Distamycin Analogues with Enhanced Lipophilicity: Synthesis and Antimicrobial Activity

J . Med. Chem. 2004, 47, 2133-2156

2133

Dista m ycin An a logu es w ith En h a n ced Lip op h ilicity: Syn th esis a n d

An tim icr obia l Activity

Abedawn I. Khalaf,^†Roger D. Waigh,^‡Allan J . Drummond,^‡Breffni Pringle,^‡Ian McGroarty,^‡

Graham G. Skellern,^‡and Colin J . Suckling*^,†

Department of Pure & Applied Chemistry and Department of Pharmaceutical Sciences, University of Strathclyde,

295 Cathedral Street, Glasgow, G1 1XL, Scotland

Received November 5, 2003

Forty-eight heterocyclic amino acid trimers, analogues of distamycin, with a number of features

that enhance lipophilicity are described. They contain alkyl or cycloalkyl groups larger than

methyl; some are N-terminated by acetamide or methoxybenzamide and are C-terminated by

dimethylaminopropyl or aliphatic heterocylic aminopropyl substituents. The ability of these

compounds to bind principally to AT tracts of DNA has been evaluated using capillary zone

electrophoresis. Significant antimicrobial activity against key organisms such as MRSA and

Candida albicans is shown by several compounds, especially those containing a thiazole.

Moreover, these compounds have low toxicity with respect to several mammalian cell lines.

In tr od u ction

of an imidazole by a thiazole would be expected to

increase log P by at least one unit.⁹This argument led

us to consider further that distamycin and netropsin,

through their methyl groups, do not take full advantage

of the available hydrophobic interactions with the

methine and methylene group forming the walls of the

DNA minor groove. Since the methyl groups point

outward from within the minor groove, larger alkyl

groups might offer increased contacts with the DNA

backbone. This might then lead to an increase in affinity

together with a beneficial effect on transport. We have

therefore synthesized a number of distamycin analogues

that embody these structural features (1-48) and have

investigated their binding to DNA using capillary zone

electrophoresis (CE) and their antimicrobial activity

against a panel of Gram-positive and -negative bacteria

and fungi. In this paper, we outline the synthesis and

discuss the emerging structure-activity relationships

with these compounds.

Analogues of distamycin and netropsin are widely

recognized as candidates for drug development based

upon their ability to bind with sequence specificity to

the minor groove of DNA.¹The feasibility of this

approach in disease-related assays has been demon-

strated for antiviral²and cytokine inhibiting activity,³

while minor-groove binders have been more broadly

considered as drugs for the future.⁴When modified at

the N-terminus by alkylating functional groups such as

R,â-unsaturated amides or chloroethylamines, distamy-

cin analogues also show antitumor activity.⁵In many

cases, large oligomers (10mers plus) are required to

show substantial biological activity and in several cases,

it has been possible to demonstrate that sequence

reading by the minor groove binder is related to biologi-

cal activity.^1-4

Linear head-to-tail oligomers have featured strongly

in drug design.⁶It is also possible to design analogues

having a head-to-head link (through the N-terminus)

and these are typically dicationic (Figure 1). While there

are potential problems in delivering large polyamides

to the mammalian nucleus,²small analogues of dista-

mycin and netropsin have been shown to have signifi-

cant antibacterial activity.⁷Typically, such compounds

contain N-alkyl pyrroles with branched or cycloalkyl

substituents in place of the methyl group found in

distamycin and netropsin and possess larger head

groups than formyl.

Design a n d Syn th esis. The structures of the mono-

mers and head and tail groups are shown in Figure 2,

and the structures of the minor-groove binders prepared

are shown in Table 1. Table 1 uses the shorthand

notation as defined in Figure 2.

N-Alk ylp yr r oles. To encompass a reasonable range

of size and shape, branched N-alkyl- and N-cycloalkyl-

pyrroles were prepared by alkylation of the potassium

salt of ethyl 3-nitropyrrole-5-carboxylate with the ap-

propriate alkyl bromide.¹⁰Examples with limited con-

formational flexibility (isopropyl and cyclopentyl) and

with substantial conformational flexibility (3-methyl-

butyl and cyclopropylmethyl) were selected. The various

nitro-carboxylate monomers were appropriate for the

synthesis of oligomers prepared as previously described

typically by alkylation of the potassium salt of ethyl

5-nitropyrrole-2-carboxylate and subsequent functional

group modification.⁶

We have drawn attention to the influence of structure

on the lipophilicity of analogues of distamycin and

netropsin.⁸As a major property that influences the

ability of a drug to reach targets by transmembrane

diffusion, the ability to control lipophilicity would be

expected to have a major effect on the biological activity

of minor-groove binders. For example, the replacement

Th ia zoles. As noted above, thiazoles can be consid-

ered as lipophilic analogues of imidazoles. Since imida-

zoles have been widely recognized as permitting binding

to GC regions,¹thiazoles can be anticipated to be very

* Corresponding author. Phone: +44 141 548 2271. Fax: +44 141

548 5743. E-mail: c.j.suckling@strath.ac.uk.

^†Department of Pure & Applied Chemistry.

^‡Department of Pharmaceutical Sciences.

Published on Web 03/05/2004

2134 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8

Khalaf et al.

F igu r e 2. Structures of components of distamycin analogues

described in this paper.

for which the nitro group acts as a latent amino group,

the thiazoles are prepared by ring synthesis¹²with an

unprotected amino group; for elaboration into oligomers,

the aminothiazoles were acylated with an N-alkyl-

pyrrole, thereby avoiding the need for further protection.

Th iop h en es. The replacement of the electronegative

nitrogen atom in pyrrole by the much more lipophilic

sulfur would also be expected to have a significant

influence on the biological activity of minor-groove

binders. Accordingly, a C-methylthiophene, ThiMe, was

prepared as one of the building blocks following pub-

lished procedures.¹⁴It was incorporated into the oligo-

mers by standard nitro/amino methodology.

Im id a zoles a n d Oxa zoles. A limited range of imi-

dazoles and oxazoles were synthesized to compare for

the activity of the more lipophilic N-alkylpyrroles and

thiazoles. N-Ethylimidazole ImiEt was prepared by

published methods.¹⁵Appropriate oxazoles proved very

difficult to prepare and we were able only to access one

example OxaMe,¹⁶which differed significantly from the

structures of the other heterocyclic building blocks by

containing an additional methylene group. This would

be expected to increase substantially the conformational

flexibility of the minor groove binder and correspond-

ingly alter its ability to bind to DNA.

F igu r e 1. Structures of prototype natural products, dista-

mycin, and netropsin together with synthetic analogues: A

head-to-head netropsin analogue⁶and a typical novel com-

pound (28) from this work with its abbreviated text code (see

Figure 2).

significant in the design of more lipophilic distamycin

analogues. N-Alkylation of a thiazole is not permitted

in this context, since this would introduce an unwanted

positive charge and also block access to GC base pairs.

There are, however, two isomeric C-alkylthiazoles to

consider. The use of C-alkyl substituents is one novel

feature of this research.¹¹The synthesis of the thiazoles

following the published procedure led to two isomers

(ThziPr and isoThziPr) with C-isopropyl substituents;¹²

Hea d Gr ou p s a n d Ta il Gr ou p s. It is possible to

modify the lipophilicity by a suitable choice of head or

tail group. In particular, the successful replacement of

the naturally occurring amidine in distamycin by a

their structures have been unambiguously determined

by X-ray crystallography.¹³Unlike the N-alkylpyrroles,

Distamycin Analogues with Enhanced Lipophilicity

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2135

Ta ble 1. Abbreviated Structures of Compounds Synthesized and Studied Together with Binding to DNA As Measured by CE

compd

head

P1

P2

P3

tail

CE to AT

Pyrrole N- and C-Alkyl Substituents

1

2

3

4

5

6

7

8

Fo

PyrMe

PyriPr

PyrMe

PyriPr

PyriPe

PyrMe₂

PyrMe

PyrEt

PyriPr

PyrCycpe

PyrCycpr

PyrMe

PyriPe

PyriPr

PyrMe

PyriPr

PyrMe

PyriPr

PyriPe

PyrMe₂

Dmap

2 to 1

1 to1

2 to 1

weak

nb^a

9

10

PyriPe

PyrMe₂

nb

Pyrrole Head Group Variations

11

12

13

14

15

16

17

18

19

20

21

22

23

Ac

DmB

PyrMe

PyriPr

PyrMe

PyrEt

PyriPr

PyrMe

PyriPe

PyrMe

PyriPe

PyrMe

Dmap

Dmapc

Dmap

1 to1

nb

DmB

MPe

nb

3MeOB

4MeOPhe

Cin

MeThi

PyrNO₂

2 to 1 then complex

2 to 1

nb

no clear stoichiometry

2 to 1

nt

Imidazoles

24

25

26

Fo

PyrMe

ImiEt

PyrMe

ImiEt

Dmap

1 to 1

nb

Thiazoles

27

28

29

30

31

32

33

Fo

Ac

3MeOB

PyrMe

ThziPr

PyrMe

ThziPr

PyrMe

ThziPr

PyrMe

isoThziPr

ThziPr

PyrMe

ThziPr

PyrMe

Dmap

nb

1 to 1

nb

2 to 1

Thiophenes

PyrMe

34

35

36

37

38

Fo

Ac

ThiMe

PyrMe

ThiMe

PyrMe

ThiMe

PyrMe

ThiMe

PyriPe

ThiMe

PyrMe

Dmap

2 to 1

nt

1 to 1

PyrMe

Oxazole

39

Fo

PyrMe

CH₂oxaMe

Dmap

nb

Pyrrole Tail Group Variations

40

41

42

43

44

45

46

47

48

Fo

Ac

3MeOB

Fo

Ac

Fo

PyrMe

PyriPe

PyriPr

PyriPe

PyrMe

PyriPr

PyrMe

Morp

Mepipp

Pyrrp

1 to 1

nb

yes

1 to 1

nb

1 to 1

2 to 1

a

nb ) no binding observed.

tertiary alkylamine in many published examples opens

the way to use other alkylamines with different phys-

icochemical characteristics. The examples described use

1-pyrrolidyl (Pyrrp), morpholine (Morp), and N-meth-

ylpiperazine (Mepipp), all linked via a propyl chain, to

cover a range of size, conformational flexibility, and

lipophilicity in the tail. Variation of head group is

synthetically the easiest of all modifications to make;

formyl (Fo), acetyl (Ac), substituted benzoyl (3MeOBz),

substituted phenylacetyl (4MeOPhe), and cinnamoyl

(Cinn) were all used. This selection again includes a

wide range of size and conformational flexibility.

Syn th esis of Oligom er s. As noted above, methods

described previously⁶served for the synthesis of the

pyrrole-only oligomers. The N-terminal pyrroles sub-

stituted by the various tail groups (Figure 2) were

prepared from the appropriate amine-substituted with

the dimethylamino or heterocyclic amino group using

the appropriate nitropyrrole acid chloride (NO₂)PyrRCl

or the trichloromethyl ketone (NO₂)PyrMeCCl₃for the

first unit. A variety of standard coupling methods was

used to prepare the oligomers, including acid chloride,

carbodiimide, and HBTU (see the Experimental Sec-

tion). Similar methods applied for the synthesis of

2136 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8

Khalaf et al.

Ta ble 2. Antibacterial and Antifungal Activity of Compounds Synthesized and Evaluated^a

antibacterial activity

antifungal activity

(MIC, M × 10⁶

(MIC, M × 10⁶)

compd

head

P1

P2

P3

tail

S.aur S.fae MRSA E.clo M.for K.aer P.vul E.col A.nig A.nid C.alb

Pyrrole N- and C-Alkyl Substituents

1

3

4

5

6

7

8

10

Fo

PyrMe PyrMe

PyrMe PyriPr

PyrMe

Dmap 10.0 0.31 na

na

75.3

164

na

nt

na

75.3

Dmap na

157

39.1 157 na

157

37.6

na

150

72

PyrMe PyrCycpe PyrMe

PyrMe PyrCycpr PyrMe

PyriPr PyrMe

PyrMe PyriPe

PyriPr PyriPr

PyrMe2 PyrMe2

Dmap 75.0 75

75.3

na

38.4 na

na na

37.5 150 na

36 144 na

77.7 153 na

na

Dmap 153

Dmap na

Dmap 150

Dmap 144

Dmap na

76.9 76.9

na 150

57.5 150

76.9 38.4

na na

150 37.5

36

na

153 76.9

150 75

na

72

PyriPr

PyrMe

PyriPr

PyrMe2

150

144

72

na

144

na

72

153

153 na

Pyrrole Head Group Variations

11

12

13

14

17

18

19

20

21

22

23

Ac

DmB

MPe

PyrMe PyriPe

PyrMe PyriPr

PyrMe PyrMe

PyrMe PyriPe

PyrMe

Dmap na

Dmap 147

Dmap 69.8 140

Dmap 16.2 16.2

Dmap 15.9 15.9 63. 6 na

73.5 na

na

147

na

147

153

80

150

147

140

na

15.9 na

70.2 70

70.8 na

na

153 na

160 na

na

73.5

153

160

na

147

140

na

15.9

140

141

15.8

na

147

153

na

40

150

na

2

160

150

147

160 160

na na

73.5 147

69.8 na

na

150

73.5

140

32.4

15.9

na

3MeOB

4MeOPhe PyrMe PyriPe

Cin

MeThi

PyrNO₂

nt

na

15.9 15.9

70.2 35.1

70.8 na

PyrMe PyrMe

PyrMe PyriPe

Dmap 8.8

Dmap na

Dmap 31.6 15.8 126

8.8

141

140

141

70.2 140

na

35.4

15.8

15.8 126 na

25.8 15.8

Imidazoles

24

25

Fo

PyrMe PyrMe

ImiEt

Dmap na

Dmap 19.0 na

na

160

na

40

na

160 na

na

160 na

ImiEt

na

Thiazoles

28

29

30

31

32

33

Fo

Ac

3MeOB

PyrMe PyrMe

PyrMe ThziPr

ThziPr PyrMe

PyrMe PyrMe

ThziPr PyrMe

ThziPr

PyrMe

ThziPr

PyrMe

Dmap 4.7

Dmap 4.8

Dmap 70.0 74.5 149.2 149 74.6

Dmap 4.3

Dmap na

Dmap 32.7 32.7 32.7

9.5

38.1

19.1

76.2 19

9.5 19

76.2 9.5

38.1 na

na

76.1

38.1

76.1 76.1

9.5 38.1

149

69.8 69.8 70

na na na

149 149 149

nt

74.6

34.9

na

4.3

74.5 na

4.3

69.8 17.5

na na

131 65.5

4.3

74.5

32.7 65.5 65.5 131

131 131

Thiophenes

34

35

36

37

38

Fo

Ac

ThiMe PyrMe

PyrMe ThiMe

ThiMe PyriPe

PyrMe ThiMe

ThiMe PyrMe

PyrMe

Dmap na

Dmap 39.8 79.6 159

Dmap 146.0 146

Dmap 78.5 na

Dmap 156.0 na

na

159

79.6 na

79.6 159 159

na

na 159

159 159

146 146

156 38.9

159 159

146 146

38.9 156

73.1

156

na

73.1 146 73.1 146

156

156 156 156

156 na

na

156

na

Oxazole

39

Fo

PyrMe PyrMe

CH₂oxaMe Dmap na

na

160

160 na

na

160

160 na

Pyrrole Tail Group Variations

40

41

42

43

44

45

46

47

48

Fo

Ac

3MeOB

Fo

Ac

Fo

PyrMe PyriPe

PyrMe PyriPr

PyrMe PyriPe

PyrMe PyrMe

PyrMe PyriPr

PyrMe

Morp

Mepipp 60

Mepipp 140

Mepipp na

Pyrrp

na

70.6

na

140

na

121

140

136

153

157

na

70.6

na

60.4 121

69.3 na

na

121 121

140 69.3

na

153 76.9

157 78.5

75.2 150

121 na

140 140 140

136 na

na na

157 157 157

150

121

30.2 121

69.3 na

136 na

153 na

157 157

150 na

na

136

na

153

na

153

1200 na

150

75.2 75.2

37.6 150 na

Controls

amoxycilin

streptomycin

fluconazole

itraconazole

0.49 0.49 16.1

10.8

4

16.1

8.1

4

>300 90.8 81.6

17.7 35.4 35.4

a

Abbreviations for microbes: S.aur ) S. aureus NCTC 6571. S.fae ) S. faecalis NCTC 775. MRSA ) MRSA PHLS M1. E.clo ) E.

cloacae NCTC 10005. M.for ) M. fortuitum NCTC 10394. K.aer ) K. aerognenes WRL CN 345. P.vul ) P. vulgaris NCTC 4175. E.col )

E. coli NCTC 9001. A.nig ) A. niger IMI17454. A.nid ) A. nidulans CABI 0160037. C.alb ) C. albicans NCPF 3179.

compounds containing thiophenes. Imidazole-containing

compounds were prepared similarly by acid chloride or

HBTU-mediated coupling. Reaction conditions were not

optimized in each case, and yields of coupling were

typically in the range 30-80%. Where necessary, in-

termediates were purified by column chromatography

and all final products were stringently purified using

preparative-scale HPLC. Full experimental details are

given for final products together with general methods

for intermediates both with full characterization details.

Bin d in g E va lu a t ion of Lip op h ilic Dist a m ycin

An a logu es. The structures of the minor-groove binders

and their properties with respect to DNA binding to a

target oligonucleotide are shown in Table 1. The anti-

microbial activity of these compounds is shown in Table

2.

The primary enquiry concerned the extent to which

nonpolar appendages were compatible with binding to

DNA. To this end, most of the present compounds

contained predominantly N-alkylpyrroles which, be-

Distamycin Analogues with Enhanced Lipophilicity

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2137

cause of the steric hindrance between the 2-hydrogen

of the pyrrole and the amino group of G in DNA, would

be expected to bind predominantly to AT-rich sequences.

Footprinting studies on a small number of selected

compounds confirmed that expectation, with the excep-

tion of 28, which showed a remarkably strong preference

for the sequence ACTAGT.^17-19The primary evaluation

therefore used the AT dodecamer AAATTATATTAT in

CE experiments. Almost all compounds synthesized

bound to this oligomer to some extent. Notable excep-

tions included compounds with variations in the head

group, alkyl substitution, and tail groups. The stoichi-

ometry of binding to this test DNA oligonucleotide was

also structure dependent, as described below. It is

important to note that while this assay identifies

acceptable features for binding to DNA, a negative

result does not mean that there is no sequence of DNA

to which that particular minor groove binder might

bind.

as in 42 and 45), binding to the test DNA sequence

under CE conditions was not observed at all.

Taken together these results clearly show that it is

possible for wide structural variations to be introduced

into the basic architecture of distamycin-like minor-

groove binders and that there is a structural dependence

for binding. The CE results are generally consistent with

information from footprinting, a substantial additional

study that will be published elsewhere. The structural

manipulability is encouraging, since it is highly desir-

able to have molecules that are both manipulable with

respect to affinity for the receptor and with respect to

the physicochemical properties that affect transport.

The calculated log P values for the neutral form of a

selected group of compounds give some insight. The

closest analogue of distamycin, 1, has an estimated

clogP of -1.98((0.47). Replacement of the central

N-methyl group by isopropyl gives compound 3, for

which clogP is -1.32. The change to a thiazole ring as

in 28 causes log P to rise to 0.61. Although no real

significance can be read into the exact values, a range

of nearly 2 orders of magnitude between members of

related structure is significant. In terms of Lipinski’s

rule of five,²²these minor-groove binders exceed the

notional limit of 500, but it is unlikely that such a broad

(but useful) generalization will apply strongly to com-

pounds of such distinct structural characteristics. How-

ever, it is possible that distamycin analogues may be

substrates for biological transporters, as are other

antibiotics. We are not aware of any studies on the

active transport of distamycin or netropsin, nor of the

flexibility of any potential transport mechanism with

regard to structural variation. Such mechanisms, if they

exist, may be limited to amidine- or guanidine-contain-

ing structures, which are closely similar to the natural

products.

With respect to the head groups, there was a signifi-

cant difference between formyl, the benzoates, and

cinnamate (in which the head group is essentially

coplanar with the amide) and those such as acetyl and

phenylacetyl, in which the adjacent atom to the carbonyl

group is tetrahedral. For example, the principal mode

of binding of the former group of compounds was 2:1

(ligand to DNA duplex, e.g. 1-5). On the other hand,

acetamides preferred 1:1 binding (e.g. 11), and bulky

amides such as dimethylbutanamide showed very weak

binding, if any, to the probe oligonucleotide (e.g. 14-

17).

Modification of the backbone heterocyclic rings also

showed significant effects. The introduction of larger

alkyl groups than methyl (e.g. 2-5) did not normally

perturb the preferred 2:1 binding mode unless more

than one large alkyl group was present (e.g. 6). If the

pyrrole contained two methyl substituents (10), binding

to the test AT oligonucleotide was not observed under

CE conditions. Compounds containing two or more

imidazoles (25, 26) and some of the thiazoles (27, 29,

31, 32) failed to bind. In the case of 28, CE experiments

were performed with the target oligonucleotide sug-

gested by the footprinting results, namely CGAC-

TAGTCG, and in this case, clear evidence for binding

was obtained. Moreover, a binding ratio of 4:1 was

observed under CE conditions.²⁰We have studied this

interaction in more detail by NMR, and while it is clear

that higher order assemblies are possible, the 2:1

stoichiometry represents a significant energy mini-

mum.¹⁸This interpretation is also supported by melting-

temperature measurements carried out on a selection

of the compounds described in this paper.²¹We therefore

attach little structural significance at this stage to the

observation of higher order associations of our com-

pounds with DNA.

Recently, the importance of molecular rigidity has

been discussed in terms of bioavailability.²³In general,

compounds with limited rotational flexibility show bet-

ter bioavailability. In the context of minor-groove bind-

ers composed of heterocyclic amino acid amides, the

principal location of flexibility resides in the tail group

and a high overall degree of rigidity exists. On the other

hand, a high polar surface area has been argued to be

a negative factor in bioavailability. The fact that in vivo

these minor-groove binders will be monocations will

have a major influence on their real distribution proper-

ties, although for aliphatic tertiary amines, a small but

significant proportion of the compound would be unpro-

tonated at pH 7. The large number of amide bonds

present will also increase the polar surface area. Over-

all, therefore, the minor-groove binders described herein

represent a class of compounds conceptually acceptable

as drugs.

Biologica l Eva lu a tion . Table 2 shows the biological

evaluation of the compounds obtained against a set of

eight bacteria and three fungi. Toxicity tests have also

been run against mammalian cell lines, namely NCTC

2544 (human keratinocytes), E6-1 J urkat cells (human

T-cell lymphoblasts), and CCI 39 (chinese hamster

lung). At concentrations of 10 µg mL^-1(equivalent to

about 16.6 µM for a compound of molecular weight 600)

over 4 and 24 h, no cytotoxic effects were observed.

A very clear-cut difference in the behavior of the tail

groups was shown between dimethylamino and pyrro-

lidino on one hand and morpholino and N-methyl-

piperazino on the other. Dimethylamino and pyrrolidino

(47, 48) both bound to the test AT oligonucleotide in a

2:1 ratio, but compounds with a six-membered ring tail

group (40, 41, 43, 44) bound only in a 1:1 ratio, as shown

by CE. Interestingly, when two features that promoted

1:1 binding were included in the same molecule (such

However at higher concentrations (up to 100 µg mL^-1

,

2138 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8

Khalaf et al.

∼166 µM) some cytotoxicity was observed in the case of

the thiazole-containing compounds 29 and 31, which

caused 50% inhibition of the growth of J urkat cells at

50 and 19 µM, respectively. These concentrations are

similar to those at which significant antibacterial and

antifungal activity was observed. There is thus some

basis for selective toxicity by these minor-groove bind-

ers; cytotoxicity was not observed with 28 under the

same conditions, for example. However as would be

expected, a careful evaluation of the balance between

antimicrobial activity and toxic effects must be main-

tained compound by compound.

With respect to the antibacterial activity,²⁴it is

noticeable that Gram-positive organisms are much more

susceptible to these compounds than Gram-negative

organisms, consistent with previous studies.⁷For Gram-

negative organisms, some compounds containing

branched or cycloalkyl substituents (3, 5, 8, 23, 29, and

48) showed similar activity to the control drug (amoxy-

cillin) in activity against Klebsiella aerogenes especially,

but this appears to be exceptional. Against Proteus

vulgaris and Escherichia coli, much weaker activity was

obtained. The greatest activity against P. vulgaris was

shown by the thiazole-containing compound 28. The

greatest activity against E. coli and P. vulgaris together

was shown by 31, also a thiazole-containing compound

and the compound with the broadest spectrum of

activity overall. It is probable that the lower activity

with respect to Gram-negative bacteria is due to the

smaller pores in the bacterial cell wall compared with

the Gram-positive bacteria. Indeed, it is possible that

poor cell penetration may account for the low toxicity

of these compounds in the mammalian cell lines tested.

Streptococcus faecalis, and K. aerogenes but against no

other organism tested. The introduction of a larger alkyl

group such as isopentyl (7) or cyclopropylmethyl (5)

expanded the range of measured activity but without

higher activity being observed.

Thiophene-containing compounds (34-38) showed

some activity against bacteria and fungi over a wider

range of species than their immediate pyrrole-contain-

ing analogues. For example, 35, which contains one

thiophene in the center of the heterocyclic trimer, was

measurably active against P. vulgaris, whereas the

prototype compound 1 was inactive against this organ-

ism. Conversely, the activity of 1 was greater than that

of 35 against S. aureus and S. faecalis. Overall, C-

methylthiophenes show no particular benefit over N-

methylpyrroles, although branched C-alkylthiophenes

remain to be tested.

As has been noted already in the case of 19, the

introduction of a planar, hydrophobic head group can

enhance activity compared, for example, with 7. The

same effect is evident with the cinnamoyl head group

in 21, which shows much broader activity than the

parent compound 1. In this series, however, a 3-meth-

oxylbenzoyl head group alone, as in 18, was insufficient

to improve the activity compared with the parent

compound 1.

Although we found a significant influence of tail group

on the stoichiometry of binding to DNA in CE experi-

ments, no substantial beneficial influence on antimi-

crobial activity was apparent in these tests. In general,

larger tail groups than dimethylamino showed de-

creased activity. Pyrrolidinyl, however, permitted mea-

surable activity (48) greater than that of the respective

dimethylamino compound (3).

Taking all of the data together, it is interesting to note

that some antimicrobial activity is found in representa-

tives of almost all of the structural variations included.

The most significant cluster of activity, however, is in

those compounds that contain thiazoles (28-33). C-

Methylthiazoles have been used before in minor-groove

binders²⁵but the C-isopropyl group seems to be a novel

and significant substructure leading to high selectivity

and affinity. The detailed structural analysis of these

effects is presented elsewhere.¹⁸The presence of one

branched alkyl substituent also promotes activity (5, 7,

19, 23), as does a nonpolar, planar head group (19, 21,

33). These features are consistent with the structures

of compounds from other groups that show significant

antibacterial activity.⁷Notably, among the distamycin

analogues, a lipophilic head group (a chloroisothiazole)

gives high activity,^7band among the netropsin ana-

logues, the cyclopropylmethyl N-substituent is promi-

nent.^7aTail groups, which are known to be significant

in binding,²⁶have so far not been significantly useful

in improving activity, although more examples need to

be tried. It will be worthwhile to make a systematic

effort to optimize the activity of any of these variations

with respect to a given organism.

For Gram-positive organisms, several of the tested

organisms are examples of infecting agents that cur-

rently have a high profile with respect to drug resis-

tance. The activity of 19 (MIC ) 2.0 µM), which contains

two hydrophobic components (isopentyl side chain and

3-methoxybenzamide), and 31 (MIC ) 4.3 µM), which

contains two thiazole residues, against MRSA was

greater than that of the control, amoxycillin (MIC ) 16.1

µM). Several other compounds showed activity on the

order of magnitude of amoxycillin. The thiazole-contain-

ing compound 29 was also similar in activity to amoxy-

icillin against Enterobacter cloacae. Both 29 and 31

showed significant activity against Mycobacterium for-

tuitum, a fast growing relative of Mycobacterium tuber-

culosis, compared with streptomycin as a control.

With respect to antifungal activity, many compounds

were vastly superior to fluconazole and also similar in

potency or better than the more potent antifungal drug,

itraconazole. Against Aspergillus niger, the most active

was 31. In this test, not only thiazole-containing com-

pounds but also the larger alkylpyrrole-containing

compounds such as 4 and 40 were active. Aspergillus

nidulans was also susceptible to 31. Finally, in the panel

tested, the thiazole-containing compounds (28-33) in

general were similarly active to fluconazole and itra-

conazole against Candida albicans.

Con clu sion

As has been noted above, the thiazole-containing

compounds showed the greatest overall activity. How-

ever it is possible to perceive structural effects from the

other modifications also. The closest relative of dista-

mycin 1 was active against Staphylococcus aureus,

Within the scope of this series of trimers, significant

antimicrobial activity is clustered around a small num-

ber of structural features, namely branched N-alkylpyr-

roles, hydrophobic N-terminal amides, and especially

Distamycin Analogues with Enhanced Lipophilicity

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2139

) 1.6 Hz), 7.63 (1H, d, J ) 1.6 Hz). IR (KBr): 3138, 2970,

C-isopropylthiazoles. These clusters of activity are all

different embodiments of the design principle of increas-

ing hydrophobicity in the compounds; it could be that

this feature gives enhanced membrane permeability for

the microbes examined. It is also clear that such

structural features have a major influence on the

binding of compounds to DNA, as shown by our recent

NMR studies¹⁸and supported by the CE results re-

ported here. While a distinction between access to cells

and enhancement of specific binding as sources of the

antimicrobial activity observed cannot be made, it is

clear, however, that a wide range of structural variation

is possible to obtain significant activity and that the

concept of preparing lipophilic analogues of distamycin

as candidates for anti-infective compounds has a sound

basis.

1707, 1539, 1510, 1487, 1426, 1389, 1321, 1259, 1204, 1130,

1097 cm^-1

.

1-Isopen tyl-4-n itr o-1H-pyr r ole-2-car boxylic acid [(NO₂)-

P yr iP e-OH] was also prepared according to a published

procedure¹⁰in 95% yield, mp 154-157 °C (lit.¹⁰mp 154-156

1

°C). H NMR (CDCl₃): δ 0.98 (6H, d, J ) 6.5 Hz), 1.62-1.75

(3H, m), 4.38 (2H, t, J ) 7.5 Hz), 7.58 (1H, d, J ) 1.9 Hz),

7.69 (1H, d, J ) 1.9 Hz). IR (KBr): 3120, 1677, 1540, 1517,

1480, 1315, 1250 cm^-1

.

Eth yl 5-m eth ylp yr r ole-2-ca r boxyla te (P yr -OEt) was

prepared according to a published procedure²⁷in 38% yield,

mp 94-96 °C (lit.²⁶mp 97-99 °C).

Eth yl 5-m eth yl-1-m eth ylpyr r ole-2-car boxylate [P yr Me₂-

OEt] was prepared according to a published procedure²⁸in

91% yield as a pale yellow oil.

Eth yl 4-n itr o-5-m eth yl-1-m eth ylp yr r ole-2-ca r boxyla te

[NO₂P yr Me₂-OEt] was prepared according to a published

procedure in 84% yield, mp 74-76 °C (lit.²⁸mp 79-80 °C).

4-Nit r o-5-m et h yl-1-m et h ylp yr r ole-2-ca r b oxylic a cid

[NO₂P yr Me₂-OH] was prepared according to the literature

procedure in 66% yield, mp 225-228 °C (lit.²⁸mp 212-213

°C).

Exp er im en ta l Section

Abbr evia tion s. br, broad; s, singlet; d, doublet; t, triplet;

q, quartet; exch, exchangeable; DMF, N,N-dimethylformamide;

HBTU, O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium

hexafluorophosphate; HPLC, high performance liquid chro-

matography; HREIMS, high-resolution electron impact mass

spectroscopy; HRFABMS, high-resolution fast atom bombard-

ment mass spectroscopy; LRESMS, low-resolution electrospray

mass spectroscopy; Pd/C, palladium on carbon; TFA, trifluo-

roacetic acid. HREIMS and HRFABMS were obtained on a J eol

J MS-AX505HA mass spectrometer. LRESMS were obtained

on a Fisons VG Platform Benchtop LC-MS. NMR spectra were

obtained on a Bruker AMX 400 spectrometer. HPLC purifica-

tion of the final compounds was carried out using a Vydac

protein and peptide C18 column on a gradient eluting system.⁶

IR spectra were run as KBr disks and liquids as films, using

a Nicolet Impact 400D. Column chromatography was per-

formed with silica gel Prolabo (200-400 mesh).

In ter m ed ia tes. P yr r oles. Acid Ch lor id e Meth od . N-[3-

(Dim eth ylam in o)pr opyl]-1-isopr opyl]-4-n itr o-1H-pyr r ole-

2-ca r boxa m id e [(NO₂)P yr iP r -Dm a p ]. 4-Nitro-N-isopropyl-

pyrrole-2-carboxylic acid (312 mg, 1.574 mmol) was dissolved

in thionyl chloride (5 mL) and heated at reflux for 4 h. The

excess thionyl chloride was removed under reduced pressure

at 50 °C to give the acid chloride as a white solid, material

that was used without further purification. 3-(Dimethylamino)-

propylamine (250 µL) was dissolved in dichloromethane (25

mL, dry) to which N-methylmorpholine (250 µL) was added

at room temperature with stirring. The acid chloride was

dissolved in dichloromethane (5 mL, dry) and added dropwise

to the amine solution at room temperature with stirring. The

reaction mixture was then left stirring at room temperature

overnight. Following this, the solvent was removed under

reduced pressure at 50 °C and then the crude product was

extracted with aqueous potassium carbonate solution (25 mL,

10% w/v) and dichloromethane (2 × 50 mL). The organic layer

was collected, dried (Na₂SO₄), and filtered, and the solvent was

removed under reduced pressure. The crude product was

purified by chromatography over silica gel using 49.5:49.5:1

methanol/ethyl acetate/triethylamine to give the product as a

Ca r bod iim id e Meth od . N-[3-(Dim eth yla m in o)p r op yl]-

1,5-d im et h yl-4-n it r o-1H -p yr r ole-2-ca r b oxa m id e (NO₂-

P yr Me₂-Dm a p ). The carboxylic acid NO₂PyrMe₂-OH (100 mg,

0.543 mmol), EDCI (208 mg, 1.085 mmol), and DMAP (166

mg, 1.359 mmol) were dissolved in DMF (2 mL, dry). N,N-

Dimethylaminopropylamine (67 mg, 69 µL, 0.656 mmol) was

added to the reaction mixture with stirring at room temper-

ature. The reaction mixture was left stirring at room temper-

ature overnight before being diluted with ethyl acetate (50 mL)

and extracted with water (50 mL). The water layer was

extracted with ethyl acetate (2 × 50 mL). The organic layers

were combined and washed with saturated solution of sodium

bicarbonate (50 mL) and then with brine (50 mL). The ethyl

acetate layer was dried (MgSO₄) and the solvent removed

under reduced pressure at 50 °C. The crude product was

purified by silica gel column chromatography using methanol/

ethyl acetate (1/4, containing 1% triethylamine). The pure

product (R_f) 0.1) was collected, and the solvent removed was

removed under reduced pressure to give pale yellow oil (108

mg, 74%). ¹H NMR (CDCl₃): δ 1.71-1.76 (2H, quintet, J )

5.9 Hz, CH₂), 2.28 (6H, s, NMe₂), 2.49-2.52 (2H, t, J ) 5.9

Hz, CH₂), 2.64 (3H, s, C-CH₃), 3.45-3.50 (2H, q, J ) 5.9 Hz,

CH₂), 3.92 (3H, s, N-CH₃), 7.00 (1H, s), 8.47 (1H,broad, CONH,

exch). IR (KBr): 3131, 2964, 1655, 1546, 1523, 1494, 1300,

1141, 1104 cm^-1. HRFABMS found: 269.16200. Calculated for

C₁₂H₂₁N₄O₃: 269.16137.

2,2,2-Tr ich lor o-1-(1-m eth yl-4-n itr o-1H-p yr r ol-2-yl)eth -

a n on e Meth od . 1-Meth yl-4-n itr o-N-[3-(1-p yr r olid in yl)-

p r op yl]-1H-p yr r ole-2-ca r boxa m id e (NO₂P yr Me-P yr r p ).

3-(1-Pyrrolidinyl)propanamine (236 mg, 1.842 mmol) was

dissolved in THF (25 mL, dry) to which a solution of 2,2,2-

trichloro-1-(1-methyl-4-nitro-1H-pyrrol-2-yl)ethanone (508 mg,

1.842 mmol) in THF (2 mL, dry) was added at room temper-

ature with stirring. The reaction mixture was left stirring at

room temperature overnight. The solvent was removed under

reduced pressure and the crude product was purified by

column chromatography using 49.5:49.5:1 ethyl acetate/

methanol/triethylamine. The product was obtained as a glassy

yellow solid, R_f) 0.1 (503 mg, 98%), mp 113-115 °C. ¹H NMR

(DMSO-d₆): δ 1.70-1.79 (6H, m), 2.48-2.51 (6H, m), 3.28-

3.51 (2H, q, J ) 6.9 Hz), 3.89 (3H, s), 7.45 (1H, d, J ) 1.6 Hz),

8.18 (1H, d, J ) 1.6 Hz), 8.51 (1H, t, J ) 5.4 Hz). IR (KBr):

3131, 2966, 2794, 1654, 1545, 1525, 1492, 1305 cm^-1. HR-

FABMS found: 281.16249.Calculated for C₁₃H₂₁N₄O₃: 281.16137.

1

pale yellow oil, R_f) 0.25, (400 mg, 90%). H NMR (CDCl₃): δ

1.46 (6H, d, J ) 6.7 Hz), 1.71-1.77 (2H, quintet, J ) 6.0 Hz),

2.32 (6H, s), 2.51 (2H, t, J ) 5.7 Hz), 3.49 (2H, quintet, J )

5.0 Hz), 5.62 (1H, qt, J ) 6.7 Hz), 6.92 (1H, d, J ) 1.6 Hz),

7.73 (1H, d, J ) 1.6 Hz), 8.58 (1H, s). IR (KBr): 3330, 3136,

2979, 1650, 1535, 1430, 1279, 1258 cm^-1. HREIMS found:

282.16908. Calculated for C₁₃H₂₂N₄O₃: 282.16919.

N-[3-(Dim eth yla m in o)p r op yl]-1-isop en tyl-4-n itr o-1H-

p yr r ole-2-ca r boxa m id e (NO₂P yr iP e-Dm a p ) was prepared

using the acid chloride method as a pale yellow solid in 95%

E t h yl 1-isop en t yl-4-n it r o-1H -p yr r ole-2-ca r b oxyla t e

[(NO₂)P yr iP e-OEt] was prepared according to a published

procedure¹⁰in 85% yield, mp 46-47 °C (lit.¹⁰mp 46-47 °C).

¹H NMR (CDCl₃): δ 0.97 (6H, d, J ) 6.5 Hz), 1.38 (3H, t, J )

7.2 Hz), 1.61-1.73 (3H, m), 4.30-4.39 (4H, m), 7.43 (1H, d, J

1

yield, mp 72-73 °C. H NMR (CDCl₃): δ 0.95 (6H, d, J ) 6.5

Hz), 1.57-1.76 (5H, m), 2.32 (6H, s), 2.51 (2H, t, J ) 10.3 Hz),

3.47-3.51 (2H, quintet, J ) 4.8 Hz), 4.40-4.44 (2H, q, J )

2140 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8

Khalaf et al.

7.5 Hz), 6.92 (1H,d, J ) 1.9 Hz), 7.56 (1H,d, J ) 1.9 Hz), 8.61

(1H, s, br, CONH). IR (KBr): 1656, 1637, 1565, 1534, 1498,

1417, 1333 cm^-1. HREIMS found: 310.20031. Calculated for

Eth yl 1-eth yl-1H-im id a zole-2-ca r boxyla te (Im iEt-OEt)

was prepared according to a published procedure²⁹in 68%

yield, bp 110-115 °C /2 mmHg. ¹H NMR (CDCl₃): δ 1.41-

1.47 (6H, 2xt, J ) 7.0 Hz, 2CH₂CH₃), 4.38-4.43 (2H, q, J )

7.0 Hz, CH₂CH₃), 4.43-4.48 (2H, q, J ) 7.0 Hz, CH₂CH₃), 7.08

(1H, d, J ) 0.6 Hz), 7.15 (1H, d, J ) 0.6 Hz). IR (liq film):

3107, 2990, 2941, 1709, 1509, 1477, 1443, 1419, 1385, 1311,

C

₁₅H₂₆O₄N₃: 310.20049.

N-Alk yla tion Meth od . Eth yl 1-(Cyclop r op ylm eth yl)-4-

n itr o-1H-pyr r ole-2-car boxylate [(NO₂)P yr CycP r -OEt]. Eth-

yl 4-nitro-1H-pyrrole-2-carboxylate (1.042 g, 5.626 mmol) was

dissolved in DMF (20 mL, dry) to which potassium metal

(0.435 g, 11.124 mmol) was added. The reaction mixture was

heated to 100 °C with stirring and it was left at that

temperature for 1 h. The reaction mixture was cooled to 50-

60 °C, then (chloromethyl)cyclopropane (1.000 g, 11.04 mmol)

(purchased from Aldrich) and potassium iodide (1.349 g, 8.127

mmol) were added, and the reaction mixture was heated again

with stirring to 80 °C for 5 h, after which time it was left

stirring overnight at room temperature. The reaction mixture

was diluted with brine (50 mL) and extracted with ethyl

acetate (3 × 100 mL). The combined organic extracts were

collected, and the solvent was removed under reduced pres-

sure. The crude product obtained (containing some DMF) was

purified by flash chromatography using silica gel eluting with

1/10 ethyl acetate/n-hexane. The product was obtained as a

white microcrystalline solid, R_f) 0.2 (1.070 g, 80%), mp 65-

1260, 1236 cm^-1

E t h yl 1-E t h yl-4-n it r o-1H -im id a zole-2-ca r b oxyla t e

.

[(NO₂)Im iEt-OEt] a n d Eth yl 1-Eth yl-5-n itr o-1H-im id a -

zole-2-ca r boxyla te. Ethyl 1-ethyl-1H-imidazole-2-carboxylate

(12.9 g, 76.8 mmol) was carefully dissolved in sulfuric acid (30

mL, concentrated) and cooled to 0 °C. Fuming nitric acid (30

mL) was slowly added while a temperature of 0 °C was

maintained. The reaction mixture was then heated under

reflux with an efficient condenser (ethyl acetate/solid carbon

dioxide) for 30 min. The reaction mixture was cooled with an

ice bath and quenched by adding ice. The resulting blue

solution was then extracted with dichloromethane (3 × 100

mL), dried over MgSO₄, and concentrated under reduced

pressure to give pale yellow, semisolid material, which was

chromatographed over silica gel to give the two isomers:

Fraction 1: 3.16 g, 19%, mp 45-47 °C. 1-Ethylimidazole-

1

66 °C. H NMR (DMSO-d₆): δ 8.34 (1H, d, J ) 1.3 Hz), 7.35

5-nitro-2-carboxylic acid ethyl ester: ¹H NMR (CDCl₃):

δ

(1H, d, J ) 1.3 Hz), 4.30-4.24 (2H, q, J ) 7.1 Hz), 4.21 (2H,

d, J ) 7.2 Hz), 1.34-1.23 (2H, t, J ) 7.1 Hz; 1H, m), 0.53-

0.45 (2H, m), 0.42-0.38 (2H, m). IR (KBr): 3142, 2996, 2921,

1704, 1502, 1310, 1270, 1225, 1184, 1091 cm^-1. HREIMS

found: 238.09585. Calculated for C₁₁H₁₄N₂O₄: 238.09536.

1.43-1.50 (3H, t, J ) 7.0 Hz, CH₂CH₃), 1.51-1.58 (3H, t, J )

7.0 Hz, CH₂CH₃), 4.45-4.56 (2H, q, J ) 7.0 Hz, CH₂CH₃),

4.90-4.95 (2H, q, J ) 7.0 Hz, CH₂CH₃), 8.04 (1H, s). IR

(KBr): 3117, 2982, 1724, 1535, 1492, 1478, 1444, 1384, 1353,

1282, 1234, 1147 cm^-1. HRFABMS found: 214.08208. Calcu-

lated for C₈H₁₂N₃O₄: 214.08278.

Ester Hyd r olysis Meth od . 1-(Cyclop r op ylm eth yl)-4-

n it r o-1H -p yr r ole-2-ca r b oxylic Acid [(NO₂)P yr CycP r -

OH]. Ethyl 1-(cyclopropylmethyl)-4-nitro-1H-pyrrole-2-car-

boxylate (482 mg, 2.023 mmol) was dissolved in ethanol (4 mL)

to which a solution of sodium hydroxide (393 mg, 9.825 mmol)

in water (10 mL) was added. The reaction mixture was heated

under reflux for 2 h and then cooled to 0 °C. Concentrated

hydrochloric acid was added dropwise with stirring until the

solution reached pH 2. The white solid material, which

precipitated was filtered off, washed with water, and dried

under reduced pressure at 60 °C overnight. The product was

obtained as a white solid material (382 mg, 90%), mp 215-

Fraction 2: 5.70 g, 35%, mp 90-92 °C. 1-Ethylimidazole-

4-nitro-2-carboxylic acid ethyl ester [(NO₂)ImiEt-OEt]: ¹H

NMR (CDCl₃): δ 1.43-1.46 (3H, t, J ) 7.0 Hz, CH₂CH₃), 1.53-

1.56 (3H, t, J ) 7.0 Hz, CH₂CH₃), 4.44-4.49 (2H, t, J ) 7.0

Hz, CH₂CH₃), 4.52-4.58 (2H, t, J ) 7.0 Hz, CH₂CH₃), 7.89

(1H, s). IR (KBr): 3140, 2993, 1725, 1535, 1496, 1389, 1346,

1320, 1270, 1231, 1138, 1080, 1015 cm^-1. HRFABMS found:

214.08296. Calculated for C₈H₁₂N₃O₄: 214.08278.

1-Eth yl-4-n itr o-1H-im id a zole 2-ca r boxylic Acid Hy-

d r och lor id e [(NO₂)Im iEt-OH]. Ethyl 1-ethyl-4-nitro-1H-

imidazole-2-carboxylate (253 mg, 1.188 mmol) was suspended

in a sodium hydroxide solution [sodium hydroxide 186 mg, 4.65

mmol and water (2 mL)]. The reaction mixture was heated

until reflux for (15 min) then cooled to 15 °C and hydrochloric

acid (dilute) was added with stirring (pH 1). The solution was

freeze-dried to give a light brown solid (175 mg, 80%). ¹H NMR

(DMSO-d₆): δ 1.31-1.35 (3H, t, J ) 7.0 Hz, CH₂CH₃), 4.45-

4.51 (2H, q, J ) 7.0 Hz, CH₂CH₃), 8.37 (1H, s). IR (KBr): 3430,

3255, 3160, 1730, 1543, 1504, 1393, 1352, 1279, 1218, 1148

1

217 °C. H NMR (DMSO-d₆): 13.14 (1H, br), 8.29 (1H, d, J )

2.4 Hz), 7.29 (1H, d, J ) 2.1 Hz), 4.20 (2H, d, J ) 2.2 Hz),

1.34-1.27 (1H, m), 0.52-0.38 (4H, m). IR (KBr): 3115, 3005,

2922, 2854, 1680, 1540, 1517, 1481, 1420, 1314 cm^-1. HREIMS

found: 210.06469. Calculated for C₉H₁₀N₂O₄: 210.06406.

Eth yl 1-cyclop en tyl-4-n itr o-1H-p yr r ole-2-ca r boxyla te

[(NO₂)P yr CycP r -OEt] was prepared by the N-alkylation

method as a pale yellow oil in 32% yield, using bromocyclo-

propane as the alkylating agent. ¹H NMR (CDCl₃): δ 7.77 (1H,

d, J ) 2.0 Hz), 7.45 (1H, d, J ) 2.0 Hz), 5.57-5.50 (1H, quintet,

J ) 6.6 Hz), 4.33 (2H, q, J ) 7.1 Hz), 2.30-2.24 (2H, m), 1.98-

1.75 (6H, m), 1.37 (3H, t, J ) 7.1 Hz). IR (KBr): 2965, 2875,

cm^-1

.

Mixed An h yd r id e Met h od . N-[3-(Dim et h yla m in o)-

p r op yl]-1-e t h yl-4-n it r o-1H -im id a zole -2-ca r b oxa m id e

[(NO₂)Im iEt-Dm a p ]. The carboxylic acid NO₂ImiEt-Dmap

(230 mg, 1.243 mmol) was dissolved in THF (25 mL, dry) to

which triethylamine (500 µL, dry) was added followed by

pivaloyl chloride (400 µL, 3.30 mmol) with stirring at room

temperature. Stirring was continued for 15 min, then N,N-

dimethylaminopropylamine (400 µL, 3.30 mmol) was added

at room temperature with stirring. Stirring was continued

overnight at room temperature. The solvent was removed

under reduced pressure and the residue was extracted with

ether and sodium hydroxide solution (40 mL, 1 N). The organic

layer was extracted with brine and dried (MgSO₄), and the

solvent was then removed under reduced pressure. The crude

product was purified by column chromatography using silica

gel, following which it was eluted with methanol/ethyl acetate

1/4 (R_f) 0.1). The product was obtained as a colorless oil,

which crystallized on standing at room temperature (208 mg,

1717, 1534, 1508, 1426, 1335, 1294, 1222, 1187, 1084 cm^-1

.

HRFABMS found: 253.11824. Calculated for C₁₂H₁₇N₂O₄:

253.11883.

1-Cyclop en t yl-4-n it r o-1H -p yr r ole-2-ca r b oxylic a cid

[(NO₂)P yr Cycp e-OH] was prepared by the ester hydrolysis

method as a white solid in 89% yield, mp 195-198 °C. ¹H NMR

(DMSO-d₆): δ 13.13 (1H, br), 8.27 (1H, d, J ) 2.0 Hz), 7.29

(1H, d, J ) 2.0 Hz), 5.45 (1H, quintet, J ) 7.1 Hz), 2.16-2.07

(2H, m), 1.88-1.78 (4H, m), 1.65-1.61 (2H, m). IR (KBr):

3152, 2964, 2880, 1683, 1508, 1430, 1331, 1292, 1078, 911

cm^-1. HREIMS found: 224.07926. Calculated for C₁₀H₁₂

N₂O₄: 224.07971.

-

All remaining pyrroles were samples prepared previously

or as previously described.⁶

Im id a zoles. 1-Eth yl-1H-im id a zole (Im iEt) was prepared

according to a published procedure¹⁶in 90% yield, bp 110 °C/

1

79%), mp 72-75 °C. H NMR (CDCl₃): δ 1.51-1.53 (3H, t, J

16 mmHg. H NMR (CDCl₃): δ 1.43-1.47 (3H, t, J ) 7.4 Hz,

) 7.0 Hz, CH₂CH₃), 1.75-1.80 (2H, quintet, J ) 7.0 Hz CH₂),

2.28 (6H, s, NMe₂), 2.40-2.44 (2H, t, J ) 7.0 Hz, CH₂), 3.45-

3.50 (2H, q, J ) 7.0 Hz, CH₂), 4.60-4.65 (2H, q, CH₂CH₃), 7.82

(1H, s), 8.42 (1H,s, CONH, exch). IR (KBr): 3099, 2947, 2821,

CH₂CH₃), 3.96-4.02 (2H, q, J ) 7.4 Hz, CH₂CH₃), 6.92 (1H, t,

J ) 1.1 Hz), 7.05 (1H, s), 7.48 (1H, s). IR (liq film): 3105, 2979,

2937, 2885, 1682, 1598, 1511, 1464, 1395, 1356, 1286, 1228,

1286 cm^-1

.

Distamycin Analogues with Enhanced Lipophilicity

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2141

1672, 1534, 1453, 1416, 1347, 1314, 1250, 1158, 833,762 cm^-1

HRFABMS found: 270.15778. Calculated for C₁₁H₂₀N₅O₃:

270.15661.

Th ia zoles. Meth yl 2-a m in o-5-isop r op yl-1,3-th ia zole-4-

ca r boxyla te (ThziPr-OMe) was prepared according to a

published procedure¹²in 41% yield, mp 151-152 °C. (lit.¹²mp

150-151 °C).

Eth yl 2-a m in o-4-isop r op yl-1,3-th ia zole-5-ca r boxyla te

(isoTh ziP r -OEt) was prepared according to a literature

procedure¹²in 49% yield, mp 176-178 °C (lit.¹²mp 176-178

°C).

.

s, Ar-CH₃), 3.42-3.49 (2H, q, J ) 7.4 Hz, CH₂NHCO), 4.45-

4.46 (2H, d, J ) 5.2 Hz, Ar-CH₂-NHCO₂), 5.16 (2H, s, CH₂O),

5.35 (1H, s, CONH, exch), 7.33-7.37 (5H, m, Ar-H), 7.61 (1H,

s, CONH, exch). IR (liquid film): 3296, 2947, 2819, 2779, 1720,

1653, 1529, 1455, 1045 cm^-1. HREIMS found: 374.195228.

Calculated for C₁₉H₂₆N₄O₄: 374.19541.

Dim er s. Sta n d a r d P r oced u r e for Red u ction of Nitr o

Gr ou p s of Mon om er s. The appropriate nitromonomer (3

mmol) was dissolved in ethanol (30 mL), and palladium on

charcoal (10%, 500 mg) was added. Hydrogenation was carried

out at room temperature and atmospheric pressure for 4 h.

The catalyst was removed by filtration through Kieselguhr and

the filtrate evaporated under reduced pressure to yield the

required amine, which was used forthwith for coupling.

Th iop h en es. Eth yl 2-Meth yl-4,5-d ih yd r o-3-th iop h en e-

ca r boxyla te. A standard procedure¹⁴was employed to give

the product as a colorless oil (96% yield).

Sta n d a r d P r oced u r e for P r ep a r in g Acid Ch lor id es of

Mon om er s. The appropriate monomer carboxylic acid (2

mmol) was dissolved in thionyl chloride (3 mL) and the solution

heated under reflux for 3 h. The solution was evaporated to

dryness and the product used directly for coupling.

Sta n d a r d P r oced u r e for Acid Ch lor id e Cou p lin g. The

appropriate acid chloride in THF or dichloromethane solution

was added to a solution of the amino monomer, prepared as

described for reduction above, in the same solvent. N-Meth-

ylmorpholine was used as the standard base and the reaction

time was typically 2-12 h.

Eth yl 2-Meth yl-3-th iop h en eca r boxyla te (Th iMe-OEt).

A standard procedure¹⁴was employed to give the product as

a colorless oil (82% yield).

2-Meth yl-3-th iop h en eca r boxylic a cid (Th iMe-OH) was

prepared according to a published method in 77% yield, mp

115-117 °C (lit.³⁰mp 116-117 °C).

2-Meth yl-5-n itr o-3-th iop h en eca r boxylic Acid [(NO₂)-

Th iMe-OH]. A mixture of concentrated nitric acid (10 mL,

specific gravity 1.42) and concentrated sulfuric acid (6 mL) was

mechanically stirred and cooled to (-10 °C) by a dry ice-

methanol bath. The temperature was kept below -5 °C while

2-methyl-3-thiophenecarboxylic acid (996 mg, 7.006 mmol) was

added in small portions. The reaction mixture was stirred at

the same temperature for 15 min and then was poured on to

ice water. The solid material that precipitated was filtered off,

washed with water, and dried to give light brown solid (883

mg, 67%), mp 177-180 °C. ¹H NMR (DMSO-d₆): δ 13.35 (1H,

br), 8.11 (1H, s), 2.76 (3H, s). IR (KBr): 1706, 1543, 1514, 1457,

1335, 1257 cm^-1. HREIMS found: 186.99431. Calculated for

C₆H₅NO₄S: 186.99393.

Oxa zoles. Meth yl 2-({[(ben zyloxy)ca r bon yl]a m in o}-

m eth yl)-5-m eth yl-1,3-oxa zole-4-ca r boxyla te was prepared

according to a literature procedure¹⁵in 74% yield as a pale

yellow crystalline solid, mp 85-86 °C (lit.¹⁵mp 86-87 °C).

2-({[(Ben zyloxy)ca r bon yl]a m in o}m eth yl)-5-m eth yl-1,3-

oxa zole-4-ca r boxylic Acid . The foregoing ester (650 mg,

2.138 mmol) was dissolved in methanol (15 mL) to which

sodium hydroxide (428 mg) in water (5 mL) was added at room

temperature with stirring. The stirring was continued over-

night at room temperature. The solution was concentrated to

a minimium amount, cooled to 0 °C, and diluted with water

(30 mL). Hydrochloric acid (concentrated) was added dropwise

with stirring at 0 °C until pH 1. The pale yellow solid was

collected and dried under reduced pressure at 60 °C overnight

to give the required product (471 mg, 76%), mp 177-180 °C.

¹H NMR (DMSO-d₆): δ 2.50 (3H, s, Me), 4.21 (2H, t, J ) 8.6

Hz, CH₂NH), 5.02 (2H, s, CH₂Ph), 7.28-7.32 (5H, m, PhH),

7.90-7.92 (1H, t, J ) 5.6 Hz, CONH, exch), 12.80 (1H, s, CO₂H,

exch). IR (KBr): 3336, 3068, 2927, 2851, 1696, 1619, 1542,

1447, 1344, 1274 cm^-1. HREIMS found: 276.07348. Calculated

for C₁₃H₁₂N₂O₅: 276.07462.

Ben zyl [4-({[3-(Dim eth yla m in o)p r op yl]a m in o}ca r bon -

yl)-5-m eth yl-1,3-oxa zol-2-yl]m eth ylca r ba m a te. Triethyl-

amine (101 µL, 0.724 mmol) and trimethylacetyl chloride (89

µL, 0.724 mmol) were added sequentially to a stirred solution

of the oxazole carboxylic acid (100 mg, 0.362 mmol) in THF

(10 mL, dry) at 0 °C. The mixture was stirred for 30 min at 0

°C, a solution of the amine (91 µL, 0.724 mmol) in THF (2 mL,

dry) was added, then the reaction was stirred for 45 min and

partitioned between ethyl acetate (30 mL) and water (30 mL).

The aqueous layer was further extracted with ethyl acetate

(2 × 30 mL). The organic extracts were combined, washed

sequentially with saturated aqueous sodium hydrogen carbon-

ate (50 mL) and brine (50 mL), dried (MgSO₄), and then

evaporated under reduced pressure before purification by flash

chromatography on silica followed by eluting with methanol/

ethyl acetate (1/3 containing 1% triethylamine, R_f) 0.2). The

product was obtained as a colorless oil (90 mg, 67%). ¹H NMR

(CDCl₃): δ 1.71-1.78 (2H, quintet, J ) 6.8 Hz, CH₂), 2.24 (6H,

s, NMe₂), 2.38-2.41 (2H, t, J ) 6.8 Hz, CH₂NMe₂), 2.61 (3H,

N-[5-({[3-(Dim eth yla m in o)p r op yl]a m in o}ca r bon yl)-1-

m eth yl-1H-p yr r ol-3-yl]1-eth yl-4-n itr o-1H-p yr r ole-2-ca r -

boxa m id e. (NO₂)PyrEt-PyrMe-Dmap was prepared using the

acid chloride method. The crude product was chromatographed

over silica gel using ethyl acetate/methanol/ammonia (49/49/

2) as eluant. The required product was obtained as a yellow

solid (499 mg, 60%), which was further purified by trituration

with a small amount of ethyl acetate/n-hexane, mp 172-174

°C. ¹H NMR (DMSO-d₆): δ 1.33-1.37 (3H, t, J ) 7.2 Hz),

1.62-1.66 (2H, m), 2.22 (6H, s), 2.33-2.36 (2H, t, J ) 6.8 Hz),

3.17-3.22 (2H, quintet, J ) 1.6 Hz), 3.81 (3H, s), 4.41-4.46

(2H, q, 7.2 Hz), 6.84 (1H, d, J ) 2.0 Hz), 7.20 (1H, d, J ) 2.0

Hz), 7.57 (1H, d, J ) 2.0 Hz), 8.09-8.12 (1H, t, 5.6 Hz), 8.23

(1H, d, J ) 2.0 Hz), 10.24 (1H, s). IR (KBr): 3428, 3275, 3135,

1658, 1624, 1570, 1539, 1496, 1439, 1307 cm^-1. HRFABMS

found: 391.20942. Calculated for C₁₈H₂₇N₆O₄: 391.20938.

N-[5-({[3-(Dim eth yla m in o)p r op yl]a m in o}ca r bon yl)-1-

m eth yl-1H-p yr r ol-3-yl]-1-isop r op yl-4-n itr o-1H-p yr r ole-2-

ca r boxa m id e. (NO₂)PyriPr-PyrMe-Dmap was prepared using

the acid chloride method. The crude product was purified by

chromatography on silica gel using ethyl acetate/methanol/

ammonia (49/49/2). Fractions with R_fvalue of 0.3 were

collected. The product was obtained as a yellow solid (640 mg,

49%), mp 205-207 °C. Found: C, 56.25, H, 6.78, N, 20.59.

Calculated for C₁₉H₂₈N₆O₄: C, 56.42, H, 6.98, N, 20.78. ¹H

NMR (DMSO-d₆): δ 1.42-1.57 (6H, d, J ) 6.7 Hz), 1.60-1.65

(2H, quintet, J ) 6.7 Hz), 2.12 (6H, s), 2.20-2.25 (2H, t, 7.0

Hz), 3.17-3.22 (2H, q, J ) 6.7 Hz), 3.81 (3H, s), 5.40-5.51

(1H, m), 6.83 (1H, s), 7.22 (1H, s), 7.48 (1H, s), 8.11-8.15 (1H,

t, J ) 5.8 Hz), 8.34 (1H, s), 10.29 (1H, s). IR (KBr): 1640, 1555,

1506, 1480, 1322, 1279, 1240, 1203 cm^-1. HRFABMS found:

405.22509. Calculated: 405.22503.

N-[5-({[3-(Dim eth yla m in o)p r op yl]a m in o}ca r bon yl)-1-

isopr opyl-1H-p yr r ol-3-yl]-1-isop r op yl-4-n itr o-1H-p yr r ole-

2-ca r boxa m id e. (NO₂)PyriPr-PyriPr-Dmap was prepared

using the acid chloride in THF solution in the presence of

N-methylmorpholine. The crude product was chromatographed

on silica gel using 50:50:0.5 methanol/ethyl acetate/triethy-

lamine. Fractions with R_fvalue of 0.1 were collected. The

solvent was removed at 50 °C under reduced pressure to give

the product as a yellow glassy material (471 mg, 77%), mp

86-90 °C (softening). ¹H NMR (CDCl₃): δ 1.42 (6H, d, 6.7 Hz),

1.75 (6H, d, J ) 6.7 Hz), 1.72-1.77 (2H, m), 2.31 (6H, s), 2.46

(2H, t, J ) 6.1 Hz), 3.44-3.49 (2H, q, J ) 6.0 Hz), 5.51-5.64

(2H, m), 6.45 (1H, d, 1.6 Hz), 7.17 (1H, d, J ) 1.6 Hz), 7.40

(1H, d, J ) 1.6 Hz), 7.69 (1H, s, br, CONH), 7.79 (1H, d, J )

1.6 Hz). IR (KBr): 2974, 2927, 1636, 1574, 1531, 1507, 1407,

1282, 1237, 1170 cm^-1. HREIMS found: 432.24833. Calculated

for C₂₁H₃₂N₆O₄: 432.24850.

2142 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8

Khalaf et al.

1-Me t h y l-N -[1-m e t h y l-5-({[3-(1-p y r r o lid in y l)p r o p -

yl]a m in o}ca r bon yl)-1H-p yr r ol-3-yl]-4-n itr o-1H-p yr r ole-2-

ca r boxa m id e. (NO₂)PyrMe-PyrMe-Pyrrp was prepared using

2,2,2-trichloro-1-(1-methyl-4-nitro-1H-pyrrol-2-yl)ethanone (Pyr-

Me-CCl₃) in THF solution. The crude product was purified by

chromatography on silica gel using 49.5:49.5:1 ethyl acetate/

methanol/triethylamine. The product was obtained as a glassy

silica gel column chromatography using methanol/ethyl acetate

(1/4, containing 1% triethylamine). The pure product (R_f) 0.1)

was collected and the solvent removed under reduced pressure

to give pale yellow oil (90 mg, 57%), mp 90-95 °C (softening).

¹H NMR (DMSO-d₆): δ 1.58-1.65 (2H, quintet, J ) 5.9 Hz,

CH₂), 2.10 (3H, s, C-CH₃), 2.19 (6H, s, NMe₂), 2.29-2.31 (2H,

t, J ) 5.9 Hz, CH₂), 2.61 (3H, s, C-CH₃), 3.15-3.20 (2H, q, J

) 5.9 Hz, CH₂), 3.77 (3H, s, NMe), 3.85 (3H, s, NMe), 6.80

(1H, s), 7.56 (1H, s), 7.94-7.97 (1H, t, J ) 5.6 Hz, CONH,

exch), 9.52 (1H, s, CONH, exch). IR (KBr): 3419, 2940, 1651,

1591, 1523, 1458, 1412, 1316, 1256, 1183 cm^-1. HRFABMS

found: 405.22659. Calculated for C₁₉H₂₉N₆O₄: 405.22503.

1

yellow material (192 mg, 56%), R_f) 0.1, mp 170-173 °C. H

NMR (DMSO-d₆) δ 1.61-1.67 (6H, m), 2.39-2.42 (6H, m),

3.18-3.23 (2H, q, J ) 6.8 Hz), 3.81 (3H, s), 3.95 (3H, s), 6.81

(1H, d, J ) 1.6 Hz), 7.18 (1H, d, J ) 1.6 Hz), 7.56 (1H, d, J )

1.6 Hz), 8.08 (1H, t, J ) 5.37 Hz), 8.16 (1H, s), 10.20 (1H,s).

IR (KBr): 1661, 1621, 1572, 1537, 1495, 1303 cm^-1. HRFABMS

found: 403.20555. Calculated for C₁₉H₂₇N₆O₄: 403.20938.

N-[5-({[3-(Dim eth yla m in o)p r op yl]a m in o}ca r bon yl)-1-

m eth yl-1H-p yr r ol-3-yl]-1-isop en tyl-4-n itr o-1H-p yr r ole-2-

ca r boxa m id e [(NO₂)P yr iP e-P yr Me-Dm a p ] was prepared

by the acid chloride method in dichloromethane solution in

the presence of N-methylmorpholine. The product was purified

by column chromatography using 1/3/0.030 methanol/ethyl

acetate/triethylamine. Fractions containing the product (R_f)

0.20) were collected and the solvents were removed under

reduced pressure at 50 °C to give the product as a yellow

N-[3-(Dim et h yla m in o)p r op yl]-1-isop en t yl-4-{[(1-iso-

p en t yl-4-n it r o-1H -p yr r ol-2-yl)ca r b on yl]a m in o}-1H -p yr -

r ole-2-ca r boxa m id e. (NO₂)PyriPe-PyriPe-Dmap was pre-

pared using the acid chloride method in THF solution in the

presence of N-methylmorpholine. The crude product was

purified by column chromatography using ethyl acetate/

methanol/triethylamine 100:100:1. Fractions containing the

required material were collected, and the solvent was then

removed under reduced pressure and coevaporated with n-

hexane to give the pure product as a glassy yellow material,

R_f) 0.34, (223 mg, 94%), mp 57-60 °C (transparent). ¹H NMR

(CDCl₃): δ 0.92616 (6H, d, J ) 4.5 Hz), 0.95 (6H, d, J ) 4.5

Hz), 1.57-1.83 (4H, m), 2.35 (6H, s), 2.52 (2H,t, J ) 6.1 Hz),

3.45-3.49 (2H, q, J ) 5.6 Hz), 4.36 (2H, t, J ) 7.4 Hz), 4.43

(2H, t, J ) 7.4 Hz), 6.51 (1H, d, J ) 1.9 Hz), 7.20 (1H, d, J )

1.9 Hz), 7.22 (1H, d, J ) 1.9 Hz), 7.64 (1H, d, J ) 1.9 Hz),

7.77 (2H, m). IR (KBr): 2953, 1633, 1592, 1573, 1539, 1525,

1503, 1312 cm^-1. HREIMS found: 488.31163. Calculated for

1

powder (347 mg, 81%), mp 148-151 °C. H NMR (DMSO-d₆):

δ 10.22 (1H, s), 8.23 (1H, d, J ) 1.8 Hz), 8.13-8.10 (1H, t, J

) 5.5 Hz), 7.54 (1H, d, J ) 1.8 Hz), 7.19 (1H, d, J ) 1.8 Hz),

6.81 (1H, d, J ) 1.8 Hz), 4.45-4.41 (2H, t, J ) 7.3 Hz), 3.81

(3H, s), 3.21-3.17 (2H, q, J ) 6.7 Hz), 2.34-2.31 (2H, t, J )

7.1 Hz), 2.20 (6H, s), 1.67-1.57 (4H, m), 1.55-1.45 (1H, m),

0.88-0.86 (6H, d, J ) 7.5 Hz). IR (KBr): 1665, 1642, 1599,

1528, 1499,1425, 1312 cm^-1. HRFABMS found: 433.25630.

Calculated for: C₂₁H₃₃N₆O₄433.25633.

1-(Cyclopr opylm eth yl)-H-[5-({[3-(dim eth ylam in o)pr op-

yl]a m in o}ca r bon yl)-1-m eth yl-1H-p yr r ol-3-yl]-4-n itr o-1H-

p yr r ole-2-ca r b oxa m id e [(NO₂)P yr CycP r -P yr Me-Dm a p ]

was prepared using HBTU in DMF solution in the presence

of N-methylmorpholine. Purification by flash column chroma-

tography over silica gel using 1/2/0.1 methanol/ethyl acetate/

triethylamine gave the required product as a glassy yellow

material, R_f) 0.5 (130 mg, 80%) with no distinct melting point.

¹H NMR (DMSO-d₆): δ 10.26 (1H, s), 8.24 (1H, d, J ) 1.9 Hz),

8.10 (1H, t, J ) 5.6 Hz), 7.57 (1H, d, J ) 1.9 Hz), 7.21 (1H, d,

J ) 1.9 Hz), 6.83 (1H, d, J ) 1.9 Hz), 4.27 (2H, d, J ) 7.2 Hz),

3.81 (3H, s), 3.21-3.16 (2H, q, J ) 6.9 Hz), 2.28 (2H, t, J )

7.1 Hz), 2.17 (6H, s), 1.65-1.59 (2H, quintet, J ) 7.1 Hz), 1.34-

1.29 (1H, m), 0.51-0.38 (4H, m). IR (KBr): 3283, 3126, 2944,

C

₂₅H₄₀O₄N₆: 488.31110.

1-Isopr opyl-N-[1-m eth yl-5-({[3-(4-m or oph olin yl)pr opyl]-

a m in o}ca r b on yl)-1H -p yr r ol-3-yl]-4-n it r o-1H -p yr r ole-2-

ca r boxa m id e. (NO₂)PyriPr-PyrMe-Morp was prepared using

HBTU in the presence of N-methylmorpholine in DMF solu-

tion. The crude product was chromatographed over flash silica

gel using 1/3 methanol/ethyl acetate. Fractions containing the

product (R_f) 0.30) were collected and the solvents were

removed under reduced pressure at 50 °C. The product was

obtained as a hygroscopic glassy yellow material (260 mg, 73%)

1

with no distinct melting point. H NMR (DMSO-d₆): δ 10.28

(1H, s), 8.33 (1H, d, J ) 1.8 Hz), 8.07-8.05 (1H, t, J ) 5.5

Hz), 7.48 (1H, d, J ) 1.8 Hz), 7.19 (1H, d, J ) 1.8 Hz), 6.85

(1H, d, J ) 1.8 Hz), 5.89-5.42 (1H, heptet, 6.7 Hz), 3.80 (3H,

s), 3.58-3.56 (4H, t, J ) 4.5 Hz), 3.21-3.16 (2H, q, J ) 6.6

Hz), 2.32-2.28 (6H, m), 1.67-1.61 (2H, quintet, J ) 7.0 Hz),

1.44-1.43 (6H, d, J ) 6.7 Hz). IR (KBr): 1661, 1582, 1540,

1441, 1389, 2326, 1284, 1242, 1116, 850, 755 cm^-1. HREIMS

found: 446.22568. Calculated for C₂₁H₃₀N₆O₅:446.22777.

1642, 1573, 1532, 1504, 1463, 1427, 1389, 1309, 1233 cm^-1

.

HRFABMS found: 417.22460. Calculated for C₂₀H₂₉N₆O₄:

417.22503.

1-Cyclopen tyl-N-[5-({[3-(dim eth ylam in o)pr opyl]am in o}-

ca r bon yl)-1-m eth yl-1H-p yr r ol-3-yl]-4-n itr o-1H-p yr r ole-2-

ca r boxa m id e [(NO₂)P yr CycP e-P yr Me-Dm a p ] was pre-

pared using HBTU in DMF in the presence of N-methyl-

morpholine. The crude product was purified by column chro-

matography using silica gel and 1/2/0.1 methanol/ethyl acetate/

triethylamine. The product was obtained as a yellow oil, R_f)

1-Isop r op yl-N-[1-m eth yl-5-({[3-(1-p yr r olid in yl)p r op yl]-

a m in o}ca r b on yl)-1H -p yr r ol-3-yl]-4-n it r o-1H -p yr r ole-2-

ca r boxa m id e. (NO₂)PyriPr-PyrMe-Pyrrp was prepared using

HBTU in the presence of N-methylmorpholine in DMF solu-

tion. The product was purified by column chromatography

using 1/2/0.1 methanol/ethyl acetate/triethylamine. The prod-

uct was obtained as a yellow material (300 mg, 86%) with no

distinct melting point. Some of this material was further

1

0.45 (157 mg, 93%). H NMR (DMSO-d₆): 10.27 (1H, s), 8.24

(1H, d, J ) 1.7 Hz), 8.11 (1H, t, J ) 5.2 Hz), 7.47 (1H, s), 7.20

(1H, d, J ) 1.7 Hz), 6.83 (1H, d, J ) 1.7 Hz), 5.48 (1H, quintet,

J ) 7.4 Hz), 3.80 (3H, s), 3.18 (2H, q, J ) 6.6 Hz), 2.66 (2H,

m), 2.30 (2H, t, J ) 7.1 Hz), 2.14 (6H, s), 2.12 (2H, m), 1.82

(4H, m), 1.62 (2H, quintet, J ) 7.1 Hz). IR (KBr): 1644, 1575,

1534, 1504, 1437, 1401, 1313, 1287, 748 cm^-1. HRFABMS

found: 431.24204. Calculated for C₂₁H₃₁N₆O₄: 431.24068.

1

purified by HPLC. H NMR (DMSO-d₆): δ 10.30 (1H, s), 9.47

(1H, br, TFA), 8.34 (1H, d, J ) 1.8 Hz), 8.21-8.18 (1H, t, J )

5.5 Hz), 7.48 (1H, d, J ) 1.8 Hz), 7.27 (1H, d, J ) 1.8 Hz),

6.92 (1H, d, J ) 1.8 Hz), 5.48-5.41 (1H, heptet, J ) 6.7 Hz),

3.82 (3H, s), 3.56-3.54 (2H, m), 3.27-3.22 (2H, q, J ) 6.6 Hz),

3.17-3.11 (2H, m), 2.99-2.96 (2H, m), 2.01 (2H, m), 1.86-

1.82 (4H, m), 1.45-1.43 (6H, d, J ) 6.7 Hz). IR (KBr): 3433,

1672, 1650, 1535, 1462, 1426, 1371, 1321, 1283, 1237, 1200,

1178, 1131 cm^-1. HRFABMS found: 430.23179. Calculated for

N-[3-(Dim eth yla m in o)p r op yl]-1-eth yl-4-{[(1-m eth yl-4-

n it r o-1H -p yr r ol-2-yl) ca r bon yl]a m in o}-1H -im id a zole-2-

ca r boxa m id e [(NO₂)P yr Me-Im iEt-Dm a p ] was prepared

using acid chloride coupling in dichloromethane solution. The

crude product was purified by reverse phase column chroma-

tography using acetonitrile/water/hydrochloric acid (25:100:

1, R_f) 0.15). Fractions containing the product were combined

and freeze-dried to give the required product as a pale yellow

solid (slightly hygroscopic) (87 mg, 55%) as the hydrochloric

C

₂₁H₃₀N₆O₄: 430.23285.

N-[3-(Dim eth ylam in o)pr opyl]-4-{[(1,5-dim eth yl-4-n itr o-

1H-pyr r ol-2-yl)car bon yl]am in o}-1,5-dim eth yl-1H-pyr r ole-

2-ca r boxa m id e [(NO₂)P yr Me₂-P yr Me₂-Dm a p ] was pre-

pared using carbodiimide coupling (EDC) in DMF solution in

the presence of DMAP. The crude product was purified by

1

acid salt with no distinct melting point. H NMR (DMSO-d₆):

δ 1.32-1.35 (3H,t, J ) 7.0 Hz, CH₂CH₃), 1.88-1.92 (2H,

quintet, J ) 7.7 Hz, CH₂), 2.73-2.74 (6H, d, J ) 4.8 Hz, NMe₂),

Distamycin Analogues with Enhanced Lipophilicity

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2143

3.02-3.07 (2H, m), 3.30-3.35 (2H, q, J ) 6.4 Hz, CH₂), 3.95

(3H, s, NMe), 4.39-4.45 (2H, q, J ) 6.4 Hz, CH₂), 7.58 (1H,

s), 7.75 (1H, d, J ) 1.4 Hz), 8.14 (1H, t, J ) 6.1 Hz, CONH,

exch), 8.20 (1H, d, J ) 1.4 Hz), 10.15 (1H, br, hydrochloric

acid, exch), 10.761 (1H, s, CONH, exch). IR (KBr): 2950, 2865,

2822, 1659, 1536, 1506, 1467, 1423, 1314 cm^-1. HRFABMS

found: 392.20579. Calculated for C₁₇H₂₆O₄N₇: 392.20463.

Eth yl 4-isop r op yl-2-{[(1-m eth yl-4-n itr o-1H-p yr r ol-2-yl-

)car bon yl]am in o}-1,3-th iazole-5-car boxylate [(NO₂)P yr Me-

isoTh ziP r -OEt] was prepared using acid chloride coupling.

The crude product was purified by column chromatography

using silica gel and 1/3 ethyl acetate/n-hexane as eluant, R_f)

0.33. The product was obtained as a pale yellow fine needles

(802 mg, 35%), mp >230 °C. ¹H NMR (DMSO-d₆): δ 1.19 (6H,

d, J ) 6.8 Hz), 1.24-1.31 (3H, t, J ) 7.1 Hz), 3.90-3.95 (1H,

m), 3.98 (3H, s), 4.22-4.28 (2H, q, J ) 7.0 Hz), 8.04 (1H, s),

8.30 (1H, s), 12.8-13.0 (1H, s, br, exch). IR (KBr): 3355, 3144,

N-[3-(Dim eth yla m in o)p r op yl]-1-eth yl-4-{[(1-eth yl-4-n i-

t r o-1H -im id a zol-2-yl)ca r b on yl]a m in o}-1H -im id a zole-2-

ca r boxa m id e [(NO₂)Im iEt-Im iEt-Dm a p ] was prepared us-

ing HBTU coupling in DMF solution in the presence of

triethylamine. The crude product was purified by reverse

phase column chromatography. Water was used first, then a

mixture of 1/4 acetonitrile/water (containing 1% hydrochloric

acid). Fractions containing the pure product were collected and

freeze-dried. The freeze-dried material was dissolved in water

(25 mL) and freeze-dried again. The product (60 mg, 46%) was

obtained as a pale yellow sticky material (hygroscopic), mp

2978, 1690, 1674, 1556, 1515, 1501, 1311, 1251, 1061 cm^-1

.

HREIMS found: 367.10959. Calculated for

367.10762.

C₁₅H₁₉N₄O₅S:

2-(1-Me t h y l-4-n it r o p y r r o le -2-c a r b o x a m id o )-4-is o -

p r op ylt h ia zole-5-ca r b oxylic Acid [(NO₂)P yr Me-iso-

Th zOH]. The above ester, (NO₂)PyrMe-isoThziPr-OEt (491

mg, 1.400 mmol), was suspended in ethanolic potassium

hydroxide (1 M, 25 mL). The reaction mixture was heated

under reflux for 4 h, and then cooled to 0 °C. Hydrochloric

acid concentrated was then added dropwise with stirring until

pH 2. The pale yellow solid obtained was filtered off, washed

with deionized water, and then dried under reduced pressure

at 45 °C overnight to give the required material (379 mg, 84%),

1

120-125 °C (transparent). H NMR (DMSO-d₆): δ 1.33-1.36

(3H, t, J ) 7.0 Hz, CH₂CH₃), 1.40-1.44 (3H, t, J ) 7.0 Hz,

CH₂CH₃), 1.86-1.91 (2H, quintet, J ) 7.7 Hz, CH₂), 2.75-

2.77 (6H, d, J ) 4.8 Hz, NMe₂), 3.04-3.09 (2H, m, CH₂), 3.28-

3.31 (2H, q, J ) 6.4 Hz, CH₂), 4.43-4.54 (4H, m, 2xCH₂CH₃),

7.61 (1H, s), 8.48-8.51 (1H, t, J ) 6.1 Hz, CONH, exch), 8.61

(1H, s), 9.55 (1H, br, hydrochloric acid, exch), 10.14 (1H, s,

CONH, exch). IR (KBr): 3414, 1665, 1542, 1476, 1380, 1349,

1150, 1088, 847 cm^-1. HRFABMS found: 407.21498. Calcu-

lated for C₁₇H₂₇N₈O₄: 407.21553.

1

mp >230 °C. H NMR (DMSO-d₆): δ 1.21 and 1.23 (6H, d, J

) 6.8 Hz), 3.92-4.03 (1H, m, 3H, s), 7.98 (1H, s), 8.20 (1H, s),

12.6-13.0 (2H, br, exch). IR (KBr): 3444, 3138, 2972, 1685,

1568, 1543, 1500,1467, 1319, 1112, 1067 cm^-1. HREIMS

found: 337.06396. Calculated for C₁₃H₁₃N₄O₅S: 337.06067.

Meth yl 5-isop r op yl-2-{[(1-m eth yl-4-n itr o-1H-p yr r ol-2-

yl)ca r b on yl]a m in o}-1,3-t h ia zole-4-ca r b oxyla t e [(NO₂)-

P yr Me-Th ziP r -OMe] was prepared using acid chloride cou-

pling in dichloromethane solution in the presence of N-methyl-

morpholine. The crude product was purified by flash chroma-

tography over silica gel [1/1 ethyl acetate/pet. ether], which

gave the required product as a white crystalline solid (460 mg,

N-[3-(Dim eth yla m in o)p r op yl]-4-isop r op yl-2-{[(1-m eth -

yl-4-n it r o-1H -p yr r ol-2-yl)ca r b on yl]a m in o}-1,3-t h ia zole-

5-ca r boxyla te [(NO₂)P yr Me-isoTh ziP r -Dm a p ] was pre-

pared by the acid chloride method. The crude product was

purified by silica gel column chromatography. The product was

eluted by methanol/ethyl acetate/triethylamine (49/49/2) and

was obtained as a yellow solid. This material was extracted

using ethyl acetate/water. The organic layer was dried (Na₂-

SO₄) and filtered, and then the solvent was removed under

reduced pressure to give the required product as a glassy solid

material (358 mg, 76%), R_f) 0.1, mp 95-100 °C (softening).

¹H NMR (CDCl₃): δ 1.26 (6H, d, J ) 6.8 Hz), 1.83 (2H, m),

2.41 (6H, s), 2.61 (2H, t, unresolved), 3.48-3.57 (2H, q, 5.6

Hz), 3.82-3.91 (1H, m), 4.09 (3H, s), 7.41 (1H, d, J ) 1.8 Hz),

7.68 (1H, d, J ) 1.8 Hz), 8.01 (1H, br, CONH, exch). IR (KBr):

1

43% yield), mp 118-120 °C (softening). H NMR (CDCl₃): δ

1.32-1.34 (6H, CH-(CH₃)₂, d, J ) 6.4 Hz), 3.89 (3H, s, CO₂-

Me), 4.08 (3H, s, NMe), 4.11-4.18(1H, m), 7.23 (1H, pyrrole,

d, J ) 1.4 Hz), 7.67 (1H, pyrrole, d, J ) 1.4 Hz), 9.85 (1H, br,

CONH, exch). IR (KBr): 3557, 3119, 2975, 1712, 1646, 1574,

1509, 1319, 1208 cm^-1. HREIMS found: 352.08579. Calculated

for C₁₄H₁₆N₄O₅S: 352.08414.

5-Isop r op yl-2-{[(1-m et h yl-4-n it r o-1H -p yr r ol-2-yl)ca r -

bon yl]am in o}-1,3-th iazole-4-car boxylic Acid [(NO₂)P yr Me-

Th ziP r -OH]. The above methyl ester (480 mg, 1.363 mmol)

was suspended in ethanolic potassium hydroxide (0.5M, 25

mL). The reaction mixture was heated under reflux for 4 h

and then cooled to 0 °C. Hydrochloric acid (concentrated) was

added dropwise with stirring until pH 2. The pale yellow solid

obtained was filtered off, washed with deionized water, and

dried under reduced pressure at 45 °C overnight to give the

required material (440 mg, 96%), mp 315-319 °C (dec). ¹H

NMR (DMSO-d₆): δ 1.23-1.25 (6H, CH-(CH₃)₂, d, J ) 6.4 Hz),

3.98 (3H, s, NMe), 4.00-4.07 (1H, m), 7.99 (1H, pyrrole, d, J

) 1.4 Hz), 8.28 (1H, pyrrole, d, J ) 1.4 Hz), 12.5-13.2 (1H,

br, CO₂H, exch). IR (KBr): 3189, 3132, 2972, 1668, 1566, 1537,

1512, 1497, 1319, 1231 cm^-1. HRFABMS found: 337.06396.

Calculated for C₁₃H₁₃O₅N₄S: 337.06067.

3438, 2985, 1668, 1634, 1543, 1526, 1421, 1319, 1279 cm^-1

.

HREIMS found: 422.17189. Calculated for

422.17363.

C₁₈H₂₆N₆O₄S:

Meth yl 5-isop r op yl-2-[(3-m eth oxyben zoyl)a m in o]-1,3-

t h ia zole-4-ca r b oxyla t e (3MeOB-Th ziP r -OMe). Methyl

2-amino-5-isopropyl-1,3-thiazole-4-carboxylate (383 mg, 1.913

mmol) was dissolved in dichloromethane (10 mL) at room

temperature with stirring. N-methylmorpholine (0.3 mL) was

added followed by 3-methoxybenzoyl chloride (326 mg, 1.913

mmol) at room temperature with stirring. The reaction mixture

was heated until reflux for 10 min and then was left stirring

at room temperature overnight. The solvent was removed

under reduced pressure and the crude product was purified

by column chromatography using silica gel and 1/2 ethyl

acetate/petroleum ether. Fractions containing the required

material (R_f) 0.4) were collected and the solvents were

removed under reduced pressure to give the product as a white

N-[3-(Dim eth yla m in o)p r op yl]-5-isop r op yl-2-{[(1-m eth -

yl-4-n it r o-1H -p yr r ol-2-yl)ca r b on yl]a m in o}-1,3-t h ia zole-

4-ca r boxa m id e [AIK-12/99] [(NO₂)P yr Me-Th ziP r -Dm a p ]

was prepared using the acid chloride method. The crude

product was purified by column chromatography using (1:1

methanol/ethyl acetate containing 1% triethylamine). The

product was obtained as a yellow glassy material (159 mg,

29%), mp 85-90 °C (softening). ¹H NMR (CDCl₃): δ 1.26-

1.28 (6H, CH-(CH₃)₂, d, J ) 6.4 Hz), 1.79-1.86 (2H, CH₂,

quintet, J ) 6.9 Hz), 2.31 (6H, s, NMe₂), 2.45-2.48 (2H, CH₂,

t, J ) 6.9 Hz), 3.43-3.50 (2H, CH₂, q, J ) 6.9 Hz), 4.09 (3H,

s, NMe), 4.37-4.44 (1H, CH(CH₃)₂), m), 7.55 (1H, pyrrole, d,

J ) 1.4 Hz), 7.62 (1H, s, CONH, exch), 7.68 (1H, pyrrole, d, J

) 1.4 Hz). IR (KBr): 3131, 2959, 1668, 1551, 1500, 1418, 1310,

1

solid (530 mg, 83%), mp 60-63 °C. H NMR (CDCl₃): δ 9.89

(1H, s), 7.45-7.38 (3H, m), 7.16-7.13 (1H, m), 4.16 (1H, heptet,

J ) 6.8 Hz), 3.86 (3H, s), 3.85 (3H, s), 1.40 (6H, d, J ) 6.8

Hz). IR (KBr): 2958, 1720, 1669, 1547, 1463, 1298, 1208, 1045,

822, 743 cm^-1. HRFABMS found: 335.10620. Calculated for

C

₁₆H₁₉N₂O₄S: 35.10655.

5-Isop r op yl-2-[(3-m eth oxyben zoyl)a m in o]-1,3-th ia zole-

4-ca r boxylic Acid (3MeOB-Th ziP r -OH). The above ester,

3MeOB-ThziPr-OMe (330 mg, 0.998 mmol), was dissolved in

ethanolic potassium hydroxide (561 mg, in 10 mL ethanol, 10

mmol). The reaction mixture was heated under reflux for 3 h,

and then it was cooled to 0 °C. Hydrochloric acid (concentrated)

was added dropwise with stirring at 0 °C, and the resulting

solid was filtered off, washed with water, and dried under

1286 cm^-1. HREIMS found: 422.17695. Calculated for C₁₈H₂₆

O₄N₆S: 422.17363.

-

2144 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8

Khalaf et al.

reduced pressure at 45 °C overnight. The product was obtained

as a white solid (290 mg, 91%), mp >230 °C (sublimation). ¹H

NMR (DMSO-d₆): δ 7.69 (2H, m), 7.44 (1H, t, J ) 3.9 Hz),

7.19 (1H, m), 4.06 (1H, heptet, J ) 6.8 Hz), 3.84 (3H, s), 1.31

(6H, d, J ) 6.8 Hz). IR (KBr): 2963, 1672, 1555, 1463, 1306,

1221, 1043, 936, 820, 736 cm^-1. HRFABMS found: 321.09056.

Calculated for C₁₅H₁₇N₂O₄S: 321.09090.

N-[3-(Dim eth yla m in o)p r op yl]-5-m eth yl-2-({[(1-m eth yl-

4-n it r o-1H -p yr r ol-2-yl)ca r b on yl]a m in o}m et h yl)-1,3-ox-

a zole-4-ca r boxa m id e [(NO₂)P yr Me-Oxa Me-Dm a p ] was

prepared in dichloromethane solution using (NO₂)PyrMe-CCl₃

as acylating agent. The crude product was purified by flash

chromatography using methanol/ethyl acetate (1/1 containing

1% triethylamine, R_f) 0.2). The product was obtained as a

light brown solid (61 mg, 69%), mp 60-63 °C. ¹H NMR

(CDCl₃): 1.74-1.81 (2H, quintet, J ) 6.8 Hz, CH₂), 2.28 (6H,

s, NMe₂), 2.43-2.46 (2H, t, J ) 7.0 Hz, CH₂NMe₂), 2.63 (3H,

s, oxazole-CH₃), 3.43-3.49 (2H, q, J ) 7.4 Hz, CONHCH₂),

4.01 (3H, s, N-Me), 4.63-4.64 (2H, d, J ) 5.2 Hz, CONH-CH₂-

oxazole), 6.73 (1H, s, CONH, exch), 7.25 (1H, d, J ) 1.4 Hz),

7.59 (1H, d, J ) 1.4 Hz), 7.63 (1H, s, CONH, exch). IR (KBr):

3410, 2946, 1654, 1528, 1419, 1311, 1262, 1128 cm^-1. HREIMS

found: 392.18101. Calculated for C₁₇H₂₄N₆O₅: 392.18082.

Tr im er s. Am in o p r ecu r sor s were prepared as for the

dimers by hydrogenation over Pd on C (10%).

N-[5-({[5-({[3-(Dim eth ylam in o)pr opyl]am in o}car bon yl)-

1-m e t h yl-1H -p yr r ol-3-yl]a m in o}ca r b on yl)-1-e t h yl-1H -

p yr r ol-3-yl]-1-m eth yl-4-n itr o-1H-p yr r ole-2-ca r boxa m id e

[(NO₂)P yr Me-P yr E t -P yr MeDm a p ] was prepared using

(NO₂)PyrMeCCl₃in THF solution. The crude product was

purified by column chromatography (silica gel, 49:49:2 ethyl

acetate/methanol/ammonia). Fractions containing the required

material (R_f) 0.1) were collected. The product was obtained

as a yellow solid (261 mg, 71%) with no distinct melting point.

¹H NMR (DMSO-d₆): δ 1.23-1.30 (3H, t, J ) 7.2 Hz), 1.68-

1.76 (2H, m), 2.39 (6H, s), 2.56-2.58 (2H, t, J ) 6.8 Hz), 3.18-

3.24 (2H, quintet, J ) 6.1 Hz), 3.79 (3H, s), 3.97 (3H, s), 4.31-

4.36 (2H, q, J ) 7.2 Hz), 6.89 (1H, d, J ) 2.0 Hz), 7.02 (1H, d,

J ) 2.0 Hz), 7.22 (1H, d, J ) 2.0 Hz), 7.33 (1H, d, J ) 2.0 Hz),

7.59 (1H, d, J ) 2.0 Hz), 8.09-8.12 (1H, t, J ) 5.8 Hz), 8.18

(1H, d, J ) 2.0 Hz), 9.93 (1H, s), 10.29 (1H, s). IR (KBr): 3419,

3122, 2929, 1647, 1579, 1533, 1502, 1398, 1307 cm^-1

.

HRFABMS found: 513.25735. Calculated for C₂₄H₃₃N₈O₅:

513.25739.

N-[5-({[3-(Dim eth yla m in o)p r op yl]a m in o}ca r bon yl)-1-

isop r op yl-1H-p yr r ol-3-yl]-1-isop r op yl-4-{[(1-isop r op yl-4-

n itr o-1H-p yr r ol-2-yl)ca r bon yl]a m in o}-1H-p yr r ole-2-ca r -

boxa m id e [(NO₂)P yr iP r -P yr iP r -P yr iP r -Dm a p ]. The nitro

dimer (NO₂)PyriPr-PyriPr-Dmap (209 mg, 0.483 mmol) was

dissolved in ethanol (20 mL) at 0 °C with stirring. Pd/C (10%,

180 mg) was added to the solution at 0 °C and under N₂with

stirring. The reaction mixture was hydrogenated for 4 h and

then the catalyst was removed by filtration over Kieselguhr

and the solvent removed under reduced pressure at 50 °C. The

carboxylic acid (92 mg, 0.462 mmol) was dissolved in thionyl

chloride (3 mL) and heated under reflux for 3 h. Excess thionyl

chloride was removed under reduced pressure at 50 °C. The

amine was dissolved in THF (20 mL, dry) to which the acid

chloride [dissolved in THF (20 mL, dry)] was added dropwise

with stirring at room temperature. The reaction mixture was

left stirring at room temperature overnight. The solvent was

removed under reduced pressure and the crude product

dissolved in dichloromethane (2 × 50 mL), after which it was

extracted with Na₂CO₃(50 mL, 5%). The organic layer was

dried and then the solvent removed. The crude product was

chromatographed over silica gel using 49.5:49.5:1 ethyl acetate/

methanol/triethylamine as eluant. The product was obtained

as a yellow glassy material, R_f) 0.15 (230 mg, 82% yield),

N-[3-(Dim eth yla m in o)p r op yl]-1-m eth yl-4-{[(2-m eth yl-

5-n itr o-3-th ien yl)ca r bon yl]a m in o}-1H-p yr r ole-2-ca r box-

a m id e [(NO₂)Th iMe-P yr Me-Dm a p ] was prepared using the

acid chloride method in dichloromethane solution in the

presence of N-methylmorpholine. The crude product was

applied to a silica gel column chromatography using methanol/

ethyl acetate/triethylamine 1/1/0.05. The product was obtained

1

as a yellow solid (440 mg, 69%), mp 180-183 °C, R_f) 0.4. H

NMR (DMSO-d₆): δ 10.27 (1H, s), 8.52 (1H, s), 8.11 (1H, t, J

) 5.6 Hz), 7.23 (1H, d, J ) 1.8 Hz), 6.84 (1H, d, J ) 1.8 Hz),

3.81 (3H, s), 3.20 (2H, q, J ) 6.7 Hz), 2.76(3H, s), 2.39 (2H, t,

J ) 4.5 Hz), 2.25 (6H, s), 1.65 (2H, quintet, J ) 7.1 Hz). IR

(KBr): 1655, 1621, 1573, 1530, 1437, 1326, 1267 cm^-1

.

HRFABMS found: 394.15481. Calculated for C₁₇H₂₄N₅O₄S:

394.15490.

1-Isopen tyl-N-[1-m eth yl-5-({[3-(4-m eth yl-1-piper azin yl)-

a m in o}ca r b on yl)-1H -p yr r ol-3-yl]-4-n it r o-1H -p yr r ole-2-

ca r boxa m id e [(NO₂)P yr iP e-P yr Me-Mep ip P ] was prepared

by the acid chloride method in dichloromethane in the presence

of N-methylmorpholine. The crude product was purified by

column chromatography using silica gel and 1/2/0.1 methanol/

ethyl acetate/triethylamine. The product was obtained as a

glassy yellow material (R_f) 0.45) (278 mg, 84%) with no

1

mp 130-133 °C (softening). H NMR (CDCl₃): δ 1.43 (6H, d,

1

J ) 6.7 Hz), 1.47 (6H, d, J ) 6.7 Hz), 1.52 (6H, d, J ) 6.7 Hz),

1.73-1.79 (2H, m), 2.32 (6H, s), 2.47-2.50 (2H, t, 6.1 Hz),

3.46-3.50 (2H, q, 6.0 Hz), 5.53-5.61 (3H, m), 6.42 (1H, d, J )

1.6 Hz), 6.60 (1H, d, J ) 1.6 Hz), 7.28 (1H, d, J ) 1.6 Hz),

7.40 (1H, d, J ) 1.6 Hz), 7.41 (1H,s), 7.44 (1H, s), 7.63 (1H, s,

br, CONH), 7.81 (1H, d, J ) 1.6 Hz), 8.02 (1H, s). IR (KBr):

2979, 2937, 1645, 1593, 1525, 1504, 1410, 1279, 1237, 1190

distinct melting point. H NMR (DMSO-d₆): δ 10.21 (1H, s),

8.23 (1H, d, J ) 1.8 Hz), 8.05 (1H, t, J ) 5.4 Hz), 7.54 (1H, d,

J ) 1.8 Hz), 7.18 (1H, d, J ) 1.8 Hz), 6.82 (1H, d, J ) 1.8 Hz),

4.43 (2H, t, J ) 7.4 Hz), 3.81 (3H, s), 3.21-3.16 (2H, q, J )

6.6 Hz), 2.32-2.28 (10H, m), 2.15 (3H, s), 1.66-1.59 (4H,

quintet, J ) 6.7 Hz), 1.55-1.47 (1H, m), 0.90 (6H, d, J ) 6.5

Hz). IR (KBr): 2951, 2805, 1642, 1575, 1532, 1506, 1437, 1312

cm^-1. HRFABMS found: 583.33440. Calculated for C₂₉H₄₃

N₈O₅: 583.33564.

-

cm^-1. HRFABMS found: 488.29707. Calculated for C₂₄H₃₈

N₇O₄: 488.29853.

-

N-[5-({[3-(Dim eth yla m in o)pr op yl]a m in o}-1-m eth yl-1H-

p yr r ol-3-yl]-1-isop r op yl-4-{[(1-m eth yl-4-{[(1-m eth yl-4-n i-

t r o-1H -p yr r ol-2-yl)ca r b on yl]a m in o}-1H -p yr r ole-2-ca r -

b oxa m id e [(NO₂)P yr Me-P yr iP r -P yr Me-Dm a p ] was pre-

pared using (NO₂)PyrMeCCl₃in THF solution. The crude

product was purified by silica gel column chromatography

ethyl acetate/methanol/ammonia (49/49/2). Fractions contain-

ing the product (R_f) 0.3) were collected. The product was

obtained as a yellow solid (221 mg, 63%) with no distinct

1-Isop en tyl-N-[1-m eth yl-5-({[3-(4-m or p h olin yl)p r op yl]-

a m in o}ca r b on yl)-1H -p yr r ol-3-yl]-4-n it r o-1H -p yr r ole-2-

ca r boxa m id e [(NO₂)P yr iP e-P yr Me-Mor p ] was prepared by

the acid chloride method in dichloromethane in the presence

of N-methylmorpholine. The crude product was purified by

column chromatography using silica gel and 1/2/0.1 methanol/

ethyl acetate/triethylamine. Fractions containing the pure

material (R_f) 0.45) were collected and the solvents removed

under reduced pressure to give yellow glassy material, which

was dissolved in small amount of ethyl acetate and precipi-

tated with n-hexane to give the required product as a yellow

1

melting point. H NMR (DMSO-d₆): δ 1.36-1.38 (6H, d, J )

6.7 Hz), 1.64-1.71 (2H, m), 2.31 (6H, s), 3.16-3.22 (2H, q, J

) 6.7 Hz), 3.81 (3H, s), 3.96 (3H, s), 5.40-5.50 (1H, m), 6.85

(1H, d, J ) 1.6 Hz), 6.93 (1H, d, J ) 1.6 Hz), 7.18 (1H, d, J )

1.6 Hz), 7.43 (1H, d, J ) 1.6 Hz), 7.58 (1H, d, J ) 1.6 Hz),

8.07-8.10 (1H, t, J ) 5.8 Hz), 8.18 (1H, d, J ) 1.6 Hz), 9.96

(1H, s), 10.29 (1H, s). IR (KBr): 1643, 1579, 1534, 1507, 1464,

1407, 1310, 1247 cm^-1. HRFABMS found: 527.27341. Calcu-

lated for C₂₅H₃₅N₈O₅: 527.27304.

1

powder (670 mg, 79%), mp 155-158 °C. H NMR (CDCl₃): δ

7.64 (1H, d, J ) 1.7 Hz), 7.60 (1H, s), 7.22 (1H, unresolved

triplet), 7.18 (1H, d, J ) 1.7 Hz), 7.07 (1H, d, J ) 1.7 Hz), 6.66

(1H, d, J ) 1.6 Hz), 4.43 (2H, t, J ) 7.5 Hz), 3.93 (3H, s), 3.77

(2H, t, J ) 4.6 Hz), 3.49 (2H, q, J ) 5.6 Hz), 2.52 (6H, m),

1.80-1.58 (5H, m), 0.97 (6H, d, J ) 6.5 Hz). IR (KBr): 1647,

1589, 1513, 1399, 1309, 1252, 1114 cm^-1. HRFABMS found:

475.26789. Calculated for C₂₃H₃₅N₆O₅: 475.26689.

N-[5-({[5-({[3-(Dim et h yla m in o)p r op yl)-1-m et h yl-1H -

Distamycin Analogues with Enhanced Lipophilicity

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2145

p yr r ol-3-yl]a m in o}ca r bon yl)-1-m eth yl-1H-p yr r ol-3-yl]-1-

m eth yl-4-n itr o-1H-p yr r ole-2-ca r boxa m id e [(NO₂)P yr Me-

P yr Me-P yr Me-Dm a p ] was prepared using (NO₂)PyrMeCCl₃

in THF solution. The crude product was chromatographed over

flash silica gel using methanol/ethyl acetate/ammonia (49/49/

2). The product was obtained as a yellow solid (1.050 g, 70%),

R_f) 0.1, mp 198-200 °C. (lit.³²mp 203-205 °C).

using HBTU in DMF in the presence of N-methylmorpholine.

The crude product was purified by column chromatography

using silica gel and 1/2/0.1 methanol/ethyl acetate/triethyl-

amine, R_f) 0.40. The product was obtained as a glassy solid

material with no distinct melting point (110 mg, 55%). ¹H NMR

(DMSO-d₆): δ 10.28 (1H, s), 9.96 (1H, s), 8.18 (1H, d, J ) 1.8

Hz), 8.07 (1H, t, J ) 5.2 Hz), 7.58 (1H, d, J ) 1.8 Hz), 7.41

(1H, d, J ) 1.8 Hz), 7.18 (1H, d, J ) 1.8 Hz), 6.93 (1H, d, J )

1.8 Hz), 6.85 (1H, d, J ) 1.8 Hz), 5.53 (1H, quintet, J ) 7.3

Hz), 3.97 (3H, s), 3.80 (3H, s), 3.20 (2H, m), 2.83 (2H, m), 2.42

(2H, t, J ) 7.2 Hz), 2.27 (6H, s), 2.09 (2H, m), 1.79-1.64 (6H,

¹H NMR (DMSO-d₆) δ 1.57-1.63(2H, t, J ) 7.2 Hz), 2.13

(6H, s), 2.20-2.26 (2H, t, J ) 6.8 Hz), 3.15-3.19 (2H, m), 3.79

(3H, s), 3.85 (3H, s), 3.96 (3H, s), 6.81 (1H, d, J ) 1.9 Hz),

7.02 (1H, d, J ) 1.9 Hz), 7.19 (1H, d, J ) 1.9 Hz), 7.27 (1H, d,

J ) 1.9 Hz), 7.59 (1H, d, J ) 1.9 Hz), 8.09 (1H, t, J ) 5.8 Hz),

8.19 (1H, d, J ) 1.9 Hz), 9.95 (1H, s), 10.30 (1H, s). IR (KBr):

m). IR (KBr): 1642, 1584, 1529, 1505, 1403, 1309 cm^-1

.

HRFABMS found: 553.28890. Calculated for C₂₇H₃₇N₈O₅:

553.28869.

1647, 1580, 1533, 1502, 1398, 1307 cm^-1

.

N-[5-({[3-(Dim eth yla m in o)p r op yl]a m in o}ca r bon yl)-1-

isop r op yl-1H-p yr r ol-3-yl]-1-isop r op yl-4-{[(1-isop r op yl-4-

n itr o-1H-p yr r ol-2-yl)ca r bon yl]a m in o}-1H-p yr r ole-2-ca r -

boxa m id e [(NO₂)P yr iP r -P yr iP r -P yr iP r -Dm a p ] was pre-

pared using the acid chloride method. The crude product was

chromatographed on silica gel using 49.5:49.5:1 ethyl acetate/

methanol/triethylamine as eluant. The product was obtained

as a yellow glassy material R_f) 0.15, (230 mg, 82%), mp 130-

133 °C (softening). ¹H NMR (CDCl₃): δ 1.43 (6H, d, J ) 6.7

Hz), 1.47 (6H, d, J ) 6.7 Hz), 1.52 (6H, d, J ) 6.7 Hz), 1.73-

1.79 (2H, m), 2.32 (6H, s), 2.47-2.50 (2H, t, 6.1 Hz), 3.46-

3.50 (2H, q, 6.0 Hz), 5.53-5.61 (3H, m), 6.42 (1H, d, J ) 1.6

Hz), 6.60 (1H, d, J ) 1.6 Hz), 7.28 (1H, d, J ) 1.6 Hz), 7.40

(1H, d, J ) 1.6 Hz), 7.41 (1H,s), 7.44 (1H, s), 7.63 (1H, s, br,

CONH), 7.81 (1H, d, J ) 1.6 Hz), 8.02 (1H, s). IR (KBr): 2979,

N-[5-({[5-({[3-(Dim eth ylam in o)pr opyl]am in o}car bon yl)-

1,2-d im et h yl-1H -p yr r ol-3-yl]a m in o}ca r b on yl)-1,2-d im e-

t h yl-1H -p yr r ol-3-yl]-1,5-d im et h yl-4-n it r o-1H -p yr r ole-2-

ca r boxa m id e [(NO₂)P yr Me₂-P yr Me₂-P yr Me₂-Dm a p ] was

prepared using EDC in DMF solution in the presence of

4-(dimethylamino)pyridine. The crude product was purified by

silica gel column chromatography using methanol/ethyl acetate

(1/4, containing 1% triethylamine). The pure product (R_f) 0.1)

was collected and the solvent removed under reduced pressure

to give a pale yellow oil (80 mg, 64%), with no distinct melting

1

point. H NMR(DMSO-d₆): δ 1.55-1.62 (2H, quintet, J ) 5.9

Hz, CH₂), 2.07 (3H, s, C-CH₃), 2.11 (6H, s, NMe₂), 2.12 (3H, s,

C-CH₃), 2.20-2.23 (2H, t, J ) 5.9 Hz, CH₂), 2.62 (3H, s, C-CH₃),

3.14-3.19 (2H, q, J ) 5.9 Hz, CH₂), 3.76 (3H, s, NMe), 3.79

(3H, s, NMe), 3.86 (3H, s, NMe), 6.76 (1H, s), 7.00 (1H, s), 7.58

(1H, s), 7.89-7.90 (1H, t, J ) 5.6 Hz, CONH, exch), 9.02 (1H,

s, CONH, exch), 9.56 (1H, s, CONH, exch). IR (KBr): 2940,

2863, 1638, 1589, 1521, 1458, 1404, 1314, 1249, 1189, 1168

2937, 1645, 1593, 1525, 1504, 1410, 1279, 1237, 1190 cm^-1

.

HRFABMS found: 583.33440. Calculated for C₂₉H₄₃N₈O₅:

583.33564.

cm^-1. HRFABMS found: 541.28620. Calculated for C₂₆H₃₇

N₈O₅: 541.28869.

-

N-[5-({[5-({[3-(Dim eth ylam in o)pr opyl]am in o}car bon yl)-

1-isop en tyl-1H-p yr r ol-3-yl]a m in o}ca r bon yl)-1-isop en tyl-

1H-p yr r ol-3-yl]-1-isop en tyl-4-n itr o-1H-p yr r ole-2-ca r box-

a m id e [(NO₂)P yr iP e-P yr iP e-P yr iP e-Dm a p ] was prepared

using the acid chloride method in THF containing N-methyl-

morpholine. The crude product was purified by column chro-

matography using methanol/ethyl acetate/triethylamine (100:

100:1) to give the product as a yellow glassy material, R_f)

N-[5-({[5-({[3-(Dim eth ylam in o)pr opyl]am in o}car bon yl)-

1-m et h yl-1H -p yr r ol-3-yl]a m in o}ca r b on yl)-1-isop en t yl-

1H -p yr r ol-3-yl]-1-m et h yl-4-n it r o-1H -p yr r ole-2-ca r b oxa -

m id e [(NO₂)P yr Me-P yr iP e-P yr Me-Dm a p ] was prepared by

the acid chloride method in dichloromethane solution in the

presence of N-methylmorpholine. The product was purified by

column chromatography using 1/3/0.03 methanol/ethyl acetate/

triethylamine. Fractions containing the product (R_f) 0.2) were

collected and the solvents were removed under reduced pres-

sure at 50 °C to give the product as a yellow solid (335 mg,

1

0.20, (223 mg, 82%). mp 110-115 °C (transparent). H NMR

(CDCl₃): δ 0.93 (6H, d, J ) 1.6 Hz), 0.95 (6H, d, J ) 5.6 Hz),

0.98 (6H, d, J ) 3.7 Hz), 1.57-1.78 (5H, m), 2.47 (6H, s), 2.70

(2H, m), 3.49 (2H, m), 4.31-4.49 (6H, m), 6.57 (1H, s), 7.28

(1H, s), 7.33 (1H, s), 7.41 (1H, s), 7.44 (1H. s), 7.61 (1H, s),

7.64 (1H, d, J ) 1.8 Hz), 7.75 (1H, s), 8.22 (1H, s). IR (KBr):

1

78%), mp 110-115 °C (transparent). H NMR (DMSO-d₆): δ

10.27 (1H, s), 9.91 (1H, s), 8.18 (1H, d, J ) 1.8 Hz), 8.10-8.07

(1H, t, J ) 5.5 Hz), 7.59 (1H, d, J ) 1.8 Hz), 7.31 (1H, d, J )

1.8 Hz), 7.17 (1H, d, J ) 1.8 Hz), 6.98 (1H, d, J ) 1.8 Hz),

6.90 (1H, d, J ) 1.8 Hz), 6.82 (1H, d, J ) 1.8 Hz), 4.35-4.31

(2H, t, J ) 7.0 Hz), 3.96 (3H, s), 3.80 (3H, s), 3.22-3.17 (2H,

q, J ) 6.7 Hz), 2.36-2.32 (2H, t, J ) 7.1 Hz), 2.21 (6H, s),

1.67-1.62 (2H, q, J ) 7.0 Hz), 1.60-1.44 (3H, m), 1.04-0.99

(2H, m), 0.89-0.88 (6H, d, J ) 6.7 Hz). IR (KBr): 3401, 3289,

1643, 1582, 1533, 1594, 1463, 1400, 1309, 1253 cm^-1. HR-

2953, 1646, 1583, 1534, 1505, 1464, 1422, 1311 cm^-1

.

HRFABMS found: 667.42932. Calculated for C₃₅H₅₅O₅N₈:

667.42954.

N-[1-(Cyclop r op ylm eth yl)-5-({[5-({[3-(d im eth yla m in o)-

pr opyl]am in o}car bon yl)-1-m eth yl-1H-pyr r ol-3-yl]am in o}-

ca r bon yl)-1H-p yr r ol-3-yl]-1-m eth yl-4-n itr o-1H-p yr r ole-2-

ca r boxa m id e [(NO₂)P yr Me-P yr Cycp r -P yr Me-Dm a p ] was

prepared using HBTU in DMF in the presence of N-methyl-

morpholine. The crude product obtained was purified by

column chromatography using silica gel and 1/2/0.1 methanol/

ethyl acetate/triethylamine. Fractions containing the pure

product (R_f) 0.4) were collected and the solvents were

removed under reduced pressure at 50 °C to give the required

product as a glassy yellow solid (110 mg, 71%) with no distinct

melting point. ¹H NMR (DMSO-d₆): δ 10.27 (1H, s), 9.94 (1H,

s), 8.18 (1H, d, J ) 1.7 Hz), 8.06 (1H, t, J ) 5.2 Hz), 7.59 (1H,

d, J ) 1.9 Hz), 7.36 (1H, d, 1.9 Hz), 7.18 (1H, d, J ) 1.9 Hz),

7.02 (1H, d, J ) 1.9 Hz), 6.83 (1H, d, J ) 1.9 Hz), 4.19 (2H, d,

J ) 7.0 Hz), 3.97 (3H, s), 3.80 (3H, s), 3.21-3.16 (2H, q, J )

6.9 Hz), 2.26 (2H, t, J ) 7.1 Hz), 2.15 (6H, s), 1.65-1.58 (2H,

quintet, J ) 7.1 Hz), 1.26-1.13 (1H, m), 0.47-0.42 (2H, m),

0.33-0.31 (2H, m). IR (KBr): 3286, 3126, 2938, 1646, 1580,

1531, 1462, 1439, 1400, 1309, 1248 cm^-1. HRFABMS found:

539.27295. Calculated for C₂₆H₃₅N₈O₅: 539.27304.

FABMS found: 555.30169. Calculated for

555.30434.

C₂₇H₃₉N₈O₅:

N-[3-(Dim eth yla m in o)p r op yl]-5-m eth yl-2-({[(1-m eth yl-

4-{[(1-m eth yl-4-n itr o-1H-p yr r ol-2-yl)ca r bon yl]a m in o}-

1H -p yr r ol-2-yl)ca r b on yl]a m in o}m et h yl)-1,3-oxa zole-4-

ca r b oxa m id e [(NO₂)P yr Me-P yr Me-Oxa Me-Dm a p ] was

prepared by the acid chloride method in dichloromethane. The

crude product was purified by flash chromatography using

methanol/ethyl acetate 1/1 containing 1% triethylamine. The

product was obtained as a pale yellow solid, R_f) 0.2 (54 mg,

71%) with no distinct melting point. ¹H NMR (CDCl₃): δ 1.74-

1.83 (2H, quintet, J ) 6.8 Hz, CH₂), 2.30 (6H, s, NMe₂), 2.45-

2.48 (2H, t, J ) 7.0 Hz, CH₂NMe₂), 2.65 (3H, s, oxazole-CH₃),

3.44-3.49 (2H, q, J ) 7.4 Hz, CONHCH₂), 3.90 (3H, s, NMe),

3.99 (3H, s, NMe), 4.61-4.63 (2H, d, J ) 5.2 Hz, CONH-CH₂-

oxazole), 6.48-6.50 (1H, t, 5.0 Hz, CONH, exch), 6.73 (1H, d,

J ) 1.4 Hz), 7.27 (1H, d, J ) 1.4 Hz), 7.32 (1H, d, J ) 1.4 Hz),

7.60-7.60 (1H, pyrrole and 1H, CONH, exch), 8.35(1H, s,

CONH, exch). IR (KBr): 3412, 3138, 2934, 1657, 1529, 1446,

1-Cyclopen tyl-N-[5-({[3-(dim eth ylam in o)pr opyl]am in o}-

ca r bon yl)-1-m eth yl-1H-p yr r ol-3-yl]-4-{[(1-m eth yl-4-n itr o-

1H -p yr r ol-2-yl)ca r b on yl]a m in o}-1H -p yr r ole-2-ca r b oxa -

m id e [(NO₂)P yr Me-P yr Cycp r -P yr Me-Dm a p ] was prepared

2146 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8

Khalaf et al.

1312, 1261 cm^-1. HRFABMS found: 515.23487. Calculated for

1640, 1535, 1466, 1433, 1412, 1311, 1261 cm^-1. HRFABMS

found: 516.20321. Calculated for C₂₃H₃₀N₇O₅S: 516.20291.

C

₂₃H₃₁N₈O₆: 515.23666.

N-[5-({[3-(Dim eth yla m in o)p r op yl]a m in o}ca r bon yl)-1-

m eth yl-1H-p yr r ol-3-yl]-1-m eth yl-4-{[(2-m eth yl-5-n itr o-3-

t h ie n yl)ca r b on yl]a m in o}-1H -p yr r ole -2-ca r b oxa m id e

[(NO₂)Th iMe-P yr Me-P yr Me-Dm a p ] was prepared using the

acid chloride method in dichloromethane solution in the

presence of N-methylmorpholine. The crude product was

applied to a silica gel column and eluted using methanol/ethyl

acetate/triethylamine (1/1/0.05). The product was obtained as

an orange solid material (R_f) 0.2) after reprecipitation from

methanol/ethyl acetate/n-hexane (468 mg, 67% yield), mp 195-

N-[3-(Dim eth yla m in o)p r op yl]-4-isop r op yl-2-{[(1-m eth -

yl-4-{[(1-m eth yl-4-n itr o-1H-p yr r ol-2-yl)ca r bon yl]a m in o}-

1H-p yr r ol-2-yl)ca r bon yl]a m in o}-1,3-th ia zole-5-ca r boxa -

m id e [(NO₂)P yr Me-P yr Me-isoTh ziP r -Dm a p ] was prepared

using (NO₂)PyrMe-CCl₃in THF solution. The crude product

was chromatographed using 49/49/2 methanol/ethyl acetate/

triethylamine. The product obtained was dissolved in dichlo-

romethane, extracted with a solution of sodium carbonate (10

mL, 5%), and then dried, and the solvent was removed under

reduced pressure. The product was obtained as a yellow solid

1

198 °C. H NMR (DMSO-d₆): 10.33 (1H, s), 9.93 (1H, s), 8.54

(140 mg, 54%) mp 240-244 °C. H NMR (CDCl₃) δ 1.27 (6H,

(1H, s), 8.06 (1H, t, J ) 5.5 Hz), 7.30 (1H, d, J ) 1.8 Hz), 7.19

(1H, d, J ) 1.8 Hz), 7.04 (1H, d, J ) 1.8 Hz), 6.83 (1H, d, J )

1.8 Hz), 3.86 (3H, s), 3.80 (3H, s), 3.21 (2H, q, J ) 6.7 Hz),

2.77 (3H, s), 2.31 (2H, t, J ) 6.9 Hz), 2.19 (6H, s), 1.62 (2H,

quintet, J ) 6.9 Hz). IR (KBr): 1638, 1541, 1465, 1434, 1405,

1330, 1263 cm^-1. HRFABMS found: 516.20501. Calculated for

d, J ) 6.8 Hz, 2Me), 1.76 (2H, m, CH₂), 2.32 (6H, s, NMe₂),

2.49 (2H, t, J ) 7.0 Hz, CH₂), 3.52 (2H, q, J ) 5.4 Hz, CH₂),

3.88 (1H, m, CH), 4.01 (3H, s, NMe), 4.05 (3H, s, NMe), 6.84

(1H, s, PyrH), 7.25 (1H, s, PyrH), 7.36 (1H, s, PyrH), 7.62 (1H,

s, PyrH), 7.84 (1H, s, CONH, exch), 7.95 (1H, t, unresolved,

CONH, exch), 9.50 (1H, br, CONH, exch). IR (KBr): 1654,

1625, 1585, 1549, 1526, 1503, 1464, 1418, 1398, 1310, 1277

C

₂₃H₃₀N₇O₅S: 516.20291.

cm^-1. HRFABMS found: 545.23168. Calculated for C₂₄H₃₃

N₈O₅S: 545.22946.

-

N-[5-({[3-(Dim eth yla m in o)p r op yl]a m in o}ca r bon yl)-1-

m eth yl-1H-p yr r ol-3-yl]-1-isop en tyl-4-{[(2-m eth yl-5-n itr o-

3-t h ien yl)ca r b on yl]a m in o}-1H -p yr r ole-2-ca r b oxa m id e

[(NO₂)Th iMe-P yr iP e-P yr Me-Dm a p ] was prepared by the

acid chloride method in dichloromethane in the presence of

N-methylmorpholine. The crude product was purified by

column chromatography using ethyl acetate/methanol/triethy-

lamine (2.5/2.5/0.6), R_f) 0.5. The product was obtained as an

amorphous yellow material (237 mg, 90%) with no distinct

melting point. ¹H NMR (DMSO-d₆): δ 10.37 (1H, s), 9.94 (1H,

s), 8.58 (1H, s), 8.12 (1H, t, J ) 5.6 Hz), 7.35 (1H, d, J ) 1.7

Hz), 7.18 (1H, d, J ) 1.7 Hz), 7.14 (1H, d, J ) 1.7 Hz), 7.01

(1H, d, J ) 1.7 Hz), 6.86 (1H, d, J ) 1.7 Hz), 4.34 (1H, t, J )

6.9 Hz), 3.80 (3H, s), 3.22 (2H, q, J ) 6.5 Hz), 2.77 (3H, s),

2.64 (2H, m), 2.44 (6H, s), 1.73 (2H, quintet, J ) 6.7 Hz), 1.59-

1.45 (3H, m), 0.90 (6H, d, J ) 6.4 Hz). IR (KBr): 1675, 1650,

N-[3-(Dim eth yla m in o)p r op yl]-5-isop r op yl-2-{[(1-m eth -

yl-4-{[(1-m eth yl-4-n itr o-1H-p yr r ol-2-yl)ca r bon yl]a m in o}-

1H-p yr r ol-2-yl)ca r bon yl]a m in o}-1,3-th ia zole-4-ca r boxa -

m id e [(NO₂)P yr Me-P yr Me-Th ziP r -Dm a p ] was prepared

using (NO₂)PyrMe-CCl₃in THF solution. The crude product

was chromatographed using 49/49/2 methanol/ethyl acetate/

triethylamine. The product obtained was dissolved in dichlo-

romethane, extracted with a solution of sodium carbonate (10

mL, 5%), and then dried, and the solvent was removed under

reduced pressure. The product was obtained as a yellow solid

(90 mg, 44%), mp 138-141 °C. ¹H NMR (DMSO-d₆) δ 1.26 (6H,

d, J ) 6.8 Hz, 2Me), 1.65 (2H, m, CH₂), 2.19 (6H, s, NMe₂),

2.32 (2H, t, J ) 5.2 Hz, CH₂), 3.26 (2H, q, J ) 5.4 Hz, CH₂),

3.90 (3H, s, NMe), 3.97 (3H, s, NMe), 4.19 (1H, m, CH), 7.36

(1H, d, J ) 1.8 Hz, PyrH), 7.47 (1H, d, J ) 1.8 Hz, pyrH), 7.62

(1H, d, J ) 1.8 Hz, PyrH), 7.81 (1H, t, 5.9 Hz, CONH exch),

8.19 (1H, d, J ) 1.8 Hz, PyrH), 10.35 (1H, s, CONH exch),

12.10 (1H, br, exch). IR (KBr): 1651, 1552, 1506, 1464, 1421,

1398, 1311, 1288, 1202 cm^-1. HRFABMS found: 545.22931.

Calculated for C₂₄H₃₃N₈O₅S: 545.22946.

1583, 1537, 1465, 1437, 1403, 1263, 1202, 1133 cm^-1

.

HRFABMS found: 572.26551. Calculated for C₂₇H₃₇N₇O₅S:

572.26580.

1-Isop r op yl-N-[1-m eth yl-5-({[3-(4-m or p h olin yl)p r op yl]-

a m in o}ca r b on yl)-1H -p yr r ol-3-yl]-4-{[(1-m et h yl-4-n it r o-

1H -p yr r ol-2-yl)ca r b on yl]a m in o}-1H -p yr r ole-2-ca r b oxa -

m id e [(NO₂)P yr Me-P yr iP r -P yr Me-Mor p ] was prepared

using HBTU in DMF solution in the presence of N-methyl-

morpholine. The crude product was chromatographed using

flash silica gel and methanol/ethyl acetate/triethylamine (1:

3:0.1), R_f) 0.30. Fractions containing the pure material were

collected and solvents were removed under reduced pressure

at 40 °C to give a semisolid glassy yellow material (170 mg,

N-[5-({[3-(Dim eth yla m in o)p r op yl]a m in o}ca r bon yl)-1-

m eth yl-1H-p yr r ol-3-yl]-5-isop r op yl-2-{[(1-m eth yl-4-n itr o-

1H-p yr r ol-2-yl)ca r bon yl]a m in o}-1,3-th ia zole-4-ca r boxa -

m id e [(NO₂)P yr Me-Th ziP r -P yr Me-Dm a p ] was prepared

using HBTU in DMF solution in the presence of N-methyl-

morpholine. The crude product obtained was chromatographed

over silica gel using 1:1:0.2 methanol/ethyl acetate/triethy-

lamine. The product was obtained as a yellow glassy material

(415 mg, 72%) with no distinct melting point. ¹H NMR (DMSO-

d₆): δ 9.59 (1H, s), 8.23 (1H, s), 8.09-8.03 (1H, t, J ) 5.5 Hz),

7.82 (1H, s), 7.24 (1H, d, J ) 1.8 Hz), 6.90 (1H, d, J ) 1.8 Hz),

4.22-4.15 (1H, heptet, J ) 6.7 Hz), 4.00 (3H, s), 3.81 (3H, s),

3.24-3.17 (2H, q, J ) 6.7 Hz), 2.37-2.34 (2H, t, J ) 3.2 Hz),

2.22 (6H, s), 1.68-1.61 (2H, quintet, J ) 7.0 Hz), 1.29 (6H, d,

J ) 6.9 Hz). IR (KBr): 1644, 1551, 1500, 1465, 1402, 1309,

1283, 1194, 1100 cm^-1. HRFABMS found: 545.22962. Calcu-

lated for C₂₄H₃₃N₈O₅S: 545.22946.

1

53%) with no distinct melting point. H NMR (DMSO-d₆): δ

10.30 (1H, s), 9.98 (1H, s), 8.19 (1H, s), 8.03-8.01 (1H, t, J )

5.5 Hz), 7.59 (1H, s), 7.44 (1H, s), 7.18 (1H, s), 6.92 (1H, s),

6.85 (1H, s), 5.48-5.42 (1H, heptet, J ) 6.4 Hz), 3.96 (3H, s),

3.79 (3H, s), 3.57-3.56 (4H, t, unresolved), 3.22-3.17 (2H, q,

J ) 6.8 Hz), 2.32 (2H, q, J ) 6.8 Hz), 1.67-1.60 (2H, quintet,

J ) 6.8 Hz), 1.37-1.36 (6H, d, J ) 6.8 Hz), 1.04-1.01 (2H,

m). IR (KBr): 3425, 2927, 1662, 1653, 1582, 1543, 1502, 1410,

1307, 1251, 1203, 1115, 848 cm^-1. HRFABMS found: 569.28147.

Calculated for C₂₇H₃₇N₈O₆: 569.28361.

N-[1-Isop r op yl-5-({[1-m et h yl-5-({[3-(1-p yr r olid in yl)-

pr opyl]am in o}car bon yl)-1H-pyr r ol-3-yl]am in o}car bon yl)-

1H -p yr r ol-3-yl]-1-m et h yl-4-n it r o-1H -p yr r ole-2-ca r b oxa -

m id e [(NO₂)P yr Me-P yr iP r -P yr Me-Mor p ] was prepared

using HBTU in DMF solution in the presence of N-methyl-

morpholine. The product was purified by column chromatog-

raphy using methanol/ethyl acetate/triethylamine (1/2/0.1).

The product was obtained as a yellow solid (239 mg, 62%) with

no distinct melting point. Some of this material was further

N-[3-(Dim eth yla m in o)p r op yl]-1-m eth yl-4-{[(2-m eth yl-

5-{[(1-m eth yl-4-n itr o-1H-p yr r ol-2-yl)ca r bon yl]a m in o}-3-

t h ie n yl)ca r b on yl]a m in o}-1H -p yr r ole -2-ca r b oxa m id e

[(NO₂)P yr Me-Th iMe-P yr Me-Dm a p ] was prepared by the

acid chloride method in dichloromethane in the presence of

N-methylmorpholine. The crude product was applied to a silica

gel column chromatography using methanol/ethyl acetate/

triethylamine (1/1/0.05). The product was obtained as a yellow

solid (170 mg, 56%), mp 150-154 °C (softening), R_f) 0.33. ¹H

NMR (DMSO-d₆): δ 11.39 (1H, s), 9.91 (1H, s), 8.21 (1H, d, J

) 1.8 Hz), 8.05 (1H, t, J ) 5.6 Hz), 7.67 (1H, d, J ) 1.8 Hz),

7.00 (1H, s), 6.77 (1H, d, J ) 1.8 Hz), 3.93 (3H, s), 3.77 (3H,

s), 3.16 (2H, q, J ) 6.4 Hz), 2.50 (3H, s), 2.29 (2H, t, J ) 6.9

Hz), 2.17 (6H, s), 1.59 (2H, quintet, J ) 6.9 Hz). IR (KBr):

1

purified by HPLC. H NMR (DMSO-d₆): δ 10.28 (1H, s), 9.97

(1H, s), 9.46 (1H, br, TFA), 8.19 (1H, s), 8.14 (1H, t, J ) 5.5

Hz), 7.58 (1H, s), 7.43 (1H, s), 7.18 (1H, s), 6.94 (2H, s), 5.45-

5.43 (1H, m), 3.97 (3H, s), 3.81 (3H, s), 3.55 (2H, m), 3.26 (2H,

m), 3.14 (2H, m), 2.99 (2H, m), 2.01 (2H, m), 1.86 (4H, m),

1.38-1.36 (6H, d, J ) 6.8 Hz). IR (KBr): 3419, 3136, 1673,

Distamycin Analogues with Enhanced Lipophilicity

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2147

1645, 1586, 1531, 1463, 1409, 1311, 1247, 1199, 1130 cm^-1

HRFABMS found: 553.29109. Calculated for C₂₇H₃₇N₈O₅:

553.28869.

.

(Dimethylamino)propyl]amino}carbonyl)-1-methyl-1H-pyrrol-

3-yl]amino}carbonyl)-1-ethyl-1H-pyrrol-3-yl]-1-methyl-4-nitro-

1H-pyrrole-2-carboxamide (93 mg, 0.181 mmol) was dissolved

in methanol (20 mL) to which Pd/C (108 mg, 10%) was added.

The reaction mixture was hydrogenated at room temperature

and atmospheric pressure for 5 h. The catalyst was removed

by filtration through Kieselguhr and the solvent was then

removed under reduced pressure to give the amine, which was

used without further purification. The amine was dissolved

in ethanol (5 mL), to which ethyl formate (20 mL) was added.

The reaction mixture was heated under reflux for 48 h. The

solvent was removed under reduced pressure to give the crude

product, which was purified by HPLC. The product was

obtained as a white solid after freeze-drying (43 mg, 30%, as

TFA salt). ¹H NMR (DMSO-d₆) δ 1.26-1.91 (2H, t, J ) 7.2

Hz), 1.81-1.29 (2H, m), 2.78-2.79 (6H, d, J ) 3.6 Hz), 3.05-

3.07 (2H, m), 3.23-3.24 (2H, q, J ) 5.9 Hz), 3.81 (3H, s), 3.84

(3H, s), 4.28-4.34 (2H, q, J ) 6.7 Hz), 6.91 (1H, s), 6.95 (1H,

s), 7.03 (1H, s), 7.17 (1H, s), 7.19 (1H, s), 7.27 (1H, s), 8.12

(1H, s), 8.15 (1H, t, J ) 5.7 Hz), 9.3 (1H, br TFA), 9.85 (1H, s),

9.91 (1H, s), 10.05 (1H, s). IR (KBr): 1673, 1644, 1582, 1540,

N -[1-Isop e n t yl-5-({[1-m e t h yl-5-({[3-(4-m e t h yl-1-p ip -

er azin yl)pr opyl]am in o}car bon yl)-1H-pyr r ol-3-yl]am in o}-

ca r bon yl)-1H-p yr r ol-3-yl]-1-m eth yl-4-n itr o-1H-p yr r ole-2-

ca r boxa m id e [(NO₂)P yr Me-P yr iP e-P yr Me-Mep ip p ] was

prepared by the acid chloride method in dichloromethane

solution in the presence of N-methylmorpholine. Column

chromatography (silica gel, 1/2/0.1 methanol/ethyl acetate/

triethylamine) was used to purify the required material, which

was obtained as a yellow glassy material (131 mg, 75%), R_f)

0.15 with no distinct melting point. ¹H NMR (DMSO-d₆): δ

10.27 (1H, s), 9.90 (1H, s), 8.18 (1H, d, J ) 1.8 Hz), 8.01 (1H,

t, J ) 5.6 Hz), 7.59 (1H, d, J ) 1.8 Hz), 7.31 (1H, d, J ) 1.8

Hz), 7.16 (1H, d, J ) 1.8 Hz), 6.98 (1H, J ) 1.8 Hz), 6.84 (1H,

d, J ) 1.8 Hz), 4.33 (1H, t, J ) 6.9 Hz), 3.96 (3H, s), 3.79 (3H,

s), 3.17 (2H, q, J ) 6.5 Hz), 2.32-2.29 (10mH, m), 2.16 (3H,

s), 1.66-1.46 (5H, m), 0.89 (6H, d, J ) 6.4 Hz). IR (KBr): 2949,

2803, 1650, 1588, 1531, 1506, 1399, 1309 cm^-1. HRFABMS

found: 610.34797. Calculated for C₃₀H₄₄N₉O₅: 610.34654.

N-[1-Isop en t yl-5-({[1-m et h yl-5-({[3-(4-m or p h olin yl)-

pr opyl]am in o}car bon yl)-1H-pyr r ol-3-yl]am in o}car bon yl)-

1H -p yr r ol-3-yl]-1-m et h yl-4-n it r o-1H -p yr r ole-2-ca r b oxa -

m id e [(NO₂)P yr Me-P yr iP e-P yr Me-Mor p ] was prepared by

the acid chloride method in dichloromethane solution in the

presence of N-methylmorpholine. The crude product was

purified by column chromatography (1/2/0.1 methanol/ethyl

acetate/triethylamine, R_f) 0.7). Fractions containing the

required material were collected and the solvents were re-

moved under reduced pressure. The yellow glassy residue was

dissolved in dichloromethane and extracted with aqueous

potassium hydroxide solution (10%, 5 mL). The organic layer

was collected and dried (MgSO₄), and the solvent was removed

under reduced pressure to give a yellow glassy material (115

mg, 67%) with no distinct melting point. ¹H NMR (acetone-

d₆): δ 9.52 (1H, s), 9.26 (1H, s), 7.95 (1H, d, J ) 1.7 Hz), 7.55

(1H, t, unresolved), 7.38 (1H, d, J ) 1.9 Hz), 7.33 (1H, d, J )

1.9 Hz), 7.20 (1H, d, J ) 1.9 Hz), 6.91 (1H, d, J ) 1.9 Hz),

6.84 (1H, d, J ) 1.9 Hz), 4.44 (2H, t, J ) 7.2 Hz), 4.08 (3H, s),

3.89 (3H, s), 3.64 (4H, m), 3.37 (2H, t, J ) 6.7 Hz), 2.42 (6H,

m), 1.77-1.56 (5H, m), 0.95 (6H, d, J ) 6.5 Hz). IR (KBr):

1640, 1588, 1524, 1464, 1399, 1310, 1252, 1114 cm^-1. HR-

FABMS 597.31332. Calculated for C₂₉H₄₁N₈O₆: 597.31491.

Distam ycin An alogu es. N-[5-({[5-({[3-(Dim eth ylam in o)-

pr opyl]am in o}car bon yl)-1-m eth yl-1H-pyr r ol-3-yl]am in o}-

ca r b on yl)-1-m et h yl-1H -p yr r ol-3-yl]-4-for m yla m in o)-1-

m et h yl-1H -p yr r ole-2-ca r b oxa m id e (F o-P yr Me-P yr Me-

p yr Me-Dm a p , 1). N-[5-({[5-({[3-(Dimethylamino)propyl)-1-

methyl-1H-pyrrol-3-yl]amino}carbonyl)-1-methyl-1H-pyrrol-3-

yl]-1-methyl-4-nitro-1H-pyrrole-2-carboxamide (104 mg, 0.208

mmol) was dissolved in methanol to which was added Pd/C

(106 mg, 10%). The reaction mixture was hydrogenated at

room temperature and atmospheric pressure for 5 h. The

catalyst was removed by filtration through Kieselguhr and the

solvent removed under reduced pressure to give the amine,

which was used without further purification. This amine was

dissolved in ethanol (5 mL), to which was added ethyl formate

(20 mL), and the reaction mixture was heated under reflux

for 48 h. The solvent was removed under reduced pressure

and the crude product purified by HPLC. The product was

obtained as a white solid after freeze-drying (73 mg, 57%, as

TFA salt). ¹H NMR (DMSO-d₆): δ 1.81-1.85 (2H, m), 2.79 (6H,

s), 3.05-3.09 (2H, q, J ) 6.7 Hz), 3.23-3.24 (2H, t, J ) 3.6

Hz), 3.81 (3H, s), 3.84 (6H, s), 6.92 (1H, s), 6.94 (1H, s), 7.06

(1H, s), 7.17 (1H, s), 7.19 (1H, s), 7.22 (1H, s), 8.12 (1H, s),

8.16 (1H, t), 9.30 (1H, br, TFA), 9.85 (1H, s), 9.91 (1H, s), 10.05

(1H, s). IR (KBr): 1673, 1644, 1582, 1540, 1470, 1441, 1402,

1204, 1139 cm^-1. HRFABMS found: 497.26255. Calculated:

497.26248.

1470, 1441, 1402, 1204, 1139 cm^-1

511.27820. Calculated: 511.27813.

. HRFABMS found:

N-[5-({[5-({[3-(Dim eth ylam in o)pr opyl]am in o}car bon yl)-

1-m et h yl-1H -p yr r ol-3-yl]a m in o}ca r b on yl)-1-isop r op yl-

1H-p yr r ol-3-yl]-4-(for m yla m in o)-1-m eth yl-1H-p yr r ole-2-

ca r boxa m id e (F o-P yr Me-P yr iP r -P yr Me-Dm a p , 3). N-[5-

({[3-(Dimethylamino)propyl]amino}-1-methyl-1H-pyrrol-3-yl]-

1-isoprop yl-4-{[(1-methyl-4-{[(1-methyl-4-nitro-1H-pyrrol-2-

yl)carbonyl]amino}-1H-pyrrole-2-carboxamide (102 mg, 0.194

mmol) was dissolved in methanol (20 mL), to which was added

Pd/C (106 mg, 10%). The reaction mixture was hydrogenated

for 5 h at room temperature and atmospheric pressure. The

catalyst was removed by filtration through Kieselguhr and

then ethyl formate (10 mL) was added to the filtrate. The

reaction mixture was then heated under reflux for 72 h. The

solvent was removed under reduced pressure and the crude

product obtained was purified by HPLC. The product was

obtained as a white solid after freeze-drying (64 mg, 51%, as

1

TFA salt). H NMR (DMSO-d₆): δ 1.35-1.37 (6H, d, J ) 6.7

Hz), 1.81-1.85 (2H, m), 2.78-2.79 (6H, d, J ) 3.6 Hz), 3.05

(2H, m), 3.23-3.25 (2H, q, J ) 5.9 Hz), 3.81 (3H, s), 3.84 (3H,

s), 5.40-5.46 (1H, m), 6.92 (1H, s), 6.94 (1H, s), 6.96 (1H, s),

7.17 (1H, s), 7.19 (1H, s), 7.38 (1H, s), 8.12 (1H, s), 8.15 (1H,

t, J ) 5.7 Hz), 9.3 (1H, br, TFA), 9.85 (1H, s), 9.91 (1H, s),

10.05 (1H, s). IR (KBr): 1673, 1644, 1582, 1540, 1470, 1441,

1402, 1204, 1139 cm^-1. HRFABMS found: 525.29372. Calcu-

lated: 525.29378.

1-Cyclopen tyl-N-[5-({[3-(dim eth ylam in o)pr opyl]am in o}-

ca r bon yl)1-m eth yl-1H-p yr r ol-3-yl]-4-({[4-(for m yla m in o)-

1-m eth yl-1H-p yr r ol-2-yl]ca r bon yl}a m in o)-1H-p yr r ole-2-

ca r b oxa m id e (F o-P yr Me-P yr Cycp e-P yr Me-Dm a p , 4).

1-Cyclopentyl-N-[5-({[3-(dimethylamino)propyl]amino}carbonyl)-

1-methyl-1H-pyrrol-3-yl]-4-{[(1-methyl-4-nitro-1H-pyrrol-2-yl)-

carbonyl]amino}-1H-pyrrole-2-carboxamide (100 mg, 0.181

mmol) was dissolved in ethanol (20 mL) at 0 °C under N₂with

stirring, to which Pd/C (10%, 92 mg) was added. The reaction

mixture was hydrogenated at room temperature and atmo-

spheric pressure for 3 h. The catalyst was removed by filtration

through Kieselguhr and ethyl formate (25 mL) was added to

the ethanolic solution. The reaction mixture was heated under

reflux for 24 h. The solvent was removed under reduced

pressure to give the crude product, which was purified by

HPLC. Fractions containing the required material were col-

lected and freeze-dried to give the product as a pale yellow

solid (21 mg, 18%) with no distinct melting point. ¹H NMR

(DMSO-d₆): δ 10.06 (1H, s), 9.95 (1H, s), 9.91 (1H, s), 9.21

(1H, br, TFA), 8.14 (1H, t, J ) 6.0 Hz), 8.13 (1H, d, J ) 1.7

Hz), 7.36 (1H, d, J ) 1.7 Hz), 7.19 (1H, d, J ) 1.7 Hz), 7.17

(1H, d, J ) 1.7 Hz), 6.97 (1H, d, J ) 1.7 Hz), 6.95 (1H, d, J )

1.7 Hz), 6.92 (1H, d, J ) 1.7 Hz), 5.52 (1H, quintet, J ) 7.5

Hz), 3.84 (3H, s), 3.81 (3H, s), 3.24 (2H, m), 3.06 (2H, m), 2.79

(6H, d, J ) 3.3 Hz), 2.08 (2H, m), 1.83 (4H, m), 1.64 (4H, m).

IR (KBr): 1674, 1647, 1582, 1536, 1463, 1440, 1406, 1201, 1131

N-[5-({[5-({[3-(Dim eth ylam in o)pr opyl]am in o}car bon yl)-

1-m eth yl-1H-pyr r ol-3-yl]am in o}car bon yl)-1-eth yl-1H-pyr -

r ol-3-yl]-4-(for m yla m in o)-1-m eth yl-1H-p yr r ole-2-ca r box-

a m id e (F o-P yr Me-P yr Et-P yr Me-Dm a p , 2). N-[5-({[5-({[3-

2148 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8

Khalaf et al.

cm^-1. HRFABMS found: 551.30950. Calculated for C₂₈H₃₉

N₈O₄: 551.30943.

-

7.15 (1H, s), 7.01 (1H, s), 6.92 (1H, s), 6.91 (1H, s), 4.31-4.29

(2H, t, J ) 6.8 Hz), 3.84 (3H, s), 3.81 (3H, s), 3.25-3.23 (2H,

m), 3.06 (2H, m), 2.79 (6H, d, J ) 3.7 Hz), 1.83-1.81 (2H, m),

1.57-1.48 (3H, m), 0.89 (6H, d, J ) 6.2 Hz). IR (KBr): 3428,

3315, 1676, 1644, 1582, 1538, 1464, 1438, 1403, 1262, 1201,

1132 cm^-1. HRFABMS found: 553.32520. Calculated for

N-[1-(Cyclop r op ylm eth yl)-5-({[5-({[3-(d im eth yla m in o)-

pr opyl]am in o}car bon yl)-1-m eth yl-1H-pyr r ol-3-yl]am in o}-

ca r bon yl)-1H-p yr r ol-3-yl]-4-(for m yla m in o)-1-m eth yl-1H-

p yr r ole-2-ca r boxa m id e

(F o-P yr Me-P yr Cycp r -P yr Me-

C

₂₈H₄₁N₈O₄: 553.32508.

Dm a p , 5). N-[1-(Cyclopropylmethyl)-5-({[5-({[3-(dimethyl-

a mino)propyl]a mino}ca rbonyl)-1-methyl-1H-pyrrol-3-yl]-

amino}carbonyl)-1H-pyrrol-3-yl]-1-methyl-4-nitro-1H-pyrrole-2-

carboxamide (100 mg, 0.186 mmol) was dissolved in ethanol

(25 mL) to which Pd/C (10%, 78 mg) was added at 0 °C under

N₂with stirring. The reaction mixture was hydrogenated for

3 h at room temperature and atmospheric pressure. The

catalyst was removed by filtration through Kieselguhr. Ethyl

formate was added to the ethanolic solution of the amine and

the reaction mixture was heated under reflux for 48 h. Ethanol

and excess ethyl formate were removed under reduced pres-

sure, and the crude product was purified by HPLC. The

product was obtained as a pale yellow solid (52 mg, 42%) after

freeze-drying with no distinct melting point. ¹H NMR (DMSO-

d₆): δ 10.04 (1H, s), 9.91 (1H, s), 9.90 (1H, s), 9.28 (1H, br,

TFA), 8.16-8.13 (2H, m), 7.31 (1H, d, J ) 1.7 Hz), 7.19 (1H,

d, J ) 1.7 Hz), 7.16 (1H, d, J ) 1.7 Hz), 7.05 (1H, d, J ) 1.7

Hz), 6.95 (1H, d, 1.7 Hz), 6.93 (1H, d, J ) 1.7 Hz), 4.18 (2H, d,

J ) 7.0 Hz), 3.85 (3H, s), 3.81 (3H, s), 3.26-3.22 (2H, q, J )

6.3 Hz), 3.07 (2H, m), 2.79 (2H, d, J ) 4.3 Hz), 1.87-1.82 (2H,

quintet, J ) 6.6 Hz), 1.23 (1H, m), 0.47-0.42 (2H, m), 0.33-

0.29 (2H, m). IR (KBr): 3410, 3294, 1674, 1649, 1582, 1533,

N-[5-({[3-(Dim eth yla m in o)p r op yl]a m in o}ca r bon yl)-1-

isop r op yl-1H -p yr r ol-3-yl]-4-({[4-(for m yla m in o)-1-iso-

p r op yl-1H-p yr r ol-2-yl]ca r bon yl}a m in o)-1-isop r op yl-1H-

pyr r ole-2-car boxam ide (Fo-P yr iP r -P yr iP r -P yr iP r -Dm ap,

8). (NO₂)PyriPr-PyriPr-PyriPr-Dmap (105 mg, 0.180 mmol)

was suspended in ethanol (20 mL) to which Pd/C (10%, 80 mg)

was added under nitrogen at 0 °C. The reaction mixture was

hydrogenated at room temperature and atmospheric pressure

for 4 h. The catalyst was removed by filtration through

Kieselguhr and the ethanolic solution was then used in the

second part of the experiment. Ethyl formate (20 mL) was

added to the ethanolic solution of the amine. The reaction

mixture was heated under reflux for 4 days. The solvents were

removed under reduced pressure at 40 °C, and then the crude

product was purified by reverse phase HPLC. The fractions

obtained were freeze-dried to give the required material as a

white solid (71 mg, 57%, as TFA salt) with no distinct melting

1

point. H NMR (DMSO-d₆): δ 1.14-1.36 (18H, m), 1.82-1.91

(2H, m), 2.78 (6H, d, J ) 4.62 Hz), 3.04-3.09 (2H, m), 3.22-

3.26 (2H, m), 5.42-5.51 (3H, m), 6.82 (1H, d, J ) 1.6 Hz), 5.89

(1H, d, J ) 1.6 Hz), 6.97 (1H, d, J ) 1.6 Hz), 7.35 (1H, d, J )

1.6 Hz), 7.34 (1H, s), 8.12-8.14 (2H, s & t), 9.25 (1H, s, br,

TFA), 9.91 (1H, s), 9.93 (1H, s), 10.03 (1H, s). IR (KBr): 3285,

2953, 1650, 1580, 1525, 1460, 1402, 1260 cm^-1. HREIMS

found: 580.35047. Calculated for C₃₀H₄₄O₄N₈: 580.34855.

1464, 1438, 1403, 1201, 1132 cm^-1

. HRFABMS found:

537.29360. Calculated for C₂₇H₃₇N₈O₄: 537.29378.

N-[5-({[5-({[3-(Dim eth ylam in o)pr opyl]am in o}car bon yl)-

1-isop r op yl-1H-p yr r ol-3yl]a m in o}ca r bon yl)-1-m eth yl-1H-

p yr r ol-3-yl]-4-(for m yla m in o)-1-isop r op yl-1H -p yr r ole-2-

ca r boxa m id e (F o-P yr iP r -P yr Me-P yr iP r -Dm a p , 6). N-[5-

({[5-({[3-(Dimethylamino)propyl]amino}carbonyl)-1-isopropyl-

1H-pyrrol-3-yl]amino}carbonyl)-1-methyl-1H-pyrrol-3-yl]-1-

isopropyl-4-nitro-1H-pyrrole-2-carboxamide (150 mg, 0.291

mmol) was dissolved in ethanol (15 mL). The solution was

cooled to 0 °C under N₂and then Pd/C (10%, 120 mg) was

added. The reaction mixture was hydrogenated at room

temperature and atmospheric pressure for 4 h. The catalyst

was removed by filtration through Kieselguhr and then ethyl

formate (20 mL) was added to the ethanolic solution. The

reaction mixture was heated under reflux overnight and then

the solvent was removed under reduced pressure at 40 °C. The

product was purified by HPLC to give the product as a white

solid (71 mg, 40%) with no distinct melting point. ¹H NMR

(DMSO-d₆): δ 10.05 (1H, s), 9.95 (1H, s), 9.89 (1H, s), 9.24

(1H, s, br, TFA), 8.15-8.13 (2H, m), 7.34 (2H, s), 7.21 (1H, s),

7.06 (1H, s), 6.89 (1H, s), 6.67 (1H, s), 5.48-5.42 (1H, m), 3.84

(3H, s), 3.24 (2H, m), 3.05 (2H, m), 2.79 (6H, d, J ) 4.7 Hz),

1.83 (2H, m), 1.36-1.33 (12H, m). IR (KBr): 3425, 3285, 2972,

1676, 1639, 1579, 1533, 1465, 1439, 1404, 1257, 1200, 1180,

N-[5-({[5-({[3-(Dim eth ylam in o)pr opyl]am in o}car bon yl)-

1-isop en tyl-1H-p yr r ol-3-yl]a m in o}ca r bon yl)-1-isop en tyl-

1H-p yr r ol-3-yl]-4-(for m yla m in o)-1-isop en tyl-1H-p yr r ole-

2-ca r b oxa m id e [(NO₂)P yr iP e-P yr iP e-P yr iP e-Dm a p , 9].

(NO₂)PyriPe-PyriPe-PyriPe-Dmap (119 mg, 0.178 mmol) was

dissolved in ethanol (20 mL) and Pd/C (10%, 104 mg) was

added at 0 °C under N₂with stirring. The reaction mixture

was hydrogenated at room temperature and atmospheric

pressure for 3 h. The catalyst was removed by filtration

through Kieselguhr, and then ethyl formate (15 mL) was added

to the ethanolic solution of the amine. The reaction mixture

was heated under reflux for 4 days. The solvent was removed

under reduced pressure and the crude product was chromato-

graphed over silica gel using ethyl acetate/methanol/triethy-

lamine (100:100:1). The product was obtained as a light brown

solid with no distinct melting point, R_f) 0.15, (84 mg, 71%).

Some of this material was purified further by HPLC for

1

analysis. H NMR (DMSO-d₆): δ 0.87-0.90 (18H, m), 1.47-

1.53 (9H, m), 1.75 (2H, m), 2.78 (6H, d, J ) 4.8 Hz), 3.06 (2H,

m), 3.25 (2H, m), 4.31 (6H, quintet, J ) 7.2 Hz), 6.88 (1H, d,

J ) 1.8 Hz), 6.92 (1H, d, J ) 1.8 Hz), 7.02 (1H, d, J ) 1.8 Hz),

7.19 (1H, d, J ) 1.8 Hz), 7.23 (2H, m), 8.13 (2H, m), 9.21 (1H,

s, br, TFA), 9.88 (1H, s), 9.89 (1H, s), 10.05 (1H, s). IR (KBr):

cm^-1. HRFABMS found: 554.33484. Calculated for C₂₈H₄₂

N₈O₄: 554.33290.

-

2953, 1650, 1580, 1525, 1461, 1402, 1260, 1221 cm^-1

.

N-[5-({[5-({[3-(Dim eth ylam in o)pr opyl]am in o}car bon yl)-

1-m et h yl-1H -p yr r ol-3-yl]a m in o}ca r b on yl)-1-isop en t yl-

1H-p yr r ol-3-yl]-4-(for m yla m in o)-1-m eth yl-1H-p yr r ole-2-

ca r boxa m id e (F o-P yr Me-P yr iP e-P yr Me-Dm a p , 7). N-[5-

({[5-({[3-(Dimethylamino)propyl]amino}carbonyl)-1-methyl-

1H-pyrrol-3-yl]amino}carbonyl)-1-isopentyl-1H-pyrrol-3-yl]-1-

methyl-4-nitro-1H-pyrrole-2-carboxamide (89 mg, 0.160 mmol)

was dissolved in ethanol (25 mL) to which Pd/C (10%, 95 mg)

was added at 0 °C under nitrogen with stirring. The reaction

mixture was hydrogenated for 2 h at room temperature and

atmospheric pressure. The catalyst was removed by filtration

through Kieselguhr. Ethyl formate (25 mL) was added to the

ethanolic solution, and then the reaction mixture was heated

under reflux overnight. Ethanol and excess ethyl formate were

removed under reduced pressure, and the crude product was

purified by HPLC to give the required material as a pale yellow

HRFABMS found: 665.44877. Calculated for C₃₆H₅₇O₄N₈:

665.45028.

N-[5-({[5-({[3-(Dim eth ylam in o)pr opyl]am in o}car bon yl)-

1,2-d im et h yl-1H -p yr r ol-3-yl]a m in o}ca r b on yl)-1,2-d im e-

t h yl-1H -p yr r ol-3-yl]-4-(for m yla m in o)-1,5-d im et h yl-1H -

p yr r ole-2-ca r b oxa m id e

(F o-P yr Me₂-P yr Me₂-P yr Me₂-

Dm a p , 10). (NO₂)PyrMe₂-PyrMe₂-PyrMe₂-Dmap (70 mg, 0.126

mmol) was dissolved in ethanol (15 mL). The solution was

cooled to 0 °C and Pd/C (10%, 67 mg) was added portionwise

with stirring. The reaction mixture was hydrogenated at room

temperature and atmospheric pressure for 4 h. The catalyst

was removed by filtration through Kieselguhr. Ethyl formate

(20 mL) was added to the solution and the reaction mixture

was then heated until reflux for 24 h. The solvent was removed

under reduced pressure and the crude product purified using

silica gel and methanol (containing 1% triethylamine). The

product was obtained as a pale yellow solid. This material was

dissolved in water (20 mL) containing TFA (30 µL) and then

1

solid (37.4 mg, 35%) with no distinct melting point. H NMR

(DMSO-d₆): δ 10.03 (1H, s), 9.89 (1H, s), 9.87 (1H, s), 9.63

(1H, br, TFA), 8.14-8.12 (2H, m), 7.26 (1H, s), 7.18 (1H, s),

Distamycin Analogues with Enhanced Lipophilicity

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2149

freeze-dried to give the required product (49 mg, 60%) with

no distinct melting point. ¹H NMR (DMSO-d₆): δ 1.57-1.61

(2H, quintet, J ) 5.9 Hz, CH₂), 2.07 (3H, s, C-CH₃), 2.11 (6H,

s, NMe₂), 2.12 (3H, s, C-CH₃), 3.16-3.17 (2H, q, J ) 5.9 Hz,

CH₂), 3.76 (3H, s, NMe), 3.77 (3H, s, NMe), 3.78 (3H, s, NMe),

6.76 (1H, s), 6.95 (1H, s), 7.15 (1H, s), 7.88 (1H, J ) 5.6 Hz,

CONH, exch), 8.13 (1H, s), 8.96 (1H, s, CONH, exch), 9.09 (1H,

s, CONH, exch), 9.47 (1H, s, CONH, exch). IR (KBr): 1676,

Dm a p , 14). N-[5-({[5-({[3-(Dimethylamino)propyl)-1-methyl-

1H-pyrrol-3-yl]amino}carbonyl)-1-methyl-1H-pyrrol-3-yl]-1-

methyl-4-nitro-1H-pyrrole-2-carboxamide (102 mg, 0.205 mmol)

was suspended in 2-propanol (30 mL) to which was added Pd/C

(102 mg, 10%). The reaction mixture was hydrogenated at

room temperature and atmospheric pressure for 3 h. The

catalyst was removed by filtration through Kieselguhr and the

solvent was then removed under reduced pressure to give the

amine, which was used without purification. The amine so

formed was dissolved in dichloromethane (10 mL, dry) to which

was added 3,3-dimethylbutanoyl chloride (35 mg, 0.260 mmol)

[35 µL was dissolved in dichloromethane (5 mL, dry)]. DMAP

(5 mg) was added to the reaction mixture at room temperature

with stirring. The reaction mixture was left stirring for 48 h

at room temperature. The solvent was removed and the crude

product chromatographed over silica gel using ethyl acetate/

methanol/ammonia (49/49/2). Fractions with an R_fvalue of 0.1

were collected, and the solvent was then removed under

reduced pressure to give the product as a pale yellow glassy

material (67 mg, 58% yield), mp 135-140 °C (softening). ¹H

NMR (CDCl₃): δ 1.03 (9H, s), 1.64-1.69 (2H, m), 2.15 (2H, s),

2.19 (6H, s), 2.32-2.37 (2H, t, J ) 6.8 Hz), 3.34-3.36 (2H,

quintet, J ) 6.1 Hz), 3.75 (3H, s), 3.78 (3H, s), 3.79 (3H, s),

6.51 (1H, s), 6.58 (2H, s), 7.07 (1H, s), 7.09 (1H, s), 7.16 (1H,

s), 7.68 (1H, t, J ) 5.8 Hz), 7.97 (1H, s), 8.49 (1H, s), 8.55 (1H,

1642, 1586, 1540, 1475, 1444, 1401, 1206, 1141 cm^-1

.

HRFABMS found: 539.30776. Calculated for C₂₇H₃₉N₈O₄:

539.30943.

4-(Acet yla m in o)-N-[5-({[5-({[3-(Dim et h yla m in o)p r op -

yl]amino}carbonyl)-1-methyl-1H-pyrrol-3-yl]amino}carbon-

yl)-1-isopentyl-1H-pyrrol-3-yl]-1-methyl-1H-pyrrole-2-carbox-

a m id e (Ac-P yr Me-P yr iP e-P yr Me-Dm a p , 11). (NO₂)PyrMe-

PyriPe-PyrMe-Dmap (150 mg, 0.271 mmol) was dissolved in

methanol (25 mL) to which was added Pd/C (10%, 100 mg) at

0 °C under N₂with stirring. The reaction mixture was

hydrogenated for 2 h at room temperature and atmospheric

pressure. The catalyst was removed by filtration through

Kieselguhr and the solvent was then removed under reduced

pressure. The amine so formed was dissolved in dichlo-

romethane (10 mL, dry) to which N-methylmorpholine (137

µL, dry) and acetyl chloride (64 µL) were added respectively

at room temperature with stirring. The stirring was continued

at room temperature overnight. Volatile materials were re-

moved under reduced pressure and the crude product was

purified by HPLC to give the required product as a white solid

(111 mg, 62%) with no distinct melting point. ¹H NMR (DMSO-

d₆): δ 9.87 (2H, s), 9.80 (1H, s), 9.35 (1H, br), 8.14 (1H, t, J )

5.5 Hz), 7.26 (1H, s), 7.15 (1H, s), 7.13 (1H, s), 7.01 (1H, s),

6.92 (1H, s), 6.86 (1H, s), 4.31 (2H, t, J ) 6.7 Hz), 3.83 (3H, s),

3.81 (3H, s), 3.25 (2H, m), 3.06 (2H, m), 2.79 (6H, d, J ) 4.0

Hz), 1.97 (3H, s), 1.83 (2H, m), 1.58-1.48 (3H, m), 0.89 (6H,

d, J ) 6.2 Hz). IR (KBr): 1674, 1643, 1581, 1538, 1464, 1438,

s). IR (KBr): 3298, 2960, 1644, 1582, 1538, 1405, 1250 cm^-1

.

HRFABMS found: 567.34079. Calculated for C₂₉H₄₃N₈O₄:

567.34073.

N-[5-({[5-({[3-(Dim eth ylam in o)pr opyl]am in o}car bon yl)-

1-m et h yl-1H -p yr r ol-3-yl]a m in o}ca r b on yl)-1-et h yl-1-H -

p yr r ol-3-yl]-4-[(3,3-d im et h ylb u t a n oyl)a m in o]-1-m et h yl-

1H-p yr r ole-2-ca r boxa m id e (Dm B-P yr Me-P yr Et-P yr Me-

Dm a p , 15). N-[5-({[5-({[3-(Dimethylamino)propyl]amino}-

carbonyl)-1-methyl-1H-pyrrol-3-yl]amino}carbonyl)-1-ethyl-1H-

pyrrol-3-yl]-1-methyl-4-nitro-1H-pyrrole-2-carboxamide (100

mg, 0.195 mmol) was suspended in methanol (20 mL) to which

was added Pd/C (137 mg, 10%). The reaction mixture was

hydrogenated overnight at room temperature and atmospheric

pressure. The catalyst was removed over Kieselguhr and the

solvent then removed under reduced pressure at 40 °C. The

amine was dissolved in dichloromethane (10 mL, dry). 3,3-

Dimethylbutanoyl chloride (55 µL, 0.409 mmol) was dissolved

in dichloromethane (5 mL, dry) and was then added dropwise

to the reaction mixture with stirring at room temperature.

Diisopropylethylamine (35 µL) was added to the reaction

mixture, and then it was left stirring at room temperature

overnight. The solvent was removed under reduced pressure

and the crude product obtained purified by column chroma-

tography (silica gel, methanol/ethyl acetate/ammonia 49/49/

2). The fractions that contained the required material (R_f)

0.1) were collected. The product obtained was dissolved in

chloroform (100 mL) and extracted with sodium hydrogen

carbonate (200 mg, dissolved in water 50 mL). The organic

layer was collected and dried (MgSO₄) and the solvent removed

under reduced pressure to give the product as a yellow glassy

solid (50 mg, 44%) with no distinct melting point. ¹H NMR

(CDCl₃): δ 1.04(9H, s), 1.25-1.32 (3H, t, J ) 7.2 Hz), 1.66-

1.71 (2H, m), 2.16 (2H, s), 2.23 (6H, s), 2.37-2.42 (2H, t, J )

6.8 Hz), 3.39-3.45 (2H, quintet, J ) 6.1 Hz), 3.83 (6H, s), 4.21-

4.27 (2H, q, J ) 7.2 Hz), 6.49 (1H, s), 6.58 (1H, s), 6.67 (1H,

s), 7.06 (1H, s), 7.19 (2H, s), 7.76 (1H, t, J ) 5.8 Hz), 7.84 (1H,

s), 8.25 (1H, s), 8.29 (1H, s). IR (KBr): 3298, 2960, 1644, 1582,

1538, 1405, 1250 cm^-1. HRFABMS found: 581.35631. Calcu-

lated for C₃₀H₄₅N₈O₄: 581.35638.

1404, 1259, 1201, 1134 cm^-1

.

HRFABMS found:

567.33800. Calculated for C₂₉H₄₃N₈O₄: 567.34073.

4-(Acet yla m in o)-N-[5-({[5-({[3-(Dim et h yla m in o)p r op -

yl]amino}carbonyl)-1-methyl-1H-pyrrol-3-yl]amino}carbon-

yl)-1-isopropyl-1H-pyrrol-3-yl]-1-methyl-1H-pyrrole-2-carbox-

a m id e (Ac-P yr Me-P yr iP r -P yr Me-Dm a p , 12). (NO₂)PyrMe-

PyriPr-PyrMe-Dmap (45 mg, 0.103 mmol) was dissolved in

methanol (25 mL) to which was added Pd/C (10%, 48 mg) at 0

°C under N₂with stirring. The reaction mixture was hydro-

genated for 2 h at room temperature and atmospheric pres-

sure. The catalyst was removed by filtration through Kiesel-

guhr and the solvent was removed under reduced pressure.

The amine so formed was dissolved in dichloromethane (10

mL, dry) to which N-methylmorpholine (100 µL, dry) and

acetyl chloride (50 µL) were added sequentially at room

temperature with stirring. Stirring was continued at room

temperature overnight. Volatile materials were removed under

reduced pressure, and the crude product was purified by HPLC

to give the required product as a white solid (21 mg, 32%) with

no distinct melting point. ¹H NMR (DMSO-d₆): δ 9.91 (1H, s),

9.87(1H, s), 9.80 (1H, s), 9.25 (1H, br, TFA), 8.14 (1H, t, J )

5.8 Hz), 7.38 (1H, d, J ) 1.8 Hz), 7.17 (1H, d, J ) 1.8 Hz),

7.13 (1H, d, J ) 1.8 Hz), 6.97 (1H, d, J ) 1.8 Hz), 6.95 (1H, d,

J ) 1.8 Hz), 6.86 (1H, d, J ) 1.8 Hz), 5.47-5.40 (1H, m), 3.83

(3H, s), 3.81 (3H, s), 3.25 (2H, q, J ) 6.4 Hz), 3.06 (2H, m),

2.79 (6H, d, J ) 4.8 Hz), 1.97 (3H, s), 1.90-1.81 (2H, m), 1.37-

1.35 (6H, d, J ) 6.7 Hz). IR (KBr): 1678, 1642, 1582, 1530,

1463, 1439, 1407, 1254, 1201, 1135 cm^-1. HRFABMS found:

539.30790. Calculated for C₂₇H₃₉N₈O₄: 539.30943.

4-(Ace t a m in o)-N -[5-({[5-({[3-(d im e t h yla m in o)p r op -

yl]a m in o}ca r b on yl)-1-m e t h yl-1H -p yr r ol-3-yl]a m in o}-

car bon yl)-1-m eth yl-1H-pyr r ol-3-yl]-1-m eth yl-1H-pyr r ole-2-

ca r boxa m id e (Ac-P yr Me-P yr Me-P yr Me-Dm a p , 13). This

material was prepared according to a published procedure³¹

in 56% yield.

N-[5-({[5-({[3-(Dim eth ylam in o)pr opyl]am in o}car bon yl)-

1-m eth yl-1H-p yr r ol-3-yl]a m in o}ca r bon yl)-1-m eth yl-1-H-

p yr r ol-3-yl]-4-[(3,3-d im et h ylbu t a n oyl)a m in o]-1-m et h yl-

1H-p yr r ole-2-ca r boxa m id e (Dm B-P yr Me-P yr Me-P yr Me-

N-[5-({[5-({[3-(Dim eth ylam in o)pr opyl]am in o}car bon yl)-

1-m et h yl-1H -p yr r ol-3-yl]a m in o}ca r b on yl)-1-isop r op yl-

1H-pyr r ol-3-yl]-4-[(3,3-dim eth ylbu tan oyl)am in o]-1-m eth yl-

1H-p yr r ole-2-ca r boxa m id e (Dm B-P yr Me-P yr Me-P yr iP r -

Dm a p , 16). N-[5-({[3-(Dimethylamino)propyl]amino}carbonyl)-

1-methyl-1H-pyrrol-3-yl]-1-isopropyl-4-nitro-1H-pyrrole-2-

carboxamide (100 mg, 0.189 mmol) was dissolved in methanol

(20 mL) to which was added Pd/C (114 mg, 10%). The reaction

mixture was hydrogenated at room temperature and atmo-

spheric pressure for 6.5 h. The catalyst was removed by

2150 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8

Khalaf et al.

filtration through Kieselguhr and the solvent removed under

reduced pressure. The amine so formed was dissolved in

dichloromethane (10 mL, dry) to which was added a solution

of 3,3-dimethylbutanoyl chloride (35 µL, 0.260 mmol), which

was dissolved in dichloromethane (5 mL, dry). The addition

was dropwise with stirring at room temperature. Di-sopropyl-

amine (35 µL) was then added in one portion to the reaction

mixture. Stirring was continued at room temperature over-

night. The solvent was removed under reduced pressure and

the crude product obtained was purified by column chroma-

tography using silica gel and ethyl acetate/methanol/ammonia

(49/49/2). The solvent was removed under reduced pressure

and the solid material was dissolved in chloroform (100 mL)

and extracted with (5%, 50 mL) sodium bicarbonate. The

organic layer was dried (Na₂SO₄) and the solvent removed to

give the required product (84 mg, 75%) as a yellow glassy

(1H, s), 9.33 (1H, br), 7.53-7.41 (3H, m), 7.32-7.24 (3H, m),

7.19-7.08 (4H, m), 6.97-6.93 (3H, m), 6.61 (1H, d, J ) 1.8

Hz), 3.92 (2H, t, J ) 6.7 Hz), 3.88 (3H, s), 3.83 (3H, s), 3.82

(3H, s), 3.71 (3H, s), 3.59 (3H, s), 3.21 (2H, m), 2.83 (6H, d, J

) 4.0 Hz), 2.85 (2H, m). IR (KBr): 1677, 1642, 1582, 1547,

1465, 1434, 1403, 1262, 1202, 1132 cm^-1. HRFABMS found:

603.30331. Calculated for C₃₁H₃₉N₈O₅: 603.30434.

N-[5-({[5-({[3-(Dim eth ylam in o)pr opyl]am in o}car bon yl)-

1-m et h yl-1H -p yr r ol-3-yl]a m in o}ca r b on yl)-1-isop en t yl-

1H-p yr r ol-3-yl]-4-[(3-m eth oxyben zoyl)a m in o]-1-m eth yl-

1H-pyr r ole-2-car boxam ide (3MeOB-P yr Me-P yr iP e-P yr Me-

Dm a p , 19). N-[5-({[5-({[3-(Dimethylamino)propyl]amino}-

carbonyl)-1-methyl-1H-pyrrol-3-yl]amino}carbonyl)-1-iso-

pentyl-1H-pyrrol-3-yl]-1-methyl-4-nitro-1H-pyrrole-2-carboxamide

(260 mg, 0.469 mmol) was dissolved in methanol (25 mL) to

which Pd/C (10%, 132 mg) was added at 0 °C under N₂with

stirring. The reaction mixture was hydrogenated for 3 h. The

catalyst was removed by filtration through Kieselguhr and the

solvent was removed under reduced pressure to give the

amine, which was dissolved in dichloromethane (5 mL). This

solution was divided in to two 2.5-mL portions. To one portion,

m-anisoyl chloride (50 mg, 0.293 mmol) was added dropwise

at room temperature with stirring. Stirring was continued

overnight. The solvent was removed under reduced pressure

and the residue was dissolved in acetonitrile containing 0.1%

TFA and purified by HPLC. Fractions containing the required

material were collected and freeze-dried to give the product

as a light pink solid with no distinct melting point (72.3 mg,

1

material with no distinct melting point. H NMR (CDCl₃): δ

1.03 (9H, s), 1.29-1.32 (6H, d, J ) 6.7 Hz), 1.68-1.83 (2H,

m), 2.15 (2H, s), 2.19 (6H, s), 2.26-2.39 (2H, t, J ) 6.2 Hz),

3.39-3.46 (2H, q, J ) 6.7 Hz), 3.83 (3H, s), 3.84 (3H, s), 5.42-

5.51 (1H, m), 6.47 (1H, d, J ) 1.4 Hz), 6.58 (1H, d, J ) 1.4

Hz), 6.67 (1H, d, J ) 1.4 Hz), 7.05 (1H, d, J ) 1.4 Hz), 7.22

(1H, d, J ) 1.4 Hz), 7.37 (1H, d, J ) 1.4 Hz), 7.78 (1H, t, J )

5.8 Hz), 7.82 (1H, s), 8.18 (1H, s), 8.32 (1H, s). IR (KBr): 3298,

2960, 1644, 1582, 1538, 1405, 1250 cm^-1. HRFABMS found:

595.37208. Calculated for C₃₁H₄₆N₈O₄: 595.37203.

N-[5-({[5-({[3-(Dim eth ylam in o)pr opyl]am in o}car bon yl)-

1-m eth yl-1H-p yr r ol-3-yl]a m in o}ca r bon yl)-1-m eth yl-1H-

p yr r ol-3-yl]-1-m eth yl-4-[(4-m eth ylp en ta n oyl)a m in o]-1H-

pyr r ole-2-car boxam ide (MP e-P yr Me-P yr Me-P yr Me-Dm ap,

17). (NO₂)PyrMe-PyrMe-PyrMe-Dmap (131 mg, 0.263 mmol)

was dissolved in ethanol (20 mL) to which Pd/C (10%, 105 mg)

was added at 0 °C under N₂with stirring. The reaction mixture

was hydrogenated for 4 h at room temperature and atmo-

spheric pressure. The catalyst was removed by filtration

through Kieselguhr and the solvent removed under reduced

pressure. The crude amine was dissolved in THF (5 mL, dry)

and added to a solution of 4-methylpentanoic acid (31 mg,

0.267 mmol) in DMF (2 mL, dry) containing HBTU (100 mg).

The reaction mixture was stirred at room temperature over-

night under N₂. Ethyl acetate (100 mL) was added and the

reaction mixture was extracted with brine. The organic layer

was dried (MgSO₄) and solvent removed under reduced pres-

sure. The crude product was purified over silica gel using ethyl

acetate/methanol/triethylamine (100:100:1). The product was

obtained as a pale yellow solid (91 mg, 61%), R_f) 0.1, mp 125-

127 °C (transparent). ¹H NMR (CDCl₃): δ 0.93 (6H, d, J ) 6.3

Hz), 1.59-1.66 (2H, m), 1.73-1.77 (2H, m), 2.29-2.35 (8H

NMe₂& CH₂, m), 2.48 (2H, t, J ) 6.3 Hz), 3.43-3.66 (2H, m),

3.90 (9H, s), 6.55 (1H, d, J ) 1.8 Hz), 6.59 (1H, d, J ) 1.7 Hz),

6.67 (1H, d, J ) 1.7 Hz), 7.06 (1H, d, J ) 1.7 Hz), 7.13 (1H, d,

J ) 1.7 Hz), 7.22 (1H, d, J ) 1.7 Hz), 7.71 (1H, s), 7.66 (1H,

s), 7.70 (1H, s), 7.96 (1H, s). IR (KBr): 2953, 1643, 1582, 1538,

1463, 1433, 1402, 1259, 1207, 1105 cm^-1. HRFABMS found:

567.34085. Calculated for C₂₉H₄₃O₄N₈: 567.34073.

1

40%). H NMR (DMSO-d₆): δ 10.29 (1H, s), 9.96 (1H, s), 9.89

(1H, s), 9.270 (1H, br, TFA), 8.15 (1H, t, J ) 5.6 Hz), 7.53-

7.37 (3H, m), 7.32 (1H, d, J ) 1.6 Hz), 7.29 (1H, d, J ) 1.6

Hz), 7.16 (1H, d, J ) 1.6 Hz), 7.14-7.10 (2H, m), 7.02 (1H, d,

J ) 1.6 Hz), 6.93 (1H, d, J ) 1.6 Hz), 4.32 (1H, t, J ) 6.9 Hz),

3.88 (3H, s), 3.83 (3H, s), 3.82 (3H, s), 3.25 (2H, q, J ) 6.2

Hz), 3.07 (2H, m), 2.79 (6H, d, J ) 4.5 Hz), 1.84 (2H, quintet,

J ) 7.3 Hz), 1.59-1.47 (3H, m), 0.90 (6H, d, J ) 6.3 Hz). IR

(KBr): 1644, 1583, 1533, 1464, 1436, 1402, 1260, 1200, 801,

778 cm^-1. HRFABMS found: 659.36784. Calculated for

C

₃₅H₄₇N₈O₅: 659.36694.

N-[5-({[5-({[3-(Dim eth ylam in o)pr opyl]am in o}car bon yl)-

1-m et h yl-1H -p yr r ol-3-yl]a m in o}ca r b on yl)-1-isop en t yl-

1H -p yr r ol-3-yl]-4-{[(4-m et h oxyp h en yl)a cet yl]a m in o}-1-

m et h yl-1H -p yr r ole-2-ca r b oxa m id e (4MeOP h e-P yr Me-

P yr iP e-P yr MeDm a p , 20). The second portion of the amine

(prepared in the previous experiment) was used in this

experiment. To the amine (2.5 mL) in dichloromethane was

added (4-methoxyphenyl)acetyl chloride (50 mg, 0.270 mmol)

at room temperature, dropwise with stirring. Stirring was

continued overnight. The solvent was removed under reduced

pressure and the crude product so formed was purified by

HPLC. Fractions containing the required material were col-

lected and freeze-dried to give a white solid (51 mg, 28%) with

1

no distinct melting point. H NMR (acetone-d₆): δ 12.15 (1H,

br), 9.18 (1H, s), 9.11 (1H, s), 9.05 (1H, s), 7.67 (1H, t,

unresolved), 7.29-7.24 (4H, m), 7.13 (1H, d, J ) 1.7 Hz), 6.89-

6.85 (4H, m), 6.78 (1H, d, J ) 1.7 Hz), 4.41 (2H, t, J ) 6.5

Hz), 3.90 (6H, s), 3.77 (3H, s), 3.58 (2H, s), 3.45 (2H, q, J )

6.1 Hz), 3.26 (2H, t, J ) 6.1 Hz), 2.93 (6H, s), 1.69-1.56 (3H,

m), 0.95 (6H, d, J ) 6.6 Hz). IR (KBr): 1650, 1588, 1515, 1465,

1402, 1251, 1203, 1133, 826, 779 cm^-1. HRFABMS found:

673.38492. Calculated for C₃₆H₄₉N₈O₅: 673.38259.

N-[5-({[5-({[3-(Dim eth ylam in o)pr opyl]am in o}car bon yl)-

1-m eth yl-1H-p yr r ol-3-yl]a m in o}ca r bon yl)-1-m eth yl-1H-

p yr r ol-3-yl]-4-[(3-m eth oxyben zoyl)a m in o]-1-m eth yl-1H-

p yr r ole-2-ca r boxa m id e (3MeOB-P yr Me-P yr Me-P yr Me-

Dm a p , 18). (NO₂)PyrMe-PyrMe-PyrMe-Dmap (52 mg, 0.105

mmol) was dissolved in methanol (25 mL) to which was added

Pd/C (10%, 46 mg) at 0 °C under N₂with stirring. The reaction

mixture was hydrogenated for 2 h at room temperature and

atmospheric pressure. The catalyst was removed by filtration

through Kieselguhr and the solvent was removed under

reduced pressure. The amine so formed was dissolved in

dichloromethane (10 mL, dry) to which N-methylmorpholine

(200 µL, dry) and m-anisoyl chloride (36 mg, 210 µL) were

added sequencially at room temperature with stirring. The

stirring was continued at room temperature overnight. Volatile

materials were removed under reduced pressure, and the crude

product was purified by HPLC to give the required product

as a white solid material (19 mg, 26%) with no distinct melting

N-[5-({[5-({[3-(Dim eth ylam in o)pr opyl]am in o}car bon yl)-

1-m eth yl-1H-p yr r ol-3-yl]a m in o}ca r bon yl)-1-m eth yl-1H-

p yr r ol-3-yl]-1-m eth yl-4-{[(2E)-3-p h en yl-2-p r op en oyl]a m i-

n o}-1H-pyr r ole-2-car boxam ide (Cin -P yr Me-P yr Me-P yr Me-

Dm a p , 21). (NO₂)PyrMe-PyrMe-PyrMe-Dmap (190 mg, 0.241

mmol) was dissolved in ethanol (20 mL) at 0 °C. Pd/C (10%,

103 mg) was added to the reaction mixture under N₂. The

reaction mixture was hydrogenated for 4 h at room tempera-

ture and atmospheric pressure. The catalyst was removed by

filtration through Kieselguhr and then the solvent was re-

moved under reduced pressure to give the amine, which was

used in the coupling reaction without further purification.

Cinnamic acid (54 mg, 0.365 mmol) was dissolved in DMF (2

1

point. H NMR (DMSO-d₆): δ 10.29 (1H, s), 9.96 (1H, s), 9.79

Distamycin Analogues with Enhanced Lipophilicity

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2151

mL, dry) to which were added HBTU (203 mg, 0.535 mmol),

N-methylmorpholine (0.200 mL, dry), and the amine, which

was dissolved in DMF (2 mL, dry) at room temperature with

stirring. The stirring was continued at room temperature

overnight. The solvent was removed under reduced pressure,

then the crude mixture was dissolved in ethyl acetate (100

mL) and extracted with brine (50 mL). The organic layer was

dried (MgSO₄), and the solvent removed under reduced pres-

sure at 60 °C. The crude product was chromatographed over

silica gel using methanol/ethyl acetate/triethylamine (100:100:

1) to give the product as a yellow glassy material (36 mg, 25%)

with no distinct melting point. This material was further

purified by HPLC to give the pure product (19 mg), as the TFA

salt. ¹H NMR (DMSO-d₆): δ 1.83 (2H, qt), 2.79 (6H, d, J ) 4.7

Hz), 3.07 (2H, m), 3.25 (2H, m), 3.81 (3H, s), 3.85 (3H, s), 3.87

(3H, s), 6.76-6.80 (1H, d, J ) 15.7 Hz), 6.95 (1H, s), 6.97 (1H,

s), 7.07 (1H, s), 7.17 (1H, s), 7.23 (1H, s), 7.31 (1H, s), 7.39-

7.46 (3H, m), 7.49-7.53 (1H, d, J ) 15.7 Hz), 7.59 (1H, s),

7.61 (1H, s), 8.15 (1H, t, J ) 5.5 Hz), 9.19 (1H, br, TFA), 9.19

(1H, s), 9.95 (1H, s), 10.18 (1H, s). IR (KBr): 1648, 1581, 1540,

added at room temperature with stirring. The acid chloride

solution was added dropwise to the amine solution with

stirring. The stirring was continued at room temperature

overnight. A solution of sodium hydroxide [sodium hydroxide

(186 mg) in water (10 mL)] was added to the reaction mixture.

The organic layer was collected and dried (MgSO₄), and the

solvent was removed under reduced pressure to give the crude

product, which was applied to a column chromatography using

silica gel and methanol/ethyl acetate/triethylamine (1/4/0.02),

R_f) 0.15. The product was obtained as a yellow glassy

material (180 mg, 46%) with no distinct melting point. Some

of this material was further purified by HPLC for analysis.

¹H NMR (DMSO-d₆): δ 10.27 (1H, s), 9.96 (1H, s), 9.88 (1H,

s), 9.31 (1H, br, TFA), 8.19 (1H, d, J ) 1.8 Hz), 8.14 (1H, t, J

) 5.7 Hz), 7.59 (1H, d, J ) 1.8 Hz), 7.29 (1H, d, J ) 1.8 Hz),

7.27 (1H, d, J ) 1.8 Hz), 7.16 (1H, d, J ) 1.8 Hz), 7.06 (1H, d,

J ) 1.8 Hz), 7.02 (1H, d, J ) 1.8 Hz), 6.93 (1H, d, J ) 1.8 Hz),

4.32 (2H, t, J ) 7.1 Hz), 3.97 (3H, s), 3.87 (3H, s), 3.82 (3H, s),

3.25 (2H, quintet, J ) 8.1 Hz), 1.59-1.47 (3H, m), 0.90 (6H,

d, J ) 6.4 Hz). IR (KBr): 1649, 1587, 1528, 1464, 1397, 1308,

1252, 1203 cm^-1. HRFABMS found: 677.35253. Calculated for

1465, 1434, 1404, 1262, 1203 cm^-1

. HRFABMS found:

599.30906. Calculated for C₃₂H₃₉N₈O₄: 599.30943.

C

₃₃H₄₅N₁₀O₆: 677.35235.

N-[5-({[3-(Dim eth yla m in o)p r op yl]a m in o}ca r bon yl)-1-

m eth yl-1H-pyr r ol-3-yl]-1-m eth yl-4-{[(1-m eth yl-4-{[(2-m eth -

yl-3-th ien yl)ca r bon yl]a m in o}-1H-p yr r ol-2-yl)ca r bon yl]-

am in o}-1H-pyr r ole-2-car boxam ide (MeTh i-P yr Me-P yr Me-

P yr Me-Dm a p , 22). 2-Methyl-3-thiophenecarboxylic acid (50

mg, 0.352 mmol) was dissolved in thionyl chloride (1 mL) and

heated until reflux for 2 h. Excess thionyl chloride was

removed under reduced pressure to give the acid chloride,

which was used without further purification. (NO₂)PyrMe-

PyrMe-PyrMe-Dmap (175 mg, 0.352 mmol) was dissolved in

methanol (25 mL) to which Pd/C (10%, 95 mg) was added at 0

°C with stirring under N₂. The reaction mixture was hydro-

genated for 2 h at room temperature and atmospheric pres-

sure. The catalyst was removed over Kieselguhr and the

solvent was removed under reduced pressure. The amine was

dissolved in dichloromethane (5 mL) to which N-methylmor-

pholine (0.2 mL) was added at room temperature with stirring.

The acid chloride was dissolved in dichloromethane (5 mL) and

then it was added to the amine solution dropwise with stirring.

The reaction mixture was left standing at room temperature

for 48 h. The volatile material was removed under reduced

pressure and the crude product was purified by column

chromatography using silica gel and methanol/ethyl acetate/

triethylamine (1/1/0.05) to give the product as a yellow solid,

R_f) 0.5 (110 mg, 53%), mp 140-143 °C (transparent). ¹H NMR

(DMSO-d₆): δ 9.97 (1H, s), 9.94 (1H, s), 9.87 (1H, s), 8.05 (1H,

t, J ) 5.5 Hz), 7.42 (1H, d, J ) 5.4 Hz), 7.35 (1H, d, J ) 5.4

Hz), 7.29 (1H, d, J ) 1.7 Hz), 7.24 (1H, d, J ) 1.7 Hz), 7.18

(1H, d, J ) 1.7 Hz), 7.06 (1H, d, J ) 1.7 Hz), 7.04 (1H, d, J )

1.7 Hz), 6.82 (1H, d, J ) 1.7 Hz), 3.87 (3H, s), 3.85 (3H, s),

3.79 (3H, s), 3.19 (2H, q, J ) 6.7 Hz), 2.66 (3H, s), 2.24 (2H, t,

J ) 7.0 Hz), 2.14 (6H, s), 1.61 (2H, quintet, J ) 7.0 Hz). IR

(KBr): 1640, 1580, 1544, 1463, 1434, 1400, 1260 cm^-1. HR-

FABMS found: 593.26448. Calculated for C₂₉H₃₇N₈O₄S:

593.26585.

N-[3-(Dim eth yla m in o)p r op yl]-1-eth yl-4-({[4-({[4-(for m -

yla m in o)-1-m et h yl-1H -p yr r ol-2-yl]ca r b on yl}a m in o)-1-

m eth yl-1H-p yr r ol-2-yl]ca r bon yl}a m in o)-1H-im id a zole-2-

ca r boxa m id e (F o-P yr Me-P yr Me-Im iEt-Dm a p , 24). (NO₂)-

PyrMe-PyrMe-ImiEt-Dmap (50 mg, 0.091 mmol) was sus-

pended in a mixture of ethanol (20 mL) and water (250 µL),

to which Pd/C (10%, 50 mg) was added at 0 °C with stirring.

The reaction mixture was hydrogenated at room temperature

and atmospheric pressure for 20 h. The catalyst was removed

by filtration through Kieselguhr and ethyl formate (20 mL)

was added to the ethanolic solution of the amine. The reaction

mixture was heated under reflux for 5d. The solvent was

removed under reduced pressure and the crude product was

purified by reverse phase HPLC. Fractions containing the

required material were collected and freeze-dried. The product

was obtained as a pale yellow solid (13 mg, 23%) with no

distinct melting point. ¹H NMR (DMSO-d₆): δ 1.33-1.36 (3H,

t, J ) 7.0 Hz, CH₂CH₃), 1.85-1.90 (2H, quintet, J ) 7.7 Hz,

CH₂), 2.77-2.79 (6H, d, J ) 4.8 Hz, NMe₂), 3.05-3.10 (2H, m,

CH₂), 3.30-3.32 (2H, m, CH₂), 3.84 (3H, s, NMe), 3.84 (3H, s,

NMe), 4.40-4.45 (2H, q, J ) 4.4 Hz, CH₂), 6.92 (1H, d, J )

1.4 Hz), 7.07 (1H, d, J ) 1.4 Hz), 7.18 (1H, d, J ) 1.4 Hz),

7.29 (1H, d, J ) 1.4 Hz), 7.55 (1H, s), 8.12 (1H, d, J ) 1.4 Hz),

8.21 (1H, t, J ) 6.1 Hz, CONH, exch), 9.22 (1H, br, hydrochloric

acid, exch), 9.91 (1H, s, CONH, exch), 10.03 (1H, s, CONH,

exch), 10.12 (1H, s, CONH, exch). IR (KBr): 3438, 3288, 1675,

1559, 1438, 1406, 1203, 1134 cm^-1

. HRFABMS found:

521.27324. Calculated for C₂₄H₃₄N₉O₄: 512.27338.

N-[2-({[2-({[3-(Dim eth ylam in o)pr opyl]am in o}car bon yl)-

1-et h yl-1H -im id a zol-4-yl]a m in o}ca r b on yl)-1-et h yl-1H -

im id a zol-4-yl]-1-et h yl-4-(for m yla m in o)-1H -im id a zole-2-

ca r b oxa m id e (F o-Im iE t -Im iE t -Im iE t -Dm a p , 25). (NO₂)-

ImiEt-ImiEt-ImiEt-Dmap (150 mg, 0.193 mmol) was dissolved

in ethanol (15 mL), and then Pd/C (10%, 99 mg) was added

with stirring at 0 °C under nitrogen. The reaction mixture was

hydrogenated at room temperature and atmospheric pressure

for 4 h. The catalyst was removed by filtration through

Kieselguhr and then ethyl formate (25 mL) was added to the

ethanolic solution. The reaction mixture was heated under

reflux for 5 d. The solvents were removed under reduced

pressure, and the crude product was purified using HPLC.

Fractions were collected and freeze-dried to give the required

product as a white solid (12 mg, 10%) with no distinct melting

point. ¹H NMR (DMSO-d₆): δ 1.33-1.40 (9H, m, 3CH₂CH₃),

1.87-1.94 (2H, quintet, J ) 6.7 Hz, CH₂), 2.72-2.73 (6H, d, J

) 4.9 Hz, NMe₂), 3.02-3.07 (2H, quintet, J ) 5.4 Hz, CH₂),

3.28-3.33 (2H, q, J ) 6.4 Hz, CH₂), 4.42-4.52 (6H, m, 3CH₂),

7.60 (1H, s), 7.61 (1H, s), 7.72 (1H, s), 8.23 (1H, s), 8.49 (1H,

t, J ) 6.1 Hz, CONH, exch), 9.20 (1H, br, TFA, exch), 9.60

(1H, s, CONH, exch), 9.75 (1H, s, CONH, exch), 10.51 (1H, s,

CONH, exch). IR (KBr): 3422, 16, 76, 1568, 1537, 1474, 1431,

N-[5-({[5-({[3-(Dim eth ylam in o)pr opyl]am in o}car bon yl)-

1-m et h yl-1H -p yr r ol-3-yl]a m in o}ca r b on yl)-1-isop en t yl-

1H-p yr r ol-3-yl]-1-m eth yl-4-{[(1-m eth yl-4-n itr o-1H-p yr r ol-

2-yl)car bon yl]am in o}-1H-pyr r ole-2-car boxam ide (P yr NO₂-

P yr Me-P yr iP e-P yr Me-Dm a p , 23). (NO₂)PyrMe-PyriPe-

PyrMe-Dmap (320 mg, 0.577 mmol) was dissolved in methanol

(25 mL) to which Pd/C (10%, 226 mg) was added at 0 °C under

N₂with stirring. The reaction mixture was hydrogenated for

4 h at room temperature and atmospheric pressure. The

catalyst was removed over Kieselguhr and the solvent was

removed under reduced pressure at 50 °C. 1-Methyl-4-nitro-

1H-pyrrole-2-carboxylic acid (98 mg, 0.577 mmol) was sus-

pended in thionyl chloride (2 mL) and heated under reflux for

4 h. Excess thionyl chloride was removed under reduced

pressure and the acid chloride so formed was dissolved in

dichloromethane (5 mL). The amine was dissolved in dichlo-

romethane (5 mL) to which N-methylmorpholine (0.2 mL) was

2152 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8

Khalaf et al.

1385, 1200, 1132, 899 cm^-1. HRFABMS found: 542.29384.

Calculated for C₂₄H₃₆N₁₁O₄: 542.29517.

4-ca r b oxa m id e (F o-P yr Me-Th ziP r -P yr Me-Dm a p , 29).

(NO₂)PyrMe-ThziPr-PyrMe-Dmap (137 mg, 0.252 mmol) was

dissolved in ethanol (15 mL), to which Pd/C (10%, 121 mg)

was added under N₂at 0 °C. The reaction mixture was

hydrogenated at room temperature and atmospheric pressure

for 2.5 h. The catalyst was removed by filtration through

Kieselguhr and then ethyl formate (25 mL) was added to the

ethanolic solution. The reaction mixture was heated under

reflux overnight, following which the solvent was removed

under reduced pressure and the product was then purified by

HPLC. The product was obtained after freeze-drying as a white

solid (41 mg, 25%), with no distinct melting point. ¹H NMR

(DMSO-d₆): δ 12.09 (1H, s), 10.14 (1H, s), 9.59 (1H, s), 9.23

(1H, br, TFA), 8.35-8.32 (1H, d, J ) 1.8 Hz and 1H, t, J ) 5.6

Hz), 7.39 (1H, s), 7.28 (1H, d, J ) 1.8 Hz), 7.20 (1H, d, J ) 1.8

Hz), 7.01 (1H, d, J ) 1.8 Hz), 4.17-4.12 (1H, m), 3.88 (3H, s),

3.82 (3H, s), 3.25 (2H, m), 3.06 (2H, m), 2.79 (6H, d, J ) 3.7

Hz, NMe₂), 1.86-1.82 (2H, m), 1.31 (6H, d, J ) 6.8 Hz). IR

N-[2-({[3-(Dim eth yla m in o)p r op yl]a m in o}ca r bon yl)-1-

et h yl-1H -im id a zol-4-yl]-1-et h yl-4-({[4-(for m yla m in o)-1-

m eth yl-1H-p yr r ol-2-yl]ca r bon yl}a m in o)-1H-im id a zole-2-

ca r boxa m id e (F o-P yr Me-Im iEt-Im iEt-Dm a p , 26). (NO₂)-

PyrMe-ImiEt-ImiEt-Dmap (20 mg, 0.038 mmol) was dissolved

in ethanol (25 mL) to which Pd/C (10%, 70 mg) was added at

0 °C under N₂. The reaction mixture was hydrogenated for 5

h at room temperature and atmospheric pressure. The catalyst

was removed by filtration through Kieselguhr and to the

ethanolic solution ethyl formate (20 mL) was added. The

mixture was heated at reflux overnight. The volatile material

was removed under reduced pressure and the residue was

purified by HPLC. Fractions containing the required material

were collected and freeze-dried to give a white solid (5 mg,

21%) which was very hygroscopic and turned to a colorless oil.

¹H NMR (DMSO-d₆): δ 10.55 (1H, s), 10.45 (1H, s), 9.36 (1H,

s), 9.20 (1H, br, TFA), 8.42 (1H, t, J ) 5.4 Hz), 7.69 (1H, s),

7.62 (1H, s), 7.20 (1H, s), 7.10 (1H, s), 4.47 (4H, m), 3.90 (3H,

s), 3.29 (2H, m), 3.08 (2H, m), 2.78 (6H, d, J ) 4.6 Hz), 1.91

(2H, m), 1.38 (4H, m). IR (KBr): 2950, 2833, 1653, 1590, 1535,

1510, 1400, 1310 cm^-1. HRFABMS found: 527.28372. Calcu-

lated for C₂₄H₃₅N₁₀O₄: 527.28427.

(KBr): 3428, 1662, 1545, 1467, 1401, 1284, 1201, 1134 cm^-1

.

HRFABMS found: 543.24988. Calculated for C₂₅H₃₅N₈O₄S:

543.25020.

2-(Acetyla m in o)-N-[5-({[5-({[3-(d im eth yla m in o)p r op yl]-

a m in o }c a r b o n y l)-1-m e t h y l-1H -p y r r o l-3-y l]a m in o }-

ca r bon yl)-1-m eth yl-1H-p yr r ol-3-yl]-5-isop r op yl-1,3-th ia -

zole-4-car boxam ide (Ac-Th ziP r -P yr Me-P yr Me-Dm ap, 30).

(NO₂)PyrMe-PyrMe-Dmap (105 mg, 0.279 mmol) was dissolved

in methanol (25 mL). The solution was cooled to 0 °C under

N₂then Pd/C (10%, 82 mg) was added. The reaction mixture

was hydrogenated at room temperature and atmospheric

pressure for 3 h. The catalyst was removed by filtration

through Kieselguhr and then the solvent was removed under

reduced pressure at 50 °C. The amine thus formed was

dissolved in DMF (2 mL, dry), to which 2-(acetylamino)-5-

isopropyl-1,3-thiazole-4-carboxylic acid (96 mg, 0.418 mmol)

was added followed by HBTU (211 mg, 0.556 mmol) and

N-methylmorpholine (300 µL, dry). The reaction mixture was

left stirring at room temperature overnight, after which it was

diluted with ethyl acetate and sodium bicarbonate solution

with stirring. The organic layer was separated and dried

(MgSO₄) and the solvent removed under reduced pressure. The

product was purified by HPLC to give the pure material as a

N-[3-(Dim eth ylam in o)pr opyl]-2-({[4-({[4-(for m ylam in o)-

1-m eth yl-1H-p yr r ol-2-yl]ca r bon yl}a m in o)-1-m eth yl-1H-

p yr r ol-2-yl]ca r bon yl}a m in o)-4-isop r op yl-1,3-th ia zole-5-

ca r boxa m id e (F o-P yr Me-P yr Me-isoTh ziP r -Dm a p , 27).

(NO₂)PyrMe-PyrMe-isoThziPr-Dmap (91 mg, 0.167 mmol) was

suspended in ethanol (20 mL) and then Pd/C (100 mg, 10%)

was added at 0 °C and under nitrogen. The reaction mixture

was hydrogenated for 4 h at room temperature and atmo-

spheric pressure. The catalyst was removed under reduced

pressure and then ethyl formate (10 mL) was added. The

reaction mixture was heated under reflux for 48 h, after which

the solvent was removed under reduced pressure. The crude

product was purified by HPLC. Fractions containing the

required material were freeze-dried to give the product as a

pale yellow solid (16 mg, 16%) with no distinct melting point.

¹H NMR (DMSO-d₆): δ 12.50 (1H, s), 10.05 (1H, s), 9.95 (1H,

s), 9.27 (1H, br, TFA), 8.13 (2H, m), 7.45 (1H, s), 7.42 (1H, d,

J ) 1.6 Hz), 7.20 (1H, d, J ) 1.6 Hz), 6.94 (1H, d, J ) 1.6 Hz),

3.88 (3H, s), 3.84 (3H, s), 3.26 (2H, q, J ) 5.6 Hz), 3.06 (2H,

m), 2.79 (6H, d, J ) 4.8 Hz), 1.90 (2H, m), 1.22 (6H, d, J ) 6.8

1

white solid (71.3 mg, 38%) with no distinct melting point. H

NMR (DMSO-d₆): δ 12.31 (1H, s), 8.87 (1H, s), 9.57 (1H, s),

9.24 (1H, br), 8.14 (1H, t, J ) 5.6 Hz), 7.26 (1H, d, J ) 1.8

Hz), 7.17 (1H, d, J ) 1.8 Hz), 7.12 (1H, d, J ) 1.8 Hz), 6.94

(1H, d, J ) 1.8 Hz), 4.19-4.12 (1H, m), 3.85 (3H, s), 3.81 (3H,

s), 3.25 (2H, m), 3.07 (2H, m), 2.79 (6H, d, J ) 3.7 Hz), 2.16

(3H, s), 1.83 (2H, quintet, J ) 7.5 Hz), 1.29 (6H, d, J ) 6.8

Hz). IR (KBr): 1676, 1644, 1550, 1465, 1435, 1404, 1202, 1136

Hz). IR (KBr): 1683, 1587, 1549, 1447, 1210, 1134 cm^-1

.

HRFABMS found: 544.25112. Calculated for C₂₅H₃₆N₈O₄S:

544.25081.

N-[3-(Dim eth ylam in o)pr opyl]-2-({[4-({[4-(for m ylam in o)-

1-m eth yl-1H-p yr r ol-2-yl]ca r bon yl}a m in o)-1-m eth yl-1H-

p yr r ol-2-yl]ca r bon yl}a m in o)-5-isop r op yl-1,3-th ia zole-4-

ca r b oxa m id e [(NO₂)P yr Me-P yr Me-Th ziP r -Dm a p , 28].

(NO₂)PyrMe-PyrMe-ThziPr-Dmap (90 mg, 0.165 mmol) was

suspended in ethanol (20 mL) and then Pd/C (97 mg, 10%)

was added at 0 °C under nitrogen. The reaction mixture was

hydrogenated at room temperature and atmospheric pressure

for 5 h. The catalyst was removed under reduced pressure,

then ethyl formate (20 mL) was added to the ethanolic solution

of the amine. The reaction mixture was heated under reflux

for 3 d, and then the solvent was removed under reduced

pressure. The crude product was dissolved in water (2 mL)

containing 0.1% TFA and then purified by HPLC. The fractions

collected were freeze-dried to give the desired product as a pale

yellow solid (55 mg, 51%) with no distinct melting point. ¹H

NMR (DMSO-d₆): δ 1.270-1.287 (6H, d, J ) 6.9 Hz), 1.86 (2H,

m), 2.78-2.79 (6H, d, J ) 4.0 Hz), 3.07 (2H, m), 3.37 (2H, m),

3.84 (3H, s), 3.88 (3H, s), 4.19 (1H, m), 6.94 (1H, d, J ) 1.6

Hz), 7.19 (1H, s), 7.38 (1H, s), 7.40 (1H, s), 7.96 (1H, t, J ) 5.6

Hz), 8.13 (1H, d, J ) 1.6 Hz), 9.30 (1H, br, TFA), 9.99 (1H, s),

10.04 (1H, s), 12.02 (1H, s). IR (KBr): 1664, 1555, 1465, 1399,

1294, 1209, 1130 cm^-1. HRFABMS found: 544.25962. Calcu-

lated for C₂₅H₃₆N₈O₄S: 544.25802.

cm^-1. HRFABMS found: 557.26442. Calculated for C₂₆H₃₇

N₈O₄S: 557.26585.

-

2-(Acetyla m in o)-N-[5-({[4-({[3-(d im eth yla m in o)p r op yl]-

a m in o}ca r b on yl)-5-isop r op yl-1,3-t h ia zol-2-yl]a m in o}-

ca r bon yl)-1-m eth yl-1H-p yr r ol-3-yl]-5-isop r op yl-1,3-th ia -

zole-4-car boxam ide (Ac-Th ziP r -P yr Me-Th ziP r -Dm ap, 31).

(NO₂)PyrMe-ThziPr-Dmap (100 mg, 0.236 mmol) was dissolved

in methanol (25 mL). The solution was cooled to 0 °C under

N₂and then Pd/C (10%, 82 mg) was added. The reaction

mixture was hydrogenated at room temperature and atmo-

spheric pressure for 3 h. The catalyst was removed by filtration

through Kieselguhr and the solvent was then removed under

reduced pressure at 50 °C. The amine so formed was dissolved

in DMF (2 mL, dry), to which 2-(acetylamino)-5-isopropyl-1,3-

thiazole-4-carboxylic acid (64 mg, 0.356 mmol) was added

followed by HBTU (135 mg, 0.356 mmol) and N-methylmor-

pholine (200 µL, dry). The reaction mixture was left stirring

at room temperature overnight, after which it was diluted with

ethyl acetate and sodium bicarbonate solution with stirring.

The organic layer was separated and dried (MgSO₄) and the

solvent removed under reduced pressure. The product was

purified by HPLC to give the pure material as a white solid

1

N-[5-({[3-(Dim eth yla m in o)p r op yl]a m in o}ca r bon yl)-1-

m et h yl-1H -p yr r ol-3-yl]-2-({[4-(for m yla m in o)-1-m et h yl-

1H-p yr r ol-2-yl]ca r bon yl}a m in o)-5-isop r op yl-1,3-th ia zole-

(78.8 mg, 47% yield) with no distinct melting point. H NMR

(DMSO-d₆): δ 12.11 (1H, s), 12.01 (1H, s), 9.64 (1H, s), 9.27

(1H, br), 7.96 (1H, t, J ) 5.6 Hz), 7.51 (1H, d, J ) 1.8 Hz),

Distamycin Analogues with Enhanced Lipophilicity

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2153

7.42 (1H, d, J ) 1.8 Hz), 4.22-4.11 (2H, m), 3.90 (3H, s), 3.34

(2H, m), 3.08 (2H, m), 2.79 (6H, d, J ) 3.7 Hz), 2.16 (3H, s),

1.91-1.85 (2H, qintet, J ) 7.5 Hz), 1.30-1.27 (12H, m). IR

mixture was heated under reflux for 3 days. The volatile

material was removed under reduced pressure and the crude

product was purified by HPLC. Fractions containing the

required material were collected and freeze-dried to give the

desired product as a pale yellow solid (25 mg, 18%) with no

(KBr): 1666, 1549, 1508, 1466, 1398, 1286, 1201, 1134 cm^-1

.

HRFABMS found: 603.25163. Calculated for C₂₇H₃₉N₈O₄S₂:

603.25357.

1

distinct melting point. H NMR (DMSO-d₆): δ 11.32 (1H, s),

9.94 (1H, s), 9.89 (1H, s), 9.23 (1H, br, TFA), 7.16 (1H, d, J )

1.7 Hz), 7.05 (1H, d, J ) 1.7 Hz), 6.95 (1H, d, J ) 1.7 Hz),

6.94 (1H, s), 3.85 (3H, s), 3.81 (3H, s), 3.25 (2H, q, J ) 6.7

Hz), 3.07 (2H, m), 2.79 (6H, d, J ) 3.8 Hz), 2.54 (3H, s), 1.83

(2H, quintet, J ) 6.7 Hz). IR (KBr): 1677, 1645, 1579, 1541,

1466, 1436, 1403 cm^-1. HRFABMS found: 514.22420. Calcu-

lated for C₂₄H₃₂N₇O₄S: 514.22365.

2-({[4-({[4-(Acet yla m in o)-1-m et h yl-1H -im id a zol-2-yl]-

ca r b on yl}a m in o)-1-m e t h yl-1H -p yr r ol-2-yl]ca r b on yl}-

a m in o)-N-[3-(d im eth yla m in o)p r op yl]-5-isop r op yl-1,3-th i-

a zole-4-ca r b oxa m id e [Ac-P yr Me-P yr Me-Th ziP r -Dm a p ,

32). (NO₂)PyrMe-ThziPr-Dmap (105 mg, 0.25 mmol) and Pd/C

(10%, 100 mg) were suspended in methanol (20 mL) and

hydrogenated for 2.5 h at room temperature. Filtration of

catalyst on to Kieselguhr under N₂, followed by removal of the

solvent under reduced pressure gave the amine as an off-white

solid, which was used without further purification. 4-(Acety-

lamino)-1-methyl-1H-imidazole-2-carboxylic acid sodium salt³³

(120 mg, 0.7 mmol) was dissolved in DMF (0.5 mL, dry), to

which was added a solution of HBTU (137 mg, 0.36 mmol) and

N-methylmorpholine (0.2 mL, dry) in DMF (1 mL, dry) at room

temperature with stirring. The stirring was continued for 30

min at room temperature. This mixture was added to the

amine with stirring at room temperature and stirring was

continued at room temperature overnight, followed by purifi-

cation by HPLC to give the desired material as the bis-TFA

salt (30 mg, 22% yield) with no distinct melting point. ¹H NMR

(DMSO-d₆): δ 1.27 (6H, d, J ) 6.8 Hz, isopropyl), 1.87 (2H,

m), 2.02 (3H, s), 2.78 (6H, s, NMe₂), 3.07 (2H,m), 3.32 (2H,m),

3.89 (3H, s, NMe), 3.95 (3H, s, COMe), 4.19 (1H, m, isopropyl),

7.44 (2H, m), 7.48 (1H, m), 7.97 (1H, m), 9.3 (1H, br), 10.00

(1H, s), 10.23 (1H, s), 12.03 (1H, s). ΙR (ΚΒr): 1667, 1550, 1470,

1288, 1198, 1133 cm^-1. HRFABMS found: 558.26332. Calcu-

lated for C₂₅H₃₆N₉O₄S: 558.26110.

N-[4-({[5-({[3-(Dim eth ylam in o)pr opyl]am in o}car bon yl)-

1-m et h yl-1H -p yr r ol-3-yl]a m in o}ca r b on yl)-5-m et h yl-2-

t h i e n y l]-4-(fo r m y la m i n o )-1-m e t h y l-1H -p y r r o le -2-

ca r boxa m id e (F o-P yr Me-Th iMe-P yr Me-Dm a p , 35). A por-

tion of the amine solution (10 mL) from the preparation of

compound 37 was used. Ethyl formate (25 mL) was added and

the reaction mixture was heated under reflux for 3 days. The

volatile material was removed under reduced pressure and the

crude product was purified by HPLC. Fractions containing the

required material were collected and freeze-dried to give the

desired product as a white solid (30 mg, 29%) with no distinct

melting point. ¹H NMR (DMSO-d₆): δ 11.05 (1H, s), 10.08 (1H,

s), 9.89 (1H, s), 9.26 (1H, br), 8.15 (2H, s & t, J ) 5.8 Hz), 7.23

(1H, d, J ) 1.7 Hz), 7.17 (1H, d, J ) 1.7 Hz), 7.08 (1H, d, J )

1.7 Hz), 6.96 (1H, s), 6.90 (1H, d, J ) 1.7 Hz), 3.84 (3H, s),

3.80 (3H, s), 3.24 (2H, q, J ) 6.7 Hz), 3.08 (2H, m), 2.77 (6H,

d, J ) 3.8 Hz), 2.50 (3H, s), 1.85 (2H, quintet, J ) 6.7 Hz). IR

(KBr): 1677, 1639, 1577, 1535, 1465, 1436, 1400, 1292, 1200,

1131 cm^-1. HRFABMS found: 514.22583. Calculated for

C

₂₄H₃₂N₇O₄S: 514.22365.

N-[5-({[5-({[3-(Dim eth ylam in o)pr opyl]am in o}car bon yl)-

1-m eth yl-1H-p yr r ol-3-yl]a m in o}ca r bon yl)-1-m eth yl-1H-

p yr r ol-3-yl]-5-isop r op yl-2-[(3-m et h oxyb en zoyl)a m in o]-

N-[5-({[3-(Dim eth yla m in o)p r op yl]a m in o}ca r bon yl)-1-

m eth yl-1H-p yr r ol-3-yl]-4-({[5-(for m yla m in o)-2-m eth yl-3-

t h ien yl]ca r b on yl}a m in o)-1-isop en t yl-1H -p yr r ole-2-ca r -

b oxa m id e (F o-Th iMe-P yr iP e-P yr Me-Dm a p , 36). (NO₂)-

ThiMe-PyriPe-PyrMe-Dmap (110 mg, 0.193 mmol) was dis-

solved in ethanol (20 mL) and cooled to 0 °C. Pd/C (10%, 120

mg) was added under N₂with stirring and the reaction was

hydrogenated at room temperature and atmospheric pressure

for 3 h. The catalyst was removed by filtration through

Kieselguhr, then ethyl formate (20 mL) was added to the

ethanolic solution. The reaction mixture was heated under

reflux for 3 d. The volatile material was then removed under

reduced pressure and the crude product was purified by HPLC.

The product was obtained as a white solid with no distinct

melting point (23 mg, 17.4%). ¹H NMR (DMSO-d₆): δ 11.32

(1H, s), 9.94 (1H, s), 9.88 (1H, s), 9.24 (1H, br, TFA), 8.29 (1H,

s), 8.14 (1H, t, J ) 5.9 Hz), 7.29 (1H, d, J ) 1.7 Hz), 6.95 (1H,

s), 6.93 (1H, d, J ) 1.7 Hz), 4.32 (1H, t, J ) 7.0 Hz), 3.81 (3H,

s), 3.25 (2H, q, J ) 5.1 Hz), 3.06 (2H, m), 2.79 (6H, d, J ) 3.5

Hz), 2.5 (3H, s), 1.83 (2H, quintet, J ) 6.7 Hz), 1.57-1.49 (3H,

m), 0.90 (6H, d, J ) 6.4 Hz). IR (KBr): 1671, 1651, 1582, 1537,

1,3-t h ia zole-4-ca r b oxa m id e

(3MeOB-Th ziP r -P yr Me-

P yr Me-Dm a p , 33). (NO₂)PyrMe-PyrMe-Dmap (113 mg, 0.300

mmol) was suspended in methanol (25 mL) at 0 °C with

stirring under N₂. Pd/C (10%, 61 mg) was added with stirring

under N₂and then the reaction mixture was hydrogenated at

room temperature and atmospheric pressure for 3 h. Filtration

of the catalyst on to Kieselguhr under N₂and removal of the

solvent under reduced pressure gave the amine, which was

used in the coupling reaction without further purification.

5-Isopropyl-2-[(3-methoxybenzoyl)amino]-1,3-thiazole-4-car-

boxylic acid (96 mg, 3.00 mmol) was dissolved in thionyl

chloride (2 mL) then heated under reflux for 3 h. The excess

thionyl chloride was removed under reduced pressure and the

acid chloride so formed was dissolved in dichloromethane (5

mL). The above amine was dissolved in dichloromethane (5

mL), to which N-methylmorpholine (0.2 mL) was added. The

acid chloride solution was added dropwise at room temperature

with stirring and then the stirring was continued overnight

at room temperature. The volatile material was removed under

reduced pressure and the crude product was purified by HPLC.

Fractions containing the required material were collected and

freeze-dried to give the product as a pale yellow solid (73 mg,

1464, 1403, 1200, 1179, 1132 cm^-1

. HRFABMS found:

570.28565. Calculated for C₂₈H₄₀N₇O₄S: 570.28625.

4-(Acet yla m in o)-N-[4-({[5-({[3-(d im et h yla m in o)p r op -

yl]amino}carbonyl)-1-methyl-1H-pyrrol-3-yl]amino}carbon-

yl)-5-m et h yl-2-t h ien yl]-1-m et h yl-1H -p yr r ole-2-ca r b ox-

a m id e (Ac-P yr Me-Th iMe-P yr Me-Dm a p , 37). (NO₂)PyrMe-

ThiMe-PyrMe-Dmap (160 mg, 0.310 mmol) was dissolved in

ethanol (20 mL) at 0 °C with stirring under N₂. Pd/C (10%,

129 mg) was added and the reaction mixture was hydroge-

nated for 2 h at room temperature and atmospheric pressure.

The catalyst was removed over Kieselguhr and the amine

solution was divided into two equal volumes. The solvent was

removed from the first (10 mL) under reduced pressure and

the amine so formed was dissolved in dichloromethane (5 mL)

to which N-methylmorpholine (0.1 mL) was added with stirring

at room temperature followed by acetyl chloride (12 µL). The

reaction mixture was stirred at room temperature overnight.

The volatile material was removed under reduced pressure and

the crude product was purified by HPLC. Fractions containing

the required material were collected and freeze-dried. The

1

32%) with no distinct melting point. H NMR (DMSO-d₆): δ

12.63 (1H, s), 9.88 (1H, s), 9.55 (1H, s), 9.29 (1H, br), 8.14 (1H,

t, 5.8 Hz), 7.69 (1H, s), 7.67 (1H, s), 7.48 (1H, t, J ) 5.9 Hz),

7.26 (1H, d, 1.7 Hz), 7.23 (1H, m), 7.18 (1H, d, J ) 1.7 Hz),

7.13 (1H, d, J ) 1.7 Hz), 6.94 (1H, d, 1.7 Hz), 4.19 (1H, heptet,

J ) 6.8 Hz), 3.86 (6H, s), 3.82 (3H, s), 3.24 (2H, q, J ) 6.7 Hz),

3.07 (2H, m), 2.79 (6H, d, J ) 3.8 Hz), 1.86 (2H, quintet, J )

6.7 Hz), 1.33 (6H, d, J ) 6.8 Hz). IR (KBr): 1660, 1547, 1466,

1436, 1403, 1286, 1199, 1137, 1043 cm^-1. HRFABMS found:

649.29379. Calculated for C₃₂H₄₁N₈O₅S: 649.29206.

N-[5-({[3-(Dim eth yla m in o)p r op yl]a m in o}ca r bon yl)-1-

m eth yl-1H-p yr r ol-3-yl]-4-({[5-(for m yla m in o)-2-m eth yl-3-

th ien yl]ca r bon yl}a m in o)-1-m eth yl-1H-p yr r ole-2-ca r box-

a m id e (F o-Th iMe-P yr Me-P yr Me-Dm a p , 34). The second

portion of the amine solution from the preparation of 38 was

used. Ethyl formate (25 mL) was added and the reaction

2154 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8

Khalaf et al.

product was obtained as a white solid (41 mg, 39%) with no

distinct melting point. H NMR (DMSO-d₆): δ 11.05 (1H, s),

9.89 (1H, s), 9.87 (1H, s), 9.56 (1H, br, TFA), 8.16 (1H, t,

unresolved), 8.12 (1H, d, J ) 1.7 Hz), 7.26 (1H, d, J ) 1.7 Hz),

7.18 (1H, d, J ) 1.7 Hz), 7.15 (1H, d, J ) 1.7 Hz), 7.02 (1H, d,

J ) 1.7 Hz), 6.93 (1H, d, J ) 1.7 Hz), 6.92 (1H, d, J ) 1.7 Hz),

4.31 (2H, t, J ) 7.1 Hz), 4.00 (2H, d, J ) 11.8 Hz), 3.84 (3H,

s), 3.81 (3H, s), 3.64 (2H, t, J ) 11.9 Hz), 3.46 (2H, d, J ) 12.8

Hz), 3.26 (2H, q, J ) 6.1 Hz), 3.12-3.06 (4H, m), 1.87 (2H,

m), 1.58-1.46 (3H, m), 0.90 (6H, d, J ) 6.3 Hz). IR (KBr):

1

9.91 (1H, s), 9.85 (1H, s), 9.26 (1H, br), 8.15 (1H, t, 5.8 Hz),

7.20 (1H, d, J ) 1.7 Hz), 7.19 (1H, d, J ) 1.7 Hz), 7.06 (1H, d,

J ) 1.7 Hz), 6.98 (1H, s), 6.92 (1H, d, J ) 1.7 Hz), 3.85 (3H,

s), 3.82 (3H, s), 3.32 (2H, q, J ) 6.7 Hz), 3.07 (2H, m), 2.79

(6H, d, J ) 3.8 Hz), 2.52 (3H, s), 1.98 (3H, s), 1.84 (2H, quintet,

J ) 6.7 Hz). IR (KBr): 1645, 1577, 1535, 1466, 1436, 1289,

1199, 1134 cm^-1. HRFABMS found: 514.22529. Calculated for

1682, 1640, 1584, 1529, 1403, 1263, 1202, 1132, 803 cm^-1

.

C

₂₄H₃₂N₇O₄S: 514.22365.

HRFABMS found: 595.33717. Calculated for C₃₀H₄₃N₈O₅:

595.33564.

4-({[5-(Ace t yla m in o)-2-m e t h yl-3-t h ie n yl]ca r b on yl}-

a m i n o )-N -[5-({[3-(d i m e t h y la m i n o )p r o p y l]a m i n o }-

ca r bon yl)-1-m eth yl-1H-p yr r ol-3-yl]-1-m eth yl-1H-p yr r ole-

2-ca r b oxa m id e (Ac-Th iMe-P yr Me-P yr Me-Dm a p , 38).

(NO₂)ThiMe-PyrMe-PyrMe-Dmap (213 mg, 0.439 mmol) was

dissolved in ethanol (20 mL) at 0 °C with stirring under N₂.

Pd/C (10%, 113 mg) was added and the reaction mixture was

hydrogenated for 6 h at room temperature and atmospheric

pressure. The catalyst was removed over Kieselguhr and the

solution was divided into two equal volumes. For the first half,

the solvent was removed under reduced pressure and the

residue was dissolved in dichloromethane (5 mL) to which

N-methylmorpholine (0.1 mL) was added with stirring at room

temperature followed by acetyl chloride (17 µL, 0.219 mmol).

The reaction mixture was stirred overnight at room temper-

ature. The volatile material was removed under reduced

pressure and the crude product was purified by HPLC.

Fractions containing the required material were combined and

freeze-dried to give a white solid (52 mg, 37%) with no distinct

melting point. ¹H NMR (DMSO-d₆): δ 11.08 (1H, s), 9.89 (1H,

s), 9.87 (1H, s), 9.25 (1H, br, TFA), 8.11 (1H, t, J ) 5.8 Hz),

7.22 (1H, d, J ) 1.7 Hz), 7.14 (1H, d, J ) 1.7 Hz), 7.02 (1H, d,

J ) 1.7 Hz), 6.92 (1H, d, J ) 1.7 Hz), 6.81 (1H, s), 3.83 (3H,

s), 3.79 (3H, s), 3.22 (2H, q, J ) 6.7 Hz), 3.05 (2H, m), 2.77

(6H, d, J ) 3.8 Hz), 2.50 (3H, s), 2.05 (3H, s), 1.82 (2H, quintet,

J ) 6.7 Hz). IR (KBr): 1638, 1578, 1542, 1465, 1435, 1403,

1201, 1133 cm^-1. HRFABMS found: 528.24004. Calculated for

4-({[4-(F or m yla m in o)-1-m et h yl-1H -p yr r ol-2-yl]ca r b -

on yl}a m in o)-1-isop r op yl-N-[1-m et h yl-5-({[3-(4-m or p h o-

li n y l)p r o p y l]a m i n o }c a r b o n y l)-1H -p y r r o l-3-y l]-1H -

p yr r ole-2-ca r boxa m id e (F o-P yr Me-P yr iP r -P yr Me-Mor p ,

41). (NO₂)PyrMe-PyriPr-PyrMe-Morp (160 mg, 0.282 mmol)

was dissolved in ethanol (25 mL) to which Pd/C (10%, 150 mg)

was added at 0 °C under nitrogen with stirring. The reaction

mixture was hydrogenated for 2 h at room temperature and

atmospheric pressure. The catalyst was removed by filtration

through Kieselguhr. Ethyl formate (25 mL) was added to the

ethanolic solution, then the reaction mixture was heated under

reflux overnight. Ethanol and excess ethyl formate were

removed under reduced pressure, and the crude product was

purified by HPLC to give the required material as a white solid

(40.1 mg, 21%) with no distinct melting point. ¹H NMR

(DMSO-d₆): δ 10.05 (1H, s), 9.93 (1H, s), 9.91 (1H, s), 9.58

(1H, br, TFA), 8.17 (1H, t, J ) 5.5 Hz), 8.12 (1H, s), 7.38 (1H,

s), 7.18 (1H, s), 7.16 (1H, s), 6.96 (1H, s), 6.94 (1H, s), 6.92

(1H, s), 3.84 (3H, s), 3.81 (3H, s), 3.65-3.42 (8H, m), 3.25-

3.24 (2H, q, J ) 5.9 Hz), 3.12-3.05 (4H, m), 1.87 (2H, m), 1.37

(6H, d, J ) 6.6 Hz). IR (KBr): 3423, 1672, 1644, 1581, 1536,

1462, 1440, 1407, 1254, 1200, 1134 cm^-1. HRFABMS found:

567.30312. Calculated for C₂₈H₃₉N₈O₅: 567.30434.

4-(Acetyla m in o)-N-[1-isop en tyl-5-({[1-m eth yl-5-({[3-(4-

m or p h olin yl)p r op yl]a m in o}ca r b on yl)-1H -p yr r ol-3-yl]-

a m in o}ca r bon yl)-1H-p yr r ol-3-yl]-1-m eth yl-1H-p yr r ole-2-

C

₂₅H₃₄N₇O₄S: 528.239.

ca r b oxa m id e

(Ac-P yr Me-P yr iP e-P yr Me-Mor p ,

42).

N-[3-(Dim eth ylam in o)pr opyl]-2-[({[4-({[4-(for m ylam in o)-

(NO₂)PyrMe-PyriPe-PyrMe-Morp (115 mg, 0.193 mmol) was

dissolved in ethanol (20 mL) to which was added Pd/C (10%,

104 mg) at 0 °C under N₂with stirring. The reaction mixture

was hydrogenated for 3 h at room temperature and atmo-

spheric pressure. The catalyst was removed by filtration

through Kieselguhr and the ethanolic solution was divided into

two equal volumes (10 mL). Ethanol was removed from one of

the fractions under reduced pressure and dichloromethane (2

mL) was added to the residue with stirring. N-Methylmor-

pholine (0.1 mL) was added followed by acetyl chloride (10 µL)

at room temperature with stirring. The stirring was continued

overnight. All volatile materials were removed under reduced

pressure, and the crude product was purified by HPLC.

Fractions containing the required material were collected and

freeze-dried to give a white solid material (25.7 mg, 35%, as

TFA salt) with no distinct melting point. ¹H NMR (DMSO-

d₆): δ 9.87 (2H, s), 9.79 (1H, s), 9.55 (1H, br, TFA), 8.16 (1H,

t, unresolved), 7.26 (1H, d, J ) 1.8 Hz), 7.15 (1H, d, J ) 1.8

Hz), 7.13 (1H, d, J ) 1.8 Hz), 7.01 (1H, d, J ) 1.8 Hz), 6.94

(1H, d, J ) 1.8 Hz), 6.87 (1H, d, J ) 1.8 Hz), 4.31 (2H, t, J )

6.9 Hz, isopentyl), 4.00 (2H, d, J ) 12.4 Hz, morpho), 3.83 (3H,

s), 3.81 (3H, s), 3.64 (2H, t, J ) 12.0 Hz, morpho), 3.45 (2H, d,

J ) 12.0 Hz, morpho), 3.24 (2H, q, J ) 6.2 Hz), 3.12-3.06 (2H,

m, side chain, 2H morpho), 1.97 (3H, s), 1.87 (2H, m, isopentyl),

1.58-1.48 (2H, m, side chain, 1H isopentyl), 0.90 (6H, d, J )

6.4 Hz, isopentyl). IR (KBr): 2925, 2858, 1655, 1583, 1526,

1-m eth yl-1H-p yr r ol-2-yl]ca r bon yl}a m in o)-1-m eth yl-1-H-

pyr r ol-2-yl]car bon yl}am in o)m eth yl]-5-m eth yl-1,3-oxazole-

4-ca r boxa m id e (F o-P yr Me-P yr Me-CH₂Oxa Me-Dm a p , 39).

N-[3-(Dimethylamino)propyl]-5-methyl-2-({[(1-methyl-4-{[(1-

methyl-4-nitro-1 H-pyrrol-2-yl)carbonyl]amino}-1H-pyrrol-2-

yl)carbonyl]amino}methyl)-1,3-oxazole-4-carboxamide (49 mg,

0.095 mmol) was dissolved in ethanol (15 mL), to which Pd/C

(10%, 51 mg) was added at 0 °C under N₂. The reaction mixture

was hydrogenated for 4 h at room temperature and atmo-

spheric pressure. The catalyst was removed by filtration

through Kieselguhr and then ethyl formate (25 mL) was added

to the ethanolic solution. The reaction mixture was heated

under reflux for 24 h, and then the solvent and excess reagent

were removed under reduced pressure at 40 °C. The product

was purified by HPLC and was obtained as a white hygroscopic

solid as TFA salt after freeze-drying (25 mg, 42%) with no

1

distinct melting point. H NMR (DMSO-d₆): δ 10.03 (1H, s),

9.89 (1H, s), 9.21 (1H, br, TFA), 8.65 (1H, t, J ) 5.5 Hz), 8.29

(1H, t, J ) 5.6 Hz), 8.12 (1H, s), 7.19 (1H, s), 7.17 (1H, s), 4.45

(2H, d, J ) 5.4 Hz), 3.26 (2H, m), 3.02 (2H, m), 2.77 (6H, d, J

) 4.5 Hz), 2.54 (3H, s), 1.83 (2H, m). IR (KBr): 1675, 1664,

1589, 1533, 1467, 1437, 1404, 1346 cm^-1. HRFABMS found:

513.25498. Calculated for C₂₄H₃₃N₈O₅: 513.25739.

4-(F or m yla m in o)-N-[1-isop en t yl-5-({[1-m et h yl-5-({[3-

(4-m or p h olin yl)p r op yl]a m in o}ca r bon yl)-1H-p yr r ol-3-yl]-

a m in o}ca r bon yl)-1H-p yr r ol-3-yl]-1-m eth yl-1H-p yr r ole-2-

ca r boxa m id e (F o-P yr Me-P yr iP e-P yr Me-Mor p , 40). To the

second half of the amine solution (10 mL) from the preparation

of compound 42, ethyl formate was added (10 mL) and the

reaction mixture was heated under reflux for 48 h. Volatile

solvents were removed under reduced pressure, and the crude

product was purified by HPLC. Fractions containing the

required material were collected and freeze-dried to give a

white solid material (22.7 mg, 32%, as TFA salt) with no

1465, 1400, 1260, 1200, 1131 cm^-1

. HRFABMS found:

609.34957. Calculated for C₃₁H₄₅N₈O₅: 609.35129.

N -[1-I s o p e n t y l-5-({[1-m e t h y l-5-({[3-(4-m e t h y l-1-

p ip e r a zin yl)p r op yl]a m in o}ca r b on yl)-1H -p yr r ol-3-yl]-

a m in o}ca r bon yl)-1H-p yr r ol-3-yl]-4-[(3-m eth oxyben zoyl)-

a m in o]-1-m et h yl-1H -p yr r ole-2-ca r b oxa m id e (3MeOB-

P yr Me-P yr iP e-P yr Me-MeP ip p , 43). The second portion of

the solution of amine used for the preparation of compound

45 was used. To this solution, 3-methoxybenzoyl chloride (26

mg, 26 µL, 0.153 mmol) was added dropwise at room temper-

1

distinct melting point. H NMR (DMSO-d₆): δ 10.03 (1H, s),

Distamycin Analogues with Enhanced Lipophilicity

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2155

ature with stirring. The stirring was continued overnight at

room temperature. The solvent was removed under reduced

pressure and the crude product was purified by HPLC. The

product was obtained as a light brown solid (60.8 mg, 48%)

hydrogenation in the preparation of the following compound,

47, and the amine obtained was dissolved in dichloromethane

(10 mL) to which N-methylmorpholine (100 µL) was added at

room temperature with stirring followed by acetyl chloride (14

µL), which was added dropwise. The stirring was continued

at room temperature overnight. Volatile material was removed

under reduced pressure and the crude product was purified

by HPLC. The product was obtained as a pale yellow solid (42

mg, 33%) with no distinct melting point. ¹H NMR (DMSO-d₆):

δ 9.89 (2H, s), 9.79 (1H, s), 9.37 (1H, br), 8.13 (1H, t, J ) 5.9

Hz), 7.21 (1H, d, J ) 1.7 Hz), 7.16 (1H, d, J ) 1.7 Hz), 7.13

(1H, d, J ) 1.7 Hz), 7.05 (1H, d, J ) 1.7 Hz), 5.95 (1H, d, J )

1.7 Hz), 5.87 (1H, d, J ) 1.7 Hz), 3.85 (3H, s), 3.84 (3H, s),

3.83 (3H, s), 3.26 (2H, q, J ) 6.1 Hz), 3.15 (2H, m), 3.00 (2H,

m), 2.01 (2H, m), 1.97 (3H, s), 1.86 (4H, m). IR (KBr): 1644,

1583, 1530, 1464, 1435, 1402, 1259, 1201, 1133 cm^-1. HR-

1

with no distinct melting point. H NMR (DMSO-d₆): δ 10.28

(1H, s), 9.95 (1H, s), 9.88 (1H, s), 8.09 (1H, t, unresolved), 7.53-

7.47 (2H, m), 7.43 (1H, t, J ) 7.8 Hz), 7.32 (1H, d, J ) 1.7

Hz), 7.29 (1H, d, J ) 1.7 Hz), 7.15-7.11 (3H, m), 7.02 (1H, d,

J ) 1.7 Hz), 6.92 (1H, d, J ) 1.7 Hz), 4.32 (2H, t, J ) 6.6 Hz),

3.88 (3H, s), 3.83 (3H, s), 3.81 (3H, s), 3.24 (2H, q, J ) 6.0

Hz), 3.10-2.40 (8H, br; 3H, s), 1.77 (2H, br), 1.59-1.47 (3H,

m), 0.91 (6H, d, J ) 6.4 Hz). IR (KBr): 1677, 1645, 1583, 1535,

1462, 1435, 1402, 1263, 1197, 1132 cm^-1. HRFABMS found:

714.40764. Calculated for C₃₈H₅₂N₉O₅: 714.40914.

4-(F or m yla m in o)-N-[1-isop en t yl-5-({[1-m et h yl-5-({[3-

(4-m eth yl-1-piper azin yl)pr opyl]am in o}car bon yl)-1H-pyr -

r ol-3-yl]a m in o}ca r b on yl)-1H -p yr r ol-3-yl]-1-m et h yl-1H -

FABMS found: 537.29348. Calculated for

537.29378.

C₂₇H₃₇N₈O₄:

p yr r ole -2-ca r b oxa m id e

(F o-P yr Me -P yr iP e -P yr Me -

4-(F or m yla m in o)-1-m eth yl-N-[1-m eth yl-5-({[1-m eth yl-

5-({[3-(1-pyr r olidin yl)pr opyl]am in o}car bon yl)-1H-pyr r ol-

3-y l]a m in o }c a r b o n y l)-1H -p y r r o l-3-y l]-1H -p y r r o le -2-

MeP ip p , 44). (NO₂)PyrMe-PyriPe-PyrMe-Mepipp (120 mg,

0.197 mmol) was dissolved in ethanol (20 mL) to which Pd/C

(10%, 77 mg) was added at 0 °C under N₂with stirring. The

reaction mixture was hydrogenated for 3 h at room tempera-

ture and atmospheric pressure. The catalyst was removed by

filtration through Kieselguhr and ethyl formate (20 mL) was

added to the ethanolic solution. The reaction mixture was

heated under reflux for 48 h. Ethanol and excess ethyl formate

were removed under reduced pressure, and the crude product

was purified by HPLC. The product was obtained as a yellow

solid with no distinct melting point (75 mg, 53%) after freeze-

drying. ¹H NMR (DMSO-d₆): δ 10.03 (1H, s), 9.89 (1H, s), 9.87

(1H, s), 8.13 (1H, d, J ) 1.6 Hz), 8.09 (1H, t, J ) 5.6 Hz), 7.26

(1H, d, J ) 1.6 Hz), 7.18 (1H, d, J ) 1.6 Hz), 7.14 (1H, d, J )

1.6 Hz), 7.01 (1H, d, J ) 1.6 Hz), 6.92 (1H, d, J ) 1.6 Hz),

6.91 (1H, d, J ) 1.6 Hz), 4.31 (2H, t, J ) 6.9 Hz), 3.83 (3H, s),

3.81 (3H, s), 3.23 (2H, q, J ) 6.5 Hz), 3.10-2.72 (8H, br; 3H,

s), 1.77 (2H, s, br), 1.58-1.46 (3H, m), 0.90 (6H, d, J ) 6.4

ca r b oxa m id e

(F o-P yr Me-P yr Me-P yr Me-P yr r p , 47).

(NO₂)PyrMe-PyrMe-PyrMe-Pyrrp (210 mg, 0.400 mmol) was

dissolved in ethanol (24 mL) at 0 °C under N₂. Pd/C (10%, 105

mg) was added and the reaction mixture was hydrogenated

for 3 h. The catalyst was removed over Kieselguhr and the

amine solution so formed was divided in half. To the first half

ethyl formate was added and the mixture was heated under

reflux for 48 h. Volatile material was removed under reduced

pressure and the crude product was purified by HPLC. The

product was obtained as a pale yellow solid (20 mg, 16%) with

1

no distinct melting point. H NMR (DMSO-d₆): δ 10.03 (1H,

s), 9.89 (1H, s), 9.88 (1H, s), 9.38 (1H, br), 8.13 (2H, d & t),

7.21 (1H, d, J ) 1.7 Hz), 7.18 (1H, d, J ) 1.7 Hz), 7.15 (1H, d,

J ) 1.7 Hz), 7.06 (1H, d, J ) 1.7 Hz), 5.95 (1H, d, J ) 1.7 Hz),

5.92 (1H, d, J ) 1.7 Hz), 3.84 (6H, s), 3.81 (3H, s), 3.54 (2H,

m), 3.26 (2H, q, J ) 6.1 Hz), 3.13 (2H, m), 3.00 (2H, m), 2.01

(2H, m), 1.85 (4H, m). IR (KBr): 1674, 1649, 1583, 1537, 1465,

1436, 1264, 1202, 1133 cm^-1. HRFABMS found: 523.27829.

Calculated for C₂₆H₃₅N₈O₄: 523.27813.

Hz). IR (KBr): 1675, 1584, 1535, 1403, 1199, 1132 cm^-1

.

HRFABMS found: 608.36634. Calculated for C₃₁H₄₆N₉O₄:

608.36728.

4-(Acetyla m in o)-N-[1-isop en tyl-5-({[1-m eth yl-5-({[3-(4-

m eth yl-1-piper azin yl)pr opyl]am in o}car bon yl)-1H-pyr r ol-

3-yl]am in o}car bon yl)-1H-pyr r ol-3-yl]-1-m eth yl-1H-pyr r ole-

2-ca r b oxa m id e (Ac-P yr Me-P yr iP e-P yr Me-MeP ip p , 45).

(NO₂)PyrMe-PyriPe-PyrMe-Mepipp (186 mg, 0.305 mmol) was

dissolved in methanol at 0 °C under N₂with stirring to which

Pd/C (10%, 136 mg) was added. The reaction mixture was

hydrogenated for 3 h at room temperature and atmospheric

pressure with stirring. The catalyst was removed by filtration

through Kieselguhr and the solvent was then removed under

reduced pressure. The amine so formed was dissolved in

dichloromethane (10 mL) and this solution was subsequently

divided in half. Acetyl chloride (12 mg, 12 µL, 0.153 mmol)

was added dropwise with stirring at room temperature to the

first half. Stirring was continued at room temperature over-

night. The solvent was removed under reduced pressure and

the crude product formed was dissolved in small amount of

acetonitrile/water containing 0.1% TFA and purified by HPLC.

Fractions containing the pure material were combined and

freeze-dried to give an off-white solid (45 mg, 40%) with no

distinct melting point. ¹H NMR (DMSO-d₆): δ 9.87 (1H, s),

9.86 (1H, s), 9.79 (1H, s), 8.09 (1H, t, unresolved), 7.26 (1H, d,

J ) 1.7 Hz), 7.14 (1H, d, J ) 1.7 Hz), 7.13 (1H, d, J ) 1.7 Hz),

7.01 (1H, d, J ) 1.7 Hz), 6.91 (1H, d, J ) 1.7 Hz), 6.86 (1H, d,

J ) 1.7 Hz), 4.31 (2H, t, J ) 6.9 Hz), 3.83 (3H, s), 3.81 (3H, s),

3.24 (2H, q, J ) 6.4 Hz), 3.10-2.60 (8H, br; 3H, s), 1.97 (3H,

s), 1.77 (2H, m), 1.58-1.46 (3H, m), 0.90 (6H, d, J ) 6.4 Hz).

IR (KBr): 1675, 1647, 1584, 1535, 1459, 1436, 1402, 1197, 1132

4-(F or m yla m in o)-N-[1-isop r op yl-5-({[1-m et h yl-5-({[3-

(1-p yr r olid in yl)p r op yl]a m in o}ca r bon yl)-1H-p yr r ol-3-yl]-

a m in o}ca r bon yl)-1H-p yr r ol-3-yl]-1-m eth yl-1H-p yr r ole-2-

ca r b oxa m id e (F o-P yr Me-P yr iP r -P yr Me-P yr r p , 48).

(NO₂)PyrMe-PyriPr-PyrMe-Pyrrp (224 mg, 0.406 mmol) was

dissolved in ethanol (25 mL) to which Pd/C (10%, 224 mg) was

added at 0 °C under nitrogen with stirring. The reaction

mixture was hydrogenated for 2.5 h at room temperature and

atmospheric pressure. The catalyst was removed by filtration

through Kieselguhr. Ethyl formate (15 mL) was added to the

ethanolic solution, and then the reaction mixture was heated

under reflux overnight. Ethanol and excess ethyl formate were

removed under reduced pressure, and the crude product was

purified by HPLC to give the required material as a white solid

(42.7 mg, 16%) with no distinct melting point. ¹H NMR

(DMSO-d₆): δ 10.07 (1H, s), 9.94 (1H, s), 9.92 (1H, s), 9.41

(1H, br, TFA), 8.16-8.12 (2H, m), 7.39 (1H, d, J ) 1.8 Hz),

7.19 (1H, d, J ) 1.8 Hz), 7.17 (1H, d, J ) 1.8 Hz), 6.97 (1H, d,

J ) 1.8 Hz), 6.94 (1H, d, J ) 1.8 Hz), 6.92 (1H, d, J ) 1.8 Hz),

3.84 (3H, s), 3.61 (3H, s), 3.55 (2H, m), 3.26-3.24 (2H, m),

3.17-3.12 (2H, m), 2.99 (2H, m), 2.01 (2H, m), 1.86 (4H, m),

1.37-1.35 (6H, d, J ) 6.7 Hz). IR (KBr): 3429, 1675, 1645,

1582, 1534, 1462, 1440, 1407, 1252, 1200, 1132 cm^-1. HR-

FABMS found: 551.31199. Calculated for

551.30943.

C₂₈H₃₉N₈O₄:

Refer en ces

cm^-1. HRFABMS found: 622.38301. Calculated for C₃₂H₄₈

N₉O₄: 622.38293.

-

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J M031089X

Article Doi

Distamycin Analogues with Enhanced Lipophilicity: Synthesis and Antimicrobial Activity

DOI: 10.1021/jm031089x

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Article abstract of DOI:10.1021/jm031089x

Full text of DOI:10.1021/jm031089x

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