Synthesis of N-benzyl- and N-phenyl-2-amino-4,5-dihydrothiazoles and thioureas and evaluation as modulators of the isoforms of nitric oxide synthase

Bioorganic & Medicinal Chemistry 11 (2003) 4189–4206

Synthesis of N-Benzyl- and N-Phenyl-2-amino-4,5-

dihydrothiazoles and Thioureas and Evaluation as Modulators of

the Isoforms of Nitric Oxide Synthase

Claire L. M. Goodyer,^aEdwin C. Chinje,^bMohammed Jaﬀar,^bIan J. Stratford^b

and Michael D. Threadgill^a,*

^aDepartment of Pharmacy & Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, UK

^bMRC Experimental Oncology Laboratory, School of Pharmacy and Pharmaceutical Sciences, University of Manchester,

Oxford Road, Manchester M13 9PL, UK

Received 20 May 2003; accepted 2 July 2003

Abstract—Inhibition of the isoforms of nitric oxide synthase (NOS) has important applications in therapy of several diseases,

including cancer. Using 1400W [N-(3-aminomethylbenzyl)acetamidine], thiocitrulline and N^d-(4,5-dihydrothiazol-2-yl)ornithine as

lead compounds, series of N-benzyl- and N-phenyl-2-amino-4,5-dihydrothiazoles and thioureas were designed as inhibitors of NOS.

Ring-substituted benzyl and phenyl isothiocyanates were synthesised by condensation of the corresponding amines with thio-

phosgene and addition of ammonia gave the corresponding thioureas in high yields. The substituted 2-amino-4,5-dihydrothiazoles

were approached by two routes. Treatment of simple benzylamines with 2-methylthio-4,5-dihydrothiazole at 180 ^ꢀC aﬀorded the

corresponding 2-benzylamino-4,5-dihydrothiazoles. For less nucleophilic amines and those carrying more thermally labile sub-

stituents, the 4,5-dihydrothiazoles were approached by acid-catalysed cyclisation of N-(2-hydroxyethyl)thioureas. This cyclisation

was shown to proceed by an S_N2-like process. Modest inhibitory activity was shown by most of the thioureas and 4,5-dihy-

drothiazoles, with N-(3-aminomethylphenyl)thiourea (IC₅₀=13 mM vs rat neuronal NOS and IC₅₀=23 mM vs rat inducible NOS)

and 2-(3-aminomethylphenylamino)-4,5-dihydrothiazole (IC₅₀=13 mM vs rat neuronal NOS and IC₅₀=19 mM vs human inducible

NOS) being the most potent. Several thioureas and 4,5-dihydrothiazoles were found to stimulate the activity of human inducible

NOS in a time-dependent manner.

Introduction

Excessive NO production by eNOS within blood vessel

walls is thought to be the basis for conditions such as

septic- and cytokine-induced circulatory shock. In these

conditions, the sGc–cGMP pathway is excessively acti-

vated, which leads to high levels of NO and so con-

tributes to profound vasodilatation and hypotension.¹

Nitric oxide (^ꢁNO) is the smallest known messenger

molecule in biological systems. It is synthesised from

l-arginine 1 by the various isoforms of nitric oxide

synthase (NOS), yielding l-citrulline 3 as a co-product.

The process comprises two separate mono-oxygenation

steps with N^G-hydroxyarginine 2 as an intermediate

(Scheme 1). Both steps require molecular oxygen (O₂)

and NADPH. There are two main groups of isoforms of

NOS, a constitutive Ca²⁺/ calmodulin-dependent type

(cNOS) and an inducible Ca²⁺/ calmodulin-indepen-

dent form (iNOS). cNOS can be further sub-divided

into endothelial and neuronal forms (eNOS and nNOS,

respectively). Underactivity and overactivity of each of

these isoforms can be associated with disease states.

*Corresponding author. Tel.: +44-1225-386840; fax: +44-1225-

386114; e-mail: m.d.threadgill@bath.ac.uk

Scheme 1. Conversion of l-arginine 1 to l-citrulline 3 and nitric oxide,

via l-N^G-hydroxyarginine 2, mediated by nitric oxide synthases.

doi:10.1016/S0968-0896(03)00451-6

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However, if too little NO is produced, this can lead to

conditions such as high blood pressure, angina and

impotence. Recently, it has been shown that a reduction

in the activity and synthesis of NO within the endothe-

lium may contribute to the initiation and progression of

atherosclerosis.²Release of NO from the pelvic nerve

neurons located in the human corpus cavernosum is

known to cause penile erection. NOS inhibitors have

been shown to prevent this action while nitric oxide

sources mimic the eﬀect.³An overexpression of nNOS

in circulating neutrophils has been found in patients

with Parkinson’s disease⁴and nNOS activity is thought

to be linked to migraine headaches; these are believed to

result from abnormal activity in large cerebral blood

vessels and high levels of nNOS occur in the vasodilator

nerves that supply the large cerebral blood vessels.⁵NO

production by iNOS is essential for the defence

mechanism of an organism; however, NO produced by

iNOS has been related to several pathological condi-

tions, including cancer,⁶arthritis⁷and diabetes.⁸Thus

selective and potent inhibition of the isoforms of NOS is

an important goal in medicinal chemistry.

esters and dipeptides,¹⁴which are also NOS inhibitors,

the ligand is a nitroguanidine; imidazoles have also been

used as ligands for haem-Fe in our non-isoform-selec-

tive inhibitor 8¹⁵and related compounds.¹⁶Substitution

on the imidazole of 8 and replacement with less elec-

tron-rich heterocycles led to weaker inhibition,¹⁵which

is consistent with their weaker potential ligation to iron.

More recent highly iNOS-selective inhibitors^17ꢂ22con-

tain cyclic amidines (e.g. 9²²) to bind to the iron but lack

the amino-acid motif. Most interesting are the N-(3-

aminomethylbenzyl)acetamidine 1400W 10 (a highly

selective inhibitor of rat iNOS)²³and the lower homo-

logue 11 (a selective inhibitor of nNOS);²⁴this pair of

compounds illustrates exquisitely the possibility of

switching isoform selectivity radically through subtle

changes in the spatial relationship between the haem-

Fe-binding group and remote functionalities. Sulfur-

containing groups have also been used as the ligands for

iron. The simplest such compound, thiocitrulline 12 is

relatively unselective,²⁵as is our potent inhibitor 13²⁶

and N-aryl-S-alkylisothioureas such as 14.²⁷The tetra-

hydrobiopterin binding site has also received some

attention in the development of isoform-selective inhi-

bitors.^28,297-Nitroindazole 15, which is reported to be

competitive with both the substrate 1 and with tetra-

hydrobiopterin, is claimed³⁰to be moderately selective

for inhibition of the nNOS isoform.

The structures of several reported inhibitors of NOS are

shown in Figure 1. Many of the early inhibitors, for

example l-NMMA 4,⁹are close analogues of the sub-

strate l-arginine 1. Isosteric replacement of the terminal

guanidine of 1 with an acetamidine gives the non-selec-

tive l-NIO 5a.¹⁰The homologue l-NIL 5b,¹¹however,

shows some selectivity for inhibition of iNOS. This

selectivity (iNOS vs eNOS) increases when the carbox-

ylate of 5b is replaced by the diol motif in 6¹²but iNOS

vs nNOS selectivity is poor. In each of these inhibitors,

a binding motif can be recognised in which the guani-

dine/amidine ligates to the haem iron at the active site

of the enzyme, while additional binding contacts recog-

nise the amine, the carboxylate and, possibly, the N–H

near the haem ligand. In N^G-nitroarginine 7¹³and its

In the present study, we sought to explore the inhibitory

activity of hybrids between the highly selective 10 and

11 and our potent dihydrothiazole 13. The structures of

the designed target compounds are shown in Figure 2.

In particular, we sought to explore whether the isoform

selectivity shown by 10 and 11 could be translated into

analogous compounds carrying diﬀerent haem-ligating

head groups. We report here the synthesis of these series

of compounds and their evaluation as inhibitors of nNOS

and iNOS. Although Collins et al.²⁴note particularly the

Figure 1. Structures of known inhibitors of the isoforms of NOS.

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4191

previously developed method,²⁶the benzylamines 21

were treated with thiophosgene in the presence of cal-

cium carbonate as an insoluble mild base in a mixed

organic/aqueous solvent system. The corresponding

isothiocyanates 22c–i,k were obtained in yields ranging

from 37 to 83%. Simple treatment of 22c–i,k with

ammonia gave the N-benzylthioureas 16c–i,k in good to

excellent yields. In some cases, deprotection or further

modiﬁcation of the substituent was necessary. Acid-

olysis removed the Boc protecting groups from 16c,d to

give the analogous N-(aminomethyl)benzylthioureas

16a,b as their bis(triﬂuoroacetate) salts. Selective reduc-

tion of the nitro groups of 16e,f with tin(II) chloride

provided the aminobenzylthioureas 16l,m, the lower

homologues of 16a,b. Hydrolysis of the protecting esters

in 16i,k with hydrobromic acid aﬀorded the required

carboxylic acids 16n,j, respectively.

Figure 2. Structures of target N-benzyl and N-phenyl thioureas and

2-amino-4,5-dihydrothiazoles.

2-Amino-4,5-dihydrothiazoles can be prepared by at

least three independent routes. Firstly, the dihy-

drothiazole can be introduced as a single unit. Stokker

et al.³¹used this approach in synthesising the corre-

sponding 2-benzylamino-4,5-dihydrothiazole by treat-

ment of 5-t-butyl-2-hydroxy-3-iodobenzylamine with

2-methylthio-4,5-dihydrothiazole 26 in boiling ethanol.

Other workers³²have used similar conditions for reac-

tions of primary amines with this electrophilic dihy-

drothiazole synthon. However, Hirashima et al.³³noted

that the reaction of 26 with substituted 2-phenylethyl-

amines gave only low yields of 2-amino-4,5-dihy-

drothiazoles, even under forcing conditions in boiling

pentan-1-ol. In our laboratory, 3-methoxybenzylamine

21g and 4-methoxybenzylamine 21h failed to react with

26 in boiling ethanol and the condensation required

strongly forcing conditions, heating the amines 21g,h

with 26 in the absence of solvent at 180 ^ꢀC for 4 h, to

achieve even moderate yields of the 2-(methox-

ybenzylamino)-4,5-dihydrothiazoles 17g,h, respectively.

Under these conditions, substituents more sensitive than

methoxy would not be expected to survive; indeed,

treatment of the mono-Boc-protected diamine 21c with

neat 26 at this temperature led only to unidentiﬁable

degradation products. Secondly, double alkylation of

N-substituted thioureas with 1,2-dibromoethane gives

2-alkylamino-4,5-dihydrothiazoles but the yields

achievable by this method are usually poor to modest.²⁶

Thirdly, Caujolle et al. reported³⁴introduction of the

CH₂CH₂unit in two steps, reaction of the iso-

thiocyanate with 2-aminoethanol to give the N-(2-

hydroxyethyl)thiourea and acid-catalysed cyclisation.

Hence, as shown in Scheme 3, the benzylisothiocyanates

22c–k were treated with 2-aminoethanol in boiling ace-

tone to give the corresponding N-benzylthioureas 24c–k

in satisfactory yields. In the cases of isothiocyanates

carrying strong electron-withdrawing groups on the

benzene ring, low yields of the 3-(benzylaminothio-

carbonyl)-2,2-dimethyltetrahydro-1,3-oxazoles 25e,f,k

were also formed. These products of formal condensa-

tion with the solvent are unlikely to have arisen from

reaction of the hydroxyethylthioureas with acetone,

since this process should not be sensitive to the sub-

stitution on the benzene ring in the manner observed. It

is more likely that 2,2-dimethyltetrahydro-1,3-oxazole is

importance of the 3-aminomethyl group for binding of

10 and 11 to NOS isoforms, we used a range of diﬀerent

substituents on the benzene ring to test whether this

substitution is still optimal when the ligand for the haem

iron is sulfur, rather than nitrogen.

Chemical Synthesis

The synthesis of the ﬁrst series of target compounds, the

N-benzylthioureas is shown in Scheme 2. The primary

strategy was to add ammonia to the electrophilic iso-

thiocyanate unit in the benzylisothiocyanates 22. A

common route was developed to prepare these from the

corresponding benzylamines 21. However, certain addi-

tional substituents on the benzyl group required prior

protection. 1,3-Bis(aminomethyl)benzene 21a was trea-

ted carefully with a sub-stoichiometric amount of di-

tert-butyl dicarbonate to give, after a simple aqueous

workup to remove excess 21a, a high yield of the crys-

talline mono-Boc derivative 21c. Similar treatment of

the analogous para diamine 21b furnished the corre-

sponding mono-Boc derivative 21d. To protect the car-

boxylic acid in the 4-aminomethylbenzoic acid 21j, the

methyl ester 21k was formed in the usual way by reac-

tion with acidic methanol. However, the corresponding

3-aminomethylbenzoic acid is not commercially avail-

able and the corresponding methyl ester 21i had to be

synthesised by an alternative route. Using a nitrile as a

synthon for the aminomethyl unit, 3-cyanobenzoic acid

20n was converted to its methyl ester 20i; the relatively

modest yield of 42% may have been due to competing

Pinner reaction of the nitrile with the acidic methanol.

Hydrogenation of 20i gave two products, the required

simple reduction product 21i and the secondary amine

23. The latter is formed by transimination of the inter-

mediate iminomethylbenzene with the primary amine

21i and subsequent hydrogenation of the new imine.

The 3-nitro-, 4-nitro- 3-methoxy- and 4-methox-

ybenzylamines 21e–h, respectively, are commercially

available;

stituted)benzylamines 21 was now in place. Using our

thus

the

set

of

(protected/sub-

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formed by reversible condensation of 2-aminoethanol

with acetone; this heterocycle is a sterically hindered

nucleophile at nitrogen (owing to the adjacent gem-

dimethyl) and reacts only with the more electrophilic

isothiocyanates.

benzylamino)-4,5-dihydrothiazole 17n. Since these

cyclisation conditions are relatively forcing and cleavage

of sensitive substituents is a risk, a milder cyclisation

was developed. N-(2-Hydroxyethyl)-N⁰-(3-methoxy-

benzyl)thiourea 24g was treated with neat triﬂuoroacetic

1

acid at ambient temperature for 2 h; direct H NMR

Treatment of the isomeric N-(2-hydroxyethyl)thioureas

24c,d with boiling hydrochloric acid for prolonged per-

iods eﬃciently closed the dihydrothiazole rings and

simultaneously removed the Boc protection, giving

17a,b in satisfactory yields as their dihydrochloride

salts. Similar treatment of the para-substituted ester 24k

again closed the dihydrothiazole ring and hydrolysed

the ester protection to aﬀord the target carboxylic acid

17j. However, application of this method to the meta

isomer 24i gave incomplete ester hydrolysis; the product

mixture was re-esteriﬁed to give 17i for puriﬁcation

before acid-catalysed hydrolytic deprotection with aqu-

eous triﬂuoroacetic acid to give the 2-(3-carboxy-

analysis showed that cyclisation to 17g was complete.

Cyclisation of the nitrobenzyl analogues 24e,f to 17e,f

with triﬂuoroacetic acid required longer reaction time.

Finally, the nitrobenzylaminodihydrothiazoles were

reduced to the corresponding aminobenzyl com-

pounds 17l,m with tin(II) chloride under neutral con-

ditions.

The preparations of the lower homologues, the N-phe-

nylthioureas 18 and the 2-phenylamino-4,5-dihy-

drothiazoles 19, followed sequences similar to those for

the benzyl compounds above, although a smaller range

of substituents was investigated (Scheme 4). The

Scheme 2. Synthesis of N-benzylthioureas 16. Reagents and conditions: (i) MeOH, SOCl₂, 4 d; (ii) MeOH, H₂, Pd/C, 16 h; (iii) Boc₂O (0.3 equiv),

Et₃N, CH₂Cl₂, 16 h; (iv) CSCl₂, CaCO₃, H₂O, CHCl₃, 16 h; (v) NH₃, CH₂Cl₂, 3.5 h, 0 ^ꢀC; (vi) CF₃CO₂H, 5 min; (vii) SnCl₂, EtOH, reﬂux, 1 h; (viii)

aq. HBr (50%), 16 h.

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4193

commercially available methoxyphenylisothiocyanates

29g,h reacted with ammonia to give the corresponding

thioureas 18g,h. Protection of the aliphatic amine

nitrogens in the nitrobenzylamines 21e,f with Boc (giv-

ing 27c,d) was followed by reduction of the nitro group

with tin(II) chloride (under neutral conditions to avoid

deprotection) aﬀorded the Boc-amino-anilines 28c,d.

The methyl esters 28q,r were formed by treatment of the

aminophenylacetic acids 28o,p with methanol and thio-

nyl chloride. As in the benzylamine series, amines

28c,d,q,r were converted eﬃciently to the iso-

thiocyanates 29c,d,q,r with thiophosgene. Again, these

reacted with ammonia to furnish the thioureas 18c,d,q,r.

Acidolytic deprotection removed the Boc groups, giving

the aminomethylphenylthioureas 18a,b as their tri-

ﬂuoroacetate salts, whereas hydrolysis with aqueous

acid yielded the carboxymethylthioureas 18o,p. As

shown in Scheme 5, the same set of phenylisothiocyan-

ates 29c,d,g,h,q,r was used to prepare the N-(2-hydroxy-

ethyl)thioureas 30c,d,g,h, q,r; in contrast to the benzyl

series, there was no evidence of formation of the acetone

adducts (the 2,2-dimethyltetrahydrooxazoles). The con-

ditions for the cyclisations were selected according to

the type of simultaneous deprotection also required.

Cyclisation/deprotection of 30c was eﬀected by both the

boiling hydrochloric acid and the triﬂuoroacetic acid

methods, giving 19aA and 19aB, respectively. The para-

substituted isomer 19b was prepared by the tri-

ﬂuoroacetic acid method only, as were the two 2-

(methoxyphenylamino)-4,5-dihydrothiazoles

19g,h.

Boiling hydrochloric acid ring-closed and deprotected

the esters 30q,r, giving the 2-(carboxymethyl-

phenylamino)-4,5-dihydrothiazoles 19o,p, respectively,

in good yield.

Scheme 3. Synthesis of N-benzyl-N⁰-(2-hydroxyethyl)thioureas 24 and 2-(benzylamino)-4,5-dihydrothiazoles 17. Reagents and conditions: (i)

H₂N(CH₂)₂OH, acetone, reﬂux 4 h; (ii) aq HCl (6 M), reﬂux, 36 h (17a,b,j); (iii) CF₃CO₂H, 16 h (17e); iv, CF₃CO₂H, reﬂux, 15 h (17f); (v) CF₃CO₂H,

2 h (17g); (vi) aq HCl (6 M), reﬂux, 40 h, then MeOH, SOCl₂, 4 d (17i); (vii) aq CF₃CO₂H (50%), reﬂux, 16 h (17n); (viii) SnCl₂, EtOH, reﬂux, 1 h;

(ix) SnCl₂, EtOH, reﬂux 1.5 h; (x) 26, 180 ^ꢀC, 4 h.

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Scheme 4. Synthesis of N-phenylthioureas 18. Reagents and conditions: (i) Boc₂O, Et₃N, CH₂Cl₂, 16 h; (ii) SnCl₂, EtOH, reﬂux, 30 min (28c);

(iii) SnCl₂, EtOH, reﬂux, 1 h (28d); (iv) MeOH, SOCl₂, 4 d; (v) CSCl₂, CaCO₃, H₂O, CHCl₃, 2 h (29c); (vi) CSCl₂, CaCO₃, H₂O, CHCl₃, 16 h

(29d,q,r); (vii) NH₃, CH₂Cl₂, 3.5 h; (viii) CF₃CO₂H, 5 min; (ix) CF₃CO₂H, 2 h; (x) aq HCl (1 M), 9 d; (xi) aq HCl (6 M), 16 h.

Scheme 5. Synthesis of N-phenyl-N⁰-(2-hydroxyethyl)thioureas 30 and 2-(phenylamino)-4,5-dihydrothiazoles 19. Reagents and conditions: (i)

H₂N(CH₂)₂OH, acetone, reﬂux, 2 h (30c,g); (ii) H₂N(CH₂)₂OH, acetone, reﬂux, 4 h (30d,q,r); (iii) H₂N(CH₂)₂OH, acetone, reﬂux, 1.5 h (30h); (iv) aq

HCl (6 M), reﬂux, 43 h (19aA); (v) CF₃CO₂H, 5 min (19aB); (vi) CF₃CO₂H, 2 h (19b); (vii) CF₃CO₂H, 3 h (19g); (viii) CF₃CO₂H, reﬂux, 15 h (19h);

(ix) aq HCl (6 M), reﬂux, 36 h (19o); (x) aq HCl (6 M), 16 h.

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the enzymic reaction by addition of [¹⁴C]-arginine. This

pre-incubation has been reported to be optimum for the

inhibitory activity of 1400W 11, which is a slow-binding

selective inhibitor of iNOS;²³this pre-incubation was

investigated since many of the test compounds can be

considered to be analogues of 11 and may also be slow-

binding. We have also noted³⁵that an amino-acid-type

inhibitor, N^e-homothiocitrulline methyl ester requires at

least 5 min pre-incubation with rat nNOS to exert its

full inhibitory potency. IC₅₀values were determined

(with 15 min pre-incubation) for compounds 18a,b and

19a, which showed consistent activity in the general

screen. Compounds 16a,h, 17a, 18a and 19a were also

evaluated at 100 mM for their inhibition of rat iNOS,

without pre-incubation.

The results of the biological evaluation of the test com-

pounds are shown in Table 1. Pre-incubation of the

compounds with the rat nNOS preparation has little or

no eﬀect on the inhibition of this isoform by most of the

compounds. Many of the compounds (16b,g,h,j,l–n,

17g,h, j,l–n, 18g,h,o,p, 19g,h,o,p, 24g,h and 30g,h) are

inactive or have only weak activity against this isoform.

Signiﬁcant activity was shown by all the new com-

pounds carrying the aminomethyl group on the benzene

ring, consistent with the view of Collins et al.²⁴that this

group is optimal in the amidine series of inhibitors, for

example 10 and 11. Thus inhibitory activity was shown

by 16a and by 17a, the N-benzylthiourea and the 2-

benzylamino-4,5-dihydrothiazole most closely related to

1400W 11 with an aminomethyl group located meta on

the benzene ring. The corresponding para-substituted

analogues 16b and 17b, respectively, were less potent.

The N-(aminomethylphenyl)thioureas 18a,b and the 2-

(aminomethylphenylamino)-4,5-dihydrothiazoles 19a,b

were also active. Again, the meta substituted isomers

were more potent than the corresponding para com-

pounds, in that 18a had IC₅₀=13 mM, whereas 18b had

IC₅₀=41 mM, when assayed with 15-min pre-incu-

bation. Interestingly, 7-nitroindazole 15, which is

claimed to be selective for nNOS inhibition,³⁰showed

IC₅₀=40 mM in this system, making compounds 18a

and 19a more potent than this lead compound in our

nNOS system and similar in potency to l-NMMA 4,

1400W 10 and thiocitrulline 12.

Scheme 6. Stereochemical outcome of acid-catalysed cyclisation to

form the 4,5-dihydrothiazole. Reagents and conditions: (i) R-1-amino-

propan-2-ol, acetone, reﬂux, 2 h; (ii) aq HCl (6 M), reﬂux, 24 h.

To investigate brieﬂy the mechanism of the acid-cata-

lysed ring-closure of the 2-hydroxyethylthioureas, the

homochiral R-N-(2-hydroxypropyl)thiourea 31 was syn-

thesised by treatment of 3-methoxyphenylisothiocyanate

29g with R-1-aminopropan-2-ol (Scheme 6). Cyclisation

with boiling hydrochloric acid gave the corresponding 2-

(3-methoxyphenylamino)-5-methyl-4,5-dihydrothiazole

20

D

32, with speciﬁc rotation ½a ¼ ꢂ32:4^ꢀ. Since the race-

mate has not been formed, the cyclisation proceeds, at

least in part, by an S_N2-like process.

Biological Evaluation

The N-benzylthioureas 16, the 2-benzylamino-4,5-dihy-

drothiazoles 17, the N-phenylthioureas 18, the 2-

phenylamino-4,5-dihydrothiazoles 19 and selected N-(2-

hydroxyethyl)thioureas 24 and 30 were evaluated for

their inhibition of the activities of the isoforms of NOS,

generally according to the method published pre-

viously.¹⁵The known inhibitors l-NMMA 4, 1400W

10, thiocitrulline 12 and 7-nitroindazole 15 were also

subjected to the test system, for comparison. Inhibition

of the activity of nNOS was measured using an enzyme

preparation from rat brain (in which the large majority

of the NOS activity is nNOS), whereas most of the

assays of activity against iNOS were performed using a

preparation of recombinant human iNOS overexpressed

in an HT1080 cell line.³⁵Selected compounds were also

evaluated for inhibition of iNOS as a crude preparation

from the lungs of rats previously treated with lipopoly-

saccharide (LPS). The assays were based on the con-

version of [¹⁴C]-arginine to [¹⁴C]-citrulline. As a screen

for inhibitory activity, all compounds were tested at

100 mM concentration against human iNOS and rat

nNOS. Assays were performed in two modes, simulta-

neous addition of the test compound to the enzyme

preparation and of [¹⁴C]-arginine (to start the enzymic

reaction) and pre-incubation of the test compound with

the enzyme preparation for 10 min before initiation of

In contrast with the results for nNOS, pre-incubation of

the test compounds with the human iNOS enzyme pre-

paration had a profound eﬀect on the inhibition. The

inhibition caused by 16g,h, 17a,g,h, 18g and 19g

appeared to decrease to a greater or lesser extent with

pre-incubation, although these compounds had only

moderate potency. In contrast, the inhibition of human

iNOS by the most potent compound, 19a, increased

with pre-incubation, suggesting that slow binding may

be involved. This eﬀect was also observed with the

weaker inhibitor 19b. The most potent inhibitor of

human iNOS was the meta-aminomethylphenylamino-

4,5-dihydrothiazole 19a, with IC₅₀=19 mM. Interest-

ingly, the corresponding thiourea 18a was much less

potent, with IC₅₀=260 mM. Again, 19a, was more

potent in this system than 7-nitroindazole 15 but less

potent than the other ‘standard’ inhibitors 4, 10 and 12.

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Most striking, however, was the strong stimulation of

the activity of human iNOS by 16j,l,m,n, 17j,m, 18o, 19p

and 24g. In each case, pre-incubation abolished this sti-

mulation and, in some cases, led to weak inhibition.

Compounds 17l,n and 24h were weaker stimulators

(with no pre-incubation) but switched to become sig-

niﬁcant inhibitors when pre-incubated with the enzyme

preparation for 10 min before initiation of the enzyme-

catalysed generation of nitric oxide. We have previously

noted¹⁵the phenomenon of stimulation of rat iNOS

activity by S-2-amino-5-(3-nitrotriazol-1-yl)pentanoic

acid and by S-2-amino-5-(3-aminotriazol-1-yl)pentanoic

acid, although the eﬀects were much weaker. A struc-

ture–activity tendency is evident for this stimulatory

eﬀect. The most eﬀective stimulators in the benzyl series

16, 17 carry either amines or carboxylic acids attached

directly to the benzene ring. In the phenyl series, only

18o and 19p are stimulatory; these carry CH₂CO₂H as

substituents on the benzene ring, giving the same dis-

tance between the carboxylate and the sulfur as in their

stimulatory isomers 16j and 16n, respectively. The

molecular origin of this stimulatory eﬀect remains

unclear, although it is consistent with a model in which

there are two binding sites for these compounds, the

substrate (arginine)-binding site at the catalytic centre

and a (possibly remote) allosteric site. To rationalise the

data in terms of this model, the binding of the com-

pounds to the arginine site would be inhibitory (and

competitive with arginine) and binding to the allosteric

site would be stimulatory, possibly through inducing a

conformational change in the enzyme protein. To ﬁt the

observed dependence on pre-incubation, binding to

the allosteric site would be fast (leading to stimula-

tion of nitric oxide synthesis without pre-incubation),

whereas inhibitory binding to the substrate-binding

site would be slow. Then the overall eﬀect observed

after pre-incubation would be the sum of the stimu-

latory and inhibitory eﬀects (leading to apparent

diminution of stimulation or switch to apparent inhi-

bition).

Table 1. Inhibition of human iNOS, rat iNOS and rat nNOS by the thioureas 16,18, the N-(2-hydroxyethyl)thioureas 24, 30, the 4,5-dihy-

drothiazoles 17, 19 and, for comparison, by the known inhibitors l-NMMA 4, 1400W 10, thiocitrulline 12 and 7-nitroindazole 15

Compd

% inhibition (human iNOS)^a

10 min

% inhibition (rat iNOS)^a

% inhibition (rat nNOS)^a,b

No

IC₅₀

(15 min

No

pre-incubation^b

No

10 min

IC₅₀(15 min

pre-incubation^bpre-incubation^b

pre-incubation^bpre-incubation^bpre-incubation)^b

pre-incubation)^b

(mM)

4

10

12

15

<4

<5

24

94ꢃ1

96ꢃ1

9

12

17

40

79ꢃ1

82ꢃ1

77ꢃ2

34ꢃ1

68ꢃ2

44ꢃ2

32ꢃ4

7ꢃ3

59ꢃ1

39ꢃ5

74ꢃ3

47ꢃ1

20ꢃ2

6ꢃ4

16a

16b

16g

16h

16j

16l

16m

16n

17a

17b

17g

17h

17j

17l

17m

17n

18a

23ꢃ2

5ꢃ3

29ꢃ5

11ꢃ6

35ꢃ2

ꢂ3ꢃ1

8ꢃ1

26ꢃ6

2ꢃ5

14ꢃ1

6ꢃ1

ꢂ56ꢃ12

ꢂ58ꢃ1

ꢂ55ꢃ11

ꢂ46ꢃ5

48ꢃ1

3ꢃ1

ꢂ0.4ꢃ0.4

ꢂ1ꢃ6

ꢂ0.3ꢃ1

14ꢃ1

9ꢃ1

9ꢃ6

12ꢃ1

7ꢃ2

12ꢃ2

ꢂ0.4ꢃ2

59ꢃ1

65ꢃ2

14ꢃ1

13ꢃ1

ꢂ11ꢃ1

17ꢃ5

12ꢃ1

13ꢃ3

67ꢃ1

26ꢃ3

51ꢃ2

8ꢃ4

5ꢃ3

55ꢃ6

ꢂ30ꢃ2

34ꢃ4

33ꢃ3

8ꢃ1

26ꢃ6

3ꢃ13

5ꢃ1

10ꢃ2

ꢂ45ꢃ1

ꢂ36ꢃ1

ꢂ47ꢃ10

ꢂ39ꢃ1

35ꢃ4

0.4ꢃ4

5ꢃ2

12ꢃ2

19ꢃ2

34ꢃ3

40ꢃ5

8ꢃ2

5ꢃ3

260

89

98ꢃ1

98ꢃ3

13

41

(IC₅₀=23 mM)

(IC₅₀=10 mM)

48ꢃ4

18b

18g

18h

18o

18p

19a

57ꢃ1

17ꢃ6

52ꢃ1

2ꢃ1

44ꢃ1

5ꢃ1

ꢂ4ꢃ1

ꢂ1ꢃ3

9ꢃ6

26ꢃ7

28ꢃ1

ꢂ3ꢃ14

13ꢃ3

86ꢃ1

4ꢃ2

ꢂ65ꢃ5

15ꢃ2

5ꢃ2

ꢂ7ꢃ8

66ꢃ1

15ꢃ6

89ꢃ1

62ꢃ2

19

34ꢃ1

13

(IC₅₀=190 mM)

(IC₅₀=21 mM)

56ꢃ1

19b

19g

19h

19o

19p

24g

24h

30g

30h

16ꢃ4

26ꢃ2

40ꢃ1

ꢂ1ꢃ12

7ꢃ3

56ꢃ1

20ꢃ2

17ꢃ1

9ꢃ3

7ꢃ1

1ꢃ1

ꢂ2ꢃ1

4ꢃ2

ꢂ1ꢃ4

ꢂ58ꢃ4

ꢂ58ꢃ7

ꢂ25ꢃ1

ꢂ10ꢃ1

6ꢃ3

ꢂ8ꢃ14

9ꢃ2

14ꢃ5

5ꢃ1

8ꢃ1

ꢂ1ꢃ4

ꢂ1ꢃ1

0ꢃ0.6

2ꢃ1

44ꢃ5

ꢂ1ꢃ1

6ꢃ2

5ꢃ1

1ꢃ1

2ꢃ3

^aConcentration of test compound 100 mM.

^bPre-incubation refers to the time between addition of the test compound and addition of [¹⁴C]-arginine to initiate the enzymic reaction.

C. L. M. Goodyer et al. / Bioorg. Med. Chem. 11 (2003) 4189–4206

4197

Examination of the activity of the most potent inhibi-

tors of the activities of the isoforms of NOS reveals

moderate selectivity. Interestingly, the claimed³⁰nNOS-

selective inhibitor 15 showed no selectivity for rat NOS

over human iNOS, although this may have been a spe-

cies eﬀect. Whereas 2-(3-aminomethylphenylamino)-4,5-

dihydrothiazole 19a was the most potent inhibitor of

human iNOS (IC₅₀=19 mM after 15 min pre-incu-

bation), it was similarly active against rat nNOS

(IC₅₀=13 mM after 15 min pre-incubation); interest-

ingly, it was much less potent against rat iNOS

(IC₅₀=190 mM with no pre-incubation) than against rat

nNOS (IC₅₀=21 mM with no pre-incubation). A diﬀer-

ent species eﬀect was seen with the corresponding N-(3-

aminomethylphenyl)thiourea 18a. Here, little selectivity

was seen between inhibition of rat iNOS (IC₅₀=23 mM

with no pre-incubation) and inhibition of rat nNOS

(IC₅₀=10 mM with no pre-incubation) but the com-

pound was 20-fold selective for inhibition of rat nNOS

(IC₅₀=13 mM with 15-min pre-incubation) over inhibi-

tion of human iNOS (IC₅₀=260 mM with 15-min pre-

incubation). The para isomer 18b, however, showed

only 2-fold selectivity in the same (rat nNOS vs human

iNOS) comparison.

mechanistic origin of this stimulation, which may have

experimental or therapeutic applications, will be the

subject of intense further study.

Experimental

NMR data were recorded on either JEOL/Varian GX

270 or EX 400 spectrometers, using solutions in CDCl₃,

unless otherwise stated. IR spectra were recorded on

samples as KBr discs, unless otherwise stated. Mass

Spectra were recorded using a VG Analytical Mass

Spectrometer in the FAB positive ion mode, unless

otherwise stated. Solutions in organic solvents were

dried with MgSO₄and solvents were evaporated under

reduced pressure. Experiments were conducted at

ambient temperature, unless otherwise stated. Melting

points were determined using a Reichert-Jung Thermo

Galen Koﬂer block.

N-(3-(Aminomethyl)phenylmethyl)thiourea bis(triﬂuoro-

acetate) salt (16a). Compound 16c (100 mg, 300 mmol)

was stirred in CF₃CO₂H (3 mL) for 5 min. Evaporation

gave 16a (140 mg, 99%) as a colourless hygroscopic

gum: IR (ﬁlm) n_max1172, 1780, 3200 cm^ꢂ1; NMR

((CD₃)₂SO) d_H4.00 (2H, m, CH₂N⁺H₃), 4.65 (2H, m,

CH₂NH), 7.15 (3H, br, N⁺H₃), 7.32 (4H, m, Ar-H₄),

8.10 (4H, br, NH+N⁺H₃); MS m/z 196.0905 (M+H)

(C₉H₁₄N₃S requires 196.0908), 179 (MꢂNH₃), 162

(Mꢂ2 ꢄ NH₃).

Conclusions

In this paper, we have described our design of novel

inhibitors of the isoforms of NOS, replacing the ami-

dines in the highly isoform-selective inhibitors 1400W

11 and its lower homologue 12 with thiourea and 4,5-

dihydrothiazole units for ligation to the haem iron; this

replacement was based on the potent activity of thioci-

trulline 13 and of the analogous dihydrothiazole 14. The

eﬀect of varying the nature and position of the sub-

stituents on the aryl ring were also examined. The

thioureas were synthesised in good yields by reaction of

the corresponding isothiocyanates with ammonia,

whereas the 2-(substituted amino)-4,5-dihydrothiazoles

were prepared by two routes, reaction of benzylamines

with 2-methylthio-4,5-dihydrothiazole under forcing

conditions or acid-catalysed cyclisation of N-(2-hydroxy-

ethyl)thioureas.

N-(4-(Aminomethyl)phenylmethyl)thiourea bis(triﬂuoro-

acetate) salt (16b). Compound 16d was treated with

CF₃CO₂H, as for the synthesis of 16a (reaction time

2 h), to give 16b (99%) as white crystals: mp 197–199 ^ꢀC;

IR n_max1188, 3293 cm^ꢂ1; NMR ((CD₃)₂SO) d_H3.99

(4H, s, 2 ꢄ CH₂), 4.64 (3H, br, NH₃), 7.29 (1H, br,

NH), 7.30 (2H, d, J=8.0 Hz, Ar 3,5-H₂), 7.37 (2H, d,

J=8.0 Hz, Ar 2,6-H₂), 8.16 (3H, br, N⁺H₃); MS m/z

196.0908 (M+H) (C₉H₁₄N₃S requires 196.0920), 179

(MꢂNH₃).

1,1-Dimethylethyl N-(3-(thioureidomethyl)phenylmethyl)-

carbamate (16c). NH₃was passed through 22c (400 mg,

1.5 mmol) in CH₂Cl₂(40 mL) for 30 min. The mixture

was stirred for 3 h at 0 ^ꢀC. Evaporation and chromato-

graphy (EtOAc/hexane 4:1) gave 16c (300 mg, 72%) as

white crystals: mp 70–72 ^ꢀC; IR n_max1164, 1608,

3308 cm^ꢂ1; NMR d_H1.40 (9H, s, Bu^t), 4.18 (2H, br,

CH₂), 4.62 (2H, br, CH₂), 5.30 (1H, br, NH), 6.01 (2H,

br, NH₂), 7.21 (5H, m, Ar-H₄+NH); MS m/z 296.1426

(M+H) (C₁₄H₂₂N₃O₂S requires 296.1433), 240

(MꢂMe₂C¼CH₂); Found C, 56.50; H, 7.11;

C₁₄H₂₁N₃O₂S requires C, 56.50; H, 7.09%.

The most potent inhibitory activity was seen where the

substituent was aminomethyl, the same substituent as in

11 and 12, although the position (meta or para) of this

substituent was of limited importance. The isoform-

selectivity for inhibition of iNOS and nNOS, where

observed, was generally limited. However, 18a showed

useful selectivity for inhibition of rat nNOS over human

iNOS. Most compounds showed modest inhibitory

potency but the activities of 18a and 19a were compar-

able to those of the widely used experimental inhibitor

thiocitrulline 13,¹⁵of 8¹⁵and of our previous o-iso-

thiourea ornithine-based inhibitors, N^d-(imino(isopro-

pylthio))methyl)ornithine,²⁶N^d-(4,5-dihydro-1,3-thiazin-

2-yl)ornithine²⁶and N^d-(4,5-dihydro-1,3-thiazol-2-yl)or-

nithine 14.²⁶Most striking, however, is the stimulation of

the activity of human iNOS by the N-(aminobenzyl) and

N-(carboxybenzyl) thioureas and 2-(aminobenzylamino)

and 2-(carboxybenzyl) 4,5-dihydrothiazoles. The

1,1-Dimethylethyl N-(4-(thioureidomethyl)phenylmethyl)-

carbamate (16d). Compound 22d was treated with

NH₃, as for the synthesis of 16c (chromatographic

eluant EtOAc), to give 16d (71%) as pale yellow crystals:

mp 104–106 ^ꢀC; IR n_max1171, 1686, 3355 cm^ꢂ1; NMR

((CD₃)₂SO) d_H1.38 (9H, s, Bu^t), 4.08 (2H, d, J=6.0 Hz,

CH₂), 4.57 (2H, br, CH₂), 7.20 (6H, m, Ar-H₄+NH₂),

7.37 (1H, br, NH), 7.98 (1H, br, NH); MS m/z 591

4198

C. L. M. Goodyer et al. / Bioorg. Med. Chem. 11 (2003) 4189–4206

(2 M+H), 296.1423 (M+H) (C₁₄H₂₂N₃O₃S requires

296.1421), 249 (MꢂMe₂C¼CH₂); Found C, 56.6: H,

7.09; N, 13.90; C₁₄H₂₁N₃O₂S requires C, 56.72; H, 7.16;

N, 14.23%.

Methyl 4-(thioureidomethyl)benzoate (16k). Compound

22k was treated with NH₃, as for the synthesis of 16h, to

give 16k (99%) as white crystals: mp 131–133 ^ꢀC; IR

n_max1179, 1711, 3409 cm^ꢂ1; NMR ((CD₃)₂SO) d_H3.82

(3H, s, Me), 4.70 (2H, s, CH₂), 7.18 (2H, br, NH₂), 7.38

(2H, d, J=8.2 Hz, Ar 3,5-H₂), 7.90 (2H, d, J=8.2 Hz,

Ar 2,6-H₂), 8.06 (1H, br NH); MS m/z 225.0690

(M+H) (C₁₀H₁₃N₂O₂S requires 225.0698).

N-(3-Nitrophenylmethyl)thiourea (16e). Compound 22e

was treated with NH₃, as for the synthesis of 16d, to

give 16e (68%) as yellow crystals: mp 143–145 ^ꢀC; IR n_max

1159, 1347, 1529, 3292 cm^ꢂ1; NMR d_H4.77 (2H, s, CH₂),

7.20 (2H, br, NH₂), 7.64 (1H, dd, J=8.2, 7.8 Hz, Ar 5-H),

7.74 (1H, d, J=7.8 Hz, Ar 4-H), 8.10 (1H, s, Ar 2-H), 8.11

(1H, d, J=8.2 Hz, Ar 6-H); MS m/z 212.0498 (M+H)

(C₈H₁₀N₃O₂S requires 212.0494), 196 (MꢂNH₂).

N-(3-Aminophenylmethyl)thiourea (16l). Compound 16e

(90 mg, 0.4 mmol) was boiled under reﬂux with

.

SnCl₂2H₂O (200 mg, 1.2 mmol) in EtOH (5 mL) for 1 h.

The solution was cooled to 0 ^ꢀC, basiﬁed with 5% aq

NaHCO₃, extracted with EtOAc and washed with satu-

rated brine. Drying, evaporation and chromatography

(EtOAc/hexane 5:1) gave 16l (40 mg, 55%) as pale buﬀ

N-(4-Nitrophenylmethyl)thiourea (16f). Compound 22f

was treated with NH₃, as for the synthesis of 16d, to

give 16f (69%) as a colourless oil: (lit.³⁶mp 113.5–

crystals: mp 141–143 ^ꢀC; IR n_max1166, 3289 cm^ꢂ1

;

115 ^ꢀC); IR (ﬁlm) n_max1159, 1344, 1563, 3213 cm^ꢂ1

;

NMR (CD₃OD) d_H3.34 (2H, s, CH₂), 4.25 (1H, br,

NH), 4.58 (2H, br, NH₂), 6.62 (3H, m, Ar 2,4,6-H₃),

6.68 (1H, br, NH), 7.05 (1H, t, J=7.8 Hz, Ar 5-H); MS

m/z 182.0757 (M+H) (C₈H₁₂N₃S requires 182.0752).

NMR d_H4.82 (2H, br, CH₂), 5.82 (2H, br, NH₂), 7.26

(1H, s, NH), 7.50 (2H, d, J=8.6 Hz, Ar 2,6-H₂), 8.21

(2H, d, J=8.6 Hz, Ar 3,5-H₂); MS m/z 422 (2 M+H),

212.0490 (M+H) (C₈H₁₀N₃O₂S requires 212.0494), 196

(MꢂNH₂).

N-(4-Aminophenylmethyl)thiourea (16m). Compound

16f was treated with SnCl₂, as for the synthesis of 16l

(reaction time 3 h; chromatographic eluant EtOAc/

AcOH/ hexane (10:1:1), to give 16m (95%) as pale

N-(3-Methoxyphenylmethyl)thiourea (16g). Compound

22g was treated with NH₃, as for the synthesis of 16d, to

give 16g (99%) as a colourless oil: (lit.³⁷mp 102 ^ꢀC); IR

(ﬁlm) n_max1046, 2835, 3273 cm^ꢂ1; NMR d_H3.80 (3H, s,

Me), 4.20 (1H, br, NH), 4.77 (2H, m, CH₂), 5.83 (2H, s,

NH₂), 6.86 (3H, m, Ar 2,4,6-H₃), 7.27 (1H, m, Ar 5-H);

MS m/z 197.0751 (M+H) (C₉H₁₃N₂OS requires

197.0749), 121 (MꢂNHCSNH₂).

orange crystals: mp >270 ^ꢀC; IR n_max1179, 3422 cm^ꢂ1

;

NMR (CD₃OD) d_H4.54 (2H, s, CH₂), 6.69 (2H, d,

J=8.2 Hz, Ar 3,5-H₂), 7.06 (2H, d, J=8.2 Hz, Ar 2,6-

H₂); MS m/z 182.0746 (M+H) (C₈H₁₂N₃S requires

182.0752), 164 (MꢂNH₃).

3-(Thioureidomethyl)benzoic acid hydrobromide (16n).

Compound 16i (80 mg, 360 mmol) was stirred in aq HBr

(50%, 5 mL) for 16 h. Evaporation gave 16n (70 mg,

98%) as a colourless hygroscopic gum: IR (ﬁlm) n_max

1704, 3298 cm^ꢂ1; NMR (CD₃OD) d_H3.89 (2H, s, CH₂),

7.45 (1H, t, J=7.8 Hz, Ar 5-H), 7.57 (1H, d, J=7.8 Hz,

Ar 4-H), 7.91 (1H, d, J=7.8 Hz, Ar 6-H), 8.08 (1H, s,

Ar 2-H); MS m/z 225.0708 (M+H) (C₁₀H₁₃N₂O₂S

requires 225.0698).

N-(4-Methoxyphenylmethyl)thiourea (16h). Compound

22h was treated with NH₃, as for the synthesis of 16c

[chromatographic eluant EtOAc/hexane (1:1)], to give

16h (99%) as white crystals: mp 124–126 ^ꢀC (lit.³⁸mp

135 ^ꢀC); IR n_max1177, 2800, 3165 cm^ꢂ1; NMR d_H

3.87 (3H, s, Me), 4.38 (2H, br, CH₂), 5.75 (2H, br,

NH₂), 6.88 (1H, br, NH), 6.90 (2H, d, J=8.4 Hz, Ar

3,5-H₂), 7.23 (2H, d, J=8.4 Hz, Ar 2,6-H₂); MS m/z

393 (2 M+H), 197.0749 (M+H) (C₉H₁₃N₂OS

requires 197.0751); Found C, 54.80: H, 6.13; N,

14.14; C₉H₁₂N₂OS requires C, 54.30; H, 6.11; N,

14.10%.

2-(3-(Aminomethyl)phenylmethylamino)-4,5-dihydrothi-

azole dihydrochloride (17a). Compound 24c (83 mg,

240 mmol) was boiled under reﬂux for 36 h in aq HCl

(6 M, 4 mL). Evaporation gave 17a (59 mg, 47%) as a

colourless hygroscopic gum: IR (ﬁlm) n_max1632,

3429 cm^ꢂ1; NMR d_H3.63 (2H, t, J=7.4 Hz, thiazole 5-

H₂), 3.99 (2H, t, J=7.4 Hz, thiazole 4-H₂), 4.06 (2H,

brq, J=5.9 Hz, CH₂N⁺H₃), 4.77 (2H, d, J=6.2 Hz,

ArCH₂Nthiazole) 7.50 (4H, m, Ar-H₄), 8.70 (3H, br,

N⁺H₃), 10.89 (2H, s, 2 ꢄ NH); MS m/z 222.1067

(M+H) (C₁₁H₁₆N₃S requires 222.1065).

Methyl 3-(thioureidomethyl)benzoate (16i). Compound

22i was treated with NH₃, as for the synthesis of 16d, to

give 16i (99%) as pale yellow crystals: mp 123–125 ^ꢀC;

IR n_max1202, 1710, 3418 cm^ꢂ1; NMR (CD₃OD) d_H3.89

(3H, s, Me), 4.88 (2H, s, CH₂), 7.44 (1H, t, J=7.8 Hz,

Ar 5-H), 7.56 (1H, d, J=7.8 Hz, Ar 4-H), 7.90 (1H, d,

J=7.8 Hz, Ar 6-H), 8.03 (1H, s, Ar 2-H); MS m/z 449

(2 M+H), 225.0708 (M+H) (C₁₀H₁₃N₂O₂S requires

225.0698), 211 (MꢂMe).

2-(4-(Aminomethyl)phenylmethylamino)-4,5-dihydrothi-

azole dihydrochloride (17b). Compound 24d (100 mg,

0.29 mmol) was boiled under reﬂux in aq HCl (6 M,

6 mL) for 36 h. Evaporation and recrystallisation

(PrⁱOH) gave 17b (40 mg, 47%) as pale yellow crystals:

mp 198–200 ^ꢀC; IR n_max1654, 3425 cm^ꢂ1; NMR

((CD₃)₂SO) d_H3.64 (2H, t, J=7.4 Hz, thiazole 5-H₂),

4.04 (2H, m, thiazole 4-H₂), 4.14 (2H, m, CH₂NHthia-

zole), 4.61 (2H, s, CH₂N⁺H₃), 7.44 (2H, m, Ar 3,5-H₂),

4-(Thioureidomethyl)benzoic acid hydrobromide (16j).

Compound 16k was treated with HBr, as for the synth-

esis of 16n, to give 16j (99%) as a colourless hygroscopic

gum: IR (ﬁlm) n_max1176, 1705, 3382 cm^ꢂ1; NMR

(CD₃OD) d_H3.88 (2H, s, CH₂), 7.41 (2H, d, J=8.3 Hz,

Ar 3,5-H₂), 7.97 (2H, d, J=8.3 Hz, Ar 2,6-H₂); MS m/z

211.0541 (M+H) (C₉H₁₁N₂O₂S requires 211.0547).

C. L. M. Goodyer et al. / Bioorg. Med. Chem. 11 (2003) 4189–4206

4199

7.53 (2H, m, Ar 2,6-H₂); MS m/z 443 (2 M+H),

222.1061 (M+H) (C₁₁H₁₆N₃S requires 222.1065), 205

(MꢂNH₃).

Methyl 3-((4,5-dihydrothiazol-2-ylamino)methyl)benzoate

hydrochloride (17i). Compound 24i (70 mg, 260 mmol)

was boiled under reﬂux for 40 h in aq HCl (6 M, 5 mL).

The evaporation residue was stirred with MeOH

(70 mL) and SOCl₂(0.5 mL) for 4 d. Evaporation gave

17i (50 mg, 67%) as a pale buﬀ gum: IR (ﬁlm) n_max

1640, 1718, 3398 cm^ꢂ1; NMR d_H3.51 (2H, br, CH₂),

3.92 (3H, s, Me), 4.02 (2H, br, CH₂), 4.54 (2H, s,

CH₂Ar), 7.47 (1H, t, J=7.4 Hz, Ar-H₅), 7.57 (1H, d,

J=7.4 Hz, Ar-H₄), 7.95 (1H, s, Ar-H₂), 7.99 (1H, d,

J=7.4 Hz, Ar-H₄), 10.40 (1H, br, NH), 10.93 (1H, br,

NH): MS m/z 251.0859 (M+H) (C₁₂H₁₄N₂O₂S requires

251.0854).

2-(3-Nitrophenylmethylamino)-4,5-dihydrothiazole

tri-

ﬂuoroacetate salt (17e). Compound 24e (120 mg,

470 mmol) was stirred for 16 h with CF₃CO₂H (5 mL).

Evaporation gave 17e (170 mg, 99%) as a colourless

hygroscopic gum: IR (ﬁlm) n_max1352, 1532, 1679,

3170 cm^ꢂ1; NMR d_H3.55 (2H, t, J=7.8 Hz, thiazole

5-H₂), 4.05 (2H, t, J=7.8 Hz, thiazole 4-H₂), 4.59

(2H, d, J=4.7 Hz, ArCH₂), 7.61 (1H, dd, J=8.2,

7.8 Hz, Ar 5-H), 7.69 (1H, d, J=7.8 Hz, Ar 6-H),

8.17 (1H, s, Ar 2-H), 8.21 (1H, d, J=8.2 Hz, Ar 4-

H), 11.94 (1H, br, NH), 12.32 (1H, br NH); MS m/z

475 (2 M+H), 238.0640 (M+H) (C₁₀H₁₂N₃O₂S

requires 238.0650).

4-(4,5-Dihydrothiazol-2-ylaminomethyl)benzoic

acid

hydrochloride (17j). Compound 24k was treated with aq

HCl, as for the synthesis of 17a, to give 17j (99%) as

pale yellow crystals: mp 138–140 ^ꢀC; IR (ﬁlm) n_max

1656, 1697, 3430 cm^ꢂ1; NMR ((CD₃)₂SO) d_H3.57 (2H,

t, J=7.4 Hz, thiazole 5-H₂), 3.92 (2H, t, J=7.4 Hz,

thiazole 4-H₂), 4.73 (2H, s, CH₂Ar), 7.46 (2H, d,

J=8.2 Hz, Ar 3,5-H₂), 7.95 (2H, d, J=8.2 Hz, Ar 2,6-

H₂), 10.66 (1H, br, NH), 13.05 (1H, br, NH); MS m/z

237.0688 (M+H) (C₁₁H₁₃N₂O₂S requires 237.0698).

2-(4-Nitrophenylmethylamino)-4,5-dihydrothiazole

tri-

ﬂuoroacetate salt (17f). Compound 24f (200 mg,

780 mmol) was boiled under reﬂux in CF₃CO₂H (5 mL)

for 15 h. Evaporation gave 17f (240 mg, 99%) as a col-

ourless hygroscopic gum: IR (ﬁlm) n_max1347, 1524,

1678, 3173 cm^ꢂ1; NMR d_H3.54 (2H, t, J=7.8 Hz, thia-

zole 5-H₂), 4.04 (2H, t, J=7.8 Hz, thiazole 4-H₂), 4.59

(2H, d, J=5.1 Hz, CH₂Ar), 7.49 (2H, d, J=8.6 Hz, Ar

2,6-H₂), 8.23 (2H, d, J=8.6 Hz, Ar 3,5-H₂), MS m/z

238.0639 (M+H) (C₁₀H₁₂N₃O₂S requires 238.0650),

222 (MꢂNH₂).

2-(3-Aminophenylmethylamino)-4,5-dihydrothiazole (17l).

Compound 17e was treated with SnCl₂, as for the

synthesis of 16l (chromatography omitted), to give 17l

(150 mg, 99%) as a colourless oil: IR (ﬁlm) n_max1618;

3391 cm^ꢂ1; NMR (CD₃OD) d_H3.30 (2H, m, thiazole 5-

H₂), 3.90 (2H, m, thiazole 4-H₂), 4.30 (2H, br,

CH₂Ar), 6.62 (2H, m, Ar 4,6-H₂), 6.67 (1H, s, Ar 2-

H), 7.04 (1H, t, J=7.4 Hz, Ar 5-H); MS m/z 208.0905

2 - (3 - Methoxyphenylmethylamino) - 4,5 - dihydrothiazole

(17g). Method A. Compound 24g (290 mg, 1.2 mmol)

was stirred in CF₃CO₂H (5 mL) for 2 h. Evaporation

and chromatography (EtOAc/MeOH 5:1) gave 17g

(200 mg, 75%) as white crystals: mp 94–96 ^ꢀC; IR (ﬁlm)

n_max1681, 3200 cm^ꢂ1; NMR d_H3.46 (2H, t, J=7.4 Hz,

thiazole 5-H₂), 3.82 (3H, s, Me), 3.95 (2H, t, J=7.8 Hz,

thiazole 4-H₂), 4.43 (2H, d, J=5.6 Hz, CH₂Ar), 6.87

(3H, m, Ar 2,4,6-H₃), 7.28 (1H, m, Ar 5-H), 12.25 (1H,

s, NH), 12.36 (1H, s, NH); NMR d_C31.1, 48.7, 51.3,

55.2, 112.9, 113.9, 119.6, 130.0, 136.5, 160.0, 174.6; MS

m/z 223.0890 (M+H) (C₁₁H₁₅N₂OS requires 223.0905).

(M+H)

(C₁₀H₁₄N₃S

requires

208.0908),

196

(Mꢂaminodihydrothiazole).

2-(4-Aminophenylmethylamino)-4,5-dihydrothiazole (17m).

Compound 17f was treated with SnCl₂, as for the

synthesis of 17l (reaction time 1.5 h), to give 17m (38%)

as a colourless oil: IR (ﬁlm) n_max1609, 3324 cm^ꢂ1

;

NMR d_H3.33 (2H, t, J=7.4 Hz, thiazole 5-H₂), 3.64

(2H, br, NH₂), 4.02 (2H, t, J=7.4 Hz, thiazole 4-H₂),

6.64 (2H, d, J=8.6 Hz, Ar 2,6-H₂), 7.11 (2H, d,

J=8.6 Hz, Ar 3,5-H₂); NMR 35.7, 49.2), 60.4, 115.4,

129.0, 129.3, 146.0, 161.9; MS m/z 208.0911 (M+H)

(C₁₀H₁₄N₃S requires 208.0908).

2-(3-Methoxyphenylmethyl)-4,5-dihydrothiazole

(17g).

Method B. 3-Methoxybenzylamine 21g was heated

with 2-methylthio-4,5-dihydrothiazole 26 (320 mg,

2.4 mmol) at 180 ^ꢀC for 4 h. Evaporation and chroma-

tography (EtOAc/ MeOH 5:1) gave 17g (163 mg, 30%)

with properties as above.

3-(4,5-Dihydrothiazol-2-ylaminomethyl)benzoic acid tri-

ﬂuoroacetate salt (17n). Compound 17i (70 mg,

280 mmol) was stirred under reﬂux in aq CF₃CO₂H

(50%, 5 mL) for 16 h. Evaporation gave 17n (70 mg,

99%) as a colourless hygroscopic gum: IR n_max1645,

1696, 3433 cm^ꢂ1; NMR (CD₃OD) d_H3.65 (2H, t,

J=7.8 Hz, thiazole 5-H₂), 4.04 (2H, t, J=7.8 Hz, thia-

zole 4-H₂), 4.60 (2H, m, CH₂Ar), 7.54 (3H, m, Ar-H₃),

8.02 (1H, m, Ar-H); MS m/z 237.0698 (M+H)

(C₁₁H₁₃N₂O₂S requires 237.0698).

2 - (4 - Methoxyphenylmethylamino) - 4,5 - dihydrothiazole

(17h). 4-Methoxybenzylamine 21h was treated with 26,

as for the synthesis of 17g (Method B) [chromato-

graphic eluant CH₂Cl₂/MeOH (10:1)] to give 17h (30%)

as white crystals: mp 77–79 ^ꢀC (lit.³³mp 84–85 ^ꢀC);

NMR d_H3.38 (2H, t, J=7.4 Hz, thiazole 5-H₂), 3.80

(3H, s, Me), 4.04 (2H, t, J=7.4 Hz, thiazole 4-H₂), 4.40

(2H, s, ArCH₂), 6.87 (2H, d, J=8.8 Hz, Ar 3,5-H₂), 7.25

(2H, d, J=8.8 Hz, Ar 2,6-H₂); IR n_max1610, 2780,

3207 cm^ꢂ1; MS m/z 223.0912 (M+H) (C₁₁H₁₅N₂OS

requires 223.0905); Found C, 58.25: H, 6.44; N, 12.35:

C₁₁H₁₄N₂OS. 0.25H₂O requires C, 58.29; H, 6.40; N,

12.36%.

N-(3-Aminomethylphenyl)thiourea bis(triﬂuoroacetate)

salt (18a). Compound 18c was treated with CF₃CO₂H,

as for the synthesis of 16a, to give 18a (99%) as a col-

ourless hygroscopic gum: IR (ﬁlm) n_max1170,

3407 cm^ꢂ1; NMR ((CD₃)₂SO) d_H4.02 (2H, brq,

4200

C. L. M. Goodyer et al. / Bioorg. Med. Chem. 11 (2003) 4189–4206

J=5.9 Hz, CH₂), 7.09 (3H, br, N⁺H₃), 7.20 (1H, m, Ar

4-H), 7.40 (2H, m, Ar 5,6-H₂), 7.50 (1H, s, Ar 2-H), 8.16

(3H, br, N⁺H₃), 9.87 (1H, s, NH); NMR ((CD₃)₂SO) d_C

42.3, 123.4, 123.5, 124.8, 129.1, 134.5, 139.5, 181.1; MS

m/z 182.0768 (M+H) (C₈H₁₂N₃S requires 182.0752),

164 (MꢂNH₃).

3-Thioureidophenylacetic acid hydrochloride (18o). Com-

pound 18q (50 mg, 220 mmol) was stirred in aq HCl

(1 M, 3 mL) for 9 d. Evaporation gave 18o (40 mg, 86%)

as white crystals: mp 159–161 ^ꢀC (lit.⁴¹mp 174–176 ^ꢀC);

IR n_max1157, 1730, 2500, 3337 cm^ꢂ1

;

NMR

(CD₃OD) d_H3.67 (2H, s, CH₂), 7.15 (1H, d,

J=7.4 Hz, Ar 4-H), 7.23 (1H, d, J=8.6 Hz, Ar 6-H),

7.25 (1H, s, Ar 2-H), 7.35 (1H, dd, J=8.6, 7.4 Hz, Ar 5-

H); MS m/z 211.0541 (M+H) (C₉H₁₁N₂O₂S requires

211.0531).

N-(4-Aminomethylphenyl)thiourea bis(triﬂuoroacetate)

salt (18b). Compound 18d was treated with CF₃CO₂H,

as for the synthesis of 16a (reaction time 2 h), to give

18b (99%) as a colourless hygroscopic gum: IR (ﬁlm)

n

_max1173, 3369 cm^ꢂ1; NMR ((CD₃)₂SO) d_H3.96 (2H, q,

4-Thioureidophenylacetic acid hydrochloride (18p). Com-

pound 18r (50 mg, 220 mmol) was stirred for 16 h in aq

HCl (6 M, 5 mL). Evaporation gave 18p (60 mg, 99%) as

white crystals: mp 198–200 ^ꢀC (lit.⁴⁰mp 200–203 ^ꢀC); IR

n_max1181, 1699, 3313 cm^ꢂ1; NMR (CD₃OD) d_H3.65

(2H, s, CH₂), 7.26–7.32 (4H, m, Ar-H₄); MS m/z

211.0541 (M+H) (C₉H₁₁N₂O₂S requires 211.0531).

J=5.6 Hz, CH₂), 7.38 (2H, d, J=8.2 Hz, Ar 2,6-H₂),

7.51 (2H, d, J=8.2 Hz, Ar 3,5-H₂), 7.51 (3H, br,

N⁺H₃), 8.11 (3H, br, N⁺H₃), 9.83 (1H, s, NH); MS m/z

182.0748 (M+H) (C₈H₁₂N₃S requires 182.0752), 164

(MꢂNH₃).

1,1-Dimethylethyl N-(3-thioureidophenylmethyl)carbamate

(18c). Compound 29c was treated with NH₃, as for the

synthesis of 16c, to give 18c (38%) as pale yellow crys-

tals: mp 198–200 ^ꢀC; IR n_max1173, 3291 cm^ꢂ1, NMR d_H

1.37 (9H, s, Bu^t), 4.01 (2H, d, J=7.4 Hz, CH₂), 6.96

(1H, d, J=7.4 Hz, NH), 7.23 (1H, d, J=7.8 Hz, Ar-H),

7.16–7.29 (5H, m, Ar-H₃₊NH₂), 9.78 (1H, s, NH); MS

m/z 282.1295 (M+H) (C₁₃H₂₀N₃O₂S requires

282.1276), 226 (MꢂMe₂C¼CH₂).

Methyl 3-thioureidophenylacetate (18q). Compound 29q

was treated with NH₃, as for the synthesis of 16h, to

give 18q (99%) as pale yellow crystals: mp 131–133 ^ꢀC;

IR n_max1160, 1730, 3409 cm^ꢂ1; NMR ((CD₃)₂SO) d_H

3.61 (3H, s, Me), 3.65 (2H, s, CH₂), 7.00 (1H, d,

J=7.4 Hz, Ar 4-H), 7.23 (1H, dd, J=8.6, 7.4 Hz, Ar 5-

H), 7.25 (1H, s, Ar 2-H), 7.33 (1H, d, J=8.6 Hz, Ar 6-

H), 7.35 (1H, br, NH), 9.71 (1H, s, NH); NMR

((CD₃)₂SO)_C40.1, 51.8, 121.6, 123.8, 125.4, 128.7,

134.9, 139.2, 171.5, 181.1; MS m/z 225.0694 (M+H)

(C₁₀H₁₃N₂O₂S) requires 225.0698).

1,1-Dimethylethyl N-(4-thioureidophenylmethyl)carbamate

(18d). Compound 29d was treated with NH₃, as for the

synthesis of 16c, to give 18d (66%) as white crystals: mp

89–91 ^ꢀC; IR n_max1187, 1690, 3298 cm^ꢂ1; NMR d_H1.46

(9H, s, Bu^t), 4.30 (2H, d, J=6.0 Hz, CH₂), 5.03 (1H, br,

NH), 6.25 (2H, br, NH₂), 7.19 (2H, d, J=8.2 Hz, Ar

3,5-H₂), 7.33 (2H, d, J=8.2 Hz, Ar 2,6-H₂), 8.39 (1H,

br, NH); MS m/z 282.1276 (M+H) (C₁₃H₂₀N₃O₂S

requires 282.1276), 226 (MꢂMe₂C¼CH₂); Found C,

54.40: H, 6.73; N, 14.20: C₁₃H₁₉N₃O₂S 0.5H₂O requires

C, 53.87; H, 6.78; N, 14.49%.

Methyl 4-thioureidophenylacetate (18r). Compound 29r

was treated with NH₃, as for the synthesis of 16d, to

give 18r (93%) as a white powder: mp 121–123 ^ꢀC; IR

n_max1718, 3168, 3341 cm^ꢂ1; NMR ((CD₃)₂SO) d_H3.59

(3H, s, Me), 3.62 (2H, s, CH₂), 7.18 (2H, d, J=8.2 Hz,

Ar 3,5-H₂), 7.31 (2H, d, J=8.2 Hz, Ar 2,6-H₂), 7.43

(2H, br, NH₂), 9.65 (1H, s, NH); MS m/z 225.0687

(M+H) (C₁₀H₁₃N₂O₂S requires 225.0698).

N-(3-Methoxyphenyl)thiourea (18g). 3-Methoxyphenyl-

isothiocyanate 29g was treated with NH₃, as for the

synthesis of 16c (chromatography omitted), to give 18g

(99%) as white crystals: mp 160–162 ^ꢀC (lit.³⁹mp

160 ^ꢀC); IR n_max1166, 3149 cm^ꢂ1; NMR d_H3.73 (3H, s,

Me), 6.67 (1H, d, J=7.6 Hz, Ar 4-H), 6.90 (1H, d,

J=7.6 Hz, Ar 6-H), 7.10 (1H, s, Ar 2-H), 7.23 (1H, t,

J=7.6 Hz, Ar 5-H), 7.53 (2H, br, NH₂) 9.62 (1H, s,

NH); NMR ((CD₃)₂SO) d_C55.1, 108.6, 110.0, 115.0,

129.6, 140.3, 159.3, 180.9; MS m/z 365 (2 M+H),

183.0598 (M+H) (C₈H₁₁N₂OS requires 183.0592);

Found C, 52.50: H, 5.53; N, 15.40; C₈H₁₀N₂OS requires

C, 52.72; H, 5.53; N, 15.37%.

2-(3-(Aminomethyl)phenylamino)-4,5-dihydrothiazole di-

hydrochloride (19aA). Compound 30c was treated with

HCl, as for the synthesis of 17a (reaction time 43 h), to

give 19aA (99%) as pale buﬀ crystals: mp 208–210 ^ꢀC;

IR n_max1640, 3432 cm^ꢂ1; NMR ((CD₃)₂SO) d_H3.65

(2H, t, J=7.7 Hz, thiazole 5-H₂), 4.01 (2H, t, J=7.7 Hz,

thiazole 4-H₂), 4.11 (2H, m, ArCH₂), 7.39 (1H, d,

J=7.5 Hz, Ar 4-H), 7.56 (2H, m, Ar 5,6-H₂), 7.62 (1H, s,

Ar 2-H), 8.66 (3H, s, N⁺H₃), 10.5 (1H, br, NH), 12.7 (1H,

br, NH); MS m/z 208.0918 (M+H) (C₁₀H₁₄N₃S requires

208.0908); Found C, 37.91: H, 6.06; N, 13.29; C₁₀H₁₃N₃S

2H₂O 2HCl requires C, 38.50; H, 5.67; N, 13.00%.

2-(3-(Aminomethylphenylamino)-4,5-dihydrothiazole bis

(triﬂuoroacetate) salt (19aB). Compound 30c was trea-

ted with CF₃CO₂H, as for the synthesis of 16a, to give

19aB (99%) as a colourless hygroscopic gum: IR (ﬁlm)

N-(4-Methoxyphenyl)thiourea (18h). 4-Methoxyphenyl-

isothiocyanate 29h was treated with NH₃, as for the

synthesis of 18g, to give 18h (99%) as white crystals:

mp 198–200 ^ꢀC (lit.⁴⁰mp 210 ^ꢀC); IR n_max1171, 2838,

3154 cm^ꢂ1; NMR d_H3.71 (3H, s, Me), 6.89 (2H, d,

J=8.8 Hz, Ar 3,5-H₂), 7.21 (2H, d, J=8.8 Hz, Ar

2,6-H₂), 7.23 (2H, br, NH₂), 9.47 (1H, s, NH); MS

n

_max1674, 3398 cm^ꢂ1; NMR ((CD₃)₂SO) d_H3.62 (2H, t,

J=7.7 Hz, thiazole 5-H₂), 4.0 (4H, m, thiazole 4-

H₂+ArCH₂), 7.30 (4H, m, Ar-H₄), 7.86 (1H, br, NH),

8.18 (3H, m, N⁺H₃), 9.73 (1H, br, NH); MS m/z

208.0915 (M+H) (C₁₀H₁₄N₃S requires 208.0908), 191

(MꢂNH₃).

m/z

183.0592).

183.0592

(M+H)

(C₈H₁₀N₂OS

requires

C. L. M. Goodyer et al. / Bioorg. Med. Chem. 11 (2003) 4189–4206

4201

2 - (4 - (Aminomethyl)phenylamino) - 4,5 - dihydrothiazole

bis(triﬂuoroacetate) salt (19b). Compound 30d was trea-

ted with CF₃CO₂H, as for the synthesis of 16a (reaction

time 2 h), to give 19b (99%) as a colourless hygroscopic

gum: IR (ﬁlm) n_max1643, 3400 cm^ꢂ1; NMR ((CD₃)₂SO)

d_H3.60 (2H, t, J=7.4 Hz, thiazole 5-H₂), 4.00 (2H, t,

J=7.4 Hz, thiazole 4-H₂), 4.03 (2H, q, J=6.0 Hz

CH₂N⁺H₃), 4.59 (1H, br, NH), 7.37 (2H, d, J=8.2 Hz,

Ar 2,6-H₂), 7.54 (2H, d, J=8.2 Hz, Ar 5,6-H₂), 8.26

(3H, br, NH₃); MS m/z 208.0914 (M+H) (C₁₀H₁₄N₃S

requires 208.0908), 191 (MꢂNH₃).

1,1-Dimethylethyl N-(3-(aminomethyl)phenylmethyl)car-

bamate (21c). Di(t-butyl) dicarbonate (1.0 g, 4.9 mmol)

was added slowly to 1,3-bis(aminomethyl)benzene 21a

(2.0 g, 15 mmol) and Et₃N (2.9 g, 29 mmol) in CH₂Cl₂

(15 mL) at 0 ^ꢀC and the mixture was stirred for 16 h. The

evaporation residue, in CH₂Cl₂, was washed with aq

NaHCO₃and dried. Evaporation gave 21c (900 mg,

78%) as white crystals: mp 61–64 ^ꢀC (lit.⁴³oil); NMR

d_H1.51 (9H, s, Bu^t), 1.67 (2H, s, NH₂), 3.90 (2H, d,

J=4.3 Hz, CH₂NH₂), 4.34 (2H, s, CH₂NHBoc), 5.10

(1H, br, NH), 7.25 (4H, m, Ar-H₄); MS m/z 237

(M+H), 181 (MꢂMe₂C¼CH₂), 164 (MꢂBu^tO).

2-(3-Methoxyphenylamino)-4,5-dihydrothiazole

(19g).

Compound 30g (200 mg, 890 mmol) was stirred in

CF₃CO₂H (5 mL) for 3 h. Evaporation and chromato-

graphy (EtOAc) gave 19g (130 mg, 70%) as pale buﬀ

1,1-Dimethylethyl N-(4-(aminomethyl)phenylmethyl)car-

bamate (21d). 1,4-Bis(aminomethyl)benzene 21b was

treated with Boc₂O, as for the synthesis of 21c, to give

21d (930 mg, 80%) as a colourless oil: (lit.⁴⁴solid) NMR

d_H1.46 (9H, s, Bu^t), 1.52 (2H, br, NH₂), 3.85 (2H, s,

CH₂NH₂), 4.29 (2H, d, J=6.0 Hz, CH₂NHBoc), 5.89

(1H, br, NH), 7.24–7.28 (4H, m, Ar-H₄); MS m/z 237

(M+H), 181 (MꢂMe₂C¼CH₂), 164 (MꢂBu^tO).

crystals: mp 80–82 ^ꢀC; IR n_max1674, 2850, 3238 cm^ꢂ1

;

NMR ((CD₃)₂SO) d_H4.17 (3H, s, Me), 3.92 (2H, t,

J=7.6 Hz, thiazole 5-H₂), 4.35 (2H, t, J=7.6 Hz, thia-

zole 4-H₂) 7.24 (1H, d, J=8.2 Hz, Ar 4-H), 7.34 (1H, d,

J=8.2 Hz, Ar 6-H), 7.41 (1H, s, NH), 7.73 (1H, t,

J=8.2 Hz, Ar 5-H), 7.74 (1H, s, Ar 2-H); MS m/z

209.0749 (M+H) (C₁₀H₁₃N₂OS requires 209.0743).

Methyl 3-(aminomethyl)benzoate (21i) and di(3-methoxy-

carbonylphenylmethyl)amine (23). Compound 20i

(900 mg, 5.6 mmol) in MeOH (30 mL) was treated with

Pd/C (10%) and H₂for 16 h. Filtration (Celite^#), eva-

poration and chromatography (EtOAc) gave 23

(150 mg, 9%) as a colourless oil: (lit.⁴⁵oil); IR (ﬁlm)

n_max1721, 3336 cm^ꢂ1; NMR d_H3.84 (4H, s, 2 ꢄ CH₂),

3.91 (6H, s, 2 ꢄ Me), 7.40 (2H, t, J=7.8 Hz, 2 ꢄ Ar 5-

H), 7.52 (2H, d, J=7.8 Hz, 2 ꢄ Ar 4-H), 7.93 (2H, d,

J=7.8 Hz, 2 ꢄ Ar 6-H), 8.02 (2H, s, 2 ꢄ Ar 2-H); MS

m/z 314 (M+H). Further elution gave 21i (330 mg,

36%) as white crystals: mp 37–39 ^ꢀC (lit.⁴⁵oil); IR (ﬁlm)

n_max1719, 3453 cm^ꢂ1; NMR d_H3.92 (3H, s, Me), 3.95

(2H, s, CH₂), 7.40 (1H, t, J=7.8 Hz, Ar 5-H), 7.53 (1H,

d, J=7.8 Hz, Ar 4-H), 7.92 (1H, d, J=7.8 Hz, Ar 6-H),

8.01 (1H, s, Ar 2-H); MS m/z 166 (M+H), 121

(MꢂNH₂).

2-(4-Methoxyphenylamino)-4,5-dihydrothiazole triﬂuoro-

acetate salt (19h). Compound 30h (200 mg, 880 mmol)

was boiled under reﬂux in CF₃CO₂H (5 mL) for 15 h.

Evaporation gave 19h (220 mg, 99%) as white crystals:

mp 101–103 ^ꢀC: IR n_max1674, 2750, 3409 cm^ꢂ1; NMR

((CD₃)₂SO) d_H2.29 (3H, s, Me), 3.60 (2H, m, CH₂) 3.75

(2H, m, CH₂), 7.02 (2H, d, J=6.9 Hz, Ar 3,5-H₂), 7.25

(2H, d, J=6.9 Hz, Ar 2,6-H₂), 7.97 (1H, br, NH); MS

m/z 209.0758 (M+H) (C₁₀H₁₃N₂OS requires 209.0749).

3-(4,5-Dihydrothiazol-2-ylamino)phenylacetic acid hydro-

chloride (19o). Compound 30q was treated with aq HCl,

as for the synthesis of 17a, to give 19o (99%) as a col-

ourless hygroscopic gum: IR (ﬁlm) n_max1633, 1714,

3450 cm^ꢂ1; NMR ((CD₃)₂SO) d_H3.55 (2H, t, J=7.6 Hz,

thiazole 5-H₂), 3.93 (2H, t, J=7.6 Hz, thiazole 4-H₂),

5.75 (2H, s, CH₂Ar), 7.25 (3H, m, Ar-H₃), 7.44 (2H, m,

Ar-H+NH); NMR ((CD₃)₂SO) d_C31.0, 40.5, 48.7,

122.0, 124.3, 129.1, 129.8, 135.8 136.8, 171.2, 173.6; MS

m/z 237.0698 (M+H) (C₁₁H₁₃N₂O₂S requires

237.0698).

Methyl 4-(aminomethyl)benzoate hydrochloride (21k). 4-

Aminomethylbenzoic acid 21j was treated with MeOH

and SOCl₂, as for the synthesis of 28q, to give 21k (2.2 g,

99%) as white crystals: mp 153–155 ^ꢀC (lit.⁴⁶mp 235–

238 ^ꢀC); NMR ((CD₃)₂SO) d_H3.84 (3H, s, Me), 4.08

(2H, s, CH₂), 7.64 (2H, d, J=8.2 Hz, Ar 3,5-H₂), 7.96

(2H, d, J=8.2 Hz, Ar 2,6-H₂), 8.62 (3H, br, NH₃); MS

m/z 166 (M+H), 150 (MꢂNH₂).

4-(4,5-Dihydrothiazol-2-ylamino)phenylacetic acid hydro-

chloride (19p). Compound 30r was treated with aq HCl,

as for the synthesis of 18p, to give 19p (99%) as a col-

ourless hygroscopic gum: IR (ﬁlm) n_max1633, 1736,

3423 cm^ꢂ1: NMR (CD₃OD) d_H3.67 (4H, s, thiazole 5-

CH₂, ArCH₂), 4.04 (2H, s, thiazole 4-CH₂), 7.30 (2H, d,

J=8.2 Hz, Ar 3,5-H₂), 7.43 (2H, d, J=8.2 Hz, Ar 2,6-

H₂); MS m/z 237.0697 (M+H) (C₁₁H₁₃N₂O₂S requires

237.0698).

1,1-Dimethylethyl N-(3-(isothiocyanatomethyl)phenylme-

thyl)carbamate (22c). Compound 21c (900 mg,

3.9 mmol), CaCO₃(400 mg, 4.0 mmol), water (3 mL),

thiophosgene (900 mg, 7.8 mmol) and CHCl₃(25 mL)

were stirred vigorously for 16 h. The mixture was

extracted with CHCl₃. Drying, evaporation and chro-

matography (EtOAc/hexane 1:3) gave 22c (400 mg,

37%) as a colourless oil (lit.⁴⁴mp 43 ^ꢀC); IR (ﬁlm) n_max

1670, 2060, 3353 cm^ꢂ1; NMR d_H1.46 (9H, s, Bu^t), 4.33

(2H, d, J=5.6 Hz, CH₂NHBoc), 4.70 (2H, s, CH₂NCS),

4.91 (1H, br, NH), 7.22–7.27 (3H, m, Ar 2,4,6-H₃), 7.34

(1H, t, J=7.8 Hz, Ar 6-H); MS m/z 279.1163 (M+H)

Methyl 3-cyanobenzoate (20i). 3-Cyanobenzoic acid 20n

was treated with MeOH and SOCl₂, as for the synthesis

of 28q, to give 20i (42%) as white crystals: mp 38–40 ^ꢀC

(lit.⁴²mp 64–65 ^ꢀC); IR n_max1720, 2228 cm^ꢂ1; NMR d_H

3.96 (3H, s, Me), 7.95 (1H, t, J=7.8 Hz, Ar 5-H), 7.84 (1H,

d, J=7.8 Hz, Ar 4-H), 8.27 (1H, d, J=7.8 Hz, Ar 6-H),

8.34 (1H, s, Ar 2-H); MS m/z 162 (M+H), 147 (MꢂMe).

(C₁₄H₁₉N₂O₂S

requires

279.1167),

223

(MꢂMe₂C¼CH₂), 179 (MꢂBoc), 164 (MꢂBocNH).

4202

C. L. M. Goodyer et al. / Bioorg. Med. Chem. 11 (2003) 4189–4206

1,1-Dimethylethyl N-(4-isothiocyanatomethyl)phenylme-

thyl)carbamate (22d). Compound 21d was treated with

thiophosgene, as for the synthesis of 22c, to give 22d

(860 mg, 81%) as pale yellow crystals: mp 80–82 ^ꢀC

(lit.⁴⁴mp 74 ^ꢀC); IR n_max1682, 2091, 3358 cm^ꢂ1; NMR

d_H1.46 (9H, s, Bu^t), 4.33 (2H, m, CH₂NHBoc), 4.70 (2H,

s, CH₂), 4.88 (1H, br, NH), 7.24–7.35 (4H, m, Ar-H₄);

MS m/z 279 (M+H), 164 (MꢂBu^tO).

(210 mg, 1.0 mmol) in acetone (1 mL) was added drop-

wise during 30 min to 2-aminoethanol (60 mg, 1.0 mmol)

in acetone (1 mL). The mixture was boiled under reﬂux

for 4 h. Evaporation and chromatography (EtOAc/hex-

ane 1:1) gave 24c (90 mg, 30%) as a colourless oil: IR

(ﬁlm) n_max1170, 1642, 3413 cm^ꢂ1; NMR ((CD₃)₂SO) d_H

1.45 (9H, s, Bu^t), 1.75 (1H, s, NHBoc), 3.75 (2H, m,

CH₂CH₂O), 4.01 (2H, t, J=6.2 Hz, CH₂O), 4.26 (2H,

m, ArCH₂thiourea), 4.85 (2H, d, J=4.7 Hz, ArCH_2-

NBoc), 5.4 (1H, br, NH or OH), 7.16–7.33 (4H, m, Ar-

H₄); MS m/z 340.1695 (M+H) (C₁₆H₂₆N₃O₃S requires

340.1697), 284 (MꢂMe₂C¼CH₂), 179, 164.

3-Nitrophenylmethylisothiocyanate (22e). 3-Nitrobenzyl-

amine 21e was treated with thiophosgene, as for the

synthesis of 22c (chromatographic eluant _ꢀEtOAc), to

give 22e (61%) as yellow crystals: mp 65–67 C (lit.⁴⁷mp

65–67 ^ꢀC); IR n_max1347, 1526, 2135 cm^ꢂ1; NMR d_H

4.82 (2H, s, CH₂), 7.62 (1H, t, J=7.8 Hz, Ar 5-H), 7.69

(1H, d, J=7.8, Hz, Ar 4-H), 8.20 (1H, s, Ar 2-H), 8.21

(1H, d, J=7.8 Hz, Ar 6-H); MS m/z 193 (MꢂH).

1,1-Dimethylethyl N-(4-(N-(2-hydroxyethyl)thioureido-

methyl)phenylmethyl)carbamate (24d). Compound 22d

was treated with 2-aminoethanol, as for the synthesis of

24c (chromatography omitted), to give 24d (41%) as an

oil: NMR d_H1.44 (9H, s, Bu^t), 3.69 (4H, m, 2 ꢄ CH₂),

4.24 (2H, d, J=5.4 Hz, ArCH₂), 4.67 (2H, s, ArCH₂),

5.04 (1H, s, OH), 6.88 (1H, br, NH), 7.18 (1H, br, NH),

7.20 (2H, d, J=7.0 Hz, Ar 3,5-H₂), 7.26 (3H, m, Ar 2,6-

H₂+NH); IR (ﬁlm) n_max1171, 1682, 3297 cm^ꢂ1; MS m/

z 340.1695 (M+H) (C₁₆H₂₆N₃O₃S requires 340.1684),

679 (2M+H), 284 (MꢂMe₂C¼CH₂).

4-Nitrophenylmethylisothiocyanate (22f). 4-Nitrobenzy-

lamine 21f was treated with thiophosgene, as for the

synthesis of 22c (chromatography omitted), to give 22f

(56%) as a colourless oil: (lit.³⁶mp 37–38 ^ꢀC); IR (ﬁlm)

n

CH₂), 7.52 (2H, d, J=8.9 Hz, Ar 2,6-H₂), 8.25 (2H, d,

J=8.9 Hz, Ar 3,5-H₂); MS m/z 195 (M+H).

_max1347, 1531, 2070, 3079 cm^ꢂ1; NMR d_H4.88 (2H, s,

N-(2-Hydroxyethyl)-N⁰-(3-nitrophenylmethyl)thiourea

(24e) and 2,2-dimethyl-3-(N-(3-nitrophenylmethyl)ami-

nothiocarbonyl)tetrahydrooxazole (25e). Compound 22e

was treated with 2-aminoethanol, as for the synthesis of

24c (chromatographic eluant EtOAc), to give 25e

(50 mg, 19%) as a colourless oil: IR (ﬁlm) n_max1144,

1346, 1534, 3413 cm^ꢂ1; NMR d_H1.80 (6H, s, 2 ꢄ Me),

3.82 (2H, br, oxazole 4-H₂), 4.05 (4H, t, J=6.6 Hz,

oxazole 5-H₂), 5.02 (2H, d, J=5.4 Hz, CH₂Ar), 5.70

(1H, br, NH), 7.55 (1H, dd, J=8.2, 7.8 Hz, Ar 5-H),

7.72 (1H, d, J=7.8 Hz, Ar 4-H), 8.15 (1H, d, J=8.2 Hz,

Ar 6-H), 8.16 (1H, s, Ar 2-H); MS m/z 296.1066

(M+H) (C₁₃H₁₈N₃O₃S requires 296.1069). Further elu-

tion gave 24e (180 mg, 70%) as pale buﬀ crystals: mp

79–81 ^ꢀC; IR n_max1144, 1348, 1531, 3402 cm^ꢂ1; NMR

3-Methoxyphenylmethylisothiocyanate (22g). 3-Methoxy-

benzylamine 21g was treated with thiophosgene, as for

the synthesis of 22c (chromatographic eluant EtOAc/

hexane (1:1)), to give 22g (40%) as a colourless oil:

(lit.³⁷oil): IR (ﬁlm) n_max2094 cm^ꢂ1; NMR d_H3.82

(3H, s, Me), 4.68 (2H, s, CH₂), 6.87 (3H, m, Ar 2,4,6-

H₃), 7.30 (1H, t, J=7.8 Hz, Ar 5-H); MS m/z 180.0460

(M+H)

(MꢂNCS).

(C₉H₁₀NOS

requires

180.0483),

121

4-Methoxyphenylmethylisothiocyanate (22h). 4-Methoxy-

benzylamine 18h was treated with thiophosgene, as for

the synthesis of 22g, to give 22h (80%) as a pale yellow

oil: (lit.⁴⁸oil); IR (ﬁlm) n_max2087 cm^ꢂ1; NMR d_H3.81

(3H, s, Me), 4.63 (2H, s, CH₂), 6.93 (2H, d, J=8.2 Hz,

Ar 3,5-H₂), 7.22 (2H, d, J=8.2 Hz, Ar 2,6-H₂); MS m/z

179 (M), 121 (MꢂNCS).

d

_H3.61 (2H, br, NCH₂), 3.82 (2H, t, J=4.7 Hz, OCH₂),

4.89 (2H, d, J=5.4 Hz, ArCH₂), 6.78 (1H, br, OH), 7.28

(1H, br, NH), 7.50 (1H, t, J=7.4 Hz, Ar 5-H), 7.77 (1H,

d, J=7.4 Hz, Ar 4-H), 8.11 (1H, d, J=7.4 Hz, Ar 6-H),

8.16 (1H, s, Ar 2-H); MS m/z 256.0753 (M+H)

(C₁₀H₁₄N₃O₃S requires 256.0756).

Methyl 4-(isothiocyanatomethyl)benzoate (22k). Com-

pound 21k was treated with thiophosgene, as for the

synthesis of 22f, to give 22k (83%) as a pale yellow oil:

(lit.⁴⁹oil); IR (ﬁlm) n_max1715, 2089 cm^ꢂ1; NMR

((CD₃)₂SO) d_H3.88 (3H, s, Me), 5.05 (2H, s, CH₂), 7.49

(2H, d, J=8.4 Hz, Ar 3,5-H₂), 7.98 (2H, d, J=8.4 Hz,

Ar 2,6-H₂); MS m/z 208 (M+H), 192 (MꢂMe).

N-(2-Hydroxyethyl)-N⁰-(4-nitrophenylmethyl)thiourea

(24f) and 2,2-dimethyl-3-(N-(4-nitrophenylmethyl)ami-

nothiocarbonyl)tetrahydrooxazole (25f). Compound

22f was treated with 2-aminoethanol, as for the synth-

esis of 24e and 25e, to give 25f (13%) as a colourless oil:

IR (ﬁlm) n_max1143, 1346, 1541, 3380 cm^ꢂ1; NMR d_H

1.79 (6H, s, 2 ꢄ Me), 3.82 (2H, m, CH₂N), 4.05 (2H, t,

J=6.2 Hz, OCH₂), 5.03 (2H, d, J=5.4 Hz, ArCH₂), 5.67

(1H, br, NH), 7.49 (2H, d, J=8.8 Hz, Ar 2,6-H₂) 8.17

(2H, d, J=8.8 Hz, Ar 3,5-H₂); MS m/z 296.1063

(M+H) (C₁₃H₁₈N₃O₃S requires 296.1069). Further elu-

tion gave 24f (34%) as a colourless oil: IR (ﬁlm) n_max

1160, 1346, 1562, 3368 cm^ꢂ1; NMR d_H3.62 (2H, br,

CH₂Ar), 3.82 (2H, t, J=5.0 Hz, NCH₂), 4.90 (2H, d,

J=5.8 Hz, OCH₂), 6.54 (1H, br, OH), 7.10 (1H, br,

NH), 7.26 (1H, br NH), 7.50 (2H, d, J=8.9 Hz, Ar

Methyl 3-(isothiocyanatomethyl)benzoate (22i). Com-

pound 21i was treated with thiophosgene, as for the

synthesis of 22e, to give 22i (51%) as a colourless oil:

(lit.⁵⁰oil); IR (ﬁlm) n_max1722, 2101 cm^ꢂ1; NMR d_H3.94

(3H, s, Me), 4.79 (2H, s, CH₂), 7.50 (1H, t, J=7.8Hz, Ar

5-H), 7.54 (1H, d, J=7.8 Hz Ar 4-H), 8.0 (1H, d,

J=7.8 Hz, Ar 6-H), 8.03 (1H, s, Ar 2-H); MS m/z 208.0434

(C₁₀H₁₀NO₂S requires 208.0432), 149 (MꢂNCS).

1,1-Dimethylethyl N-(3-(N⁰-(2-hydroxyethyl)thioureidome-

thyl)phenylmethyl)carbamate (24c). Compound 22c

C. L. M. Goodyer et al. / Bioorg. Med. Chem. 11 (2003) 4189–4206

4203

2,6-H₂), 8.19 (2H, d, J=8.9 Hz, Ar 3,5-H₂); MS m/z

256.0755 (M+H) (C₈H₁₀N₃O₂S requires 256.0756).

evaporation residue, in CH₂Cl₂, was washed with aq

NaHCO₃and dried. Evaporation and chromatography

(CH₂Cl₂) gave 27c (900 mg, 56%) as white crystals: mp

124–126 ^ꢀC (lit.⁵¹mp 75–76 ^ꢀC); NMR d_H1.39 (9H, s,

Bu^t), 4.35 (2H, d, J=6.1 Hz, CH₂), 5.23 (1H, br, NH),

7.40 (1H, dd, J=8.9, 7.8 Hz, Ar 5-H), 7.54 (1H, d,

J=7.8 Hz, Ar 6-H), 8.03 (1H, d, J=8.9 Hz, Ar 4-H),

8.04 (1H, s, Ar 2-H); MS m/z 253.1184 (M+H)

(C₁₂H₁₇N₂O₄requires 253.1188), 197 (MꢂMe₂C¼CH₂);

Found C, 57.30: H, 6.35; N, 11.20; C₁₂H₁₅N₂O₄

requires C, 57.14; H, 6.35; N, 11.11%.

N-(2-Hydroxyethyl)-N⁰-(3-methoxyphenylmethyl)thio-

urea (24g). Compound 22g was treated with 2-ami-

noethanol, as for the synthesis of 24e and 25e, to give

24g (85%) as a colourless oil: NMR ((CD₃)₂SO) d_H3.36

(4H, m, 2 ꢄ CH₂), 3.67 (3H, s, Me), 4.55 (2H, m,

CH₂Ar), 4.78 (1H, s, OH), 6.80 (1H, dd, J=7.4, 2.1 Hz,

Ar 6-H), 6.86 (1H, dd, J=7.4, 2.1 Hz, Ar 4-H), 6.85

(1H, s, Ar 2-H), 7.23 (1H, dd, J=7.4 Hz, Ar 5-H), 7.52

(1H, br, NH), 7.92 (1H, br, NH); NMR ((CD₃)₂SO) d_C

46.7, 48.5, 55.3, 112.9, 113.2, 119.7, 129.7, 159.6; MS m/z

241.1009 (M+H) (C₁₁H₁₇N₂O₂S requires 241.1011).

1,1-Dimethylethyl

N-(4-nitrophenylmethyl)carbamate

(27d). 4-Nitrobenzylamine 21f was treated with Boc₂O,

as for the synthesis of 27c, to give 27d (91%) as white

crystals: mp 111–114 ^ꢀC (lit.⁵²mp 109–110 ^ꢀC); NMR

d_H1.53 (9H, s, Bu^t), 4.42 (2H, d, J=5.9 Hz, CH₂), 5.05

(1H, br, NH), 7.44 (2H, d, J=8.6 Hz, Ar 2,6-H₂), 8.19

(2H, d, J=8.6 Hz, Ar 3,5-H₂); MS m/z 505 (2 M+H),

406 (2 M+HꢂBoc), 275 (M+Na), 253 (M+H), 197

(MꢂMe₂C¼CH₂), 180 ( MꢂBu^tO).

N-(2-Hydroxyethyl)-N⁰-(4-methoxyphenylmethyl)thio-

urea (24h). 3-Methoxyphenylisothiocyanate 22h was

treated with 2-aminoethanol, as for the synthesis of 24e

and 25e (reaction time 2 h), to give 24h (47%) as a col-

ourless oil: IR (ﬁlm) n_max1171, 3336 cm^ꢂ1; NMR d_H

3.49 (3H, s, Me), 3.66 (2H, br, CH₂N), 3.81 (2H, d,

J=4.8 Hz, CH₂O), 4.58 (2H, s, ArCH₂), 6.48 (1H, br,

NH), 6.74 (1H, br, NH), 6.87 (2H, d, J=8.6 Hz, Ar 3,5-

H₂), 7.25 (2H, d, J=8.6 Hz, Ar 2,6-H₂); MS m/z

241.1011 (M+H) (C₁₁H₁₇N₂O₂S requires 241.1018).

1,1-Dimethylethyl N-(3-aminophenylmethyl)carbamate

(28c). Compound 27c was treated with SnCl₂, as for

the synthesis of 28d (reaction time 30 min), to give 28c

(31%) as a pale buﬀ oil (lit.⁵³oil); NMR d_H1.40

(9H, s, Bu^t), 3.59 (2H, br, NH₂), 4.15 (2H, d,

J=5.7 Hz, CH₂), 4.70 (1H, br, NH), 6.55 (3H, m, Ar

2,4,6-H₃), 7.06 (1H t, J=7.7 Hz, Ar 5-H); MS m/z

222 (M), 167 (MꢂMe₂C¼CH₂), 121 (MꢂBoc), 106

(MꢂBocNH).

Methyl 3-(N⁰-(2-hydroxyethyl)thioureidomethyl)benzoate

(24i). Compound 22i was treated with 2-aminoethanol,

as for the synthesis of 24e and 25e (reaction time 2.5 h),

to give 24i (77%) as a colourless oil: IR (ﬁlm) n_max1197,

1715, 3355 cm^ꢂ1; NMR (CD₃OD) d_H3.60 (2H, m,

CH₂), 3.66 (2H, m, CH₂), 3.89 (3H, s, Me), 4.80 (2H, s,

CH₂Ar), 7.44 (1H, t, J=7.8 Hz, Ar-H₅), 7.56 (1H, d,

J=7.8 Hz, Ar-H₄), 7.88 (1H, d, J=7.8 Hz, Ar-H₄), 7.96

(1H, s, Ar-H₂); MS m/z 537 (2 M+H), 269.0955

(M+H) (C₁₂H₁₇N₂O₃S requires 269.0960).

1,1-Dimethylethyl N-(4-aminophenylmethyl)carbamate

(28d). Compound 27d was treated with SnCl₂, as for

the synthesis of 16l (chromatography omitted), to give

28d (31%) as a colourless oil: (lit.⁵⁴mp 75–76 ^ꢀC);

NMR d_H1.45 (9H s Bu^t), 3.8 (2H, br, NH₂), 4.18 (2H,

d, J=5.1 Hz, CH₂), 4.73 (1H, br, NH), 6.64 (2H, d,

J=8.2 Hz, Ar 2,6-H₂), 7.07 (2H, d, J=8.2 Hz, Ar 3,5-

H₂); MS m/z 445 (2 M+H), 222 (M+H), 165

(MꢂMe₂C¼CH₂).

Methyl 4-(N⁰-(2-hydroxyethyl)thioureidomethyl)benzoate

(24k) and 2,2-dimethyl-3-(N-(4-methoxycarbonylphenyl-

methyl)aminothiocarbonyl)tetrahydrooxazole

(25k).

Compound 22k was treated with 2-aminoethanol, as

for the synthesis of 24e and 25e, to give 25k (280 mg,

43%) as white crystals: mp 105–107 ^ꢀC; IR n_max1199,

1701, 3359 cm^ꢂ1; NMR ((CD₃)₂SO) nd_H1.69 (6H, s,

Me₂C), 3.82 (3H, s, OMe), 3.64 (2H, t, J=6.3 Hz, oxa-

zole 4-CH₂), 3.96 (2H, t, J=6.3 Hz, oxazole 5-H₂), 4.82

(2H, d, J=5.5 Hz, ArCH₂), 7.39 (2H, d, J=8.2 Hz, Ar

3,5-H₂), 7.88 (2H, d, J=8.2 Hz, Ar 2,6-H₂), 7.84 (1H,

br, NH); MS m/z 309.1263 (M+H) (C₁₅H₂₁N₂O₃S

requires 309.1273). Further elution gave 24k (64%) as

pale buﬀ crystals: mp 75–77 ^ꢀC; IR (ﬁlm) n_max1194,

1714, 3351 cm^ꢂ1; NMR ((CD₃)₂SO) d_H3.47 (4H, m, 2 ꢄ

CH₂), 3.82 (3H, s, Me), 4.74 (3H, m, CH₂Ar+OH),

7.38 (2H, d, J=8.2 Hz, Ar 3,5-H₂), 7.89 (2H, d,

J=8.2 Hz, Ar 2,6-H₂), 7.58 (1H, br, NH), 8.01 (1H, br,

NH); MS m/z 269.0961 (M+H) (C₁₂H₁₆N₂O₃S requires

269.0960).

Methyl 3-aminophenylacetate hydrochloride (28q). 3-

Aminophenylacetic acid 28o (2.0 g, 13.2 mmol) was stir-

red with MeOH (350 mL) and SOCl₂(20 mL) for 4 d.

Evaporation gave 28q (2.6 g, 99%) as a colourless

hygroscopic gum: (lit.⁵⁵mp 167–170 ^ꢀC); NMR

((CD₃)₂SO) d_H3.60 (2H, s, CH₂), 3.41 (3H, br, N⁺H₃),

3.74 (3H, s, Me), 7.15 (3H, m, Ar-H₃), 7.41 (1H, m, Ar

5-H); MS m/z 166 (M+H).

Methyl 4-aminophenylacetate hydrochloride (28r). 4-

Aminophenylacetic acid 28p was treated with MeOH

and SOCl₂, as for the synthesis of 28q, to give 28r (99%)

as oﬀ-white crystals: mp 118–120 ^ꢀC (lit.⁵⁶mp 197–

199 ^ꢀC); NMR ((CD₃)₂SO) d_H3.47 (3H, br, NH₃), 3.59

(3H, s, Me), 3.68 (2H, s, CH₂), 7.19 (2H, d, J=8.6 Hz,

Ar 3,5-H₂), 7.30 (2H, d, J=8.6 Hz, Ar 2,6-H₂); MS m/z

166 (M+H), 121 (MꢂCO₂H).

1,1-Dimethylethyl

N-(3-nitrophenylmethyl)carbamate

(27c). Di-tert-butyl dicarbonate (1.7 g, 7.8 mmol) was

added slowly to 3-nitrobenzylamine 21e (1.0 g,

6.6 mmol) and Et₃N (1.1 g, 11 mmol) in CH₂Cl₂(25 mL)

at 0 ^ꢀC and the mixture was stirred for 16 h. The

1,1-Dimethylethyl N-(3-isothiocyanatophenylmethyl)car-

bamate (29c). Compound 28c was treated with thio-

phosgene, as for the synthesis of 22c (reaction time 2 d),

4204

C. L. M. Goodyer et al. / Bioorg. Med. Chem. 11 (2003) 4189–4206

to give 29c (70%) as white crystals: mp 70–72 ^ꢀC; IR

n_max1675, 2100, 3180 cm^ꢂ1; NMR ((CD₃)₂SO) d_H1.39

(9H, s, Bu^t), 4.12 (2H, d, J=5.9 Hz, CH₂), 7.23 (3H, m,

Ar 2,4,6-H₃), 7.43 (1H, t, J=7.7 Hz, Ar 5-H), 7.46 (1H,

br, NH); MS m/z 265.1012 (M+H) (C₁₃H₁₇N₂O₂S

requires 265.1011), 209 (MꢂMe₂C¼CH₂); Found C,

58.50; H, 6.02; N, 10.40; C₁₃H₁₆N₂O₂S requires C,

59.00; H, 6.06; N, 10.60%.

N-(2-Hydroxyethyl)-N⁰-(3-methoxyphenyl)thiourea (30g).

3-Methoxyphenylisothiocyanate 29g was treated with

2-aminoethanol, as for the synthesis of 24d (reaction

time 2 h), to give 30g (84%) as white crystals: mp 129–

131 ^ꢀC; IR n_max1149, 2900, 3186 cm^ꢂ1

;

NMR

((CD₃)₂SO) d_H3.52 (4H, m, 2 ꢄ CH₂), 3.70 (3H, s, Me),

4.80 (1H, br, OH), 6.65 (1H, d, J=8.2 Hz, Ar 6-H), 6.90

(1H, d, J=7.4 Hz, Ar 4-H), 7.20 (2H, m, Ar 2,5-H₂),

7.71 (1H, br, NH), 9.41 (1H, s, NH); MS m/z 452

(2 M+H), 227.0846 (M+H) (C₁₀H₁₅N₂O₂S requires

227.0854); Found C, 53.4: H, 6.28; N, 12.36;

C₁₀H₁₄N₂O₂S requires C, 53.08; H, 6.24; N, 12.38%.

1,1-Dimethylethyl N-(4-isothiocyanatophenylmethyl)car-

bamate (29d). Compound 28d was treated with thio-

phosgene, as for the synthesis of 22c, to give 29d (26%)

as a pale yellow powder: mp 113–115 ^ꢀC; IR n_max1683,

2123, 3366 cm^ꢂ1; NMR d_H1.46 (9H, s, Bu^t), 4.29 (2H,

d, J=5.6 Hz, CH₂), 4.91 (1H, br, NH), 7.18 (2H, d,

J=8.6 Hz, Ar 2,6-H₂), 7.27 (2H, d, J=8.6 Hz, Ar 3,5-

H₂); MS m/z 529 (2 M+H), 265.1006 (M+H)

N-(2-Hydroxyethyl)-N⁰-(4-methoxyphenyl)thiourea (30h).

4-Methoxyphenylisothiocyanate 29h was treated with

2-aminoethanol, as for the synthesis of 24e and 25e

(reaction time 1.5 h), to give 30h (88%) as pale buﬀ

crystals: mp 147 ^ꢀC (lit.⁵⁸mp 146–147 ^ꢀC); IR n_max1165,

2835, 3189, 3646 cm^ꢂ1; NMR ((CD₃)₂SO) d_H3.35 (3H,

s, Me), 3.73 (4H, s, 2 ꢄ CH₂), 4.79 (1H, br, OH), 6.88

(2H, d, J=8.6 Hz, Ar 3,5-H₂), 7.24 (2H, d, J=8.6 Hz,

Ar 2,6-H₂), 7.46 (1H, br, NH), 9.41 (1H, s, NH); MS

m/z 227.0850 (M+H) (C₁₀H₁₅N₂O₂S requires

227.0854); Found C, 53.0; H, 6.17; N, 12.2;

C₁₁H₁₄N₂O₂S requires C, 53.08; H, 6.24; N, 12.38%.

(C₁₃H₁₇N₂O₂S

requires

265.1011),

209

(MꢂMe₂C¼CH₂); Found C, 58.10: H, 5.92; N, 10.30;

C₁₃H₁₆N₂O₂S 0.25H₂O requires C, 58.08; H, 6.19; N,

10.42%.

Methyl 3-isothiocyanatophenylacetate (29q). Compound

28q was treated with thiophosgene, as for the synthesis

of 22g, to give 29q (77%) as a pale yellow liquid: IR

(ﬁlm) n_max1738, 2119 cm^ꢂ1; NMR ((CD₃)₂SO) d_H3.61

(2H, s, CH₂), 3.71 (3H, s, Me), 7.26 (3H, m, Ar-H₃),

7.30 (1H, t, J=7.8 Hz, Ar 5-H); MS m/z 208.0432

(M+H) (C₁₀H₁₀NO₂S requires 208.0432), 192

(M+HꢂMe), 148 (M+HꢂNCS).

Methyl 3-(N⁰-(2-hydroxyethyl)thioureido)phenylacetate

(30q). Compound 29q was treated with 2-aminoetha-

nol, as for the synthesis of 24e and 25e, to give 30q

(64%) as a colourless oil: IR (ﬁlm) n_max1061, 1732,

3293 cm^ꢂ1; NMR ((CD₃)₂SO) d_H3.34 (2H, m, CH₂N),

3.52 (2H, br, CH₂O), 3.59 (3H, s, Me), 3.63 (2H, m,

CH₂Ar), 4.70 (1H, s, OH), 6.97 (1H, d, J=7.4 Hz, Ar 4-

H) 7.24 (1H, dd, J=7.8, 6.3 Hz, Ar 5-H), 7.28 (1H, s, Ar

2-H), 7.33 (1H, d, J=6.3 Hz, Ar 6-H), 7.69 (1H, s, NH),

9.60 (1H, br, NH); MS m/z 537 (2 M+H), 269.0953

(M+H) (C₁₂H₁₇N₂O₃S requires 269.0960).

Methyl 4-isothiocyanatophenylacetate (29r). Compound

28r was treated with thiophosgene, as for the synthesis

of 22f, to give 29r (83%) as pale buﬀ oil: (lit.⁵⁷mp 168–

170 ^ꢀC); IR n_max1738, 2120 cm^ꢂ1; NMR ((CD₃)₂SO) d_H

3.61 (3H, s, Me), 3.73 (2H, s, CH₂), 7.34 (2H, d,

J=8.6 Hz, Ar 2,6-H₂), 7.39 (2H, d, J=8.6 Hz, Ar 3,5-

H₂); MS m/z 208 (M+H), 192 (MꢂMe).

1,1-Dimethylethyl N-(3-(N⁰-(2-hydroxyethyl)thioureido)-

phenylmethyl)carbamate (30c). Compound 28c was

treated with 2-aminoethanol, as for the synthesis of 24e

and 25e (reaction time 2 h), to give 30c (43%) as a col-

ourless oil; IR n_max(ﬁlm) 1164, 1693, 3380 cm^ꢂ1; NMR

((CD₃)₂SO) d_H1.39 (9H, s, Bu^t), 3.50 (2H, br, CH₂NH),

3.52 (2H, m, CH₂O), 4.04 (2H, d, J=6.6 Hz,

CH₂NHBoc), 4.80 (1H, s, OH), 6.96 (1H, d, J=7.4 Hz,

Ar 4-H), 7.23 (2H, m, NH+Ar 2-H), 7.36 (2H, m, Ar

5,6-H₂), 7.66 (1H, br, NH), 9.60 (1H, br, NH); MS m/z

651 (2 M+H), 326.1541 (M+H) (C₁₅H₂₄N₃O₃S

requires 326.1538), 270 (MꢂMe₂C¼CH₂).

1,1-Dimethylethyl N-4-(N⁰-(2-hydroxyethyl)thioureido-

phenylmethyl)carbamate (30d). Compound 29d was

treated with 2-aminoethanol, as for the synthesis of 24e

and 25e, to give 30d (45%) as a colourless oil; IR (ﬁlm)

n_max1166, 1689, 3323 cm^ꢂ1; NMR ((CD₃)₂SO) d_H1.39

(9H, s, Bu^t), 3.32 (2H, m, CH₂), 3.52 (2H, m, CH₂), 4.07

(2H, d, J=6.2 Hz, CH₂NHBoc), 4.94 (1H, br, OH), 7.15

(2H, d, J=8.2 Hz, Ar 3,5-H₂), 7.33 (2H, d, J=8.2 Hz,

Ar 2,6-H₂), 7.38 (1H, br, NH), 7.65 (1H, br, NH), 9.57

(1H, br, NH); MS m/z 326.1552 (M+H) (C₁₅H₂₄N₃O₃S

requires 326.1538).

Methyl 4-(N⁰-(2-hydroxyethyl)thioureido)phenylacetate

(30r). Compound 29r was treated with 2-aminoethanol,

as for the synthesis of 24e and 25e, to give 30r (27%) as

pale yellow crystals: mp 53–55 ^ꢀC; IR n_max1169, 1730,

3325, 3480 cm^ꢂ1; NMR d_H2.35 (1H, br, OH), 3.64 (2H,

s, CH₂), 3.72 (3H, s, Me), 3.80 (4H, m, 2 ꢄ CH₂), 6.56

(1H, br, NH), 7.20 (2H, d, J=7.8 Hz, Ar 3,5-H₂), 7.33

(2H, d, J=7.8 Hz, Ar 2,6-H₂), 7.94 (1H, br, NH); MS

m/z 537 (2 M+H), 269.0933 (M+H) (C₁₂H₁₆N₂O₃S

requires 269.0960).

(R)-N-(2-Hydroxypropyl)-N⁰-(3-methoxyphenyl)thiourea

(31). Compound 29g (500 mg, 3.0 mmol) in acetone

(2.1 mL) was added dropwise during 30 min to (R)-1-

aminopropan-2-ol (420 mg, 4.0 mmol) in acetone

(2.1 mL). The mixture was boiled under reﬂux for 2 h.

Evaporation and chromatography (EtOAc/hexane 1:1)

gave 31 (270 mg, 38%) as a colourless oil: NMR d_H1.22

(3H, d, J=6.3 Hz, Me), 3.46 (1H, m, CHNH), 3.81 (3H,

s, OMe), 3.94 (1H, m, CHNH), 4.02 (1H, CHOH),

6.64 (1H, br, NH), 6.78 (3H, m, Ar 2,4,6-H₃), 7.33

(1H, t, J=8.2 Hz, Ar 5-H), 7.76 (1H, br, NH); IR

20

D

, MeOH); MS m/z 241.1007 (C₁₁H₁₇N₂O₂S

(ﬁlm) n_max1180, 3369 cm^ꢂ1; ½a ¼ ꢂ7:2^ꢀ(c 1.4 mg

mL^ꢂ1

requires 241.1011).

C. L. M. Goodyer et al. / Bioorg. Med. Chem. 11 (2003) 4189–4206

4205

(S)-2-(3-Methoxyphenylamino)-5-methyl-4,5-dihydrothi-

Acknowledgements

azole hydrochloride (32). Compound 31 (80 mg,

0.3 mmol) was boiled under reﬂux for 24 h in aq HCl

(6 M, 4 mL). Evaporation gave 32 (70 mg, 99%) as a

colourless hygroscopic gum; NMR d_H1.51 (3H, d,

J=5.9 Hz, thiazole-Me), 3.72 (1H, m, 4-H), 3.81

(3H, s, OMe), 4.11 (2H, m, 4-H+5-H), 6.81 (1H, m,

Ar 6-H), 6.88 (1H, d, J=8.2 Hz, Ar 4-H), 7.30 (1H,

s, Ar 2-H), 7.32 (1H, t, J=8.2 Hz, Ar 5-H), 10.67

We are very grateful to the Association for Interna-

tional Cancer Research for a Project Grant which gen-

erously supported this work. We also thank Dr. S. J.

Black (University of Bath) for the NMR spectra and

Mr. C. Cryer (University of Bath) for the mass spectra.

(1H, br, NH), 12.29 (1H, br, NH); IR (ﬁlm) n_max

20

½ꢀ ¼ ꢂ32:4^ꢀ(c 2.6 mg mL^ꢂ1

1629, 3434 cm^ꢂ1

MeOH); MS m/z 223.08963 (C₁₁H₁₆N₂OS requires

223.0905).

;

,

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Article Doi

Synthesis of N-benzyl- and N-phenyl-2-amino-4,5-dihydrothiazoles and thioureas and evaluation as modulators of the isoforms of nitric oxide synthase

DOI: 10.1016/S0968-0896(03)00451-6

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