Synthesis of analogues of Congo red and evaluation of their anti-prion activity

J . Med. Chem. 2004, 47, 5515-5534

5515

Syn th esis of An a logu es of Con go Red a n d Eva lu a tion of Th eir An ti-P r ion

Activity

Shane Sellarajah,^†Tamuna Lekishvili,^‡Claire Bowring,^‡Andrew R. Thompsett,^‡Helene Rudyk,^†

Christopher R. Birkett,^§David R. Brown,^‡and Ian H. Gilbert*^,†

Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff CF10 3XF, U.K., Department of Biology and

Biochemistry, University of Bath, Bath, BA2 7AY, U.K., and Institute for Animal Health, Compton Laboratory, Compton,

Newbury, Berkshire RG20 7NN, U.K.

Received J anuary 28, 2004

No cure as of yet exists for any of the transmissible spongiform encephalopathies. In this paper,

we describe the synthesis of analogues of Congo red and evaluation against a cellular model of

infection, the SMB (scrapie mouse brain) persistently infected cell line, for their ability to inhibit

the infectivity of the abnormal form of prion protein (PrP-res). The compounds have also been

tested for their ability to inhibit the polymerization of PrP^Cby PrP-res. A number of analogues

showed inhibition of PrP-res infectivity at nanomolar concentrations. Several analogues show

promise; the most active compound, 2a , inhibits the formation of PrP-res in SMB cells with an

EC₅₀of 25-50 nM.

In tr od u ction

We were particularly interested in the diazo dye

Congo red, which has been shown to have anti-prion

activity in a cell free polymerization assay,¹³in cellular

assays,^14,15and in vivo activity against scrapie-infected

golden Syrian hamsters.¹⁶

Transmissible spongiform encephalopathies (TSE) are

a group of rare neurological degenerative disorders.¹The

diseases can arise spontaneously, have a genetic origin,

or can occur by infection. In humans, the TSEs are

Creutzfeldt J akob Disease (CJ D), Gertmann-Strauss-

ler-Scheinker disease (GSS), Fatal Familial Insomnia

(FFI), Kuru, and more recently, a new form of the

disease called variant CJ D.^2,3TSEs affect other organ-

isms; diseases of note include bovine spongiform en-

cephalopathy (BSE) in cattle and scrapie in sheep. There

is no cure currently available for these diseases, which

are fatal.

The molecule itself has a number of shortfalls:¹⁷(i) it

has a lack of specificity, (ii) it does not have significant

permeability through the blood-brain barrier^18,19(pre-

sumably because of its charged nature, primarily caused

by the presence of two sulfonate groups), and (iii) the

diazo bonds in Congo red can be cleaved by enzymes

present in the mammalian gut and intestines,^20,21giving

rise to benzidine (Figure 1), a highly carcinogenic

compound strongly associated with urinary bladder

cancer.²²However, unlike many of the other compounds

reported to have activity in various models of TSEs,

Congo red is a small molecule, which should be ame-

nable to chemical synthesis and structure-activity

relationship studies, with the aim of producing a low-

molecular-weight therapeutic agent. We have previously

reported some structure-activity relationship studies

with Congo red,^13-15which taken together with other

work^17,23have allowed us to come to the following

principal conclusions:

• Although Congo red is a symmetrical molecule, both

“halves” of the molecule are required for activity.

• It is possible to exchange the sulfonate groups for

carboxylic acid groups without a significant effect on the

activity.

• It also should be possible to vary the location of the

acidic group on the aromatic ring.

• The amino group may not be necessary for activity.

• Some variation in the linker (biphenyl) is possible.

In this paper, we address the following:

The causative agent of these diseases is thought to

be or to contain an abnormally folded protein, the prion

protein. During the course of the disease, there is an

accumulation of the misfolded prion protein form (PrP-

res).⁴The normal cellular prion protein (PrP^C) is a

glycolipid-anchored membrane protein and is easily

degraded by proteases, whereas PrP-res is highly re-

sistant to degradation.

A number of compounds have shown anti-prion activ-

ity in models of TSE infection.^5-8Compounds shown to

have anti-prion activity include sulfated polysaccharides

such as pentosan polysulfate,⁹Congo red and other

diazo dyes, amphotericin B and analogues, anthracy-

clines, phthalocyanines and porphyrins, inorganic ions,

branched polyamines, and antagonists of the N-methyl-

D-aspartate (NMDA) receptor such as memantine.⁶

Recently, there has been interest in quinacrine and

other acridine derivatives.^10,11In addition, further

screening methodologies have recently been reported to

allow screening of larger numbers of compounds in

vitro.¹²

• Replacement of the diazo group with amide bonds

and sulfonamide bonds. These are bioisoteres of the

diazo group and will prevent the molecule from being

metabolized to benzidine.

• To enable blood-brain barrier penetration, we will

choose nonpolar terminator groups or, alternatively,

* Author to whom correspondence should be addressed. Tel: +44

29 2087 5800. Fax: +44 29 2087 4149. E-mail: gilbertih@cf.ac.uk.

^†Cardiff University.

^‡University of Bath.

^§Institure for Animal Health.

Published on Web 09/29/2004

5516 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22

Sellarajah et al.

F igu r e 1. Structures of Congo red and benzidine.

The amines utilized in the reactions were either

readily commercially available or synthesized via one-

step esterifications (for example, see Scheme 2).

The biphenyl amide analogues (series 2) were syn-

thesized either by the reaction of the corresponding acid

chloride with the appropriate aniline derivative in the

presence of triethylamine or by using TBTU, a uronium-

based peptide-coupling reagent (Scheme 3).

F igu r e 2. Active mixture.

The monophenyl analogues (series 3) were synthe-

sized using terephthaloyl chloride and the appropriate

aniline.

The imine analogues (series 4 and 5) were made by

the condensation of the corresponding aldehyde with the

appropriate amine. The corresponding imines were

generally insoluble in ethanol and precipitated from

solution; filtration and washing generally provided com-

pounds in good yields. The biphenyl analogues proved

not only to be less soluble but also had longer reaction

times. The monophenyl imines (series 5) were synthe-

sized from commerically available terephthalaldehyde,

whereas the biphenyl analogues (series 4) required 4,4′-

biphenyldialdehyde to be made first (Scheme 5).

The amine analogues (series 6) were synthesized by

the reduction of the imines with sodium borohydride.

The only limiting factor was the initial solubility of the

imines, many of which proved to be insoluble in any

solvent once formed. Generally, the reduction was

favorable in a methanol/dichloromethane mixture.

The alkenes (series 7) were prepared using the Heck

reaction, which gave trans stereochemistry (Scheme 7).

The reagent 1,4-divinylbenzene could be purchased

commercially but only as a mixture with 1,2-divinyl-

benzene; therefore, this compound was prepared from

terephthaldehyde using a Wittig reaction.

Biologica l Assa ys. Cellu la r Assa ys. The synthe-

sized compounds were tested in the SMB cell line assay.

The SMB cells are persistently scrapie-infected mouse

cells cloned from a scrapie-infected mouse brain but of

non-neuronal origin.^14,15,24,25These cells are highly

phagocytic and may mimic cells involved in the initial

uptake and replication of the agent in peripheral

infection.¹⁴In addition, these cells show stable persis-

tent scrapie infection over many passages, suggesting

that they are suitable for drug screening.²⁵We have

never observed the SMB cells to lose their infection or

“self-cure”. Indeed, we have successfully used this cell

line to screen many derivatives of Congo red.^14,15

Compounds were initially screened at 10 µM to see the

effect at this concentration. Those for which there was

no detectable PrP-res at this concentration are marked

as “+ve” in the tables. Several of the compounds were

also evaluated at 50 µM. The percentage of PrP-res

compared to control is indicated in the tables; the lower

this value, the more potent the compounds. The results

F igu r e 3. Structure of compound 2a .

ester-masked polar carboxylic acids, which should be

hydrolyzed by esterases present in the brain.

Preliminary results from our group were obtained

from a mixture of two compounds, A and B (Figure 2).

In an assay conducted in persistently scrapie-infected

SMB cells, the mixture reduced levels of PrP-res to 55%

of control levels at 10 µM.

Resu lts a n d Discu ssion

Ch em istr y. The active mixture of A and B (Figure

2) proved to be difficult to separate by normal purifica-

tion techniques such as recrystallization or column

chromatography. This was mainly due to the low

solubility of compounds A and B in any organic solvent.

Thus, the initial molecule envisaged for synthesis was

very similar to diamide A, with the added solubilizing

effect of two extra hydroxy groups (compound 2a , Figure

3).

As discussed below, 2a eventually proved to be our

most active compound in the SMB cellular model.

Therefore, different molecules based on the molecular

architecture of 2a were designed for synthesis and

testing (Figure 4). Essentially, in these compounds, the

linker was kept as either biphenyl or phenyl; the diazo

connector was replaced by the sulfonamide, amide,

imine, aminomethylene, and alkene connectors; the

structure of the terminator was varied. The replace-

ments for the diazo bond are all isosteres of this bond

(Figure 4).

Sulfonamide analogues (series 1) could be synthesized

from the readily available 4,4′-biphenyldisulfonyl chlo-

ride 8 and the appropriate amine utilizing DMAP as a

catalyst and pyridine as the solvent.

Synthesis and Evaluation of Analogues of Congo Red

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22 5517

F igu r e 4. General structures of the target compounds.

Sch em e 1. Sulfonamide Coupling

Sch em e 3. Amide Coupling

For the sulfonamides (Table 1), none of the com-

pounds showed inhibition of PrP-res at 10 µM, although

some of the compounds (1a and 1c) showed some

activity at 50 µM. Interestingly, compound 1a , which

has the same terminator as the lead compound, 2a ,

showed some activity at this higher concentration.

Several of the amides with a biphenyl core (Table 2)

showed good activity. In particular, compounds 2a , 2b,

and 2h showed the prevention of PrP-res formation. In

2a and 2h , there is a free hydroxyl group, and in

compound 2b, a methoxy group attached to the phenyl

ring; the hydroxyl group of 2a and 2h could act as an

H-bond donor or acceptor, whereas the methoxy group

of 2b could act as an H-bond acceptor. Compound 2g,

which is a regioisomer of 2a and 2h , was inactive. This

suggests that the relative orientations of the ester and

the hydroxyl group are important in the terminator.

Compound 2e, which is the analogue of 2b with the free

carboxylate, did not show activity, indicating that the

ester has a role. Often esters are subject to cleavage by

Sch em e 2. Synthesis of Amines

from these assays are shown in Tables 1-7 for the seven

series of compounds: the sulfonamides (Table 1); amides

with a biphenyl linker (Table 2); amides with a phenyl

linker (Table 3); imines with a biphenyl linker (Table

4); imines with a phenyl linker (Table 5); amines with

a phenyl linker (Table 6); and alkenes with a phenyl

linker (Table 7). An example of a Western Blot is given

in Figure 5.

5518 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22

Sch em e 4. Imine Formation

Sellarajah et al.

Sch em e 5. Synthesis of Biphenyl Dialdehyde^a

a

(i) BH₃SMe₂, THF; (ii) Pyridinium Dichromate, Dichloromethane

Sch em e 6. Reduction of Imines

been unable to prepare the analogues of 2b or 2h in

sufficient purity, which would provide a more interest-

ing comparison with the data for the biphenyl ana-

logues.

For the biphenyl series of imines (series 4, Table 4),

two of the compounds showed an inhibition of PrP-res

formation in the cellular assay (4c and 4e). In the

terminator of 4c, there is a free carboxylic acid and a

hydroxyl group, whereas 4e contains only a hydroxyl

group. Interestingly, amides or sulfonamides with these

terminator groups were inactive. Compound 4b, which

has the same terminator group as 2a , was inactive at

10 µM but showed some effect at 50 µM.

Sch em e 7. Alkene Coupling

In Table 5, we present the data for the series of

compounds with a phenyl linker and an imine (series

5). None of these compounds showed any significant

activity in the cellular assay, with the exception of 5s.

In this compound, there is a nitrile substituent on the

terminator group, which can act as a hydrogen-bond

acceptor if it is not charged.²⁶

The aminomethylene compounds of series 6 (Table 6)

all showed activity except 6c. Compound 6b contains

the same terminator group as 2a .

Finally, a series of compounds were prepared in which

the diazo bond was replaced by an alkene (series 7,

Table 7). Compound 7a is named X-34, a compound used

by Klunk et al. for diagnosing Alzheimer’s disease.^27,28

This compound showed no activity at 10 µM but weak

activity at 50 µM. Analogue 7b, in which the carboxylic

acid functionality has been removed from the termina-

tor, shows potent activity. Compound 7b showed activity

at 2 µM; however, the mechanism of action is unknown,

but it is possibly different from that of the other

molecules in this paper. This molecule is an interesting

lead and could be acting through an antioxidant mode

of action.

esterases; if this is the case here, then the ester may be

acting as a prodrug to allow the delivery of compounds.

Other compounds with the methyl carboxylate removed

or replaced (2c, 2d , 2e, 2f, and 2i) were inactive in the

cell culture assay, further indicating that the ester may

have a role in the activity of the molecule; by altering

the structure of the ester it may be possible to modify

the potency and physicochemical properties of the

compounds.

The compounds of series 3 were the amides with a

phenyl core (Table 3). Compounds 3b and 3h showed

activity, and following a fuller dose-response experi-

ment, the ED₅₀values were found to be 250 and 5000

nM, respectively. None of the other compounds in this

series showed activity in the cellular assays. This may

be due to the replacement of the biphenyl core with a

smaller core. Compound 3h is the analogue of 2a , which

has maintained some but not all of the activity of 2a ,

suggesting not only that the biphenyl moiety plays an

important part in the activity of 2a but also that the

terminator functionality is important. So far, we have

For compounds showing activity at 10 µM, a full

dose-response study was undertaken to get a more

accurate indication of the potency (Table 8). The most

active compound, 2a , showed an ED₅₀of 25-50 nM. A

number of other compounds showed potent inhibition,

including 2b, 3b, 4e, 5s, 6a , and 6b, which all showed

an ED₅₀of <500 nM. The two compounds that showed

the most potent activity, 2a and 6b, have the same

terminator group. Compound 3h has the same termina-

tor group but has lower activity.

Synthesis and Evaluation of Analogues of Congo Red

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22 5519

Ta ble 1. Sulfonamides: Synthetic Yield, Inhibition of PrP-res Formation in SMB Cells at 10 µM, and Inhibition of Polymerization at

100 µM

P olym er iza tion Assa ys. To try to understand the

mode of action of these compounds, we carried out

investigations to determine if these compounds could

inhibit the polymerization of PrP^Cby PrP-res. In this

assay, the seed for polymerization was recombinant

mouse PrP that was refolded and aggregated using

Mn²⁺ions. This was then used to seed the aggregation

of recombinant mouse PrP (which was refolded in the

absence of metal ions).²⁹The aggregation was monitored

by looking at the scattering of UV light. The effect of

compounds on inhibiting aggregation was determined.

It was discovered that some of the compounds degraded

when kept in the UV beam, so the UV beam was not

left on continually. Some of the compounds caused

precipitation prior to the addition of the seed. This

means that the interpretation of the data related to

these compounds is unreliable. Where this is the case,

it is marked in the tables.

For the sulfonamides (Table 1), several compounds

showed an inhibition of polymerization. In particular,

compounds 1a and 1c, which showed weak activity in

the cellular assays, also showed an inhibition of polym-

erization. Compound 1b, which was not active in the

cellular assay, showed some activity in the polymeri-

zation assay. Compound 1a , which showed the greatest

inhibition of polymerization, contains the same termi-

nator groups as 2a , the lead compound.

erization. These compounds also showed activity in the

cellular assays. However, compound 2h , which showed

an inhibition of PrP-res formation in the cellular assays,

actually seemed to stimulate aggregation in the polym-

erization assay. Similarly, the other compounds assayed

in this series appeared to stimulate aggregation.

In the third series of compounds investigated, the

amides with a phenyl core (Table 3), compounds 3e, 3g,

and 3h appeared to cause the prevention of polymeri-

zation. The other compounds assayed were either inac-

tive or caused precipitation.

In series 4, where compounds contained a biphenyl

linker and an imine bond (Table 4), 4b and 4c showed

some inhibition of polymerization. None of the other

compounds investigated showed inhibition. The two

compounds that inhibited polymerization are the two

compounds that also have some effect on cell culture

assays, indicating that the mode of action of these

compounds may include the inhibition of polymeriza-

tion. The terminator group on 4b is the same as that

on 2a , and that on 4c differs only in the carboxylate

ester being replaced by a free carboxylate group.

The compounds of series 5 are those with a phenyl

linker and an imine bond (Table 5). A number of these

showed an inhibition of PrP^Cpolymerization: 5c, 5d ,

5f, 5g, 5h , and 5q. The only compound in this series

that was active in the cellular assay (5s) had a very

small effect on polymerization. Also, the terminator

group found in 2a is not active in this series of

For the amides with a biphenyl core (Table 2),

compounds 2a and 2b showed an inhibition of polym-

5520 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22

Sellarajah et al.

Ta ble 2. Amides: Synthetic Yield, Inhibition of PrP-res Formation in SMB Cells at 10 µM, and Inhibition of Polymerization at 100

µM^a

a

Inhibition of polymerization at 100 µM.

compounds (compound 5a ). The presence of a carboxy-

late group (either as the free acid or as an ester) seemed

to be important for activity. Compounds not containing

these functionalities were not active in this assay.

Compound 5e is a direct homologue of 5d (one of the

most active compounds), except that it has an extra

methylene between the terminator and the imine but

is inactive, suggesting that conjugation with the termi-

nator is an important feature.

For the amine-linked compounds (series 6, Table 6),

several of the compounds showed the inhibition of PrP-

res formation, 6a and 6c. However, the other two

compounds show no activity. Of particular note here is

6b, which stimulated polymerization but was one of the

most potent compounds in the cellular assay. This may

indicate that there is more than one mechanism of

action for these compounds. Finally, 7a (Table 7) shows

some inhibition of polymerization.

Deta ch m en t Assa ys. Another possible mode of ac-

tion of the compounds would be detaching PrP^Cfrom

the surface of the cells. Therefore, the supernatant was

examined to determined if there were increased levels

of PrP^Cin treated cells compared to control levels.

However, none was detected, suggesting that the pri-

mary mode of action of 2a is not the removal of the PrP^C

from the surface of the cell.

Toxicity Assa ys. It is important that the compounds

are not toxic to neuronal cells. Therefore, a toxicity

assay was done using cultures of neurons isolated from

mice with the lead compound (2a ) (Figure 6). The

compound was found to be nontoxic at 0.1 µM. There

was a very low level of toxicity at 0.5 µM and an IC₅₀of

around 30-40 µM. This indicates that 2a causes selec-

tive action against TSE-infected cells. For comparison,

the toxicity of compound 3g was investigated. This

compound had no effect on levels of PrP-res in the cell

Synthesis and Evaluation of Analogues of Congo Red

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22 5521

Ta ble 3. Phenyl Amides: Synthetic Yield, Inhibition of PrP-res Formation in SMB Cells at 10 µM, and Inhibition of Polymerization

at 100 µM

culture assays and also showed a higher toxicity to

neurons compared to 2a .

significant effects in an animal model of infection.³²

Work from Prusiner’s group has led to the development

of some bis-acridines, which show very good activity in

cellular models, although no in vivo data has been

reported on these to date.¹¹The anthracycline IDX has

also been shown to have activity in animal models of

infection; however, it has poor blood-brain barrier

permeability, which would limit its therapeutic value.³³

Porphyrins and phthalocyanines have also been inves-

tigated and have shown the ability to increase the

incubation period of scrapie-infected mice.³⁴Congo red

and other azo dyes have been investigated as potential

agents against prion diseases. Congo red has been

shown to increase the incubation time of the disease in

mouse models of infection,¹⁶but it has very low blood-

brain barrier permeability and is also unsuitable as a

drug as previously described.

Discu ssion

As outlined in the Introduction, the scientific litera-

ture contains reports on a number of compounds that

have been investigated for the treatment of prion

diseases. Some of these have been tested in animal

models of infection. These include the sulfated polysac-

charides (pentosan polysulfate being the most studied

example), which can protect mice from scrapie infection

when injected intraperitoneally immediately after infec-

tion but has poor blood-brain barrier permeability.⁹

Amphotericin B has shown some promise in animal

studies,³⁰being able to extend the incubation time for

the disease, even given some time post-infection, but

was disappointing when used in a human case.³¹In

addition, the drug is very toxic. Recently, quinacrine and

chlorpromazine have been highlighted as potential

agents for the treatment of these diseases.¹⁰However,

quinacrine suffers from toxicity problems and had no

Unfortunately, of those tested in animal models of

disease, all of the compounds have limitations, and there

is an urgent need for the development of therapeutic

agents for these diseases.³⁵This lack of therapeutic

5522 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22

Sellarajah et al.

Ta ble 4. Biphenyl Imines: Synthetic Yield, Inhibition of PrP-res Formation in SMB Cells at 10 µM, and Inhibition of Polymerization

at 100 µM

agents has lead us to explore Congo red further as a

drug lead.

assay employed on its own also has limitations. The

assay does not identify all compounds that prevent

polymerization. These latter compounds may be a useful

source of information for future rounds of drug design.

The compounds described in this paper can be cat-

egorized in four ways as a result of these assays:

(i) Compounds that are active in both the cellular and

polymerization assay. These compounds may be pre-

venting PrP-res formation in cells by preventing polym-

erization of PrP^C(2a , 2b, 3h , 4c, 5s, and 6a ).

(ii) Compounds that are active in the polymerization

assay but not in the cellular assay. Either these

compounds are too weak at inhibiting polymerization

to be active in cellular assays, or they are inactivated

in a cellular system, or they are unable to access the

molecular target. It is possible that some structural

modification can be used to give activity in cellular

models (1a , 1b, 1c, 3e, 3g, 4b, 5d , 5f, 5g, 5h , 5q, 6c,

and 7a ).

(iii) Compounds that are active in the cellular assay

but not in the polymerization assay. These compounds

probably are preventing PrP-res formation in the SMB

cells by a mode of action other than the inhibition of

polymerization. Further investigation is required to

determine their mode of action (2h , 3b, 4e, 6b, and 6d ).

(iv) Compounds that are active in neither assay. They

are unsuitable for further investigation (1d , 1e, 1f, 2c,

2d , 2e, 2f, 2g, 2i, 3a , 3c, 3d , 3f, 4d , 4f, 5a , 5b, 5c, 5e,

5i, 5j, 5l, 5m , 5n , 5o, and 5r ).

Effect of th e Con n ector Gr ou p . A number of

different connector groups have been investigated: sul-

fonamide, amide, imine, aminomethylene, and alkene.

There appears to be some room for variation of the

connector. The most promising results in the cellular

assay were obtained with amide or aminomethylene

connectors. Some of the imines were also active in these

assays. The sulfonamide compounds were inactive in a

cellular model of infection. There is too little data on

the alkene linker to make conclusions.

The sulfonamides (series 1) appeared less active than

the biphenyl amides (series 2). In general, the sulfon-

amide is a bioisostere of the amide group. However, in

the case of the sulfonamides, the NH is ionizable and

may well be ionized at physiological pH. Although

ionization may well increase the solubility of these

compounds, interaction with the prion protein may be

also be prevented. However, some of the sulfonamides

showed weak activity in a cellular model and an

inhibition of polymerization.

The biphenyl amides showed particular promise, with

compounds 2a , 2b and 2h being active in the cellular

assay. Compound 2a is of great interest. For the

biphenyl amides, there seems to be some correlation

between the prevention of PrP-res formation in the cell

culture and the prevention of the polymerization of

PrP^C. This suggests that the anti-TSE activity of at least

some of the compounds is related to their ability to

prevent polymerization.

Both the cellular and polymerization assays provide

useful information in the context of a drug discovery

program. The polymerization assay provides informa-

tion on the possible modes of action of compounds and

can identify most, but not all, molecules that reduce

PrP-res formation in cellular assays. On its own,

however, it is not a useful screen to select out com-

pounds for cellular assays. In addition, the cellular

Imines are normally readily subject to hydrolysis.

However in this case, the imines are highly conjugated,

Synthesis and Evaluation of Analogues of Congo Red

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22 5523

Ta ble 5. Phenyl Imines: Synthetic Yield, Inhibition of PrP-res Formation in SMB Cells at 10 µM, and Inhibition of Polymerization

at 100 µM

5524 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22

Sellarajah et al.

Ta ble 6. Phenyl Amines: Synthetic Yield, Inhibition of PrP-res Formation in SMB cells at 10 µM, and Inhibition of Polymerization

at 100 µM

Ta ble 7. Phenyl Alkenes: Synthetic Yield, Inhibition of PrP-res Formation in SMB Cells at 10 µM, and Inhibition of Polymerization

at 100 µM

compounds with carboxylates on the terminator: 5d ,

and 5f, 5g, 5h .

The aminomethylene compounds (series 6) show some

very promising activity not only in the cellular assay

but also in the polymerization assay.

Effect of th e Ter m in a tor Gr ou p . Structure-activ-

ity relationships indicate that the most active com-

pounds in cellular assays have a methyl carboxylate

F igu r e 5. Western blot showing the effects of varying

attached to the terminator and either a hydroxy or

concentrations of compound 2a on PrP-res formation in SMB

methoxy group. Clearly, the terminator found in com-

Cells. Ctl ) untreated control. DMSO ) treated with the

pound 2a is active in both the cellular and polymeriza-

DMSO at the concentration used with 2a . PK ) proteinase K.

tion assays. This terminator group is generally active

which stablizes them to hydrolysis. They should repre-

sent a very good mimic of the diazo bond found in Congo

red. By ensuring that the nitrogen of the imine is

attached to the terminator rather than the linker, any

hydrolysis should not give rise to benzidene or its

analogues. The biphenyl imines (series 4) showed higher

activity than the corresponding monophenyl imines

(series 5) in the cellular assay, although several of the

monophenyl imines showed a potent inhibition of po-

lymerization. These latter compounds tended to be

for other series of compounds in either the cellular or

polymerization assays (1a , 3h , 4b, and 6b but not 5a ).

However, other regioisomers are also active (2b and 2h ).

Some compounds containing a carboxylic acid on the

terminator are active in the inhibition of polymerization

but not in the cellular assay. Conversely, compounds

that are active in the cellular assay and contain a

methyl ester are not active in the polymerization assay

(2h , 3b, and 6b). This could imply, assuming that the

mechanism of action of these compounds is the inhibi-

Synthesis and Evaluation of Analogues of Congo Red

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22 5525

Ta ble 8. ED₅₀Values of Compounds in SMB Cells Showing Activity <10 µM

5526 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22

Sellarajah et al.

F igu r e 8. Most potent compounds in the cellular assays.

F igu r e 9. Most potent compounds in the polymerization

assays.

F igu r e 6. Cell viability of cerebellar neurones when incubated

with 2a and 3g.

tract. In addition, we have carried out a series of

structure-activity studies with the terminator group

and have prepared analogues with a much lower charge

than is found in Congo red, which should facilitate

delivery of the compounds across biological barriers.

Compounds were assayed in a persistently infected

scrapie cell line, the SMB cell line, and also in a cellular

polymerization assay. The structure-activity relation-

ships for the cellular model are shown in Figure 8, and

those for the polymerization assay, in Figure 9.

F igu r e 7. Terminator groups investigated for activity.

Our work has yielded a number of compounds show-

ing potent activity in a cellular model of prion diseases.

The most potent compound is 2a , which shows strong

inhibition of PrP-res formation, with an ED₅₀in the

range of 25-50 nM in the SMB cells. The compound

appears to have a good selectivity index against neu-

ronal cells, with an IC₅₀of 30-40 µM and a selectivity

index of approximately 1000-fold, indicating that it

could have a therapeutic window. In addition to com-

pound 2a , there are a variety of other analogues that

show potent activity, with six other compounds showing

an ED₅₀of <0.5 µM. These compounds represent excit-

ing leads in this area and are significantly more potent

than the starting material for this project, Congo red.

They are low-molecular-weight compounds that prob-

ably have sufficient lipophilicity to cross the blood-

brain barrier. They are also amenable to further struc-

ture-activity relationship studies.

The mode or modes of action of these compounds are

not fully established but are probably due to preventing

the formation of PrP-res. Detachment of PrP from the

cellular surface has been discounted.

The approach that we adopted has yielded some

interesting lead molecules that are worthy of further

study. Even though the mechanisms of action of any of

these compounds have not been elucidated, the compila-

tion presents an array of compounds with structure-

activity relationships. This work gives us a sound

platform from which to launch further investigations.

tion of polymerization, that the methyl esters are

metabolized to the active form, the carboxylic acid, by

the cells and then inhibit polymerization. Some evidence

exists for this: 6c, the demethylated analogue of 6b, is

active in the polymerization assay. The compounds that

contain free carboxylic acids may not be active in the

cellular assays because they cannot access the site of

action. In addition, there are several compounds that

are active in cellular and/or polymerization assays that

do not have a carboxylate substituent, implying that this

is not an essential substituent for activity.

To investigate the effect of the terminator group, we

carried out assays with two of the terminator groups

not attached to anything else. The terminator groups

investigated were methyl-4-aminobenzoate (8a ) and

methyl-4-amino-3-hydroxybenzoate (8b), the terminator

group of 2a (Figure 7). Both of these compounds showed

ED₅₀values of about 1 µM in the cellular assay,

suggesting that the terminator has an important role

in the anti-prion activity of the compounds.

Effect of th e Lin k er . The linkers we used were the

biphenyl and phenyl. In general, where it is possible to

compare, the biphenyl compounds were more active

than the phenyl compounds in cellular assays. This

could be due to some kind of intermolecular interactions

between the linker (the biphenyl group) and the molec-

ular target or the linker maintaining the terminators

at the correct distance.

Con clu sion s

Exp er im en ta l Section

In this paper, we have described the preparation of a

number of compounds in which we have replaced the

diazo bond of Congo red with a variety of other bonds,

including imine, aminomethylene, and alkene. It is

important to replace this bond because metabolism of

this bond gives rise to benzidine in the gastrointestinal

Gen er a l P r oced u r es. ¹H and ¹³C NMR spectra were

recorded for solutions (in specified solvents) on a Bruker

Advance DPX300 spectrometer at 300 and 75 MHz, respec-

tively. Chemical shifts are quoted in ppm downfield with

respect to tetramethylsilane (δ ) 0.00 ppm). Coupling con-

stants (J ) are quoted in Hertz to the nearest 0.5 Hz. Low-

Synthesis and Evaluation of Analogues of Congo Red

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22 5527

δ 3.57 (6H, s,OMe), 3.80 (6H, s, COOMe), 7.01 (2H, d, J ) 8.7

Hz, C_5′′-H), 7.78-7.90 (12H, m, C_2′′-H + C_6′′-H + C₃-H +

C₂-H), 9.89 (2H, s, NH); ¹³C NMR (75 MHz, DMSO-d₆) δ 52.3

(COOMe), 56.1 (OMe), 112.0 (C_5′′), 122.0 (C_2′′), 125.6 (C_6′′), 126.4

(C_1′′), 127.7 (C₃), 127.9 (C₂), 128.8 (CNH), 140.3 (C₄), 142.7 (C₁),

156.5 (C_4′′), 165.8 (COO); ES^-m/z 638.8 (M^-); high-resolution

ES⁺m/z found 658.1531 C₃₀H₃₂N₃O₁₀S₂(M + NH₄) requires

658.1529. Anal. (C₃₀H₂₈N₂O₁₀S₂) C, H, N.

Bis-3-({[4′-(a m in osu lfon yl)-1,1′-bip h en yl-4-yl]su lfon yl}-

a m in o)-4-m et h oxyb en zoic a cid (1c). To compound 1b

(0.100 g, 0.156 mmol) was added dropwise an aqueous solution

of NaOH (4 mL of a 0.15 M solution, 0.625 mmol). The mixture

was left stirring at room temperature for 2 h. HCl (5 mL, 2 N)

was added dropwise, and a white precipitate was formed. The

solid was filtered and dried in vacuo. Product 1c was isolated

as a white solid (0.030 g, 0.076 mmol, 49%); mp >360 °C

(decomp.); ¹H NMR (300 MHz, DMSO-d₆) δ 3.53 (6H, s, OMe),

7.00 (2H, d, J ) 8.6 Hz, C_5′′-H), 7.72-7.90 (12H, m, C_2′′-H +

C_6′′-H + C₃-H + C₂-H), 9.84 (2H, s, NH); ¹³C NMR (75 MHz,

DMSO-d₆) δ 56.1 (OMe), 111.8 (C_5′′), 123.2 (C_2′′), 125.4 (C_6′′),

126.7 (C_1′′), 127.7 (C₃), 127.9 (C₂), 128.9 (CNH), 140.4 (C₄),

142.7 (C₁), 156.3 (C_4′′), 166.9 (COOH); ES^-m/z 610.8 (M - H)^-,

305.0 ({M - 2H}^-/2); high-resolution ES^-m/z found 633.0606

F igu r e 10. Numbering system used for assigning NMR

spectra.

resolution ES mass spectrometry was performed on a Fisons

VG platform II electrospray mass spectrometer at the Welsh

School of Pharmacy, Cardiff University. Low-resolution EI, CI,

and FAB and all high-resolution mass spectra were recorded

on a VG ZAB spectrometer at the Engineering and Physical

Sciences Research Council (EPSRC) Mass Spectrometry Cen-

ter at the University College of Wales, Swansea. IR spectra

were recorded on an Perkin-Elmer 1600 series spectropho-

tometer using sodium chloride plates for liquid samples and

potassium bromide for solid samples. Elemental analyses were

obtained from MEDAC Analytical and Chemical Consultancy

Services, Ltd. Melting points are uncorrected values and were

determined using a Gallenkamp melting-point apparatus.

All reactions were performed in an oven-dried apparatus

under an atmosphere of nitrogen. All solvents were of reagent

grade. Anhydrous diethyl ether, methanol, and tetrahydrofu-

ran were purchased from Fluka. Anhydrous N,N-dimethylacet-

amide, N,N-dimethylformamide, and 1,4-dioxane were pur-

chased from Aldrich Chemical Company. Reaction temperatures

of -78 and 0 °C were achieved by the use of acetone/dry ice

baths and water/ice baths, respectively. Thin-layer chroma-

tography (TLC) was performed on Fluka aluminum-backed

silica plates. Compounds were visualized under a UV lamp

with aqueous potassium permanganate solution and/or phos-

phomolybdic acid in ethanolic solution. Column chromatogra-

phy was performed on Fluka silica gel (with the solvent

mixtures specified).

C

₂₈H₂₂N₂O₁₀S₂Na requires 633.0614.

Bis-4-({[4′-(a m in osu lfon yl)-1,1′-bip h en yl-4-yl]su lfon yl}-

a m in o)-3-h yd r oxyben zoic a cid (1d ). An identical procedure

to the one employed for 1c was used. 1a (0.086 g, 0.14 mmol)

was reacted with aqueous NaOH (4 mL of a 1.4 M solution,

0.023 g, 0.56 mmol), and product 1d was isolated as a light-

brown solid (0.045 g,0.094 mmol, 55%); mp >360 °C (decomp.);

¹H NMR (300 MHz, DMSO-d₆) δ 7.32 (6H, br, s, C_2′′-H +

C_5′′-H + C_6′′-H), 7.89-8.04 (8H, br, m, C₂-H + C₃-H), 9.76

(2H, s, OH), 10.14 (2H, s, NH), 12.77 (2H, br, s, CO₂H); ¹³C

NMR (75 MHz, DMSO-d₆) δ 116.5 (C_5′′), 118.5 (C_2′′), 122.7 (C_1′′),

124.1 (C_6′′), 129.6 (C₃), 129.9 (C₂), 130.9 (C₄), 142.2 (CNH),

144.5 (C₁), 150.9 (COH), 168.9 (COOH); ES^-m/z 582.5 (M -

H)^-, 291.1 ({M - 2H}^-/2); high-resolution ES^-m/z found

583.0494 C₂₆H₁₉N₂O₁₀S₂(M - H) requires 583.0487; Anal.

(C₂₆H₁₆N₂Na₄O₁₀S₂‚1.5H₂O) C, H, N.

Bis-d im eth yl 5-({[4′-(Am in osu lfon yl)-1,1′-bip h en yl-4-

yl]su lfon yl}a m in o)isop h th a la te (1e). An identical proce-

dure to the one employed for 1a was used. DMAP (0.024 g,

0.199 mmol) and dimethyl-5-aminoisophthalate (0.917 g, 4.38

mmol) were reacted with 4,4′-biphenyldisulfonyl chloride

(0.700 g, 1.99 mmol) in anhydrous pyridine (15 mL). The

required product (the tetramethylester) was not formed;

instead, it was hydrolyzed to 1e during the workup. Product

1e was isolated as an off-white solid (0.0618 g, 0.96 mmol,

48%); mp 278-281 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 7.83-

7.95 (12H, m, C_4′′-H + C_6′′-H + C₂-H + C₃-H), 8.12 (2H, s,

C_2′′-H), 10.89 (2H, s, NH), 13.34 (4H, br, s, COOH); ¹³C NMR

(75 MHz, DMSO-d₆) δ 124.3 (C_2′′), 125.7 (C_4′′), 127.6 (C₃), 128.6

(C₂), 132.7 (C_3′′), 138.8 (C₄), 139.2 (C₁), 142.9 (CNH), 166.3

(COOH); ES^-m/z 638.5(M^-), 319.0 (m/2z); high-resolution ES^-

m/z found 639.0373 C₂₈H₁₉N₂O₁₂S₂(M - H) requires 639.0379;

Anal. (C₂₈H₂₀N₂O₁₂S₂‚2.5H₂O) C, H, N.

The assignment of spectra was done according to the

numbering system shown in Figure 10. The numbering of

terminators was done according to the normal IUPAC rules

except using the symbol ′′ to differentiate from the numbering

of the core.

Syn th esis of Su lfon am ide An alogu es. Bis-m eth yl 4-({[4′-

(Am in osu lfon yl)-1,1′-bip h en yl-4-yl]su lfon yl}a m in o)-3-h y-

d r oxyben zoa te (1a ). To DMAP (0.012 g, 0.099 mmol) and

methyl-4-amino-3-hydroxybenzoate (0.366 g, 2.2 mmol) in

anhydrous pyridine (10 mL) at 0 °C was added 4,4′-biphenyldi-

sulfonyl chloride (0.350 g, 0.99 mmol). The reaction was stirred

at room temperature overnight (14 h). HCl (50 mL, 2 N) was

added, and the organic phase was extracted with ethyl acetate

(2 × 25 mL). The organic layer was separated, dried (MgSO₄),

and evaporated in vacuo. Column chromatography in a 4:1

ethyl acetate/petroleum ether mixture yielded product 1a as

a white solid (0.169 g, 0.276 mmol, 28%); mp 289-290 °C; ¹H

NMR (300 MHz, DMSO-d₆) δ 3.77 (6H, s, OMe), 7.35 (6H, m,

C_2′′-H + C_5′′-H +C_6′′-H), 7.89 (8H, br, s, C₂-H + C₃-H), 9.97

(4H, br, s, NH + OH); ¹³C NMR (75 MHz, pyridine-d₅) δ 54.2

(OMe), 119.2 (COH), 123.9 (C_6′′), 125.0 (C_1′′), 129.7 (C_2′′), 130.3

(C₃), 130.5 (C₂), 133.7 (CNH), 143.4 (C₄), 145.5 (C₁), 152.4 (C_5′′),

168.9 (COO); ES^-m/z 610.9 (M^-); high-resolution ES^-m/z

found 611.0787 C₂₈H₂₃N₂O₁₀S₂(M - H) requires 611.0794.

Anal. (C₂₈H₂₄N₂O₁₀S₂‚0.2C₅H₅N) C, H, N.

Bis-p h en yl 4-({[4′-(Am in osu lfon yl)-1,1′-bip h en yl-4-yl]-

su lfon yl}a m in o)-2-h yd r oxyben zoa te (1f). An identical pro-

cedure to the one employed for 1a was used. DMAP (0.026 g,

0.216 mmol) and phenyl-4-aminosalicylate (1.09 g, 4.76 mmol)

were reacted with 4,4′-biphenyldisulfonyl chloride (0.760 g,

2.16 mmol) in anhydrous pyridine (15 mL). Product 1f was

isolated as a white solid (0.065 g, 0.088 mmol, 4%); mp 266-

1

270 °C; H NMR (300 MHz, DMSO-d₆) δ 6.77-6.81 (4H, m,

C_3′′-H + C_5′′-H), 7.21-7.46 (10H, m, Ar-H), 7.87 (2H, d, J )

9.3 Hz, C_6′′-H), 7.96 (8H, s, C₂-H + C₃-H), 10.39 (2H, s, NH),

11.28 (2H, br, s, OH); ¹³C NMR (75 MHz, DMSO-d₆) δ 103.9

(C_3′′), 106.4 (C_5′′), 108.1 (C_1′′), 120.4 (Ar-C), 124.5 (Ar-C), 125.9

(C₃), 126.7 (C₂), 127.9 (Ar-C), 130.8 (C_6′′), 137.5 (C₄), 141.2

(C₁), 148.5 (Ar-C + CNH), 159.7 (COH), 164.7 (COO); ES^-

m/z 734.7 (M^-); high-resolution ES^-m/z found 735.1100

C₃₈H₂₇N₂O₁₀S₂(M - H) requires 735.1107; Anal. (C₃₈H₂₈N₂O₁₀S₂‚

0.2H₂O) C, H, N.

Bis-m eth yl 3-({[4′-(Am in osu lfon yl)-1,1′-bip h en yl-4-yl]-

su lfon yl}a m in o)-4-m eth oxyben zoa te (1b). An identical

procedure to the one employed for 1a was used. DMAP (0.017

g, 0.142 mmol) and methyl-3-amino-4-methoxybenzoate (0.567

g, 3.13 mmol) were reacted with 4-4′-biphenyldisulfonyl

chloride (0.350 g, 0.99 mmol) in anhydrous pyridine (10 mL).

Product 1b was isolated as an off-white solid (0.458 g, 0.71

1

mmol, 50%); mp 265-268 °C; H NMR (300 MHz, DMSO-d₆)

5528 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22

Sellarajah et al.

Syn th esis of Am id e An a logu es. Bis-m eth yl 4-({[4′-

(Am in oca r bon yl)-1,1′-bip h en yl-4-yl]ca r bon yl}a m in o)-3-

h yd r oxyben zoa te (2a ). To a mixture of biphenyl 4,4′ dicar-

boxylic acid (0.300 g, 1.24 mmol) and TBTU (0.875 g, 2.72

mmol) in anhydrous DMA (15 mL) was added DIPEA (0.7 mL,

3.71 mmol). After stirring at room temperature for 15 min,

methyl-4-amino-3-hydroxybenzoate (0.455 g, 2.72 mmol) was

dissolved in DMA (5 mL) in a separate flask and added

dropwise to the reaction flask. The reaction mixture was left

stirring for 48 h at room temperature. HCl (40 mL, 2 N) was

added, and the organic product was extracted with ethyl

acetate (2 × 40 mL). The organic phase was dried (MgSO₄)

and then evaporated in vacuo to leave the crude product as

an off-white solid. Pure compound 2a was obtained after

recrystallization from ethyl acetate (0.070 g, 0.13 mmol, 10%);

(M + Na); high-resolution ES⁺m/z found 447.1319 C₂₆H₂₀N₂O₄-

Na requires 447.1321; Anal. (C₂₆H₂₀N₂O₄.0.09HCl) C, H; N

calcd 6.55%, N found 6.11%.

N,N′-Dip h en yl-1,1′-bip h en yl-4,4′-d ica r boxa m id e (2d ).

4,4′-Biphenyldicarboxyl chloride (0.300 g, 1.07 mmol) and

aniline (0.2 mL, 2.15 mmol) in anhydrous THF (15 mL) were

stirred for 1 h at room temperature, and a solid precipitated,

which was filtered and washed with water (30 mL) and hexane

(20 mL) to leave product 2d as a white solid. Yield 0.202 g

(0.51 mmol, 48%); mp >360 °C (decomp.); ¹H NMR (300 MHz,

DMSO-d₆) δ 7.12 (2H, m, C_4′′-H), 7.35 (4H, m, C_{3′′,5′′}-H), 7.80

(4H, d, J ) 7.7 Hz, C_{2′′,6′′}-H), 7.93 (4H, d, J ) 8.3 Hz, C₂-H),

8.09 (4H, d, J ) 8.3 Hz, C₃-H), 10.34 (2H, s, NH). ¹³C NMR

could not be done because of insolubility. Anal. (C₂₆H₂₀N₂O₂‚

0.5HCl) C, H, N.

Bis-4-({[4′-(am in ocar bon yl)-1,1′-biph en yl-4-yl]car bon yl}-

a m in o)-2-m eth oxyben zoic Acid (2e). An identical procedure

to the one employed for 1c was used. 2b (0.100 g, 0.176 mmol)

was reacted with aqueous NaOH (0.070 g, 1.76 mmol) in 1

mL of water and THF (5 mL). Product 2e was isolated as a

brown solid (0.030 g, 0.05 mmol, 32%); mp 280 °C (decomp.);

¹H NMR (300 MHz, DMSO-d₆) δ 3.84 (6H, s, OMe), 7.54 (2H,

d, J ) 8.6 Hz, C_5′′-H), 7.86-8.16 (12H, m, C_3′′-H + C_6′′-H +

C₂-H + C₃-H), 10.60 (2H, s, NH), 12.70 (2H, br, s, COOH);

¹³C NMR (75 MHz, DMSO-d₆) δ 55.9 (OMe), 104.0 (C_3′′), 111.6

(C_1′′), 115.6 (C_5′′), 127.3 (C₂), 127.5 (C_3′′), 130.6 (C_6′′), 132.5 (C₄),

142.5 (C₁), 144.3 (CNH), 159.6 (C_2′′), 165.7 (CONH₉), 167.4

(COO); ES⁺m/z 563.3 (M + Na); high-resolution ES⁺m/z found

563.1407 C₃₀H₂₄N₂O₈Na requires 563.1430.

1

mp 286-288 °C (decomp.); H NMR (300 MHz, DMSO-d₆) δ

3.75 (6H, br, s, OMe), 6.08 (4H, br s, NH + OH), 6.80 (2H, br,

s, C_2′′-H), 7.58 (4H, br, s, C_5′′-H + C_6′′-H), 8.00 (4H, br, s,

C₂-H), 8.27 (4H, br, s, C₃-H); ¹³C NMR (75 MHz, DMSO-d₆)

δ 52.2 (OMe), 115.4 (C_2′′), 116.6 (C_5′′), 124.8 (C_6′′), 127.9 (C_1′′),

129.1 (C₂), 129.6 (C₃), 131.6 (CNH), 136.1 (C₄), 144.4 (C₁), 146.5

(COH), 164.9 (CONH), 166.5 (COO); ES^-m/z 538.9 (M^-); high-

resolution ES⁺m/z found 558.1878 C₃₀H₂₈N₃O₈(M + NH₄)

requires 558.1876; Anal. (C₃₀H₂₄N₂O₈) C, H, N.

Bis-m eth yl 4-({[4′-(Am in oca r bon yl)-1,1′-bip h en yl-4-yl]-

ca r b on yl}a m in o)-3-h yd r oxyb en zoa t e (2a ) (Alt er n a t ive

P r oced u r e). A mixture of methyl-4-amino-3-hydroxybenzoate

(0.438 g, 2.15 mmol) and triethylamine (0.45 mL, 3.22 mmol)

in anhydrous THF (15 mL) was cooled to 0 °C. After 10 min

at 0 °C, 4,4′-biphenyldicarboxyl chloride (0.300 g, 1.07 mmol)

was added to the reaction mixture, and a solid formed

immediately. The mixture was left stirring at room tempera-

ture for 18 h. Water (50 mL) was added, and the organic

product was extracted with ethyl acetate (2 × 40 mL). The

organic phase was washed with 2 N HCl (50 mL), NaHCO₃

(50 mL), and brine (50 mL). The organic layer was then dried

(MgSO₄) and concentrated in vacuo. Column chromatography

(1:1 ethyl acetate/hexane) afforded product 2a as a slightly

off-white solid (0.080 g, 0.15 mmol, 14%). Characterization

data was identical to that obtained with the initial experi-

mental procedure.

Bis-N,N′-(2-h yd r oxy-4-n itr op h en yl)-1,1′-bip h en yl-4,4′-

d ica r boxa m id e (2f). This compound was made identically to

2a (alternative procedure). 2-Amino-5-nitrophenol (0.331 g,

2.15 mmol) and triethylamine (0.45 mL, 3.21 mmol) were

reacted with 4,4′-biphenyldicarboxyl chloride (0.300 g, 1.07

mmol) in anhydrous THF (10 mL). An identical workup

procedure was employed, and product 2f was isolated as an

orange/red solid (0.406 g, 0.79 mmol, 74%); mp 302-304 °C;

¹H NMR (300 MHz, DMSO-d₆) δ 6.81 (2H, s, C_6′′-H), 6.87 (2H,

d, J ) 9.0 Hz, C_3′′-H), 7.98 (2H, d, J ) 9.0 Hz, C_4′′-H), 8.05

(4H, d, J ) 8.2 Hz, C₂-H), 8.30 (4H, d, J ) 8.2 Hz, C₃-H); ¹³

C

NMR (75 MHz, DMSO-d₆) δ 119.9 (C_6′′), 114.3 (C_4′′), 120.2 (C₃),

127.6 (C₂), 128.9 (C₃), 131.4 (CNH), 134.9 (C₄), 135.4 (C₁), 144.1

(C_5′′), 148.7 (COH), 164.4 (CONH); Anal. (C₂₆H₁₈N₄O₈‚0.3H₂O)

C, H, N.

Bis-m eth yl 4-({[4′-(Am in oca r bon yl)-1,1′-bip h en yl-4-yl]-

ca r bon yl}a m in o)-2-m eth oxyben zoa te (2b). Compound 2b

was made identically to 2a (alternative procedure). Methyl-

4-amino-2-methoxybenzoate (0.260 g, 1.43 mmol) and triethyl-

amine (0.2 mL, 1.42 mmol) were reacted with 4,4′-biphenyl-

dicarboxyl chloride (0.200 g, 0.71 mmol) in anhydrous THF

(10 mL). An identical workup procedure was employed, and

product 2b was isolated as an off-white solid (0.186 g, 0.33

Bis-m eth yl 4-({[4′-(Am in oca r bon yl)-1,1′-bip h en yl-4-yl]-

ca r bon yl}a m in o)-2-h yd r oxyben zoa te (2g). This compound

was made identically to 2a (alternative procedure). Methyl-

4-amino-2-hydroxybenzoate (0.238 g, 1.43 mmol) and triethyl-

amine (0.30 mL, 2.13 mmol) were reacted with 4,4′-biphenyl-

dicarboxyl chloride (0.200 g, 0.71 mmol) in anhydrous THF

(10 mL). An identical workup procedure was employed, and

product 2g was isolated as a white solid (0.230 g, 0.43 mmol,

1

mmol, 46%); mp 280-283 °C; H NMR (300 MHz, DMSO-d₆)

δ 3.77 (6H, s, OMe), 3.84 (6H, s, COOMe), 7.55 (2H, d, J ) 6.0

Hz, C_5′′-H), 7.74 (4H, m, C_3′′-H + C_6′′-H), 7.98 (4H, d, J )

6.9 Hz, C₂-H), 8.13 (4H, d, J ) 6.9 Hz, C₃-H), 10.58 (2H, s,

NH); ¹³C NMR (75 MHz, DMSO-d₆) δ 51.9 (COOMe), 55.9

(OMe), 103.9 (C_3′′), 111.6 (C_1′′), 114.5 (C_5′′), 127.3 (C₂), 128.9

(C₃), 130.3 (C_6′′), 132.4 (C₄), 134.2 (C₁), 142.5 (CNH), 144.6 (C_2′′),

159.6 (CONH), 165.7 (COO); EI m/z 568.3 (M⁺); high-resolution

EI m/z found 568.1842 C₃₂H₂₈N₂O₈requires 568.1840; Anal.

(C₃₂H₂₈N₂O₈‚0.5H₂O) C, H, N.

1

60%); mp 246-250 °C; H NMR (300 MHz, DMSO-d₆) δ 3.85

(6H, s, OMe), 7.39 (2H, d, J ) 8.3 Hz, C_5′′-H), 7.87-8.35 (12H,

m, C_3′′-H + C_6′′-H + C₂-H + C₃-H); 10.75 (2H, s, NH), 10.90

(2H, br, s, OH); ¹³C NMR (75 MHz, DMSO-d₆) δ 51.9 (OMe),

106.9 (C_3′′), 111.8 (C_5′′), 115.0 (C_1′′), 127.3 (C₂), 127.6 (C₃), 130.3

(C_6′′), 131.4 (C₄), 144.9 (C₁), 151.3 (CNH), 156.4 (COH), 163.2

(CONH), 166.0 (COO); Anal. (C₃₀H₂₄N₂O₈‚0.4H₂O) C, H, N.

Bis-N,N′-(2-h yd r oxyp h en yl)-1,1′-b ip h en yl-4,4′-d ica r -

boxa m id e (2c). Compound 2c was made identically to 2a

(alternative procedure). 2-Aminophenol (0.234 g, 2.15 mmol)

and triethylamine (0.45 mL, 3.21 mmol) were reacted with 4,4′-

biphenyldicarboxyl chloride (0.300 g, 1.07 mmol) in anhydrous

THF (10 mL). An identical workup procedure was employed,

and product 2c was isolated as an off-white solid (0.065 g,

Bis-m eth yl 5-({[4′-(Am in oca r bon yl)-1,1′-bip h en yl-4-yl]-

ca r bon yl}a m in o)-2-h yd r oxyben zoa te (2h ). This compound

was made identically to 2a (alternative procedure). Methyl-

3-amino-5-hydroxybenzoate (0.357 g, 2.15 mmol) and triethyl-

amine (0.45 mL, 3.21 mmol) were reacted with 4,4′-biphenyl-

dicarboxyl chloride (0.300 g, 1.07 mmol) in anhydrous THF

(10 mL). An identical workup procedure was employed, and

product 2h was isolated as a white solid (0.294 g, 0.54 mmol,

1

0.153 mmol, 14%); mp 260-263 °C (decomp.); H NMR (300

1

MHz, DMSO-d₆) δ 6.87 (2H, t, J ) 7.5 Hz, C_6′′-H), 6.95 (2H,

d, J ) 7.9 Hz, C_5′′-H), 7.07 (2H, t, J ) 7.5 Hz, C_4′′-H), 7.71

(2H, d, J ) 7.9 Hz, C_3′′pH), 7.95 (4H, d, J ) 8.2 Hz, C₃-H),

8.12 (4H, d, J ) 8.2 Hz, C₂-H), 9.64 (2H, s, NH), 9.78 (2H, br,

s, OH); ¹³C NMR (75 MHz, DMSO-d₆) δ 116.3 (C_6′′), 119.4 (C_4′′),

124.7 (C_3′′), 126.2 (CNH), 127.3 (C_5′′), 128.7 (C₂), 134.1 (C₃),

142.3 (C₄), 149.9 (C₁), 159.9 (C_1′′), 165.1 (CONH); ES⁺m/z 447.2

51%); mp 290-292 °C; H NMR (300 MHz, DMSO-d₆) δ 3.93

(6H, s, OMe), 7.03 (2H, d, J ) 8.9 Hz, C_3′′-H), 8.11 (4H, d, J

) 7.4 Hz, C₃-H), 7.94 (6H, m, C_4′′-H + C₂-H), 8.35 (2H, s,

C_6′′-H), 10.37 (4H, s, OH + NH); ¹³C NMR (75 MHz, DMSO-

d₆) δ 52.9 (OMe), 112.8 (C_3′′), 117.8 (C_1′′), 121.8 (C_6′′), 127.2 (C_4′′),

128.7 (C₂), 128.9 (C₃), 131.4 (CNH), 134.8 (C₄), 142.3 (C₁), 156.7

(COH), 165.1 (CONH), 169.4 (COO); EI m/z 540.2 (M + H)⁺;

Synthesis and Evaluation of Analogues of Congo Red

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22 5529

high-resolution EI m/z found 541.1605 C₃₀H₂₅N₂O₈(M + H)

requires 541.1604; Anal. (C₃₀H₂₄N₂O₈‚0.3H₂O) C, H, N.

mmol) were reacted in anhydrous THF (20 mL). Product 3e

was isolated as a white solid (0.719 g, 2.27 mmol, 92%); mp

340-342 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 7.14 (2H, t, J )

7.4 Hz, C_4′′-H), 7.39 (4H, t, J ) 7.9 Hz, C_{3′′,5′′}-H), 7.81 (4H, d,

J ) 7.9 Hz, C_{2′′,6′′}-H), 8.10 (4H, s, C₂-H), 10.42 (2H, s, NH);

¹³C NMR (75 MHz, DMSO-d₆) δ 120.8 (C_{2′′,6′′}), 124.2 (C_4′′), 128.1

(C₂), 129.0 (C_{3′′,5′′}), 137.8 (C₁), 139.3 (CNH), 165.2 (CONH);

Anal. (C₂₀H₁₆N₂O₂) C, H, N.

Bis-N,N′-(4-cya n op h en yl)-1,1′-bip h en yl-4,4′-d ica r box-

a m id e (2i). This compound was made identically to 2a

(alternative procedure). 4-Aminobenzonitrile (0.254 g, 2.15

mmol) and triethylamine (0.5 mL, 3.21 mmol) were reacted

with 4,4′-biphenyldicarboxyl chloride (0.300 g, 1.07 mmol) in

anhydrous THF (20 mL). An identical workup procedure was

employed, and product 2i was isolated as a white solid (0.300

g, 0.68 mmol, 63%); mp 335-338 °C; ¹H NMR (300 MHz,

DMSO-d₆) δ 7.84-8.13 (16H, m, C_{2′′,6′′}-H + C_{3′′,5′′}-H + C₂-H

+ C₃-H), 10.74 (2H, s, NH); ¹³C NMR (75 MHz, DMSO-d₆) δ

105.8 (C_1′′), 120.6 (CN), 127.5 (C_3′′), 129.0 (C₂), 130.4 (C₃), 133.5

(C_2′′), 134.1 (C₄), 142.7 (C₁), 143.8 (CNH), 166.1 (CONH); ES^-

m/z 441.1 (M - H^-); high-resolution ES^-m/z found 441.1343

N,N′-Bis-(2-h yd r oxy-4-n it r op h en yl)t er ep h t h a la m id e

(3f). This compound was made identically to 2a (alternative

procedure), with the exception of adding DMAP as a catalyst

(0.009 g, 0.689 mmol). 2-Amino-5-nitrophenol (1.062 g, 6.89

mmol) and triethylamine (0.45 mL, 3.21 mmol) were reacted

with terephthaloyl chloride (0.700 g, 3.44 mmol) in anhydrous

THF (15 mL). An identical workup procedure was employed,

and product 3f was isolated as an orange solid (1.362 g, 3.1

C

₂₈H₁₇N₄O₂requires 441.1352; Anal. (C₂₈H₁₈N₄O₂‚0.6H₂O) C,

1

H; N calcd 12.36%, N found 10.60%.

mmol, 90%); mp 283-286 °C; H NMR (300 MHz, DMSO-d₆)

δ 7.71 (2H, s, C_3′′-H), 7.73 (2H, d, J ) 9.4 Hz, C_6′′-H), 8.08

(4H, s, C₂-H), 8.26 (2H, d, J ) 9.4 Hz, C_5′′-H), 9.86 (2H, br,

s, OH); ¹³C NMR (75 MHz, DMSO-d₆) δ 109.8 (C_3′′), 114.0 (C_5′′),

121.4 (C_6′′), 128.2 (C₂), 133.6 (CNH), 137.2 (C₁), 143.9 (C_4′′),

150.4 (COH), 164.7 (CONH); Anal. (C₂₀H₁₄N₄O₈‚0.4H₂O.0.4Et₃N)

C, H, N.

Meth yl 3-[(4-{[(2-Meth oxy-5-(m eth oxycar bon yl)ph en yl)-

am in o]car bon yl}ben zoyl)am in o]-4-m eth oxyben zoate (3a).

This compound was made identically to 2a (alternative

procedure). Methyl-3-amino-4-methoxybenzoate (0.892 g, 4.92

mmol) and triethylamine (1.1 mL, 7.38 mmol) were reacted

with terephthaloyl chloride (0.500 g, 2.46 mmol) in anhydrous

THF (10 mL). An identical workup procedure was employed,

and product 3a was isolated as an off-white solid (1.006 g, 2.04

N,N′-Bis-(4-cya n op h en yl)ter ep h th a la m id e (3g). Com-

pound 3g was made identically to 2a (alternative procedure).

4-Aminobenzonitrile (0.349 g, 2.95 mmol) and triethylamine

(0.6 mL, 4.44 mmol) were reacted with terephthaloyl chloride

(0.300 g, 1.48 mmol) in anhydrous THF (20 mL). An identical

workup procedure was employed, and product 3g was isolated

as a white solid (0.351 g, 0.96 mmol, 65%); mp 343-346 °C;

1

mmol, 83%); mp 266-268 °C; H NMR (300 MHz, DMSO-d₆)

δ 3.89 (6H, s, OMe), 3.98 (6H, s, COOMe), 7.27 (2H, d, J ) 8.6

Hz, C_5′′-H), 7.88 (2H, d, J ) 8.7 Hz, C_6′′-H), 8.15 (4H, s, C₂-

H), 8.48 (2H, s, C_2′′-H), 9.85 (2H, s, NH); ¹³C NMR (75 MHz,

DMSO-d₆) δ 52.3 (COOMe), 56.5 (OMe), 111.7 (C_5′′), 121.8 (C_2′′),

125.7 (CNH), 128.1 (C₂), 129.9 (C_6′′), 137.2 (C₁), 155.8 (C_4′′),

164.9 (CONH), 166.1 (COO); Anal. (C₂₆H₂₄N₂O₈‚0.2H₂O) C, H,

N.

¹H NMR (300 MHz, DMSO-d₆) δ 7.86 (4H, d, J ) 8.8 Hz, C_{2′′,6′′}

-

H), 8.03 (4H, d, J ) 8.8 Hz, C_{3′′,5′′}-H), 8.13 (4H, s, C₂-H), 10.82

(2H, s, NH); ¹³C NMR (75 MHz, DMSO-d₆) δ 106.0 (C_1′′), 119.4

(CN), 120.7 (C_{3′′,5′′}), 128.4 (C₂), 133.6 (C_{2′′,6′′}), 137.6 (C₁), 143.6

(CNH), 165.8 (CONH); Anal. (C₂₂H₁₄N₄O₂.0.2Et₃N‚0.1H₂O) C,

H, N.

Meth yl 3-[(4-{[(3-(Meth oxyca r bon yl)-5-(m eth oxyca r -

bon yl)p h en yl)a m in o]ca r bon yl}ben zoyl)a m in o]-5-(m eth -

oxyca r bon yl)ben zoa te (3b). This compound was made

identically to 2a (alternative procedure). Dimethyl-5-amino-

isophthalate (1.030 g, 4.92 mmol) and triethylamine (1.1 mL,

7.38 mmol) were reacted with terephthaloyl chloride (0.500 g,

2.46 mmol) in anhydrous THF (15 mL). An identical workup

procedure was employed, and product 3b was isolated as a

white solid (1.266 g, 2.31 mmol, 94%); mp 303-306 °C; ¹H

NMR (300 MHz, CDCl₃) δ 3.92 (12H, s, OMe), 8.15 (4H, s, C₂-

H), 8.22 (2H, s, C_2′′-H), 8.74 (4H, s, C_{4′′,6′′}-H), 10.80 (2H, s,

NH). ¹³C NMR could not be done because of the insolubility of

the compound. Anal. (C₂₈H₂₄N₂O₁₀‚1H₂O) C, H, N.

3-[(4-{[(3-Ca r boxy-5-ca r boxyp h en yl)a m in o]ca r bon yl}-

ben zoyl)a m in o]-5-ca r boxyben zoic Acid (3c). An identical

procedure to the one employed for 1c was used. 3b (0.250 g,

0.456 mmol) was reacted with aqueous NaOH (0.291 g, 0.729

mmol) in water (4 mL), and product 3c was isolated as a light-

gray solid (0.172 g, 0.35 mmol, 77%); mp 320 °C (decomp.); ¹H

NMR (300 MHz, CDCl₃) δ 8.17 (4H, s, C₂-H), 8.24 (2H, s, C_2′′-

H), 8.68 (4H, s, C_{4′′,6′′}-H), 10.79 (2H, s, NH), 13.31 (4H, br, s,

COOH); ¹³C NMR (75 MHz, DMSO-d₆) δ 125.1 (C_{4′′,6′′}), 125.5

(C_2′′), 128.2 (C₂), 132.0 (C_3′′), 137.4 (C₁), 140.0 (CNH), 165.4

(COOH), 166.8 (CONH); Anal. (C₂₄H₁₆N₂O₁₀.2HCl‚2H₂O) C, H,

N.

Met h yl 4-[(4-{[(4-(Met h oxyca r b on yl)p h en yl)a m in o]-

ca r bon yl}ben zoyl)a m in o]ben zoa te (3d ). This compound

was made identically to 2a (alternative procedure). Methyl-

4-aminobenzoate (0.551 g, 2.95 mmol) and triethylamine (1.1

mL, 7.38 mmol) were reacted with terephthaloyl chloride

(0.300 g, 1.48 mmol) in anhydrous THF (10 mL). An identical

workup procedure was employed, and product 3d was isolated

as a white solid (0.303 g, 0.70 mmol, 47%); mp 340-343 °C;

¹H NMR (300 MHz, DMSO-d₆) δ 3.85 (3H, s, OMe), 7.99 (8H,

m, C_{3′′,5′′}-H + C_{2′′,6′′}-H), 8.12 (4H, s, C₂-H), 10.74 (2H, s, NH);

¹³C NMR (75 MHz, DMSO-d₆) δ 52.3 (OMe), 120.0 (C_3′,5′′), 124.8

(C_1′′), 128. (C_{2′′,6′′}), 130.5 (C₂), 137.6 (C₁), 143.7 (CNH), 165.6

(CONH), 166.1 (COO); Anal. (C₂₄H₂₀N₂O₆) C, H, N.

Meth yl 4-{[4-({[2-Hydr oxy-4-(m eth oxycar bon yl)ph en yl]-

am in o}car bon yl)ben zoyl]am in o}3-h ydr oxyben zoate (3h ).

Compound 3h was made identically to 2a (alternative proce-

dure). Methyl-4-amino-4-hydroxybenzoate hydrogen sulfate

salt (0.522 g, 1.97 mmol) and triethylamine (1.4 mL, 9.85

mmol) were reacted with terephthaloyl chloride (0.200 g, 0.98

mmol) in anhydrous THF (3 mL). An identical workup

procedure was employed, and product 3h was isolated as a

light-brown solid (0.258 g, 0.56 mmol, 56%); mp 287-290 °C;

¹H NMR (300 MHz, DMSO-d₆) δ 3.82 (6H, s, OMe), 7.48 (2H,

d, J ) 8.3 Hz, C_5′′-H), 7.51 (2H, s, C_2′′-H), 8.01 (2H, d, J )

8.3 Hz, C_6′′-H), 8.09 (4H, s, C₂-H), 9.66 (2H, s, NH), 10.45

(2H, br, s, OH); ¹³C NMR (75 MHz, DMSO-d₆) δ 52.4 (OMe),

116.1 (C_2′′), 120.8 (C_5′′), 123.0 (C_6′′), 126.5 (C₂), 128.2 (C_1′′), 130.9

(CNH), 137.3 (C₁), 148.9 (COH), 164.9 (CONH), 166.3 (COO);

Anal. (C₂₄H₂₀N₂O₈‚0.5H₂O) C, H, N.

Syn th esis of Im in es. Bis-m eth yl 3-{[(1Z)-1,1′-Bip h en yl-

4-ylm et h ylen e]a m in o}-4-m et h oxyb en zoa t e (4a ). Com-

pound 4a was prepared with an identical procedure to the one

employed for 5a . 4,4′-Biphenyldialdehyde (0.100 g, 0.47 mmol)

and methyl-3-amino-4-methoxybenzoate (0.181 g, 0.99 mmol)

were reacted in ethanol (5 mL). Product 4a was isolated as a

yellow solid (0.035 g, 0.065 mmol, 14%); mp 188-190 °C;¹H

NMR (300 MHz, DMSO-d₆) δ 3.85 (6H, s, OMe), 3.91 (6H, s,

COOMe-H), 7.22 (2H, d, J ) 8.6 Hz, C_5′′-H), 7.62 (2H, d, J )

2.0 Hz, C_2′′-H), 7.87 (2H, dd, J ) 2.0 Hz and 8.6 Hz, C_6′′-H),

7.96 (4H, d, J ) 8.3 Hz, C₂-H), 8.08 (4H, d, J ) 8.3 Hz, C₃-

H), 8.67 (2H, s, CHdN); ¹³C NMR (75 MHz, DMSO-d₆) δ 52.3

(COOMe), 56.3 (OMe), 112.1 (C_5′′), 120.9 (C_1′′), 122.4 (C_2′′), 127.6

(C₂), 128.7 (C_6′′), 129.9 (C₃), 135.9 (C₄), 141.6 (C_3′′), 142.4 (C₁),

156.3 (C_4′′), 162.1 (CHdN), 166.3 (COO); Anal. (C₃₂H₂₈N₂O₆)

C, H, N.

Bis-m eth yl 4-{[(1Z)-1,1′-Biph en yl-4-ylm eth ylen e]am in o}-

3-h yd r oxyben zoa te (4b). An identical procedure to the one

employed for 5a was used. 4,4′-Biphenyldialdehyde (0.044 g,

0.209 mmol) and methyl-4-amino-3-hydroxybenzoate (0.070 g,

0.418 mmol) were reacted in ethanol (10 mL). Product 4b was

isolated as a yellow solid (0.048 g, 0.094 mmol, 45%); mp 221°C

N,N′-Dip h en ylter ep h th a la m id e (3e). An identical pro-

cedure to the one employed for 2d was carried out. Aniline

(0.5 mL, 4.92 mmol) and terephthaloyl chloride (0.500 g, 2.46

5530 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22

Sellarajah et al.

(decomp.);¹H NMR (300 MHz, DMSO-d₆) δ 3.85 (6H, s, OMe),

7.27 (2H, d, J ) 7.9 Hz, C_5′′-H), 7.48-7.51 (4H, m, C_2′′-H +

C_6′′-H), 7.98 (4H, d, J ) 8.1 Hz, C₂-H), 8.15 (4H, d, J ) 8.1

Hz, C₃-H), 8.78 (2H, s, CHdN), 9.63 (2H, s, OH); ¹³C NMR

(75 MHz, DMSO-d₆) δ 51.6 (OMe), 112.8 (C_2′′), 114.8 (C_5′′), 116.5

(C_6′′), 122.9 (C₂), 128.3 (C₃), 130.6 (C₄), 136.2 (C_1′′), 142.7 (C₁),

143.1 (C_4′′), 144.7 (COH), 160.1 (CHdN), 166.8 (COO); ES^-m/z

507.2 (M - H^-); high-resolution ES^-m/z found 507.1560

NMR (300 MHz, DMSO-d₆) δ 3.84 (6H, s, OMe), 7.26 (2H, d,

J ) 8.1 Hz, C_5′′-H), 7.50 (4H, m, C_2′′-H + C_6′′-H), 8.16 (4H,

s, C₂-H), 8.78 (2H, s, CHdN), 9.68 (2H, br, s, OH); ¹³C NMR

(75 MHz, DMSO-d₆) δ 52.4 (OMe), 116.9 (C_2′′), 120.4 (C_5′′), 121.1

(C_6′′), 128.3 (C₂), 129.6 (C_1′′), 138.9 (C₁), 143.1 (C_4′′), 150.9

(COH), 161.6 (CHdN), 166.2 (COO); Anal. (C₂₄H₂₀N₂O₆‚

0.1H₂O) C, H, N.

P h en yl 2-Hydr oxy-4-({(1Z)-[4-((Z)-{[3-h ydr oxy-4-(p h en -

yloxyca r b on yl)p h e n yl]im in o}m e t h yl)p h e n yl]m e t h yl-

id en e}a m in o)ben zoa te (5b). An identical procedure to the

one employed for 5a was used. Terephthalaldehyde (0.500 g,

3.72 mmol) and phenyl-4-aminosalicylate (1.708 g, 7.45 mmol)

were reacted in ethanol (25 mL). Product 5b was isolated as

a yellow solid (0.291 g, 0.52 mmol, 14%); mp 185-186 °C;¹H

NMR (300 MHz, DMSO-d₆) δ 6.92 (4H, m, C_3′′-H + C_5′′-H),

7.35 (6H, m, Ar-H), 7.50 (4H, m, Ar-H), 8.06 (2H, d, J ) 8.3

Hz, C_6′′-H), 8.14 (4H, s, C₂-H), 8.76 (2H, s, CHdN), 10.5 (2H,

br s, OH); ¹³C NMR (75 MHz, DMSO-d₆) δ 109.4 (C_3′′), 110.7

(C_5′′), 113.4 (C_1′′), 122.3 (Ar-H), 126.5 (Ar-H), 129.8 (Ar-H),

129.9 (C₂), 132.3 (C_6′′), 138.7 (C₁), 150.5 (Ar-H), 158.5 (C_4′′),

161.7 (COH), 162.7 (CHdN), 167.0 (COO); Anal. (C₃₄H₂₄N₂O₆)

C, H, N.

C

₃₀H₂₃N₂O₆requires 507.1556; Anal. (C₃₀H₂₄N₂O₆‚0.25H₂O) C,

H; (N calcd 5.46%, N found 4.74%).

Bis-4-{[(1Z)-1,1′-bip h en yl-4-ylm eth ylen e]a m in o}-3-h y-

d r oxyben zoic Acid (4c). An identical procedure to the one

employed for 5a was used. 4,4′-Biphenyldialdehyde (0.060 g,

0.285 mmol) and 4-amino-3-hydroxybenzoic acid (0.087 g, 0.570

mmol) were reacted in ethanol (10 mL). The product 4c was

isolated as a yellow solid (0.039 g, 0.081 mmol, 28%); mp >360

1

°C; H NMR (300 MHz, DMSO-d₆) δ 7.25 (2H, d, J ) 8.0 Hz,

C_5′′-H), 7.45-7.50 (4H, m, C_2′′-H + C_6′′-H), 7.97 (4H, d, J )

7.9 Hz, C₂-H), 8.16 (4H, d, J ) 7.9 Hz, C₃-H), 8.78 (2H, s,

CHdN), 9.53 (2H, s, OH); ¹³C NMR (75 MHz, DMSO-d₆) δ

112.8 (C_2′′), 115.1 (C_5′′), 117.8 (C_6′′), 122.9 (C₂), 128.3 (C₃), 130.6

(C₄), 136.1 (C_1′′), 142.3 (C₁), 143.1 (C_4′′), 144.7 (COH), 160.1

(CHdN), 168.0 (COO); ES^-m/z 479.2 (M - H^-); high-

resolution ES^-m/z found 479.1250 C₂₈H₁₉N₂O₆requires

479.1243; Anal. (C₂₈H₂₀N₂O₆) C, H; N calcd 5.83%, N found

5.23%.

Meth yl 4-Meth oxy-3-({(1Z)-[4-((Z)-{[2-m eth oxy-5-(m eth -

oxyca r bon yl)p h en yl]im in o}m eth yl)p h en yl]m eth ylen e}-

a m in o)ben zoa te (5c). An identical procedure to the one

employed for 5a was used. Terephthalaldehyde (0.500 g, 3.72

mmol) and methyl-3-amino-4-methoxybenzoate (1.350 g, 7.45

mmol) were reacted in ethanol (25 mL). Product 5c was

isolated as a yellow solid (0.764 g, 1.65 mmol, 45%); mp 182-

2-{[(1Z)-(4′-{(Z)-[(2-Hyd r oxyp h en yl)im in o]m eth yl}-1,1′-

bip h en yl-4-yl)m eth ylen e]a m in o}p h en ol (4d ). An identical

procedure to the one employed for 5a was used. 4,4′-Biphen-

yldialdehyde (0.100 g, 0.47 mmol) and 2-aminophenol (0.103

g, 0.95 mmol) were reacted in ethanol (15 mL). Product 4d

was isolated as a yellow solid (0.129 g, 0.33 mmol, 70%); mp

226-230 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 6.84-6.94 (4H,

m, C_5′′-H + C_6′′-H), 7.10 (2H, t, J ) 7.1 Hz, C_4′′-H), 7.26 (2H,

d, J ) 7.8 Hz, C_3′′-H), 7.95 (4H, d, J ) 8.3 Hz, C₂-H), 8.16

(4H, d, J ) 8.3 Hz, C₃-H), 8.79 (2H, s, CHdN), 9.07 (2H, br,

s, OH); ¹³C NMR (75 MHz, DMSO-d₆) δ 116.4 (C_6′′), 119.4 (C_4′′),

119.8 (C_3′′), 127.4 (C₂), 127.9 (C_5′′), 129.9 (C₃), 136.3 (C₄), 138.1

(C₁), 142.0 (C_2′′), 151.7 (COH), 158.9 (CHdN); Anal. (C₂₆H₂₀N₂O₂)

C, H, N.

4-{[(1Z)-(4′-{(Z)-[(4-Hyd r oxyp h en yl)im in o]m eth yl}-1,1′-

bip h en yl-4-yl)m eth ylen e]a m in o}p h en ol (4e). An identical

procedure to the one employed for 5a was used. 4,4′-Biphen-

yldialdehyde (0.100 g, 0.47 mmol) and 4-aminophenol (0.103

g, 0.95 mmol) were reacted in ethanol (15 mL). Product 4e

was isolated as a yellow solid (0.127 g, 0.32 mmol, 69%); mp

>360 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 6.82 (4H, d, J ) 8.6

Hz, C_{2′′,6′′}-H), 7.25 (4H, d, J ) 8.6 Hz, C_{3′′,5′′}-H), 7.90 (4H, d,

J ) 8.2 Hz, C₂-H), 8.01 (4H, d, J ) 8.2 Hz, C₃-H). 8.69 (2H,

s, CHdN), 9.56 (2H, s, OH); ¹³C NMR (75 MHz, DMSO-d₆) δ

116.1 (C_{2′′,6′′}), 122.9 (C_{3′′,5′′}), 127.4 (C₂), 129.3 (C₃), 136.3 (C₄),

141.7 (C₁), 142.9 (C_4′′), 156.8 (COH), 156.9 (CHdN); Anal.

1

183 °C; H NMR (300 MHz, CDCl₃) δ 3.97 (6H, s, OMe), 4.03

(6H, s, COOMe), 7.03 (2H, d, J ) 8.6 Hz, C_5′′-H), 7.78 (2H, d,

J ) 2.1 Hz, C_2′′-H), 7.99 (2H, dd, J ) 2.1 Hz and 8.6 Hz, C_6′′-

H), 8.10 (4H, s, C₂-H), 8.63 (2H, s, CHdN). ¹³C could not be

carried out because of solubility problems. Anal. (C₂₆H₂₄N₂O₆‚

0.2H₂O) C, H, N.

4-{[(1Z)-(4-{(Z)-[(4-Ca r b oxyp h e n yl)im in o]m e t h yl}-

p h en yl)m eth ylen e]a m in o}ben zoic a cid (5d ). An identical

procedure to the one employed for 5a was used. Terephthal-

aldehyde (0.300 g, 2.23 mmol) and 4-aminobenzoic acid (0.628

g, 4.58 mmol) were reacted in ethanol (20 mL). Product 5d

was isolated as a yellow solid (0.647 g, 1.73 mmol, 78%); mp

>360 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 7.37 (4H, d, J ) 8.5

Hz, C_{3′′,5′′}-H), 8.00 (4H, d, J ) 8.5 Hz, C_{2′′,6′′}-H), 8.12 (4H, s,

C₂-H), 8.74 (2H, s, CHdN), 12.93 (2H, br, s, COOH); ¹³C NMR

(75 MHz, DMSO-d₆) δ 121.5 (C_{3′′,5′′}), 128.6 (C₂), 129.7 (C_1′′),

130.9 (C_{2′′,6′′}), 138.8 (C₁), 155.5 (C_4′′), 162.2 (CHdN), 167.3

(COOH); Anal. (C₂₂H₁₆N₂O₄‚0.1H₂O) C, H, N.

4-({[(1Z)-(4-{(Z)-[(4-Ca r b oxyb e n zyl)im in o]m e t h yl}-

p h en yl)m eth ylen e]a m in o}m eth yl)ben zoic Acid (5e). An

identical procedure to the one employed for 5a was used.

Terephthalaldehyde (0.250 g, 1.86 mmol) and 4-(aminomethyl)-

benzoic acid (0.563 g, 3.72 mmol) were reacted in ethanol (15

mL). Product 5e was isolated as a white solid (0.475 g, 1.19

mmol, 64%); mp >235 °C (decomp.);¹H NMR (300 MHz,

C

₂₆H₂₀N₂O₂‚0.3H₂O) C, H, N.

N-((1Z)-{4′-[(Z)-(P h en ylim in o)m eth yl]-1,1′-bip h en yl-4-

yl}m eth ylen e)a n ilin e (4f). An identical procedure to the one

employed for 5a was used. 4,4′-Biphenyldialdehyde (0.200 g,

0.95 mmol) and aniline (0.17 mL, 1.90 mmol) were reacted in

ethanol (20 mL). Product 4f was isolated as a yellow solid

(0.252 g, 0.69 mmol, 74%); mp 237-238 °C; ¹H NMR (300 MHz,

DMSO-d₆) δ 7.26-7.33 (6H, m, C_{2′′,6′′}-H + C_4′-H), 7.45 (4H,

t, J ) 7.5 Hz, C_3′′,5′-H), 7.96 (4H, d, J ) 8.3 Hz, C₂-H), 8.08

(4H, d, J ) 8.3 Hz, C₃-H), 8.71 (2H, s, CHdN); ¹³C NMR (75

MHz, DMSO-d₆) δ 121.4 (C_{2′′,6′′}), 126.0 (C_4′′), 127.6 (C₂), 129.6

(C₃), 129.7 (C_{3′′,5′′}), 135.9 (C₄), 142.2 (C₁), 151.7 (C_1′′), 160.5

(CHdN); Anal. (C₂₆H₂₀N₂) C, H, N.

Meth yl 3-Hydr oxy-4-({(1Z)-[4-((Z)-{[2-h yd r oxy-4-(m eth -

oxyca r bon yl)p h en yl]im in o}m eth yl)p h en yl]m eth ylen e}-

a m in o)ben zoa te (5a ). Terephthalaldehyde (0.200 g, 1.49

mmol) and methyl-4-amino-3-hydroxy-benzoate (0.498 g, 2.98

mmol) were added to ethanol (15 mL), and the reaction

mixture was heated to 55 °C for 5 h. The precipitate that

formed was filtered and washed with ethanol (40 mL). Product

5a was isolated as a yellow powder. No further purification

was necessary (0.491 g, 1.13 mmol, 76%); mp 263-266 °C; ¹H

DMSO-d₆) δ 4.88 (4H, s, CH₂), 7.46 (4H, d, J ) 8.2 Hz, C_{3′′,5′′}-

H), 7.89 (4H, s, C₂-H), 7.92 (4H, d, J ) 8.2 Hz, C_{2′′,6′′}-H), 8.59

(2H, s, CHdN), 12.95 (2H, br, s, COOH); ¹³C NMR (75 MHz,

DMSO-d₆) δ 63.8 (CH₂), 128.2 (C_1′′), 128.7 (C_{3′′,5′′}), 129.7 (C₂),

129.8 (C_{2′′,6′′}), 138.2 (C₁), 144.8 (C_4′′), 162.4 (CHdN), 167.6

(COOH); Anal. (C₂₄H₂₀N₂O₄‚0.2H₂O) C, H, N.

3-{[(1Z)-(4-{(Z)-[(5-Ca r boxy-2-m eth oxyp h en yl)im in o]-

m eth yl}ph en yl)m eth ylen e]am in o}-4-m eth oxyben zoic Acid

(5f). An identical procedure to the one employed for 5a was

used. Terephthalaldehyde (0.080 g, 0.596 mmol) and 3-amino-

4-methoxybenzoic acid (0.192 g, 1.19 mmol) were reacted in

ethanol (10 mL). Product 5f was isolated as a yellow solid

1

(0.216 g, 0.50 mmol, 84%); mp >335 °C (decomp.); H NMR

(300 MHz, DMSO-d₆) δ 3.90 (6H, s, OMe), 7.20 (2H, d, J ) 8.6

Hz, C_5′′-H), 7.62 (2H, d, J ) 2.0 Hz, C_2′′-H), 7.85 (2H, dd, J

) 2.0 Hz and 8.6 Hz, C_6′′-H), 8.09 (4H, s, C₂-H), 8.69 (2H, s,

CHdN), 12.76 (2H, br, s, COOH); ¹³C NMR (75 MHz, DMSO-

d₆) δ 56.3 (OMe), 112.0 (C_5′′), 121.2 (C_1′′), 123.6 (C_2′′), 129.5 (C₂),

138.8 (C_6′′), 141.2 (C₁), 156.0 (C_3′′), 161.8 (C_4′′), 167.3 (CHdN),

172.3 (COOH); Anal. (C₂₄H₂₀N₂O₆‚1H₂O) C, H, N.

Synthesis and Evaluation of Analogues of Congo Red

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22 5531

5-{[(1Z)-(4-{(Z)-[(3-Ca r boxy-4-h yd r oxyp h en yl)im in o]-

m eth yl}ph en yl)m eth ylen e]am in o}-2-h ydr oxyben zoic Acid

(5g). An identical procedure to the one employed for 5a was

used. Terephthalaldehyde (0.200 g, 1.49 mmol) and 5-amino-

salicylic acid (0.456 g, 2.98 mmol) were reacted in ethanol (10

mL). Product 5g was isolated as a gray solid (0.400 g, 0.99

mmol, 66%); mp >360 °C;¹H NMR (300 MHz, DMSO-d₆) δ 7.03

(2H, d, J ) 8.8 Hz, C_3′′-H), 7.60 (2H, dd, J ) 2.7 Hz and 8.8

Hz, C_4′′-H), 7.78 (2H, d, J ) 2.7 Hz, C_6′′-H), 8.06 (4H, s, C₂-

H), 8.78 (2H, s, CHdN); ¹³C NMR (75 MHz, DMSO-d₆) δ 113.7

(C_3′′), 118.3 (C_1′′), 122.8 (C_6′′), 129.1 (C_4′′), 129.4 (C₂), 138.7 (C₁),

142.6 (C_5′′), 158.7 (C_2′′), 160.3 (CHdN), 172.0 (COOH); Anal.

(C₂₂H₁₆N₂O₆‚0.5H₂O) C, H, N.

4-{[(1Z)-(4-{(Z)-[(4-Ca r boxy-2-h yd r oxyp h en yl)im in o]-

m eth yl}ph en yl)m eth ylen e]am in o}-3-h ydr oxyben zoic Acid

(5h ). An identical procedure to the one employed for 5a was

used. Terephthalaldehyde (0.100 g, 0.74 mmol) and 4-amino-

3-hydroxybenzoic acid (0.239 g, 1.56 mmol) were reacted in

ethanol (10 mL). Product 5h was isolated as a brown solid

(0.220 g, 0.54 mmol, 74%); mp >360 °C;¹H NMR (300 MHz,

DMSO-d₆) δ 7.24 (2H, d, J ) 8.1 Hz, C_5′′-H), 7.44 (4H, m,

C_2′′-H + C_6′′-H), 8.17 (4H, s, C₂-H), 8.79 (2H, s, CHdN), 9.57

(2H, br, s, OH), 12.79 (2H, br, s, COOH); ¹³C NMR (75 MHz,

DMSO-d₆) δ 117.1 (C_2′′), 120.2 (C_5′′), 121.2 (C_6′′), 129.6 (C₂),

129.8 (C_1′′), 138.9 (C₁), 142.7 (C_4′′), 150.8 (COH), 161.3 (CHd

N), 167.4 (COOH); Anal. (C₂₂H₁₆N₂O₆‚0.2H₂O) C, H, N.

2-{[(1Z)-(4-{(Z)-[(2-H yd r oxyp h e n yl)im in o]m e t h yl}-

p h en yl)m eth ylen e]a m in o}p h en ol (5i). An identical proce-

dure to the one employed for 5a was used. Terephthalaldehyde

(0.300 g, 2.23 mmol) and 2-aminophenol (0.488 g, 4.47 mmol)

were reacted in ethanol (15 mL). Product 5i was isolated as a

yellow solid (0.387 g, 1.22 mmol, 55%); mp 188-190 °C;¹H

NMR (300 MHz, DMSO-d₆) δ 6.84-6.93 (4H, m, C_5′′-H + C_6′′-

H), 7.12 (2H, t, J ) 6.8 Hz, C_4′′-H), 7.27 (2H, d, J ) 6.8 Hz,

C_3′′-H), 8.17 (4H, s, C₂-H), 8.81 (2H, s, CHdN), 9.11 (2H, s,

OH); ¹³C NMR (75 MHz, DMSO-d₆) δ 116.5 (C_6′′), 119.5 (C_4′′),

119.9 (C_3′′), 128.1 (C_5′′), 129.4 (C₂), 137.9 (C₁), 138.9 (C_2′′), 151.8

(COH), 160.0 (CHdN); Anal. (C₂₀H₁₆N₂O₂) C, H, N.

2-{[(1Z)-(4-{(Z)-[(2-H yd r oxy-4-n it r op h e n yl)im in o]-

m eth yl}p h en yl)m eth ylen e]a m in o}-5-n itr op h en ol (5m ).

An identical procedure to the one employed for 5a was used.

Terephthalaldehyde (0.300 g, 2.2 mmol) and 2-amino-5-nitro-

phenol (0.690 g, 4.4 mmol) were reacted in ethanol (15 mL).

Product 5m was isolated as a dark-yellow solid (0.573 g, 1.41

1

mmol, 64%); mp 268-270 °C; H NMR (300 MHz, DMSO-d₆)

δ 7.35 (2H, d, J ) 8.4 Hz, C_3′′-H), 7.76 (4H, m, C_4′′-H + C_6′′-

H), 8.18 (4H, s, C₂-H), 8.78 (2H, s, CHdN), 10.32 (2H, br, s,

OH); ¹³C NMR (75 MHz, DMSO-d₆) δ 110.9 (C_6′′), 115.5 (C_4′′),

121.0 (C_3′′), 129.9 (C₂), 138.9 (C₁), 145.6 (C_2′′), 145.9 (C_5′′), 151.0

(COH), 163.0 (CHdN); Anal. (C₂₀H₁₄N₄O₆0‚2H₂O‚0.1Et₃N) C,

H, N.

Meth yl 2-Hydr oxy-4-({(1Z)-[4-((Z)-{[3-h ydr oxy-4-(m eth -

oxyca r bon yl)p h en yl]im in o}m eth yl)p h en yl]m eth ylen e}-

a m in o)ben zoa te (5n ). An identical procedure to the one

employed for 5a was used. Terephthalaldehyde (0.200 g, 1.49

mmol) and methyl-4-amino-2-hydroxybenzoate (0.49 g, 2.98

mmol) were reacted in ethanol (13 mL). Product 5n was

isolated as a yellow solid (0.267 g, 0.62 mmol, 41%); mp 198-

201 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 3.84 (6H, br, s, OMe),

6.78 (4H, br, s, C_3′′-H + C_5′′-H), 7.77 (2H, br, s, C_6′′-H), 8.03

(4H, br, s, C₂-H), 8.65 (2H, br, s, CHdN), 10.63 (2H, br, s,

OH). ¹³C could not be done because of a problem with solubility.

Anal. (C₂₄H₂₀N₂O₆‚0.5H₂O) C, H, N.

E t h yl 2-H yd r oxy-4-({(1Z)-[4-((Z)-{[3-h yd r oxy-4-(et h -

oxyca r bon yl)p h en yl]im in o}m eth yl)p h en yl]m eth ylen e}-

a m in o)ben zoa te (5o). An identical procedure to the one

employed for 5a was used. Terephthalaldehyde (0.060 g, 0.44

mmol) and ethyl-4-amino-2-hydroxybenzoate (0.161 g, 0.88

mmol) were reacted in ethanol (5 mL). Product 5o was isolated

as a yellow solid (0.060 g, 0.13 mmol, 30%); mp 190-191 °C;

¹H NMR (300 MHz, DMSO-d₆) δ 1.37 (6H, t, J ) 7.1 Hz, CH₃-

H), 4.39 (4H, q, J ) 7.1 Hz, CH₂-H), 6.85 (2H, s, C_3′′-H), 6.88

(2H, d, J ) 8.1 Hz, C_5′′-H), 7.86 (2H, d, J ) 8.1 Hz, C_6′′-H),

8.11 (4H, s, C₂-H), 8.73 (2H, s, CHdN), 10.78 (2H, s, OH).

¹³C could not be done because of a problem with solubility.

Anal. (C₂₆H₂₄N₂O₆‚0.4H₂O) C, H, N.

Meth yl 2-Hydr oxy-5-({(1Z)-[4-((Z)-{[4-h ydr oxy-3-(m eth -

oxyca r bon yl)p h en yl]im in o}m eth yl)p h en yl]m eth ylen e}-

a m in o)ben zoa te (5p ). An identical procedure to the one

employed for 5a was used. Terephthalaldehyde (0.100 g, 0.75

mmol) and methyl-5-amino-2-hydroxybenzoate (0.250 g, 1.49

mmol) were reacted in ethanol (7 mL). Product 5p was isolated

as a yellow solid (0.070 g, 0.16 mmol, 22%); mp 207-210 °C;

¹H NMR (300 MHz, DMSO-d₆) δ 3.88 (6H, s, OMe), 7.04 (2H,

dd, J ) 1.2 Hz and 8.8 Hz, C_4′′-H), 7.59 (2H, d, J ) 8.8 Hz,

C_3′′-H), 7.72 (2H, s, C_6′′-H), 8.02 (4H, s, C₂-H), 8.74 (2H, s,

CHdN), 10.47 (2H, s, OH). ¹³C could not be done because of a

lack of solubility. Anal. C₂₄H₂₀N₂O₆) C, H, N.

4-{[(1Z)-(4-{(Z)-[(4-H yd r oxyp h e n yl)im in o]m e t h yl}-

p h en yl)m eth ylen e]a m in o}p h en ol (5j). An identical proce-

dure to the one employed for 5a was used. Terephthalaldehyde

(0.300 g, 2.33 mmol) and 4-aminophenol (0.496 g, 4.47 mmol)

were reacted in ethanol (15 mL). Product 5j was isolated as a

yellow solid (0.244 g, 0.77 mmol, 33%); mp 260-262 °C; ¹H

NMR (300 MHz, DMSO-d₆) δ 6.81 (4H, d, J ) 8.6 Hz, C_{2′′,6′′}

-

H), 7.25 (4H, d, J ) 8.6 Hz, C_{3′′,5′′}-H), 8.01 (4H, s, C₂-H), 8.69

(2H, s, CHdN), 9.58 (2H, s, OH); ¹³C NMR (75 MHz, DMSO-

d₆) δ 116.1 (C_{2′′,6′′}), 123.0 (C_{3′′,5′′}), 128.9 (C₂), 138.7 (C₁), 142.7

(C_4′′), 156.7 (C_1′′), 160.2 (CHdN); Anal. (C₂₀H₁₆N₂O₂‚0.1H₂O)

C, H, N.

E t h yl 2-H yd r oxy-5-({(1Z)-[4-((Z)-{[4-h yd r oxy-3-(et h -

oxyca r bon yl)p h en yl]im in o}m eth yl)p h en yl]m eth ylen e}-

a m in o)ben zoa te (5q). An identical procedure to the one

employed for 5a was used. Terephthalaldehyde (0.050 g, 0.37

mmol) and ethyl-5-amino-2-hydroxybenzoate (0.134 g, 0.74

mmol) were reacted in ethanol (7 mL). Product 5q was isolated

as a yellow solid (0.117 g, 0.25 mmol, 69%); mp 171-173 °C;

¹H NMR (300 MHz, DMSO-d₆) δ 1.33 (6H, t, J ) 7.1 Hz, CH₃),

4.36 (4H, q, J ) 7.1 Hz, CH₂), 7.04 (2H, d, J ) 8.8 Hz, C_3′′-H),

7.60 (2H, d, J ) 8.8 Hz, C_4′′-H), 7.72 (2H, s, C_6′′-H), 8.03 (4H,

s, C₂-H), 8.74 (2H, s, CHdN), 10.55 (2H, s, OH). ¹³C could

not be carried out because of the insolubility of the compound.

Anal. (C₂₆H₂₄N₂O₆‚0.2H₂O) C, H, N.

N -((1Z)-{4-[(Z)-(P h e n ylim in o)m e t h yl]p h e n yl}m e t h -

ylen e)a n ilin e (5k ). An identical procedure to the one em-

ployed for 5a was used. Terephthalaldehyde (0.200 g, 1.49

mmol) and aniline (0.27 mL, 2.98 mmol) were reacted in

ethanol (15 mL). Product 5k was isolated as a yellow solid

(0.313 g, 1.1 mmol, 74%); mp 158-160 °C; ¹H NMR (300 MHz,

DMSO-d₆) δ 7.25-7.34 (6H, m, C_2′,6′′-H + C_4′′-H), 7.45 (4H,

t, J ) 8.1 Hz, C_{3′′,5′′}-H), 8.09 (4H, s, C₂-H), 8.72 (2H, s, CHd

N); ¹³C NMR (75 MHz, DMSO-d₆) δ 121.5 (C_{2′′,6′′}), 126.7 (C_4′′),

129.4 (C₂), 129.6 (C_{3′′,5′′}), 138.8 (C₁), 151.5 (C_1′′), 160.4 (CHd

N); Anal. (C₂₀H₁₆N₂) C, H, N.

4-{[(1Z)-(4-{(Z)-[(4-C y a n o p h e n y l)im in o ]m e t h y l}-

n a p h th yl)m eth ylen e]a m in o}n a p h th on itr ile (5l). An iden-

tical procedure to the one employed for 5a was used. Tereph-

thalaldehyde (0.050 g, 0.372 mmol) and 4-amino-1-naphthalene

carbonitrile (0.125 g, 0.745 mmol) were reacted in ethanol (6

mL). Product 5l was isolated as a yellow solid (0.020 g, 0.046

mmol, 12%); ¹H NMR (300 MHz, DMSO-d₆, 313K) δ 7.46 (2H,

d, J ) 7.8 Hz, C_3′′-H), 7.80 (2H, t, J ) 7.3 Hz, C_7′′-H), 7.91

(2H, t, J ) 7.3 Hz, C_6′′-H), 8.20 (2H, d, J ) 8.5 Hz, C_5′′-H),

8.26 (2H, d, J ) 7.6 Hz, C_8′′-H), 8.34 (4H, s, C₂-H), 8.47 (2H,

d, J ) 7.8 Hz, C_2′′-H), 8.92 (2H, s, CHdN). ¹³C could not be

done because of a lack of solubility.

2-{[(1Z)-(4-{(Z)-[(2-H yd r oxy-4-m et h ylp h en yl)im in o]-

m eth yl}p h en yl)m eth ylen e]a m in o}-5-m eth ylp h en ol (5r ).

An identical procedure to the one employed for 5a was used.

Terephthalaldehyde (0.300 g, 2.2 mmol) and 2-amino-5-meth-

ylphenol (0.550 g, 4.4 mmol) were reacted in ethanol (15 mL).

Product 5r was isolated as a yellow solid (0.649 g, 1.88 mmol,

1

86%); mp 205-207 °C; H NMR (300 MHz, DMSO-d₆) δ 2.26

(6H, s, CH₃), 6.68 (2H, d, J ) 8.1 Hz, C_4′′-H), 6.75 (2H, s, C_6′′-

H), 7.23 (2H, d, J ) 8.1 Hz, C_3′′-H), 8.15 (4H, s, C₂-H), 8.81

(2H, s, CHdN), 8.99 (2H, s, OH); ¹³C NMR (75 MHz, DMSO-

d₆) δ 21.3 (CH₃), 117.0 (C_6′′), 118.9 (C_4′′), 120.6 (C_3′′), 129.3 (C₂),

5532 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22

Sellarajah et al.

135.1 (C₁), 138.0 (C_2′′), 138.9 (C_5′′), 151.9 (COH), 157.4 (CHd

N); Anal. (C₂₂H₂₀N₂O₂‚0.2H₂O) C, H, N.

ethyl acetate/hexane) to leave 6d as a light-orange solid (0.020

g, 0.062 mmol, 10%); ¹H NMR (300 MHz, DMSO-d₆) δ 4.25

(4H, d, J ) 6.0 Hz, CH₂); 5.19 (2H, t, J ) 6.0 Hz, NH), 6.36

(4H, m, C_3′′-H + C_5′′-H), 6.51 (2H, t, J ) 7.3 Hz, C_4′′-H), 6.63

(2H, d, J ) 7.8 Hz, C_6′′-H), 7.28 (4H, s, C₂-H), 9.25 (2H, s,

OH); ¹³C NMR (75 MHz, DMSO-d₆) δ 46.6 (CH₂), 110.4 (C_3′′),

113.7 (C_6′′), 116.0 (C_5′′), 119.8 (C_4′′), 127.3 (C₂), 137.5 (C_2′′), 139.1

(C₈₁), 144.3 (COH); EI⁺m/z 320.1 (M⁺); high-resolution EI⁺

m/z found 320.1535 C₂₀H₂₀N₂O₂requires 320.1525; Anal.

(C₂₀H₂₀N₂O₂‚0.2HCl) C, H: N calcd 8.55%, N found 7.95%.

Syn th esis of Alk en e An a logu es. 5-((E)-2-{4-[(E)-2-(3-

Car boxy-4-h ydr oxyph en yl)vin yl]ph en yl}vin yl)-2-h ydr oxy-

ben zoic Acid (7a ). A mixture of DMF (17 mL) and water (3

mL) was added to 1,4-divinylbenzene (0.200 g, 1.53 mmol),

5-iodosalicylic acid (0.811 g, 3.07 mmol), 10% palladium acetate

(0.034 g, 0.153 mmol), and potassium carbonate (0.424 g, 3.07

mmol). The reaction mixture was heated to 100 °C. After 18

h, water (75 mL) was added followed by 2 M HCl (10 mL).

Upon addition of the acid, a solid precipitated, which was

filtered. The solid was recrystallized from ethyl acetate (80

mL), filtered, and washed with petroleum ether (20 mL).

Product 7a was isolated as a light-green crystalline solid (0.080

g, 0.19 mmol, 13%); mp >300 °C; ¹H NMR (300 MHz, DMSO-

d₆) δ 6.98 (1H, d, J ) 8.6 Hz, C_3′′-H), 7.11 (1H, d, J ) 16.4

Hz, CHdCH), 7.25 (1H, d, J ) 16.4 Hz, CHdCH), 7.59 (2H, s,

C₂-H), 7.81 (1H, dd, J ) 1.9 Hz and 8.7 Hz, C_4′′-H), 7.99 (1H,

d, J ) 1.9 Hz, C_6′′-H), 11.44 (1H, br, s, COOH); ¹³C NMR (75

MHz, DMSO-d₆) δ 113.5 (C_3′′), 118.0 (C_1′′), 126.8 (CHdCH),

126.9 (CHdCH), 127.4 (C₂), 128.8 (C_5′), 128.9 (C_6′′), 133.4 (C_4′′),

136.6 (C₁), 161.0 (COH), 172.1 (COOH); Anal. (C₂₄H₁₈O₆) C,

H.

4-{[(1Z)-(4-{(Z)-[(4-Cyan oph en yl)im in o]m eth yl}ph en yl)-

m eth ylen e]a m in o}ben zon itr ile (5s). An identical procedure

to the one employed for 5a was used. Terephthalaldehyde

(0.200 g, 1.49 mmol) and 4-amino-benzonitrile (0.352 g, 2.98

mmol) were reacted in ethanol (10 mL). Product 5s was

isolated as a yellow solid (0.207 g, 0.62 mmol, 42%); mp 209-

1

210 °C; H NMR (300 MHz, DMSO-d₆) δ 7.46 (4H, d, J ) 8.2

Hz, C_{3′′,5′′}-H), 7.92 (4H, d, J ) 8.2 Hz, C_{2′′,6′′}-H), 8.13 (4H, s,

C₂-H), 8.74 (2H, s, CHdN); ¹³C NMR (75 MHz, DMSO-d₆) δ

95.9 (C_1′′), 113.8 (CN), 122.5 (C_3′′,5′), 130.4 (C₂), 133.8 (C_{2′′,6′′}),

133.9 (C₁), 140.1 (C₄), 153.4 (CHdN); Anal. (C₂₂H₁₄N₄‚0.2H₂O)

C, H, N.

Syn th esis of Am in es. Bis-m eth yl 3-{[4-(Am in om eth yl)-

ben zyl]am in o}-4-m eth oxyben zoate (6a). Anhydrous metha-

nol (8 mL) was added slowly dropwise to sodium borohydride

(0.100 g, 2.6 mmol); effervescence occured. In a separate flask,

5c (0.200 g, 0.42 mmol) was dissolved in anhydrous dichlo-

romethane (8 mL) and added slowly dropwise to the main

reaction vessel. After stirring at room temperature for 12 h, a

precipitate formed, which was filtered and washed with 2 N

HCl (10 mL), water (10 mL), methanol (20 mL), and dichlo-

romethane (20 mL). Product 6a was obtained as a white solid

without the need for further purification (0.044 g, 0.095 mmol,

1

23%); mp 218-220 °C; H NMR (300 MHz, DMSO-d₆) δ 3.71

(6H, s, OMe), 3.88 (6H, s, COOMe), 4.30 (4H, d, J ) 5.9 Hz,

CH₂), 5.77 (2H, t, J ) 5.9 Hz, NH), 6.89 (4H, m, C_2′′-H + C_5′′-

H), 7.22 (6H, m, C_6′′-H + C₂-H). ¹³C could not be done

becaouse of a lack of solubility. ES⁺m/z 462.9 (M⁺); Anal.

(C₂₆H₂₈N₂O₆.0.7HCl) C, H, N.

4-((E)-2-{4-[(E)-2-(4-Hydr oxyph en yl)vin yl]ph en yl}vin yl)-

p h en ol (7b). An identical procedure to the one employed for

7a was used. 1,4-Divinylbenzene (0.240 g, 1.84 mmol), 4-io-

dophenol (0.811 g, 3.68 mmol), 10% palladium acetate (0.042

g, 0.184 mmol), and potassium carbonate (0.509 g, 3.68 mmol)

were reacted together in DMF (15 mL) and water (5 mL). After

work up, the crude was columned in a 1:1 ethyl acetate/hexane

mixture. Product 7b was isolated as a gray-green solid (0.025

g, 0.079 mmol, 4%); ¹H NMR (300 MHz, MeOD-d₄) δ 6.81 (4H,

d, J ) 8.6 Hz, C_{2′′,6′′}-H), 7.08 (2H, d, J ) 16.4 Hz, CHdCH),

7.33 (2H, d, J ) 16.4 Hz, CHdCH), 7.47 (4H, C₂-H), 7.88 (4H,

d, J ) 8.6 Hz, C_{3′′,5′′}-H); ¹³C NMR (75 MHz, DMSO-d₆) δ 117.0

(C_{2′′,5′′}), 125.7 (CHdCH), 128.0 (CHdCH), 129.9 (C₂), 130.1

(C_4′′), 131.6 (C_{3′′,5′′}), 133.9 (C₁), 159.7 (COH); ES^-m/z 312.7

(M^-); high-resolution ES⁺m/z found 315.1382 C₂₂H₁₈O₂(M +

H⁺) requires 315.1380.

Meth yl 4-Am in oben zoa te (8a ). Concentrated sulfuric acid

(7 mL) was added dropwise slowly to 4-aminobenzoic acid (10

g, 72.90 mmol) in methanol (150 mL). The reaction mixture

was heated to reflux for 36 h. The solution remaining was

evaporated in vacuo to dryness. Water (100 mL) was added,

and the pH of the solution was adjusted to 3 using a 2 M

solution of NaOH. The precipitate formed, was filtered, and

was washed with water (50 mL). Pure product 8a was

isolatedas an off-white solid (7.980 g, 52.80 mmol, 72%); mp

107-110 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 3.77 (3H, s,

OMe), 6.60 (2H, d, J ) 8.7 Hz, C_{3′′,5′′}-H), 7.68 (2H, d, J ) 8.7

Hz, C_{2′′,6′′}-H); ¹³C NMR (75 MHz, DMSO-d₆) δ 51.5 (OMe),

113.1 (C_{3′′,5′′}), 116.2 (C_1′′), 131.4 (C_{2′′,6′′}), 153.8 (C_4′′), 166.7 (COO);

ES⁺m/z 174.0 (M + Na⁺), 325.1 (2M + Na⁺).

Meth yl 4-Am in o-3-h yd r oxyben zoa te (8b). Concentrated

sulfuric acid (3.5 mL) was added dropwise slowly to 4-amino-

3-hydroxybenzoic acid (5.0 g, 32.65 mmol) in methanol (140

mL). The reaction mixture was heated to reflux for 36 h. The

solution remaining was evaporated in vacuo to dryness. Ice

water (125 mL) was added, and the pH of the solution was

adjusted to 5 using a 2 M NaOH solution. The brown

precipitate that was formed was filtered and washed with

water (50 mL). Product 8b was isolated as a brown solid (3.360

g, 20.10 mmol, 62%); mp 116-117 °C; ¹H NMR (300 MHz,

MeOD-d₄) δ 3.82 (3H, s, OMe), 6.67 (1H, d, J ) 8.20 Hz, C_6′′-

H), 7.32 (1H, d, J ) 1.89 Hz, C_2′′-H), 7.36 (1H, d, J ) 1.89

and 8.20 Hz, C_5′′-H); ¹³C NMR (75 MHz, MeOD-d₄) δ 52.4

Bis-m eth yl 4-{[4-(Am in om eth yl)ben zyl]a m in o}-3-h y-

d r oxyben zoa te (6b). An identical procedure to the one

employed for 6a was used. 5a (0.300 g, 0.693 mmol) in

dichloromethane (10 mL) was reacted with sodium borohydride

(0.158 g, 4.16 mmol) in methanol (5 mL). Water (20 mL) was

added to precipitate the product, which was washed further

with water (20 mL) and dichloromethane (20 mL). Product 6b

was isolated as a light-brown solid (0.148 g, 0.34 mmol, 49%);

mp 239-240 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 3.71 (6H, s,

OMe), 4.33 (4H, d, J ) 6.2 Hz, CH₂), 6.15 (2H, t, J ) 6.2 Hz,

NH), 6.36 (2H, d, J ) 8.7 Hz, C_5′′-H), 7.23 (8H, m, C₂-H +

C_2′′-H + C_6′′-H), 9.74 (2H, br, s, OH); ¹³C NMR (75 MHz,

DMSO-d₆) δ 45.7 (OMe), 51.5 (CH₂), 108.6 (C_5′′), 113.5 (C_2′′),

116.1 (C_1′′), 123.0 (C_6′), 127.3 (C₂), 138.5 (C_4′′), 142.2 (C₁), 143.5

(C_3′′), 166.7 (COO); ES⁺m/z 459.5 (100%, M + Na⁺); high-

resolution ES⁺m/z found 459.1538 C₂₄H₂₄N₂O₆Na (M + Na⁺)

requires 459.1532; Anal. (C₂₄H₂₄N₂O₆‚0.7H₂O) C, H, N.

4-[(4-{[(4-Ca r b oxy-2-h yd r oxyp h en yl)a m in o]m et h yl}-

ben zyl)a m in o]-3-h yd r oxyben zoic Acid (6c). A solution of

NaOH (0.070 g, 1.71 mmol) in water (6 mL) was added

dropwise to 6b (0.075 g, 0.171 mmol), and the reaction mixture

was stirred at room temperature for 45 min. HCl (8 mL, 2 N)

was added dropwise to the reaction mixture until a neutral

pH was achieved. A solid precipitated, which was filtered and

washed with water (20 mL). Product 6c was obtained as a

white solid without further need for purification (0.015 g, 0.037

mmol, 21%); mp >360 °C (decomp.); ¹H NMR (300 MHz,

DMSO-d₆) δ 4.34 (4H, br, s, CH₂), 6.08 (2H, br, s, NH), 6.38

(2H, br, s, C_5′′-H), 7.21-7.30 (8H, m, C₂-H + C_2′-H + C_6′-

H), 9.67 (2H, br, s, OH); ¹³C NMR (75 MHz, DMSO-d₆) δ 53.6

(CH₂), 115.9 (C_5′′), 118.1 (C_2′′), 119.6 (C_1′′), 122.4 (C_6′′), 129.8

(C₂), 132.1 (C_4′′), 140.6 (C₁), 143.6 (COH), 169.9 (COOH); Anal.

(C₂₂H₂₀N₂O₆‚0.8HCl) C, H, N.

2-[(4-{[(2-Hydr oxyph en yl)am in o]m eth yl}ben zyl)am in o]-

p h en ol (6d ). An identical procedure to the one employed for

6a was used. 5i (0.200 g, 0.63 mmol) was dissolved in

dichloromethane (10 mL) and reacted with sodium borohydride

(0.158 g, 4.16 mmol) in methanol (10 mL). After stirring for

12 h, dichloromethane (20 mL) was added, and the solution

was washed with 2 N HCl (20 mL) and saturated sodium

bicarbonate solution (20 mL). The organic phase was sepa-

rated, dried (MgSO₄), and evaporated in vacuo. The crude

product was then purified by column chromatography (1:1

Synthesis and Evaluation of Analogues of Congo Red

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22 5533

(OMe), 114.9 (C_5′′), 116.3 (C_2′′), 119.8 (C_6′′), 124.6 (C_1′′), 143.7

(COH), 145.2 (CNH₂), 169.8 (COO); ES⁺m/z 167.5 (M⁺), 189.9

(M + Na⁺).

Cell Cu ltu r e Assa ys. Cell Cu ltu r e. SMB cells were grown

in tissue culture-treated flasks in 199 medium (Gibco) supple-

mented with 10%(v/v) fetal calf serum, 5%(v/v) newborn calf

serum (Gibco), 100 U/mL penicillin, and 100 µg/mL strepto-

mycin at 37 °C and 5% CO₂.

Ack n ow led gm en t. We acknowledge the Medical

Research Council for funding and the EPSRC National

Mass Spectrometry Centre, Swansea for accurate mass

spectrometry.

Su p p or tin g In for m a tion Ava ila ble: Elemental analysis

data for all compounds. This material is available free of

charge via the Internet at http://pubs.acs.org.

Dr u g Tr ea tm en t. Drugs were resuspended in DMSO at a

stock concentration of 10 mM. To assess the effects of a drug,

we plated SMB cells at 50% confluency per well in 6-well

plates. Cells were left for 24 h to allow for attachment. The

media were then replaced with fresh media containing the

appropriate dilution of the 10 mM drug stock. Four days after

the addition of the drug, the media were removed and the

protein was extracted.

Refer en ces

(1) Bounias, M.; Purdey, M. Transmissible spongiform enceph-

alopathies: a family of etiologically complex diseasessa review.

Sci. Total Environ. 2002, 297, 1-19.

(2) Dormont, D. Prion diseases: pathogenesis and public health

concerns. FEBS Lett. 2002, 529, 17-21.

(3) Hill, A. F.; Desbruslais, M.; J oiner, S. The same prion strain

causes vCJ D and BSE. Nature 1997, 389, 448-450.

(4) Prusiner, S. B. Prions. Proc. Natl. Acad. Sci. U.S.A. 1998, 95,

13363-13383.

(5) Supattapone, S.; Nishina, K.; Rees, J . R. Pharmacological

approaches to prion research. Biochem. Pharmacol. 2002, 63,

1383-1388.

(6) Muller, W. E. G.; Laplanche, J . L.; Ushijima, H.; Schroder, H.

C. Novel approaches in diagnosis and therapy of Creutzfeldt-

J akob disease. Mech. Aging Dev. 2000, 116, 193-218.

(7) Gilbert, I. H.; Rudyk, H. Inhibitors of protease resistant prion

formation. Int. Antiviral News 1999, 7, 78-82.

(8) Koster, T.; Singh, K.; Zimmermann, M.; Gruys, E. Emerging

therapeutic agents for transmissible spongiform encephalopa-

thies: a review. J . Vet. Pharmacol. Ther. 2003, 26, 315-326.

(9) Farquhar, C.; Dickinson, A.; Bruce, M. Prophylactic potential

of pentosan polysulphate in transmissible spongiform enceph-

alopathies. Lancet 1999, 353, 117-117.

(10) Korth, C.; May, B. C. H.; Cohen, F. E.; Prusiner, S. B. Acridine

and phenothiazine derivatives as pharmacotherapeutics for

prion disease. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 9836-9841.

(11) May, B. C. H.; Fafarman, A. T.; Hong, S. B.; Rogers, M.; Deady,

L. W. et al. Potent inhibition of scrapie prion replication in

cultured cells by bis-acridines. Proc. Natl. Acad. Sci. U.S.A. 2003,

100, 3416-3421.

(12) Kocisko, D. A.; Baron, G. S.; Rubenstein, R.; Chen, J .; Kuizon,

S. et al. New inhibitors of scrapie-associated prion protein

formation in a library of 2000 drugs and natural products. J .

Virol. 2003, 77, 10288-10294.

(13) Kirby, L.; Birkett, C. R.; Rudyk, H.; Gilbert, I. H.; Hope, J . In

vitro cell-free conversion of bacterial recombinant PrP to Prp-

(res) as a model for conversion. J . Gen. Virol. 2003, 84, 1013-

1020.

(14) Rudyk, H.; Vasiljevic, S.; Hennion, R. M.; Birkett, C. R.; Hope,

J . et al. Screening Congo red and its analogues for their ability

to prevent the formation of PrP-res in scrapie-infected cells. J .

Gen. Virol. 2000, 81, 1155-1164.

(15) Rudyk, H.; Knaggs, M. H.; Vasiljevic, S.; Hope, J .; Birkett, C. et

al. Synthesis and evaluation of analogues of congo red as

potential compounds against transmissible spongiform. enceph-

alopathies. Eur. J . Med. Chem. 2003, 38, 567-579.

(16) Ingrosso, L.; Ladogana, A.; Pocchiari, M. Congo red prolongs the

incubation period in scrapie-infected hamsters. J . Virol. 1995,

69, 506-508.

(17) Demaimay, R.; Harper, J .; Gordon, H.; Weaver, D.; Chesebro,

B. et al. Structural aspects of Congo red as an inhibitor of

protease- resistant prion protein formation. J . Neurochem. 1998,

71, 2534-2541.

P r otein Extr a ction . Cells were lysed in PBS containing

1% Triton-X 100 and 1% Igepal CA-630 for 20 min at 37 °C.

Cell lysates were either placed on ice or treated with 80 µg/

mL proteinase k for 1 h at 37 °C. Proteins were concentrated

from the total cell lysate by methanol precipitation, and the

protein pellet was resuspended and denatured by boiling for

5 min in 8 M urea.

Wester n Blottin g. Samples were electrophoresed on a 12%

acrylamide gel and transferred electrophoretically to the PVDF

membrane (Immobilon-P, Millipore). PrP was detected using

the primary antibody DR1 as previously described³⁶and an

HRP conjugated secondary antibody (Dako). Specific protein

bands were visualized using ECL Plus chemiluminescent

reagent (Amersham Pharmacia Biotech) followed by autora-

diography. Autoradiographs were analyzed using Scion Image

densitometric software (Scion Corporation).

In Vitr o Aggr ega tion Assa y. All measurements were

performed on a Cary 100Bio UV-vis spectrophotometer at 325

nm using a quartz cuvette of 5-mm path length. The substrate

recombinant mouse PrP (rPrP) was refolded in the absence of

metal ions, and the seed for aggregation was aged manganese-

refolded recombinant mouse PrP (MnPrP) prepared as previ-

ously described.^29,37Briefly, a seed of MnPrP induces the

immediate aggregation of substrate rPrP, which is observed

as an increase in solution turbidity. The resultant scattering

of UV light at 325 nm results in an increased absorbance

measurement. The abilities of the potential anti-TSE com-

pounds to prevent this turbidity increase were measured, and

the results were expressed as a percentage of the turbidity

observed with a DMSO control. rPrP (50 µg) and anti-TSE

compound (100 µM) were preincubated in 500 µL of H₂O at

pH 6.5 for 30 min to provide a zero for the measurement.

MnPrP seed (10 µg) from a 400 µg mL^-1stock was added to

the drug/rPrP mixture, and an initial reading was obtained

immediately. A second reading was measured after 5 min, and

the increase in absorbance over 5 min was recorded. Time in

the spectrophotometer beam was minimized due to the sen-

sitivity to UV light of some Congo red derivatives.

(18) Klunk, W. E.; Debnath, M. L.; Koros, A. M. C.; Pettegrew, J . W.

Chrysamine-G, a lipophilic analogue of Congo red, inhibits A

beta-induced toxicity in PC12 cells. Life Sci. 1998, 63, 1807-

1814.

(19) Klunk, W. E.; Debnath, M. L.; Pettegrew, J . W. Development of

small-molecule probes for the beta-amyloid protein of alz-

heimers-disease. Neurobiol. Aging 1994, 15, 691-698.

(20) Gray, L. E.; Ostby, J . S. The effects of prenatal administration

of azo dyes on testicular development in the mousesa structure

activity profile of dyes derived from benzidine, dimethylbenzi-

dine, or dimethoxybenzidine. Fundam. Appl. Toxicol. 1993, 20,

177-183.

(21) Bos, R. P.; Koopman, J . P.; Theuws, J . L. G.; Henderson, P. T.

The essential role of the intestinal flora in the toxification of

orally-administered benzidine-based dyessinternal exposure of

rats to benzidine after intestinal azo reduction. Mutat. Res. 1987,

181, 327-327.

Toxicity Assa y. Cerebellar neuronal cultures were pre-

pared from 6-day-old mice 129SV. Cultures were prepared as

described previously.³⁸Briefly, the cerebella were dissociated

in Hanks’ media (Sigma) containing 0.5% trypsin (Sigma) and

plated at (1-2) × 10⁶cells/cm²in 24-well trays (Falcon) coated

with poly-^D-lysine (50 µg/mL, Sigma). Cultures were main-

tained in Dulbecco’s minimal essential media (Sigma) supple-

mented with 10% fetal calf serum, 2 mM glutamine, and 1%

antibiotics (penicillin, streptomycin) (Sigma). The cultures

were maintained at 37 °C with 5% CO₂. Compounds were

prepared in DMSO (Sigma). After 4 days of treatment, the

survival of the cerebellar cells was determined. MTT (3,[4,5

dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide, Sigma)

was diluted to 200 µM in Hanks’ solution and added to cultures

for 30 min at 37 °C. The MTT formazan product was released

from cells by the addition of dimethyl sulfoxide (Sigma) and

measured at 570 nm in a spectrophotometer (Bio50, Cary).

Relative survival in comparison to the control treated with the

DMSO vehicle could then be determined.

(22) Talaska, G. Aromatic amines and human urinary bladder

cancer: Exposure sources and epidemiology. J . Environ. Sci.

Health, Part C 2003, 21, 29-43.

(23) Demaimay, R.; Chesebro, B.; Caughey, B. Inhibition of formation

of protease-resistant prion protein by Trypan blue, Sirius red

and other Congo red analogues. Arch. Virol. 2000, 277-283.

5534 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22

Sellarajah et al.

(24) Clarke, M. C.; Haig, D. A. Evidence for the multiplication of

scrapie agent in cell culture. Nature 1970, 225, 100-101.

(25) Birkett, C. R.; Hennion, R. M.; Bembridge, D. A.; Clarke, M. C.;

Chree, A. et al. Scrapie strains maintain biological phenotypes

on propagation in a cell line in culture. EMBO J . 2001, 20,

3351-3358.

(26) Ziao, N.; Graton, J .; Laurence, C.; Le Questel, J .-Y. Amino and

cyano N atoms in competitive situations: which is the best

hydrogen bond acceptor? A crystallographic database investiga-

tion. Acta Crystallogr., Sect. B 2001, 57, 850-858.

(27) Link, C. D.; J ohnson, C. J .; Fonte, V.; Paupard, M. C.; Hall, D.

H. et al. Visualization of fibrillar amyloid deposits in living,

transgenic Caenorhabditis elegans animals using the sensitive

amyloid dye, X-34. Neurobiol. Aging 2001, 22, 217-226.

(28) Styren, S. C.; Hamilton, R. L.; Styren, G. C.; Klunk, W. K. X-34,

a fluorescent derivative of Congo red: A novel histochemical

stain for Alzheimer’s disease pathology. J . Histochem. Cytochem.

2000, 48, 1223-1232.

(29) Brown, D. R.; Hafiz, F.; Glasssmith, L. L.; Wong, B. S.; J ones, I.

M. et al. Consequences of manganese replacement of copper for

prion protein function and proteinase resistance. EMBO J . 2000,

19, 1180-1186.

(31) Masullo, C.; Macchi, G.; Xi, Y. G.; Pocchiari, M. Failure to

Ameliorate Creutzfeldt-J akob Disease with Amphotericin-B

Therapy. J . Infect. Dis. 1992, 165, 784-785.

(32) Barret, A.; Tagliavini, F.; Forloni, G.; Bate, C.; Salmona, M. et

al. Evaluation of quinacrine treatment for prion diseases. J .

Virol. 2003, 77, 8462-8469.

(33) Tagliavini, F.; McArthur, R. A.; Canciani, B.; Giaccone, G.; Porro,

M. et al. Effectiveness of anthracycline against experimental

prion disease in Syrian hamsters. Science 1997, 276, 1119-1122.

(34) Priola, S. A.; Raines, A.; Caughey, W. S. Porphyrin and phthalo-

cyanine antiscrapie compounds. Science 2000, 287, 1503-1506.

(35) Aguzzi, A.; Haass, C. Games played by rogue proteins in prion

disorders and Alzheimer’s disease. Science 2003, 302, 814-818.

(36) Brown, D. R. PrPSc-like prion protein peptide inhibits the

function of cellular prion protein. Biochem. J . 2000, 352, 511-

518.

(37) Wong, B. S.; Wang, H.; Brown, D. R.; J ones, I. M. Selective

oxidation of methionine residues in prion proteins. Biochem.

Biophys. Res. Commun. 1999, 259, 352-355.

(38) Brown, D. R. Prion protein peptide neurotoxicity can be mediated

by astrocytes. J . Neurochem. 1999, 73, 1105-1113.

(30) Demaimay, R.; Adjou, K. T.; Beringue, V.; Demart, S.; Lasmezas,

C. I. et al. Late treatment with polyene antibiotics can prolong

the survival time of scrapie-infected animals. J . Virol. 1997, 71,

9685-9689.

J M049922T

Article Doi

Synthesis of analogues of Congo red and evaluation of their anti-prion activity

DOI: 10.1021/jm049922t

Source and publish data:

Authors:

Article abstract of DOI:10.1021/jm049922t

Full text of DOI:10.1021/jm049922t

Products guided by the article

R&D Labs maybe for 796049-30-4

Relevant to this article

Hot Product