Design, synthesis and preliminary biological evaluation of acridine compounds as potential agents for a combined targeted chemo-radionuclide therapy approach to melanoma

Bioorganic & Medicinal Chemistry 16 (2008) 7671–7690

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

journal homepage: w ww.elsevier.com/locate/bmc

Design, synthesis and preliminary biological evaluation of acridine

compounds as potential agents for a combined targeted chemo-radionuclide

therapy approach to melanoma

Nicolas Desbois ^a,b,c, Maryline Gardette ^a,b,c, Janine Papon ^a,b,c, Pierre Labarre ^a,b,c, Aurélie Maisonial ^a,b,c

,

Philippe Auzeloux ^a,b,c, Claire Lartigue ^a,b,c, Bernadette Bouchon ^a,b,c, Eric Debiton ^a,b,c, Yves Blache ^d,

Olivier Chavignon ^a,b,c, Jean-Claude Teulade ^a,b,c, Jean Maublant ^a,b,c, Jean-Claude Madelmont ^a,b,c

,

Nicole Moins ^a,b,c, Jean-Michel Chezal ^a,b,c,

*

^aUniv Clermont 1, UFR Pharmacie, Clermont-Ferrand F-63001, France

^bInserm U484, Clermont-Ferrand F-63005, France

^cCentre Jean-Perrin, Clermont-Ferrand F-63011, France

^dLaboratoire MAPIEM EA 3834, UFR Sciences et Techniques, Université du Sud Toulon-Var, BP 20132, 83957 La Garde Cedex, France

a r t i c l e i n f o

a b s t r a c t

Article history:

Various iodo-acridone and acridine carboxamides have been prepared and evaluated as agents for tar-

geted radionuclide and/or chemotherapy for melanoma, due to their structural similarity to benzamides

which are known to possess speciﬁc afﬁnity for melanin. Three of these carboxamides selected for their in

vitro cytotoxic properties were radioiodinated with [¹²⁵I]NaI at high speciﬁc activity. Biodistribution

studies carried out in B16F0 murine melanoma tumour-bearing mice highlighted that acridone 8f and

acridine 9d, presented high, long-lasting tumour concentrations together with an in vivo kinetic proﬁle

favourable to application in targeted radionuclide therapy.

Received 11 March 2008

Revised 29 June 2008

Accepted 4 July 2008

Available online 10 July 2008

Keywords:

Acridine

Melanoma treatment

Targeted therapy

Radionuclide therapy

1. Introduction

or agents with a high afﬁnity for melanin. This fourth group consti-

tutes an heterogeneous drug family that includes polyamines,⁸

Malignant melanoma is the most serious forms of skin cancer

and has a high rate of dissemination. Patients with distant metasta-

ses show a very low response rate to chemotherapy, monochemo-

therapy or polychemotherapeutic regimens, and there has been

no progress in systemic palliative melanoma treatment for three

decades.¹Multiple randomised studies have been unable to demon-

strate any signiﬁcant advantage for survival.²Melanoma is de-

scribed as intrinsically resistant to many chemotherapeutic drugs,

which makes it exceptionally difﬁcult to treat this neoplasm. The

increasing incidence of skin melanoma and the lack of effective

therapy have intensiﬁed the search for new methods of early dis-

ease detection and treatment. One approach explored is to use

selective tissue-targeted chemotherapy or radionuclide therapy,

which has led to the development of new carriers suitable for tar-

geting melanoma. The carriers already investigated can be grouped

into four classes: monoclonal antibodies (MAbs),³alpha-melano-

benzamides^9–12and polycyclic aromatic compounds.¹³

Drug–melanin binding is a complex process involving various

interactions. The polyanionic feature of melanin is an important

factor in the afﬁnity of these drugs.^8,14Moreover, in vitro studies

of the binding of many compounds to melanin¹³showed that

several polycyclic aromatic compounds, especially those with

coplanar fused rings, bind strongly to melanin. This class of com-

pounds has been used for melanoma targeted chemotherapy or

radionuclide therapy. Wolf et al., in a study on N-(2-diethylamino-

ethyl)-benzamides conjugated with alkylating cytostatics, reported

higher melanin afﬁnity and toxicity against B16F0 melanoma cells

than with the parent drugs.¹⁵Otherwise, radiolabelled methylene

blue (MTB) with astatine-211, an alpha emitter, has also shown

promising results for the treatment of cutaneous tumours and their

metastases.¹⁶

Our institution has recently developed aromatic and heteroaro-

matic analogues of N-(2-diethylaminoethyl)-4-iodobenzamide

(1, BZA) (Fig. 1), which is a radiotracer already successfully used

in humans for the diagnosis of malignant melanoma,¹⁷as part of

a targeted radionuclide therapy approach to melanoma treatment.

cyte stimulating hormone (a

-MSH),^4,5false melanin precursors^6,7

* Corresponding author. Tel.: +33 4 73 15 08 00; fax: +33 4 73 15 08 01.

E-mail address: j-michel.chezal@u-clermont1.fr (J.-M. Chezal).

doi:10.1016/j.bmc.2008.07.015

7672

N. Desbois et al. / Bioorg. Med. Chem. 16 (2008) 7671–7690

O

N

X

NEt₂

, HCl

N

H

, HCl

, 2HCl

I

H

N

I

N

, 2HCl

I

H

CONHR

1

9

BZA

2

3

X = CH

X = N

8

MeO

NHSO₂Me

Figure 1.

HN

CONHR

Y

X, Y = H, I

X

, 2HCl

CONHR

In most cases, the aromatic moiety was changed without loss of

speciﬁc tumoural afﬁnity. Quinoline 2 and quinoxaline 3 analogues

(Fig. 1) showed signiﬁcantly higher tumoural uptakes after 72 h,

respectively, peaking at 7- and 16-fold higher values than with

the BZA parent compound.¹⁸

N

11

10

R = (CH₂)₂NEt₂

Figure 3. Chemical structures proposed.

These results led us to attempt to combine the beneﬁts of che-

motherapy and radionuclide therapy by conjugating an effective

radionuclide to cytotoxic heteroaromatic analogues of compounds

1–3. Such compounds could be used for radionuclide therapy using

a beta or Auger electron emitter and/or for chemotherapy of mel-

anoma. We selected an acridine analogue, a decision based on sev-

eral considerations: (a) these molecules are well known as

cytotoxic DNA-intercalating agents^19–21; (b) the acridine deriva-

tives such as N-(2-dimethylaminoethyl)-acridine-4-carboxamide

(DACA: NSC 601316) (4), SN 16713 (5) and the acridone derivative

6 possess a basic side chain similar to the BZA compound (Fig. 2);

(c) the acridine moiety shows structural similarity with com-

pounds 2 and 3, and acridine orange (5) has a documented afﬁnity

for melanin and has been reported to bind strongly to synthetic

melanin (98% binding with synthetic or uveal melanin).²²

Furthermore, DACA (4) showed toxicity against human mela-

noma cell lines and their tumour xenografts in mice.^23,24These ef-

fects are in accordance with low capacity of DACA to induce any

signiﬁcant resistance compared with other anti-tumoural agents.²⁵

Moreover, DNA-intercalating agents would appear suitable for

application in radionuclide therapy using iodine-125, an Auger

electron emitter, and it has been demonstrated that intracellular

¹²⁵I has a much higher cell-killing effect when located in the close

vicinity of DNA than when in the cytoplasm.^26–28The range of the

Auger electron (1–1.5 nm) causes double-strand breaks in DNA and

induces less surrounding tissue damage than ¹³¹I because of that

short-range biological effectiveness.²⁹The beta particles emitted

from ¹³¹I have a maximum range of 3 mm and are better dedicated

to neoadjuvant therapy, whereas ¹²⁵I, which is only highly toxic if

it is located inside the cell nucleus, appears more suitable for adju-

vant therapy or for the treatment of melanoma micrometastases.

In the present work, we describe the method for the synthesis of

a panel of iodo-acridone or iodo-acridine-carboxamide compounds

8–11 (Fig. 3) and report the results of preliminary in vitro and in

vivo biological tests designed to select a leading candidate for

use in radionuclide therapy and/or chemotherapy of melanoma.

The structure–activity relationships and particularly the positions

of iodine and carboxamide substituents were evaluated while the

lipophilic side chain was kept identical to the parent compound

BZA.

Given that we working towards an approach to melanoma

treatment combining targeted chemotherapy and radionuclide

therapy, this pharmacomodulation program gave rise to two initial

questions: (i) is the toxicity of the chemical compounds con-

served? (ii) Is there a speciﬁc afﬁnity for melanoma tumour with

a kinetic proﬁle favourable to application in targeted radionuclide

therapy when administered in vivo? Biological experiments were

developed using in vitro and in vivo tumour models to assess these

issues.

2. Results and discussion

2.1. Chemistry

Acridones are important starting materials for the synthesis of a

wide range of acridine derivatives. Furthermore, acridine com-

pounds 9 and 9-anilino derivatives 10 were prepared from the

same acridones 8, whereas acridine-9-carboxamide products 11

were synthesised from the commercially available acridine-9-car-

boxylic acid hydrate.

2.1.1. Synthesis of acridone derivatives 8a–g

The (1-3)-iodo-acridones 18a–c were achieved by a method re-

ported previously,³⁰starting from methyl iodoanthranilate 14

(Schemes 1 and 2). Methyl 5-iodoanthranilate (14b) was prepared

following the procedure described by Sy.³¹Methyl 4- and 6-iodo-

anthranilates 14a and 14c, previously described by Allison³²and

Seltzman,³³were prepared by a more convenient procedure from

4 and 6-iodoisatin (12a–b)³⁴obtained from the same commercially

MeO

HN

NHSO₂Me

CO₂Me

I

NH₂

N

CONHR

14a

: 4-iodo

14c: 6-iodo

H

N

4

CO₂H

NH₂

: DACA

O

5: SN 16713

6

I

O

i

ii

I

or iii

4

I

CO₂Me

12a

12b

13a

: 6-iodo

: 4-iodo

13b: 6-iodo

N

Me₂N

NMe₂

NHMe

H

CONHR

6

7: acridine orange

15: from 13b

R = (CH₂)₂NMe₂

Scheme 1. Reagents and conditions: (i) a—1.5 N NaOH, 30% H₂O₂, 50 °C; b—6.5 N

HCl; (ii) 17% HCl, MeOH, reﬂux from 13a; (iii) NaH, DMF, MeI, rt, from 13b.

Figure 2. Structures of compounds studied in the text.

N. Desbois et al. / Bioorg. Med. Chem. 16 (2008) 7671–7690

7673

O

1

4

CO₂H ^5'

N

CO₂

I

i

ii

X

I

+

3

6

N

H

3'

H₂N

CO₂Me

H

CO₂Me

16

14a: 4-iodo

17a: 5'-iodo

18a: X = 1-iodo

14b

14c

17b

17c

18b

18c

19 :

: 5-iodo

: 6-iodo

: 4'-iodo

: 3'-iodo

: X = 2-iodo

: X = 3-iodo

X = H (from

17b

)

Scheme 2. Reagents and conditions: (i) Cu(OAc)₂, DMF, 90–100 °C; (ii) H₂SO₄, 80 °C.

available precursor 3-iodoaniline (Scheme 1). Oxidation of 6-iodoi-

satin (12a) with alkaline hydrogen peroxide afforded 4-iodoanth-

ranilic acid (13a) according to the procedure describe by Snow³⁵

for the 6-iodoanthranilic acid (13b). Esteriﬁcation of the 4-iodo-

anthranilic acid (13a) with anhydrous methanol in the presence

of a 17% HCl/MeOH solution afforded methyl anthranilate 14a. In

the case of acid 13b, esteriﬁcation using the usual conditions of

17% HCl/MeOH or SOCl₂/MeOH gave an unequivocal decarboxyl-

ation reaction to yield 3-iodoaniline. We then developed a less sen-

sitive reaction by treating acid 13b with sodium hydride and

methyl iodide in dry dimethylformamide. Using these conditions,

esteriﬁcation gave the expected compound 14c (44%) which was

accompanied with methyl N-methyl-6-iodoanthranilate (15) in

13% yield.

substitutedinthe3⁰-position, twoavailabledirectionsofringclosure

were possible, and a mixture of both iodo-isomers 23b and 23d (1/1

as observed by ¹H NMR) was obtained. These isomers were sepa-

rated by chromatographic procedures at the next step as ester deriv-

atives 18e and 18g obtained from the same methodology developed

for compound 14b. We also prepared methyl 9,10-dihydro-5 and

7-iodo-9-oxoacridine-4-carboxylates (18d and 18f) by known

sequential treatment with thionyl chloride and methanol from an

equimolar mixture of acids 23a and 23c.

The basic side chain was easily installed on the carboxylate

function of 18a–g by condensation of N,N-diethylethylenediamine

in the presence of trimethylaluminium to give amides 24a–g in

38–98% yields (Scheme 4). In order to carry out the in vitro and

in vivo biological evaluation of these compounds, the free base

forms of 24a–g were converted into their water-soluble hydrochlo-

ride salts 8a–g.

According to the reaction described by Rewcastle,²⁸diphenyliod-

onium-2-carboxylate (16)³⁶reacted with methyl iodoanthranilates

14a–c to give 2-(iodo-2⁰-methoxycarbonylphenyl-amino)benzoic

acids 17a–c with good yields (59–80%) (Scheme 2). Ring closure

of acids 17a–c was performed using sulphuric acid, as previously

described,³⁷to give methyl 9,10-dihydro-iodo-9-oxoacridine-

4-carboxylates 18a–c. During the synthesis of compound 18b,

the deiodinated by-product 19 was isolated with moderated

yield (8%).

Preparation of (5–8)-iodo-9-oxoacridine-4-carboxylates 18d–g

required a different synthetic approach starting from 2-(iodophe-

nylamino)isophthalic acids 22a–c (Scheme 3) prepared via a

Jourdan–Ullmann reaction. The cross-coupling reaction of o-iodoi-

sophthalic^38,39acid (21) with iodoanilines 20a–c gave compounds

22a–c, which were then cyclised using polyphosphoric acid (PPA)

to yield 9,10-dihydro-9-oxoacridine-4-carboxylic acids 23a–d,

according to the procedure described by Rewcastle et al.³⁶

Diacids 22a and 22c substituted in the 2⁰- or 4⁰-position cyclised

unequivocally in PPA to give the 5-iodo-9,10-dihydro-9-oxoacri-

dine-4-carboxylic acid (23a) and 7-iodo-9,10-dihydro-9-oxoacri-

dine-4-carboxylic acid (23c), respectively. For diacid 22b

2.1.2. Synthesis of acridine derivatives 9a–g

There are several methods available that can be adapted to the

synthesis of DACA analogues (27). The acridine 27 already reported

was generally prepared⁴⁰from acridine-4-carboxylic acids 26 gen-

erated by reduction of the corresponding acridones 25 by alumin-

ium/mercury amalgam,⁴¹followed by oxidation of the resulting

acridans with FeCl₃(Scheme 5). It was noted that dehalogenation

took place with the chlorinated compounds.⁴⁰

This prompted us to develop an original method for yield iodo

analogues of DACA using borane tetrahydrofuran complex as

reducing agent. A solution of BH₃–THF was added to reﬂux the

tetrahydrofuran solution of compounds 18a–g (Scheme 6). The

reactions were maintained at reﬂux for 45 min before being hydro-

lysed. The resulting acridans 28a–g were oxidised to acridines

29a–g with FeCl₃in yields ranging from 87% to 97%. These esters

O

8

5

1

a

b

: 1-iodo

: 2-iodo

ii

, HCl

NEt₂

I

c : 3-iodo

d: 5-iodo

e

f

N

H

N

H

HO₂C

I

O

R

O

N

H

: 6-iodo

: 7-iodo

: 8-iodo

8a-g

18a-g

R = OMe

CO₂H

i

g

O

HO₂C

¹N^{' 1}

8

5

1

21

+

24a-g

R = NHCH₂CH₂NEt₂

4'

ii

CO₂H

i

I

^3'I ^2'

N

H

Scheme 4. Reagents and conditions: (i) AlMe₃, NH₂(CH₂)₂NEt₂, CH₂Cl₂or PhMe,

0 °C then reﬂux; (ii) 2 N HCl, ether, rt.

H

CO₂R

22a

22b

22c

: 2'-iodo

: 3'-iodo

: 4'-iodo

23a

23b

23c

: 5-iodo

: 6-iodo

: 7-iodo

NH₂

R = H

20a

: 2-iodo

20b: 3-iodo

23d: 8-iodo

O

20c

iii

: 4-iodo

i

ii

CO₂H Me₂N

18d: 5-iodo

18e: 6-iodo

R

R = Me

N

H

: 7-iodo

: 8-iodo

18f

CO₂H

N

H

O

18g

25

26

27

Scheme 3. Reagents and conditions: (i) CuCl, N-ethylmorpholine, butan-2,3-diol,

benzene, 120 °C; (ii) PPA, 120 °C; (iii) a—SOCl₂, DMF,

23a,c; NaH, MeI, DMF, rt, from 23b,d.

D; b—MeOH, CaCO₃from

Scheme 5. Reagents and conditions: (i) a—Al(Hg); b—FeCl₃; (ii) SOCl₂,

NH₂(CH₂)₂NEt₂.

7674

N. Desbois et al. / Bioorg. Med. Chem. 16 (2008) 7671–7690

O

i

ii

I

a

b

: 1-iodo

: 2-iodo

N

H

N

H

18a-g

CO₂Me

29a-g

CO₂Me

c: 3-iodo

d: 5-iodo

28a-g

e

f

g

: 6-iodo

: 7-iodo

: 8-iodo

8

5

1

iii

iv

NEt₂

I

, 2HCl

N

O

N

NEt₂

CO₂Me

O

N

H

N

H

29a, b, d-g

30a, b, d-g

9a, b, d-g

Scheme 6. Reagents and conditions: (i) BH₃–THF (1 M), THF, reﬂux; (ii) FeCl₃, H₂O, EtOH, 50 °C; (iii) AlMe₃, NH₂(CH₂)₂NEt₂, CH₂Cl₂or PhMe, 0 °C then reﬂux; (iv) 2 N HCl,

ether, rt.

29a–g were very unstable to oxidation and afforded, in our case,

the corresponding acridones 18a–g. For this reason, the esters

29a,b,d–g had to be immediately treated with N,N-diethylethyl-

enediamine to give stable amides 30a,b,d–g before being con-

verted into dihydrochloride salts 9a,b,d–g using the same

procedure as developed for compounds 8a–g.

compound 36 (Scheme 8). One problem, as mentioned earlier for the

synthesis of compound 30c from 29c, was the lability of the halogen

substituent in the presence of thionyl chloride.⁴³This prompted us

not to use the 1, 3 and 6-iodo derivatives 18a,c,e and the unstable

8-iodo compound18 g for thisseries. In thecase of 2-iodo compound

18b, conversion in acid 23e was performed using a methanolic 2 N

sodium hydroxide solution. The iodo-9-chloroacridine carboxam-

ides 35a–c were prepared in two steps from 9,10-dihydroiodo-9-

oxoacridine-4-carboxylic acids 23a,c,e. Treatment of acids 23a,c,e

with thionyl chloride afforded 9-chloroacridine-4-carbonyl chlo-

rides 34a–c. Reacting these crude compounds with N,N-diethyleth-

ylenediamine in anhydrous dichloromethane gave excellent yields

(80–96%) of the unstable carboxamide derivatives 35a–c. Typically,

m-AMSA analogues 10a–c were obtained from 35a–c by amine 36 ⁴⁴

substitution at the 9-position under acidic conditions followed by

treatment with a 2 N HCl/ether solution. There are two tautomeric

forms of 9-substituted acridines,^45–47but no isomers have been ob-

served for 9-aminoacridine dihydrochloride salts 10a–c.

Some difﬁculties were encountered with the more hindered

methyl 3-iodoacridine-4-carboxylate (29c). Attempts to prepare

compound 30c by treatment of ester 29c under the usual condi-

tions (previously reported for compounds 24 and 30a,b,d–g) only

recovered starting material. This prompted us to follow another

route for synthesising amide 30c via acid intermediate 31 as out-

lined in Scheme 7. Treatment of acid 31, obtained by saponiﬁcation

of ester 29c, with thionyl chloride and N,N-diethylethylenediamine

afforded the undesired chloramide 32. One unexpected problem

was the lability of iodine group in the presence of an ortho elec-

tron-withdrawing group, and we observed iodine/chloride ex-

change during activation with thionyl chloride. An alternative

route to obtain amide 30c was the reduction of the corresponding

acridone-4-carboxamide 24c with borane tetrahydrofuran com-

plex, followed by FeCl₃oxidation of the resulting acridan 33. It

was noted that secondary amides could be reduced rapidly and

quantitatively into the corresponding amines by borane in reﬂuxed

tetrahydrofuran.⁴²In our case, the presence of hindered 3-iodo-

group prevents probably the reduction of 4-amido function. Final-

ly, the dihydrochloride salt 9c was obtained by treatment with a

2 N HCl/ether solution, as described for compounds 8 with an over-

all yield of 35% from 24c.

Preparation of m-AMSA derivative 10d was performed with N-

(2-diethylaminoethyl)-9-chloroacridine-4-carboxamide⁴⁸(37) as

previously reported, and iodoamino derivative 38 by a similar

procedure to that used for compounds 10a–c (Scheme 9). Interme-

diate derivative 38 was prepared from ethyl N-(2-methoxy-4-ami-

nobenzene)carbamate⁴⁴(39) using

a three steps procedure.

Monoiodinated aniline 40 was obtained in good yield by the reac-

tion of benzyltriethylammonium dichloroiodate⁴⁹with aniline 39

in the presence of calcium carbonate and methanol. Mesylation

of the amino group afforded compound 41 and basic hydrolysis

of the ethyl carbamate protecting group gave anilino compound

38 in good yield (89%). Regiospeciﬁc iodination reaction was con-

ﬁrmed by NMR studies (HSQC and HMBC ¹H–¹³C NMR sequences

performed on sulfamide 41).

2.1.3. m-AMSA derivatives 10a–d syntheses

To obtain m-AMSA derivatives, the more convenient method was

directnucleophilicsubstitutionof9-choroacridines35a–cbyanilino

ii

i

N

I

N

I

N

I

NEt₂

CO₂Me

CO₂H

O

N

H

ii

29c

31

30c

N

Cl

O

N

H

32

O

v

iii

iv

, 2HCl

I

N

I

N

H

I

N

I

NEt₂

H

NEt₂

O

N

H

O

N

H

O

N

H

O

N

H

24c

33

30c

9c

Scheme 7. Reagents and conditions: (i) a—4 N NaOH, MeOH, reﬂux; b—AcOH; (ii) a—SOCl₂, DMF, reﬂux; b—5% NEt₃/THF, NH₂(CH₂)₂NEt₂, rt; c—10% Na₂CO₃; (iii) BH₃–THF

(1 M), THF, reﬂux; (iv) a—FeCl₃, H₂O, EtOH, 50 °C; (v) 2 N HCl, ether, rt.

N. Desbois et al. / Bioorg. Med. Chem. 16 (2008) 7671–7690

7675

MeO

HN

NHSO₂Me

Cl

O

8

5

I ⁷

iv

2

iii

I

ii

, 2HCl

NEt₂

I

N

NHSO₂Me

N

H

5

Et₂N

COCl

CO₂R

N

H

O

N

H

34a: 2-iodo

23a: 5-iodo; R = H

OMe

NH₂

36

35a: 2-iodo

35b: 5-iodo

35c: 7-iodo

10a: 2-iodo

10b: 5-iodo

10c: 7-iodo

: 5-iodo

: 7-iodo

23c

18b

34b

34c

: 7-iodo; R = H

: 2-iodo; R = Me

i

23e: 2-iodo; R = H

Scheme 8. Reagents and conditions: (i) a—2 N NaOH, MeOH, reﬂux; b—AcOH; (ii) SOCl₂, DMF, reﬂux; (iii) a—CH₂Cl₂, NH₂(CH₂)₂Et₂, rt; b—10% Na₂CO₃; (iv) a—EtOH, HCl,

reﬂux, b—2 N HCl, ether, rt.

DLD-1 colon adenocarcinoma), a murine cell line (B16F0 mela-

noma) and human ﬁbroblasts in primary culture, using amsacrine

and DACA (4) as reference compounds. Table 1 gives the results for

acridone and acridine derivatives (8, 9 and 11) and Table 2 gives

the results for amsacrine derivatives 10.

NHSO₂Me

MeO

I

Cl

N

NHSO₂Me

i

HN

I

7

2

, 2HCl

OMe

NH₂

38

The results reported in Table 1 highlighted variation of IC₅₀val-

ues depending on the position of iodo substituent. Substitution of

iodine at the 3-position (8c and 9c) decreased the cytotoxicity

compared with parent compound 4 in all cell lines. Ortho substitu-

tion to the carboxamide function is likely to interfere with the side

chain conformation, as was observed in the DACA series where 3-

substitution led to a marked drop in DNA binding and cytotoxic-

ity.⁴⁰Analogues bearing a side chain at the 9-position (11a,b) were

also less potent than DACA (4). In contrast, substitution of iodine at

the 5-position (9d) led to markedly increased potency, especially

on human melanoma cell line M4Beu, with a ﬁvefold lower IC₅₀va-

lue than DACA. It should be underlined that such results have been

shown with compounds of the DACA series.⁵¹For the other acri-

dine compounds studied (9), there were no marked differences in

effects compared with the parent compound. Among acridone ana-

logues (8), all the compounds except 8c exhibited a globally higher

cytotoxicity than DACA on human melanoma but globally lower

cytotoxicity on the other cell lines.

N

Et₂N

N

O

Et₂N

N

H

10d

O

H

37

NHR

iv

NHSO₂Me

NH₂

I

ii

OMe

NHCO₂Et

OMe

NH₂

OMe

NHCO₂Et

38

39

40: R = H

41: R = SO₂Me

iii

Scheme 9. Reagents and conditions: (i) a—EtOH, HCl, reﬂux; b—2 N HCl, ether, rt;

(ii) BTEAICl₂, CaCO₃, CH₂Cl₂, MeOH; (iii) CH₃SO₂Cl, pyridine, 0 °C; (iv) 10% NaOH,

reﬂux.

2.1.4. Acridine derivatives syntheses 11a–b

The dihydrochloride iodoacridine-9-carboxamide salts 11a,b

were prepared in three steps from methyl 9-acridinecarboxylate

(42)⁵⁰(Scheme 10). 2- and 2,7-diodoacridine 43a,b (the more

favourable position for electrophilic aromatic substitution) were

directly synthesised from acridine 42 by the reaction with iodine

and periodic acid dihydrate. The method used was the only one

which gave a positive result. Regiospeciﬁc iodination was conﬁrmed

by NMR studies (¹H–¹H COSY, ¹H–¹³C HSQC and ¹H–¹³C HMBC NMR

sequences) performed for monoiodinated compound 43a. Coupling

esters 43a,b with N,N-diethylethylenediamine to give amides 44a,b

and the synthesis of dihydrochloride salts as reported previously for

compounds 8 gave the expected amides 11a,b.

Our results (Table 2) showed that the compounds 10a–d exhib-

ited a signiﬁcant in vitro cytotoxic activity, with IC₅₀values rang-

ing from 0.61 to 3.5 lM on M4Beu cells. The most cytotoxic of

these compounds was 10c, which exhibited a similar effect to

the amsacrine compound. However, all of these compounds

showed lower activity was than amsacrine on Jurkat leukaemia,

murine melanoma B16F0 and colon adenocarcinoma DLD-1 cell

lines.

To conclude, from acridone derivatives 8, which with the excep-

tion of 8c expressed the same range of efﬁcacy, we chose to select

compound 8f for several reasons. First, due to the chemical stabil-

ity of its carbon-iodine bond comparatively to isomeric derivatives

8a, f and g for which a deiodation took place in solution as previ-

ously observed by Denny⁴³for 6- and 8-halo-4-carboxyacridones.

Second, compound 8f was obtained with better overall yield than

8d and in few steps than 8b. Based on their cytotoxic activity on

human cell line M4Beu, the acridine compounds 9d and 10c were

also selected as leads for further biological evaluation.

2.2. Cytotoxic activity

IC₅₀values were determined for compounds 8–11 in a panel of

human cancer cell lines (M4Beu melanoma, Jurkat leukaemia and

H

O

N

2.3. Radiolabelling of stannane compounds

CO₂Me

N

NEt₂

I

2

7

R₁

R₂

X

ii

The selected compounds 8f, 9d and 10c were radioiodinated

using electrophilic substitution methods with high speciﬁc activ-

ity, as depicted in Scheme 11. The iodo derivatives 24f, 30d and

48 (obtained from hydrochloride salt 10c) were converted to the

corresponding tributylstannane compounds 45, 46 and 49 using

a palladium(0)-catalysed cross-coupling reaction. It was noted that

for compound 30d a dehalogenation reaction took place and the

desired stannane 46 was obtained with the by-product compound

N

44a: X = H

42

: R_1,R₂= H

44b

: X = I

11a: X = H, 2HCl

i

iii

43a

: R₁= H, R₂= I

43b: R_1,R₂= I

11b

: X = I, 2HCl

Scheme 10. Reagents and conditions: (i) HIO₄dihydrate, I₂, AcOH, H₂O, H₂SO₄,

reﬂux; (ii) AlMe₃, NH₂(CH₂)₂NEt₂, PhMe, 0 °C then reﬂux; (iii) 2 N HCl, ether, rt.

7676

N. Desbois et al. / Bioorg. Med. Chem. 16 (2008) 7671–7690

Table 1

Cytotoxicity of compounds 8, 9 and 11 in comparison to DACA (4)

a

Compound

Iodine position

IC₅₀

(lM)

M4Beu^b

4.5 0.6

B16F0^c

DLD-1^d

JURKAT^e

Fibroblast^f

DACA (4)

8a

0.42 0.3

(0.19 0.1)

3.1 0.3

(2.3 1.6)

0.56 0.2

(0.3 0.1)

4.4 0.4

(7.1 0.2)

(1.8 0.2)

1

2.9 0.04

(3.9 0.04)

2.2 0.6

(1.8 0.4)

2.6 1.0

(2.3 0.8)

0.80 0.06

(1.2 0.04)

2.3 0.8

(2.4 0.3)

8b

2

3.5 1.4

(3.5 1.0)

1.6 0.4

(1.5 0.1

3.1 0.5

(2.2 0.1)

1.5 0.1

(1.4 0.2)

3.7 2.1

(1.8 0.2)

8c

3

11 1.6

14 5.9

37 5.1

5.0 1.9

23 8.7

(29 4.5)

(12 5.4)

(35 7.4)

(4.4 0.4)

(54 2.1)

8d

8e

5

5.4 0.5

(3.9 0.5)

3.6 1.1

(3.0 1.3)

7.0 1.0

(4.1 0.5)

1.8 1.2

(2.6 0.3)

4.2 0.1

(3.6 0.2)

6

2.7 0.3

1.9 0.8

9.4 0.2

1.3 0.1

2.0 1.0

(3.4 0.4)

(1.6 0.6)

(6.2 0.1)

(0.85 0.1)

(2.7 1.4)

8f

7

4.7 0.5

4.1 1.8

5.4 0.2

2.8 0.6

4.2 0.1

(3.8 0.5)

(3.7 1.4)

(3.9 0.6)

(0.88 0.5)

(3.1 0.2)

8g

8

3.4 0.4

6.3 0.5

7.5 0.3

2.7 1.3

9.8 0.2

(3.4 0.3)

(5.3 0.1)

(3.3 2.7)

(7.7 0.5)

(9.7 0.2)

9a

1

1.5 0.2

1.4 0.8

4.4 1.0

1.1 0.1

2.0 0.3

(2.4 0.2)

(1.1 0.9)

(2.3 0.2)

(0.9 0.1)

(2.2 0.6)

9b

2

1.5 0.1

0.49 0.2

2.4 0.5

1.3 0.2

1.8 0.1

(1.8 0.2)

(0.14 0.1)

(1.4 0.2)

(0.6 0.1)

(1.2 0.05)

9c

3

52.9 16.2

(52.0 11.3)

39.3 0.2

(22.5 8.9)

79.2 5.6

(47.6 11.0)

22.9 4.3

(32.3 10.2)

27.1 2.8

nd

9d

9e

5

0.9 0.05

(0.76 0.2)

0.6 0.2

(0.42 0.2)

1.1 0.4

(0.27 0.1)

0.4 0.1

(0.6 0.1)

0.9 0.1

(0.69 0.6)

6

2.4 0.1

1.8 0.2

2.2 0.4

1.2 0.1

2.3 1.2

(3.6 0.4)

(1.4 0.4)

(1.8 0.1)

(1.4 0.3)

(2.1 1.1)

9f

7

3.2 0.3

2.7 0.8

2.4 0.4

3.4 1.4

2.6 0.1

(2.7 0.3)

(2.0 0.3)

(1.8 0.4)

(4.5 0.5)

(3.1 0.2)

9g

8

2.1 0.2

1.9 0.8

4.5 0.4

1.7 0.2

2.5 0.7

(3.0 0.3)

(1.1 0.5)

(2.2 0.1)

(1.1 0.4)

(2.8 1.1)

11a

11b

a

2

40 16.3

(28 5.1)

11 0.9

(9.5 1.4)

16 0.9

(9.8 0.5)

23 5.4

(3.4 3.1)

32 11.8

nd

2 and 7

5.9 0.9

(5.6 1.3)

20 1.3

(21 1.4)

5.3 1.4

(4.3 1.2)

6.3 0.8

(7.5 0.9)

6.7 0.7

(3.9 0.5)

Results are expressed as IC₅₀obtained with the resazurin test with the Hoechst test in brackets. The values given are averages of at least two independent determinations.

b

c

d

e

f

Human melanoma.

Murine melanoma.

Human colon adenocarcinoma.

Human leukaemia.

Normal human ﬁbroblasts.

Table 2

Cytotoxicity of compounds 10 in comparison to amsacrine

a

Compound

Iodine position

IC₅₀

(lM)

M4Beu^b

B16F0^c

DLD-1^d

JURKAT^e

Fibroblast^f

Amsacrine

0.74 0.3

0.08 0.01

0.57 0.4

0.08 0.05

4.2 0.1

(0.40 0.2)

(0.04 0.001)

(0.08 0.02)

(0.02 0.006)

(2.0 0.3)

10a

10b

10c

10d

a

2

5

7

5⁰

1.9 1.1

(1.9 0.9)

0.81 0.2

(0.95 0.4)

2.9 1.4

(2.5 0.6)

0.72 0.1

(1.1 0.1)

2.0 1.0

(1.9 1.6)

3.5 0.3

(3.0 0.2)

0.58 0.3

(0.42 0.2)

3.0 2.2

(1.7 0.8)

0.24 0.01

(0.15 0.4)

1.2 0.7

(0.83 0.7)

0.61 0.1

(0.65 0.1)

1.3 0.5

(0.98 0.3)

1.7 1.1

(1.2 0.6)

0.89 0.3

(0.37 0.01)

1.4 0.5

(1.0 0.4)

2.5 0.2

(2.3 0.2)

1.2 0.5

(0.99 0.4)

2.4 0.3

(2.0 0.2)

1.4 0.1

(0.49 0.5)

1.9 0.2

(1.5 0.2)

Results are expressed as IC₅₀obtained with the rezazurin test with the Hoechst test in brackets. The values given are averages of at least two independent determinations.

b

c

d

e

f

Human melanoma.

Murine melanoma.

Human colon adenocarcinoma.

Human leukaemia.

Normal human ﬁbroblasts.

N. Desbois et al. / Bioorg. Med. Chem. 16 (2008) 7671–7690

7677

Table 4

O

¹²⁵I

In vitro binding to melanin and logP of 8f, 9d and 10c derivatives compared to BZA

24f

: 7-iodo

i

ii

, HCl

NEt₂

45

: 7-SnBu₃

Compounds

% Bound to synthetic melanin

H₂O/PBS

logP^a

N

H

O

N

H

BZA^b

8f

9d

78.6/13.8

50.6/33.3

69.2/56.8

27.2/6.3

1.34

0.27

0.8

125

8f

I]

[

30d: 5-iodo

46: 5-SnBu₃

47: 5-H

10c

0.64

i

, 2HCl

NEt₂

N

a

(from 46)

P, partition coefﬁcient between octanol and phosphate buffer solution (pH 7.4).

BZA values were obtained from previous research.^[52]

¹²⁵I

b

O

¹²⁵I]9d

N

H

[

MeO₂SHN

: 7-iodo, 2HCl

OMe

NH

philicity values (logP) of the studied compounds (summarised in

Table 4) were all lower than that obtained for BZA.

10c

iii

i

¹²⁵I

48

: 7-iodo

49: 7-SnBu₃

, 2HCl

NEt₂

ii

2.5. Biodistribution studies of compounds 8f, 9d and 10c

N

O

N

The three selected compounds also presented major differences

in excretion (Table 5). Eliminations were almost complete in 72 h

for 9d with fairly equivalent rates for both urinary and faecal

routes. Compounds 8f and 10c were mainly eliminated very slowly

by faecal route, and after 72 h only 49% and 37% of radioactivity,

respectively, had been excreted. The radioactivity present in one

mouse 8 days after 8f administration was still equal to 19% of

the injected dose. This is an original pattern of kinetic behaviour

compared with BZA and with the majority of benzamide analogues

previously studied, which are mainly eliminated via the urinary

tract and are almost completely eliminated at 72 h.

H

125

10c

I]

[

Scheme 11. Reagents and conditions: (i) Pd(PPh₃)₄, (Bu₃Sn)₂, PhMe, reﬂux; (ii) a—

chloramine-T, [¹²⁵I]NaI, 1% AcOH/EtOH for 45 and 49 or citrate buffer solution for

46; b—2 N HCl, ether, rt; (iii) saturated Na₂CO₃.

Table 3

HPLC data, radiochemical yields and purities for compounds [¹²⁵I]8f, [¹²⁵I]9d and

[

¹²⁵I]10c

Entry CAT

(mg/

Retention time

(min)

Retention time

(min)Stannane

compounds

Yield^aPurity^b

(%)

For two [¹²⁵I]-labelled compounds, 8f and 9d, tissue distribu-

tion evaluated in melanoma-bearing mice rapidly demonstrated

a high tumoural activity, whereas there was no signiﬁcant tumour-

al uptake for compound 10c. This result was almost certainly re-

lated to the low afﬁnity measured for synthetic melanin (Table

4). No melanoma targeting was observed and the weak tumoural

uptake value remained stable throughout the study, while higher

concentrations were measured in the liver and the elimination

organs. This result is unexpected, given the high afﬁnity for mela-

notic tumour tissue already showed with m-AMSA.⁵³

mL)

[

¹²⁵I]compounds

1

2

3

0.4

0.5

0.25

Rt [¹²⁵I]8f = 9.8

Rt 45 = 17.8

Rt 46 = 17.3

Rt 49 = 18.5

57

66

53

99

97

Rt [¹²⁵I]9d = 8 11.9

Rt [¹²⁵I]10c = 9.9

a

Radiochemical yields were calculated by dividing the radioactivity in the ﬁnal

HPLC product by the initial amount of radioactive sodium iodide.

b

Radiochemical purities were determined by HPLC.

47 in 25% and 29% yields, respectively. The radioiodo-destannyla-

tion of compounds 45, 46 and 49 with [¹²⁵I]NaI was performed

using chloramine-T (CAT) as oxidant to give, after HPLC puriﬁca-

tion and conversion into hydrochloride salts, the desired [¹²⁵I]8f,

With [¹²⁵I]8f and [¹²⁵I]9d, there was uptake in B16F0 melanoma

graft cells from 1 h post-injection (pi) (>12.6% ID/g) which went on

to peak at between 6 and 24 h and was still very high after 72 h.

Tumoural concentrations of 9d and 8f at 72 h post-injection were

9- and 23-fold the BZA values, respectively. These values are sug-

gestive of strong tumoural irradiation if used in doses geared to

radionuclide therapy. The kinetic proﬁle of compound 8f justiﬁed

the large amount of radioactivity present in the mouse body after

a long delay.

[

¹²⁵I]9d and [¹²⁵I]10c labelled compounds with high speciﬁc activ-

ity (81.4 TBq/mmol). The parameters such as amount of substrate,

amount of oxidant and reaction time were all optimised for each

reaction. The [¹²⁵I]-labelled compounds were obtained with good

radiochemical conversion (53–66%) in a 10-min reaction time

according to radio-TLC analysis. The radiochemical data of the

three compounds [¹²⁵I]8f, [¹²⁵I]9d and [¹²⁵I]10c are summarised

in Table 3.

Compared with our reference [¹²⁵I]BZA, the tumoural ﬁxation

values (% ID/g) of compounds [¹²⁵I]8f and [¹²⁵I]9d appeared not

only signiﬁcantly higher at each timepoint in the study, but

also much more durable. While many organs and tissues concen-

trated the radioactivity at 1 h pi, a preferential afﬁnity for the

tumour compared with liver or lungs was observed from 3 h pi,

2.4. In vitro binding to melanin and partition coefﬁcients for

compounds 8f, 9d and 10c

In our approach, the melanin afﬁnity of the studied compounds

had to be a consistent parameter. Synthesised (hetero)aromatic

analogues 8f and 9d displayed the same strong afﬁnity to synthetic

melanin in water as BZA (see Table 4). When melanin was sus-

pended in PBS, the melanin afﬁnity of 8f and 9d was slightly less

modiﬁed than with BZA. While this decrease in value for the parent

compound BZA⁵²is probably due to the ionic strength of PBS,¹⁴the

lower variations in PBS for compounds 8f and 9d, with identical N-

alkyl side chains to BZA, could result from a higher pi stacking

interaction between the aromatic rings of melanin and acri-

dine(one) derivatives, which was independent of both pH and ionic

strength. In contrast, compound 10c exhibited a weak melanin

afﬁnity that was highly dependent on the ionic charges. The lipo-

Table 5

Urinary and faecal excretions^aof radioactivity following injection of [¹²⁵I]compounds

8f, 9d and 10c in B16F0 melanoma-bearing mice compared to BZA

Compound

Urinary % ID

0–72 h

Faecal % ID

0–72 h

BZA^b

83.1

10.4

52.8

4.2

4.8

38.4

46.1

33.2

[

¹²⁵I]8f

¹²⁵I]9d

¹²⁵I]10c

a

Cumulative excretions (two mice for each compound).

BZA values were obtained from previous research.⁵²

b

7678

N. Desbois et al. / Bioorg. Med. Chem. 16 (2008) 7671–7690

as illustrated in Table 6 and Figure 4. From 24 h p.i., only the

tumour remained labelled, due to its selective afﬁnity and

the long-lasting binding, whereas the radioactivity had been

eliminated from the other tissues.

Based on this in vivo data, melanoma targeting was conﬁrmed

for the compounds 8f and 9d, with favourable kinetic proﬁles for

application in radionuclide therapy. The biological half life within

the tumour, calculated using MIRD software was much higher with

8f and 9d, at 269 h and 43 h, respectively, than the 19.6 h for BZA.

The kinetic proﬁle of 8f in one mouse is illustrated by the scinti-

graphic imaging reported in Figure 5, showing the speciﬁc long-

lasting afﬁnity for the tumour and the rapid clearance from non-

target organs.

3. Conclusion

We synthesised 20 novel iodo-acridone or iodoa-cridine–

carboxamide compounds designed by pharmacomodulation of

known anti-tumoural agents. Three of these compounds were

selected for their in vitro cytotoxicity, which was close to

the parent molecules, and for their chemical stability. Their

in vivo distribution proﬁles from studies performed on murine

B16F0 melanoma-bearing mice after ¹²⁵I-radiolabelling showed:

(1) that the 7-iodo amsacrine 10c derivative displayed a lack

of afﬁnity for melanoma tumour; (2) that the 7-iodo-acridone

8f and the 5-iodo acridine 9d derivatives possesses a speciﬁc

and durable in vivo afﬁnity for melanoma, with for 8f,

a

14-fold longer biological half life within the tumour than

BZA that was compatible with clinical application in targeted

radionuclide therapy. The next step will be to study the indi-

vidual in vivo anti-tumoural effects, the chemotoxicity of the

stable compound, the radiotoxicity of the labelled compound,

and the association of these two factors with

a

view to

designing

molecule.

a

bi-modal treatment using the same, single

4. Experimental

4.1. Chemistry

Column chromatography was performed with Merck neutral

aluminium oxide 90 standardised (63–200

lm) or silica gel A nor-

mal phase (35–70 m). Thin layer chromatography was performed

l

on Merck neutral aluminium oxide 60 F₂₅₄plates or Merck silica

gel 60 F₂₅₄plates. The plates were visualised with UV light

(254 nm). Melting points were determined on an electrothermal

IA9300 (capillary) or a Reichert-Jung-Kofﬂer apparatus and were

not corrected. NMR spectra (400 or 200 MHz for ¹H or 100 or

50 MHz for ¹³C) were recorded on a Bruker Avance 400 or Bruker

AM 200 instruments using CDCl₃, CD₃OD or DMSO-d₆as solvent.

Infrared spectra were recorded in KBr pellets or in CCl₄on a FTIR

Nicolet impact 410 or an FT vector 22 instrument (m expressed in

cm^ꢀ1). Mass spectra (MS) were obtained in electron impact mode

on 5989A instruments (Agilent Technologies). Electrospray ioniza-

tion mass spectra (ESI-MS) were obtained on an Esquire-LC spec-

trometer (Bruker Daltonics). The samples were analysed in

CH₃OH/H₂O (1:1, containing 1% HCOOH) at a ﬁnal concentration

of 2–10 pmol/lL. Each ESI-MS spectrum was recorded by averag-

ing 10 spectra. For organostannane compounds, theoretical masses

were calculated based on the mass of the most abundant isotope

(

¹²⁰Sn, isotopic abundance, 32.59%). Microanalyses were per-

formed by the Analytical Laboratory of the CNRS (Vernaison,

France) for the elements indicated and were within 0.4% of the the-

oretical values unless indicated. All air-sensitive reactions were

run under argon atmosphere. All solvents were dried using com-

mon techniques.⁵⁴

N. Desbois et al. / Bioorg. Med. Chem. 16 (2008) 7671–7690

7679

35

30

25

20

15

10

5

35

A

B

30

B 16

25

liver

B 16

liver

lung

brain

20

lung

brain

15

10

5

0

BZA

8f

9d

10c

BZA

8f

9d

10c

Figure 4. Concentrations of compounds [¹²⁵I]8f, [¹²⁵I]9d and [¹²⁵I]10c compared with BZA in selected tissues after iv administration in B16F0 melanoma-bearing mice. (A) At

3 h. (B) At 72 h. Mean SD (two mice, n determinations for each compound at each timepoint). BZA values were obtained from previous research.⁵⁰

Figure 5. In vivo kinetics of compound [¹²⁵I]8f in a B16F0 melanoma-bearing mouse illustrated by repeated planar scintigraphic images using a

animal imaging, at 1 h (A), 3 h (B), 6 h (C), 24 h (D), 72 h (E) and 8d (F) after iv injection of a 3 MBq dose (acquisition time: 10 min).

c imager dedicated to small

4.1.1. 4-Iodoanthranilic acid⁵⁴(13a)

(1.03 g, 25.67 mmol, 60% in mineral oil) was added, and the mix-

ture was stirred for 30 min. Methyl iodide (1.60 mL, 25.7 mmol)

was then added and the reaction mixture was stirred for 19 h at

room temperature. The solvent was evaporated and water was

added (50 mL). The reaction mixture was made alkaline to pH 8

with an aqueous saturated sodium carbonate solution and ex-

tracted with ethyl acetate (3ꢁ 80 mL). The organic layers were

dried (MgSO₄), ﬁltered and concentrated under vacuum. The crude

product was chromatographed (SiO₂and CH₂Cl₂), to give in order

of elution: methyl N-methyl-6-iodoanthranilate (15); yield 13%;

R_f= 0.73 (SiO₂and CH₂Cl₂); mp 48–50 °C; IR (KBr) 3413, 2947,

1703, 1588, 1562, 1504, 1259 cm^ꢀ1; MS m/z 291 (M⁺, 100), 259

(74), 231 (73), 104 (100), 77 (84); ¹H NMR (CDCl₃, 400 MHz) d

2.82 (s, 3 H), 3.92 (s, 3H), 6.00 (m, 1H), 6.66 (d, 1H, J = 8 Hz), 6.94

(t, 1H, J = 8 Hz), 7.24 (d, 1H, J = 8 H); ¹³C NMR (CDCl₃, 100 MHz) d

30.2, 51.8, 94.8, 110.6, 120.2, 128.4, 132.9, 149.7, 168.6. Methyl 6-

iodoanthranilate³³(14c) as an oil; yield 44%; R_f= 0.51 (SiO₂and

dichloromethane); ¹H NMR (CDCl₃, 200 MHz) d 4.08 (s, 3H), 6.80

(d, 1H, J = 8 Hz), 6.98 (t, 1H, J = 8 Hz), 7.43 (d, 1H, J = 8 Hz).

To a mechanically stirred solution of 6-iodoisatin³⁴(12a)

(1.01 g, 3.70 mmol) in 1.5 N sodium hydroxide solution (8.6 mL)

heated at 50 °C, was added an aqueous 30% hydrogen peroxide

solution (0.9 mL) (kept temperature below 65 °C). After standing

overnight at room temperature, the reaction mixture was carefully

neutralised with an aqueous 12 N hydrochloric acid solution, trea-

ted with charcoal, and ﬁltered through a pad of Celite^Ò545. The

volume was reduced to 7 mL under vacuum and acidiﬁed to pH 4

with an aqueous 6.5 M hydrochloric acid solution. The resulting

precipitate was collected by ﬁltration, taken in dry ethanol

(50 mL) and dried under vacuum. Yield 84%; mp 204–206 °C

(lit.⁵⁵214 °C).

4.1.2. Methyl 4-iodoanthranilate³²(14a)

A solution of 4-iodoantranilic acid (13a) (0.50 g, 1.90 mmol) in

anhydrous methanol (5 mL) was stirred, at room temperature, un-

der argon gas, for 10 min. An anhydrous 17% HCl/MeOH solution

(5 mL) was added and heated gently under reﬂux for 21 h. After

cooling to room temperature, the reaction mixture was made alka-

line with a saturated aqueous sodium carbonate solution (30 mL)

and extracted with dichloromethane (3ꢁ 50 mL). The organic lay-

ers were dried (MgSO₄), ﬁltered and concentrated under vacuum.

The product was used without puriﬁcation. Yield 40%; mp 71–

73 °C; ¹H NMR (CDCl₃, 200 MHz) d 3.84 (s, 3H), 6.98 (dd, 1H,

J = 8.5, 1.5 Hz), 7.10 (d, 1H, J = 1.5 Hz), 7.50 (d, 1H, J = 8.5 Hz).

4.1.4. General procedure for the synthesis of 2-(iodo-2⁰-metho-

xycarbonylphenylamino)benzoic acids 17a–c

A mixture of methyl iodoanthranilate 14a–c (0.50 g, 1.80 mmol)

diphenyliodonium-2-carboxylate³⁶(16) (0.88 g, 2.72 mmol), and

copper(II) acetate monohydrate (12 mg) was suspended in dimeth-

ylformamide (30 mL) and heated at 90–100 °C for 12 h. The solvent

was removed under vacuum and the oily residue was dissolved in

ethyl acetate (18 mL). This solution was washed with an aqueous

0.1 N hydrochloric acid solution (18 mL). The organic layers were

extracted with an aqueous 0.1 N ammonia solution (2ꢁ 9 mL)

and the aqueous extract was acidiﬁed to pH 6 with an aqueous

4.1.3. Methyl 6-iodoanthranilate³³(14c)

6-Iodoanthranilic acid³⁵(13b) (4.50 g, 17.1 mmol) was dis-

solved in dry dimethylformamide (10 mL), sodium hydride

7680

N. Desbois et al. / Bioorg. Med. Chem. 16 (2008) 7671–7690

0.1 N hydrochloric acid solution. The precipitated product was ﬁl-

tered and washed well with hot water (5 mL). The precipitate was

taken in dry ethanol (50 mL) and was dried under vacuum.

4.1.5.3. Methyl 9,10-dihydro-3-iodo-9-oxoacridine-4-carboxylate

(18c). From 17c. Yield 65%; R_f= 0.41 (SiO₂, CH₂Cl₂/EtOH, 99.5:0.5,

v/v); mp 202–204 °C; IR (KBr) 3600–3000, 1617, 1583, 1431,

1281 cm^ꢀ1; MS m/z 379 (M⁺, 60), 347 (100), 164 (54), 139 (25),

63 (29); ¹H NMR (CDCl₃, 200 MHz) d 4.07 (s, 3H), 7.31 (m, 2H),

7.70 (t, 1H, J = 7 Hz), 7.88 (d, 1H, J = 8.5 Hz), 8.23 (d, 1H,

J = 8.5 Hz), 8.41 (d, 1H, J = 8 Hz), 10.56 (br s, 1H); ¹³C NMR

(DMSO-d₆, 100 MHz) d 53.3, 102.0, 118.1, 120.4, 120.5, 122.2,

125.8, 127.8, 128.9, 131.1, 134.1, 137.4, 140.7, 167.0, 176.1.

4.1.4.1. 2-(5⁰-Iodo-2⁰-methoxycarbonylphenylamino)benzoic acid

(17a). From 14a.³²Yield 80%; mp 206–208 °C; IR (KBr) 3600–3000,

1685, 1575, 1254, 1220 cm^ꢀ1; MS m/z 397 (M⁺, 100), 321 (88), 194

(59), 166 (95), 139 (62), 63 (77), 55 (61); ¹H NMR (DMSO-d₆,

400 MHz) 3.83 (s, 3H), 7.04 (t, 1H, J = 7.5 Hz), 7.30 (d, 1H,

J = 8.5 Hz), 7.47 (m, 2H), 7.62 (d, 1H, J = 8.5 Hz), 7.74 (s, 1H), 7.92

(d, 1H, J = 7.5 Hz), 10.70 (s, 1H), 13.20 (m, 1H); ¹³C NMR (DMSO-

d₆, 100 MHz) d 52.1, 101.9, 115.6, 118.2 (2C), 121.1, 125.3, 128.5,

131.8, 132.8, 133.5, 142.4, 144.4, 166.5, 168.3.

4.1.6. General procedure for the synthesis of 2-(iodophenyl-

amino)isophthalic acids 22a–c

A mixture of 2-iodoisophthalic acid³⁸(21) (6.00 g, 20.5 mmol),

commercial iodoaniline (20a–c) (6.74 g, 30.8 mmol), copper (I)

chloride (2.03 g, 20.5 mmol), dry 2,3-butanediol (25 mL) and dry

benzene (20 mL) was heated and stirred until the benzene being

allowed to boil off. When the internal temperature reached

100 °C, distilled N-ethylmorpholine (5.88 mL, 45.9 mmol) was

added, and the mixture was stirred at 120 °C for 4 h before being

diluted with an aqueous 0.5 M ammonia solution (100 mL) and

treated with charcoal (6 g). After ﬁltration through Celite^Ò521

(washing with water (2ꢁ 50 mL)), the dark coloured solution was

acidiﬁed with an aqueous 2 N hydrochloric acid solution (80 mL)

and extracted with ethyl acetate (2ꢁ 100 mL). An insoluble inor-

ganic precipitate was removed by ﬁltration through Celite^Ò521

and the organic layers were extracted with an aqueous 0.5 M

ammonia solution (2ꢁ 100 mL). The aqueous extract was acidiﬁed

with concentrated hydrochloric acid and concentrated to 100 mL

under vacuum. The product was collected by ﬁltration and washed

with hot water (15 mL). The precipitate was taken in dry ethanol

(50 mL) and was dried under vacuum.

4.1.4.2.

2-(4⁰-Iodo-2⁰-methoxycarbonylphenylamino)benzoic

acid (17b). From 14b.³¹Yield 59%; mp 230–232 °C; IR (KBr)

3200–2800, 1719, 1683, 1577, 1508, 1269, 1209 cm^ꢀ1; MS m/z

397 (M⁺, 100), 320 (67), 194 (54), 166 (77), 139 (59), 63 (72); ¹H

NMR (DMSO-d₆, 400 MHz) d 3.86 (s, 3H), 7.00 (m, 1H), 7.33 (d,

1H, J = 9 Hz), 7.46 (m, 2H), 7.74 (d, 1H, J = 9 Hz), 7.93 (d, 1H,

J = 8 Hz), 8.14 (s, 1H), 10.78 (s, 1H), 13.21 (m, 1H); ¹³C NMR

(DMSO-d₆, 100 MHz) d 52.2, 81.5, 117.8, 118.0, 118.7, 119.9,

120.7, 131.7, 133.4, 139.2, 141.8, 142.7, 143.1, 165.5, 168.4.

4.1.4.3.

2-(3⁰-Iodo-2⁰-methoxycarbonylphenylamino)benzoic

acid (17c). From 14c.³³Yield 67%; mp 191–193 °C; IR (KBr)

3318, 3200–2700, 1740, 1657, 1575, 1444, 1269, 1247 cm^ꢀ1; MS

m/z 397 (M⁺, 73), 321 (100), 194 (36), 166 (41), 139 (18); ¹H

NMR (DMSO-d₆, 200 MHz) d 3.85 (s, 3 H), 6.85 (t, 1H, J = 8 Hz),

7.18 (m, 2H), 7.40 (t, 1H, J = 8 Hz), 7.50 (d, 1H, J = 8 Hz), 7.59 (d,

1H, J = 8 Hz), 7.90 (d, 1H, J = 8 Hz), 9.93 (br s, 1H, NH); ¹³C NMR

(DMSO-d₆, 100 MHz) d 52.6, 93.6, 113.6, 114.4, 118.7, 121.1,

131.8, 131.9, 133.0, 133.4, 134.2, 139.0, 145.8, 167.6, 169.9.

4.1.6.1. 2-(2⁰-Iodophenylamino)isophthalic acid³⁸(22a). From

20a. Yield 36%; mp 225–227 °C (lit.³⁸235–238 °C).

4.1.5. General procedure for the synthesis of methyl 9,10-

dihydro-iodo-9-oxoacridine-4-carboxylates 18a–c

4.1.6.2. 2-(3⁰-Iodophenylamino)isophthalic acid (22b). From

20b. Yield 26%; mp 214–216 °C; IR (KBr) 3200–2800, 1693, 1665,

1584, 1432, 1249 cm^ꢀ1; MS m/z 383 (M⁺, 100), 321 (38), 194

(48), 166 (53), 139 (21); ¹H NMR (DMSO-d₆, 400 MHz) d 6.89 (d,

1 H, J = 8 Hz), 6.98 (t, 1H, J = 8 Hz), 7.08 (t, 1 H, J = 8 Hz), 7.21 (d,

1H, J = 8 Hz), 7.25 (s, 1H), 7.95 (d, 2H, J = 8 Hz), 9.54 (br s, 1H),

13.10 (m, 2H); ¹³C NMR (DMSO-d₆, 100 MHz) d 95.1, 116.4,

120.2, 122.0 (2C), 125.2, 129.6, 130.9, 135.0 (2C), 142.8, 145.5,

168.4 (2C).

2-(Iodo-2⁰-methoxycarbonylphenylamino)benzoic acids 17a–c

(1.00 g, 2.52 mmol) was stirred in concentrated sulphuric acid

(2 mL) at 80 °C for 30 min. The cooled mixture was diluted with

water (15 mL) and the resulting precipitate was collected and well

washed with water (4 mL). The solid was taken in ethanol (50 mL)

and dried under vacuum. The crude product was chromatographed

(SiO₂, CH₂Cl₂/EtOH, 99.5:0.5, v/v).

4.1.5.1. Methyl 9,10-dihydro-1-iodo-9-oxoacridine-4-carboxylate

(18a). From 17a. Yield 85%; R_f= 0.65 (SiO₂, CH₂Cl₂/EtOH, 99.5:0.5,

v/v); mp 189–191 °C; IR (KBr) 3217, 1685, 1640, 1613, 1508,

1273 cm^ꢀ1; MS m/z 379 (M⁺, 71), 347 (81), 192 (24), 164 (100),

127 (24), 88 (24), 76 (24), 63 (24); ¹H NMR (CDCl₃, 400 MHz) d

4.01 (s, 3 H), 7.29 (m, 2H), 7.67 (t, 1H, J = 8.5 Hz), 7.91 (m, 2H),

8.42 (d, 1H, J = 8.5 Hz), 12.07 (br s, 1H, NH); ¹³C NMR (CDCl₃,

100 MHz) d 52.8, 102.4, 113.4, 117.3, 119.8, 121.4, 122.9, 127.5,

134.1, 135.1, 135.3, 138.7, 142.4, 168.6, 176.2.

4.1.6.3. 2-(4⁰-Iodophenylamino)isophthalic acid (22c). From

20c. Yield 62%; mp 241–243 °C; IR (KBr) 3200–2800, 1689, 1592,

1505, 1408, 1243 cm^ꢀ1; MS m/z 383 (M⁺, 100), 194 (86), 166

(35), 139 (22); ¹H NMR (DMSO-d₆, 200 MHz) d 6.75 (d, 2H,

J = 9 Hz), 7.04 (t, 1H, J = 8 Hz), 7.49 (d, 2H, J = 9 Hz), 7.94 (d, 2H,

J = 8 Hz), 9.60 (s, 1H); ¹³C NMR (DMSO-d₆, 100 MHz) d 83.8,

119.7 (2C), 119.8, 121.4 (2C), 135.1 (2C), 137.4 (2C), 143.3, 143.9,

168.5 (2C).

4.1.5.2. Methyl 9,10-dihydro-2-iodo-9-oxoacridine-4-carboxylate

(18b) and methyl 9,10-dihydro-9-oxoacridine-4-carboxylate

(19).³⁰From 17b afforded in order of elution: compound 18b.

Yield 85%; R_f= 0.41 (SiO₂, CH₂Cl₂/EtOH, 99.5:0.5, v/v); mp 275–

277 °C; IR (KBr) 3262, 1694, 1508, 1430, 1282 cm^ꢀ1; MS m/z 379

(M⁺, 100), 347 (52), 164 (30); ¹H NMR (CDCl₃, 200 MHz) d 4.03

(s, 3H), 7.32 (t, 1H, J = 8.5 Hz), 7.38 (d, 1H, J = 8.5 Hz), 7.70 (t, 1 H,

J = 8.5 Hz), 8.42 (d, 1H, J = 8.5 Hz), 8.65 (d, 1H, J = 2 Hz), 8.99 (d,

1H, J = 2 Hz), 11.65 (br s, 1H); ¹³C NMR (CDCl₃, 100 MHz) d 52.9,

81.9, 115.6, 117.8, 121.7, 122.8, 124.2, 127.3, 134.4, 139.9, 140.9,

142.4, 144.5, 167.3, 176.5. Compound 19. Yield 8%; R_f= 0.21

(SiO₂, CH₂Cl₂/EtOH, 99.5:0.5, v/v); mp 170–172 °C (lit.³⁰172 °C).

4.1.7. General procedure for the synthesis of 9,10-dihydro-iodo-

9-oxoacridine-4-carboxylic acids 23a–d

2-(iodophenylamino)isophthalic acid 22a–c (0.89 g, 23.2 mmol)

was dissolved in polyphosphoric acid (PPA) (26 g) at 120 °C, and

the resulting solution was stirred at this temperature for 2 h and

then poured into boiling water (86 mL). The yellow precipitate

was collected by ﬁltration and dissolved in a mixture of methanol

(70 mL) and an aqueous 1 N sodium hydroxide solution (70 mL).

The hot solution (60 °C) was ﬁltered, acidiﬁed with glacial acetic

acid (pH 5), concentrated, and cooled to 0 °C. The resulting precip-

itate was collected by ﬁltration and washed with water. The pure

N. Desbois et al. / Bioorg. Med. Chem. 16 (2008) 7671–7690

7681

product was ﬁnally obtained as yellow solid after recrystallization

from methanol.

8.41 (d, 1H, J = 8 Hz), 8.67 (d, 1H, J = 8 Hz), 8.72 (d, 1H, J = 2 Hz),

11.76 (s, 1H); ¹³C NMR (CDCl₃, 100 MHz) d 52.7, 85.4, 113.9,

119.6, 120.4, 122.5, 123.2, 134.0, 136.0, 136.9, 139.3, 141.6,

142.3, 168.4, 176.5.

4.1.7.1. 9,10-Dihydro-5-iodo-9-oxoacridine-4-carboxylic acid³⁸

38

(23a). From 22a. Yield 77%; mp > 400 °C (lit.³⁸> 360 °C).

4.1.8.3. Methyl 9,10-dihydro-6-iodo-9-oxoacridine-4-carboxylate

(18e) and methyl 9,10-dihydro-8-iodo-9-oxoacridine-4-carboxylate

(18g). Method B, from a mixture of 23 b and 23d afforded in order of

elution: compound 18g; yield 51%; R_f= 0.58 (SiO₂and CH₂Cl₂); mp

239–241 °C; IR (KBr) 2961, 2926, 1692, 1611, 1592, 1261 cm^ꢀ1; MS

m/z 379 (M⁺, 79), 347 (82), 220 (28), 192 (34), 164 (100), 75 (43), 63

(33); ¹H NMR (CDCl₃, 400 MHz) d 4.01 (s, 3H), 7.23 (m, 2H), 7.34 (d,

1H, J = 8 Hz), 7.92 (d, 1H, J = 7.5 Hz), 8.37 (d, 1H, J = 7 Hz), 8.67 (d, 1H,

J = 7.5 Hz), 11.64 (br s, 1H); ¹³C NMR (CDCl₃, 100 MHz) d 52.6, 92.5,

113.3, 118.6, 119.0, 120.5, 122.3, 133.8, 134.4, 136.6, 137.2, 140.7,

141.3, 168.4, 176.1. Compound 18e; yield 32%; R_f= 0.38 (SiO₂and

CH₂Cl₂); mp 258–260 °C; IR (KBr) 3271, 1685, 1640, 1609, 1589, 1280

cm^ꢀ1; MS m/z 379 (M⁺, 95), 347 (100), 220 (32), 192 (29), 164 (99),

137 (26), 75 (58), 63 (42); ¹H NMR (CDCl₃, 200 MHz) d 4.03 (s, 3H),

7.30 (t, 1H, J = 8 Hz), 7.61 (d, 1H, J = 8.5 Hz), 7.85 (s, 1H), 8.13 (d, 1H,

J = 8.5 Hz), 8.46 (d, 1H, J = 7.5 Hz), 8.71 (d, 1H, J = 7.5 Hz), 11.77 (br s,

1H); ¹³C NMR (CDCl₃, 50 MHz) d 52.7, 101.5, 120.5, 120.8, 121.3,

122.7, 126.4, 128.5, 131.6, 134.0, 136.9, 140.7, 141.7, 168.5, 174.6.

4.1.7.2. 9,10-Dihydro-7-iodo-9-oxoacridine-4-carboxylic acid

(23c). From 22c. Yield 63%; mp 355–357 °C; IR (KBr) 3200–2800,

1693, 1609, 1518, 1446, 1148 cm^ꢀ1; MS m/z 365 (M⁺, 95), 347

(100), 319 (23), 164 (33); ¹H NMR (DMSO-d₆, 200 MHz) d 7.25 (t,

1H, J = 8 Hz), 7.46 (d, 1H, J = 9 Hz), 7.86 (d, 1H, J = 9 Hz), 8.34 (m,

3H), 11.93 (br s, 1H); ¹³C NMR (DMSO-d₆, 100 MHz) d 85.8,

115.9, 120.4, 121.0, 121.4, 122.2, 131.9, 134.0, 136.9, 139.0,

140.9, 141.6, 169.0, 175.1.

4.1.7.3. 9,10-Dihydro-6-iodo-9-oxoacridine-4-carboxylic acid

(23b) and 9,10-dihydro-8-iodo-9-oxoacridine-4-carboxylic acid

(23d). From 22b which gave a mixture of acid 23b and 23d in ratio

of 1:1 observed by ¹H NMR spectroscopy.

4.1.8. General procedure for the synthesis of methyl 9,10-

dihydro-iodo-9-oxoacridine-4-carboxylates 18d–g

Method A:

A sample of the appropriate acid 23 (1.00 g,

27.4 mmol) was warmed in thionyl chloride (30 mL) in the pres-

ence of three drops of dry dimethylformamide until total dissolu-

tion. The solution was maintained at the boiling temperature of

thionyl chloride for 15 min. The solvent was evaporated under re-

duced pressure and the residue was suspended in dry toluene

(20 mL). After evaporation to dryness, the product was dissolved

in dry methanol (30 mL) and stirred at room temperature with cal-

cium carbonate (1.20 g) for 3 h. The excess of calcium carbonate

was ﬁltered off and the methanol was evaporated under reduce

pressure. An aqueous 1 N hydrochloric acid solution (25 mL) was

added to the residue and the mixture was extracted with dichloro-

methane (3ꢁ 50 mL). The organic layers were dried (Na₂SO₄), con-

centrated under vacuum and the crude product was

chromatographed (Al₂O₃and CH₂Cl₂).

4.1.9. General procedure for the synthesis of N-(2-diethylamino-

ethyl)-9,10-dihydro-iodo-9-oxoacridine-4-carboxamides 24a–g

Diethylethylenediamine (77 lL, 0.54 mmol) and trimethylalu-

minium (2.0 M solution in hexanes) (0.36 mL, 0.72 mmol) were

added dropwise, at 0 °C, to a stirred solution of dry dichloro-

methane or dry toluene (10 mL). After 10 min, the appropriate

iodo compound 18 (0.15 g, 0.40 mmol) in dry dichloromethane

solution (2 mL) or dry toluene solution (4 mL) was added and

the mixture was reﬂuxed. After cooling to room temperature,

water (15 mL) was added and the mixture was made alkaline

with a saturated aqueous sodium carbonate solution (20 mL)

and extracted with dichloromethane (3ꢁ 30 mL). The organic

layers were dried (Na₂SO₄) and concentrated under vacuum.

The crude product was chromatographed (Al₂O₃, CH₂Cl₂/EtOH,

99:1, v/v).

Method B: A mixture of acids 23b and 23d (95.0 mg, 0.26 mmol)

was dissolved in dry dimethylformamide (5 mL), sodium hydride

(10.4 mg, 0.26 mmol, 60% in mineral oil) was added, and the mix-

ture was stirred for 30 min. Methyl iodide (16.2 lL, 0.26 mmol)

4.1.9.1.

N-(2-Diethylaminoethyl)-9,10-dihydro-1-iodo-9-oxo-

was added and the reaction mixture was stirred for 18 h at room

temperature. The solvent was evaporated and water (10 mL) was

added. The resulting solution was made alkaline with an aqueous

saturated sodium carbonate solution and extracted with ethyl ace-

tate (3ꢁ 20 mL). The organic layers were dried (MgSO₄), ﬁltered

and concentrated under vacuum. The crude product was chro-

matographed (SiO₂and CH₂Cl₂).

acridine-4-carboxamide (24a). From 18a. The mixture was re-

ﬂuxed in dry dichloromethane for 5 h. Yield 64%; R_f= 0.40 (Al₂O₃,

CH₂Cl₂/EtOH, 99:1, v/v); mp 248–250 °C; IR (KBr) 3281, 1615,

1513 cm^ꢀ1; MS m/z 463 (M⁺, 1), 86 (100), 58 (16); ¹H NMR (CDCl₃,

400 MHz) d 1.04 (t, 6H, J = 6.5 Hz), 2.60 (q, 4H, J = 6.5 Hz), 2.70 (m,

2H); 3.48 (m, 2H), 7.19 (t, 1H, J = 7 Hz), 7.25 (d, 1H, J = 8.5 Hz), 7.36

(d, 1H, J = 8 Hz), 7.57 (m, 2H), 7.81 (d, 1H, J = 8 Hz), 8.34 (d, 1H,

J = 8 Hz), 12.75 (s, 1H); ¹³C NMR (CDCl₃, 100 MHz) d 11.7 (2C),

37.1, 46.9 (2C), 51.2, 99.0, 117.5 (2C), 119.9, 121.3, 122.5, 127.4,

130.9, 134.0, 134.8, 139.1, 142.1, 168.4, 176.5.

4.1.8.1. Methyl 9,10-dihydro-5-iodo-9-oxoacridine-4-carboxylate

(18d). Method A, from 23a.³⁸Yield 57%; R_f= 0.78 (Al₂O₃and

CH₂Cl₂); mp 174–176 °C; IR (KBr) 3197, 1608, 1518, 1430, 1279,

1141 cm^ꢀ1; MS m/z 379 (M⁺, 80), 347 (100), 164 (99), 75 (71), 63

(40); ¹H NMR (CDCl₃, 400 MHz) d 4.07 (s, 3H), 7.06 (t, 1H,

J = 7.5 Hz), 7.30 (t, 1H, J = 8 Hz), 8.17 (d, 1H, J = 7.5 Hz), 8.46 (m,

2 H), 8.69 (d, 1H, J = 8 Hz), 12.11 (s, 1H); ¹³C NMR (CDCl₃,

100 MHz) d 52.9, 86.2, 114.3, 120.8, 121.9, 122.5, 123.6, 127.7,

133.8, 137.0, 140.7, 141.5, 144.0, 168.0, 177.6.

4.1.9.2. N-(2-Diethylaminoethyl)-9,10-dihydro-2-iodo-9-oxoac-

ridine-4-carboxamide (24b). From 18b. The mixture was re-

ﬂuxed in dry toluene for 1.5 h. Yield 88%; R_f= 0.43 (Al₂O₃,

CH₂Cl₂/EtOH, 99:1, v/v); mp 250–252 °C; IR (KBr) 3422, 1617,

1512 cm^ꢀ1; MS m/z 463 (M⁺, 6), 348 (18), 320 (14), 193 (11),

164 (11), 86 (100), 58 (23); ¹H NMR (CDCl₃, 200 MHz) d 1.08

(t, 6H, J = 7 Hz), 2.67 (q, 4H, J = 7 Hz), 2.75 (t, 2H, J = 6 Hz), 3.56

(m, 2H), 7.21 (t, 1H, J = 8 Hz), 7.30 (d, 1H, J = 8 Hz), 7.61 (t, 1H,

J = 8 Hz), 7.65 (br s, 1H), 8.08 (d, 1H, J = 2 Hz), 8.32 (d, 1H,

J = 8 Hz), 8.75 (d, 1H, J = 2 Hz), 12.15 (br s, 1H); ¹³C NMR (CDCl₃,

100 MHz) d 11.8 (2C), 37.4, 46.9 (2C), 51.2, 82.0, 117.9, 120.0,

121.2, 122.3, 124.0, 126.8, 134.0, 139.7, 139.8, 140.0 (2C),

166.9, 176.5.

4.1.8.2. Methyl 9,10-dihydro-7-iodo-9-oxoacridine-4-carboxylate

(18f). Method A, from 23c. Yield 60%; R_f= 0.85 (Al₂O₃and CH₂Cl₂);

mp 224–226 °C; IR (KBr) 3247, 1692, 1614, 1589, 1518, 1440,

1137 cm^ꢀ1; MS m/z 379 (M⁺, 91), 347 (100), 220 (29), 164 (94),

75 (44), 63 (34); ¹H NMR (CDCl₃, 200 MHz) d 4.01 (s, 3H), 7.14

(d, 1H, J = 8.5 Hz), 7.25 (t, 1H, J = 8 Hz), 7.89 (dd, 1H, J = 8.5, 2 Hz),

7682

N. Desbois et al. / Bioorg. Med. Chem. 16 (2008) 7671–7690

4.1.9.3.

N-(2-Diethylaminoethyl)-9,10-dihydro-3-iodo-9-

trated under vacuum and started again with dry ether (3 mL). The

suspension was stirred overnight and ﬁltered.

oxoacridine-4-carboxamide (24c). From 18c. The mixture was

reﬂuxed in dry dichloromethane for 6 h. Yield 38%; R_f= 0.40

(Al₂O₃, CH₂Cl₂/EtOH, 99:1, v/v); mp 215–217 °C; IR (KBr) 3230–

2960, 1614, 1556 cm^ꢀ1; MS m/z 463 (M⁺, 1), 86 (100), 58 (12);

¹H NMR (CDCl₃, 200 MHz) d 1.49 (t, 6H, J = 7 Hz), 3.37 (m, 4H),

3.52 (m, 2H), 4.08 (m, 2H), 7.00 (d, 1H, J = 8 Hz), 7.19 (t, 1H,

J = 8 Hz), 7.59 (m, 3H), 8.21 (d, 1H, J = 8 Hz), 8.77 (br s, 1H), 10.09

(s, 1H); ¹³C NMR (DMSO-d₆, 100 MHz) d 10.8 (2C), 39.5, 46.8

(2C), 50.3, 102.3, 118.2, 120.3, 120.4, 121.9, 125.7, 127.6, 130.9,

132.0, 133.9, 137.2, 140.9, 166.5, 176.3.

4.1.10.1. N-(2-Diethylaminoethyl)-9,10-dihydro-1-iodo-9-oxoac-

ridine-4-carboxamide hydrochloride salt (8a). From 24a. Yield

81%; mp 269–271 °C; IR (KBr) 3500–3100, 3057, 1613, 1577,

1513 cm^ꢀ^{1 1}H NMR (DMSO-d₆, 400 MHz) d 1.26 (t, 6H, J = 7 Hz),

;

3.22 (m, 4H), 3.33 (m, 2H, J = 7 Hz), 3.75 (m, 2H), 7.33 (t, 1H,

J = 7 Hz), 7.62 (d, 1H, J = 8 Hz), 7.76 (d, 1H, J = 7 Hz), 7.96 (m, 2H),

8.20 (d, 1H, J = 8 Hz), 9.49 (br s, 1H), 10.39 (br s, 1H), 12.70 (br s,

1H); ¹³C NMR (DMSO-d₆, 100 MHz) d 8.3 (2C), 34.3, 46.5 (2C),

49.6, 99.0, 117.7, 118.0, 118.8, 120.4, 122.3, 126.3, 133.0, 134.1,

134.3, 138.6, 141.3, 168.2, 175.2. Anal. Calcd for C₂₀H₂₂IN₃O₂,

HCl, 3H₂O: C, 43.38; H, 5.28; N, 7.59. Found: C, 43.74; H, 4.99; N,

7.68.

4.1.9.4. N-(2-Diethylaminoethyl)-9,10-dihydro-5-iodo-9-oxoac-

ridine-4-carboxamide (24d). From 18d. The mixture was reﬂuxed

in dry toluene for 2 h. Yield 73%; R_f= 0.23 (Al₂O₃, CH₂Cl₂/EtOH,

99:1, v/v); mp 148–150 °C; IR (KBr) 3500–3200, 1610, 1521 cm^ꢀ1

;

MS m/z 463 (M⁺, 1), 86 (100), 58 (7); ¹H NMR (CDCl₃, 400 MHz) d

1.05 (t, 6H, J = 7 Hz), 2.61 (q, 4H, J = 7 Hz), 2.72 (m, 2H), 3.56 (m,

2H), 6.95 (t, 1H, J = 8 Hz), 7.21 (t, 1H, J = 8 Hz), 7.58 (br s, 1H), 7.92

(d, 1H, J = 8 Hz), 8.08 (d, 1H, J = 8 Hz), 8.36 (d, 1H, J = 8 Hz), 8.53 (d,

1H, J = 8 Hz), 12.80 (s, 1H); ¹³C NMR (CDCl₃, 100 MHz) d 11.8 (2C),

37.3, 46.9 (2C), 51.2, 86.3, 118.1, 120.6, 122.2, 122.3, 123.2, 127.6,

131.8, 132.0, 141.1, 141.2, 143.9, 167.9, 177.8.

4.1.10.2. N-(2-Diethylaminoethyl)-9,10-dihydro-2-iodo-9-oxoac-

ridine-4-carboxamide hydrochloride salt (8b). From 24b. Yield

71%; mp 298–300 °C; IR (KBr) 3400–3200, 1617, 1560, 1517,

1300, 1168 cm^ꢀ^{1 1}H NMR (DMSO-d₆, 400 MHz) d 1.27 (m, 6H),

;

3.23 (m, 4H), 3.33 (m, 2H) 3.75 (m, 2H), 7.33 (t, 1H, J = 7 Hz),

7.75 (m, 2H), 8.23 (d, 1H, J = 8 Hz), 8.60 (s, 1H), 8.68 (s, 1H), 9.42

(br s, 1H), 10.27 (br s, 1H), 12.19 (br s, 1H); ¹³C NMR (DMSO-d₆,

100 MHz) d 8.3 (2C), 34.2, 46.4 (2C), 49.5, 83.2, 118.6, 120.5 (2C),

122.3, 123.3, 125.9, 134.3, 138.2, 139.3, 139.8, 140.7, 166.8,

175.3. Anal. Calcd for C₂₀H₂₂IN₃O₂, HCl, 1.75H₂O: C, 45.21; H,

5.03; N, 7.91. Found: C, 44.91; H, 4.66; N, 8.07.

4.1.9.5. N-(2-Diethylaminoethyl)-9,10-dihydro-6-iodo-9-oxoacri-

dine-4-carboxamide (24e). From 18e. The mixture was reﬂuxed in

dry dichloromethane for 2 h. Yield 65%; R_f= 0.25 (Al₂O₃, CH₂Cl₂/EtOH,

99:1, v/v); mp 208–210 °C; IR (KBr) 3343, 2966, 1610, 1580, 1560,

1522, 1449, 1301 cm^ꢀ1; MS m/z 463 (M⁺, 1), 86 (100), 58 (12); ¹H

NMR (CDCl₃, 400 MHz) d 1.25 (t, 6H, J = 7 Hz), 2.95 (m, 4H), 3.04 (m,

2H), 3.72 (m, 2H), 7.22 (t, 1H, J = 7.5 Hz), 7.47 (d, 1H, J = 8.5 Hz), 7.70

(s, 1H), 8.00 (d, 1H, J = 8.5 Hz), 8.25 (d, 1H, J = 7 Hz), 8.51 (d, 1H,

J = 8 Hz), 8.67 (br s, 1H), 12.40 (s, 1H); ¹³C NMR (CDCl₃, 100 MHz) d

9.9 (2C), 35.9, 47.9 (2C), 52.5, 101.3, 116.7, 120.5, 120.6, 122.7, 126.5,

128.3, 131.0, 132.0, 133.0, 140.7, 141.2, 168.7, 177.7.

4.1.10.3. N-(2-Diethylaminoethyl)-9,10-dihydro-3-iodo-9-oxoac-

ridine-4-carboxamide hydrochloride salt (8c). From 24c. Yield

87%; mp 262–264 °C; IR (KBr) 3400–3200, 1618, 1590, 1427

cm^ꢀ^{1 1}H NMR (DMSO-d₆, 400 MHz) d 1.27 (t, 6H, J 8 7 Hz),

;

3.23 (m, 4H), 3.32 (m, 2H), 3.76 (m, 2H), 7.32 (t, 1H, J 8 8 Hz),

7.75 (m, 2H), 7.95 (d, 1H, J 8 8.5 Hz), 7.99 (d, 1H, J = 8.5 Hz),

8.21 (d, 1H, J = 8 Hz), 9.05 (m, 1H), 10.17 (br s, 1H), 10.79 (br

s, 1H); ¹³C NMR (DMSO-d₆, 100 MHz) d 8.4 (2C), 34.5, 46.6

(2C), 49.3, 102.6, 118.3, 120.4 (2C), 121.9, 125.7, 127.8, 130.9,

131.6, 133.8, 137.2, 141.0, 166.9, 176.3. Anal. Calcd for

4.1.9.6. N-(2-Diethylaminoethyl)-9,10-dihydro-7-iodo-9-oxoac-

ridine-4-carboxamide (24f). From 18f. The mixture was reﬂuxed

in dry dichloromethane for 6 h. Yield 69%; R_f= 0.27 (Al₂O₃, CH₂Cl₂/

EtOH, 99:1, v/v); mp 140–142 °C; IR (KBr) 3500–2800, 1647, 1613,

1516, 1300 cm^ꢀ1; MS m/z 463 (M⁺, 4), 86 (100), 58 (7); ¹H NMR

(CDCl₃, 400 MHz) d 1.30 (t, 6H, J = 7 Hz), 3.01 (m, 4H), 3.10 (m,

2H), 3.76 (m, 2H), 7.07 (d, 1H, J = 9 Hz), 7.24 (t, 1H, J = 8 Hz), 7.81

(d, 1H, J = 9 Hz), 8.25 (d, 1H, J = 8 Hz), 8.53 (d, 1H, J = 8 Hz), 8.65

(m, 2H), 12.48 (s, 1H); ¹³C NMR (CDCl₃, 100 MHz) d 10.0 (2C),

35.9, 47.6 (2C), 52.2, 84.9, 116.8, 119.7, 120.4, 122.4, 122.8,

131.9, 132.9, 135.6, 139.2, 141.0, 141.9, 168.6, 176.5.

C₂₀H₂₂IN₃O₂, HCl, H₂O: C, 46.39; H, 4.87; N, 8.12. Found: C,

46.59; H, 4.89; N, 8.06.

4.1.10.4. N-(2-Diethylaminoethyl)-9,10-dihydro-5-iodo-9-oxoac-

ridine-4-carboxamide hydrochloride salt (8d) from 24d. From

24d. Yield 82%; mp 251–253 °C; IR (KBr) 3400–3200, 2946, 2650,

1611, 1522, 1428, 1301 cm^ꢀ^{1 1}H NMR (DMSO-d₆, 400 MHz) d

;

1.27 (t, 6H, J = 7 Hz), 3.24 (m, 4H), 3.36 (m, 2H), 3.79 (m, 2H),

7.12 (t, 1H, J = 8 Hz), 7.43 (t, 1H, J = 7.5 Hz), 8.26 (d, 1H, J = 8 Hz),

8.32 (d, 1H, J = 7 Hz), 8.46 (d, 1H, J = 8 Hz), 8.55 (d, 1H, J = 7 Hz),

9.55 (br s, 1H), 10.34 (br s, 1H), 12.97 (br s, 1H); ¹³C NMR

(DMSO-d₆, 100 MHz) d 8.3 (2C), 34.2, 46.5 (2C), 49.8, 87.5, 117.4,

120.7, 121.1, 121.3, 123.5, 126.5, 130.8, 133.8, 140.5 (2C), 144.0,

168.1, 176.3. Anal. Calcd for C₂₀H₂₂IN₃O₂, HCl, H₂O: C, 46.39; H,

4.87; N, 8.12. Found: C, 46.03; H, 4.80; N, 8.06.

4.1.9.7. N-(2-Diethylaminoethyl)-9,10-dihydro-8-iodo-9-oxoac-

ridine-4-carboxamide (24g). From 18g. The mixture was reﬂuxed

in dry toluene for 4 h. Yield 63% (unstable); IR (KBr) 3500–2800,

1645, 1606, 1514, 1298 cm^ꢀ1; MS m/z 463 (M⁺, 1), 86 (100), 58

(12); ¹H NMR (CDCl₃, 200 MHz) d 1.15 (t, 6H, J = 7 Hz), 2.76 (q,

4H, J = 7 Hz), 2.86 (t, 2H, J = 6 Hz), 3.59 (m, 2H), 7.17 (m, 2H),

7.31 (d, 1H, J = 8 Hz), 7.80 (br s, 1H), 7.85 (d, 1H, J = 7.5 Hz), 7.95

(d, 1H, J = 7.5 Hz), 8.54 (d, 1H, J = 7 Hz), 11.30 (br s, 1H); ¹³C NMR

(CDCl₃, 100 MHz) d 11.3 (2C), 36.6, 47.0 (2C), 51.3, 92.2, 116.8,

118.5, 118.7, 120.3, 122.4, 131.8, 132.1, 133.6, 136.6, 140.1,

141.3, 168.1, 176.1.

4.1.10.5. N-(2-Diethylaminoethyl)-9,10-dihydro-6-iodo-9-oxoac-

ridine-4-carboxamide hydrochloride salt (8e). From 24e. Yield

46%; mp 266-268 °C; IR (KBr) 3450-3250, 3066, 1611, 1577,

1523, 1450, 1309 cm^ꢀ^{1 1}H NMR (DMSO-d₆, 400 MHz) d 1.27 (t,

;

6H, J = 7 Hz), 3.23 (m, 4H), 3.34 (m, 2H), 3.76 (m, 2H), 7.37 (t, 1H,

J = 7.5 Hz), 7.61 (t, 1H, J = 8.5 Hz), 7.93 (d, 1H, J = 8.5 Hz), 8.26 (s,

1H), 8.38 (d, 1H, J = 7.5 Hz), 8.42 (d, 1H, J = 8 Hz), 9.43 (m, 1H),

10.41 (br s, 1H), 12.21 (br s, 1H); ¹³C NMR (DMSO-d₆, 100 MHz)

d 8.3 (2C), 34.2, 46.5 (2C), 49.7, 102.2, 118.6, 119.5, 120.4, 121.7,

126.8, 127.4, 130.2, 130.6, 133.6, 139.9, 140.6, 167.9, 176.2. Anal.

Calcd for C₂₀H₂₂IN₃O₂, HCl, 1.5H₂O: C, 45.60; H, 4.97; N, 7.98.

Found: C, 46.80; H, 4.71; N, 7.92.

4.1.10. General procedure for the synthesis of N-(2-diethylamino-

ethyl)-9,10-dihydro-iodo-9-oxoacridine-4-carboxamides

hydrochloride salts 8a–g

Compound 24 (100 mg, 0.22 mmol) was diluted with dry

dichloromethane (0.5 mL) and treated with an anhydrous 2 N

hydrochloric acid–ether solution (2 mL). The solution was concen-

N. Desbois et al. / Bioorg. Med. Chem. 16 (2008) 7671–7690

7683

4.1.10.6. N-(2-Diethylaminoethyl)-9,10-dihydro-7-iodo-9-oxoac-

ridine-4-carboxamide hydrochloride salt (8f). From 24f. Yield

100 MHz) d 31.2, 51.8, 91.7, 114.7, 118.1, 119.4, 121.7 (2C), 127.3,

128.2, 132.2, 132.5, 138.5, 142.4, 168.1.

99%; mp 220–222 °C; IR (KBr) 3400–3200, 1617, 1517, 1303 cm^ꢀ1

;

¹H NMR (DMSO-d₆, 400 MHz) d 1.27 (t, 6H, J = 7 Hz), 3.23 (m,

4H), 3.34 (m, 2H), 3.76 (m, 2H), 7.38 (t, 1H, J = 8 Hz), 7.63 (d, 1H,

J = 9 Hz), 8.00 (d, 1H, J = 9 Hz), 8.44 (m, 2H), 8.49 (s, 1H), 9.46 (br

s, 1H), 10.40 (br s, 1H), 12.38 (br s, 1H); ¹³C NMR (DMSO-d₆,

100 MHz) d 8.3 (2C), 34.2, 46.5 (2C), 49.7, 85.7, 118.5, 120.4,

121.0, 121.6, 122.2, 130.4, 133.7, 134.1, 139.2, 140.1, 141.7,

168.0, 175.2. Anal. Calcd for C₂₀H₂₂IN₃O₂, HCl, 2H₂O: C, 44.83; H,

5.08; N, 7.84. Found: C, 44.67; H, 4.43; N, 7.76.

4.1.11.4. Methyl 5-iodoacridan-4-carboxylate (28d). From 18d.

Yield 98%; R_f= 0.94 (SiO₂and CH₂Cl₂); mp 105–107 °C; IR (KBr)

3260, 2924, 1689, 1491, 1443, 1259 cm^ꢀ1; MS m/z 365 (M⁺, 100),

332 (56), 206 (53), 177 (81), 151 (63), 103 (36), 89 (37), 75 (46);

¹H NMR (CDCl₃, 400 MHz) d 3.97 (s, 3H), 4.08 (s, 2H), 6.61 (t, 1H,

J = 7.5 Hz), 6.83 (t, 1H, J = 7.5 Hz), 7.03 (d, 1H, J = 7 Hz), 7.24 (d,

1H, J = 7 Hz), 7.58 (d, 1H, J = 8 Hz), 7.84 (d, 1H, J = 8 Hz), 10.16 (br

s, 1H); ¹³C NMR (CDCl₃, 100 MHz) d 32.2, 52.2, 84.3, 111.3, 119.6,

121.1, 121.7, 122.7, 128.3, 129.5, 133.3, 137.3, 140.1, 143.0, 168.5.

4.1.10.7. N-(2-Diethylaminoethyl)-9,10-dihydro-8-iodo-9-oxoac-

ridine-4-carboxamide hydrochloride salt (8g). From 24g. Yield

91%; mp 228–230 °C; IR (KBr) 3400–3200, 2925, 1607, 1560,

4.1.11.5. Methyl 6-iodoacridan-4-carboxylate (28e). From 18e.

Very instable precipitate; Yield 76%; IR (KBr) 3342, 2942, 1684,

1517, 1458, 1295 cm^ꢀ1

;

¹H NMR (DMSO-d₆, 400 MHz) d 1.25 (t,

1594, 1507, 1464, 1429, 1266 cm^ꢀ^{1 1}H NMR (CDCl₃, 400 MHz) d

;

6H, J = 7 Hz), 3.30 (m, 6H), 3.71 (m, 2H), 7.37 (m, 2H), 7.71 (d,

1H, J = 8 Hz), 7.90 (d, 1H, J = 7.5 Hz), 8.28 (d, 1H, J = 7.5 Hz), 8.43

(d, 1H, J = 7 Hz), 9.23 (br s, 1H), 9.44 (m, 1H), 12.26 (br s, 1H);

¹³C NMR (DMSO-d₆, 100 MHz) d 8.3 (2C), 34.2, 46.5 (2C), 49.7,

92.4, 117.5, 117.8, 119.3, 120.4, 121.6, 130.9, 133.4, 134.2, 136.3,

139.3, 141.2, 168.0, 175.0. Anal. Calcd for C₂₀H₂₂IN₃O₂, HCl,

0.5H₂O: C, 47.21; H, 4.74; N, 8.26. Found: C, 47.70; H, 5.12; N, 8.29.

3.94 (s, 3H), 4.01 (s, 2H), 6.80 (m, 2H), 7.14 (s, 1H), 7.18 (d, 1H,

J = 8 Hz), 7.22 (d, 1H, J = 7.5 Hz), 7.79 (d, 1H, J = 8 Hz), 9.82 (br s,

1H); ¹³C NMR (CDCl₃, 100 MHz) d 31.2, 52.0, 91.6, 110.8, 119.2,

119.5, 121.3, 123.3, 129.4, 129.9, 130.2, 133.6, 140.5, 143.0, 168.8.

4.1.11.6. Methyl 7-iodoacridan-4-carboxylate (28f). From 18f.

Yield 95%; R_f= 0.90 (SiO₂and CH₂Cl₂); mp 122–124 °C; IR (KBr)

3320, 2948, 1682, 1594, 1502, 1466, 1430, 1269, 1194 cm^ꢀ1; MS

m/z 365 (M⁺, 100), 333 (50), 206 (40), 177 (65), 151 (44), 127

(30), 103 (29), 89 (33), 75 (42); ¹H NMR (CDCl₃, 400 MHz) d 3.91

(s, 3H), 4.04 (s, 2 H), 6.54 (d, 1H, J = 8.5 Hz), 6.79 (t, 1H,

J = 7.5 Hz), 7.22 (d, 1H, J = 7 Hz), 7.37 (m, 2H), 7.77 (d, 1H,

J = 7.5 Hz), 9.83 (br s, 1H); ¹³C NMR (CDCl₃, 100 MHz) d 30.4,

52.0, 82.9, 110.7, 116.7, 119.1, 121.1, 122.4, 129.4, 133.7, 136.0,

136.8, 138.8, 143.1, 168.9.

4.1.11. General procedure for the synthesis of methyl

iodoacridan-4-carboxylates 28a–g

The appropriate methyl 9,10-dihydro-iodo-9-oxoacridine-4-

carboxylate 18 (0.26 g, 0.69 mmol) was dissolved in anhydrous

tetrahydrofuran (10 mL) under a slowly ﬂowing argon gas. A 1 M

borane tetrahydrofuran complex solution (0.82 mL, 0.82 mmol)

was added dropwise to the stirred reﬂux reaction. The mixture

was reﬂuxed for 45 min. After cooling to room temperature, an

aqueous 3 N hydrochloric acid solution (5 mL) was added and the

reaction mixture was made alkaline with an aqueous saturated

sodium carbonate solution (20 mL). The mixture was extracted

with dichloromethane (3ꢁ 20 mL). The organic phases collected

were dried (MgSO₄), ﬁltered and the solvent removed under reduce

pressure. The crude product was chromatographed (SiO₂and

CH₂Cl₂) except for unstable compounds 28c and 28e which were

used in the next step without puriﬁcation.

4.1.11.7. Methyl 8-iodoacridan-4-carboxylate (28g). From 18g.

Yield 92%; R_f= 0.97 (SiO₂and CH₂Cl₂); mp 108–110 °C; IR (KBr)

3319, 2960, 1685, 1603, 1501, 1442, 1262 cm^ꢀ1; MS m/z 365 (M⁺,

100), 333 (53), 332 (55), 206 (81), 178 (90), 177 (95), 151 (71),

127 (42), 103 (42), 89 (32), 75 (66), 63 (42); ¹H NMR (CDCl₃,

400 MHz) d 3.90 (s, 3H), 4.09 (s, 2 H), 6.69 (d, 1H, J = 8 Hz), 6.77

(m, 2H), 7.24 (d, 1H, J = 7.5 Hz), 7.36 (d, 1H, J = 8 Hz), 7.77 (d, 1H,

J = 8 Hz), 9.77 (br s, 1H); ¹³C NMR (CDCl₃, 100 MHz) d 37.5, 51.9,

101.2, 110.3, 114.8, 115.7, 119.0, 122.5, 128.9, 129.4, 131.4,

133.9, 139.5, 142.8, 168.9.

4.1.11.1. Methyl 1-iodoacridan-4-carboxylate (28a). From 18a.

Yield 75%; R_f= 0.91 (SiO₂and CH₂Cl₂); mp 119–121 °C; IR (KBr)

3304, 2948, 1690, 1585, 1498, 1431, 1269, 1252 cm^ꢀ1; MS m/z

365 (M⁺, 100), 332 (78), 206 (52), 178 (53), 151 (23); ¹H NMR

(CDCl₃, 400 MHz) d 3.91 (s, 3H), 4.13 (s, 2H), 6.74 (d, 1H,

J = 7.5 Hz), 6.88 (t, 1H, J = 7.5 Hz), 7.10 (m, 2H), 7.26 (d, 1H,

J = 8.5 Hz), 7.41 (d, 1H, J = 8.5 Hz), 9.93 (br s, 1H); ¹³C NMR (CDCl₃,

100 MHz) d 38.1, 52.0, 109.0, 109.8, 114.5, 119.5, 121.8, 124.3,

127.6, 128.6, 128.8, 130.1, 137.9, 143.6, 168.8.

4.1.12. General procedure for the synthesis of methyl

iodoacridine-4-carboxylates 29a–g

Methyl iodoacridan-4-carboxylate 28 (140 mg, 0.38 mmol) was

stirred in a mixture of ethanol (10 mL), water (2 mL) and iron chlo-

ride hexahydrate (0.30 g) for 30 min at 50 °C. After cooling to room

temperature, an aqueous saturated sodium bicarbonate solution

(20 mL) was added and the mixture was extracted with dichloro-

methane (3ꢁ 30 mL). The organic layers were dried (MgSO₄) and

ﬁltered. Evaporation of the solvent gave a very instable precipitate

which was used without puriﬁcation.

4.1.11.2. Methyl 2-iodoacridan-4-carboxylate (28b). From 18b.

Yield 58%; R_f= 0.93 (SiO₂and CH₂Cl₂); mp 148–150 °C; IR (KBr)

3344, 2941, 1676, 1501, 1263, 1253 cm^ꢀ1; MS m/z 365 (M⁺, 100),

332 (23), 178 (58), 151 (20), 127 (22); ¹H NMR (CDCl₃, 400 MHz)

d 3.92 (s, 3H), 4.06 (s, 2H), 6.77 (d, 1H, J = 7.5 Hz), 6.90 (t, 1H,

J = 7.5 Hz), 7.06 (d, 1H, J = 7.5 Hz), 7.12 (t, 1H, J = 7.5 Hz), 7.48 (s,

1H), 8.08 (s, 1H), 9.77 (br s, 1H); ¹³C NMR (CDCl₃, 100 MHz) d

31.0, 52.2, 79.2, 112.3, 114.9, 119.4, 121.9, 124.4, 127.5, 128.4,

137.5, 138.3, 141.3, 143.3, 167.7.

4.1.12.1. Methyl 1-iodoacridine-4-carboxylate (29a). From 28a.

Yield 87%; ¹H NMR (CDCl₃, 400 MHz) d 4.00 (s, 3H), 7.53 (t, 1 H,

J = 7 Hz), 7.66 (d, 1H, J = 7 Hz), 7.76 (t, 1H, J = 7 Hz), 7.99 (d, 1H,

J = 8 Hz), 8.07 (d, 1H, J = 7 Hz), 8.21 (d, 1H, J = 8 Hz), 8.91 (s, 1H).

4.1.12.2. Methyl 2-iodoacridine-4-carboxylate (29b). From 28 b.

Yield 93%; ¹H NMR (CDCl₃, 200 MHz) d 4.14 (s, 3H), 7.62 (t, 1H,

J = 7.5 Hz), 7.88 (t, 1H, J = 7.5 Hz), 8.05 (d, 1H, J = 8 Hz), 8.36 (m,

2H), 8.59 (s, 1H), 8.86 (s, 1H).

4.1.11.3. Methyl 3-iodoacridan-4-carboxylate (28c). From 18c.

Very instable precipitate; Yield 78%; IR (KBr) 3442, 2946, 1730,

1234 cm^ꢀ^{1 1}H NMR (CDCl₃, 400 MHz) d 3.98 (s, 3H), 4.00 (s, 2H),

;

6.72 (d, 1H, J = 8 Hz), 6.80 (d, 1H, J = 8 Hz), 6.91 (t, 1H, J = 7.5 Hz),

4.1.12.3. Methyl 3-iodoacridine-4-carboxylate (29c). From 28c.

Yield 88%; ¹H NMR (CDCl₃, 400 MHz) d 4.19 (s, 3H), 7.55 (t, 1H,

7.10 (m, 2H), 7.39 (d, 1 H, J = 8 Hz), 8.33 (br s, 1H); ¹³C NMR (CDCl₃,

7684

N. Desbois et al. / Bioorg. Med. Chem. 16 (2008) 7671–7690

J = 7.5 Hz), 7.68 (d, 1H, J = 8.5 Hz), 7.77 (m, 2H), 7.93 (d, 1H,

J = 8.5 Hz), 8.20 (d, 1H, J = 8.5 Hz), 8.69 (s, 1H).

4.1.13.4. N-(2-Diethylaminoethyl)-6-iodoacridine-4-carboxamide

(30e). From 29e. The mixture was reﬂuxed in dry dichloromethane

for 4 h. Yield 52%; R_f= 0.37 (Al₂O₃, CH₂Cl₂/EtOH, 99:1, v/v); mp 80–

82 °C; IR (KBr) 3500–3300, 2924, 1654, 1559, 1458 cm^ꢀ1; MS m/z

447 (M⁺, 3), 177 (9), 86 (100), 58 (15), ¹H NMR (CDCl₃, 200 MHz) d

1.16 (t, 6H, J = 7 Hz), 2.79 (m, 6H), 3.77 (q, 2H, J = 6 Hz), 7.65 (dd,

1H, J = 8.5, 7 Hz), 7.70 (d, 1H, J = 9 Hz), 7.78 (d, 1H, J = 9 Hz), 8.06

(d, 1H, J = 8.5 Hz), 8.78 (s, 1H), 8.81 (s, 1 H), 8.97 (d, 1H, J = 7 Hz),

11.90 (br s, 1H); ¹³C NMR (CDCl₃, 50 MHz) d 12.0 (2C), 38.0, 47.0

(2C), 51.9, 98.1, 124.6, 126.0, 127.0, 128.9, 129.0, 132.1, 134.8,

135.8, 137.6, 138.4, 146.6, 147.7, 165.5.

4.1.12.4. Methyl 5-iodoacridine-4-carboxylate (29d). From 28d.

Yield 97%; ¹H NMR (CDCl₃, 200 MHz) d 4.20 (s, 3H), 7.17 (dd, 1H,

J = 8.5, 7 Hz), 7.51 (dd, 1H, J = 8.5, 7 Hz), 7.87 (d, 1H, J = 8.5 Hz),

8.03 (d, 1H, J = 8.5 Hz), 8.11 (d, 1H, J = 7 Hz), 8.39 (d, 1H, J = 7 Hz),

8.60 (s, 1H).

4.1.12.5. Methyl 6-iodoacridine-4-carboxylate (29e). From 28e.

Yield 88%; ¹H NMR (CDCl₃, 200 MHz) d 4.12 (s, 3H), 7.54 (dd, 1H,

J = 8.5, 7 Hz), 7.67 (m, 2H), 8.06 (d, 1H, J = 8.5 Hz), 8.12 (d, 1H,

J = 7 Hz), 8.70 (s, 1H), 8.77 (s, 1H).

4.1.13.5. N-(2-Diethylaminoethyl)-7-iodoacridine-4-carboxamide

(30f). From 29f. The mixture was reﬂuxed in dry dichloromethane

for 6 h. Yield 53%; R_f= 0.33 (Al₂O₃, CH₂Cl₂/EtOH, 99:1, v/v); mp

176–178 °C; IR (KBr) 3500–3300, 2965, 1665, 1562, 1515, 1457 cm

^ꢀ1; MS m/z 447 (M⁺, 2), 332 (10), 305 (12), 177 (8), 86 (100), 58

(8); ¹H NMR (CDCl₃, 200 MHz) d 1.26 (t, 6H, J = 7 Hz), 2.98 (m, 6H),

3.98 (m, 2 H), 7.70 (dd, 1H, J = 8.5, 7 Hz), 8.06 (d, 1H, J = 9 Hz), 8.16

(m, 2H), 8.47 (s, 1H), 8.78 (s, 1H), 8.98 (d, 1H, J = 7 Hz), 12.01 (br s,

1H); ¹³C NMR (CDCl₃, 100 MHz) d 10.8 (2C), 37.2, 47.2 (2C), 51.6,

92.2, 126.1, 127.0, 127.5, 128.3, 130.9, 132.6, 135.8, 136.4, 136.8,

139.8, 146.3, 146.5, 166.1.

4.1.12.6. Methyl 7-iodoacridine-4-carboxylate (29f). From 28f.

Yield 95%; ¹H NMR (CDCl₃, 200 MHz) d 4.13 (s, 3H), 7.48 (dd, 1H,

J = 8.5, 7 Hz), 7.98 (m, 3H), 8.09 (d, 1H, J = 7 Hz), 8.29 (s, 1H), 8.53

(s, 1H).

4.1.12.7. Methyl 8-iodoacridine-4-carboxylate (29g). From 26 g.

Yield 90%; ¹H NMR (CDCl₃, 200 MHz) d 4.11 (s, 3H), 7.51 (dd, 1H,

J = 8.5, 7 Hz), 7.62 (dd, 1H, J = 8.5, 7 Hz), 8.22 (m, 3H), 8.32 (d,

1 H, J = 8.5 Hz), 9.02 (s, 1H).

4.1.13. N-(2-diethylaminoethyl)-iodoacridine-4-carboxamides

30a,b,d–g

Were prepared as described for the synthesis of carboxamides

24a–g.

4.1.13.6. N-(2-Diethylaminoethyl)-8-iodoacridine-4-carboxamide

(30g). From 29g. The mixture was reﬂuxed in dry dichloromethane for

16 h. Yield 55%; R_f= 0.35 (Al₂O₃, CH₂Cl₂/EtOH, 99:1, v/v); mp 78–80 °C;

IR (KBr) 3200, 2925, 1654, 1546, 1508 cm^ꢀ1; MS m/z 447 (M⁺, 1), 177

(11), 86 (100), 58 (11); ¹H NMR (CDCl₃, 400 MHz) d 1.16 (t, 6H,

J = 7 Hz), 2.80 (q, 4H, J = 7 Hz), 2.91 (m, 2H), 3.85 (m, 2H), 7.50 (t, 1H,

J = 8 Hz), 7.69 (t, 1H, J = 7.5 Hz), 8.16 (m, 2H), 8.27 (d, 1H, J = 8.5 Hz),

8.96 (s, 1H), 8.98 (d, 1H, J = 7 Hz), 11.76 (br s, 1H); ¹³C NMR (CDCl₃,

100 MHz) d 11.4 (2C), 37.7, 47.1 (2C), 51.8, 98.4, 126.1, 127.5, 127.7,

128.2, 130.2, 131.6, 132.4, 135.9, 137.4, 142.4, 146.7, 147.4, 165.6.

4.1.13.1. N-(2-Diethylaminoethyl)-1-iodoacridine-4-carboxamide

(30a). From 29a. The mixture was reﬂuxed in dry dichlorometh-

ane for 4 h. Yield 80%; R_f= 0.40 (Al₂O₃, CH₂Cl₂/EtOH, 99:1, v/v);

mp 103–105 °C; IR (KBr) 3500–3300, 3186, 2967, 1654, 1649,

1517 cm^ꢀ1; MS m/z 447 (M⁺, 8), 332 (18), 177 (17), 86 (100), 58

(15); ¹H NMR (CDCl₃, 400 MHz) d 1.15 (t, 6H, J = 7 Hz), 2.78 (q,

4H, J = 7 Hz), 2.88 (t, 2H, J = 7 Hz), 3.83 (m, 2H), 7.63 (t, 1H,

J = 7 Hz), 7.85 (t, 1H, J = 8 Hz), 8.07 (d, 1H, J 8 8.5 Hz), 8.24 (d, 1H,

J = 8 Hz), 8.29 (d, 1H, J = 8.5 Hz), 8.60 (d, 1H, J = 7.5 Hz), 9.04 (s,

1H), 11.82 (br s, 1 H); ¹³C NMR (CDCl₃, 100 MHz) d 11.6 (2C),

38.0, 47.1 (2C), 51.9, 103.8, 126.9, 127.0, 128.0, 128.3, 128.7,

129.4, 131.9, 135.4, 137.1, 142.6, 145.8, 148.1, 165.4.

4.1.14. 3-Iodoacridine-4-carboxylic acid (31)

The ester 29c (0.15 g, 0.36 mmol) was suspended in a mixture

of methanol (30 mL) and an aqueous 4 N sodium hydroxide solu-

tion (30 mL). The solution was heated under reﬂux for 12 h to give

a clear yellow solution which was ﬁltered and acidiﬁed to pH 5

with glacial acetic acid. Concentration and cooling to 0 °C gave a

yellow precipitate which was collected by ﬁltration and washed

with water (2 mL). The precipitate was taken in dry ethanol

(50 mL) and dried under vacuum to give 3-iodoacridine-4-carbox-

ylic acid (31). Yield 44%; mp 139–141 °C; IR (KBr) 3500–3200,

1577, 1421 cm^ꢀ1; ESI-MS m/z 349.4 [M+H]⁺; ¹H NMR (DMSO-d₆,

400 MHz) d 7.61 (t, 1H, J = 7 Hz), 7.68 (d, 1H, J = 8.5 Hz), 7.82 (m,

2H), 8.07 (d, 1H, J = 8 Hz), 8.12 (d, 1H, J = 8.5 Hz), 9.02 (s, 1H); ¹³C

NMR (DMSO-d₆, 50 MHz) d 93.1, 125.1, 125.9 (2C), 126.3, 128.3,

129.0, 130.5, 135.2, 136.0, 145.4, 147.9, 149.6, 177.3.

4.1.13.2. N-(2-diethylaminoethyl)-2-iodoacridine-4-carboxamide

(30b). From 29b. The mixture was reﬂuxed in dry dichlorometh-

ane for 4 h. Yield 80%; R_f= 0.41 (Al₂O₃, CH₂Cl₂/EtOH, 99:1, v/v);

mp 108–110 °C; IR (KBr) 3182, 2965, 1637, 1516 cm^ꢀ1; MS m/z

447 (M⁺, 1), 177 (8), 86 (100), 58 (13); ¹H NMR (CDCl₃, 400 MHz)

d 1.19 (t, 6H, J = 7 Hz), 2.85 (m, 4H), 2.96 (m, 2H), 3.88 (m, 2H),

7.61 (t, 1H, J = 7 Hz), 7.87 (t, 1 H, J = 8 Hz), 8.01 (d, 1H, J = 8.5 Hz),

8.29 (d, 1H, J = 8.5 Hz), 8.51 (s, 1H), 8.71 (s, 1H), 9.10 (s, 1H),

11.88 (s, 1H); ¹³C NMR (CDCl₃, 100 MHz) d 11.2 (2C), 37.7, 47.2

(2C), 51.7, 91.0, 126.2, 127.1, 128.2, 128.4, 129.4, 129.6, 131.8,

136.3, 140.7, 143.4, 145.0, 147.9, 164.8.

4.1.15. N-(2-Diethylaminoethyl)-3-chloroacridine-4-carboxamide

(32)

4.1.13.3. N-(2-Diethylaminoethyl)-5-iodoacridine-4-carboxamide

(30d). From 29d. The mixture was reﬂuxed in dry toluene for 3 h.

Yield 73%; R_f= 0.17 (Al₂O₃, CH₂Cl₂/EtOH, 99:1, v/v); mp 64–66 °C;

IR (KBr) 3500–3300, 3200, 2925, 1654, 1648 cm^ꢀ1; MS m/z 447

(M⁺, 1), 177 (9), 86 (100), 58 (12); ¹H NMR (CDCl₃, 200 MHz) d

1.11 (t, 6H, J = 7 Hz), 2.69 (q, 4H, J = 7 Hz), 2.92 (m, 2H), 3.82 (m,

2H), 7.19 (dd, 1H, J = 8.5, 7 Hz), 7.56 (dd, 1H, J = 8.5, 7 Hz), 7.86

(d, 1H, J = 8.5 Hz), 7.98 (d, 1H, J = 8.5 Hz), 8.32 (d, 1H, J = 7 Hz),

8.60 (s, 1H), 8.88 (d, 1H, J = 7 Hz), 11.76 (s, 1H); ¹³C NMR (CDCl₃,

100 MHz) d 11.8 (2C), 38.4, 47.5 (2C), 52.4, 102.6, 125.8, 126.1,

127.0, 127.1, 128.2, 129.5, 132.9, 136.0, 138.6, 141.6, 145.8,

146.3, 165.4.

A suspension of 3-iodoacridine-4-carboxylic acid (31) (40 mg,

0.11 mmol) in thionyl chloride (5 mL) containing dimethylform-

amide (1 drop) was heated gently under reﬂux with stirring until

homogeneous and then for a further 45 min. The solution was

evaporated to dryness under vacuum below 40 °C, and residual

traces of thionyl chloride were removed by addition of dry toluene

(5 mL) and complete reevaporation of all solvents. The above prod-

uct in dry dichloromethane solution (2 mL) was cooled to ꢀ5 °C

and was added an ice-cold solution of N,N-diethylethylenediamine

(20 lL, 0.14 mmol) in dry dichloromethane (1 mL). The solution

was stirred at room temperature for 2 h. The organic layers were

washed twice with a 10% aqueous sodium carbonate solution

N. Desbois et al. / Bioorg. Med. Chem. 16 (2008) 7671–7690

7685

(20 mL) and once with saturated aqueous sodium chloride solution

(20 mL). The organic phase was dried (MgSO₄), ﬁltered and evapo-

rated to dryness. The product 32 obtained was chromatographed

(Al₂O₃, EtOAc/cyclohexane, 8:2, v/v). Yield 79%; R_f= 0.40 (Al₂O₃,

EtOAc/cyclohexane, 8:2, v/v); mp 229–231 °C; IR (KBr) 3500–

3300, 2924, 1654 cm^ꢀ1; MS m/z 357 (M⁺+2, 3), 355 (M⁺, 1), 240

(11), 177 (15), 86 (100), 58 (12); ¹H NMR (CDCl₃, 400 MHz) d

1.03 (t, 6H, J = 6.5 Hz), 2.68 (m, 4H), 2.89 (m, 2H), 3.80 (m, 2H),

7.05 (br s, 1H), 7.42 (d, 1H, J = 8.5 Hz), 7.52 (t, 1H, J = 7 Hz), 7.75

(t, 1H, J = 7 Hz), 7.88 (d, 1H, J = 9 Hz), 7.93 (d, 1H, J = 8 Hz), 8.16

(d, 1H, J = 9 Hz), 8.68 (s, 1H); ¹³C NMR (CDCl₃, 50 MHz) d 11.3

(2C), 37.3, 47.0 (2C), 51.8, 124.7, 126.4, 126.5, 127.3, 128.2,

130.0, 130.1, 130.9, 133.3, 134.8, 136.1, 146.5, 149.6, 166.6.

1H, J = 8.5 Hz), 8.52 (d, 1H, J = 8.5 Hz), 8.79 (s, 1H), 8.90 (s, 1H),

9.27 (s, 1H), 10.49 (m, 1H), 11.33 (br s, 1H); ¹³C NMR (DMSO-d₆,

100 MHz) d 8.5 (2C), 34.5, 46.9 (2C), 49.6, 91.2, 125.8, 127.4,

128.0, 128.6, 128.7, 129.1, 132.4, 138.0, 141.0, 142.0, 143.5,

146.9, 164.5. Anal. Calcd for C₂₀H₂₂IN₃O, 2HCl, 1.5H₂O: C, 43.90;

H, 4.97; N, 7.68. Found: C, 43.77; H, 4.77; N, 7.54.

4.1.18.3. N-(2-Dethylaminoethyl)-3-iodoacridine-4-carboxamide

dihydrochloride salt (9c). From 30c. Yield 83%; mp 134–136 °C;

IR (KBr) 3500–3200, 2924, 1654, 1636, 1560, 1458 cm^ꢀ^{1 1}H

;

NMR (CD₃OD, 200 MHz) d 1.47 (t, 6 H, J = 7 Hz), 3.49 (q, 4H,

J = 7 Hz), 3.62 (t, 2H, J = 7 Hz), 3.97 (t, 2H, J = 7 Hz), 7.79 (m, 1H),

8.06 (m, 3H), 8.35 (m, 2H), 9.40 (s, 1H); ¹³C NMR (DMSO-d₆,

100 MHz) d 8.8 (2C), 34.1, 46.9 (2C), 49.4, 97.9, 124.9, 126.2,

126.6, 128.7, 128.9, 130.0, 131.6, 135.0, 137.0, 142.3, 145.6,

148.3, 169.0. Anal. Calcd for C₂₀H₂₂IN₃O, 2HCl, H₂O: C, 44.63; H,

4.87; N, 7.81. Found: C, 44.58; H, 4.92; N, 7.42.

4.1.16. N-(2-Diethylaminoethyl)-3-iodoacridan-4-carboxamide

(33)

Compound 33 was prepared from compound 24c as described

for the synthesis of methyl iodoacridan-4-carboxylates 28. The

product was chromatographed (Al₂O₃, CH₂Cl₂/EtOH, 99.5:0.5,

v/v). Yield 60%; R_f= 0.27 (Al₂O₃, CH₂Cl₂/EtOH, 99.5:0.5, v/v); mp

106–108 °C; IR (KBr) 3268, 2346, 1636, 1449, 1164 cm^ꢀ1; MS m/z

449 (M⁺, 1), 86 (100), 58 (10); ¹H NMR (CDCl₃, 200 MHz) d 1.26

(t, 6H, J = 7 Hz), 2.99 (m, 6H), 3.91 (q, 2 H, J = 6.5 Hz), 3.98 (s, 2H),

6.48 (m, 1H), 6.71 (d, 1H, J = 8 Hz), 6.79 (t, 1H, J = 8 Hz), 6.89 (m,

1H), 7.09 (m, 2H), 7.26 (m, 2H); ¹³C NMR (CDCl₃, 50 MHz) d 8.5

(2C), 31.1, 35.1, 53.4 (2C), 57.0, 90.1, 114.6, 119.2, 121.3, 121.5,

124.5, 127.3, 128.3, 130.9, 131.3, 138.8, 139.6, 168.8.

4.1.18.4. N-(2-Diethylaminoethyl)-5-iodoacridine-4-carboxamide

dihydrochloride salt (9d). From 30d. Yield 69%; mp 144–146 °C;

IR (KBr) 3500–3300, 2928, 2660, 1654, 1578, 1546 cm^ꢀ^{1 1}H

;

NMR (DMSO-d₆, 400 MHz) d 1.27 (t, 6 H, J = 7 Hz), 3.26 (m, 4H),

3.43 (m, 2H), 4.10 (m, 2H), 7.48 (t, 1H, J = 7.5 Hz), 7.83 (t, 1H,

J = 7.5 Hz), 8.27 (d, 1H, J = 8.5 Hz), 8.46 (d, 1H, J = 8 Hz), 8.60 (d,

1H, J = 7 Hz), 8.84 (d, 1H, J = 7 Hz), 9.36 (s, 1H), 10.72 (br s, 1H),

12.27 (br s, 1H); ¹³C NMR (DMSO-d₆, 100 MHz) d 8.4 (2C), 34.8,

46.4 (2C), 49.9, 104.0, 125.9, 126.0, 126.9, 127.0, 127.8, 129.3,

133.2, 135.7, 140.1, 141.8, 145.6, 145.8, 165.2. Anal. Calcd for

4.1.17. N-(2-Diethylaminoethyl)-3-iodoacridine-4-carboxamide

(30c)

C₂₀H₂₂IN₃O, 2HCl, H₂O: C, 44.63; H, 4.87; N, 7.81. Found: C,

44.80; H, 4.73; N, 7.63.

was prepared from 33 as described for the synthesis of methyl

iodoacridine-4-carboxylates 29. The product was chromato-

graphed (Al₂O₃, EtOAc/pentane, 5:5, v/v). Yield 73%; R_f= 0.13

(Al₂O₃, EtOAc/pentane, 5/5, v/v); mp 81–83 °C; IR (KBr) 3500–

3200, 2925, 1654, 1458 cm^ꢀ1; MS m/z 447 (M⁺, 1), 177 (11), 86

(100), 58 (12); ¹H NMR (CDCl₃, 400 MHz) d 1.04 (t, 6H, J = 7 Hz),

2.69 (m, 4H), 2.91 (m, 2H), 3.81 (m, 2H), 6.95 (br s, 1H), 7.54 (d,

1H, J = 8 Hz), 7.65 (d, 1H, J = 9 Hz), 7.76 (t, 1H, J = 8 Hz), 7.80 (d,

1H, J = 9 Hz), 7.94 (d, 1H, J = 8.5 Hz), 8.17 (d, 1H, J = 9 Hz), 8.68 (s,

1H); ¹³C NMR (CDCl₃, 100 MHz) d 11.3 (2C), 37.2, 46.8 (2C), 51.6,

97.1, 125.3, 126.5, 126.6, 128.0, 129.5, 130.1, 130.7, 135.1, 136.0,

142.5, 146.2, 149.2, 169.2.

4.1.18.5. N-(2-Diethylaminoethyl)-6-iodoacridine-4-carboxamide

dihydrochloride salt (9e). From 30e. Yield 72%; mp 201–203 °C; IR

(KBr) 3500–3300, 2970, 1637, 1609 cm^ꢀ^{1 1}H NMR (DMSO-d₆,

;

400 MHz) d 1.27 (t, 6 H, J = 7 Hz), 3.24 (m, 4 H), 3.38 (m, 2H), 3.97 (q,

2H, J = 6 Hz), 7.79 (dd, 1H, J 8 8.5, 7 Hz), 7.95 (d, 1H, J = 9 Hz), 8.02 (d,

1H, J = 9 Hz), 8.40 (d, 1H, J = 8.5 Hz), 8.77 (d, 1H, J = 7 Hz), 9.11 (s,

1H), 9.37 (s, 1H), 10.47 (br s, 1H), 11.33 (t, 1H, J = 6 Hz); ¹³C NMR

(DMSO-d₆, 100 MHz) d 8.5 (2C), 34.4, 46.9 (2C), 49.8, 100.5, 124.5,

125.6, 126.5, 127.7, 129.8, 133.4, 134.9, 135.5, 137.2, 139.5, 145.3,

147.2, 165.6. Anal. Calcd for C₂₀H₂₂IN₃O, 2HCl, 0.5H₂O: C, 45.39; H,

4.76; N, 7.94. Found: C, 45.23; H, 4.70; N, 7.72.

4.1.18. N-(2-Ddiethylaminoethyl)iodoacridine-4-carboxamides

dihydrochloride salts 9a–g

Compounds 9a–g were prepared as described for the synthesis

of hydrochloride salts 8a–g.

4.1.18.6. N-(2-Diethylaminoethyl)-7-iodoacridine-4-carboxamide

dihydrochloride salt (9f). From 30f. Yield 67%; mp 213–215 °C; IR

(KBr) 3500–3200, 3222, 2953, 2665, 1628, 1585, 1403 cm^ꢀ^{1 1}H

;

NMR (DMSO-d₆, 400 MHz) d 1.27 (t, 6H, J = 7 Hz), 3.25 (m, 4H),

3.41 (m, 2H), 3.92 (m, 2H), 7.79 (t, 1H, J = 7.5 Hz), 8.19 (d, 1H,

J = 9 Hz), 8.36 (d, 1H, J = 9 Hz), 8.41 (d, 1H, J = 8.5 Hz), 8.74 (m,

2H), 9.28 (s, 1H), 10.57 (br s, 1H), 11.32 (br s, 1H); ¹³C NMR

(DMSO-d₆, 100 MHz) d 8.5 (2C), 34.4, 46.9 (2C), 49.7, 93.3, 125.8,

126.5, 127.2, 128.0, 130.6, 133.3, 135.1, 136.7, 137.8, 139.8,

145.3, 145.7, 165.6. Anal. Calcd for C₂₀H₂₂IN₃O, 2HCl, 2.5H₂O: C,

42.50; H, 5.17; N, 7.43. Found: C, 42.78; H, 4.96; N, 7.35.

4.1.18.1. N-(2-Diethylaminoethyl)-1-iodoacridine-4-carboxamide

dihydrochloride salt (9a). From 30a. Yield 71%; mp 219–221 °C;

IR (KBr) 3500–3200, 2975, 2638, 2472, 1624, 1577, 1549,

1394 cm^ꢀ^{1 1}H NMR (DMSO-d₆, 400 MHz) d 1.27 (t, 6 H, J = 7 Hz),

;

3.27 (m, 4 H), 3.43 (m, 2 H), 4.00 (m, 2 H), 7.80 (t, 1 H, J = 7.5 Hz),

8.06 (d, 1H, J = 7.5 Hz), 8.40 (d, 1H, J = 7.5 Hz), 8.46 (m, 2H), 8.58

(d, 1 H, J = 9 Hz), 9.34 (s, 1H), 10.25 (s, 1H), 11.30 (s, 1H); ¹³C

NMR (DMSO-d₆, 100 MHz) d 8.5 (2C), 34.5, 46.9 (2C), 54.9, 105.4,

126.6, 127.4, 127.5, 128.3, 128.8, 132.7, 134.8, 136.8, 142.9,

144.7, 147.4, 165.4, one quaternary carbon not observed. Anal.

Calcd for C₂₀H₂₂IN₃O, 2HCl: C, 46.18; H, 4.65; N, 8.08. Found: C,

46.38; H, 4.79; N, 7.99.

4.1.18.7. N-(2-Diethylaminoethyl)-8-iodoacridine-4-carboxamide

dihydrochloride salt (9g). From 30g. Yield 72%; mp 128–130 °C;

IR (KBr) 3500–3200, 1653, 1559, 1540, 1507 cm^ꢀ1

;

¹H NMR

(DMSO-d₆, 400 MHz) d 1.28 (t, 6 H, J = 7 Hz), 3.25 (m, 4 H), 3.41

(m, 2H), 3.99 (m, 2H), 7.71 (t, 1H, J = 7.5 Hz), 7.82 (t, 1H, J = 7 Hz),

8.33 (d, 1H, J = 7 Hz), 8.60 (m, 2H), 8.76 (d, 1H, J = 7 Hz), 9.25 (s,

1H), 10.73 (m, 1H), 11.26 (br s, 1H); ¹³C NMR (DMSO-d₆,

100 MHz) d 8.5 (2C), 34.4, 46.9 (2C), 49.7, 99.2, 125.9, 127.1,

127.3, 127.6, 130.0, 132.6, 133.4, 135.3, 137.8, 142.7, 145.6,

147.1, 165.5. Anal. Calcd for C₂₀H₂₂IN₃O, 2HCl, 1.5H₂O: C, 43.90;

H, 4.97; N, 7.68. Found: C, 43.87; H, 4.77; N, 7.38.

4.1.18.2. N-(2-Diethylaminoethyl)-2-iodoacridine-4-carboxamide

dihydrochloride salt (9b). From 30b. Yield 76%; mp 215–217 °C;

IR (KBr) 3500–3200, 2975, 2651, 1649, 1625, 1572 cm^ꢀ^{1 1}H NMR

;

(DMSO-d₆, 200 MHz) d 1.26 (t, 6H, J = 7 Hz), 3.24 (m, 4H), 3.41

(m, 2H), 3.83 (m, 2H), 7.73 (m, 1H), 8.01 (t, 1H, J = 8 Hz), 8.23 (d,

7686

N. Desbois et al. / Bioorg. Med. Chem. 16 (2008) 7671–7690

4.1.19. 9,10-Dihydro-2-iodo-9-oxoacridine-4-carboxylic acid

(23e)

drous 2 N hydrochloric acid–ether solution (2 mL). The solution

was concentrated under vacuum started again with dry ether

(3 mL). The suspension was stirred overnight and ﬁltered.

Compound 23e was prepared as described for the synthesis of

3-iodoacridine-4-carboxylic acid (31) with an aqueous 2 N sodium

hydroxide solution and heated under reﬂux for 10 min. Yield 90%;

mp > 400 °C; IR (KBr) 3415–3000, 1615, 1514, 1165 cm^ꢀ1; MS m/z

365 (M⁺, 5), 164 (10), 84 (27), 69 (64), 55 (100); ¹H NMR (DMSO-d₆,

400 MHz) d 7.31 (t, 1H, J = 7.5 Hz), 7.64 (d, 1H, J = 7.5 Hz), 7.77 (t,

1H, J = 7.5 Hz), 8.25 (d, 1H, J = 7.5 Hz), 8.59 (m, 2H), 14.45 (br s,

1 H); ¹³C NMR (DMSO-d₆, 100 MHz) d 83.3, 118.4, 120.4, 121.5,

123.1, 124.8, 126.0, 133.9, 136.5, 139.9, 140.7, 143.2, 167.8, 175.5.

4.1.21.1. N-(2-Diethylaminoethyl)-2-iodo-9-(4⁰-methanesulfo-

namido-2⁰-methoxyanilino)acridine-4-carboxamide dihydro-

chloride salt (10a). From 35a. Yield 28%; mp 235–237 °C; IR (KBr)

3500–3200, 2933, 1618, 1510, 1323, 1150 cm^ꢀ1; MS (base) m/z

661 (M⁺, 1), 582 (11), 509 (11), 86 (100), 58 (21); ¹H NMR

(DMSO-d₆, 400 MHz) d 1.29 (t, 6H, J = 7 Hz), 3.14 (s, 3H), 3.24 (m,

4H), 3.37 (m, 2H), 3.48 (s, 3H), 3.80 (m, 2H), 7.04 (d, 1H,

J = 8.5 Hz), 7.09 (s, 1H), 7.46 (t, 1H, J = 7.5 Hz), 7.52 (d, 1H,

J = 8 Hz), 8.00 (t, 1H, J = 7.5 Hz), 8.16 (d, 1H, J = 8.5 Hz), 8.20 (br s,

1H), 8.85 (s, 1H), 8.89 (br s, 1H), 9.80 (br s, 1H), 10.20 (s, 1H),

10.75 (br s, 1H), 11.75 (m, 1H), 13.83 (br s, 1H); ¹³C NMR

(DMSO-d₆, 50 MHz) d 8.5 (2C), 34.7, 39.5, 46.6 (2C), 49.8, 56.0,

87.0, 104.1, 112.0, 113.7, 116.0, 120.4, 121.8, 123.8, 124.8, 125.2,

127.7, 136.2, 137.3, 137.6, 139.1, 140.4, 142.7, 153.6, 155.3,

166.3. Anal. Calcd for C₂₈H₃₂IN₅O₄S, 2HCl, 2.5H₂O: C, 43.14; H,

5.04; N, 8.98. Found: C, 43.06; H, 4.74; N, 8.90.

4.1.20. General procedure for the synthesis of N-(2-

diethylaminoethyl)-9-chloro-iodoacridine-4-carboxamides

35a–c

A suspension of 9,10-dihydro-iodo-9-oxoacridine-4-carboxylic

acid 23 (100 mg, 0.27 mmol) in thionyl chloride (5 mL) containing

dimethylformamide (1 drop) was heated gently under reﬂux with

stirring until homogeneous and then for a further 45 min. The solu-

tion was evaporated to dryness under vacuum below 40 °C, and

residual traces of thionyl chloride were removed by addition of

dry toluene (5 mL) and complete reevaporation of all solvents to

give 3-chloroacridine-4-carbonyl chloride 34a–c. The above prod-

uct in a 5% NEt₃/THF solution (2 mL) was cooled to ꢀ5 °C and to

this was added in one portion an ice-cold solution of N,N-diethyl-

4.1.21.2. N-(2-Diethylaminoethyl)-5-iodo-9-(4⁰-methanesulfo-

namido-2⁰-methoxyanilino)acridine-4-carboxamide dihydro-

chloride salt (10b). From 35b. Yield 38%; mp 212–214 °C; IR

(KBr) 3500–3200, 3100–2900, 1613, 1585, 1513, 1325, 1151 cm

^ꢀ1; MS (base) m/z 661 (M⁺, 1), 582 (9), 509 (15), 446 (13), 383

(12), 86 (100); ¹H NMR (DMSO-d₆, 400 MHz) d 1.28 (t, 6H,

J = 7 Hz), 3.13 (s, 3H), 3.25 (q, 4H, J = 7 Hz), 3.40 (m, 2H), 3.48 (s,

3H), 3.85 (m, 2H), 7.02 (m, 2H), 7.21 (t, 1H, J = 8 Hz), 7.55 (m,

2H), 8.39 (m, 1H), 8.54 (m, 2H), 8.89 (d, 1H, J = 7 Hz), 10.06 (s,

1H), 10.25 (br s, 1H), 10.81 (m, 1H), 12.00 (br s, 1H), 14.70 (br s,

1H); ¹³C NMR (DMSO-d₆, 100 MHz) d 8.3 (2C), 34.4, 39.5, 46.4

(2C), 49.5, 55.7, 89.5, 103.7, 111.6, 113.8, 114.2, 117.8, 122.7,

123.5, 125.2, 125.4, 127.5, 129.6, 135.9, 138.9, 139.3, 140.0,

146.0, 153.2, 156.9, 167.6. Anal. Calcd for C₂₈H₃₂IN₅O₄S, 2HCl,

3H₂O: C, 42.65; H, 5.11; N, 8.88. Found: C, 42.72; H, 4.81; N, 8.68.

ethylenediamine (40 lL, 0.28 mmol) in dry dichloromethane

(1 mL). The solution was stirred at room temperature for 2 h. The

organic layers were washed twice with an aqueous 10% sodium

carbonate solution (20 mL), once with a saturated aqueous sodium

chloride solution (20 mL) and evaporated under vacuum. The

unstable precipitate obtained was used without puriﬁcation.

4.1.20.1. N-(2-Diethylaminoethyl)-9-chloro-2-iodoacridine-4-

carboxamide (35a). From 23e. Yield 80%; ¹H NMR (CDCl₃,

200 MHz) d 1.23 (t, 6H, J = 7 Hz), 2.95 (m, 6H), 3.93 (m, 2H), 7.70

(t, 1H, J = 7.5 Hz), 7.91 (t, 1H, J = 7.5 Hz), 8.38 (m, 2H), 8.98 (d,

1H, J = 2 Hz), 9.12 (d, 1H, J = 2 Hz), 11.77 (br s, 1H).

4.1.21.3. N-(2-Diethylaminoethyl)-7-iodo-9-(4’-methanesulfo-

namido-2⁰-methoxyanilino)acridine-4-carboxamide dihydro-

chloride salt (10c). From 35c. Yield 33%; mp 208–210 °C; IR

(KBr) 3500–3300, 2926, 1618, 1586, 1511, 1324, 1150 cm^ꢀ1; MS

(base) m/z 661 (M⁺, 7), 582 (47), 509 (62), 466 (29), 439 (21),

383 (17), 86 (100), 58 (13); ¹H NMR (DMSO-d₆, 400 MHz) d 1.28

(t, 6H, J = 7 Hz), 3.13 (s, 3H), 3.24 (m, 4H), 3.37 (m, 2H), 3.49 (s,

3H), 3.80 (m, 2H), 7.06 (d, 1H, J = 8.5 Hz), 7.09 (s, 1H), 7.54 (m,

2H), 7.97 (d, 1H, J = 9 Hz), 8.19 (d, 1H, J = 9 Hz), 8.58 (m, 2H),

8.70 (d, 1H, J = 7.5 Hz), 9.95 (t, 1H, J = 6 Hz), 10.26 (s, 1H), 10.85

(m, 1H), 11.80 (m, 1H), 14.04 (br s, 1H); ¹³C NMR (DMSO-d₆,

100 MHz) d 8.3 (2C), 34.3, 39.5, 46.4 (2C), 49.5, 55.7, 89.2, 103.7,

111.7, 114.2, 114.8, 119.9, 121.9, 122.7, 123.4, 127.5, 129.1,

133.3, 135.7, 137.9, 138.2, 140.2, 143.2, 153.3, 155.0, 167.2. Anal.

Calcd for C₂₈H₃₂IN₅O₄S, 2HCl, 2H₂O: C, 43.65; H, 4.97; N, 9.09.

Found: C, 43.37; H, 4.64; N, 8.93.

4.1.20.2. N-(2-Diethylaminoethyl)-9-chloro-5-iodoacridine-4-

carboxamide (35b). From 23a Yield 93%; ¹H NMR (CDCl₃,

200 MHz) d 1.38 (t, 6H, J = 7 Hz), 3.17 (q, 4H, J = 7 Hz), 3.37 (m,

2H), 4.22 (m, 2H), 7.41 (dd, 1H, J = 9, 7 Hz), 7.78 (dd, 1H, J = 8.5,

7 Hz), 8.44 (d, 1H, J = 8.5 Hz), 8.51 (d, 1H, J = 7 Hz), 8.60 (d, 1H,

J = 8.5 Hz), 8.99 (d, 1H, J = 7 Hz), 12.40 (m, 1H).

4.1.20.3. N-(2-Diethylaminoethyl)-9-chloro-7-iodoacridine-4-

carboxamide (35c). From 23c. Yield 96%; ¹H NMR (CDCl₃,

200 MHz) d 1.12 (t, 6H, J 8 7 Hz), 2.75 (m, 6 H), 3.77 (q, 2H,

J = 6 Hz), 7.72 (dd, 1H, J = 8.5, 7 Hz), 8.00 (m, 2H), 8.49 (d, 1H,

J = 8.5 Hz), 8.74 (s, 1H), 8.98 (d, 1H, J = 7 Hz), 11.60 (m, 1H).

4.1.21. General procedure for the synthesis of N-(2-diethyla-

minoethyl)-iodo-9-(4⁰-methanesulfonamido-2⁰-methoxy-

anilino)acridine-4-carboxamides dihydrochloride salt 10a–c

To a solution of the appropriate 9-chloroacridine 35 (0.22 g,

0.46 mmol) in dry ethanol (10 mL), under argon atmosphere, was

added 4-methanesulfonamido-2-methoxyaniline⁴³(36) (97 mg,

4.1.22. Ethyl N-(4-amino-5-iodo-2-methoxybenzene)carbamate

(40)

To

a solution of N-(2-methoxy-4-aminobenzene)carbamate

(39)⁴⁴(0.40 g, 1.90 mmol) in a mixture of methanol (15 mL) and

dichloromethane (40 mL) was added benzyltriethylammonium

dichloroiodate (BTEAICl₂)⁴⁹(0.81 g, 2.09 mmol) and calcium car-

0.45 mmol) and concentrated hydrochloric acid (40 lL). The mix-

ture was reﬂuxed for 17 h and allowed to room temperature. An

aqueous saturated sodium carbonate solution (20 mL) was added

and the mixture was extracted with dichloromethane (3ꢁ

50 mL). The combinated organic layers were dried (MgSO₄), ﬁl-

tered and evaporated under vacuum. The product was chromato-

graphed (Al₂O₃, CH₂Cl₂/EtOH, 99:1, v/v). The red precipitate was

diluted with dichloromethane (0.5 mL) and treated with an anhy-

bonate (0.32 mg, 3.08 mmol). The solution was stirred at room

3

temperature for

h. After return back to room temperature, the

4

mixture was ﬁltered on Celite^Ò521 and concentrated to 1/3. An

aqueous 5% sodium bicarbonate solution (10 mL) was added and

the layers were separated. The organic phase was washed with

an aqueous saturated sodium bicarbonate solution (10 mL), water

N. Desbois et al. / Bioorg. Med. Chem. 16 (2008) 7671–7690

7687

(10 mL) and an aqueous saturated sodium chloride solution

(10 mL). The organic layers were dried (MgSO₄), ﬁltered and evap-

orated under vacuum. The product was chromatographed (Al₂O₃,

EtOAc/pentane, 7:3, v/v). Yield 16%; R_f= 0.79 (Al₂O₃, EtOAc/pen-

tane, 7:3, v/v); viscous oil; IR (CCl₄) 3500–3200, 1529, 1221

cm^ꢀ1; MS m/z 336 (M⁺, 100), 290 (16), 264 (18), 249 (18), 108

(32), 94 (20), 52 (20); ¹H NMR (CDCl₃, 200 MHz) d 1.29 (t, 3H,

J = 7 Hz), 3.78 (s, 3H), 4.01 (s, 2H), 4.19 (q, 2H, J = 7 Hz), 6.31 (s,

1H), 6.84 (br s, 1H), 8.25 (br s, 1H); ¹³C NMR (CDCl₃, 100 MHz) d

14.7, 55.8, 61.2, 72.8, 97.8, 120.5, 128.4, 142.8, 149.8, 153.7.

water (320 lL) and glacial acetic acid (1 mL), were added periodic

acid dihydrate (76 mg, 0.33 mmol) and iodine (216 mg,

0.85 mmol). The mixture was reﬂuxed for 8 h. After cooling to

room temperature, the solution was made alkaline with an aque-

ous saturated sodium carbonate solution and extracted with

dichloromethane (3ꢁ 50 mL). The organic phases were dried

(MgSO₄), ﬁltered and the solvent removed under reduced pressure.

The crude product was chromatographed (SiO₂, CH₂Cl₂) to give in

order of elution. Methyl 2,7-diiodoacridine-9-carboxylate (43b);

yield 6%; R_f= 0.17 (SiO₂and CH₂Cl₂); mp 201–203 °C; IR (KBr)

1720, 1220 cm^ꢀ1; MS m/z 489 (M⁺, 100), 458 (29), 430 (18), 347

(10), 303 (17), 164 (14); ¹H NMR (CDCl₃, 400 MHz) d 4.24 (s, 3H),

8.04 (m 4H), 8.42 (d, 2H, J = 2 Hz); ¹³C NMR (CDCl₃, 100 MHz) d

53.5, 94.7 (2C), 123.9 (2C), 130.8 (2C), 134.1 (2C), 139.8, 146.8

(2C), 166.7. Methyl 2-iodoacridine-9-carboxylate (43a); yield 16%;

R_f= 0.11 (SiO₂and CH₂Cl₂); mp 106–108 °C; IR (KBr) 1728,

1218 cm^ꢀ1; MS m/z 363 (M⁺, 100), 332 (33), 304 (28), 177 (33);

¹H NMR (CDCl₃, 400 MHz) d 4.20 (s, 3H), 7.58 (m, 1H), 7.79 (m,

1H), 7.95 (m, 2H), 7.98 (d, 1H, J = 8 Hz), 8.20 (d, 1H, J = 8.5 Hz),

8.39 (s, 1H); ¹³C NMR (CDCl₃, 100 MHz) d 53.2, 93.6, 122.5,

123.7, 125.3, 127.8, 130.0, 130.7, 131.3, 134.0, 135.4, 138.9,

147.1, 148.8, 167.4.

4.1.23. Ethyl N-(5-iodo-4-methanesulfonamido-2-

methoxybenzene)carbamate (41)

To a solution of amine 40 (0.20 g, 1.90 mmol) in dry pyridine

(5 mL), was added over 30 min, at ꢀ15 °C, under argon gas, meth-

ane sulfonyl chloride (0.16 mL, 2.09 mmol) keeping the tempera-

ture below ꢀ5 °C. The mixture was stored at 4 °C overnight. The

solution was concentrated to 2/3. The excess of pyridine was neu-

tralised by concentrated hydrochloric acid. The aqueous solution

was extracted with dichloromethane (3ꢁ 30 mL). The combinated

organic layers were dried (MgSO₄), ﬁltered and evaporated under

vacuum. The crude product was chromatographed (Al₂O₃, CH₂Cl₂).

Yield 55%; R_f= 0.08 (Al₂O₃and CH₂Cl₂); mp 153–155 °C; IR (KBr)

3353, 3195, 1708, 1526, 1337, 1150 cm^ꢀ1; MS m/z 414 (M⁺, 40),

335 (100), 291 (13), 263 (40), 136 (12); ¹H NMR (CDCl₃,

400 MHz) d 1.31 (t, 3H, J = 7 Hz), 2.94 (s, 3H), 3.87 (s, 3H), 4.21

(q, 2H, J = 7 Hz), 6.45 (s, 1H), 7.14 (s, 1H), 7.19 (s, 1H), 8.53 (br s,

1H); ¹³C NMR (CDCl₃, 100 MHz) d 14.5, 39.8, 56.1, 61.6, 82.5,

106.8, 126.9, 127.5, 132.2, 148.6, 153.3.

4.1.27. N-(2-Diethylaminoethyl)iodoacridine-9-carboxamides

44a,b

Compound 44a,b were prepared as described for the synthesis

of carboxamides 24a–g.

4.1.27.1. N-(2-Diethylaminoethyl)-2-iodoacridine-9-carboxamide

(44a). From 43a. Yield 58%; R_f= 0,61 (Al₂O₃, EtOAc/EtOH, 99:1,

v/v); mp 83–85 °C; IR (KBr) 3400–3200, 2956, 1636 cm^ꢀ1; MS

m/z 447 (M⁺, 1), 177 (3), 86 (100), 58 (9); ¹H NMR (CDCl₃,

400 MHz) d 1.00 (t, 6H, J = 7 Hz), 2.58 (q, 4H, J = 7 Hz), 2.78 (t,

2H, J = 6 Hz), 3.79 (q, 2H, J = 6 Hz), 6.85 (m, 1H), 7.55 (m, 1H),

7.80 (m, 1H), 7.91 (d, 1H, J = 9 Hz), 7.96 (d, 1H, J = 9 Hz), 8.08

(d, 1H, J = 9 Hz), 8.17 (d, 1H, J = 8.5 Hz), 8.48 (s, 1H); ¹³C NMR

(CDCl₃, 50 MHz) d 11.6 (2C), 37.6, 46.8 (2C), 51.8, 93.1, 122.3,

123.6, 125.6, 127.3, 129.7, 130.7, 131.1, 134.2, 138.9, 140.1,

147.2, 148.7, 166.5.

4.1.24. 5-Iodo-4-methanesulfonamido-2-methoxyaniline (38)

A solution of carbamate 41 (0.60 g, 1.45 mmol) in an aqueous

10% sodium hydroxide solution was reﬂuxed for 1.5 h. After cool-

ing to room temperature the reaction mixture was acidiﬁed to

pH 7–8 with concentrated hydrochloric acid and extracted with

dichloromethane (4ꢁ 50 mL). The organic layers were dried

(MgSO₄) and evaporated under vacuum to dryness to give the ani-

lino compound 38 which was used without puriﬁcation. Yield 89%;

mp 110–112 °C; IR (KBr) 3430, 3356, 1723, 1529, 1221 cm^ꢀ1; MS

m/z 342 (M⁺, 19), 263 (100), 136 (18), 121 (12); ¹H NMR (DMSO-d₆,

400 MHz) d 2.96 (s, 3H), 3.75 (s, 3H), 5.07 (s, 2H), 6.76 (s, 1H), 7.08

(s, 1H), 8.94 (s, 1H); ¹³C NMR (DMSO-d₆, 100 MHz) d 40.9, 55.5,

89.7, 111.7, 121.7, 126.0, 138.8, 146.3.

4.1.27.2. N-(2-Diethylaminoethyl)-2,7-diiodoacridine-9-carboxamide

(44b). From 43b. Yield 57%; R_f= 0,72 (Al₂O₃, EtOAc/EtOH, 99:1, v/v); mp

209–211 °C; IR (KBr) 3500-3300, 2924, 1637 cm^ꢀ1; MS m/z 573 (M⁺, 1),

1

458 (2), 430 (3), 303 (7), 86 (100), 58 (12); H NMR (CDCl₃, 400 MHz)

4.1.25. N-(2-Diethylaminoethyl)-9-(5⁰-iodo-4⁰-methanesulfo-

namido-2⁰-methoxyanilino)acridine-4-carboxamide hydro-

chloride salt (10d)

d 1.03 (t, 6H, J = 7 Hz), 2.63 (q, 4H, J = 7 Hz), 2.80 (t, 2H, J– 6 Hz), 3.81

(q, 2H, J = 6 Hz), 6.90 (m, 1H), 7.88 (d, 2H, J = 9 Hz), 7.98 (d, 2H,

J = 9 Hz), 8.46 (s, 2H); ¹³C NMR (CDCl₃, 100 MHz) d 11.7 (2C), 37.8, 46.7

(2C), 51.7, 94.1 (2C), 123.6 (2C), 130.9 (2C), 134.4 (2C), 138.9, 139.3

(2C), 147.1 (2C), 165.9.

This compound was prepared from 37 ⁴⁸and 38 by similar pro-

cedure using for 10a–c. Yield 33%; mp 213–215 °C; IR (KBr) 3500–

3300, 2927, 1623, 1572, 1522, 1320, 1151 cm^ꢀ1; MS (base) m/z 661

(M⁺, 2), 582 (24), 509 (25), 456 (74), 383 (73), 340 (40), 128 (27),

86 (100), 58 (19); ¹H NMR (DMSO-d₆, 400 MHz) d 1.28 (t, 6H,

J = 7 Hz), 3.16 (s, 3H), 3.24 (q, 4H, J = 7 Hz), 3.38 (m, 2H), 3.47 (s,

3H), 3.82 (m, 2H), 7.15 (s, 1H), 7.48 (m, 1H), 7.57 (m, 1H), 8.02

(m, 1H), 8.11 (s, 1H), 8.19 (d, 1H, J = 8 Hz), 8.28 (d, 1H, J = 8 Hz),

8.61 (d, 1H, J = 8 Hz), 8.74 (d, 1H, J = 7 Hz), 9.50 (br s, 1H), 9.95

(br s, 1H, NH), 10.86 (br s, 1H), 11.80 (br s, 1H), 14.20 (br s, 1H);

¹³C NMR (DMSO-d₆, 50 MHz) d 8.3 (2C), 34.3, 41.4 (2C), 46.4,

49.5, 56.0, 86.4, 111.1, 113.6, 114.5, 119.7, 120.2, 122.6, 124.5,

124.9, 128.0, 129.1, 133.6, 135.9 (2C), 138.3, 138.8, 139.2, 153.0,

156.0, 167.3. Anal. Calcd for C₂₈H₃₂IN₅O₄S, 2HCl, 2H₂O: C, 43.65;

H, 4.97; N, 9.09. Found: C, 43.51; H, 4.76; N, 9.03.

4.1.28. N-(2-Diethylaminoethyl)iodoacridine-9-carboxamides

dihydrochloride salts 11a,b

Copmound 11a,b were prepared as described for the synthesis

of hydrochloride salts 8a–g.

4.1.28.1. N-(2-Diethylaminoethyl)-2-iodoacridine-9-carboxamide

dihydrochloride salt (11a). From 44a. Yield 78%; mp 121–123 °C;

IR (KBr) 3500–3200, 2976, 2600–2400, 1654, 1464, 1420 cm^ꢀ^{1 1}H

;

NMR (DMSO-d₆, 400 MHz) d 1.29 (t, 6H, J = 7 Hz), 3.22 (m, 4H), 3.41

(m, 2H), 3.96 (m, 2H), 7.79 (m, 1H), 8.06 (t, 1H, J = 7.5 Hz), 8.13 (m,

2H), 8.24 (d, 1H, J = 9 Hz), 8.33 (d, 1H, J = 9 Hz), 8.45 (s, 1H), 9.55 (t,

1H, J = 6 Hz), 10.79 (m, 1H); ¹³C NMR (DMSO-d₆, 50 MHz) d 8.5

(2C), 34.3, 46.6 (2C), 49.3, 94.6, 122.0, 123.2, 126.2, 126.7, 128.8,

128.5, 133.0, 133.9, 140.6, 142.9, 144.4, 146.0, 165.3. Anal. Calcd

for C₂₀H₂₂IN₃O, 2HCl, 1.5H₂O: C, 43.90; H, 4.97; N, 7.68. Found:

C, 43.73; H, 4.72; N, 7.58.

4.1.26. Iodation of methyl acridine-9-carboxylate (42)

To a solution of methyl acridine-9-carboxylate⁵⁰(42) (400 mg,

1.69 mmol) in a mixture of concentrated sulfuric acid (80 lL),

7688

N. Desbois et al. / Bioorg. Med. Chem. 16 (2008) 7671–7690

4.1.28.2. N-(2-Diethylaminoethyl)-2,7-diiodoacridine-9-carbox-

amide dihydrochloride salt (11b). From 44b. Yield 92%; mp 224–

4.2. Radiolabelling

226 °C; IR (KBr) 3500–3200, 2600–2300, 2575, 1663, 1420 cm^ꢀ1

;

4.2.1. General methods

¹H NMR (DMSO-d₆, 200 MHz) d 1.31 (t, 6H, J = 7 Hz), 3.24 (m,

4H), 3.42 (m, 2H), 3.96 (m, 2H), 8.03 (d, 2H, J = 9 Hz), 8.21 (d, 2H,

J = 9 Hz), 8.42 (s, 2H), 9.56 (m, 1H), 10.74 (br s, 1H); ¹³C NMR

(DMSO-d₆, 50 MHz) d 8.6 (2C), 34.4, 46.6 (2C), 49.3, 95.3 (2C),

123.4 (2C), 129.6 (2C), 133.9 (3C), 140.3 (2C), 145.5 (2C), 165.0.

Anal. Calcd for C₂₀H₂₂IN₃O, 2HCl: C, 37.18; H, 3.59; N, 6.50. Found:

C, 37.57; H, 3.76; N, 6.18.

[ lg) was purchased from

¹²⁵I]NaI (3.7 GBq/mL, 644 MBq/

Amersham Int. Plc. (Little Chalfont, Buckinghamshire, UK) as a

‘no carrier added’ solution in reductant free 0.1 N aqueous sodium

hydroxide solution. Extrelut and citrate buffer solution (pH 4) were

purchased from Merck (Darmstadt, Germany). The radio-TLC strips

(Merck neutral aluminium oxide 60F₂₅₄plates) were developed

with (Al₂O₃and EtOAc) for [¹²⁵I]8f, (Al₂O₃, EtOAc/pentane, 5:5,

v/v) for [¹²⁵I]9d or (Al₂O₃, EtOAc/EtOH, 96:4, v/v) for [¹²⁵I]10c,

and measured on an AMBIS 400 system (Scanalytics, CSPI, San

Diego, CA, USA). Analytical HPLC was run on a system consisting

of an HP1100 chromatograph (Hewlett–Packard, Les Ulis, France)

and a Flow one A₅₀₀Radiomatic detector (Packard, Canberra,

Australia). Separation was carried out on a C₁₈column (Purospher

4.1.29. General procedure for the synthesis of tributylstannyl

compounds 45, 46 and 49

A mixture of the appropriate iodo compound 24f, 30d or 48

(0.22 mmol) in dry toluene (5 mL) was degassed and hexabutyld-

itin (351 lL, 0.66 mmol) and freshly prepared tetrakis(triphenyl-

phosphine)palladium(0) (Pd(PPh₃)₄)⁵⁶(20 mg) were added under

an argon atmosphere. The resulting solution was stirred under re-

ﬂux, until no starting material was observed by TLC. The black

reaction mixture was evaporated.

RP₁₈e, 5 lm) under the following conditions: gradient time =

10 min, ﬂow rate = 0.5 mL/min, H₂O/MeOH/(30:70 ? 0:100)

(NH₄OH 0.2%), k = 254 nm. HPLC puriﬁcation was performed on a

system including a Shimadzu LC 6A pump, an SLC 6B controller,

a CR₅A integrator, a SPD 6AV UV detector and a ﬂow-through

Raytest Stefﬁ gamma detector. The separation was carried out on

a C₁₈column (ZORBAX 80 Å, 4.6ꢁ150 mm) under the following

conditions: gradient time = 10 min, ﬂow rate = 1 mL/min, H₂O/

MeOH/(30:70 ? 0:100) (NH₄OH 0.2%), k = 254 nm. All radio-

labelled compounds were shown identical by TLC or HPLC to the

authentic non-radioactive material and to be free of signiﬁcant

chemical and radiochemical impurities.

4.1.29.1. N-(2-Diethylaminoethyl)-9,10-dihydro-9-oxo-7-tributyl-

stannylacridine-4-carboxamide (45). From 24f. Reaction time at

reﬂux 4 h. The crude product was chromatographed (Al₂O₃and

EtOAc). Yield 69%; R_f= 0.20 (Al₂O₃and EtOAc); viscous oil; IR

(CCl₄) 2960, 2928, 1652, 1608, 1508, 1150 cm^ꢀ1; ESI-MS m/z

628,2 [M+H]⁺; ¹H NMR (CDCl₃, 200 MHz) d 0.88 (t, 9H, J = 7 Hz),

1.05 (m, 12H), 1.24 (m, 6H), 1.55 (m, 6H), 2.57 (q, 4H, J = 7 Hz),

2.70 (t, 2H, J = 7 Hz), 3.52 (m, 2H), 7.19 (t, 1H, J = 8 Hz), 7.37 (d,

1H, J = 8 Hz), 7.52 (br s, 1H), 7.75 (d, 1H, J = 8 Hz), 7.90 (d, 1H,

4.2.2. Preparation of radioiodinated compounds [¹²⁵I]8f,

[

¹²⁵I]9d and [¹²⁵I]10c

J = 8 Hz), 8.54 (s, 1H), 8.65 (d, 1H, J = 8 Hz), 12.40 (br s, 1H);¹³

NMR (CDCl₃, 50 MHz) d 9.9, 12.1 (2C), 13.7, 27.4 (³J

C

=

The selected [¹²⁵I]-labelled compounds were prepared using

¹¹⁹Sn—¹³

C

iododestannylation reactions with tributyltin precursors 45, 46

and 49. In a closed vial containing the appropriate stannane

20 Hz), 29.2 (²J

= 20 Hz), 37.3, 46.9 (2C), 51.1, 117.2,

¹¹⁹Sn—¹³

C

117.8, 119.5, 121.1, 123.3, 131.3, 131.8, 134.8, 135.1, 140.4,

141.3, 141.4, 168.4, 178.2.

(0.12 mg) in EtOH (30

1% acetic acid/ethanol solution (30

or a citrate buffer solution pH 4 (30

¹²⁵I]NaI (20–50

L, 55.5–138.8 MBq) and an aqueous solution of

chloramine-T monohydrate (15 L, 0.4 mg/mL for 45, 0.5 mg/mL

l

L) were added, in the following order: a

L) (for compound 45 or 49)

L) (for compound 46),

l

4.1.29.2. N-(2-Diethylaminoethyl)-5-tributylstannylacridine-4-

carboxamide (46). From 30d. Reaction time at reﬂux 32 h. The

crude product was chromatographed (Al₂O₃, EtOAc/pentane, 5:5,

v/v) to give in order of elution: stannane 46; yield 25%; R_f= 0.79

(Al₂O₃, EtOAc/pentane, 5:5, v/v); viscous oil; IR (CCl₄) 2959, 2927,

1662, 1286 cm^ꢀ1; ESI-MS m/z 612,3 [M+H]⁺; ¹H NMR (CDCl₃,

200 MHz) d 0.79 (t, 9H, J = 7 Hz), 1.3 (m, 18H), 1.45 (m, 6H), 2.76

(m, 4H), 2.88 (m, 2H), 3.73 (m, 2H), 7.54 (t, 1H, J = 7.5 Hz), 7.64 (dd,

1H, J = 7.5, 8.5 Hz), 8.00 (m, 2H), 8.15 (d, 1H, J = 8.5 Hz), 8.88 (s,

1H), 8.93 (d, 1H, J = 7.5 Hz), 10.59 (br s, 1H); ¹³C NMR (CDCl₃,

[

l

for 46 and 0.25 mg/mL for 49). The resulting solution was vortexed

at room temperature for 10 min. The reaction was quenched with

an aqueous 0.1 N NaOH solution (20 lL). The mixture was vortexed

for 5 min and the vial cap and septum were removed. The reaction

mixture was transferred to an Extrelut^Òcolumn and the vial was

rinsed with a solution of H₂O/EtOH (1:1, v/v, 2ꢁ 100

lL). After

10 min, the column was eluted with dichloromethane (5ꢁ 2 mL).

The organic extracts were collected were evaporated under

50 MHz)

¹¹⁹Sn—¹³

d

10.1, 12.2, 13.7, 27.4 (³J

= 60 Hz), 29.3

¹¹⁹Sn—¹³C

reduced pressure, taken up with methanol (200 lL), and puriﬁed

(²J

= 20 Hz), 39.1, 47.8, 52.1, 125.1, 126.0, 126.5, 127.0,

C

by HPLC at a ﬂow rate of 1 mL/min. The fractions containing the

product were collected, evaporated to dryness and re-dissolved

in dichloromethane (1 mL). An anhydrous 2 N HCl/ether solution

(2 mL) was added and the total solution was evaporated to yield

the expected [¹²⁵I]compound. Radiochemical yields based on TLC

of exchange reaction mixture and radiochemical purities are given

in Table 3.

128.8, 129.3, 132.7, 135.3, 139.3, 141.0, 145.0, 146.4, 153.0, 166.7.

N-(2-diethylaminoethyl)-acridine-4-carboxamide⁴⁰(47); yield

29%; R_f= 0.33 (Al₂O₃, EtOAc/pentane, 5:5, v/v)); mp 148–150 °C.

4.1.29.3. N-(2-Diethylaminoethyl)-7-tributylstannyl-9-(4⁰-metha-

nesulfonamido-2⁰-methoxyanilino)acridine-4-carboxamide (49). The

dihydrochloride 10c (0.13 g, 0.50 mmol) was diluted with water

(5 mL), and treated with an aqueous saturated sodium carbonate

solution (20 mL). The aqueous solution was extracted with

dichloromethane (3ꢁ 10 mL). The organic layers were dried

(Na₂SO₄) and concentrated under vacuum to give compound 48.

Product 49 was obtained from 48 according to the procedure

developed for compound 45 with reaction time at reﬂux 4.5 h.

The crude product was chromatographed (Al₂O₃, EtOAc/EtOH,

96:4, v/v). Yield 34%; R_f= 0.48 (Al₂O₃, EtOAc/EtOH, 96:4, v/v),

4.3. Pharmacology

4.3.1. Partition coefﬁcient measurements

The partition coefﬁcient between n-octanol and phosphate buf-

fer was determined for each molecule. The measurement was per-

formed by shaking 2 mL of [¹²⁵I]molecule solution (50

lM in

phosphate buffer solution, PBS, pH 7.4) with 2 mL of n-octanol.

The activity of each phase was measured. P was calculated as the

ratio of activities (n-octanol/buffer), and its logarithm was deter-

mined in order to express lipophilicity.

mp 139–141 °C; IR (KBr) 2924, 1592, 1507, 1457, 1152 cm^ꢀ1

ESI-MS m/z 826.5 [M+H]⁺.

;

N. Desbois et al. / Bioorg. Med. Chem. 16 (2008) 7671–7690

7689

4.3.2. In vitro binding to melanin

1 mM EDTA and 2 M NaCl) were added to each well. The plates

were maintained in dark conditions and incubated in this solution

on a plate shaker for 1 h at room temperature. Fluorescence was

then measured at 360/460 nm on a microplate ﬂuorescence reader

and the IC₅₀was calculated as described above.

An in vitro experiment was performed to evaluate the binding

afﬁnity of new compounds to melanin using synthetic tyrosine-

melanin (Sigma) suspended in two different media: H₂O or PBS.

The general procedure used was as follows: [¹²⁵I]compound was

added to the melanin suspension (0.5 mg/10 mL). The reaction

mixture was incubated at room temperature for 24 h with stirring.

After incubation, the tubes were centrifuged at 35,000g for 20 min,

and aliquots of the supernatants were counted on the gamma

4.3.4. In vivo distribution

All experiments were carried out in compliance with the French

laws governing animal experimentation. The biodistribution of

radioiodinated compounds was studied in C57BL6 male mice bear-

ing the B16F0 murine melanoma.

counter (Packard, Minaxi

c 5530, Rungis, France).

4.3.3. Cytotoxicity assays

4.3.3.1. Cell culture. Normal human ﬁbroblasts were purchased

from Biopredic International (Rennes, France). This frozen culture

was obtained from abdominal surgical waste from a 36-year-old

female and the cells used in this work were from the 7th to

12th passage of the culture. M4Beu, a human melanoma cell line,

was established in the laboratory of Dr. J. F. Doré (INSERM, Unit

218, Lyon, France) from metastatic biopsy specimens and main-

tained in cell culture for roughly 15 years. Colon adenocarcinoma

DLD-1 cells, Jurkat leukaemia cells, and B16F0 murine melanoma

cell line cells were purchased from the European Collection of Cell

Cultures (ECACC; Salisbury, United Kingdom). Stock cell cultures

were maintained as monolayers in 75-cm²culture ﬂasks in

Eagle’s minimum essential medium with Earle’s salts (MEM;

Gibco–BRL, Invitrogen, Cergy-Pontoise, France) or Dulbecco mod-

iﬁed MEM (DMEM, Gibco) for B16F0 supplemented with 10% foe-

tal calf serum (Sigma), and 5 mL of a 100ꢁ solution of vitamins

(Gibco), 5 mL of 100 mM sodium pyruvate (Gibco), 5 mL of 100ꢁ

non-essential amino acids (Gibco) and 2 mg of gentamicin base

(Gibco). Cells were grown in a humidiﬁed 37 °C incubator con-

taining 5% CO₂.

4.3.4.1. Tumoural model. For transplantation, an aliquot was

grown in a monolayer culture to conﬂuence, after which the cells

were trypsinised and washed with phosphate buffer saline (PBS)

before being resuspended in PBS. Each mouse was subcutaneously

administered 0.1 mL (3 ꢁ 10⁵cells) on the left ﬂank. Ten days later,

the tumours became palpable, with 98–100% of tumours taken.

4.3.4.2. Quantitative biodistribution on whole body slides. The

[

¹²⁵I]molecule was administered intravenously via a tail vein

(0.74–0.92 MBq/animal) in 10 animals for each compound. After

the injections, two mice were sacriﬁced by CO₂inhalation and

quickly frozen in liquid nitrogen at different times after adminis-

tration (1, 3, 6, 24 or 72 h). These frozen animals were cryosec-

tioned using the technique described by Ullberg.⁵⁸Slices of

40 lm were obtained using a Reichert-Jung cryopolycut (Leica

Instruments, Rueil Malmaison, France) at ꢀ22 °C and dehydrated

for 48 h in the cryochamber. Eight slices per mouse and per time-

point were selected from a total number of more than 500 slices,

for the analyses on distant slices and the section of the organs of

interest. The radioactivity contained in the slices was analysed

using an AMBIS 4000 detector (Scanalytics, CSPI, San Diego, CA),

which is a computer-controlled multi-wire proportional counter

validated for evaluation of iodinated agents in mice.⁵⁹Measure-

ments were performed following a 1000-min acquisition time.

The quantiﬁcation of radioactivity in different organs was made

after contouring a suitable area on the 2D image of the slice dis-

played on the computer screen for analysis. Radioactivity per unit

area (net cpm/mm²) was converted into radioactive concentration

(kBq/g) and expressed as percentage of injected dose/g of tissue (%

ID/g). For comparison of tumour (T) uptake with other tissues, ra-

tios of radioactive concentrations (T/organ) were determined to

illustrate the image contrast.

4.3.3.2. Cell survival assays. Two test were used: the resazurin

reduction test (RRT) for measuring cellular redox potential, and

the Hoechst dye 33342 test for DNA quantization. Cells were plated

at a density of 5 ꢁ 10³cells per well in 96-well microplates

(Nunclon^TM, Nunc, Roskilde, Denmark) in 100

lL of culture medium

and were allowed to adhere for 16 h before treatment with each

drug. Stock solution (200ꢁ) of each drug was prepared in DMSO

(Sigma) and kept at ꢀ20 °C until use. Each set of experimental con-

ditions was tested in triplicate. After 48 h of continuous drug expo-

sure, the anti-proliferative effect of each drug was assessed by the

RRT and then, cells were frozen and assessed by the Hoechst test as

previously described.⁵⁷

For two animals, urine and faeces were collected until 72 h and

counted to determine the cumulative urinary and faecal

excretions.

RRT. Brieﬂy, RRT was carried out as follows: plates were rinsed

with 200

of a 25

l

L PBS (37 °C, Gibco) at 37 °C to which was added 150

lL

l

g/mL (20 L of a 275 g/mL for Jurkat cells) solution of

l

resazurin in MEM without phenol red. The plates were incubated

for approximately 1 h (6 h for Jurkat cells) at 37 °C in a humidiﬁed

atmosphere with 5% CO₂for ﬂuorescence development by living

cells. Fluorescence was then measured on the Fluoroskan Ascent

FL^TM(Labsystems) automated 96-well plate reader using an excita-

tion wavelength of 590 nm and an emission wavelength of 630 nm.

In the conditions used here, ﬂuorescence was proportional to the

number of living cells in the well. Cell survival percentage is de-

ﬁned as the ratio of ﬂuorescence in drug-treated wells to ﬂuores-

cence in control wells, after subtraction of blank values. After

reading, the plates were emptied and stored at ꢀ80 °C for Hoechst

dye test.

4.3.4.3. Scintigraphic imaging. For each selected compound, the

in vivo kinetic proﬁle was tracked in two mice by repeated planar

scintigraphic imaging (3 MBq, acquisition time: 10 min) using a

gamma camera dedicated for small animals (Biospace, Paris,

France). This study made it possible to follow tumoural uptake

kinetics in the same animal.

Acknowledgments

We thank the French ‘Agence Nationale de la Recherche’ and the

‘Ligue Régionale contre le Cancer’ for their ﬁnancial support.

Hoechst dye 33342 test. Plates were thawed at room tempera-

ture for 10 min. A 0.01% (m/v) SDS solution (100 lL) in distilled

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Article Doi

Design, synthesis and preliminary biological evaluation of acridine compounds as potential agents for a combined targeted chemo-radionuclide therapy approach to melanoma

DOI: 10.1016/j.bmc.2008.07.015

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Article abstract of DOI:10.1016/j.bmc.2008.07.015

Full text of DOI:10.1016/j.bmc.2008.07.015

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