (eluent: cyclohexane/

AcOEt 80/20 (v/v)) to give 22 mg (31%) of 6g as an

1

amorphous white powder. H NMR(CDCl ₃): d 6.98

(s, 1H, H2), 6.66 (d, 1H, J = 8.5 Hz, H5⁰), 6.62 (dd,

1H, J = 1, 8.5 Hz, H6⁰), 6.54 (s, 2H, H2⁰⁰,H6⁰⁰), 6.48 (d,

1H, J = 2 Hz, H2⁰), 6.03 (bs, 1H, –NH), 3.86 (s, 3H,

4⁰⁰-OCH₃), 3.76 (s, 3H, COOCH₃), 3.74 (s, 3H, 4⁰-

OCH₃), 3.73 (s, 3H, 3⁰⁰,5⁰⁰-OCH₃), 0.90 (s, 9H, C–

CH₃), 0.00 (s, 6H, Si–CH₃); ¹³C NMR(CDCl ₃): d

154.2 (–NHCOOCH₃), 153.5 (C3⁰⁰,C5⁰⁰), 149.6 (C4⁰),

144.4 (C3⁰), 138.3 (C4⁰⁰), 133.1 (C1), 132.4 (C1⁰⁰), 129.4

(C1⁰), 122.9 (C6⁰), 121.2 (C2⁰), 115.4 (C2), 111.6 (C5⁰),

106.4 (C2⁰⁰,C6⁰⁰), 60.9 (4⁰⁰-OCH₃), 56.1 (3⁰⁰,5⁰⁰-OCH₃),

55.5 (4⁰-OCH₃), 52.3 (–COOCH₃), 25.6 (C–CH₃in

TBDMS), 18.3 (Si–C), ꢀ0.7 (Si–CH₃); MS(ES) m/

5.7. Cell growth inhibition assay

MTT colorimetric assay was employed according to the

established procedure.²⁸The human cell lines MCF-7

(breast adenocarcinoma), KB-3-1 (epidermoid carci-

noma) and IGROV (ovary adenocarcinoma) were main-

tained in DMEM medium (Eurobio) supplemented with

10% fetal calf serum, 2 mM ^L-glutamine, 50 UI/mL pen-

icillin and 50 UI/mL streptomycin. Cells were grown at

37 °C and 5% CO₂in humidiﬁed atmosphere.

10,000 cells/well were seeded in 200 lL of growth med-

ium in 96-well microtitre plates (ATGC Biotechnol-

ogies, France) and incubated for 24 h prior to addition

of experimental drugs (ﬁnal dimethylsulfoxide concen-

tration, 0.1%). The cells were treated with eleven con-

centrations of test compounds in a CO₂incubator for

72 h. At the end of this period, 20 lL of 5 mg/mL 3-

(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-

mide (MTT, Sigma) was added to each well and the

plates were incubated for 3 h at 37 °C. The medium

was aspirated and the formazan solubilized by 100 L

of DMSO. The UV absorbance of the solubilized forma-

zan crystals was measured spectrophotometrically

(Metermech R960 Microplate Reader, Bioblock Scien-

tiﬁc, France). The IC₅₀(concentration reducing by

50% the absorbance at 570 nm) was calculated by a

z = 526 [M+Na]⁺; UV (MeOH)

k

(nm) 205

(loge = 4.70), 207 (loge = 4.69), 242 (loge = 4.17), 288

(loge = 4.12), 314 (loge = 4.07); IR(NaCl ﬁlm)

m⁰

(cm^ꢀ1) 3312, 2939, 1773, 1676, 1587, 1499, 1463, 1416,

1339, 1250, 1174, 1127, 1028, 1002, 836, 775. Anal.

Calcd

N = 2.78%. Found C = 61.77%, H = 7.62%, N = 2.75%.

(C₂₆H₃₇NO₇Si)

C = 62.00%,

H = 7.40%,

5.5.8.

(E)-Ethyl{5⁰-[2-methylcarbamoyl-2-(3⁰⁰,4⁰⁰,5⁰⁰-tri-

methoxyphenyl)ethen-1-yl]-2⁰-methoxyphenyl}carbonate

(6h). The general procedure was performed with 3b

(50 mg, 0.14 mmol). The reaction mixture was heated

for 4 h 30 min and the residue was puriﬁed by ﬂash col-

umn chromatography (eluent: cyclohexane/AcOEt 70/30

(v/v)) to give 23 mg (36%) of 6b as a white powder. Mp

(CH₂Cl₂/n-hexane) 180–185 °C; ¹H NMR(CDCl ₃): d

7.00 (s, 1H, H1), 6.84 (dd, 1H, J = 2, 8.5 Hz, H4⁰),

6.74 (d, 1H, J = 8.5 Hz, H3⁰), 6.72 (d, 1H, J = 2 Hz,

3864

C. Borrel et al. / Bioorg. Med. Chem. 13 (2005) 3853–3864

14. Kirwan, I. G.; Loadman, P. M.; Swaine, D. J.; Anthoney,

D. A.; Pettit, G. R.; Lippert, J. W.; Shnyder, S. D.;

Cooper, P. A.; Bibby, M. C. Clin. Cancer Res. 2004, 10,

1446.

linear regression performed on the linear zone of the

dose–response curve.

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Article Doi

New antitubulin derivatives in the combretastatin A4 series: Synthesis and biological evaluation

DOI: 10.1016/j.bmc.2005.02.039

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Full text of DOI:10.1016/j.bmc.2005.02.039

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