Protein structure-based design, synthesis, and biological evaluation of 5-thia-2,6-diamino-4(3H)-oxopyrimidines: Potent inhibitors of glycinamide ribonucleotide transformylase with potent cell growth inhibition

2502

J . Med. Chem. 1997, 40, 2502-2524

P r otein Str u ctu r e-Ba sed Design , Syn th esis, a n d Biologica l Eva lu a tion of

5-Th ia -2,6-d ia m in o-4(3H)-oxop yr im id in es: P oten t In h ibitor s of Glycin a m id e

Ribon u cleotid e Tr a n sfor m yla se w ith P oten t Cell Gr ow th In h ibition

Michael D. Varney,* Cindy L. Palmer, William H. Romines III, Theodore Boritzki, Stephen A. Margosiak,

Robert Almassy, Cheryl A. J anson, Charlotte Bartlett, Eleanor J . Howland, and Rosanne Ferre

Agouron Pharmaceuticals, Inc., 3565 General Atomics Court, San Diego, California 92121

Received October 25, 1996^X

The design, synthesis, biochemical, and biological evaluation of a novel series of 5-thia-2,6-

diamino-4(3H)-oxopyrimidine inhibitors of glycinamide ribonucleotide transformylase (GART)

are described. The compounds were designed using the X-ray crystal structure of human GART.

The monocyclic 5-thiapyrimidinones were synthesized by coupling an alkyl thiol with 5-bromo-

2,6-diamino-4(3H)-pyrimidinone, 20. The bicyclic compounds were prepared in both racemic

and diastereomerically pure forms using two distinct synthetic routes. The compounds were

found to have human GART K_is ranging from 30 µM to 2 nM. The compounds inhibited the

growth of both L1210 and CCRF-CEM cells in culture with potencies down to the low nanomolar

range and were found to be selective for the de novo purine biosynthesis pathway. The most

potent inhibitors had 2,5-disubstituted thiophene rings attached to the glutamate moiety.

Placement of a methyl substituent at the 4-position of the thiophene ring to give compounds

10, 18, and 19 resulted in inhibitors with significantly decreased mFBP affinity.

In tr od u ction

modes of the inhibitors was available to guide structure-

activity studies. We, along with other investigators,¹⁰

have recently reported the solution of the X-ray crystal

structure of the Escherichia coli GART in both its apo

and complexed form, and in addition, we have reported

the cloning and expression of an active soluble form of

the human enzyme.¹¹Herein, we report the use of the

X-ray crystal structure of both the E. coli and the human

GART enzymes, complexed with a number of inhibitors,

in the design of a class of 5-thiapyrimidinone inhibitors

and the synthesis and biological evaluation of these

inhibitors as antitumor agents.

The design of small-molecule antagonists of folic acid,

acting at a number of critical folate binding enzymes,

has been an active area of drug discovery research for

over 50 years and has resulted in the development of a

number of clinically useful anticancer agents.¹Only

relatively recently however have the two folate-utilizing

enzymes in the de novo purine biosynthesis pathway,

glycinamide ribonucleotide transformylase (GART, EC

2.1.2.2) and aminoimidazole carboxamide ribonucleotide

transformylase (AICART, EC.2.1.2.3), been recognized

as useful targets in the search for new, potent, and

selective antitumor agents.^1b

Design

The first of the folate dependent enzymes in the

pathway, GART, catalyzes the formylation of the glycine

nitrogen of glycinamide ribonucleotide 1 using N-10-

formyltetrahydrofolic acid 2 as the cofactor.²Validation

of GART as a legitimate anticancer target came in the

mid 1980s with the discovery of the first potent and

selective inhibitor, 5,10-dideaza-5,6,7,8-tetrahydrofolic

acid (DDATHF, 3a ).³The C-6 R isomer of DDATHF

3b,⁴referred to as Lometrexol, eventually entered

clinical trials and demonstrated antitumor activity

against a wide range of solid tumors.⁵Additional

investigations have examined many of the structure-

activity relationships of the DDATHF core.⁶The most

potent compound reported to date in this class is the

thiophene derivative 3c.⁷In addition to the bicyclic

pyrimidinones 3a -c, a family of monocyclic pyrimidi-

nones represented by the structures 4a ,b have also been

reported to have selective GART inhibition.⁸

The starting point for our design effort was the X-ray

crystal structure of E. coli GART complexed with the

potent inhibitor 5-deaza-5,6,7,8-tetrahydrofolic acid

(5dTHF).^10aAnalysis of the E. coli structure reveals

that there are 20 residues that contribute to the folate

binding pocket (this analysis excluded the glutamate

binding region since this is at the solvent interface and

all of the designs were expected to contain the glutamate

moiety), and of these, 12 use at least part of their side

chain to form the binding site. An overlap of the

sequences of the human and the E. coli enzymes in this

active site region revealed that all of the side chains

that contribute to folate binding are identical in both

enzymes except for the conservative change of residue

143 (E. coli numbering) from a Leu in E. coli to a Val in

human, thus lending credence to the usefulness of the

E. coli structure as a surrogate for the human enzyme

in the initial designs.

The antitumor activity of the previous GART inhibi-

tors, and for that matter most glutamate containing

antifolates, has been shown to be dependent upon three

properties: enzyme inhibition, active cellular transport,

and polyglutamation.⁹Previously, however, no struc-

tural information regarding the protein or the binding

Initial design work focused on the region of the active

site surrounding the methylene group at the 5-position

of 5dTHF. An overlap of the van der Waals surfaces of

the inhibitor and the protein showed that this area of

the active site is hydrophobic and is not completely filled

by the CH₂group. It was felt that a somewhat bulkier

sulfur atom might better fill the available space. In

addition, introducing the sulfur atom onto the pyrimi-

^XAbstract published in Advance ACS Abstracts, J uly 1, 1997.

Glycinamide Ribonucleotide Transformylase Inhibitors

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16 2503

difference in the hydrophilic interactions between pro-

tein and ligand are seen at the protein solvent interface

where the glutamate moiety is located. In contrast to

the E. coli case, in the complex of 7 with the human

enzyme, there is a pair of hydrogen bonds between the

two oxygens of the R-carboxylate of the glutamate and

one of the guanidine NH groups of the Arg 64 side chain

and the backbone NH of Ile 91. All of the hydrophobic

residues that make up the active site in the human

protein are in essentially the same positions to that seen

in E. coli.

After a detailed analysis of this structure, we drew

the following conclusions: (1) Given that all of the

available hydrogen-bonding groups on the pyrimidinone

portion of the molecule are complemented by the

protein, making changes in this region would likely not

be productive, (2) since the R-carboxylate of the glutamate

moiety appears to make a set of strong hydrogen bonds

with the protein, and since the glutamate is critical for

membrane transport and intracellular activity due to

polyglutamation, making changes in this region would

also not likely be productive, and (3) the most striking

aspect of the structure is that the atoms of the inhibitor

that link the sulfur at the 5-position of the pyrimidinone

to the glutamate portion are flexible and do not opti-

mally fill the available space. As a result, all of the

designed derivatives were intended to investigate ways

to more efficiently fill this region of the active site and

to reduce the number of conformations each inhibitor

has available.

As can be seen in Figure 1, there are hydrophobic

residues Val 143 and Met 89 that make up the opening

of the active site on both sides of the thiophene ring. It

was felt that small hydrophobic substituents (such as

methyl or ethyl) could be placed at either the 3- or the

4-position of the thiophene ring that could make favor-

able interactions with these residues. A methyl and an

ethyl group were placed at the 3-position to give

compounds 9 and 11, and these two compounds showed

small but measurable increases in GART inhibition.

Compound 10, with a methyl at the 4-position, was

equipotent with the parent compound 7. In an attempt

to fill more of the space and to restrict the conforma-

tional flexibility of the linker, compound 13 was de-

signed. As a result of the fact that compound 13 has a

dinone ring might confer some synthetic advantage over

the methylene group. A search of the chemical litera-

ture revealed that no glutamate-containing 5-thia an-

tifolates had been reported.¹²To test this design

hypothesis, the monocyclic 5-thiapyrimidinones 5-8

(see Table 1) were prepared and tested for human GART

inhibition.¹³Compounds 5 and 7 (with the four atom

linker) showed good GART inhibition (confirming that

the sulfur was a suitable substitute for the CH₂), while

the compounds 6 and 8 (with one less linker atom) were

significantly less active.

Crystals of human GART were grown in the presence

of the substrate GAR 1 and compound 7, and the X-ray

structure of this complex was solved by the molecular

replacement technique¹⁴using the coordinates of the

previously solved E. coli enzyme^10aand is shown in

Figure 1. The structure shows that from the standpoint

of the protein active site, the human enzyme is es-

sentially identical to that of E. coli. In addition, with

the exception of the additional water molecules in the

human structure due to higher resolution and more

extensive refinement, all of the hydrophilic interactions

with the pyrimidine portion of 7 are identical to those

made to 5dTHF in the E. coli structure.^10aThe only

new chiral center, and that the S-CH₂

substituent can

be situated in either a pseudoaxial or equatorial orien-

tation, models of the four possible binding isomers were

built and evaluated in the active site of the protein. The

modeling study indicated that only two of the four

isomers could fit into the active site without extensive

overlap with the protein. Molecular mechanics calcula-

tions were performed in which these two isomers were

minimized in the protein in order to try to predict the

likely binding mode.¹⁴The calculations indicated that

the energy of these two binding modes were similar with

the pseudoaxial isomer being lower by 1-2 kcal/mol.

Compound 13 was synthesized, and it was found to be

equipotent with the parent compound 7 in terms of

GART inhibition. The X-ray crystal structure of human

GART, as a ternary complex with GAR 1 and compound

13, was solved and is shown in Figure 2. Compound

13 is bound with the pyrimidinone and the R-carboxy-

late essentially overlapping the compound 7 structure,

and the substituent on the 6-membered ring is in a

2504 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16

Ta ble 1. Structure-Activity Data of Monocyclic 5-Thiapyrimidinones

Varney et al.

F igu r e 1. Stereodrawing showing the X-ray structure of human GART complexed with GAR 1 and compound 7. The linker and

the thiophene ring are shown exiting the active site flanked by the Val 143 side chain on the left and the Met 89 side chain on

the right. The R-carboxyl of the glutamate moiety is shown hydrogen bonding to the Arg 64 side chain.

pseudoequatorial orientation. The bound isomer is one

of the isomers that by modeling looked like it would

have extensive overlap with the protein, and careful

analysis shows that in this crystal structure, in order

to relieve this unfavorable interaction, the ligand has

pushed the side chain of Val 143 and its local peptide

backbone back by as much as 1 Å from the position seen

in the compound 7 structure. It would appear that the

energy cost of moving this backbone segment is com-

pensated by a more favorable hydrophobic interaction.

In our experience, this type of large change in protein

structure is difficult to anticipate and underscores the

value of iterative structural information.

The final set of designed compounds were the bicyclic

5-thiapyrimidinones 14-19. These were prepared to

test the concept of ligand preorganization as with

Glycinamide Ribonucleotide Transformylase Inhibitors

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16 2505

F igu r e 2. Stereodrawing showing the X-ray structure of human GART complexed with GAR 1 and compound 13. The linker

attached to the six-membered ring of the tetrahydrobenzthiophene is in an equatorial orientation. The Val 143 side chain has

shifted to the left roughly 1 Å from that seen in the structure of compound 7.

F igu r e 3. Stereodrawing showing the X-ray structure of human GART complexed with GAR 1 and compound 17. The linker

attached to the six-membered ring of the pyrimidothiazine is in an equatorial orientation. The substrate GAR 1 (residue 222) is

on the lower right.

Sch em e 1.^aGeneral Synthesis of Monocyclic

5-Thiapyrimidinones

compound 13. In addition, in previous GART inhibitor

series,^3-8the bicyclic compounds, as a rule, showed

increased activity over the monocyclic pyrimidinones.

The 2,5-thienyl compounds 16-19 showed tighter bind-

ing than did the 1,4-phenylene 14. We believe this is

due, at least in part, to the better orientation of the

glutamate toward Arg 64 and Ile 91, and this is

consistent with the 1,3-phenylene 15 having superior

inhibition. The X-ray crystal structure of human GART,

as a ternary complex with GAR 1 and compound 17,

was solved and is shown in Figure 3. The most

significant observation from this structure is that the

orientation of the C-6 S linker (which is analogous to

the natural configuration) is equatorial. This is con-

sistent with the lower GART inhibition of this compound

when compared to the C-6 R isomer 16 because the

methylene groups of the linker can no longer make a

favorable interaction with the side chain of Val 143. In

addition, because the linker now runs along the bottom

portion of the active site, where the catalytic Asp 144

and His 108 residues reside, the nitrogen of the sub-

strate GAR (residue 222) has been rotated from the

previous structures by roughly 90°.^10aThis is presum-

a

(a) DIEA, DMF, ∆; (b) NaOH; (c) L-glutamate diethyl ester,

ably to avoid a steric clash, but the energetic conse-

quences of this movement are unknown. Finally, as can

be seen with compounds 18 and 19, placement of a

methyl group at the 4-position of the thiophene ring

conferred no increased GART inhibition.

coupling agent; (d) NaOH.

Ch em istr y

Shown in Scheme 1 is the general strategy for the

synthesis of the monocyclic 5-thiapyrimidinones 5-13.

2506 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16

Sch em e 2.^aGeneral Synthesis of Thiols 21a -b

Varney et al.

a

(a) Propargyl alcohol, (Ph₃P)₂PdCl₂, CuI, Et₂NH; (b) H₂, Pd/C, EtOH; (c) Ph₃P, CBr₄, CH₂Cl₂; (d) KSAc, acetone, ∆; (e) 1 N HCl,

MeOH; (f) Ph₃P, DEAD, HSAc, THF.

Sch em e 3.^aSynthesis of Thiol 21i

reaction to prepare compound 14 as a mixture of

diastereomers.¹⁹The synthesis began with the coupling

of the lithium anion of TMS acetylene with glyoxal

dimethyl acetal followed by the palladium-catalyzed

coupling of the resulting terminal acetylene with methyl

4-iodobenzoate.^8aSubsequent reduction of the alkyne

gave compound 31. Initially, we felt that conversion of

the secondary alcohol to a small leaving group would

allow for introduction of the sulfur atom by simple

displacement, and as a result, the mesylate of compound

31 was prepared. In the event, we were not able to find

any nucleophile that would displace the mesylate group

even under forcing conditions. One possible explanation

for this unexpected result is the proximity of the two

adjacent oxygen atoms of the dimethyl acetal which

precludes any positive charge from building up during

the transition state making the activation barrier too

high. The solution to the problem was to hydrolyze the

acetal to the aldehyde, displace the mesylate with a

protected sulfur atom, and reintroduce the acetal in the

form of a dioxolane group. Removal of the sulfur

protecting group²⁰provided the thiol 32. Base-initiated

coupling followed by removal of the acetal under acidic

conditions provided the aldehyde which spontaneously

cyclized to a diastereomeric mixture of hemiaminals.^8b

The reduction of the hemiaminal to give the thiazine

ring containing compound 33 proved difficult. Eventu-

ally it was found that sodium cyanoborohydride in the

presence of BF₃‚Et₂O could be used successfully.²¹The

synthesis was completed as described for the earlier

compounds.

a

(a) NaH, (CH₃O)₂CO, DMF; (b) NaBH₄, THF, MeOH; (c)

Et₃SiH, BF₃‚Et₂O, CH₂Cl₂; (d) LAH, THF; (e) TBSCl, TEA, CH₂Cl₂;

(f) BuLi, THF, CO₂; (g) L-glutamate diethyl ester, EDC, HOBT,

DIEA, DMF; (h) TBAF, THF; (i) CBr₄, Ph₃P, CH₂Cl₂; (j) KSAc,

acetone, ∆; (k) 0.5 N HCl, EtOH.

The key reaction in the sequence is the base-initiated

coupling of the alkyl thiols 21a -i with the 5-bromo-2,6-

diamino-4(3H)-pyrimidinone (20)¹⁵to give the thioethers

22a -i. The reaction proceeds under relatively mild

conditions in yields ranging from 67% with the simple

thiols like 21d to 19% for the more complex thiol 21i

and is compatible with both the aromatic ester func-

tionality and the diethyl glutamate. Displacements of

this type have only been reported previously with

aromatic thiols.¹⁶

The thiols 21a -h were prepared as shown in Scheme

2. The intermediate alcohols either were prepared by

coupling of propargyl alcohol with an aromatic halide^8a

followed by reduction or were purchased. The thiols

were generated from the intermediate thioacetates by

acid hydrolysis. The thioacetates were prepared by

either displacing the corresponding bromides or directly

using thioacetic acid under Mitsunobu conditions.¹⁷

The synthesis of the thiol 21i is shown in Scheme 3.

The synthesis begins with the known ketone 25¹⁸and

proceeds by one-carbon homologation and deoxygena-

tation. Once the alcohol was protected as a tert-

butyldimethylsilyl ether, the carboxyl group was intro-

duced by selective lithiation of the 5-position of the

thiophene. Standard peptide coupling followed by in-

troduction of the thiol went without event.

An alternate synthetic route, shown in Scheme 5, was

used to prepare the optically pure thiophene derivatives

16-19. This strategy, which is based in part on work

described for 5-thiapterin ring systems by Benkovic,²²

has as the key reaction the guanidine cyclization of the

lactams 37a -d to produce the bicyclic pyrimidothiazine

ring system. The chiral centers for both the C-6(R) and

C-6(S) isomers (using folic acid numbering) were ob-

tained from the known optically pure acetylene 34²³

prepared from D-(R)-glyceraldehyde acetonide.²⁴The

synthesis began with the palladium-catalyzed coupling

of the optically pure form of the acetylene 34 with both

2-bromothiophene-5-carboxylic acid ethyl ester^8cand the

3-methyl derivative²⁵followed by reduction and acid

hydrolysis to afford the diol intermediates 35a ,b. For

the C-6(R) compounds, the nitrogen atom at the 8-posi-

tion was introduced by monotosylation of the primary

alcohol followed by displacement with sodium azide. For

the C-6(S) compounds, the hydroxy group was inverted

by protecting the primary alcohol as a silyl ether,

mesylating the secondary alcohol, removing the protect-

ing group, and forming the inverted epoxide with

Two distinct synthetic strategies were developed to

prepare the bicyclic 5-thiapyrimidinones 14-19.¹²The

first route, shown in Scheme 4, utilized the coupling of

thiol 32 with the bromopyrimidinone 20 as the key

Glycinamide Ribonucleotide Transformylase Inhibitors

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16 2507

Sch em e 4.^aSynthesis of Pyrimidothiazine 14

a

(a) Methyl 4-iodobenzoate, (Ph₃P)₂PdCl₂, CuI, Et₂NH; (b) H₂, 5% Pd/C, EtOH; (c) MsCl, Et₃N, CH₂Cl₂; (d) TFA, H₂O, CHCl₃; (e)

(4-methoxyphenyl)methanethiol, DIEA, DMF; (f) ethylene glycol, PPTS; (g) Hg(OAc)₂, CH₂Cl₂, TFA; (h) 20, DIEA, DMF, ∆; (i) 2 N HCl,

THF, H₂O; (j) BF₃‚Et₂O, NaBH₃CN, THF; (k) NaOH; (l) L-glutamate diethyl ester, EDC, HOBT, DIEA, DMF; (m) NaOH.

Sch em e 5.^aSynthesis of Pyrimidothiazines 16-19

a

(a) Bromothiophene ethyl ester, (Ph₃P)₂PdCl₂, CuI, Et₃N, CH₃CN; (b) H₂, 5% Pd/C, EtOH; (c) TsOH, EtOH; (d) TsCl, Et₃N, CH₂Cl₂;

(e) NaN₃, DMF or CH₃CN, ∆; (f) (BOC)₂O, H₂, 5% Pd/C, THF; (g) TBSCl, Et₃N, CH₂Cl₂; (h) MsCl, Et₃N, CH₂Cl₂; (i) TBAF, THF; (j) NaH,

THF; (k) MsCl, Et₃N, CH₂Cl₂; (l) KSAc, acetone; (m) (CH₃OCO)₂CHCl, K₂CO₃, MeOH; (n) TFA, CH₂Cl₂; (o) (CH₃)₃O‚BF₄, CH₂Cl₂; (p)

guanidine hydrochloride, NaOEt, EtOH; (q) NaOH; (r) ^L-glutamate diethyl ester, EDC, HOBT, DIEA, DMF; (s) NaOH.

sodium hydride. The epoxide was opened selectively at

the less hindered primary position with sodium azide.²⁶

The azido alcohols were reduced in the presence of BOC

anhydride to give the alcohols 36a -d . The sulfur atom

at the 5-position was introduced by the displacement

of the corresponding mesylate with KSAc.²⁷The acetyl

protecting group was removed under basic conditions

in the presence of dimethyl chloromalonate, and once

the BOC protecting group was removed, the free amine

spontaneously cyclized to the lactams 37a -d . The

lactam was alkylated with trimethyloxonium tetrafluo-

roborate, and the resultant lactim ether was treated

with 3 equiv of free-base guanidine in refluxing ethanol

to give the 2-amino-4(3H)-oxopyrimido[5,4-b][1,4]-

thiazines. Hydrolysis of the ethyl esters, peptide cou-

pling, and hydrolysis completed the synthesis of the

glutamic acids 16-19.

The compounds were evaluated for cell growth inhibi-

tion against three cell lines: L1210 murine leukemia;³⁰

CCRF-CEM,³⁰a human lymphoblastic leukemia line of

T-cell origin; and L1210/CI920, a cell line that was

developed for resistance to Fostriecin which lacks the

reduced folate carrier function.³¹Results are reported

as IC₅₀values (in µM). As a measure of the selectivity

of the compounds for the de novo purine biosynthesis

pathway, cell growth inhibition protection experiments

were performed with and without the addition of 100

µM hypoxanthine. The results are reported as the ratio

of the IC₅₀with 100 µM hypoxanthine to the IC₅₀

without hypoxanthine added. Larger numbers are

indicative of increased specificity.

The ability of the inhibitors to serve as substrates for

folyl-polyglutamate synthetase (FPGS) was measured

using the enzyme prepared from rat liver.³⁵The results

are reported as the ratio of the velocity V_mdivided by

the K_mof the test compound. Larger numbers indicate

that the compounds are better substrates.

Bioch em istr y a n d Biology

The compounds shown in Tables 1 and 2 were all

evaluated for their inhibition of recombinant human

GART using N ¹⁰-formyl-5,8-dideazafolate (FDDF) as

the cofactor and were found to be competitive with

respect to FDDF.²⁸Results are reported as K_ivalues

(in µM).²⁹

Selected compounds were also measured for their

ability to function as transport substrates for the human

folate-binding protein (mFBP). The assay was a com-

petitive binding assay in which membrane-associated

mFBP was prepared from cultured KB cells³⁸and the

2508 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16

Ta ble 2. Structure-Activity Data of Bicyclic 5-Thiapyrimidinones

Varney et al.

individual compounds were allowed to compete against

[³H]folic acid. The results are reported as K_dvalues (in

nM).

the bicyclic compounds in Table 2 showed superior

activity over the monocyclic compounds in Table 1. The

one exception is compound 15 which showed poor cell

growth inhibition. The most potent compound in the

5-thiapyrimidinone series is compound 17 with cell

growth inhibition IC₅₀s of 4 nM against both L1210 and

CCRF-CEM cell lines. As described below, the cell

growth inhibition activity is a composite of a number

factors.

Discu ssion

The in vitro cell growth inhibition activity of these

glutamate-containing GART inhibitors is largely de-

pendent upon three measurable properties: GART

enzyme inhibition, FPGS substrate activity, and the

ability of the compounds to function as substrates for

either one or both of folate transporters (the reduced

folate transporter or the mFBP). In addition, although

a more complicated situation to interpret, in vivo

activity is also influenced by the same set of properties.⁹

As can be seen from Tables 1 and 2, the 5-thiapyrim-

idinones 5-19 have GART inhibition constants ranging

from 30 µM to 2 nM. The bicyclic compounds 14-19

are, in general, more potent as enzyme inhibitors than

are the monocyclic compounds 5-13. We believe this

is due in part to preorganization of the more rigid

bicyclic compounds. Among the bicyclic compounds, the

unnatural C-6(R) compounds 16 and 18 are 7-10 times

more potent than the corresponding natural C-6(S)

isomers 17 and 19. This increased potency of the

unnatural C-6(R) isomers is believed to occur because

the linker connecting the thiazine ring to the aromatic

thiophene ring on these compounds is oriented in a

pseudoaxial conformation which positions the two meth-

ylene groups nicely against the side chain of Val 143.

As shown in Figure 3, for the natural C-6(S) isomer

compound 17, the linker is in a pseudoequatorial

conformation which does not allow this compound to

make the same favorable interactions.

Hypoxanthine shift experiments showed that the cell

growth inhibition for all the compounds was due to a

block in the de novo purine biosynthesis pathway. In

particular, compounds 16-19 were very selective in this

regard with protection ratios of over 1500.

Folyl polyglutamate synthetase (FPGS)³⁵is the in-

tracellular enzyme that extends the glutamate chain on

monoglutamated cofactors by sequentially adding

glutamate moieties to the γ-carboxylic acid as a mech-

anism for enhancing the binding to target enzymes and

retaining the cofactors inside the cell. Many of the

known folate analogues also function as substrates for

this enzyme which serves to trap the inhibitors inside

the cells and build them to high concentrations. As a

result, we tested compounds 5-19 for their ability to

serve as substrates for the readily available rat enzyme,

and as can be seen from Tables 1 and 2, a number of

the compounds are good substrates. In general, the

compounds that are good substrates are also potent

inhibitors of cell growth, whereas poor substrates like

compounds 11, 15, and 18 tend to also have poor cell

growth inhibition activity.

The relative activity of individual compounds 5-19

against the L1210/CI920 cell line versus L1210 is an

indirect measure of the dependence on the reduced

folate carrier. As evidenced by the observed poor cell

growth inhibition against the L1210/CI920 cell line, all

the compounds are dependent to some extent upon the

The compounds 5-19 were tested for their cell growth

inhibition and were found to have IC₅₀values ranging

from the low micromolar to the low nanomolar range.

Again, as was the case with GART enzyme inhibition,

Glycinamide Ribonucleotide Transformylase Inhibitors

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16 2509

or MHW Laboratories, Phoenix, AZ, and gave results for the

elements stated with (0.4% of the theoretical values. N,N-

Dimethylformamide (DMF) was dried over activated (250 °C)

4-Å molecular sieves. Tetrahydrofuran (THF) was distilled

from sodium benzophenone ketyl under nitrogen. Et₂O refers

to diethyl ether, DIEA refers to diisopropylethylamine, TBSCl

refers to tert-butyldimethylsilyl chloride, TEA refers to tri-

ethylamine, HOBT refers to 1-hydroxybenzotriazole hydrate,

EDC refers to 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide

hydrochloride and DMAP refers to 4-(dimethylamino)pyridine.

Flash chromatography was performed using silica gel 60

(Merck Art 9385). Thin layer chomatograph (TLC) were

performed on precoated sheets of silica 60 F₂₅₄(Merck Art

reduced folate carrier for in vitro activity. This is an

important observation since a number of human tumors

are known to have the reduced folate carrier.³⁹

The membrane folate binding protein (mFBP) is a

high-affinity low-flux transporter which has a prefer-

ence for folic acid.³⁹One of the unique properties of the

benchmark GART inhibitor, DDATHF, is the fact that

it is transported by both the reduced folate transporter

and the mFBP into cells.⁴⁰Investigators have shown

that concurrent folic acid treatment in both low folate

animal models and in humans, which is believed to

block the mFBP, can attenuate the toxicity of DDATHF.⁴¹

As a result, the compounds 5-19 were tested for their

binding affinity to the mFBP. All of the compounds

have at least moderate affinity for the mFBP, and

compounds 14, 16, and 17 are very tight binders to the

mFBP with K_dvalues in the picomolar range. In

addition, a relationship between the substitution pat-

tern on the aromatic ring and the mFBP affinity was

discovered. That is, a simple replacement of the hy-

drogen at the 4-position of the thiophene ring with a

methyl group decreases mFBP binding by a factor of

889, 1900, and 215 for the three pairs of compounds 7

and 10, 16 and 18, and 17 and 19, respectively, without

significantly affecting enzyme inhibition. In addition,

compound 19 is still a good substrate for FPGS and

retains much of the cell growth inhibition of the parent

compound 17.⁴²

5719). Melting points were determined on

a Mel-TeMp

apparatus and are uncorrected. mFBP refers to human folate-

binding protein; FPGS, folyl-polyglutamate synthetase; BSA,

bovine serum albumin; FDDF, N ¹⁰-formyl-5,8-dideazafolate;

HEPES, N-(2-hydroxyethyl)piperazine-N ′-2-ethanesulfonic acid;

Tris, tris(hydroxymethyl)aminomethane; EDTA, ethylenedi-

aminetetraacetic acid; PMSF, phenylmethanesulfonyl fluoride;

₂₉₄, extinction coeficient at 294 nm; K_i, inhibition constant;

K_i,app, apparent inhibition constant; K_d, equilibrium disociation

constant; K_m, Michaelis constant; V_max, maximal velocity.

Bioch em ica l Assa ys. GART activity was measured using

a modification of the method of Young.²⁸Reaction mixtures

contained the catalytic domain of the human GART,¹¹20 µM

R,â-GAR, 10 or 20 µM FDDF, a variable concentration of GART

inhibitor, 50 mM HEPES-KOH, pH 7.5, and 50 mM KCl. The

reaction was initiated with the addition of enzyme and followed

by monitoring the increase in absorbance at 294 nm at 20 °C

(

₂₉₄) 18.9 mM^-1cm^-1). GART inhibition constants (K_i) were

determined from the dependence of the steady-state catalytic

rate upon inhibitor and substrate concentration. The type of

inhibition observed was determined to be competitive with

Con clu sion

respect to FDDF by the dependence of the apparent K_i(K_i,app

)

We have described a series of 5-thiapyrimidinone

GART inhibitors that were designed using the X-ray

crystal structure of human GART complexed with a

number of inhibitors. The most potent compounds have

on the concentration of FDDF and was shown to be described

by K_i,app) K_i+ (K_i/K_m)[FDDF]. The Michaelis constant for

FDDF, K_m, was determined independently by the dependence

of the catalytic rate upon FDDF concentration and shown to

be 0.6 µM. Data for both the K_mand K_ideterminations were

fitted by nonlinear methods to the Michaelis equation or the

Michaelis equation for competitive inhibition as appropriate.

Data resulting from tight-binding inhibition was analyzed, and

K_ivalues were determined by fitting the data to the tight-

binding equation of Morrison²⁹by nonlinear methods.

inhibition constants in the low nanomolar range.

A

synthetic route to the monocyclic 5-thiapyrimidinone

compounds 5-13 was developed in which the key

reaction was the coupling of an alkyl thiol with 5-bromo-

2,6-diamino-4(3H)-pyrimidinone (20). The bicyclic com-

pounds 14-19 were prepared using two distinct syn-

thetic routes which generated both the racemic products

14 and 15 and the diastereomerically pure compounds

16-19. A number of the compounds were found to have

cell growth IC₅₀values in the low nanomolar range, and

all compounds, as evidenced by hypoxanthine protec-

tion, were found to selectively inhibit cell growth by

blocking the de novo purine biosynthesis pathway.

Placement of a methyl substituent at the 4-position of

the thiophene ring of compounds 7, 16, and 17 gave

inhibitors with significantly decreased mFBP affinity.

From this series of 5-thiapyrimidinone GART inhibitors,

compound 17 has shown excellent in vivo antitumor

activity⁴³and has been chosen for clinical evaluation.

The ability of GART inhibitors to serve as substrates for

FPGS was measured using the enzyme prepared from rat liver.

FPGS was partially purified from rat liver essentially by the

method of Moran and Colman.³⁵Sprague-Dawley rats were

killed by CO₂inhalation. The liver was removed and perfused

though the portal vein with 20 mL of ice-cold 20 mM HEPES,

pH 7.4; 250 mM sucrose; 50 mM 2-mercaptoethanol. The

tissue was kept at 4 °C or on ice for the remainder of the

procedure. Livers were rinsed in fresh perfusion buffer,

weighed, and then blended in 2 times the wet-weight volume

of fresh perfusion buffer containing 0.5 mM PMSF; 50 µg/mL

soybean trypsin inhibitor; and 0.5 mM EDTA until smooth.

The homogenate was filtered though cheesecloth and the

filtrate centrifuged at 130000g for 60 min. The resulting

supernatant was filtered through fine glass wool to remove

suspended fats, and ATP was added to 2.5 mM. Protein was

precipitated with 30% saturated ammonium sulfate and

centrifuged at 30000g for 15 min. The resulting pellet was

resuspended in a small volume of 20 mM Tris-Cl, pH 7.4, 50

mM 2-mercaptoethanol, 5 mM MgCl₂, 5 mM ATP, and 25 µg/

mL soybean trypsin inhibitor and then centrifuged as before.

Suspended fats were removed from the supernatant, and

proteins were precipitated with 50% saturated ammonium

sulfate and then centrifuged as before to obtain the pellet.

Protein pellets precipitated in this manner were stable, when

stored at -70 °C, for more than 4 months. Since FPGS is quite

unstable in solution, a frozen FPGS pellet was resuspended

for assay as described below and used immediately. The pellet

was thawed and resuspended in a minimal amount of buffer

(20 mM Tris-Cl, pH 7.4, 50 mM 2-mercaptoethanol, 5 mM

MgCl₂, 5 mM ATP, and 25 µg/mL soybean trypsin inhibitor)

Exp er im en ta l Section

¹H NMR spectra were determined using a General Electric

QE-300 spectrometer operating at a field strength of 300 MHz.

Chemical shifts are reported in parts per million (δ) and setting

the references such that in CDCl₃the CHCl₃is at 7.26 ppm

and in DMSO-d₆the DMSO is at 2.49 ppm. Standard and

peak multiplicities are designated as follows: s, singlet; d,

doublet; dd, doublet of doublets; t, triplet; brs, broad singlet;

brd, broad doublet; br, broad signal; m, multiplet. Mass

spectra were determined at the Scripps Reaseach Institute

Mass Spectrometry Center. Infrared absorption spectra were

taken on a MIDAC Corporation FTIR. Elemental microanaly-

ses were performed by Atlantic Microlab Inc., Norcross, GA,

2510 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16

Varney et al.

and centrifuged to remove residual solids. The supernatant

was desalted by passage though a Sephadex G25 column

equilibrated in 20 mM Tris, pH 7.4, 50 mM 2-mercaptoethanol,

and 5 mM MgCl₂. Fractions containing protein were pooled

and brought to 5 mM ATP. Protein concentrations were

determined by the Bradford method with BSA as a protein

standard.³⁶Typical protein concentrations were 3 mg/mL. The

typical assay mixture contained 0.7 mg of partially purified

FPGS protein along with 200 mM Tris-Cl, pH 8.5, a variable

concentration of GART inhibitor or folic acid, 5 mM ATP, 10

mM MgCl₂, 30 mM KCl, 50 mM 2-mercaptoethanol, 1 mM

glutamic acid, and 2 µCi [³H]glutamic acid in a volume of 0.5

mL. Reactions were initiated with the addition of enzyme and

incubated for 60 min at 37 °C. The reaction was then

quenched with 0.5 mL of charcoal solution and placed on ice

to complete product absorption. (Preparation of the charcoal

solution and the important purification of [³H]glutamic acid

was accomplished as described previously.³⁷) The charcoal was

separated from the reaction mixture by centrifugation and the

resulting pellet was washed four times with 1 mL of 10 mM

glutamate, pH 6.8, and 10 mM 2-mercaptoethanol by repeated

resuspension and centrifugation. The products are finally

eluted from the charcoal with 1 mL of ethanolic ammonia (3

M NH₄OH; 60% ethanol) by resuspension and centrifugation.

The radioactivity in 1 mL of eluent supernatant containing

the tritiated product was measured by scintillation counting

after the addition of 1 mL of water. The kinetic parameters

K_mand V_maxwere determined by nonlinear fitting of the

substrate dependence of product formation to the Michaelis

equation.

The dissociation constant (K_d) mFBP was determined for

GART inhibitors with a competitive binding assay using

membrane-associated FBP prepared from cultured KB cells.³⁸

Adherent KB cells were scraped from flasks, washed once in

ice-cold PBS, and centrifuged at 5000g for 5 min at 4 °C. KB

cell pellets were stored at -70 °C, and mFBP activity was

stable for at least 6 months. Cell pellets containing ap-

proximately 2 × 10⁸cells were resuspended in 10 mL of

suspension buffer (KH₂PO₄-KOH, pH 7.4:10 mM EDTA:10

mM 2-mercaptoethanol), sonicated briefly to complete cell lysis,

and centrifuged at 12000g for 10 min at 4 °C. The pellet was

stripped of endogenous bound folate by resuspension in 20 mL

of acidic buffer (50 mM KH₂PO₄-KOH, pH 3.5:10 mM EDTA:

10 mM 2-mercaptoethanol) and centrifuged as before. The

pellet was then resuspended in 20 mL of the suspension buffer

at pH 7.4 and centrifuged as before. The pellet was resus-

pended in 5 mL of suspension buffer at pH 7.4 lacking EDTA.

Protein content was quantitated using the Bradford method

with BSA as a protein standard.³⁶Typical yields for this

procedure were 4-5 mg of total membrane protein per 2 ×

10⁸cells. This final suspension was used as a source of

membrane-associated human FBP. In this competitive bind-

ing assay, competitor ligand was allowed to compete against

[³H]folic acid for binding to mFBP. Reactions mixtures

contained 50-100 µg of cell membrane protein containing 3-6

pmol (3-6 nM) of FBP, 17.25 pmol of [³H]folic acid (17.25 nM,

0.5 µCi), and various concentrations of competitor ligand, in 1

mL of 50 mM KH₂PO₄-KOH, pH 7.4:10 mM 2-mercapto-

ethanol. Binding reactions were performed at 25 °C. Because

of the rapid binding and very slow release of bound [³H]folic

acid, the competitor ligand was prebound for 30 min in the

absence of [³H]folic acid. [³H]Folic acid was then added, and

the mixtures were allowed to equilibrate for 2.5 h. The full

reaction mixtures were drawn though nitrocellulose filters

under vacuum to trap the cell membranes with bound [³H]-

folic acid. The trapped membranes were then washed four

times with 1 mL of reaction buffer. The amount of bound [³H]-

folic acid was measured by scintillation counting of the

nitrocellulose membrane. The data obtained were nonlinearly

fitted as described above. The mFBP K_dfor [³H]folic acid, used

to calculate the competitor K_d, was obtained by direct titration

of mFBP with [³H]folate and subsequent nonlinear fitting of

the data to a tight-binding K_dequation and found to be 60 (

14 pM.

Culture Collection. The L1210/CI920 cell line was developed

for resistance to Fostriecin³¹and lacks reduced folate carrier

function. L1210 cells were grown in RPMI-1640 medium³²and

supplemented with 5% dialyzed fetal calf serum.³³The CEM

and L1210/CI920 cell lines were grown in RPMI medium

supplemented with 10% dialyzed fetal calf serum. All cultures

were maintained at 37 °C, 5% air-CO₂in a humidified

incubator. Cell growth inhibition was measured by a modi-

fication of the method of Mosmann.³⁴Mid-log phase cells of

each cell line were diluted to 18 500 cells/mL in fresh RPMI-

1640 growth medium supplemented with dialyzed fetal calf

serum and then aliquoted into columns 2-12 of 96-well

microtiter plates. Column 1 was filled with the same volume,

135 mL, of fresh medium, without cells, for use as a blank.

The plates were then placed in a 37 °C, 5% air-CO₂incubator.

After 4 h, plates were removed from the incubator followed

by addition of drug solution, 15 mL/well in binary dilutions,

to columns 12 to 4. Wells containing test compound were

prepared in quadruplicate on each plate. To the wells in

columns 1 and 2 of the plates was added 15 mL of media,

without test compound. Column 3 received 15 µL of drug

diluent. The cells were then returned to the incubator for 72

(L1210 and CI920 cell lines) or 120 h (CEM cell line). For

reversal experiments media was supplemented with 100 µM

hypoxanthine. After drug exposure, 50 mL of 0.8 mg/mL of

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

(MTT) dissolved in tissue culture medium was added to each

well of all plates after which cells were returned to the

incubator. After 4 h all plates were removed from the

incubator and centrifuged at 1200 rpm for 7 min. Media was

siphoned off, and 150 mL of DMSO was added to each well of

all plates. Plates were then mixed at slow speed on a vortex

mixer for 1 h in the dark at room temperature. The extent of

metabolized MTT was measured spectrophotometrically at 540

nm on a Molecular Devices Vmax kinetic microplate reader.

The concentration of drug required to reduce cell growth by

50% as measured by MTT metabolism was determined by

interpolation between the OD (minus blank) immediately

above and below 50% of control OD (minus blank).

Molecu la r Mech a n ics Ca lcu la tion s on Com p ou n d 13.

These calculations were performed using the batchmin tool

within the program Macromodel version 3.5.⁴⁴Models of the

pseudoequatorial and pseudoaxial orientations of compound

13 were built, and charges were generated using MOPAC 6.⁴⁵

The ligands were each then placed in the active site of the

enzyme by overlaying the pyrimidine and glutamate portions

on top of the crystal structure of compound 5, and the

substructure was minimized using the AMBER forcefield⁴⁶

with the implicit solvent model.⁴⁷

Cr ysta llogr a p h y on Com p ou n d s 7, 13, a n d 17.⁵⁰The

GART domain (residues 808-1010) of the trifunctional human

GARS-AIRS-GART enzyme was purified as described previ-

ously.¹¹Following purification, GART was concentrated to 20

mg/mL in a buffer containing 25 mM Tris pH 7.0 and 1 mM

DTT. Crystallization was done by hanging-drop vapor diffu-

sion, mixing the protein and reservoir solution (38-44% MPD,

0.1 M Hepes, pH 7.2-7.6) in a 1:1 ratio, and equilibrating at

13 °C. Crystals would typically grow within 3 days and

measure 0.2 × 0.25 × 0.3 mm.

X-ray diffraction data were collected from ternary complex

crystals of GART, GAR 1 and inhibitor at 4 °C using a San

Diego Multiwire Systems 2-panel area detector and a Rigaku

AFC-6R monochomatic Cu KR X-ray source and goniostat

(Table 3). The space group was determined to be P3₂21, with

the cell constants shown below. The crystal structures of both

compound 7 and 13 complexes were solved by molecular

replacement using MERLOT.^14aThe search model consisted

of residues 1-209 from an E. coli GART ternary complex

structure (Protein Data Bank accession number 1cde). The

highest peak in the cross rotation function^14bwas used in

3-dimensional translation functions,^14cin search of Harker

vectors. The top peak in all five searches (i.e. from one

molecule to each of the five symmetry related molecules)

produced a consistent set of vectors that positioned the model.

After initial refinement with XPLOR,⁴⁹density was seen for

the substrate GAR 1 and the inhibitor. The final structures

Mea su r em en t of Tissu e Cu ltu r e IC₅₀’s. The L1210 and

CCRF-CEM cell lines were obtained from the American Type

Glycinamide Ribonucleotide Transformylase Inhibitors

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16 2511

(broad), 2953, 2838, 1717, 1607, 1437, 1310, 1283 cm^-1

¹H

NMR (CDCl₃) δ 2.46 (brs, 1H), 3.55 (s, 3H), 3.56 (s, 3H), 3.92

(s, 3H), 4.45 (d, 1H, J ) 5.4 Hz), 4.60 (d, 1H, J ) 5.3 Hz), 7.52

(d, 2H, J ) 8.3 Hz), 7.98 (d, 2H, J ) 8.3 Hz). Anal. (C₁₄H₁₆O₅)

C, H.

Ta ble 3. Summary of X-ray Data and Refinement for

Compounds 7, 13, and 17

;

7

13

17

resolution (Å)

cell (a, Å)

8.5-2.6

76.8

8.4-2.4

76.8

7.7-2.5

77.0

3-(3-H yd r oxy-4,4-d im et h oxyb u t -1-yn yl)b en zoic a cid

m eth yl ester : from alkyne 30; yellow oil; ¹H NMR (CDCl₃) δ

3.55 (3H, s), 3.56 (3H, s), 3.92 (3H, s), 4.44 (1H, d, J ) 5.4

Hz), 4.59 (1H, d, J ) 5.4 Hz), 7.39 (1H, t, J ) 7.8 Hz), 7.64

(1H, dt, J ) 7.8, 1.5 Hz), 7.98 (1H, dt, J ) 7.8, 1.5 Hz), 8.14

(1H, t, J ) 1.5 Hz). Anal. (C₁₄H₁₆O₅‚0.3H₂O) C, H.

5-[(2,2-Dim eth yl[1,3]dioxolan -4(S)-yl)eth yn yl]th ioph en e-

2-car boxylic acid eth yl ester : from 4(S)-ethynyl-2,2-dimethyl-

[1,3]dioxolane, 34;²³amber oil; [R]₅₈₉+36.6° (c ) 0.88, MeOH);

cell (c, Å)

101.5

9.0

101.5

6.1

101.9

6.8

R_merge(%)^a

total refls

51851

10684

17.5

50

0.017

3.0

53387

13534

17.9

64

0.017

3.1

38594

11545

15.5

67

0.016

3.0

unique refls

R factor (%)^b

no. solvent

bond dev (Å)^c

angle dev (deg)^c

a

IR (neat) 2986, 2226, 1715, 1451, 1255, 1223 cm^-1; H NMR

R_merge: 100 × ∑_h∑_i|I(h)i - I(h) |/∑_h∑_iI(h)i where I(h)i is the

1

ith measurement of reflection h and I(h)i is the mean intensity

(CDCl₃) δ 1.34 (t, 3H, J ) 7.0 Hz), 1.42 (s, 3H), 1.53 (s, 1H),

4.03 (dd, 1H, J ) 6.2, 1.8 Hz), 4.34 (dd, 1H, J ) 6.4, 1.6 Hz),

4.34 (q, 2H, J ) 7.1 Hz), 4.95 (dd, 1H, J ) 6.4, 0 Hz), 7.16 (d,

1H, J ) 3.9 Hz), 7.63 (d, 1H, J ) 3.9 Hz). Anal. (C₁₄H₁₆O₄S)

C, H, S.

b

from N measurements of reflection h. R factor: ∑||F_o| - |F_c||/

∑|F_o|. ^cAverage deviation from ideal values.

were obtained by manual model building in 2F_o- F_cand F_o-

F_celectron density maps followed by further refinement with

XPLOR (Table 3).

5-[(2,2-Dim eth yl[1,3]d ioxola n -4(S)-yl)eth yn yl]-4-m eth -

ylth iop h en e-2-ca r boxylic a cid eth yl ester : starting with

4(S)-ethynyl-2,2-dimethyl[1,3]dioxolane (34) and 2-bromo-3-

methylthiophene-5-carboxylic acid ethyl ester;²⁵orange oil;

[R]₅₈₉+36.7° (c ) 1.04, MeOH); IR (neat) 2986, 2936, 2222,

Gen er a l Meth od for Acetylen e Cou p lin g to Ar yl Ha -

lid es. 4-(3-Hyd r oxyp r op -1-yn yl)ben zoic Acid Meth yl Es-

ter . A mixture of methyl 4-iodobenzoate 23a (9.00 g, 34.4

mmol), cuprous iodide (65 mg, 0.34 mmol), propargyl alcohol

(1.93 g, 34.4 mmol), and bis(triphenylphosphine)palladium

chloride (121 mg, 0.17 mmol) in diethylamine (90 mL) was

stirred under an argon atmosphere overnight at room tem-

perature. After the solvent was removed in vacuo, the residue

was diluted with water (200 mL) and then extracted with C₆H₆

(3 × 100 mL) and EtOAc (75 mL). The combined organic

extracts were dried (Na₂SO₄) and concentrated to give a brown

solid which was purified by flash chromatography with hexane/

EtOAc (2:1) to give 5.44 g (83%) of the product as a yellow

solid: mp 81-82 °C; ¹H NMR (CDCI₃) δ 3.92 (3H, s), 4.52 (2H,

s), 7.49 (2H, d, J ) 8.3 Hz), 7.98 (2H, d, J ) 8.3 Hz). Anal.

(C₁₁H₁₀O₃) C, H.

1

1711, 1441, 1283, 1244 cm^-1; H NMR (CDCl₃) δ 1.36 (t, 3H,

J ) 7.0 Hz), 1.42 (s, 3H), 1.53 (s, 3H), 2.29 (s, 3H), 4.04 (dd,

1H, J ) 6.2, 1.8 Hz), 4.26 (dd, 1H, J ) 6.2, 1.8 Hz), 4.32 (q,

2H, J ) 7.0 Hz), 4.99 (t, 1H, J ) 6.0 Hz), 7.50 (s, 1H). Anal.

(C₁₅H₁₈O₄S) C, H, S.

Gen er a l P r oced u r e for H yd r ogen a t ion of Cou p led

Acetylen es. 4-(3-Hyd r oxyp r op yl)ben zoic Acid Meth yl

Ester (24a ). A mixture of methyl 4-(3-hydroxypropynyl)-

benzoate (3.0 g, 15.8 mmol) and 5% Pd/C (0.3 g, 10% wt equiv)

in ethanol (200 mL) was shaken in a Parr flask under 45 psi

of H₂for 3.5 h. The crude reaction mixture was filtered though

a pad of Celite, and the filtrate was concentrated to give a

green oil which was purified by flash chromatography with

hexane/EtOAc (2:1) to give 2.9 g (95%) of the product as a

yellow oil: ¹H NMR (CDCl₃) δ 1.91 (2H, tt, J ) 6.4, 7.5 Hz),

2.77 (2H, t, J ) 7.5 Hz), 3.68 (2H, t, J ) 6.4 Hz), 3.90 (3H, s),

7.27 (2H, d, J ) 8.3 Hz), 7.96 (2H, d, J ) 8.3 Hz). Anal.

(C₁₁H₁₄O₃‚0.3H₂O) C, H.

5-(3-Hyd r oxyp r op -1-yn yl)th iop h en e-2-ca r boxylic a cid

m eth yl ester : yellow solid; mp 66-68 °C; ¹H NMR (CDCl₃) δ

1.84 (1H, broad), 3.88 (3H, s), 4.52 (2H, broad), 7.15 (1H, d, J

) 3.9 Hz), 7.64 (1H, d, J ) 3.9 Hz). Anal. (C₉H₈O₃S) C, H, S.

2(S)-[[[5-(3-Hyd r oxyp r op -1-yn yl)-3-m eth ylth iop h en e-

2-yl]ca r b on yl]a m in o]p en t a n ed ioic a cid d iet h yl est er :

yellow oil; IR (neat) 3366, 2982, 2250, 1738, 1640, 1545, 1516,

2(S)-[[[5-(3-Hyd r oxyp r op yl)-3-m eth ylth iop h en e-2-yl]-

ca r bon yl]a m in o]p en ta n ed ioic a cid d ieth yl ester (24d ):

colorless oil; IR (neat) 3354, 2980, 2930, 1732, 1634, 1514,

1

1445, 1377 cm^-1; H NMR (CDCl₃) δ 1.24 (3H, t, J ) 7.1 Hz),

1

1447, 1377 cm^-1; H NMR (CDCl₃) δ 1.23 (3H, t, J ) 7.1 Hz),

1.30 (3H, t, J ) 7.1 Hz), 1.80 (1H, broad t), 2.04-2.46 (4H,

m), 2.47 (3H, s), 4.12 (2H, q, J ) 7.0 Hz), 4.24 (2H, q, J ) 7.1

Hz), 4.50 (2H, d, J ) 5.2 Hz), 4.73 (1H, m), 6.63 (1H, d, J )

7.3 Hz), 6.96 (1H, s). Anal. (C₁₈H₂₃NO₆S) C, H, N, S.

2(S)-[[[5-(3-Hyd r oxyp r op -1-yn yl)-4-m eth ylth iop h en e-

2-yl]ca r b on yl]a m in o]p en t a n ed ioic a cid d iet h yl est er :

yellow oil; IR (neat) 3329, 2980, 2222, 1738, 1634, 1557, 1532,

1.30 (3H, t, J ) 7.1 Hz), 1.93 (2H, quintet, J ) 7.2 Hz), 2.08-

2.46 (4H, m), 2.47 (3H, s), 2.88 (2H, t, J ) 7.6 Hz), 3.70 (2H,

t, J ) 6.3 Hz), 4.11 (2H, q, J ) 7.1 Hz), 4.23 (2H, q, J ) 7.1

Hz), 4.75 (1H, m), 6.47 (1H, d, J ) 7.3 Hz), 6.62 (1H, s). Anal.

(C₁₈H₂₇NO₆S) C, H, N, S.

2(S)-[[[5-(3-Hyd r oxyp r op yl)-4-m eth ylth iop h en e-2-yl]-

ca r bon yl]a m in o]p en ta n ed ioic a cid d ieth yl ester (24e):

colorless oil; IR (neat) 3337, 2980, 2938, 1738, 1632, 1560,

1

1447 cm^-1; H NMR (CDCl₃) δ 1.23 (3H, t, J ) 7.1 Hz), 1.29

(3H, t, J ) 7.2 Hz), 2.04 (3H, s), 2.07-2.50 (4H, m), 4.14 (2H,

q, J ) 7.1 Hz), 4.24 (2H, q, J ) 7.1 Hz), 4.54 (2H, s), 4.71 (1H,

m), 6.88 (1H, d, J ) 7.4 Hz), 7.27 (1H, s). Anal. (C₁₈H₂₃NO₆S)

C, H, N, S.

1

1530, 1449 cm^-1; H NMR (CDCl₃) δ 1.23 (3H, t, J ) 7.2 Hz),

1.30 (3H, t, J ) 7.1 Hz), 1.89 ((2H, quintet, J ) 7.2 Hz), 2.06-

2.49 (4H, m) 2.16 (3H, s), 2.85 (2H, t, J ) 7.5 Hz), 3.70 (2H, t,

J ) 6.3 Hz), 4.11 (2H, q, J ) 7.1 Hz), 4.23 (2H, q, J ) 7.2 Hz),

4.74 (1H, m), 6.67 (1H, d, J ) 7.6 Hz), 7.26 (1H, s). Anal.

(C₁₈H₂₇NO₆S) C, H, N, S.

2(S)-[[[5-(3-Hyd r oxyp r op -1-yn yl)-3-et h ylt h iop h en e-2-

yl]car bon yl]am in o]pen tan edioic acid dieth yl ester : brown

oil; IR (neat) 3364 (broad), 2980, 2224, 1732, 1638, 1543, 1516,

1206 cm^-1; ¹H NMR (CDCl₃) δ 1.25 (2 overlapping triplets, 6H),

1.30 (t, 3H, J ) 7.1 Hz), 2.12 (m, 1H), 2.25-2.54 (m, 3H), 2.90

(q, 2H, J ) 7.5 Hz), 4.12 (q, 2H, J ) 7.1 Hz), 4.23 (q, 2H, J )

7.1 Hz), 4.50 (s, 2H), 4.73 (m, 1H), 6.61 (d, 1H, J ) 7.3 Hz),

7.05 (s, 1H). Anal. (C₁₉H₂₅NO₆S) C, H, N, S.

2(S)-[[[5-(3-Hyd r oxyp r op yl)-3-eth ylth iop h en e-2-yl]ca r -

bon yl]a m in o]p en ta n ed ioic a cid d ieth yl ester (24f): color-

less oil; IR (neat) 3374 (broad), 2973, 2936, 1738, 1634, 1514,

1454, 1206 cm^-^{1 1}H NMR (CDCl₃) δ 1.23 (2 overlapping

;

triplets, 6H), 1.30 (t, 3H, J ) 7.1 Hz), 1.93 (m, 2H), 2.10 (m,

1H), 2.35 (m, 1H), 2.43 (m, 2H), 2.90 (t, 2H, J ) 7.7 Hz), 2.92

(t, 2H, J ) 7.8 Hz), 3.72 (t, 2H, J ) 6.6 Hz), 4.12 (q, 2H, J )

7.1 Hz), 4.23 (q, 2H, J ) 7.1 Hz), 4.74 (m, 1H), 6.45 (d, 1H, J

) 7.4 Hz), 6.70 (s, 1H). Anal. (C₁₉H₂₉NO₆S) C, H, N, S.

2(S)-[[3-(3-H yd r oxyp r op yl)b en zoyl]a m in o]p en t a n e-

d ioic a cid d ieth yl ester (24g): colorless oil; ¹H NMR (CDCl₃)

δ 1.22 (3H, t, J ) 7.1 Hz), 7.67 (1H, s), 1.31 (3H, t, J ) 7.1

Hz), 1.91 (2H, tt, J ) 6.3, 7.4 Hz), 2.19-2.08 (1H, m), 2.38-

2.26 (1H, m), 2.58-2.40 (2H, m), 2.77 (2H, t, J ) 7.4 Hz), 3.67

(2H, t, J ) 6.3 Hz), 4.11 (2H, q, J ) 7.1 Hz), 4.24 (2H, q, J )

2(S)-[[3-(3-H yd r oxyp r op -1-yn yl)b en zoyl]a m in o]p en -

ta n ed ioic a cid d ieth yl ester : orange oil; ¹H NMR (CDCl₃) δ

1.22 (3H, t, J ) 7.0 Hz), 1.30 (3H, t, J ) 7.1 Hz), 2.19-2.04

(1H, m), 2.39-2.26 (1H, m), 2.56-2.41 (2H, m), 4.11 (2H, q, J

) 7.0 Hz), 4.24 (2H, q, J ) 7.1 Hz), 4.49 (2H, s), 4.81 (1H,

ddd, J ) 4.8, 7.6, 12.5 Hz), 7.11 (1H, d, J ) 7.6 Hz), 7.38 (1H,

t, J ) 7.7 Hz), 7.53 (1H, d, J ) 7.7 Hz), 7.78 (1H, d, J ) 7.7

Hz), 7.86 (1H, s). Anal. (C₁₉H₂₃NO₆‚0.2H₂O) C, H, N.

4-(3-H yd r oxy-4,4-d im et h oxyb u t -1-yn yl)b en zoic a cid

m eth yl ester : from alkyne 30; orange oil; IR (neat) 3451

2512 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16

7.1 Hz), 4.79 (1H, ddd, J ) 4.7, 7.7, 12.4 Hz), 7.04 (1H, d, J )

Varney et al.

methyl 4-(3-bromopropyl)benzoate (1.29 g, 5.0 mmol) and KSAc

(1.14 g, 10 mmol) in acetone (40 mL) was heated at reflux for

40 min. After being cooled to room temperature, the reaction

mixture was filtered and the filtrate was concentrated. The

residue was partitioned between Et₂O and water (25 mL each).

The layers were separated, and the aqueous phase was

extracted with Et₂O (25 mL). The combined organic extracts

were dried (Na₂SO₄) and concentrated to give a red oil which

was purified by flash chromatography with hexane/EtOAc (9:

1) to give 1.22 g (97%) of the product as an amber oil: ¹H NMR

(CDCl₃) δ 1.91 (2H, tt, J ) 7.2, 7.7 Hz), 2.34 (3H, s), 2.74 (2H,

t, J ) 7.7 Hz), 2.88 (2H, t, J ) 7.2 Hz), 3.90 (3H, s), 7.24 (2H,

d, J ) 8.3 Hz), 7.95 (2H, d, J ) 8.3 Hz). Anal. (C₁₃H₁₆O₃S) C,

H, S.

4-[2-(Acetylsu lfa n yl)eth yl]ben zoic Acid Meth yl Ester .

Meth od A: yellow solid; mp 68-69 °C; ¹H NMR (CDCl₃) δ 2.33

(3H, s), 2.92 (2H, t, J ) 7.3 Hz), 3.13 (2H, t, J ) 7.3 Hz), 3.91

(3H, s), 7.29 (2H, d, J ) 8.2 Hz), 7.97 (2H, d, J ) 8.2 Hz).

Anal. (C₁₂H₁₄O₃S) C, H, S.

5-[3-(Acetylsu lfan yl)pr opyl]th ioph en e-2-car boxylic Acid

Meth yl Ester . Meth od A: yellow oil; ¹H NMR (CDCl₃) δ 1.97

(2H, pentet, J ) 7.3 Hz), 2.34 (3H, s), 2.91 (4H, t, J ) 7.3 Hz),

3.86 (3H, s), 6.81 (1H, d, J ) 3.7 Hz), 7.63 (1H, d, J ) 3.7 Hz).

Anal. (C₁₁H₁₄O₃S₂) C, H, S.

5-[2-(Acetylsu lfan yl)eth yl]th ioph en e-2-car boxylic Acid

Meth yl Ester . Meth od A: yellow solid; mp 67-68 °C; ¹H

NMR (CDCl₃) δ 2.35 (3H, s), 3.19-3.06 (4H, m), 3.86 (3H, s),

6.85 (1H, d, J ) 3.7 Hz), 7.64 (1H, d, J ) 3.7 Hz). Anal.

(C₁₀H₁₂O₃S₂) C, H, S.

2(S)-[[3-[3-(Acetylsu lfa n yl)p r op yl]ben zoyl]a m in o]p en -

ta n ed ioic Acid Dieth yl Ester . Meth od A: orange solid; mp

83-85 °C; H NMR (CDCl₃) δ 1.22 (3H, t, J ) 7.1 Hz), 1.30

(2H, t, J ) 7.1 Hz), 1.93 (2H, tt, J ) 7.3, 7.6 Hz), 2.20-2.08

(1H, m), 2.34 (3H, s), 2.38-2.26 (1H, m), 2.55-2.40 (2H, m),

2.74 (2H, t, J ) 7.6 Hz), 2.88 (2H, t, J ) 7.3 Hz), 4.11 (2H, q,

J ) 7.1 Hz), 4.24 (2H, q, J ) 7.1 Hz), 4.79 (1H, ddd, J ) 4.8,

7.8, 12.6 Hz), 7.06 (1H, d, J ) 7.8 Hz), 7.38-7.30 (2H, m),

7.65-7.61 (2H, m). Anal. (C₂₁H₂₉NO₆S) C, H, N, S.

2(S)-[[[6-[(Acetylsu lfan yl)m eth yl]-4,5,6,7-tetr ah ydr oben -

zo[b]th iop h en e-2-yl]ca r bon yl]a m in o]p en ta n ed ioic Acid

Dieth yl Ester . Meth od A: orange oil; H NMR (CDCl₃) δ

7.7 Hz), 7.37-7.34 (2H, m), 7.64-7.60 (1H, m). Anal. (C₁₉H₂₇

-

NO₆) C, H, N.

4-(3-Hyd r oxy-4,4-d im eth oxybu tyl)ben zoic a cid m eth yl

ester (31): yellow oil; IR (neat) 3495 (broad), 2953, 1721, 1611,

1437, 1283 cm^-1; ¹H NMR (CDCl₃) δ 1.72-1.93 (m, 2H), 2.75-

2.93 (m, 2H), 3.39 (s, 3H), 3.44 (s, 3H), 3.58 (m, 1H), 3.90 (s,

3H), 4.13 (d, 1H, J ) 6.1 Hz), 7.29 (d, 2H, J ) 8.1 Hz), 7.95 (d,

2H, J ) 8.2 Hz). Anal. (C₁₄H₂₀O₅‚0.20H₂O) C, H.

3-(3-Hyd r oxy-4,4-d im eth oxybu tyl)ben zoic a cid m eth yl

1

ester : yellow oil; H NMR (CDCl₃) δ 1.74 (1H, dddd, J ) 5.0,

9.3, 9.7, 17.0 Hz), 1.91 (1H, dddd, J ) 3.2, 7.0, 10.0, 17.0 Hz),

2.75 (1H, ddd, J ) 7.0, 9.7, 13.8 Hz), 2.93 (1H, ddd, J ) 5.0,

10.0, 13.8 Hz), 3.39 (3H, s), 3.45 (3H, s), 3.59 (1H, ddd, J )

3.2, 6.1, 9.3 Hz), 3.91 (3H, s), 4.14 (1H, d, J ) 6.1 Hz), 7.34

(1H, t, J ) 7.6 Hz), 7.42 (1H, dt, J ) 7.6, 1.4 Hz), 7.86 (1H, dt,

J ) 7.6, 1.4 Hz), 7.91 (1H, t, J ) 1.4 Hz). Anal. (C₁₄H₂₀O₅‚

0.2H₂O) C, H.

5-[2-(2,2-Dim eth yl[1,3]dioxolan -4(S)-yl)eth yl]th ioph en e-

2-ca r boxylic a cid eth yl ester : colorless oil; [R]₅₈₉-12.1° (c

) 0.78, MeOH); IR (neat) 2984, 2938, 2874, 1709, 1462, 1263

cm^-1; ¹H NMR (CDCl₃) δ 1.36 (t, 3H, J ) 7.0 Hz), 1.37 (s, 3H),

1.43 (s, 3H), 1.94 (m, 2H), 2.96 (m, 2H), 3.55 (t, 1H, J ) 7.3

Hz), 4.04 (dd, 1H, J ) 6.1, 1.5 Hz), 4.12 (m, 1H), 4.32 (q, 2H,

J ) 7.0 Hz), 6.82 (d, 1H, J ) 3.7 Hz), 7.63 (d, 1H, J ) 4.0 Hz).

Anal. (C₁₄H₂₀O₄S) C, H, S.

5-[2-(2,2-Dim eth yl[1,3]d ioxola n -4(S)-yl)eth yl]-4-m eth -

ylth iop h en e-2-ca r boxylic a cid eth yl ester : colorless oil;

[R]₅₈₉-12.6° (c ) 0.94, MeOH); IR (neat) 2984, 2936, 2872,

1

1707, 1456, 1371, 1246 cm^-1; H NMR (CDCl₃) δ 1.35 (t, 3H,

J ) 7.0 Hz), 1.36 (s, 3H), 1.43 (s, 3H), 1.88 (m, 2H), 2.16 (s,

3H), 2.80-2.91 (m, 2H), 3.55 (dd, 1H, J ) 6.6, 0.7 Hz), 4.03

(dd, 1H, J ) 5.9, 1.8 Hz), 4.12 (m, 1H), 4.31 (q, 2H, J ) 7.0

Hz), 7.50 (s, 1H). Anal. (C₁₅H₂₂O₄S) C, H, S.

1

Gen er a l P r oced u r e for th e P r ep a r a tion of Alk yl Br o-

m id es. 4-(3-Br om op r op yl)ben zoic Acid Meth yl Ester . A

solution of Ph₃P (5.67 g, 21.6 mmol) in CH₂Cl₂(25 mL) was

added dropwise to a solution of methyl 4-(3-hydroxypropyl)-

benzoate (3.49 g, 18 mmol) and CBr₄(7.16 g, 21.6 mmol) in

CH₂Cl₂(40 mL) at 0 °C. The resultant reaction mixture was

stirred for 30 min at 0 °C and then overnight at room

temperature. The solvent was removed, and the residue

obtained was purified by flash chromatography with hexane/

EtOAc (9:1) to give 4.38 g (95%) of the product as a yellow oil:

¹H NMR (CDCl₃) δ 2.18 (2H, tt, J ) 6.5, 7.4 Hz), 2.84 (2H, t,

J ) 7.4 Hz), 3.39 (2H, t, J ) 6.5 Hz), 3.91 (3H, s), 7.27 (2H, d,

J ) 8.2 Hz), 7.97 (2H, d, J ) 8.2 Hz). Anal. (C₁₁H₁₃O₂Br) C,

H, Br.

1

1.23 (3H, t, J ) 7.1 Hz), 1.29 (3H, t, J ) 7.1 Hz), 1.57-1.46

(1H, m), 2.15-1.96 (3H, m), 2.35-2.22 (1H, m), 2.36 (3H, s),

2.74-2.38 (5H, m), 3.02-2.92 (3H, m), 4.11 (2H, q, J ) 7.1

Hz), 4.22 (2H, q, J ) 7.1 Hz), 4.72 (1H, ddd, J ) 4.8, 7.6, 12.6

Hz), 6.69 (1H, d, J ) 7.6 Hz), 7.21 (1H, s). Anal. (C₂₁H₂₉

NO₆S₂‚0.5H₂O) C, H, N, S.

-

Meth od B. 2(S)-[[[5-[3-(Acetylsu lfa n yl)p r op yl]-3-m eth -

ylth iop h en e-2-yl]ca r bon yl]a m in o]p en ta n ed ioic Acid Di-

eth yl Ester . To a stirred solution of triphenylphosphine (210

mg, 0.79 mmol) and diethyl azodicarboxylate (126 µL, 0.79

mmol) in THF (5 mL) at 0 °C was added dropwise a solution

of the above alcohol (154 mg, 0.39 mmol) and thioacetic acid

(57 µL, 0.79 mmol) in THF (5 mL) over a 15 min period. After

1 h at 0 °C, the solvent was removed in vacuo. The crude

residue was flash chromatographed with CH₂Cl₂/EtOAc (20:

1) to give 118 mg (67%) of the desired thioacetate as a colorless

oil: IR (neat) 3366, 2980, 2936, 1736, 1692, 1649, 1510, 1447,

5-(3-Br om op r op yl)th iop h en e-2-ca r boxylic a cid m eth yl

ester : yellow oil; ¹H NMR (CDCl₃) δ 2.22 (2H, tt, J ) 6.4, 7.2

Hz), 3.03 (2H, t, J ) 7.2 Hz), 3.43 (2H, t, J ) 6.4 Hz), 3.86

(3H, s), 6.85 (1H, d, J ) 3.7 Hz). 7.64 (1H, d, J ) 3.7 Hz).

Anal. (C₉H₁₁O₂SBr) C, H, S, Br.

5-(2-Br om oeth yl)th iop h en e-2-ca r boxylic a cid m eth yl

1

ester : yellow oil; H NMR (CDCl₃) δ 3.38 (2H, t, J ) 7.1 Hz),

3.58 (2H, t, J ) 7.1 Hz), 3.87 (3H, s), 6.89 (1H, d, J ) 3.8 Hz),

7.66 (1H, d, J ) 3.8 Hz). Anal. (C₈H₉O₂SBr) C, H, S, Br.

2(S)-[[3-(3-Br om opr op-1-yl)ben zoyl]am in o]pen tan edio-

ic a cid d ieth yl ester : colorless oil; H NMR (CDCl₃) δ 1.23

1377 cm^-1; H NMR (CDCl₃) δ 1.23 (3H, t, J ) 7.2 Hz), 1.30

1

(3H, t, J ) 7.1 Hz), 1.31 (3H, t, J ) 7.1 Hz), 2.19-2.08 (3H,

m), 2.38-2.21 (1H, m), 2.58-2.40 (2H, m), 2.84 (2H, t, J ) 7.4

Hz), 3.39 (2H, t, J ) 6.5 Hz), 4.12 (2H, q, J ) 7.1 Hz), 4.24

(2H, q, J ) 7.1 Hz), 4.79 (1H, ddd, J ) 4.7, 7.6, 12.5 Hz), 7.03

(1H, d, J ) 7.6 Hz), 7.39-7.34 (2H, m), 7.66-7.62 (1H, m),

7.68 (1H, s). Anal. (C₁₉H₂₆NO₅Br) C, H, N, Br.

(3H, t, J ) 7.1 Hz), 1.95 (2H, pentet, J ) 7.3 Hz), 2.04-2.48

(4H, m), 2.34 (3H, s), 2.47 (3H, s), 2.83 (2H, t, J ) 7.4 Hz),

2.91 (2H, t, J ) 7.2 Hz), 4.11 (2H, q, J ) 7.1 Hz), 4.23 (2H, q,

J ) 7.1 Hz), 4.75 (1H, m), 6.47 (1H, d, J ) 7.4 Hz), 6.61 (1H,

s).

2(S)-[[[5-[3-(Acetylsu lfan yl)pr opyl]-4-m eth ylth ioph en e-

2-yl]ca r bon yl]a m in o]p en ta n ed ioic Acid Dieth yl Ester .

Meth od B: yellow oil; IR (neat) 3337, 2982, 2938, 1736, 1694,

2(S)-[[[6-(Br om om eth yl)-4,5,6,7-tetr ah ydr oben zo[b]th io-

p h en e-2-yl]ca r b on yl]a m in o]p en t a n ed ioic a cid d iet h yl

ester : colorless gum; H NMR (CDCl₃) δ 1.23 (3H, t, J ) 7.2

1634, 1526, 1449, 1206 cm^-1; H NMR (CDCl₃) δ 1.20 (3H, t,

1

Hz), 1.29 (3H, t, J ) 7.2 Hz), 1.67-1.53 (1H, m), 2.79-2.03

(9H, m), 3.04 (1H, dd, J ) 5.0, 16.8 Hz), 3.49-3.41 (2H, m),

4.10 (2H, q, J ) 7.2 Hz), 4.23 (2H, q, J ) 7.2 Hz), 4.73 (1H,

ddd, J ) 4.8, 7.6, 12.6 Hz), 6.73 (1H, d, J ) 7.6 Hz), 7.23 (1H,

s). Anal. (C₁₉H₂₆NO₅SBr) C, H, N, S, Br.

J ) 7.1 Hz), 1.30 (3H, t, J ) 7.1 Hz), 1.91 (2H, pentet, J ) 7.4

Hz), 2.08-2.49 (4H, m), 2.15 (3H, s), 2.34 (3H, s), 2.80 (2H, t,

J ) 7.4 Hz), 2.91 (2H, t, J ) 7.2 Hz), 4.13 (2H, q, J ) 7.1 Hz),

4.24 (2H, q, J ) 7.2 Hz), 4.74 (1H, m), 6.67 (1H, d, J ) 7.6

Hz), 7.26 (1H, s). Anal. (C₂₀H₂₉NO₆S₂) C, H, N, S.

Gen er a l P r oced u r es for th e F or m a tion of Th ioa cetyl

Com p ou n d s. Meth od A. 4-[3-(Acetylsu lfa n yl)p r op yl]-

ben zoic Acid Met h yl E st er . Met h od A. A mixture of

2(S)-[[[5-[3-(Acetylsu lfa n yl)p r op yl]-3-eth ylth iop h en e-

2-yl]ca r bon yl]a m in o]p en ta n ed ioic Acid Dieth yl Ester .

Meth od B: yellow oil; ¹H NMR (CDCl₃) δ 1.24 (2 overlapping

Glycinamide Ribonucleotide Transformylase Inhibitors

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16 2513

triplets, 6H), 1.30 (t, 3H, J ) 7.1 Hz), 1.96 (m, 2H), 2.26-2.56

(m, 3H), 2.35 (5, 3H), 2.85 (t, 2H, J ) 7.4 Hz), 2.92 (m, 4H),

4.12 (q, 2H, J ) 7.1 Hz), 4.23 (q, 2H, J ) 7.1 Hz), 4.74 (m,

1H), 6.45 (d, 1H, J ) 7.4 Hz), 6.68 (s, 1H).

8.2 Hz), 2.83 (2H, dt, J ) 8.2, 7.1 Hz), 3.14 (2H, t, J ) 7.1 Hz),

3.87 (3H, s), 6.86 (1H, d, J ) 3.7 Hz), 7.66 (1H, d, J ) 3.7 Hz).

Anal. (C₈H₁₀O₂S₂) C, H, S.

2(S)-[[[5-(3-Mer ca p top r op yl)-3-m eth ylth iop h en e-2-yl]-

ca r bon yl]a m in o]p en ta n ed ioic a cid d ieth yl ester (21e):

used as a crude oil.

2(S)-[[[5-(3-Mer ca p top r op yl)-4-m eth ylth iop h en e-2-yl]-

ca r bon yl]a m in o]p en t a n ed ioic a cid d iet h yl est er (21f):

used as a crude oil.

2(S)-[[[5-(3-Mer captopr opyl)-3-eth ylth ioph en e-2-yl]car -

bon yl]a m in o]p en ta n ed ioic a cid d ieth yl ester (21g): used

as a crude oil.

2(S)-[[3-(3-Mer ca p t op r op yl)b en zoyl]a m in o]p en t a n e-

d ioic a cid d ieth yl ester (21h ): orange oil; ¹H NMR (CDCl₃)

δ 1.22 (3H, t, J ) 7.1 Hz), 1.31 (3H, t, J ) 7.1 Hz), 1.37 (1H,

t, J ) 7.9 Hz), 1.95 (2H, pentet, J ) 7.4 Hz), 2.20-2.08 (1H,

m), 2.40-2.26 (1H, m), 2.45 (2H, t, J ) 6.9 Hz), 2.54 (2H, dt,

J ) 7.9, 7.4 Hz), 2.78 (2H, t, J ) 7.4 Hz), 4.11 (2H, q, J ) 7.1

Hz), 4.24 (2H, q, J ) 7.1 Hz), 4.79 (1H, ddd, J ) 4.8, 7.7, 12.5

Hz), 7.02 (1H, d, J ) 7.7 Hz), 7.40-7.32 (2H, m), 7.62 (1H, dt,

J ) 6.5, 2.0 Hz), 7.66 (1H, brs). Anal. (C₁₉H₂₇NO₅S) C, H, N,

S.

Meth od C. 5-[3(R)-(Acetylsu lfa n yl)-4-[(ter t-bu toxyca r -

b on yl)a m in o]b u t yl]t h iop h en e-2-ca r b oxylic Acid E t h yl

Ester . To a stirred solution of 5-[3(S)-[(methylsulfonyl)oxy]-

4-[(tert-butoxycarbonyl)amino]butyl]thiophene-2-carboxylic acid

ethyl ester (38.36 g, 91.0 mmol) in acetone (650 mL) was added

KSAc (41.57 g, 364 mmol). After 14 h, the starting mesylate

had disappeared by TLC and the reaction mixture was a red-

brown color and thick with precipitate. The crude reaction

was filtered, washing the precipitate with acetone and Et₂O

until the filter cake was beige colored. The filtrate was

concentrated in vacuo to a volume of 500 mL, diluted with

EtOAc, washed with brine, dried (MgSO₄), and concentrated.

The crude oil was purified by flash chromatography on 250 g

of silica gel, eluting hexanes/EtOAc (1:1) to remove baseline

colored impurities yielding 39.77 g (109%) of the thioacetate

as a red oil which was used without further purification: [R]₅₈₉

+7.6° (c ) 0.66, MeOH); IR (neat) 3376, 2978, 2932, 1712,

1

1684, 1520, 1462, 1263 cm^-1; H NMR (CDCl₃) δ 1.35 (t, 3H,

J ) 7.0 Hz), 1.44 (s, 9H), 1.90 (m, 1H), 2.04 (m, 1H), 2.36 (s,

3H), 2.95 (m, 2H), 3.36 (m, 2H), 3.60 (m, 1H), 4.32 (q, 2H, J )

7.0 Hz), 4.78 (1H, br t), 6.78 (d, 1H, J ) 3.7 Hz), 7.61 (d, 1H,

J ) 3.7 Hz). Anal. (C₁₈H₂₇NO₅S₂) C, H, N, S.

2(S)-[[[6-(Mer ca p tom eth yl)-4,5,6,7-tetr a h yd r oben zo[b]-

th iop h en e-2-yl]ca r bon yl]a m in o]p en ta n ed ioic a cid d i-

1

eth yl ester (21i): orange oil; H NMR (CDCl₃) δ 1.23 (3H, t,

J ) 7.1 Hz), 1.30 (3H, t, J ) 7.1 Hz), 1.38 (1H, t, J ) 8.3 Hz),

1.59-1.48 (1H, m), 2.15-1.92 (3H, m), 2.77-2.23 (8H, m), 3.04

(1H, dd, J ) 5.0, 16.6 Hz), 4.12 (2H, q, J ) 7.1 Hz), 4.23 (2H,

q, J ) 7.1 Hz), 4.73 (1H, ddd, J ) 4.8, 7.6, 12.6 Hz), 6.69 (1H,

d, J ) 7.6 Hz), 7.23 (1H, s). Anal. (C₁₉H₂₇NO₅S₂‚0.25H₂O) C,

H, N, S.

5-[3(S)-(Acetylsu lfan yl)-4-[(ter t-bu toxycar bon yl)am in o]-

bu tyl]th iop h en e-2-ca r boxylic Acid Eth yl Ester . Meth od

C: orange oil; [R]₅₈₉-4.9° (c ) 0.61, MeOH); IR (neat) 3374,

2978, 2932, 1715, 1695, 1518, 1460, 1264 cm^-^{1 1}H NMR

;

(CDCl₃) δ 1.35 (t, 3H, J ) 7.0 Hz), 1.44 (s, 9H), 1.90 (m, 1H),

2.03 (m, 1H), 2.36 (s, 3H), 2.95 (m, 2H), 3.35 (m, 2H), 3.59 (m,

1H), 4.31 (2H, q, J ) 7.0 Hz), 4.74 (br t, 1H), 6.79 (d, 1H, J )

3.7 Hz), 7.62 (d, 1H, J ) 3.7 Hz). Anal. (C₁₈H₂₇NO₅S₂) C, H,

N, S.

Gen er a l P r oced u r e for th e P r ep a r a tion of Su lfid es.

4-[3-[(2,4-Dia m in o-6-oxo-1,6-d ih yd r op yr im id in -5-yl)su l-

fa n yl]p r op yl]ben zoic Acid Meth yl Ester (22a ). Argon was

bubbled through a slurry of 5-bromo-2,6-diamino-4(3H)-pyrim-

idinone (20) (1.01 g, 4.9 mmol) in DMF (15 mL). To this slurry

was added a solution of thiol 21a (1.11 g, 5.3 mmol) in DMF

(10 mL) and DIEA (1.8 mL, 10.3 mmol). The resultant reaction

mixture was heated at 100 °C for 30 min and then poured over

ice (100 g).¹³The precipitate that formed was collected by

filtration and washed with water (2 × 30 mL) and then with

Et₂O (2 × 30 mL) to provide 711 mg (43%) of the product as

5-[3(R)-(Acetylsu lfa n yl)-4-[(ter t-bu toxyca r bon yl)a m i-

n o]b u t yl]-4-m et h ylt h iop h en e-2-ca r b oxylic Acid E t h yl

Ester . Meth od C: orange oil; [R]₅₈₉-2.8° (c ) 0.78, MeOH);

1

IR (neat) 3376, 2978, 2932, 1699, 1516, 1454, 1250 cm^-1; H

NMR (CDCl₃) δ 1.34 (t, 3H, J ) 7.0 Hz), 1.44 (s, 9H), 1.80-

2.04 (m, 2H), 2.13 (s, 3H), 2.37 (s, 3H), 2.85 (m, 2H), 3.38 (m,

2H), 3.60 (m, 1H), 4.30 (q, 2H, J ) 7.0 Hz), 4.88 (br t, 1H),

7.48 (s, 1H). Anal. (C₁₉H₂₉NO₅S₂) C, H, N, S.

5-[3(S)-(Acetylsu lfan yl)-4-[(ter t-bu toxycar bon yl)am in o]-

bu tyl]-4-m eth ylth iop h en e-2-ca r boxylic Acid Eth yl Ester .

Meth od C: orange oil; [R]₅₈₉+2.8° (c ) 0.93, MeOH); ¹H NMR

(CDCl₃) δ 1.34 (t, 3H, J ) 7.0 Hz), 1.44 (s, 9H), 1.78-1.89 (m,

1H), 1.93-2.04 (m, 1H), 2.13 (s, 3H), 2.37 (s, 3H), 2.75-2.94

(m, 2H), 3.26-3.45 (m, 2H), 3.56-3.64 (m, 1H), 4.30 (q, 2H, J

) 7.0 Hz), 4.75 (broad, 1H), 7.48 (s, 1H). Anal. (C₁₉H₂₉NO₅S₂)

C, H, N, S.

Gen er a l P r oced u r e for th e P r ep a r a tion of Th iols 21a -

i. 4-(3-Mer ca p top r op yl)ben zoic Acid Meth yl Ester (21a ).

A solution of 4-[3-(acetylsulfanyl)propyl]benzoic acid methyl

ester (1.01 g, 4 mmol) in 1 N HCl in MeOH (15 mL) was heated

at reflux for 2 h. The reaction mixture was diluted with water

(10 mL), the MeOH was removed in vacuo, and the aqueous

residue was extracted with Et₂O (2 × 25 mL). The combined

organic extracts were dried (Na₂SO₄) and concentrated to

provide 818 mg (97%) of the product as a yellow oil: ¹H NMR

(CDCl₃) δ 1.37 (1H, t, J ) 7.9 Hz), 2.00 (2H, m), 2.57-2.50

(2H, m), 2.79 (2H, t, J ) 7.6 Hz), 3.90 (3H, s), 7.25 (2H, d, J

) 8.2 Hz), 7.96 (2H, d, J ) 8.2 Hz). Anal. (C₁₁H₁₄O₂S) C, H,

S.

1

an off-white powder: mp 248-251 °C dec; H NMR (DMSO-

d₆) δ 1.70 (2H, tt, J ) 7.0, 7.2 Hz), 2.42 (2H, t, J ) 7.0 Hz),

2.76 (2H, t, J ) 7.2 Hz), 3.82 (3H, s), 6.28 (4H, broad), 7.33

(2H, d, J ) 8.2 Hz), 7.84 (2H, d, J ) 8.2 Hz), 9.95 (1H, s).

Anal. (C₁₅H₁₈N₄O₃S‚0.3H₂O) C, H, N, S.

4-[2-[(2,4-Dia m in o-6-oxo-1,6-d ih yd r op yr im id in -5-yl)-

su lfa n yl]eth yl]ben zoic a cid m eth yl ester (22b): white

1

solid; mp 286-288 °C dec; H NMR (DMSO-d₆) δ 2.70 (2H, t,

J ) 7.3 Hz), 2.81 (2H, t, J ) 7.3 Hz), 3.82 (3H, s), 6.33 (4H,

broad), 7.36 (2H, d, J ) 8.2 Hz), 7.85 (2H, d, J ) 8.2 Hz), 9.98

(1H, brs). Anal. (C₁₄H₁₆N₄O₃S) C, H, N, S.

5-[3-[(2,4-Dia m in o-6-oxo-1,6-d ih yd r op yr im id in -5-yl)-

su lfa n yl]p r op yl]th iop h en e-2-ca r boxylic a cid m eth yl es-

ter (22c): off-white solid; mp 196 °C dec; ¹H NMR (DMSO-d₆)

δ 1.74 (2H, tt, J ) 6.8, 7.5 Hz), 2.45 (2H, t, J ) 6.8 Hz), 2.97

(2H, t, J ) 7.5 Hz), 3.77 (3H, s), 6.29 (4H, broad), 6.93 (1H, d,

J ) 3.8 Hz), 7.61 (1H, d, J ) 3.8 Hz), 9.96 (1H, brs). Anal.

(C₁₃H₁₆N₄O₃S₂) C, H, N, S.

5-[2-[(2,4-Dia m in o-6-oxo-1,6-d ih yd r op yr im id in -5-yl)-

su lfa n yl]eth yl]th iop h en e-2-ca r boxylic a cid m eth yl ester

(22d ): off-white solid; mp 228-229 °C; ¹H NMR (DMSO-d₆) δ

2.72 (2H, t, J ) 7.1 Hz), 2.98 (2H, t, J ) 7.1 Hz), 3.77 (3H, s),

6.33 (4H, brs), 7.02 (1H, d, J ) 3.6 Hz), 7.62 (1H, d, J ) 3.6

Hz), 9.98 (1H, s). Anal. (C₁₂H₁₄N₄O₃S₂) C, H, N, S.

4-(2-Mer ca p toeth yl)ben zoic a cid m eth yl ester (21b):

1

yellow oil; H NMR (CDCl₃) δ 1.37 (1H, t, J ) 7.9 Hz), 2.81

(2H, dt, J ) 7.9, 7.3 Hz), 2.98 (2H, t, J ) 7.3 Hz), 3.91 (3H, s),

7.27 (2H, d, J ) 8.2 Hz), 7.98 (2H, d, J ) 8.2 Hz). Anal.

(C₁₀H₁₂O₂S) C, H, S.

2(S)-[[[5-[3-[(2,4-Dia m in o-6-oxo-1,6-d ih yd r op yr im id in -

5-yl)su lfa n yl]p r op yl]-3-m eth ylth iop h en e-2-yl]ca r bon yl]-

a m in o]p en ta n ed ioic a cid d ieth yl ester (22e): white solid;

mp 164-165 °C; IR (KBr) 3329, 2930, 1734, 1636, 1597, 1518,

5-(3-Mer captopr opyl)th ioph en e-2-car boxylic acid m eth -

yl ester (21c): orange oil; ¹H NMR (CDCl₃) δ 1.36 (1H, t, J )

8.0 Hz), 1.99 (2H, tt, J ) 7.1, 7.4 Hz), 2.57 (2H, dt, J ) 8.0,

7.1 Hz), 2.98 (2H, t, J ) 7.4 Hz), 3.86 (3H, s), 6.81 (1H, d, J )

3.7 Hz), 7.63 (1H, d, J ) 3.7 Hz). Anal. (C₉H₁₂O₂S₂) C, H, S.

5-(2-Mer ca p toeth yl)th iop h en e-2-ca r boxylic a cid m eth -

yl ester (21d ): yellow oil; ¹H NMR (CDCl₃) δ 1.50 (1H, t, J )

1441 cm^-^{1 1}H NMR (DMSO-d₆) δ 1.16 (3H, t, J ) 7.1 Hz),

;

1.18 (3H, t, J ) 7.0 Hz), 1.71 (2H, m), 1.90-2.10 (2H, m), 2.32

(3H, s) 2.40 (4H, m), 2.86 (2H, t, J ) 7.4 Hz), 4.07 (4H, m),

4.32 (1H, m), 6.30 (4H, brs), 6.67 (1H, s), 8.13 (1H, d, J ) 7.5

Hz), 9.95 (1H, s). Anal. ( C₂₂H₃₁N₅O₆S₂‚1.0H₂O) C, H, N, S.

2514 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16

Varney et al.

2(S)-[[[5-[3-[(2,4-Dia m in o-6-oxo-1,6-d ih yd r op yr im id in -

5-yl)su lfa n yl]p r op yl]-4-m eth ylth iop h en e-2-yl]ca r bon yl]-

a m in o]p en ta n ed ioic a cid d ieth yl ester (22f): white solid;

mp 159-160 °C; IR (KBr) 3324, 2980, 1734, 1657, 1632, 1603,

J ) 8.2 Hz), 8.51 (1H, d, J ) 7.7 Hz), 9.96 (1H, broad), 12.34

(2H, broad). Anal. (C₁₉H₂₃N₅O₆S) C, H, N, S.

4-[3-[(2,4-Dia m in o-6-oxo-1,6-d ih yd r op yr im id in -5-yl)-

su lfa n yl]eth yl]ben zoic a cid : white solid; mp 291-292 °C;

¹H NMR (DMSO-d₆) δ 2.69 (2H, t, J ) 7.3 Hz), 2.80 (2H, t, J

) 7.3 Hz), 6.31 (4H, broad), 7.32 (2H, d, J ) 8.2 Hz), 7.82 (2H,

d, J ) 8.2 Hz), 9.98 (1H, brs), 12.78 (1H, brs). Anal.

(C₁₃H₁₄N₄O₃S‚0.5H₂O) C, H, N, S.

2(S)-[[4-[2-[(2,4-Dia m in o-6-oxo-1,6-d ih yd r op yr im id in -

5-yl)su lfa n yl]et h yl]b en zoyl]a m in o]p en t a n ed ioic a cid

(6): white solid; mp 177-181 °C; ¹H NMR (DMSO-d₆) δ 1.89-

2.09 (2H, m), 2.33 (2H, t, J ) 7.3 Hz), 2.69 (2H, t, J ) 6.8 Hz),

2.78 (2H, t, J ) 6.8 Hz), 4.35 (1H, ddd, J ) 5.3, 7.6, 13.0 Hz),

6.33 (4H, broad), 7.30 (2H, d, J ) 8.1 Hz), 7.77 (2H, d, J ) 8.1

Hz), 8.49 (1H, d, J ) 7.6 Hz), 10.01 (1H, brs). Anal.

(C₁₈H₂₁N₅O₆S‚1.05H₂O) C, H, N, S.

1

1466, 1206 cm^-1; H NMR (DMSO-d₆) δ 1.15 (3H, t, J ) 7.1

Hz), 1.17 (3H, t, J ) 7.1 Hz), 1.68 (2H, m), 2.01 (2H, m), 2.11

(3H, s), 2.43 (4H, m), 2.85 (2H, t, J ) 7.3 Hz), 4.04 (2H, q, J )

7.1 Hz), 4.12 (2H, q, J ) 7.1 Hz), 4.34 (1H, m), 6.31 (4H, brs),

7.55 (1H, s), 8.52 (1H, d, J ) 7.5 Hz), 9.95 (1H, s). Anal.

(C₂₄H₃₁N₅O₆S) C, H, N, S.

2(S)-[[[5-[3-[(2,4-Dia m in o-6-oxo-1,6-d ih yd r op yr im id in -

5-yl)su lfan yl]pr opyl]-3-eth ylth ioph en e-2-yl]car bon yl]am i-

n o]p en ta n ed ioic a cid d ieth yl ester (22g): light yellow

solid; mp 166.6 °C dec; IR (KBr) 3324 (broad), 1734, 1638,

1

1601, 1443, 1206 cm ^-1; H NMR (DMSO-d₆) δ 1.09 (t, 3H, J

) 7.5 Hz), 1.17 (2 overlapping triplets, 6H), 1.74 (m, 2H), 1.91-

2.07 (m, 2H), 2.39 (t, 2H, J ) 7.5 Hz), 2.47 (t, 2H, partially

obscured by DMSO), 2.76 (q, 2H, J ) 7.5 Hz), 2.88 (t, 2H, J )

7.4 Hz), 4.03 (q, 2H, J ) 7.1 Hz), 4.10 (q, 2H, J ) 7.1 Hz), 4.31

(m, 1H), 6.32 (brs, 4H), 6.75 (s, 1H), 8.19 (d, 1H, J ) 7.5 Hz),

9.95 (s, 1H). Anal. (C₂₃H₃₃N₅O₆S₂) C, H, N, S.

5-[3-[(2,4-Dia m in o-6-oxo-1,6-d ih yd r op yr im id in -5-yl)-

su lfa n yl]p r op yl]th iop h en e-2-ca r boxylic a cid : yellow pow-

1

der; mp 254 °C dec; H NMR (DMSO-d₆) δ 1.73 (2H, tt, J )

6.9, 7.4 Hz), 2.46 (2H, t, J ) 6.9 Hz), 2.95 (2H, t, J ) 7.4 Hz),

6.30 (4H, brs), 6.89 (1H, d, J ) 3.7 Hz), 7.53 (1H, d, J ) 3.7

Hz), 10.02 (1H, broad), 12.86 (1H, broad). Anal. (C₁₂H₁₄N₄-

O₃S₂‚0.5H₂O) C, H, N, S.

2(S)-[[3-[3-[(2,4-Dia m in o-6-oxo-1,6-d ih yd r op yr im id in -

5-yl)su lfa n yl]p r op yl]b en zoyl]a m in o]p en t a n ed ioic a cid

d ieth yl ester (22h ): yellow amorphous powder; ¹H NMR

(DMSO-d₆) δ 1.15 (3H, t, J ) 7.1 Hz), 1.18 (3H, t, J ) 7.1 Hz),

1.72 (2H, tt, J ) 7.0, 7.5 Hz), 2.16-1.95 (2H, m), 2.43 (2H, t,

J ) 7.4 Hz), 2.44 (2H, t, J ) 7.0 Hz), 2.73 (2H, t, J ) 7.5 Hz),

4.03 (2H, q, J ) 7.1 Hz), 4.10 (2H, q, J ) 7.1 Hz), 4.41 (1H,

ddd, J ) 5.5, 7.3, 9.4 Hz), 6.28 (4H, brs), 7.37-7.32 (2H, m),

7.69-7.64 (2H, m), 8.68 (1H, d, J ) 7.3 Hz), 9.94 (1H, s). Anal.

(C₂₃H₃₁N₅O₆S) C, H, N, S.

2-[[[6-[[(2,4-Dia m in o-6-oxo-1,6-d ih yd r op yr im id in -5-yl)-

su lfa n yl]m eth yl]-4,5,6,7-tetr a h yd r oben zo[b]th iop h en e-2-

yl]ca r bon yl]a m in o]p en ta n ed ioic a cid d ieth yl ester (22i):

yellow solid; mp 122-128 °C; ¹H NMR (DMSO-d₆) δ 1.15 (3H,

t, J ) 7.1 Hz), 1.17 (3H, t, J ) 7.1 Hz), 1.48-1.37 (1H, m),

1.83-1.73 (1H, m), 2.09-1.89 (3H, m), 2.40 (2H, t, J ) 7.4

Hz), 2.66-2.43 (5H, m), 3.07 (1H, dd, J ) 4.4, 16.6 Hz), 4.03

(2H, q, J ) 7.1 Hz), 4.08 (2H, q, J ) 7.1 Hz), 4.34 (1H, ddd, J

) 5.3, 7.5, 9.4 Hz), 6.28 (4H, brs), 7.54 (1H, s), 8.53 (1H, d, J

) 7.5 Hz), 9.92 (1H, s). Anal. (C₂₃H₃₁N₅O₆S₂) C, H, N, S.

4-[3-[(2,4-Dia m in o-6-oxo-1,6-d ih yd r op yr im id in -5-yl)-

su lfa n yl]-3-[1,3]d ioxola n -2-ylp r op yl]ben zoic a cid m eth yl

ester : off-white solid; mp 206-208 °C dec; IR (KBr) 3439,

3341, 3154, 1701, 1636, 1591, 1470, 1447, 1287 cm^-1; ¹H NMR

(DMSO-d₆) δ 1.64 (m, 1H), 1.84 (m, 1H), 2.58 (m, 1H), 2.79

(m, 1H), 3.19 (m, 1H), 3.82 (s, 3H), 3.85 (m, 4H), 4.83 (d, 1H,

J ) 4.3 Hz), 6.33 (brs, 4H), 7.33 (d, 2H, J ) 8.1 Hz), 7.83 (d,

2H, J ) 8.1 Hz), 10.03 (s, 1H). Anal. (C₁₈H₂₂N₄O₅S) C, H, N,

S.

2(S)-[[[5-[3-[(2,4-Dia m in o-6-oxo-1,6-d ih yd r op yr im id in -

5-yl)su lfan yl]pr opyl]th ioph en e-2-yl]car bon yl]am in o]pen -

ta n ed ioic a cid (7): yellow powder; mp 171-173 °C; ¹H NMR

(DMSO-d₆) δ 1.73 (2H, tt, J ) 6.8, 7.2 Hz), 1.95-1.83 (1H, m),

2.11-2.00 (1H, m), 2.32 (2H, t, J ) 7.3 Hz), 2.46 (2H, t, J )

6.8 Hz), 2.92 (2H, t, J ) 7.2 Hz), 4.36-4.28 (1H, m), 6.39 (4H,

broad), 6.85 (1H, d, J ) 3.6 Hz), 7.66 (1H, d, J ) 3.6 Hz), 8.49

(1H, d, J ) 7.7 Hz), 10.09 (1H, broad), 12.41 (2H, broad). Anal.

(C₁₇H₂₁N₅O₆S₂‚0.3H₂O) C, H, N, S.

5-[2-[(2,4-Dia m in o-6-oxo-1,6-d ih yd r op yr im id in -5-yl)-

su lfa n yl]eth yl]th iop h en e-2-ca r boxylic a cid : yellow pow-

der; mp 273 °C dec; ¹H NMR (DMSO-d₆) δ 2.72 (2H, t, J ) 7.3

Hz), 2.97 (2H, t, J ) 7.3 Hz), 6.34 (4H, brs), 6.97 (1H, d, J )

3.7 Hz), 7.53 (1H, d, J ) 3.7 Hz), 9.99 (1H, brs), 12.86 (1H,

broad). Anal. (C₁₁H₁₂N₄O₃S₂) C, H, N, S.

2(S)-[[5-[2-[(2,4-Dia m in o-6-oxo-1,6-d ih yd r op yr im id in -

5-yl)su lfa n yl]eth yl]th iop h en e-2-yl]ca r bon yl]a m in o]p en -

ta n ed ioic a cid (8): off-white powder; mp 228-230 °C dec;

¹H NMR (DMSO-d₆) δ 1.95-1.85 (1H, m), 2.12-2.00 (1H, m),

2.32 (2H, t, J ) 7.1 Hz), 2.70 (2H, t, J ) 7.3 Hz), 2.94 (2H, t,

J ) 7.3 Hz), 4.36-4.28 (1H, m), 6.36 (4H, brs), 6.94 (1H, d, J

) 3.3 Hz), 7.67 (1H, d, J ) 3.3 Hz), 8.51 (1H, d, J ) 7.7 Hz),

10.02 (1H, brs), 12.41 (2H, broad). Anal. (C₁₆H₁₉N₅O₆S₂) C,

H, N, S.

2(S)-[[[5-[3-[(2,4-Dia m in o-6-oxo-1,6-d ih yd r op yr im id in -

5-yl)su lfa n yl]p r op yl]-3-m eth ylth iop h en e-2-yl]ca r bon yl]-

a m in o]p en ta n ed ioic a cid (9): white solid; mp 217-220 °C;

Gen er a l P r oced u r es for th e Hyd r olysis of Meth yl a n d

Dieth yl Glu ta m a te Ester s. 4-[3-[(2,4-Dia m in o-6-oxo-1,6-

d ih yd r op yr im id in -5-yl)su lfa n yl]p r op yl]ben zoic Acid . A

suspension of ester 22a (669 mg, 2 mmol) in a 1 N NaOH

solution (30 mL) was stirred overnight at room temperature

and then filtered. The filtrate was acidified to ∼pH 5 by

addition of AcOH (∼3 mL). The precipitate that formed was

collected by filtration and washed with water (3 × 5 mL) to

give 589 mg (92%) of the product as an off-white powder: mp

IR (KBr) 3341, 3200, 2922, 1709, 1620, 1516, 1468, 1263 cm^-1

;

¹H NMR (DMSO-d₆) δ 1.71 (2H, pentet, J ) 7.2 Hz), 1.74-

2.05 (2H, m), 2.31 (2H, t, J ) 7.5 Hz), 2.33 (3H, s), 2.46 (2H,

t, J ) 6.9 Hz), 2.86 (2H, t, J ) 6.9 Hz), 4.28 (1H, m), 6.33 (4H,

brs), 6.66 (1H, s), 7.96 (1H, d, J ) 7.7 Hz), 9.96 (1H, brs). Anal.

(C₁₈H₂₃N₅O₆S₂‚1.3H₂O) C, H, N, S.

2(S)-[[[5-[3-[(2,4-Dia m in o-6-oxo-1,6-d ih yd r op yr im id in -

5-yl)su lfa n yl]p r op yl]-4-m eth ylth iop h en e-2-yl]ca r bon yl]-

a m in o]p en ta n ed ioic a cid (10): white solid; mp 154-158 °C;

1

262-263 °C; H NMR (DMSO-d₆) δ 1.70 (2H, tt, J ) 7.0, 7.2

1

IR (KBr) 3322, 3179, 2922, 1705, 1632, 1564, 1445 cm^-1; H

Hz), 2.43 (2H, t, J ) 7.0 Hz), 2.74 (2H, t, J ) 7.2 Hz), 6.29

(4H, broad), 7.29 (2H, d, J ) 8.1 Hz), 7.82 (2H, d, J ) 8.1 Hz),

9.97 (1H, broad), 12.72 (1H, broad). Anal. (C₁₄H₁₆N₄O₃S) C,

H, N, S.

NMR (DMSO-d₆) δ 1.68 (2H, m), 1.92-2.01 (2H, m), 2.10 (3H,

s), 2.31 (2H, t, J ) 7.4 Hz), 2.47 (2H, t, J ) 7.4 Hz), 2.82 (2H,

t, J ) 7.4 Hz), 4.29 (1H, m), 6.29 (4H, brs), 7.53 (1H, s), 8.36

(1H, d, J ) 7.7 Hz), 9.90 (1H, brs). Anal. (C₁₈H₂₃N₅O₆-

S₂‚0.4H₂O) C, H, N, S.

2(S)-[[[5-[3-[(2,4-Dia m in o-6-oxo-1,6-d ih yd r op yr im id in -

5-yl)su lfan yl]pr opyl]-3-eth ylth ioph en e-2-yl]car bon yl]am i-

n o]p en ta n ed ioic a cid (11): off-white solid; mp 137 °C dec;

IR (KBr) 3337 (broad), 1709, 1640, 1601, 1514, 1451 cm^-1; ¹H

NMR (DMSO-d₆) δ 1.10 (t, 3H, J ) 7.5 Hz), 1.72 (m, 2H), 1.95

(m, 2H), 2.30 (t, 2H, J ) 7.3 Hz), 2.46 (t, 2H, partially obscured

by DMSO), 2.77 (q, 2H, J ) 7.5 Hz), 2.87 (t, 2H, J ) 7.4 Hz),

4.26 (m, 1H), 6.26 (brs, 4H), 6.74 (s, 1H), 7.98 (d, 1H, J ) 7.4

Hz), 10.03 (brs, 1H), 12.50 (brs, 2H). Anal. (C₁₉H₂₅N₅-

O₆S₂‚1.7H₂O) C, H, N, S.

2(S)-[[4-[3-[(2,4-Dia m in o-6-oxo-1,6-d ih yd r op yr im id in -

5-yl)su lfa n yl]p r op yl]ben zoyl]a m in o]p en ta n ed ioic Acid

(5). A solution of the glutamate diethyl ester (192 mg, 0.4

mmol) in a 1 N NaOH solution (15 mL) was stirred at room

temperature for 70 h. After the pH was adjusted to ∼3-4 with

6 N HCl, the precipitate that formed was collected by filtration

and washed with water (3 × 10 mL) to give 147 mg (86%) of

the product as a white solid: mp 205-206 °C; ¹H NMR

(DMSO-d₆) δ 1.69 (2H, tt, J ) 6.9, 7.2 Hz), 1.99-1.89 (1H, m),

2.11-2.01 (1H, m), 2.34 (2H, t, J ) 7.9 Hz), 2.43 (2H, t, J )

6.9 Hz), 2.73 (2H, t, J ) 7.2 Hz), 4.37 (1H, ddd, J ) 4.8, 7.7,

9.7 Hz), 6.29 (4H, brs), 7.27 (2H, d, J ) 8.2 Hz), 7.76 (2H, d,

Glycinamide Ribonucleotide Transformylase Inhibitors

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16 2515

2(S)-[[3-[3-[(2,4-Dia m in o-6-oxo-1,6-d ih yd r op yr im id in -

5-yl)su lfa n yl]p r op yl]b en zoyl]a m in o]p en t a n ed ioic a cid

(12): yellow solid; mp 207-209 °C dec; ¹H NMR (DMSO-d₆) δ

1.72 (2H, tt, J ) 7.0, 7.5 Hz), 2.00-1.90 (1H, m), 2.14-2.02

(1H, m), 2.34 (2H, t, J ) 7.4 Hz), 2.44 (2H, t, J ) 7.0 Hz), 2.73

(2H, t, J ) 7.5 Hz), 4.38 (1H, ddd, J ) 5.1, 7.7, 9.5 Hz), 6.30

(4H, brs), 7.36-7.31 (2H, m), 7.70-7.63 (2H, m), 8.54 (1H, d,

1458, 1352 cm^-1; ¹H NMR (DMSO-d₆) δ 1.72 (m, 1H), 1.89 (m,

1H), 2.80-3.03 (m, 2H), 3.16-3.52 (m, 3H, partially obscured

by H₂O), 6.09 (s, 2H), 6.80 (s, 1H), 6.92 (d, 1H, J ) 3.7 Hz),

7.52 (d, 1H, J ) 3.7 Hz), 10.20 (s, 1H), 12.80 (brs, 1H); HMS

calcd for C₁₃H₁₄N₄O₃S_s(M + Na⁺) 361.0405, found 361.0390.

2(S)-[[[5-[2-(2-Am in o-4-oxo-4,6,7,8-tetr a h yd r o-3H-p yr -

im id o[5,4-b][1,4]th ia zin -6(S)-yl)eth yl]th iop h en e-2-yl]ca r -

bon yl]a m in o]p en ta n ed ioic a cid (17): off-white solid; mp

220 °C dec; [R]₅₈₉-57.1° (c ) 0.61, 1 N NaOH); IR (KBr) 3353,

3094, 2926, 1711, 1641, 1605, 1559, 1454, 1400, 1333, 1279

cm^-1; ¹H NMR (DMSO-d₆) δ 1.70-2.05 (m, 4H), 2.29 (t, 2H, J

) 7.4 Hz), 2.87 (m, 2H), 3.15-3.48 (m, 3H, partially obscured

by H₂O), 4.29 (m, 1H), 6.03 (s, 2H), 6.66 (s, 1H), 6.89 (d, 1H,

J ) 3.7 Hz), 7.65 (d, 1H, J ) 3.7 Hz), 8.50 (d, 1H, J ) 7.7 Hz),

10.05 (s, 1H), 12.50 (brs, 2H). Anal. (C₁₈H₂₁N₅O₆S₂‚1.4H₂O)

C, H, N, S.

J

) 7.7 Hz), 10.00 (1H, broad), 12.41 (2H, broad). Anal.

(C₁₉H₂₃N₅O₆S‚0.5H₂O) C, H, N, S.

2(S)-[[[6-[[(2,4-Dia m in o-6-oxo-1,6-d ih yd r op yr im id in -5-

yl)su lfan yl]m eth yl]-4,5,6,7-tetr ah ydr oben zo[b]th ioph en e-

2-yl]ca r bon yl]a m in o]p en ta n ed ioic a cid (13): white solid;

mp 227-230 °C dec; ¹H NMR (DMSO-d₆) δ 1.46-1.37 (1H,

m), 2.10-1.77 (4H, m), 2.31 (2H, t, J ) 7.3 Hz), 2.66-2.36 (5H,

m), 3.07 (1H, dd, J ) 4.3, 16.6 Hz), 4.30 (1H, ddd, J ) 4.6, 7.8,

12.6 Hz), 6.44 (4H, brs), 7.53 (1H, s), 8.42 (1H, d, J ) 7.8 Hz),

10.08 (1H, broad), 12.38 (2H, broad). Anal. (C₁₉H₂₃N₅O₆-

S₂‚0.7H₂O) C, H, N, S.

5-[2-(2-Am in o-4-oxo-4,6,7,8-t et r a h yd r o-3H -p yr im id o-

[5,4-b][1,4]t h ia zin -6(R)-yl)et h yl]-4-m et h ylt h iop h en e-2-

ca r boxylic a cid : off-white solid; mp 253 °C dec; [R]₅₈₉+60.7°

(c ) 0.29, 1 N NaOH); IR (KBr) 3339 (broad), 2922, 1641, 1539,

4-[2-(2-Am in o-4-oxo-4,6,7,8-t et r a h yd r o-3H -p yr im id o-

[5,4-b][1,4]th ia zin -6-yl)eth yl]ben zoic a cid : white solid; mp

>310 °C dec; IR (KBr) 3285, 3086, 2928, 1698, 1642, 1611,

1

1451, 1346, 1269 cm^-1; H NMR (DMSO-d₆) δ 1.64 (m, 1H),

1

1576, 1449, 1348 cm^-1; H NMR (DMSO-d₆) δ 1.72 (m, 1H),

1.82 (m, 1H), 2.09 (s, 3H), 2.78-2.94 (m, 2H), 3.10-3.55 (m,

3H, partially obscured by H₂O), 6.10 (s, 2H), 6.69 (s, 1H), 7.42

(s, 1H), 10.15 (s, 1H), 12.75 (brs, 1H); HMS calcd for

C₁₄H₁₆N₄O₃S₂(M + Na⁺) 375.0562, found 375.0575.

1.89 (m, 1H), 2.78 (m, 3H), 3.20 (m, 1H), 3.48 (m, 1H), 6.07 (s,

2H), 6.68 (s, 1H), 7.33 (d, 2H, J ) 8.1 Hz), 7.85 (d, 2H, J ) 8.1

Hz), 10.11 (s, 1H), 12.77 (brs, 1H). Anal. (C₁₅H₁₆N₄O₃-

S‚1.20H₂O) C, H, N, S.

2(S)-[[[5-[2-(2-Am in o-4-oxo-4,6,7,8-tetr a h yd r o-3H-p yr -

im ido[5,4-b][1,4]th iazin -6(R)-yl)eth yl]-4-m eth ylth ioph en e-

2-yl]ca r bon yl]a m in o]p en ta n ed ioic a cid (18): off-white

solid; mp 210 °C dec; [R]₅₈₉+64.4° (c ) 0.45, 1 N NaOH); IR

2(S)-[[4-[2-(2-Am in o-4-oxo-4,6,7,8-tetr ah ydr o-3H-pyr im i-

d o [5,4-b ][1,4]t h ia zin -6-y l)e t h y l]b e n zo y l]a m in o ]p e n -

ta n ed ioic a cid (14): off-white solid; mp 188-190 °C; IR (KBr)

3348 (broad), 2930, 1717, 1642, 1539, 1505, 1348 cm^-^{1 1}H

;

(KBr) 3341 (broad), 2928, 1701, 1638, 1536, 1449, 1340 cm^-1

;

NMR (DMSO-d₆) δ 1.71 (m, 1H), 1.92 (m, 2H), 2.08 (m, 1H),

2.34 (t, 2H, J ) 7.4 Hz), 2.79 (m, 3H), 3.20 (m, 1H), 3.55 (m,

1H), 4.38 (m, 1H), 6.07 (s, 2H), 6.68 (s, 1H), 7.31 (d, 2H, J )

8.1 Hz), 7.80 (d, 2H, J ) 8.2 Hz), 8.53 (d, 1H, J ) 7.7 Hz),

10.11 (s, 1H), 12.40 (brs, 2H). Anal. (C₂₀H₂₃N₅O₆S‚1.5H₂O)

C, H, N, S.

¹H NMR (DMSO-d₆) δ 1.78-2.05 (m, 4H), 2.10 (s, 3H), 2.28 (t,

2H, J ) 7.0 Hz), 2.82 (m, 2H), 3.45 (m, 3H, partially obscured

by H₂O), 4.25 (m, 1H), 5.98 (s, 2H), 6.65 (s, 1H), 7.54 (s, 1H),

8.38 (d, 1H, J ) 7.7 Hz), 10.05 (s, 1H), 12.5 (brs, 2H). Anal.

(C₁₉H₂₃N₅O₆S₂‚0.7H₂O) C, H, N, S.

5-[2-(2-Am in o-4(3H )-oxo-5,6,7,8-t et r a h yd r op yr im id o-

[5,6-b][1,4]t h ia zin -6(S)-yl)et h yl]-4-m et h ylt h iop h en e-2-

ca r boxylic a cid : [R]₅₈₉-77.9° (c ) 0.58, 1 N NaOH); ¹H NMR

(DMSO-d₆) δ 1.62-1.73 (m, 1H), 1.79-1.92 (m, 1H), 2.12 (s,

3H), 2.81-2.98 (m, 3H), 3.16-3.26 (m, 1H), 3.50-3.58 (m, 1H),

6.21 (brs, 2H), 6.76 (brs, 1H), 7.45 (s, 1H), 10.24 (broad, 1H),

12.76 (broad, 1H). Anal. (C₁₄H₁₆N₄O₃S₂‚1.4H₂O) C, H, N, S.

3-[2-(2-Am in o-4-oxo-4,6,7,8-t et r a h yd r o-3H -p yr im id o-

[5,4-b][1,4]th ia zin -6-yl)eth yl]ben zoic a cid : beige powder;

mp 253 °C dec; ¹H NMR (DMSO-d₆) δ 1.75-1.63 (1H, m),

1.94-1.82 (1H, m), 2.88-2.70 (3H, m), 3.25-3.16 (1H, m),

3.57-3.48 (1H, m), 6.02 (2H, s), 6.66 (1H, s), 7.48-7.35 (2H,

m), 7.78-7.72 (2H, m), 10.07 (1H, brs), 12.83 (1H, brs). Anal.

(C₁₅H₁₆N₄O₃S‚0.5H₂O) C, H, N, S.

2(S)-[[[5-[2-(2-Am in o-4-oxo-4,6,7,8-tetr a h yd r o-3H-p yr -

im ido[5,4-b][1,4]th iazin -6(S)-yl)eth yl]-4-m eth ylth ioph en e-

2-yl]ca r bon yl]a m in o]p en ta n ed ioic a cid (19): [R]₅₈₉-36.8°

2(S)-[[3-[2-(2-Am in o-4-oxo-4,6,7,8-tetr ah ydr o-3H-pyr im i-

d o [5,4-b ][1,4]t h ia zin -6-y l)e t h y l]b e n zo y l]a m in o ]p e n -

1

ta n ed ioic a cid (15): off-white soild; mp 188-189 °C dec; H

1

(c ) 0.57, 1 N NaOH); H NMR (DMSO-d₆) δ 1.61-1.72 (m,

NMR (DMSO-d₆) δ 1.77-1.67 (1H, m), 1.98-1.83 (2H, m),

2.11-2.02 (1H, m), 2.34 (2H, t, J ) 7.3 Hz), 2.87-2.72 (3H,

m), 3.25-3.17 (1H, m), 3.58-3.51 (1H, m), 4.38 (1H, ddd, J )

5.3, 7.6, 12.8 Hz), 6.02 (2H, s), 6.67 (1H, s), 7.44-7.34 (2H,

m), 7.77-7.65 (2H, m), 8.56 (1H, d, J ) 7.6 Hz), 10.07 (1H,

brs), 12.42 (2H, broad). Anal. (C₂₀H₂₃N₅O₆S‚1.1H₂O) C, H,

N, S.

1H), 1.76-1.92 (m, 2H), 1.99-2.08 (m, 1H), 2.12 (s, 3H), 2.31

(t, 2H, J ) 7.0 Hz), 2.79-2.94 (m, 3H), 3.17-3.28 (m, 1H),

3.49-3.56 (m, 1H), 4.30 (ddd, 1H, J ) 5.7, 7.7, 9.8 Hz), 6.08

(s, 2H), 6.70 (s, 1H), 7.58 (s, 1H), 8.44 (d, 1H, J ) 7.7 Hz),

10.12 (brs, 1H), 12.43 (broad, 2H). Anal. (C₁₉H₂₃N₅O₆-

S₂‚0.75H₂O) C, H, N, S.

5-[2-(2-Am in o-4-oxo-4,6,7,8-t et r a h yd r o-3H -p yr im id o-

[5,4-b][1,4]th ia zin -6(R)-yl)eth yl]th iop h en e-2-ca r boxylic

a cid : off-white solid; mp 283-285 °C dec; [R]₅₈₉+71.0° (c )

0.60, 1 N NaOH); IR (KBr) 3256 (broad) 2942, 1707, 1641,

Gen er a l P r oced u r es for th e Cou p lin g of L-Glu ta m ic

Acid Dieth yl Ester . Meth od A. 2(S)-[[4-[3-[(2,4-Dia m in o-

6-oxo-1,6-dih ydr opyr im idin -5-yl)su lfan yl]pr opyl]ben zoyl]-

a m in o]p en ta n ed ioic Acid Dieth yl Ester . A solution of

4-[3-[(2,6-diamino-4(3H)-oxopyrimidin-5-yl)sulfanyl]propyl]-

benzoic acid (577 mg, 1.8 mmol), 4-methylmorpholine (l mL,

9.1 mmol), and phenyl N-phenylphosphoramidochloridate (723

mg, 2.7 mmol) in 1-methyl-2-pyrrolidinone (40 mL) was stirred

for 1 h prior to addition of of ^L-glutamic acid diethyl ester

hydrochloride (863 mg, 3.6 mmol). The reaction mixture was

stirred overnight at room temperature. After removal of the

solvent in vacuo, the residue was partitioned between CHCl₃

(30 mL) and water (30 mL). The layers were separated, and

the aqueous phase was extracted with CHCl₃(30 mL). The

combined organic extracts were dried (MgSO₄) and concen-

trated to give a yellow gum which was purified by flash

chromatography with 5% MeOH in EtOAc to give 212 mg

(23%) of the product as a white solid: mp 78-81 °C; ¹H NMR

(CDCl₃) δ 1.21 (3H, t, J ) 7.1 Hz), 1.29 (3H, t, J ) 7.1 Hz),

1.83 (2H, tt, J ) 7.0, 7.3 Hz), 2.21-2.09 (2H, m), 2.57-2.25

(4H, m), 2.64 (2H, t, J ) 7.3 Hz), 4.09 (2H, q, J ) 7.1 Hz),

4.21 (2H, q, J ) 7.1 Hz), 4.78 (1H, ddd, J ) 5.0, 8.0, 12.8 Hz),

5.84 (2H, brs), 6.64 (2H, brs), 7.16 (2H, d, J ) 8.1 Hz), 7.28

1

1612, 1464, 1364 cm^-1; H NMR (DMSO-d₆) δ 1.72 (m, 1H),

1.89 (m, 1H), 2.81-3.04 (m, 2H), 3.16-52 (m, 3H, partially

obscured by H₂O), 6.08 (s, 2H), 6.68 (s, 1H), 6.92 (d, 1H, J )

4.0 Hz), 7.52 (d, 1H, J ) 3.7 Hz), 10.12 (s, 1H), 12.80 (brs,

1H). Anal. (C₁₃H₁₄N₄O₃S₂‚0.60H₂O) C, H, N, S.

2(S)-[[[5-[2-(2-Am in o-4-oxo-4,6,7,8-tetr a h yd r o-3H-p yr -

im id o[5,4-b][1,4]th ia zin -6(R)-yl)eth yl]th iop h en e-2-yl]ca r -

bon yl]a m in o]p en ta n ed ioic a cid (16): off-white solid; mp

191-194 °C foams; [R]₅₈₉+61.9° (c ) 0.65, 1 N NaOH); IR

(KBr) 3389, 3235, 3086, 2924, 1701, 1624, 1545, 1340 cm^-1

;

¹H NMR (DMSO-d₆) δ 1.70-2.04 (m, 4H), 2.29 (t, 2H, J ) 7.3

Hz), 2.90 (m, 2H), 3.13-3.53 (m, 3H, partially obscured by

H₂O), 4.29 (m, 1H), 6.30 (s, 2H), 6.77 (s, 1H), 6.89 (d, 1H, J )

3.7 Hz), 7.66 (d, 1H, J ) 3.7 Hz), 8.50 (d, 1H, J ) 8.1 Hz),

10.30 (brs, 1H). Anal. (C₁₈H₂₁N₅O₆S₂‚1.8H₂O) C, H, N, S.

5-[2-(2-Am in o-4-oxo-4,6,7,8-t et r a h yd r o-3H -p yr im id o-

[5,4-b][1,4]t h ia zin -6(S)-yl)et h yl]t h iop h en e-2-ca r b oxylic

a cid : off-white solid; mp 258-261 °C dec; [R]₅₈₉-81.3° (c )

0.63, 1 N NaOH); IR (KBr) 3254 (broad), 2918, 1692, 1635,

2516 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16

Varney et al.

(1H, d, J ) 8.0 Hz), 7.67 (2H, d, J ) 8.1 Hz). Anal.

7.8 Hz), 7.76 (1H, dd, J ) 1.7, 7.8 Hz), 7.83 (1H, dd, J ) 1.7,

7.8 Hz), 8.15 (1H, t, J ) 1.7 Hz). Anal. (C₁₆H₂₀NO₅I) C, H,

N, I.

(C₂₃H₃₁N₅O₆S) C, H, N, S.

2(S)-[[4-[2-[(2,4-Dia m in o-6-oxo-1,6-d ih yd r op yr im id in -

5-yl)su lfa n yl]eth yl]ben zoyl]a m in o]p en ta n ed ioic Acid Di-

eth yl Ester . Meth od A: white solid; mp 105-107 °C; ¹H

NMR (acetone-d₆) δ 1.18 (3H, t, J ) 7.1 Hz), 1.23 (3H, t, J )

7.1 Hz), 2.15-2.05 (1H, m), 2.29-2.17 (1H, m), 2.50 (2H, t, J

) 7.3 Hz), 2.93-2.78 (4H, m), 4.07 (2H, q, J ) 7.1 Hz), 4.15

(2H, q, J ) 7.1 Hz), 4.65 (1H, ddd, J ) 5.3, 8.0, 13.3 Hz), 6.11

(2H, s), 6.62 (2H, s), 7.31 (2H, d, J ) 7.8 Hz), 7.80 (2H, d, J )

7.8 Hz), 7.87 (1H, d, J ) 8.0 Hz), 10.72 (1H, s). Anal.

(C₂₂H₂₉N₅O₆S) C, H, N, S.

2(S)-[[4-[2-(2-Am in o-4-oxo-4,6,7,8-t et r a h yd r o-3H -p yr -

im id o[5,4-b][1,4]t h ia zin -6-yl)et h yl]b en zoyl]a m in o]p en -

ta n ed ioic Acid Dieth yl Ester . Meth od B: orange solid;

mp 132-136 °C; IR (KBr) 3333, 1732, 1645, 1572, 1535, 1449,

1

1343, 1203 cm^-1; H NMR (DMSO-d₆) δ 1.15 (t, 3H, J ) 7.3

Hz), 1.17 (t, 3H, J ) 7.3 Hz), 1.72 (m, 1H), 1.88-2.10 (m, 3H),

2.42 (t, 2H, J ) 7.4 Hz), 2.79 (m, 3H), 3.22 (m, 1H), 3.50 (m,

1H), 4.02 (q, 2H, J ) 7.3, 14.5 Hz), 4.09 (q, 2H, J ) 7.2, 14.3

Hz), 4.41 (m, 1H), 6.21 (s, 2H), 6.74 (s, 1H), 7.32 (d, 2H, J )

8.0 Hz), 7.80 (d, 2H, J ) 8.0 Hz), 8.64 (d, 1H, J ) 7.4 Hz),

10.24 (s, 1H). Anal. (C₂₄H₃₁N₅O₆S) C, H, N, S.

2(S)-[[[5-[2-[(2,4-Dia m in o-6-oxo-1,6-d ih yd r op yr im id in -

5-yl)su lfa n yl]eth yl]th iop h en e-2-yl]ca r bon yl]a m in o]p en -

ta n ed ioic Acid Dieth yl Ester . Meth od A: yellow solid; mp

95-96 °C; ¹H NMR (DMSO-d₆) δ 1.15 (3H, t, J ) 7.1 Hz), 1.17

(3H, t, J ) 7.1 Hz), 2.00-1.91 (1H, m), 2.13-2.01 (1H, m),

2.40 (2H, t, J ) 7.4 Hz), 2.70 (2H, t, J ) 7.3 Hz), 2.94 (2H, t,

J ) 7.3 Hz), 4.03 (2H, q, J ) 7.1 Hz), 4.09 (2H, q, J ) 7.1 Hz),

4.40-4.33 (1H, m), 6.32 (4H, brs), 6.94 (1H, d, J ) 3.0 Hz),

7.67 (1H, d, J ) 3.0 Hz), 8.61 (1H, d, J ) 7.5 Hz), 9.98 (1H, s).

Anal. (C₂₀H₂₇N₅O₆S₂‚0.5H₂O) C, H, N, S.

2(S)-[[3-[2-(2-Am in o-4-oxo-4,6,7,8-t et r a h yd r o-3H -p yr -

im id o[5,4-b][1,4]t h ia zin -6-yl)et h yl]b en zoyl]a m in o]p en -

ta n ed ioic Acid Dieth yl Ester . Meth od B: white solid; mp

69-72 °C; ¹H NMR (DMSO-d₆) δ 1.18 (3H, t, J ) 7.1 Hz), 1.23

(3H, t, J ) 7.1 Hz), 1.89-1.79 (1H, m), 2.00-1.92 (1H, m),

2.11-2.05 (1H, m), 2.29-2.18 (1H, m), 2.50 (2H, t, J ) 7.6

Hz), 2.97-2.77 (3H, m), 3.44-3.36 (1H, m), 3.76-3.70 (1H,

m), 4.07 (2H, q, J ) 7.1 Hz), 4.15 (2H, q, J ) 7.1 Hz), 4.66

(1H, ddd, J ) 5.1, 7.7, 13.0 Hz), 6.07 (2H, s), 6.12 (1H, s), 7.44-

7.33 (2H, m), 7.73 (1H, d, J ) 7.7 Hz), 7.82 (1H, s), 8.01-7.95

(1H, m), 10.43 (1H, brs). Anal. (C₂₄H₃₁N₅O₆S‚0.5H₂O) C, H,

N, S.

2(S)-[[[5-[3-[(2,4-Dia m in o-6-oxo-1,6-d ih yd r op yr im id in -

5-yl)su lfan yl]pr opyl]th ioph en e-2-yl]car bon yl]am in o]pen -

ta n ed ioic Acid Dieth yl Ester . Meth od A: off-white solid;

1

mp 136-138 °C; H NMR (CDCl₃) δ 1.21 (3H, t, J ) 7.1 Hz),

2(S)-[[[5-[2-(2-Am in o-4-oxo-4,6,7,8-tetr a h yd r o-3H-p yr -

im id o[5,4-b][1,4]th ia zin -6(R)-yl)eth yl]th iop h en e-2-yl]ca r -

bon yl]a m in o]p en ta n ed ioic Acid Dieth yl Ester . Meth od

B: white solid; mp 108-112 °C; [R]₅₈₉+35.0° (c ) 0.68,

DMSO); IR (KBr) 3343 (broad) 2930, 1732, 1634, 1545, 1450,

1.29 (3H, t, J ) 7.1 Hz), 1.89 (2H, quintet, J ) 7.2 Hz), 2.18-

2.08 (1H, m), 2.34-2.22 (1H, m), 2.45 (2H, t, J ) 7.2 Hz), 2.55

(2H, t, J ) 7.0 Hz), 2.89 (2H, t, J ) 7.2 Hz), 4.10 (2H, q, J )

7.1 Hz), 4.22 (2H, q, J ) 7.1 Hz), 4.72 (1H, ddd, J ) 4.8, 7.8,

12.6 Hz), 5.55 (2H, broad), 6.29 (2H, broad), 6.72 (1H, d, J )

3.7 Hz), 7.02 (1H, d, J ) 7.8 Hz), 7.36 (1H, d, J ) 3.7 Hz),

11.24 (1H, brs). Anal. (C₂₁H₂₉N₅O₆S₂) C, H, N, S.

1

1344, 1207 cm^-1; H NMR (DMSO-d₆) δ 1.13 (t, 3H, J ) 7.0

Hz), 1.14 (t, 3H, J ) 7.0 Hz), 1.91 (m, 4H), 2.37 (t, 2H, J ) 7.3

Hz), 2.89 (m, 2H), 3.23 (m, 2H), 3.49 (m, 1H), 4.00 (q, 2H, J )

7.0 Hz), 4.06 (q, 2H, J ) 7.0 Hz), 4.34 (m, 1H), 6.01 (s, 2H),

6.63 (s, 1H), 6.90 (d, 1H, J ) 3.7 Hz), 7.66 (d, 1H, J ) 3.7 Hz),

Meth od B. 2(S)-[[(5-Br om o-3-m eth ylth iop h en e-2-yl]-

ca r bon yl]a m in o]p en ta n ed ioic Acid Dieth yl Ester (23c).

To a stirred solution of of 5-bromo-3-methylthiophene-2-

carboxylic acid⁴⁸(10.86 g, 49.1 mmol), HOBT (6.97 g, 51.6

mmol), DIEA (9.0 mL, 51.7 mmol), and ^L-glutamic acid diethyl

ester hydrochloride (12.36 g, 51.6 mmol) in DMF (70 mL) was

added EDC (9.89 g, 51.6 mmol). The reaction mixture was

stirred under argon for 18 h, poured into H₂O, and extracted

with ethyl acetate. The organic layer was washed sequentially

with 0.5 N HCl, saturated NaHCO₃, and brine, dried (MgSO₄),

and then concentrated in vacuo. This residue was purified

by flash chromatography, eluting with CH₂Cl₂-EtOAc (20:1)

to give 19.70 g (99%) of the desired product as a colorless oil:

IR (neat) 3329, 2982, 1738, 1651,1545, 1514, 1417, 1377, 1258,

8.60 (d, 1H, J ) 7.3 Hz), 10.07 (s, 1H). Anal. (C₂₂H₂₉

N₅O₆S₂‚1.0H₂O) C, H, N, S.

-

2(S)-[[[5-[2-(2-Am in o-4-oxo-4,6,7,8-tetr a h yd r o-3H-p yr -

im id o[5,4-b][1,4]th ia zin -6(S)-yl)eth yl]th iop h en e-2-yl]ca r -

bon yl]a m in o]p en ta n ed ioic Acid Dieth yl Ester . Meth od

B: off-white solid; mp 105-110 °C (with foaming); [R]₅₈₉-55.5°

(c ) 0.53, DMSO); IR (Br) 3345, 2930, 1734, 1653, 1636, 1541,

1

1456, 1345 cm^-1; H NMR (DMSO-d₆) δ 1.12 (t, 3H, J ) 7.0

Hz), 1.14 (t, 3H, J ) 7.0 Hz), 1.75-2.03 (m, 4H), 2.37 (t, 2H,

J ) 7.4 Hz), 2.88 (m, 2H), 3.17 (m, 2H), 3.47 (m, 1H), 4.01 (q,

2H, J ) 7.0 Hz), 4.07 (q, 2H, J ) 7.0 Hz), 4.34 (m, 1H), 6.00

(s, 2H), 6.64 (s, 1H), 6.89 (d, 1H, J ) 3.7 Hz), 7.65 (d, 1H, J )

3.7 Hz), 8.60 (d, 1H, J ) 7.3 Hz), 10.05 (s, 1H). Anal.

(C₂₂H₂₉N₅O₆S₂‚1.0H₂O) C, H, N, S.

1

1206 cm^-1; H NMR (CDCl₃) δ 1.24 (3H, t, J ) 7.1 Hz), 1.30

(3H, t, J ) 7.1 Hz), 2.04-2.45 (4H, m), 2.48 (3H, s), 4.12 (2H,

q, J ) 7.1 Hz), 4.23 (2H, q, J ) 7.1 Hz), 4.71 (1H, ddd, J )

12.3, 7.2, 4.8 Hz), 6.56 (1H, d, J ) 7.3 Hz), 6.87 (1H, s). Anal.

(C₁₅H₂₀BrNO₅S) C, H, Br, N, S.

2(S)-[[[5-[2-(2-Am in o-4-oxo-4,6,7,8-tetr a h yd r o-3H-p yr -

im ido[5,4-b][1,4]th iazin -6(R)-yl)eth yl]-4-m eth ylth ioph en e-

2-yl]ca r bon yl]a m in o]p en ta n ed ioic Acid Dieth yl Ester .

Meth od B: off-white solid; mp 124 °C (with foaming); [R]₅₈₉

+33.6° (c ) 0.50, DMSO); IR (KBr) 2996, 2860, 1734, 1653,

2(S)-[[(5-Br om o-4-m eth ylth iop h en e-2-yl)ca r bon yl]a m i-

n o]p en ta n ed ioic Acid Dieth yl Ester (23d ). Meth od B:

yellow oil; IR (neat) 3339, 2984, 1738, 1634, 1562, 1527, 1425,

1

1636, 1559, 1456, 1206 cm^-1; H NMR (DMSO-d₆) δ 1.11 (t,

3H, J ) 7.0 Hz), 1.15 (t, 3H, J ) 7.0 Hz), 1.75-2.04 (m, 4H),

2.10 (s, 3H), 2.37 (t, 2H, J ) 7.4 Hz), 2.85 (m, 2H), 3.12 (m,

2H), 3.50 (m, 1H), 4.00 (q, 2H, J ) 7.0 Hz), 4.06 (q, 2H, J )

7.0 Hz), 4.31 (m, 1H), 6.01 (s, 2H), 6.65 (s, 1H), 7.55 (s, 1H),

8.53 (d, 1H, J ) 7.7 Hz), 10.08 (s, 1H); HRMS calcd for

C₂₃H₃₁N₅O₆S₂(M + Cs⁺) 670.0770, found 670.0742. Anal.

(C₂₃H₃₁N₅O₆S₂) C, H, N, S.

1

1209 cm^-1; H NMR (CDCl₃) δ 1.24 (3H, t, J ) 7.1 Hz), 1.30

(3H, t, J ) 7.1 Hz), 2.04-2.52 (4H, m), 2.20 (3H, s), 4.12 (2H,

q, J ) 7.3 Hz), 4.23 (2H, q, J ) 7.3 Hz), 4.70 (1H, ddd, J )

12.3, 7.7, 4.8 Hz), 6.84 (1H, d, J ) 7.4 Hz), 7.22 (1H, s). Anal.

(C₁₅H₂₀BrNO₅S) C, H, Br, N, S.

2(S)-[[(5-Br om o-3-eth ylth ioph en e-2-yl)car bon yl]am in o]-

p en ta n ed ioic Acid Dieth yl Ester (23e). Meth od B: yellow

oil; IR (neat) 3324, 2978, 2936, 1738, 1651, 1543, 1512, 1204

2(S)-[[[5-[2-(2-Am in o-4-oxo-4,6,7,8-tetr a h yd r o-3H-p yr -

im ido[5,4-b][1,4]th iazin -6(S)-yl)eth yl]-4-m eth ylth ioph en e-

2-yl]ca r bon yl]a m in o]p en ta n ed ioic Acid Dieth yl Ester .

Meth od B: amorphous solid; [R]₅₈₉-54.1° (c ) 0.61, DMSO);

¹H NMR (DMSO-d₆) δ 1.15 (t, 3H, J ) 7.1 Hz), 1.17 (t, 3H, J

) 7.1 Hz), 1.61-1.72 (m, 1H), 1.77-2.07 (m, 3H), 2.13 (s, 3H),

2.40 (t, 2H, J ) 7.5 Hz), 2.77-2.94 (m, 3H), 3.18-3.28 (m, 1H),

3.50-3.56 (m, 1H), 4.03 (q, 2H, J ) 7.1 Hz), 4.08 (q, 2H, J )

7.1 Hz), 4.34 (ddd, 1H, J ) 5.4, 7.7, 9.6 Hz), 6.02 (s, 2H), 6.67

(s, 1H), 7.58 (s, 1H), 8.55 (d, 1H, J ) 7.7 Hz), 10.06 (s, 1H).

Anal. (C₂₃H₃₁N₅O₆S₂‚0.5H₂O) C, H, N, S.

1

cm ^-1; H NMR (CDCl₃) δ 1.22-1.33 (3 overlapping triplets,

9H), 2.11 (m, 1H), 2.30 (m, 1H), 2.44 (m, 2H), 2.90 (q, 2H, J )

7.5 Hz), 4.12 (q, 2H, J ) 7.1 Hz), 4.23 (q, 2H, J ) 7.1 Hz), 4.71

(m, 1H), 6.54 (d, 1H, J ) 7.2 Hz), 6.94 (s, 1H). Anal. (C₁₆H₂₂

BrNO₅S) C, H, Br, N, S.

-

2(S)-[(3-Iod oben zoyl)a m in o]p en ta n ed ioic Acid Dieth yl

Ester (23f). Meth od B: white solid; mp 65-66 °C; ¹H NMR

(CDCl₃) δ 1.23 (3H, t, J ) 7.1 Hz), 1.30 (3H, t, J ) 7.1 Hz),

2.19-2.06 (1H, m), 2.36-2.24 (1H, m), 2.56-2.39 (2H, m), 4.12

(2H, q, J ) 7.1 Hz), 4.23 (2H, q, J ) 7.1 Hz), 4.75 (1H, ddd, J

) 4.8, 7.4, 12.5 Hz), 7.09 (1H, d, J ) 7.4 Hz), 7.17 (1H, d, J )

Gen er a l P r oced u r e for th e F or m a tion of a TBS Eth er .

ter t-Bu tyld im eth yl(2-th iop h en e-2-yleth oxy)sila n e. To a

Glycinamide Ribonucleotide Transformylase Inhibitors

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16 2517

solution of TBSCl (26.38 g, 0.18 mol), TEA (25 mL, 0.18 mol),

and DMAP (300 mg, 2.5 mmol) in CH₂Cl₂(200 mL) under an

argon atmosphere at -5 °C was added, dropwise, 2-(2-thienyl)-

ethanol (18 mL, 0.16 mol). The resultant mixture was stirred

at 0 °C for 30 min and then at room temperature for 18 h.

The crude mixture was poured into water (300 mL), and the

layers were separated. The organic phase was washed with

0.5 N HCl (200 mL) and then with brine (200 mL), dried (Na₂-

SO₄), and concentrated. The crude residue was purified by

flash chromatography with hexane/EtOAc (95:5) to give 38.73

g (99%) of the product as a yellow oil: ¹H NMR (CDCl₃) δ 0.03

(6H, s), 0.89 (9H, s), 3.03 (2H, t, J ) 6.7 Hz), 3.82 (2H, t, J )

6.7 Hz), 6.83 (1H, d, J ) 3.3 Hz), 6.92 (1H, dd, J ) 3.3, 5.1

Hz), 7.13 (1H, d, J ) 5.1 Hz). Anal. (C₁₂H₂₂OSSi) C, H, S.

t er t -Bu t yld im e t h yl(4,5,6,7-t e t r a h yd r ob e n zo[b]t h io-

ph en e-6-ylm eth oxy)silan e (28): colorless oil; ¹H NMR (CDCl₃)

δ 0.06 (6H, s), 0.91 (9H, s), 1.53-1.41 (1H, m), 2.03-1.92 (2H,

m), 2.65-2.42 (2H, m), 2.76-2.68 (1H, m), 2.89 (1H, dd, J )

5.2, 16.2 Hz), 3.60 (2H, d, J ) 6.2 Hz), 6.75 (1H, d, J ) 5.1

Hz), 7.05 (1H, d, J ) 5.1 Hz). Anal. (C₁₅H₂₆OSSi) C, H, S.

5-[4-[(ter t-Bu tyld im eth ylsila n yl)oxy]-3(S)-h yd r oxybu -

tyl]th iop h en e-2-ca r boxylic a cid eth yl ester : colorless oil;

[R]₅₈₉-26.6° (c ) 0.82, MeOH); IR (neat) 3486 (broad) 2953,

in MeOH (1 L) containing concentrated H₂SO₄(10 mL), and

this solution was heated under refluxed for 18 h. After the

solvent was removed by concentration, the residue was

partitioned between saturated NaHCO₃(300 mL) and Et₂O

(300 mL). The layers were separated and the aqueous phase

extracted with EtOAc (2 × 300 mL). The combined organic

extracts were dried (Na₂SO₄). The crude residue was purified

by flash chromatography with hexane/EtOAc (2:1) to give 12.31

g (44%) of the product as a yellow oil: ¹H NMR (CDCl₃) δ 3.09

(2H, t, J ) 6.2 Hz), 3.86 (3H, s), 3.89 (2H, t, J ) 6.2 Hz), 6.88

(1H, d, J ) 3.7 Hz), 7.66 (1H, d, J ) 3.7 Hz). Anal. (C₈H₁₀O₃S)

C, H, S.

5-Br om o-3-eth ylth iop h en e-2-ca r boxylic Acid Meth yl

Ester . To a stirred solution of 3-ethylthiophene (10.55 g, 94

mmol) and DMF (9.5 mL, 123 mmol) at 0 °C was added POCl₃

(11.0 mL, 123 mmol). When the addition was complete, the

reaction mixture was heated at 70 °C for about 30 min, cooled

to room temperature, poured into H₂O, and extracted with

Et₂O. The aqueous layers were made basic with 1 N aqueous

NaOH and extracted with Et₂O until free from any UV active

compound by TLC. The combined Et₂O layers were washed

once with saturated NaHCO₃solution, dried (MgSO₄), and

concentrated to 13.16 g (quantitative) of an orange oil as a

mixture of 3-ethyl 2-carboxaldehyde and 4-ethyl 2-carboxal-

dehyde in a 5:1 ratio. To this crude mixture dissolved in HOAc

was added 5.03 mL (103 mmol) of bromine. After 44 h at 45

°C, the mixture was cautiously poured into saturated NaHCO₃

solution and extracted with EtOAc. The combined organic

layers were washed with brine solution, dried (MgSO₄), and

concentrated to give 17.71 g of a crude mixture of bromo

carboxaldehydes which were subjected to oxidation conditions

without purification. To a stirred solution of silver nitrate

(27.46 g, 162 mmol) in H₂O (350 mL) was added a solution of

NaOH (12.93 g, 323 mmol) in H₂O (50 mL). The resulting

thick brown slurry was stirred at room temperature while the

crude aldehyde was added neat dropwise. After 3 h at room

temperature, the reaction mixture was filtered washing the

filter cake with 2 N NaOH. The filtrate was acidified with 12

N HCl and cooled, and the precipitate was collected, washed

with H₂O, and dried. The residue was slurried in hexanes,

filtered, and dried to give 6.27 g (28% overall, from 3-ethyl-

thiophene) of 5-bromo-3-ethylthiophene-2-carboxylic acid as a

light gray solid: IR (KBr) 2961 (broad), 1674, 1651, 1423, 1279

cm^-1; ¹H NMR (CDCl₃) δ 1.23 (t, 3H, J ) 7.5 Hz), 2.99 (q, 2H,

J ) 7.5 Hz), 6.70 (s, 1H).

1

2930, 2859, 1709, 1462, 1259 cm^-1; H NMR (CDCl₃) δ 0.07

(s, 6H), 0.90 (s, 9H), 1.36 (t, 3H, J ) 7.2 Hz), 1.78 (m, 2H),

2.96 (m, 2H), 3.43 (m, 1H), 3.64 (m, 2H), 4.32 (q, 2H, J ) 7.0

Hz), 6.82 (d, 1H, J ) 3.7 Hz), 7.62 (d 1H, J ) 4.0 Hz). Anal.

(C₁₇H₃₀O₄SSi) C, H, S.

5-[4-[(ter t-Bu tyld im eth ylsilyl)oxy]-3(S)-h yd r oxybu tyl]-

4-m eth ylth iop h en e-2-ca r boxylic a cid eth yl ester : color-

less oil; [R]₅₈₉-23.5° (c ) 1.19, MeOH); ¹H NMR (CDCl₃) δ

0.07 (s, 6H), 0.90 (s, 9H), 1.35 (t, 3H, J ) 7.1 Hz), 1.61-1.81

(m, 2H), 2.17 (s, 3H), 2.79-3.01 (m, 2H), 3.43 (dd, 1H, J )

7.0, 9.6 Hz), 3.63 (dd, 1H, J ) 3.3, 9.6 Hz), 3.66-3.71 (m, 1H),

4.31 (q, 2H, J ) 7.1 Hz), 7.50 (s, 1H). Anal. (C₁₈H₃₂O₄SSi) C,

H, S.

4-(2-Br om oeth yl)ben zoic Acid Meth yl Ester . This was

prepared using 4-(2-bromoethyl)benzoic and CH₂N₂: yellow oil;

¹H NMR (CDCl₃) δ 3.23 (2H, t, J ) 7.4 Hz), 3.59 (2H, t, J )

7.4 Hz), 3.92 (3H, s), 7.30 (2H, d, J ) 8.3 Hz), 8.01 (2H, d, J

) 8.3 Hz). Anal. (C₁₀H₁₁O₂Br) C, H, Br.

5-Br om ot h iop h en e-2-ca r b oxylic Acid Met h yl E st er

(23b). This was prepared using CH₂N₂from 5-bromo-

thiophene-2-carboxylate: yellow solid; mp 58-59 °C; ¹H NMR

(CDCl₃) δ 3.87 (3H, s), 7.07 (1H, d, J ) 4.0 Hz), 7.55 (1H, d, J

) 4.0 Hz). Anal. (C₆H₅O₂SBr) C, H, S, Br.

7-Oxo-4,5,6,7-tetr a h yd r oben zo[b]th iop h en e-6-ca r boxy-

lic Acid Meth yl Ester . A solution of 7-oxo-4,5,6,7-tetrahy-

drobenzothiophene (25) (1.37 g, 9 mmol) in DMF (10 mL) was

added dropwise, under an argon atmosphere, to a suspension

of NaH (800 mg, 20 mmol) in DMF (6 mL). The resultant,

purple solution was stirred at ambient temperature for 15 min

and then cooled to 0 °C prior to the dropwise addition of

dimethyl carbonate (5.35 g, 59 mmol). The resultant reaction

mixture was stirred at ambient temperature for 90 min, then

poured into water (150 mL), and extracted with Et₂O (3 × 50

mL) and EtOAc (50 mL). The combined organic extracts were

dried (MgSO₄) and concentrated. The crude residue was

purified by flash chromatography with hexane/EtOAc (4:1) to

give 1.28 g (68%) of the product as a yellow oil: ¹H NMR

(CDCl₃) δ 2.43-2.33 (1H, m), 2.62-2.50 (1H, m), 2.87 (1H, ddd,

J ) 4.9, 8.2, 17.1 Hz), 3.05 (1H, ddd, J ) 4.9, 6.5, 17.1 Hz),

3.61 (1H, dd, J ) 4.8, 9.0 Hz), 3.77 (3H, s), 6.97 (1H, d, J )

4.8 Hz), 7.66 (1H, d, J ) 4.8 Hz). Anal. (C₁₀H₁₀O₃S) C, H, S.

7-Hyd r oxy-4,5,6,7-tetr a h yd r oben zo[b]th iop h en e-6-ca r -

boxylic Acid Meth yl Ester (26) a n d 6-(Hyd r oxym eth yl)-

4,5,6,7-tetr a h yd r oben zo[b]th iop h en e-7-ol (27). To a so-

lution of methyl 7-oxo-4,5,6,7-tetrahydrobenzothiophene-6-

carboxylate (2.57 g, 12.2 mmol) in THF (15 mL) and MeOH

(10 mL) at 0 °C was added, portionwise, NaBH₄(465 mg, 12.2

mmol). The resultant reaction mixture was stirred for 2 h,

gradually warming to 15 °C, and then poured into saturated

NH₄Cl (30 mL). The layers were separated, and the aqueous

phase was extracted with EtOAc (30 mL). The combined

organic extracts were dried (MgSO₄) and concentrated. The

crude residue was purified by flash chromatography with

hexane/EtOAc (3:2) to give two separate products. The faster-

5-(3-Hydr oxypr opyl)th ioph en e-2-car boxylic Acid Meth -

yl Ester (24b). To a solution of the above alkyne (7.41 g, 38

mmol) in THF (140 mL) was added 2,4,6-triisopropylbenzene-

sulfonyl hydrazide (96.13 g, 0.32 mol) in four portions at 90

min intervals. After the reaction had been heated at reflux

for a total of 6.5 h, the solvent was removed and the residue

was partitioned between 0.5 N NaOH (700 mL) and Et₂O (500

mL). The layers were separated, and the aqueous phase was

extracted with Et₂O (250 mL). The combined organic extracts

were washed with 0.5 N NaOH (2 × 150 mL), dried (Na₂SO₄),

and concentrated to give an oil which was purified by flash

chromatography with hexane/EtOAc (2:1) to give 3.67 g (48%)

of the product as a yellow oil: ¹H NMR (CDCl₃) δ 1.96 (2H, tt,

J ) 6.2, 7.6 Hz), 2.96 (2H, t, J ) 7.6 Hz), 3.71 (2H, t, J ) 6.2

Hz), 3.86 (3H, s), 6.82 (1H, d, J ) 3.8 Hz), 7.64 (1H, d, J ) 3.8

Hz). Anal. (C₉H₁₂O₃S) C, H, S.

5-(2-Hyd r oxyeth yl)th iop h en e-2-ca r boxylic Acid Meth -

yl Ester (24c). To a solution of 2-[2-[(tert-butyldimethylsilyl)-

oxy]ethyl]thiophene (36.16 g, 0.15 mol) in THF (350 mL) under

argon at -75 °C was added a 1.6 M solution of n-butyllithium

in hexane (140 mL, 0.22 mol). After the mixture was stirred

for 1 h at -70 °C, dry CO₂was then bubbled though this

solution, for 40 min at -60 °C, and then for 60 min at -5 °C

and an additional 75 min while warming to room temperature.

The crude reaction mixture was poured into a mixture of

saturated NH₄Cl (600 mL) and ice (600 g) and extracted with

Et₂O (300 mL) and then with EtOAc (2 × 300 mL). The

combined organic extracts were dried (Na₂SO₄) and concen-

trated to give 47 g a yellow solid which was used without

further purification. The above product (47 g) was dissolved

2518 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16

Varney et al.

eluting product was a yellow oil, 0.91 g (35%). The following

analyses indicated that this product was compound 26: ¹H

NMR (CDCl₃) δ 2.38-2.05 (2H, m), 2.90-2.57 (3H, m), 3.09

(1H, broad), 3.78 (3H, s), 5.23-5.16 (1H, m), 6.78 (1H, d, J )

5.1 Hz), 7.25 (1H, d, J ) 5.1 Hz). Anal. (C₁₀H₁₂O₃S) C, H, S.

The slower-eluting product was a milky-white gum, 1.28 g

(57%). The following analyses indicated that this second

product was diol 27: ¹H NMR (CDCl₃) δ 1.63-1.48 (1H, m),

1.77-1.71 (1H, m), 1.91-1.80 (1H, m), 2.07-1.94 (1H, m),

2.86-2.54 (3H, m), 3.96-3.79 (2H, m), 5.03-4.85 (1H, m), 6.79

(1H, d, J ) 5.1 Hz), 7.24 (1H, d, J ) 5.1 Hz). Anal. (C₉H₁₂O₂S)

C, H, S.

4,5,6,7-Tetr ah ydr oben zo[b]th ioph en e-6-car boxylic Acid

Meth yl Ester . To a solution of methyl 7-hydroxy-4,5,6,7-

tetrahydrobenzothiophene-6-carboxylate (459 mg, 2.2 mmol)

and Et₃SiH (510 mg, 4.4 mmol) in CH₂Cl₂(10 mL), under argon

at -5 °C, was added BF₃‚Et₂O (0.55, 4.5 mmol). The resultant

reaction mixture was stirred for 3 h, gradually warming to 15

°C, and then poured into saturated NaHCO₃(30 mL). After

addition of K₂CO₃(∼1 g), the layers were separated and the

aqueous phase was extracted with CH₂Cl₂(10 mL) and Et₂O

(2 × 15 mL). The combined organic extracts were dried

(MgSO₄) and concentrated. The crude residue was purified

by flash chromatography with hexane/EtOAc (95:5) to give 300

mg (71%) of the product as a colorless oil: ¹H NMR (CDCl₃) δ

1.94-1.81 (1H, m), 2.27-2.18 (1H, m), 2.85-2.59 (3H, m),

3.11-2.94 (2H, m), 3.73 (3H, s), 6.75 (1H, d, J ) 5.1 Hz), 7.07

(1H, d, J ) 5.1 Hz). Anal. (C₁₀H₁₂O₂S) C, H, S.

4,5,6,7-Tet r a h yd r ob en zo[b]t h iop h en e-6-yl)m et h a n ol.

Met h od A. A solution of methyl 4,5,6,7-tetrahydroben-

zothiophene-6-carboxylate (209 mg, 1.1 mmol) in THF (6 mL)

was added to a slurry of LiAlH₄(50 mg, 1.3 mmol) in THF (3

mL). The resultant reaction mixture was heated at reflux for

3 h. After being cooled to room temperature, the crude

reaction mixture was diluted with saturated NH₄Cl (20 mL).

The layers were separated, and the aqueous phase was

extracted with Et₂O (10 mL) and then with EtOAc (2 × 10

mL). The combined organic extracts were dried (MgSO₄) and

concentrated to give the 167 mg (93%) of product as a colorless

oil.

Meth od B. To a solution of 988 mg (5.4 mmol) of 7-hydroxy-

6-(hydroxymethyl)-4,5,6,7-tetrahydrobenzothiophene and 1.31

g (11.3 mmol) of Et₃SiH in CH₂Cl₂(25 mL), under argon at

-5 °C, was added 1.62 g (11.3 mmol) of BF₃‚Et₂O. The

resultant reaction mixture was stirred for 3 h, gradually

warming to 10 °C, and then poured into saturated NaHCO₃

(50 mL). After addition of K₂CO₃(1.5 g), the layers were

separated and the aqueous phase was extracted with Et₂O (2

× 40 mL). The combined organic extracts were dried (MgSO₄),

and the crude residue was purified by flash chromatography

with hexane/EtOAc (2:1) to give 593 mg (66%) of the product

as a colorless oil. The following analyses indicated that the

product was 6-(hydroxymethyl)-5,5,6,7-tetrahydrobenzothio-

phene: ¹H NMR (CDCl₃) δ 1.54-1.46 (1H, m), 2.11-1.96 (2H,

m), 2.68-2.46 (2H, m), 2.79-2.71 (1H, m), 2.94 (1H, dd, J )

5.2, 16.2 Hz), 3.66 (2H, d, J ) 6.4 Hz), 6.76 (1H, d, J ) 5.1

Hz), 7.06 (1H, d, J ) 5.1 Hz). Anal. (C₉H₁₂OS) C, H, S.

2(S )-[[[6-[[(t er t -Bu t yld im e t h ylsila n yl)oxy]m e t h yl]-

4,5,6,7-tetr a h yd r oben zo[b]th iop h en e-2-yl]ca r bon yl]a m i-

n o]p en ta n ed ioic Acid Dieth yl Ester (29). To a solution

of 6-[[(tert-butyldimethylsilyl)oxy]methyl]-4,5,6,7-tetrahydroben-

zothiophene (7.69 g, 27.2 mmol) in THF (100 mL), under argon

at -70 °C, was added a 2.5 M solution of n-butyllithium in

hexane (12 mL, 30 mmol). The resultant reaction mixture was

stirred for an additional 40 min at -70 °C and then at -10 °C

for 45 min while dry CO₂was bubbled though the solution.

The crude reaction mixture was subsequently poured into

saturated NH₄Cl (300 mL). The layers were separated, and

the aqueous phase was extracted with Et₂O (2 × 150 mL). The

combined organic extracts were dried (Na₂SO₄) and concen-

trated to give 8.24 g of a yellow solid. This intermediate was

employed in the subsequent reaction without any further

purification. The glutamate was coupled using method B

described above to give 6.63 g (48%) of the product as a yellow

oil: ¹H NMR (CDCl₃) δ 0.06 (6H, s), 0.90 (9H, s), 1.23 (3H, t,

J ) 7.1 Hz), 1.29 (3H, t, J ) 7.1 Hz), 1.56-1.42 (1H, m), 2.15-

1.91 (3H, m), 2.75-2.23 (6H, m), 2.89 (1H, dd, J ) 5.1, 16.7

Hz), 3.59 (2H, d, J ) 6.2 Hz), 4.11 (2H, q, J ) 7.1 Hz), 4.22

(2H, q, J ) 7.1 Hz), 4.73 (1H, ddd, J ) 4.8, 7.7, 12.6 Hz), 6.67

(1H, d, J ) 7.7 Hz), 7.23 (1H, s). Anal. (C₂₅H₄₁NO₆SSi) C, H,

N, S.

2(S)-[[[6-(Hyd r oxym eth yl)-4,5,6,7-tetr a h yd r oben zo[b]-

th iop h en e-2-yl]ca r bon yl]a m in o]p en ta n ed ioic Acid Di-

eth yl Ester . This material was prepared using the procedure

described for 5-(2-hydroxyethyl)thiophene-2-carboxylic acid

methyl ester: yellow gum; ¹H NMR (CDCl₃) δ 1.23 (3H, t, J )

7.1 Hz), 1.29 (3H, t, J ) 7.1 Hz), 1.57-1.44 (1H, m), 2.15-

1.97 (3H, m), 2.77-2.22 (6H, m), 2.94 (1H, dd, J ) 5.0, 16.9

Hz), 3.66 (2H, d, J ) 6.3 Hz), 4.11 (2H, q, J ) 7.1 Hz), 4.22

(2H, q, J ) 7.1 Hz), 4.73 (1H, ddd, J ) 4.8, 7.6, 12.6 Hz), 6.70

(1H, d, J ) 7.6 Hz), 7.23 (1H, s). Anal. (C₁₉H₂₇NO₆S‚0.5H₂O)

C, H, N, S.

1,1-Dim eth oxybu t-3-yn -2-ol (30). To a stirred solution of

TMS acetylene (1.037 g, 10.55 mmol) in dry THF (50 mL)

under argon at -78 °C was added dropwise 1.6 M n-butyl-

lithium (6.6 mL). After 10 min at -78 °C, a solution of glyoxal

dimethyl acetal (1.21 g, 10.46 mmol) in THF (5 mL) was added

dropwise. After 1 h at -78 °C, the reaction was quenched with

H₂O (1 mL), and the mixture was allowed to warm to room

temperature, diluted with EtOAc, and washed with brine. The

aqueous layer was reextracted with EtOAc, and the combined

organic layers were dried (MgSO₄) and concentrated to give

785 mg of the crude addition product as a yellow oil. The crude

product was dissolved in THF, and to it was added 5.8 mL of

1.0 M tetrabutylammonium fluoride in THF. After the

mixture was heated for 1 h at 50 °C, the volatiles were

evaporated and the residue was flash chromatographed on

silica with CH₂Cl₂-EtOAc (9:1) to give 412 mg (60% overall)

of the alkyne-alcohol 30 as a colorless oil: IR (neat) 3441

1

(broad), 3277, 2944, 2839, 1636, 1450 cm^-1; H NMR (CDCl₃)

δ 2.42 (bs, 1H), 2.49 (s, 1H), 3.51 (s, 3H), 3.53 (s, 3H), 4.36

(bs, 2H). Anal. (C₆H₁₀O₃‚0.35H₂O) C, H.

4-[3-[(Meth ylsu lfon yl)oxy]-4,4-d im eth oxybu tyl]ben zo-

ic Acid Meth yl Ester . To a stirred solution of alcohol 31 (206

mg, 0.77 mmol) and TEA (0.16 mL, 1.15 mmol) in CH₂Cl₂(5

mL) at 0 °C was added methanesulfonyl chloride (0.07 mL,

0.85 mmol). After 20 min at 0 °C, another 0.02 mL more

methanesulfonyl chloride was added. After 30 min more, the

reaction mixture was poured into saturated NaHCO₃and

extracted with 2 × CH₂Cl₂. The combined organic layers were

dried (MgSO₄). This material was sufficiently pure for use in

the next step. An analytical sample was obtained by flash

chromatography on silica, eluting with CH₂Cl₂/EtOAc (20:1)

to give the mesylate as a colorless oil: IR (neat) 2949, 2839,

1

1719, 1611, 1437, 1352, 1283 cm^-1; H NMR (CDCl₃) δ 2.04

(m, 2H), 2.77-2.91 (m, 2H), 3.09 (s, 3H), 3.41 (s, 3H), 3.45 (s,

3H), 3.90 (s, 3H), 4.38 (d, 1H, J ) 5.5 Hz), 4.64 (m, 1H), 7.29

(d, 2H, J ) 8.2 Hz), 7.96 (d, 2H, J ) 8.2 Hz). Anal.

(C₁₅H₂₂O₇S) C, H, S.

4-[3-[(Met h ylsu lfon yl)oxy]-4-oxob u t yl]b en zoic Acid

Meth yl Ester . To a stirred solution of the crude dimethyl

acetal mesylate from above (600 mg, 1.73 mmol) in CHCl₃(5

mL) at 0 °C was added H₂O (1 mL) and trifluoroacetic acid (1

mL). The reaction was warmed to room temperature and then

heated under reflux for 24 h. The cooled reaction mixture was

diluted with EtOAc, washed sequentially with saturated NaCl,

saturated NaHCO₃, and then again with saturated NaCl, and

dried (MgSO₄), and the volatiles were removed in vacuo. This

material was used immediately in the next step: ¹H NMR

(CDCl₃) δ 2.20 (m, 2H), 2.85 (m, 2H), 3.17 (s, 3H), 3.91 (s, 3H),

4.95 (dd, 1H, J ) 4.2, 8.4 Hz), 7.29 (d, 2H, J ) 8.1 Hz), 7.99

(d, 2H, J ) 8.2 Hz), 9.59 (s, 1H).

4-[3-[(4-Meth oxyben zyl)th io]-4-oxobu tyl]ben zoic Acid

Meth yl Ester . To a stirred solution of the mesylate aldehyde

prepared above (686 mg, 2.28 mmol) and DIEA (0.40 mL, 2.29

mmol) in DMF was added 4-methoxy-R-toluenethiol (0.48 mL,

3.44 mmol). After 3 h at room temperature, the reaction

mixture was poured into 0.5 N HCl and extracted with EtOAc.

The combined organic layers were washed with brine, dried

(MgSO₄), and concentrated in vacuo. The material obtained

was sufficiently pure to use without further purification in the

next step: IR (KBr) 2930, 1715, 1703, 1611, 1512, 1282, 1244

Glycinamide Ribonucleotide Transformylase Inhibitors

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16 2519

1

cm^-1; ¹H NMR (CDCl₃) δ 1.80-2.16 (m, 2H), 2.75 (m, 2H), 2.99

(m, 1H), 3.53 (AB, 2H, J ) 13.4 Hz), 3.81 (s, 3H), 3.90 (s, 3H),

6.83 (d, 2H, J ) 8.5 Hz), 7.12 (d, 2H, J ) 8.2 Hz), 7.19 (d, 2H,

J ) 8.6 Hz), 7.90 (d, 2H, J ) 8.2 Hz), 9.27 (d, 1H, J ) 4.2 Hz).

4-[3-[1,3]Dioxola n -2-yl-3-[(4-m eth oxyben zyl)su lfa n yl]-

p r op yl]ben zoic Acid Meth yl Ester . A flask containing the

above aldehyde (301 mg, 0.84 mmol), ethylene glycol (94 µL,

1.68 mmol), and pyridinium p-toluenesulfonate (42 mg, 0.17

mmol) in C₆H₆(30 mL) was heated at reflux, removing the

generated water with a Dean-Stark trap. After 3 h, the

reaction mixture was poured into brine and extracted twice

with EtOAc. The combined organic layers were dried (MgSO₄),

and the crude residue was flash chromatographed on silica

eluting hexanes-EtOAc (5:1) to give an 84% overall of the title

compound from the mesylate dimethyl acetal: IR (neat) 2949,

cm^-1; H NMR (DMSO-d₆) δ 1.72 (m, 1H), 1.90 (m, 1H), 2.80

(m, 3H), 3.22 (m, 1H), 3.52 (m, 1H), 3.82 (s, 3H), 6.00 (s, 2H),

6.65 (s, 1H), 7.37 (d, 2H, J ) 8.1 Hz), 7.87 (d, 2H, J ) 8.1 Hz),

10.05 (s, 1H). Anal. (C₁₆H₁₈N₄O₃S) C, H, N, S.

3-[3-[(Meth ylsu lfon yl)oxy]-4,4-d im eth oxybu tyl]ben zo-

ic Acid Meth yl Ester . This compound was prepared accord-

ing to the general procedure described for 4-[3-[(methylsulfonyl)-

oxy]-4,4-dimethoxybutyl]benzoic acid methyl ester to give 20.69

g (99%) of a yellow oil: ¹H NMR (CDCl₃) δ 2.13-1.96 (2H, m),

2.77 (1H, ddd, J ) 6.6, 10.0 14.0 Hz), 2.90 (1H, ddd, J ) 6.0,

10.1, 14.0 Hz), 3.09 (3H, s), 3.42 (3H, s), 3.45 (3H, s), 3.91 (3H,

s), 4.38 (1H, d, J ) 5.4 Hz), 4.66 (1H, ddd, J ) 3.8, 5.4, 8.8

Hz), 7.36 (1H, t, J ) 7.5 Hz), 7.42 (1H, dt, J ) 7.5, 1.4 Hz),

7.90-7.85 (2H, m). Anal. (C₁₅H₂₂O₇S) C, H, S.

3-[3-[(Met h ylsu lfon yl)oxy]-4-oxob u t yl]b en zoic Acid

Meth yl Ester . This compound was prepared according to the

general procedure described for 4-[3-[(methylsulfonyl)oxy]-4-

oxobutyl]benzoic acid methyl ester to give 15.42 g (87%) of a

yellow oil: ¹H NMR (CDCl₃) δ 2.33-2.09 (2H, m), 2.90-2.83

(1H, m), 3.15-3.08 (1H, m), 3.18 (3H, s), 3.92 (3H, s), 4.95 (1H,

dd, J ) 4.3, 8.5 Hz), 7.44-7.36 (2H, m), 7.93-7.86 (2H, m),

9.59 (1H, s). Anal. (C₁₃H₁₆O₆S‚0.6H₂O) C, H, S.

2886, 1721, 1611, 1510, 1435, 1279, 1248 cm^-^{1 1}H NMR

;

(CDCl₃) δ 1.67 (m, 1H), 1.99 (m, 1H), 2.57 (m, 2H), 2.84 (m,

1H), 3.72-4.03 (m, 6H), 3.81 (s, 3H), 3.90 (s, 3H), 4.97 (d, 1H,

J ) 4.6 Hz), 6.83 (d, 2H, J ) 8.6 Hz), 7.09 (d, 2H, J ) 8.1 Hz),

7.23 (d, 2H, J ) 8.5 Hz), 7.88 (d, 2H, J ) 8.1 Hz). Anal.

(C₂₂H₂₆O₅S) C, H, S.

4-(3-[1,3]Dioxola n -2-yl-3-m er ca p top r op yl)ben zoic Acid

Meth yl Ester (32). A stirred solution of the above benzyl

thioether (5.70 g, 14.16 mmol) and mercuric acetate (5.42 g,

17.00 mmol) in CH₂Cl₂cooled to 0 °C was added dropwise to

trifluoroacetic acid (5 mL). After 3 h at 0 °C, hydrogen sulfide

saturated MeOH was added, and stirring was continued at 0

°C for 20 min. The reaction mixture was poured into brine

and extracted with CH₂Cl₂. The combined organic layers were

dried (MgSO₄) and concentrated at reduced pressure. The

crude residue was partially suspended in MeOH, and to this

was added 1.07 g (28.28 mmol) of NaBH₄in portions. After

about 30 min, the reaction mixture was poured into EtOAc

and 0.5 N HCl, and the layers were separated. The metallic

mercury that formed in the reaction was washed with EtOAc.

The combined organic layers were washed with brine and dried

(MgSO₄), and the crude residue was flash chomatographed on

silica, eluting with hexanes-EtOAc (4:1) to give 1.99 g (50%)

of the thiol as a yellow oil: IR (neat) 2951, 2886, 1719, 1611,

1435, 1281 cm^-1; ¹H NMR (CDCl₃) δ 1.64 (d, 1H, J ) 7.8 Hz),

1.74 (m, 1H), 2.15 (m, 1H), 2.80 (m, 2H), 3.00 (m, 1H), 3.90 (s,

3H), 3.97 (m, 4H), 4.91 (d, 1H, J ) 4.0 Hz), 7.28 (d, 2H, J )

8.1 Hz), 7.95 (d, 2H, J ) 8.1 Hz). Anal. (C₁₄H₁₈O₄S) C, H.

4-[2-(2-Am in o-7-h yd r oxy-4-oxo-4,6,7,8-tetr a h yd r o-3H-

pyr im ido[5,4-b][1,4]th iazin -6-yl)eth yl]ben zoic Acid Meth -

yl Ester . A stirred suspension of the above dioxolane (1.20

g, 2.85 mmol) and 2 N HCl (4 mL) in THF (20 mL) was heated

under reflux for 2.5 h. The homogeneous solution was poured

slowly into saturated NaHCO₃, and the precipitate which

formed was collected. The filtrate was extracted with EtOAc.

A precipitate (58 mg) formed between layers, and it was

collected and combined with the first precipitate. The EtOAc

layer was dried (MgSO₄) and the solvent removed in vacuo.

The residue (42 mg) was also combined with the original

precipitate to give 984 mg (98%) of the carbinolamine as an

orange solid: mp 213-216 °C; IR (KBr) 3351, 3441, 1705, 1638,

3-[3-[(4-Met h oxyb en zyl)su lfa n yl]-4-oxob u t yl]b en zoic

Acid Meth yl Ester . This compound was prepared according

to the general procedure described for 4-[3-[(4-methoxybenzyl)-

sulfanyl]-4-oxobutyl]benzoic acid methyl ester to give 7.88 g

(93%) of a yellow oil: ¹H NMR (CDCl₃) δ 1.95-1.83 (1H, m),

2.16-2.04 (1H, m), 2.84-2.65 (2H, m), 3.00 (1H, ddd, J ) 4.3,

7.4, 11.7 Hz), 3.49 (1H, d, J ) 13.3 Hz), 3.59 (1H, d, J ) 13.3

Hz), 3.80 (3H, s), 3.91 (3H, s), 6.83 (2H, d, J ) 8.6 Hz), 7.19

(2H, d, J ) 8.6 Hz), 7.35-7.28 (2H, m), 7.88-7.82 (2H, m),

9.22 (1H, d, J ) 4.3 Hz). Anal. (C₂₀H₂₂O₄S) C, H, S.

3-[3-[1,3]Dioxola n -2-yl-3-[(4-m eth oxyben zyl)su lfa n yl]-

p r op yl]ben zoic Acid Meth yl Ester . This compound was

prepared according to the general procedure described for 4-[3-

[1,3]dioxola n -2-yl-3-[(4-m et h oxyben zyl)su lfa n yl]pr opyl]-

benzoic acid methyl ester to give 10.68 g (95%) of a yellow oil:

¹H NMR (CDCl₃) δ 1.75-1.61 (1H, m), 2.07-1.95 (1H, m),

2.64-2.54 (2H, m), 2.89 (1H, ddd, J ) 4.4, 9.5, 14.2 Hz), 3.80

(3H, s), 3.90 (3H, s), 4.05-3.75 (6H, m), 4.98 (1H, d, J ) 4.4

Hz), 6.83 (2H, d, J ) 8.7 Hz), 7.24 (2H, d, J ) 8.7 Hz), 7.31-

7.26 (2H, m), 7.86-7.82 (2H, m). Anal. (C₂₂H₂₆O₅S) C, H, S.

3-(3-[1,3]Dioxola n -2-yl-3-m er ca p top r op yl)ben zoic Acid

Meth yl Ester . This compound was prepared according to the

general procedure described for 4-(3-[1,3]dioxolan-2-yl-3-mer-

captopropyl)benzoic acid methyl ester to give 3.58 g (47%) of

a colorless oil: ¹H NMR (CDCl₃) δ 1.65 (1H, d, J ) 7.8 Hz),

1.81-1.67 (1H, m), 2.22-2.07 (1H, m), 2.91-2.73 (2H, m),

3.05-2.96 (1H, m), 3.91 (3H, s), 4.04-3.92 (4H, m), 4.92 (1H,

d, J ) 4.1 Hz), 7.43-7.31 (2H, m), 7.90-7.84 (2H, m). Anal.

(C₁₄H₁₈O₄S) C, H, S.

3-[3-[(2,4-Dia m in o-6-oxo-1,6-d ih yd r op yr im id in -5-yl)-

su lfa n yl]-3-[1,3]d ioxola n -2-ylp r op yl]ben zoic Acid Meth yl

Ester . This compound was prepared according to the general

procedure described for 4-[3-[(2,4-diamino-6-oxo-1,6-dihydro-

pyrimidin-5-yl)sulfanyl]-3-[1,3]dioxolan-2-ylpropyl]benzoic acid

methyl ester to give 3.12 g (55%) of a white solid: mp 146-

1609, 1557, 1470, 1289 cm^-^{1 1}H NMR as a single pair of

;

diastereomers (DMSO-d₆) δ 1.39 and 1.96 (m, m, 1H), 1.70 (m,

1H), 2.56-2.89 (m, 3H), 3.82 (s, 3H), 4.71 and 4.84 (m, m, 1H),

5.37 and 5.40 (d, d, 1H, J ) 6.6 Hz), 6.06 (s, 2H), 7.20 (d, 1H,

J ) 4.5 Hz), 7.31 and 7.36 (d, d, 2H, J ) 8.1 Hz), 7.86 and

7.88 (d, d, 2H, J ) 8.0 Hz), 10.16 and 10.19 (s, s, 1H). Anal.

(C₁₆H₁₈N₄O₄S‚1.7H₂O) C, H, N, S.

4-[2-(2-Am in o-4(3H)-oxo-5,6,7,8-tetr a h yd r o[5,6-b][1,4]-

th ia zin -6-yl)eth yl]ben zoic Acid Meth yl Ester (33). To a

0 °C suspension of the carbinolamine from above (1.126 g, 3.1

mmol) in THF was added boron trifluoride etherate (2.3 mL,

18.64 mmol). When the addition was complete, NaCNBH₃

(0.586 g, 9.32 mmol) was added in portions over 5 min. After

an additional 30 min, ammonia-saturated MeOH (5 mL) was

added, the reaction mixture was diluted with EtOAc and

washed with brine. The organic layer was dried (MgSO₄), and

the crude residue was flash chromatographed on silica, eluting

with CH₂Cl₂/MeOH (9:1) to give 542 mg (50%) of the dehy-

drated product as an orange solid: mp 245-246 °C dec; IR

(KBr) 3358, 2936, 1721, 1644, 1595, 1537, 1447, 1346, 1281

1

148 °C; H NMR (DMSO-d₆) δ 1.67-1.56 (1H, m), 1.91-1.79

(1H, m), 2.57 (1H, ddd, J ) 3.8, 8.0, 9.6 Hz), 2.79 (1H, ddd, J

) 6.7, 9.6, 13.6 Hz), 3.12 (1H, ddd, J ) 4.4, 9.2, 14.1 Hz), 3.81-

3.74 (2H, m), 3.83 (3H, s), 3.91-3.84 (2H, m), 4.83 (1H, d, J )

4.4 Hz), 6.33 (4H, broad), 7.38 (1H, t, J ) 7.7 Hz), 7.48 (1H, d,

J

) 7.7 Hz), 7.76-7.72 (2H, m), 10.00 (1H, s). Anal.

(C₁₈H₂₂N₄O₅S‚0.6H₂O) C, H, N, S.

3-[2-(2-Am in o-7-h yd r oxy-4-oxo-4,6,7,8-tetr a h yd r o-3H-

pyr im ido[5,4-b][1,4]th iazin -6-yl)eth yl]ben zoic Acid Meth -

yl Ester . This compound was prepared according to the

general procedure described for 4-[2-(2-amino-7-hydroxy-4-oxo-

4,6,7,8-tetrahydro-3H-pyrimido[5,4-b][1,4]thiazin-6-yl)ethyl]-

benzoic acid methyl ester to give 1.23 g (48%) of an orange

solid: mp 105-108 °C; ¹H NMR (DMSO-d₆) δ 1.55-1.35 (1H,

m), 1.78-1.60 (1H, m), 2.88-2.54 (3H, m), 3.83 (3H, s), 4.74-

4.69 (1H, m), 5.38 (1H, d, J ) 6.6 Hz), 5.41, 6.06 (2H, s), 7.21

(1H, d, J ) 4.8 Hz), 7.52-7.39 (2H, m), 7.80-7.74 (2H, m),

10.20 (1H, s). Anal. (C₁₆H₁₈N₄O₄S‚0.5H₂O) C, H, N, S.

2520 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16

Varney et al.

Meth yl 3-[2-(2-Am in o-4(3H)-oxo-5,6,7,8-tetr a h yd r op yr -

im id o[5,6-b][1,4]th ia zin -6-yl)eth yl]ben zoa te. This com-

pound was prepared according to the general procedure

described for methyl 4-[2-(2-amino-4(3H)-oxo-5,6,7,8-tetrahy-

dro[5,6-b][1,4]thiazin-6-yl)ethyl]benzoate to give 308 mg (46%)

of an orange solid: mp 120 °C dec; ¹H NMR (DMSO-d₆) δ 1.75-

1.64 (1H, m), 1.94-1.82 (1H, m), 2.89-2.71 (3H, m), 3.23-

3.16 (1H, m), 3.56-3.38 (1H, m), 3.83 (3H, s), 6.01 (2H, s), 6.66

(1H. s), 7.53-7.41 (2H, m), 7.80-7.76 (2H, m), 10.06 (1H, s).

Anal. (C₁₆H₁₈N₄O₃S‚0.7H₂O) C, H, N, S.

crude mixture was filtered though a pad of Celite and the

filtrate concentrated. The residue was purified by flash

chromatography on silica gel, eluting with 6-50% EtOAc in

CH₂Cl₂to give 35.04 g (84%) of the desired product as a

colorless oil: [R]₅₈₉-24.8° (c ) 0.86, MeOH); IR (neat) 3391

(broad), 2980, 1723, 1674, 1537, 1462, 1368, 1283 cm^-^{1 1}H

;

NMR (CDCl₃) δ 1.36 (t, 3H, J ) 7.0 Hz), 1.45 (s, 9H), 1.81 (m,

2H), 2.94-3.07 (m, 3H), 3.26 (m, 1H), 3.75 (m, 1H), 4.32 (q,

2H, J ) 7.1 Hz), 4.9 (brs, 1H), 6.82 (d, 1H, J ) 3.7 Hz), 7.62

(d, 1H, J ) 4.0 Hz). Anal. (C₁₆H₂₅NO₅S) C, H, N, S.

5-(3(S),4-Dih yd r oxybu tyl)th iop h en e-2-ca r boxylic Acid

Eth yl Ester (35a ). To a stirred solution of 5-[2-(2,2-dimethyl-

[1,3]dioxolan-4(S)-yl)ethyl]thiophene-2-carboxylic acid ethyl

ester (65.98 g, 232 mmol) in EtOH (300 mL) was added

p-toluenesulfonic acid monohydrate (17.65 g, 93 mmol). The

reaction mixture was heated at 65 °C for 3 h, concentrated in

vacuo, and redissolved in EtOH, and heating was continued.

This procedure was repeated until starting material had

disappeared by TLC. The crude reaction was diluted with

EtOAc, washed with saturated NaHCO₃and then with satu-

rated NaCl, dried (MgSO₄), and concentrated in vacuo. The

residue was recrystallized from Et₂O/hexanes, yielding 49.90

g of the desired diol. The mother liquors were flash chomato-

graphed on silica gel, eluting with 30-100% EtOAc in CH₂-

Cl₂to give an additional 5.81 g of product. The combined yield

was 55.71 g (98%): [R]₅₈₉-28.7° (c ) 0.82, MeOH); IR (neat)

5-[4-[(ter t-Bu toxyca r bon yl)a m in o]-3(S)-[(m eth ylsu lfo-

n yl)oxy]bu tyl]th iop h en e-2-ca r boxylic Acid Eth yl Ester .

To an ice-cold solution of the alcohol 36a (34.34 g, 100 mmol)

and TEA (20.9 mL, 150 mmol) in CH₂Cl₂(250 mL) was added

methanesulfonyl chloride (9.3 mL, 120 mmol). After 30 min

the reaction mixture was poured into 0.5 N HCl, and the layers

were separated. The organic layer was washed with saturated

NaHCO₃and then with brine, dried (MgSO₄), and concentrated

to give 38.36 g (91%) of the crude mesylate as a yellow oil,

which solidified on standing. This material was used without

further purification: ¹H NMR (CDCl₃) δ 1.36 (t, 3H, J ) 7.0

Hz), 1.44 (s, 9H), 2.09 (m, 2H), 3.00 (m, 2H), 3.08 (s, 3H), 3.34-

3.56 (m, 2H), 4.32 (q, 2H, J ) 7.0 Hz), 4.79 (m, 1H), 4.95 (br

t, 1H), 6.82 (d, 1H, J ) 3.7 Hz), 7.62 (d, 1H, J ) 3.8 Hz).

2-[[1-[[(ter t-Bu t oxyca r b on yl)a m in o]m et h yl]-3(R)-[5-

(eth oxycar bon yl)th ioph en e-2-yl]pr opyl]th io]m alon ic Acid

Dim eth yl Ester . To an ice-cold stirred solution of the crude

thiolacetate from above (39.77 g, 99.0 mmol) and dimethyl

chloromalonate (13.91 mL, 109.0 mmol) in MeOH (350 mL)

was added K₂CO₃(27.38 g, 198.1 mmol). After 3 h at 0 °C,

the reaction mixture was stirred at room temperature for 2 h.

After the mixture was poured into H₂O and extracted with

EtOAc (3×), the combined organic layers were washed with

brine, dried (MgSO₄), and concentrated in vacuo to yield,

instead of the desired malonate, 49.80 g of the crude disulfide.

To a stirred solution of this disulfide (11.44 g, 15.96 mmol) in

EtOH under argon was added NaBH₄(1.81 g, 47.85 mmol).

After 4 h, the reaction was quenched with 0.5 N HCl, and the

mixture was diluted with EtOAc and washed with more 0.5

N HCl. The organic layer was washed twice with brine, dried

(MgSO₄), and concentrated in vacuo to yield 10.88 g of the

crude thiol/disulfide mixture, which was dissolved in degassed

MeOH. To this stirred solution were added dimethyl chloro-

malonate (5.79 mL, 45.36 mmol) and potassium carbonate

(8.36 g, 60.49 mmol). After 30 min of stirring under argon,

the reaction mixture was diluted with EtOAc, washed twice

with brine, dried (MgSO₄), and concentrated. The residue was

purified by flash chromatography on silica gel eluting hexanes/

EtOAc (3:1) to give 4.20 g of disulfide which was recycled and

6.96 g (45%) of the desired malonate as a light yellow oil: [R]₅₈₉

+31.9° (c ) 0.64, MeOH); IR (neat) 3397, 2978, 2938, 1759,

1

3393 (broad), 2936, 1705, 1462, 1287 cm^-1; H NMR (CDCl₃)

δ 1.36 (t, 3H, J ) 7.2 Hz), 1.7 (brs, 2H), 1.76 (m, 2H), 2.99 (m,

2H), 3.48 (dd, 1H, J ) 7.4, 3.5 Hz), 3.68 (dd, 1H, J ) 7.6, 3.2

Hz), 3.77 (m, 1H), 4.32 (q, 2H, J ) 7.2 Hz), 6.82 (d, 1H, J )

3.9 Hz), 7.63 (d, 1H, J ) 3.7 Hz). Anal. (C₁₁H₁₆O₄S) C, H, S.

5-[3(S)-Hyd r oxy-4-[[p-tolylm eth yl)su lfon yl]oxy]bu tyl]-

th iop h en e-2-ca r boxylic Acid Eth yl Ester . To an ice-cold

solution of diol 35a (45.42 g, 186 mmol) and TEA (31.1 mL,

223 mmol) in CH₂Cl₂(400 mL) was added p-toluenesulfonyl

chloride (38.99 g, 204 mmol). The ice bath was removed, the

reaction mixture was stirred at room temperature for 18 h and

poured into 0.5 N HCl, and the layers were separated. The

aqueous layer was reextracted with CH₂Cl₂. The combined

organic layers were washed with brine and dried (MgSO₄). The

crude residue was purified by flash chromatography on silica

gel, eluting with 4-100% EtOAc in CH₂Cl₂until product eluted

and then 0-10% MeOH in EtOAc to elute the starting diol.

In this manner there were recovered 7.43 g of diol 35a and

49.23 g (66%) of the monotosylate as a lightly colored oil which

solidified on standing: [R]₅₈₉-8.2° (c ) 0.74, MeOH); IR (neat)

3507 (broad) 2947, 1705, 1462, 1362, 1287 cm^-^{1 1}H NMR

;

(CDCl₃) δ 1.36 (t, 3H, J ) 6.9 Hz), 1.78 (m, 2H), 2.46 (s, 3H),

2.95 (m, 2H), 3.90 (m, 2H), 4.03 (m, 1H), 4.32 (q, 2H, J ) 7.0

Hz), 6.78 (d, 1H, J ) 3.7 Hz), 7.36 (d, 2H, J ) 8.2 Hz), 7.61 (d,

1H, J ) 3.7 Hz), 7.79 (d, 2H, J ) 8.2 Hz). Anal. (C₁₈H₂₂O₆S₂)

C, H, S.

1

1715, 1505, 1454, 1275 cm^-1; H NMR (CDCl₃) δ 1.36 (t, 3H,

5-(4-Azid o-3(S)-h yd r oxyb u t yl)t h iop h en e-2-ca r b oxyl-

ic Acid Eth yl Ester . To a stirred solution of the monotosylate

prepared above (42.94 g, 108 mmol) in DMF (250 mL) was

added NaN₃(31.52 g, 485 mmol). The solution was heated

under argon for 4 h at 80 °C. The cooled reaction mixture

was poured into 600 mL of brine, and the oil was separated.

The aqueous solution was extracted three times with Et₂O.

The Et₂O extracts were combined with the oil, washed once

with brine, dried (MgSO₄), and concentrated to yield 33.0 g of

crude azide which was sufficiently pure to use in the next step.

An analytical sample was obtained by flash chromatography

on silica gel eluting CH₂Cl₂/EtOAc (20:1) to yield a colorless

oil: [R]₅₈₉-18.8° (c ) 0.84, MeOH); IR (neat) 3445 (broad),

J ) 7.0 Hz), 1.46 (s, 9H), 1.86 (m, 1H), 2.03 (m, 1H), 3.04 (m,

3H), 3.31 (m, 2H), 3.81 (s, 6H), 4.24 (s, 1H), 4.34 (q, 2H, J )

7.0 Hz), 5.13 (brs, 1H), 6.83 (d, 1H, J ) 3.7 Hz), 7.64 (d, 1H,

J ) 3.7 Hz). Anal. (C₂₁H₃₁NO₈S₂) C, H, N, S.

6-[2-[5-(E t h oxyca r b on yl)t h iop h en e-2-yl]et h yl]-3(R)-

oxoth iom or p h olin e-2-ca r boxylic Acid Eth yl Ester (37a ).

To an ice-cold solution of the malonate prepared above (29.18

g, 59.60 mmol) in CH₂Cl₂(225 mL) was added trifluoroacetic

acid (35 mL). After 1 h at 0 °C, the reaction mixture was

diluted with CH₂Cl₂and continuously washed with saturated

NaHCO₃, making sure the aqueous layer was alkaline. The

organic layer was washed with brine, dried (MgSO₄), and

concentrated. The crude amine was dissolved in MeOH and

stirred for 2 h at room temperature. The crude lactam was

flash chromatographed on silica gel, eluting with CH₂Cl₂/

EtOAc (2:1) to give 17.74 g (83%) of the desired product as an

amber oil: [R]₅₈₉+30.9° (c ) 0.92, MeOH); IR (neat) 3314,

1

2926, 2099, 1705, 1539, 1460, 1281 cm^-1; H NMR (CDCl₃) δ

1.36 (t, 3H, J ) 7.1 Hz), 1.85 (m, 2H), 2.99 (m, 2H), 3.35 (m,

2H), 3.79 (m, 1H), 4.32 (q, 2H, J ) 7.1 Hz), 6.82 (d, 1H, J )

3.6 Hz), 7.63 (d, 1H, J ) 4.0 Hz). Anal. (C₁₁H₁₅N₃O₃S₂) C, H,

N, S.

1

3242, 2951, 1732, 1660, 1462, 1294 cm^-1; H NMR (CDCl₃) δ

5-[4-[(ter t-Bu toxyca r bon yl)a m in o]-3(S)-h yd r oxybu tyl]-

th iop h en e-2-ca r boxylic Acid Eth yl Ester (36a ). A Parr

flask containing the azide prepared above (32.70 g, 121 mmol),

di-tert-butyl dicarbonate (27.83 g, 127 mmol), 5% Pd/C (4.5 g),

and THF (300 mL) was shaken under 30 psi of H₂. The

exothermic reaction was kept below 45 psi. After 2 h, the

1.36 (t, 3H, J ) 7.0 Hz), 1.94 (m, 2H), 2.95-3.25 (m, 2H), 3.42-

3.67 (m, 3H), 3.79 and 3.82 (s, s, 3H), 4.14 and 4.25 (s, s, 1H),

4.32 (q, 2H, J ) 7.0 Hz), 6.23 (m, 1H), 6.82 (d, 1H, J ) 3.7

Hz), 7.63 (d, 1H, J ) 3.7 Hz). Anal. (C₁₅H₁₉NO₅S₂) C, H, N,

S.

Glycinamide Ribonucleotide Transformylase Inhibitors

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16 2521

6-[2-[5-(E t h oxyca r b on yl)t h iop h en e-2-yl]et h yl]-3(R)-

m eth oxy-5,6-d ih yd r o-2H-[1,4]th ia zin e-2-ca r boxylic Acid

Eth yl Ester . To a stirred solution of lactam 37a (11.8 g, 33.01

mmol) in CH₂Cl₂(70 mL) was added trimethyloxonium tet-

rafluoroborate (6.835 g, 46.21 mmol). After 1 h of stirring

under argon at room temperature all the starting lactam was

consumed. The reaction mixture was cooled, and 50% aqueous

K₂CO₃was added until the pH was alkaline. The KBF₄was

filtered off, and the aqueous layer was extracted with CH₂-

Cl₂. The combined organic layers were washed with brine,

dried (Na₂SO₄), and concentrated to give 11.55 g (94%) of the

crude lactim ether which was used without further purifiction.

5-[2-(2-Am in o-4-oxo-4,6,7,8-t et r a h yd r o-3H -p yr im id o-

[5,4-b]th ia zin -6(R)-yl)eth yl]th iop h en e-2-ca r boxylic Acid

Eth yl Ester . A solution of sodium ethoxide was prepared by

dissolving sodium metal (0.845 g, 36.75 mmol) in degassed

absolute ethanol under argon. To this solution was added

guanidine hydrochloride (3.63 g, 38.00 mmol). After 15 min

of stirring, a solution of the lactim ether prepared above (4.55

g, 12.25 mmol) in degassed absolute EtOH was added, and

the mixture was heated at reflux for 1 h. The cooled reaction

was neutralized with 0.5 N HCl, diluted with EtOAc, and

extracted repeatedly. The combined EtOAc layers were washed

with brine, dried (MgSO₄), and concentrated. The solid residue

was slurried in hot EtOH, cooled, and filtered to give 1.75 g

(33%) of the desired product as a light yellow solid: mp 166

°C foams; [R]₅₈₉+57.7° (c ) 0.62, DMSO); IR (KBr) 3349

graphed on silica gel, eluting with CH₂Cl₂to give 322 mg (82%)

of the desired epoxide as a colorless oil: [R]₅₈₉+19.6° (c ) 1.14,

MeOH); IR (neat) 2984, 1707, 1460, 1283, 1262 cm^-1; ¹H NMR

(CDCl₃) δ 1.36 (t, 3H, J ) 7.0 Hz), 1.80-2.05 (m, 2H), 2.51

(dd, 1H, J ) 4.8, 2.6 Hz), 2.78 (dd, 1H, J ) 4.8, 4.1 Hz), 2.99

(m, 3H), 4.32 (q, 2H, J ) 7.0 Hz), 6.82 (d, 1H, J ) 3.7 Hz),

7.63 (d, 1H, J ) 3.7 Hz). Anal. (C₁₁H₁₄O₃S) C, H, S.

5-(4-Azid o-3(R)-h yd r oxyb u t yl)t h iop h en e-2-ca r b oxyl-

ic Acid Eth yl Ester . To a stirred solution of the above

epoxide (200 mg, 0.88 mmol) in CH₃CN (10 mL) was added

magnesium perchlorate (296 mg, 1.33 mmol). After 10 min,

the reaction mixture became homogeneous and NaN₃(144 mg,

2.21 mmol) was added. The reaction mixture was heated at

75 °C. After 18 h, NaN₃(25 mg, 0.39 mmol) was added, and

heating was continued for 18 h more. The cooled reaction

mixture was diluted with EtOAc and washed with brine. The

aqueous layer was reextracted with EtOAc. The combined

organic layers were washed again with brine and dried

(MgSO₄), and the crude residue was flash chromatographed

on silica gel, eluting with 20-25% EtOAc in hexanes to give

187 mg (79%) of the azide as a colorless oil: [R]₅₈₉+18.9° (c )

0.62, MeOH); IR (neat) 3462 (broad) 2982, 2936, 2101, 1705,

1682, 1462, 1269 cm^-1; ¹H NMR (CDCl₃) δ 1.36 (t, 3H, J ) 7.0

Hz), 1.85 (m, 2H), 2.99 (m, 2H), 3.30 (m, 2H), 3.79 (m, 1H),

4.32 (q, 2H, J ) 7.0 Hz), 6.82 (d, 1H, J ) 3.7 Hz), 7.63 (d, 1H,

J ) 3.7 Hz). Anal. (C₁₁H₁₅N₃O₃S) C, H, N, S.

5-[4-[(ter t-Bu toxycar bon yl)am in o]-3(R)-h ydr oxybu tyl]-

th iop h en e-2-ca r boxylic Acid Eth yl Ester (36b). This

compound was prepared as described for compound 36a :

colorless oil; [R]₅₈₉+26.3° (c ) 0.76, MeOH); IR (neat) 3378

(broad) 2926, 1701, 1640, 1603, 1537, 1458, 1344, 1285 cm^-1

;

¹H NMR (DMSO-d₆) δ 1.23 (t, 3H, J ) 7.0 Hz), 1.80 (m, 2H),

2.82-3.00 (m, 2H), 3.15-3.52 (m, 3H), 4.21 (q, 2H, J ) 7.0

Hz), 6.00 (s, 2H), 6.64 (s, 1H), 6.95 (d, 1H, J ) 3.7 Hz), 7.59

(d, 1H, J ) 3.7 Hz), 10.04 (s, 1H); HMS calcd for C₁₅H₁₈N₄O₃S₂

(M + Na⁺) 389.0718, found 389.0731.

(broad) 2980, 2934, 1715, 1682, 1516, 1462, 1294 cm^-^{1 1}H

;

NMR (CDCl₃) δ 1.33 (t, 3H, J ) 7.0 Hz), 1.44 (s, 9H), 1.79 (m,

2H), 2.22 (brs, 1H), 3.04 (m, 3H), 3.25 (m, 1H), 3.73 (m, 1H),

4.32 (q, 2H, J ) 7.0 Hz), 6.81 (d, 1H, J ) 3.7 Hz), 7.62 (d, 1H,

J ) 3.7 Hz). Anal. (C₁₆H₂₅NO₅S) C, H, N, S.

5-[4-[(ter t-Bu toxyca r bon yl)a m in o]-3(R)-[(m eth ylsu lfo-

n yl)oxy]bu tyl]th iop h en e-2-ca r boxylic Acid Eth yl Ester .

Starting with alcohol 36b, the title compound was prepared

in 94% crude yield according to the general procedure de-

scribed for 5-[4[(-tert-butoxycarbonyl)amino]-3(S)-[(methylsul-

fonyl)oxy]butyl]thiophene-2-carboxylic acid ethyl ester: ¹H

NMR (CDCl₃) δ 1.36 (t, 3H, J ) 7.0 Hz), 1.45 (s, 9H), 2.07 (m,

2H), 3.00 (m, 2H), 3.06 (s, 3H), 3.37-3.50 (m, 2H), 4.32 (q,

2H, J ) 7.0 Hz), 4.78 (m, 1H), 4.91 (br t, 1H), 6.84 (d, 1H, J )

4.0 Hz), 7.62 (d, 1H, J ) 4.0 Hz).

5-[4-[(ter t-Bu tyld im eth ylsila n yl)oxy]-3(S)-[(m eth ylsu l-

fon yl)oxy]bu tyl]th iop h en e-2-ca r boxylic Acid Eth yl Es-

ter . To an ice-cold stirred solution of the mono TBS alcohol

prepared above (23.19 g, 64.68 mmol) and TEA (13.5 mL, 96.86

mmol) in CH₂Cl₂(200 mL) was added methanesulfonyl chloride

(6.0 mL, 77.52 mmol). After 30 min at 0 °C, the reaction was

warmed to room temperature. After an additional 30 min, the

reaction mixture was diluted with CH₂Cl₂and washed se-

quentially with 0.5 N HCl, saturated NaHCO₃, and brine. The

organic layer was dried (MgSO₄) and the solvent removed to

give 26.61 g (94%) of the desired mesylate as a colorless oil

which was used without further purification: ¹H NMR (CDCl₃)

δ 0.11 (s, 3H), 0.13 (s, 3H), 0.92 (s, 9H), 1.39 (t, 3H, J ) 7.0

Hz), 2.08 (m, 2H), 3.03 (m, 2H), 3.10 (s, 3H), 3.80 (m, 2H),

4.36 (q, 2H, J ) 7.0 Hz), 4.75 (m, 1H), 6.88 (d, 1H, J ) 3.7

Hz), 7.66 (d 1H, J ) 3.7 Hz).

2-[[1-[[(ter t-Bu t oxyca r b on yl)a m in o]m et h yl]-3(S)-[5-

(eth oxycar bon yl)th ioph en e-2-yl]pr opyl]th io]m alon ic Acid

Dim eth yl Ester . To a stirred solution of the thioacetate

prepared above (8.44 g, 21.0 mmol) and dimethyl chloromalo-

nate (3.22 mL, 25.2 mmol) in degassed MeOH (75 mL) at 0 °C

was added K₂CO₃(5.81 g, 42.0 mmol). The reaction mixture,

under an Ar atmosphere, was allowed to stir for 1 h at 0 °C

and then for 1.5 h at room temperature, poured into brine,

and extracted twice with EtOAc. The combined organic layer

was washed twice more with brine solution and dried (MgSO₄).

The crude residue was purified by flash chromatography on

silica gel, eluting with hexanes/EtOAc (3:1) to give 9.678 g

(94%) of the desired malonate as a yellow oil: [R]₅₈₉-32.8° (c

) 0.67, MeOH); IR (neat) 3395 (broad), 2978, 1755, 1715, 1699,

5-[4-H yd r oxy-3(S)-[(m e t h ylsu lfon yl)oxy]b u t yl]t h io-

p h en e-2-ca r boxylic Acid Eth yl Ester . To a stirred solution

of the above mesylate (26.56 g, 60.83 mmol) in THF (200 mL)

was added a 1.0 M solution of tetrabutylammonium fluoride

in THF (67 mL, 67.00 mmol). After 1.5 h of stirring at room

temperature, the reaction mixture was diluted with EtOAc and

washed with 0.5 N HCl. The aqueous layer was reextracted

with EtOAc. The combined organic layers were washed twice

with brine and dried (MgSO₄), and the crude residue was flash

chromatographed on silica gel, eluting with 0-20% EtOAc in

CH₂Cl₂to give 12.465 g (64%) of the desired alcohol as a

colorless oil which solidified on standing: [R]₅₈₉-4.3° (c ) 0.78,

MeOH); IR (neat) 3520, 2982, 2940, 1705, 1684, 1462, 1345,

1

1462, 1261 cm^-1; H NMR (CDCl₃) δ 1.36 (t, 3H, J ) 7.0 Hz),

1.44 (s, 9H), 1.86 (m, 1H), 2.05 (m, 1H), 3.02 (m, 3H), 3.31 (m,

2H), 3.79 (s, 6H), 4.23 (s, 1H), 4.33 (q, 2H, J ) 7.0 Hz), 6.81

(d, 1H, J ) 3.7 Hz), 7.62 (d, 1H, J ) 3.7 Hz). Anal. (C₂₁H₃-

NO₈S₂) C, H, N, S.

1

1287 cm^-1; H NMR (CDCl₃) δ 1.36 (t, 3H, J ) 7.0 Hz), 2.09

(m, 2H), 3.01 (m, 2H), 3.12 (s, 3H), 3.80 (m, 2H), 4.32 (q, 2H,

J ) 7.0 Hz), 4.81 (m, 1H), 6.86 (d, 1H, J ) 3.7 Hz), 7.63 (d,

1H, J ) 3.7 Hz). Anal. (C₁₂H₁₈O₆S₂) C, H, S.

6-[2-[5-(E t h oxyca r b on yl)t h iop h en e-2-yl]et h yl]-3(S)-

oxoth iom or p h olin e-2-ca r boxylic Acid Eth yl Ester (37b).

Starting with the malonate prepared above, compound 37b

was prepared in 86% yield according to the general procedure

described for compound 37a : [R]₅₈₉-30.5° (c ) 1.01, MeOH);

5-[2(R)-Oxir an yleth yl]th ioph en e-2-car boxylic Acid Eth -

yl Ester . To an ice-cold stirred solution of the mesylate

alcohol prepared above (559 mg, 1.73 mmol) in THF (10 mL)

was added a 60% dispersion of NaH in mineral oil (76 mg,

1.90 mmol). After 1 h, the ice bath was removed, and the

reaction mixture was stirred at room temperature. After 18

h, additional 60% dispersion NaH (35 mg, 0.88 mmol) was

added. After 4 days more, the reaction mixture was diluted

with EtOAc, washed with 0.5 N HCl and then with brine, and

dried (MgSO₄), and the crude residue was flash chomato-

IR (neat) 3320, 3229, 2951, 1738, 1703, 1669, 1460, 1281 cm^-1

;

¹H NMR (CDCl₃) δ 1.36 (t, 3H, J ) 7.0 Hz), 1.91-2.04 (m,

2H), 2.95-3.25 (m, 2H), 3.42-3.65 (m, 3H), 3.79 and 3.82 (s,

s, 3H), 4.13 and 4.25 (s, s, 1H), 4.32 (q, 2H, J ) 7.0 Hz), 6.26

(m, 1H), 6.82 (d, 1H, J ) 3.7 Hz), 7.64 (d, 1H, J ) 3.7 Hz).

Anal. (C₁₅H₁₉NO₅S₂) C, H, N, S.

2522 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16

Varney et al.

6-[2-[5-(E t h oxyca r b on yl)t h iop h en e-2-yl]et h yl]-3(S)-

m eth oxy-5,6-d ih yd r o-2H-[1,4]th ia zin e-2-ca r boxylic Acid

Eth yl Ester . Starting with lactam 37b, the title compound

was prepared in quantitative crude yield according to the

general procedure described for compound 6-[2-[5-(ethoxycar-

bonyl)thiophene-2-yl]ethyl]-3(R)-methoxy-5,6-dihydro-2H-[1,4]-

thiazine-2-carboxylic acid ethyl ester and was used in the next

step without purification.

5-[2-(2-Am in o-4-oxo-4,6,7,8-t et r a h yd r o-3H -p yr im id o-

[5,4-b]th ia zin -6(S)-yl)eth yl]th iop h en e-2-ca r boxylic Acid

Eth yl Ester . Starting with the lactim ether prepared above,

the title compound was prepared in 45% yield according to

the general procedure described for compound 5-[2-(2-amino-

4-oxo-4,6,7,8-tetrahydro-3H-pyrimido[5,4-b]thiazin-6(R)-yl)eth-

yl]thiophene-2-carboxylic acid ethyl ester: mp 181-184 °C;

[R]₅₈₉-59.7° (c ) 0.38, DMSO); IR (KBr) 3335, 2926, 1705,

1632, 1593, 1456, 1335, 1260 cm^-1; ¹H NMR (DMSO-d₆) δ 1.23

(t, 3H, J ) 7.0 Hz), 1.72 (m, 1H), 1.88 (m, 1H), 2.81-3.00 (m,

2H), 3.16-3.52 (m, 3H), 4.22 (q, 2H, J ) 7.0 Hz), 6.00 (s, 2H),

6.65 (s, 1H), 6.96 (d, 1H, J ) 3.7 Hz), 7.60 (d, 1H, J ) 3.7 Hz),

10.04 (s, 1H). Anal. (C₁₅H₁₈N₄O₃S₂‚0.4H₂O‚0.5EtOH) C, H,

N, S.

5-(3(S),4-Dih yd r oxyb u t yl)-4-m et h ylt h iop h en e-2-ca r -

boxylic Acid Eth yl Ester (35b). Starting with the dioxolane

prepared above, the title compound was prepared in 92% yield

according to the general procedure described for compound

35a : [R]₅₈₉-24.3° (c ) 0.61, MeOH); IR (KBr) 3264 (broad),

2924, 1707, 1458, 1447, 1260 cm^-1; ¹H NMR (CDCl₃) δ 1.35 (t,

3H, J ) 7.0 Hz), 1.76 (m, 2H), 2.17 (s, 3H), 2.89 (m, 2H), 3.48

(dd, 1H, J ) 7.4, 3.3 Hz), 3.68 (dd, 1H, J ) 7.7, 3.3 Hz), 3.75

(m, 1H), 4.30 (q, 2H, J ) 7.0 Hz), 7.50 (s, 1H). Anal.

(C₁₂H₁₈O₄S) C, H, S.

2-[[1-[[(ter t-Bu t oxyca r b on yl)a m in o]m et h yl]-3(R)-[5-

(eth oxyca r bon yl)-3-m eth ylth iop h en e-2-yl]p r op yl]th io]-

m a lon ic Acid Dim eth yl Ester . Starting with the thioacetate

prepared above, the title compound was prepared in 90% yield

according to the general procedure described for compound

2-[[1-[[(tert-butoxycarbonyl)amino]methyl]-3(S)-[5-(ethoxycar-

bonyl)thiophene-2-yl]propyl]sulfanyl]malonic acid dimethyl

ester: [R]₅₈₉+22.7° (c ) 0.67, MeOH); IR (neat) 3397, 2978,

1755, 1714, 1514, 1454, 1250 cm^-1; ¹H NMR (CDCl₃) δ 1.34 (t,

3H, J ) 7.0 Hz), 1.44 (s, 9H), 1.92 (m, 2H), 2.15 (s, 3H), 2.95-

3.40 (m, 5H), 3.80 and 3.82 (s, s, 6H), 4.23 (s, 1H), 4.31 (q, 2H,

J ) 7.0 Hz), 5.18 (brs, 1H), 7.49 (s, 1H). Anal. (C₂₂H₃₃NO₈S)

C, H, N, S.

6-[2-[5-(Eth oxycar bon yl)-3-m eth ylth ioph en e-2-yl]eth yl]-

3(R)-oxoth iom or p h olin e-2-ca r boxylic Acid Meth yl Ester

(37c). Starting with the malonate prepared above, compound

37c was prepared in 87% yield according to the general

procedure described for compound 37a : mp 72-73 °C; [R]₅₈₉

+19.1° (c ) 0.67, MeOH); IR (thin film) 2954, 1738, 1694, 1651,

1

1464, 1296 cm^-1; H NMR (CDCl₃) δ 1.35 (t, 3H, J ) 7.0 Hz),

1.90 (m, 2H), 2.17 (s, 3H), 2.88 (m, 2H), 3.21-3.62 (m, 3H),

3.80 and 3.82 (s, s, 3H), 4.14 and 4.25 (s, s, 1H), 4.31 (q, 2H,

J ) 7.0 Hz), 6.22 (m, 1H), 7.50 (s, 1H). Anal. (C₁₆H₂₁NO₅S₂)

C, H, N, S.

5-[2-(2-Am in o-4-oxo-4,6,7,8-t et r a h yd r o-3H -p yr im id o-

[5,4-b]t h ia zin -6(R)-yl)et h yl]-4-m et h ylt h iop h en e-2-ca r -

boxylic Acid Eth yl Ester . Starting with the lactam pre-

pared above, the title compound was prepared in 27% yield

according to the general procedure described for compound

5-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimido[5,4-b]thi-

azin-6(R)-yl)ethyl]thiophene-2-carboxylic acid ethyl ester: mp

120-140 °C dec; [R]₅₈₉+57.1° (c ) 0.67, DMSO); IR (KBr) 3337,

2926, 1701, 1653, 1635, 1599, 1449, 1250 cm^-^{1 1}H NMR

;

5-(3(S)-Hydr oxy-4-[[(p-tolylm eth yl)su lfon yl]oxy]bu tyl)-

4-m eth ylth iop h en e-2-ca r boxylic Acid Eth yl Ester . Start-

ing with diol 35b, the title compound was prepared in 60%

yield according to the general procedure described for com-

pound 5-[3(S)-hydroxy-4-[[(p-tolylmethyl)sulfonyl]oxy]butyl]-

thiophene-2-carboxylic acid ethyl ester: [R]₅₈₉-5.7° (c ) 0.74,

MeOH); IR (neat) 3491 (broad), 2982, 2928, 1703, 1449, 1360,

(DMSO-d₆) δ 1.22 (t, 3H, J ) 7.0 Hz), 1.60-1.75 (m, 1H), 1.80-

1.92 (m, 1H), 2.10 (s, 3H), 2.86 (m, 2H), 3.12-3.52 (m, 3H),

4.20 (q, 2H, J ) 7.0 Hz), 6.02 (s, 2H), 6.65 (s, 1H), 7.50 (s,

1H), 10.08 (s, 1H); HMS calcd for C₁₆H₂₀N₄O₃S₂(M + H⁺)

381.1055, found 381.1067. Anal. (C₁₆H₂₀N₄O₃S₂‚0.8H₂O) C,

H, N, S.

1

1250 cm^-1; H NMR (CDCl₃) δ 1.35 (t, 3H, J ) 7.0 Hz), 1.71

5-[4-[(ter t-Bu tyld im eth ylsilyl)oxy]-3(S)-[(m eth ylsu lfo-

n yl)oxy]bu tyl]-4-m eth ylth ioph en e-2-car boxylic Acid Eth -

yl Ester . Starting with the alcohol prepared above, the title

compound was prepared in 84% yield according to the general

procedure described for compound 5-[4-[(tert-butyldimethylsi-

lyl)oxy]-3(R)-[(methylsulfonyl)oxy]butyl]-4-methylthiophene-2-

carboxylic acid ethyl ester: [R]₅₈₉+3.9° (c ) 1.48, MeOH); ¹H

NMR (CDCl₃) δ 0.09 (s, 6H), 0.90 (s, 9H), 1.35 (t, 3H, J ) 7.0

Hz), 1.97-2.05 (m, 2H), 2.16 (s, 3H), 2.82-3.01 (m, 2H), 3.07

(s, 3H), 3.68-3.82 (m, 2H), 4.31 (q, 2H, J ) 7.0 Hz), 4.69-

4.77 (m, 1H), 7.50 (s, 1H). Anal. (C₁₉H₃₄O₆S₂Si) C, H S.

5-[4-Hyd r oxy-3(S)-[(m eth ylsu lfon yl)oxy]bu tyl]-4-m eth -

ylth iop h en e-2-ca r boxylic Acid Eth yl Ester . Starting with

the silyl ether prepared above, the title compound was

prepared in 39% yield according to the general procedure

described for compound 5-[4-hydroxy-3(R)-[(methylsulfonyl)-

oxy]butyl]-4-methylthiophene-2-carboxylic acid ethyl ester:

[R]₅₈₉+14.2° (c ) 0.97, MeOH); ¹H NMR (CDCl₃) δ 1.35 (t,

3H, J ) 7.0 Hz), 1.96-2.12 (m, 2H), 2.17 (s, 3H), 2.85-2.94

(m, 2H), 3.12 (s, 3H), 3.77 (dd, 1H, J ) 6.6, 12.5 Hz), 3.86 (dd,

1H, J ) 2.9, 12.5 Hz), 4.31 (q, 2H, J ) 7.0 Hz), 4.78-4.87 (m,

1H), 7.50 (s, 1H). Anal. (C₁₃H₂₀O₆S₂) C, H, S.

4-Meth yl-5-(2(R)-oxir a n yleth yl)th iop h en e-2-ca r boxyl-

ic Acid Eth yl Ester . Starting with the mesylate prepared

above, the title compound was prepared in 70% yield according

to the general procedure described for compound 5-[3(S),4-

epoxybutyl]-4-methylthiophene-2-carboxylic acid ethyl ester:

[R]₅₈₉+23.5° (c ) 0.80, MeOH); ¹H NMR (CDCl₃) δ 1.35 (t,

3H, J ) 7.1 Hz), 1.74-1.86 (m, 1H), 1.89-2.01 (m, 1H), 2.17

(s, 3H), 2.51 (dd, 1H, J ) 2.6, 14.8 Hz), 2.78 (dd, 1H, J ) 4.0,

14.8 Hz), 2.86-3.00 (m, 3H), 4.31 (q, 2H, J ) 7.1 Hz), 7.50 (s,

1H). Anal. (C₁₂H₁₆O₃S) C, H, S.

(m, 2H), 2.13 (s, 3H), 2.46 (s, 3H), 2.85 (m, 2H), 3.88-4.03 (m,

3H), 4.31 (q, 2H, J ) 7.0 Hz), 7.36 (d, 2H, J ) 8.1 Hz), 7.48 (s,

1H), 7.79 (d, 2H, J ) 8.4 Hz). Anal. (C₁₉H₂₄O₆S₂) C, H, S.

5-(4-Azid o-3(S)-h yd r oxyb u t yl)-4-m et h ylt h iop h en e-2-

ca r boxylic Acid Eth yl Ester . Starting with the tosylate

prepared above, the title compound was prepared in 96% crude

yield according to the general procedure described for com-

pound 5-(4-azido-3(S)-hydroxybutyl)thiophene-2-carboxylic acid

ethyl ester and used without purification: ¹H NMR (CDCl₃) δ

1.36 (t, 3H, J ) 7.0 Hz), 1.80 (m, 2H), 2.16 (s, 3H), 2.83-2.96

(m, 2H), 3.29 (dd, 1H, J ) 12.1, 7.4 Hz), 3.40 (dd, 1H, J )

12.1, 3.7 Hz), 3.78 (m, 1H), 4.30 (q, 2H, J ) 7.0 Hz), 7.50 (s,

1H).

5-[4-[(ter t-Bu toxyca r bon yl)a m in o]-3(S)-h yd r oxybu tyl]-

4-m eth ylth iop h en e-2-ca r boxylic Acid Eth yl Ester (36c).

Starting with the azide prepared above, compound 36c was

prepared in 89% yield according to the general procedure

described for compound 36a : [R]₅₈₉-21.7° (c ) 0.71, MeOH);

IR (neat) 3385 (broad), 2978, 2932, 1715, 1682, 1520, 1454,

1

1254 cm^-1; H NMR (CDCl₃) δ 1.35 (t, 3H, J ) 7.0 Hz), 1.44

(s, 9H), 1.75 (m, 2H), 2.16 (s, 3H), 2.80-2.94 (m, 2H), 3.08 (dd,

1H, J ) 14.3, 7.4 Hz), 3.28 (dd, 1H, J ) 14.3, 3.0 Hz), 3.74 (m,

1H), 4.30 (q, 2H, J ) 7.0 Hz), 7.49 (s, 1H). Anal. (C₁₇H₂₇

NO₅S) C, H, N, S.

-

5-[4-[(ter t-Bu toxyca r bon yl)a m in o]-3(S)-[(m eth ylsu lfo-

n yl)oxy]bu tyl]-4-m eth ylth ioph en e-2-car boxylic Acid Eth -

yl Ester . Starting with alcohol 36c, the title compound was

prepared in 94% yield according to the general procedure

described for compound 5-[4-[(tert-butoxycarbonyl)amino]-3(S)-

[(methylsulfonyl)oxy]butyl]thiophene-2-carboxylic acid ethyl

ester: mp 75-76 °C; [R]₅₈₉+7.8° (c ) 0.60, MeOH); IR (KBr)

3362, 2982, 1699, 1680, 1530, 1350, 1278 cm^-1

;

¹H NMR

5-[4-Azid o-3(R)-h yd r oxyb u t yl]-4-m et h ylt h iop h en e-2-

ca r boxylic Acid Eth yl Ester . Starting with the epoxide

prepared above, the title compound was prepared in 79% yield

according to the general procedure described for compound

5-[4-azido-3(S)-hydroxybutyl]-4-methylthiophene-2-carboxyl-

(CDCl₃) δ 1.35 (t, 3H, J ) 7.0 Hz), 1.45 (s, 9H), 2.01 (m, 2H),

2.16 (s, 3H), 2.89 (m, 2H), 3.07 (s, 3H), 3.30-3.48 (m, 2H), 4.31

(q, 2H, J ) 7.0 Hz), 4.80 (m, 1H), 4.95 (br t, 1H), 7.50 (s, 1H).

Anal. (C₁₈H₂₉NO₇S₂) C, H, N, S.

Glycinamide Ribonucleotide Transformylase Inhibitors

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16 2523

1

(2) (a) Lehninger, A. L. Biochemistry, 2nd ed.; Worth Publishers,

Inc.: New York, 1975; pp 729-732. (b) Dev, I. K.; Harvey, R. J .

N10-Formyltetrahydrofolate is the Formyl Donor for Glycinam-

ide Ribotide Transformylase in Escherichia coli. J . Biol. Chem.

1978, 253, 4242-4244. (c) Smith, G. K.; Mueller, W. T.; Slieker,

W. T.; DeBrosse, C. W.; Benkovic, S. J . Direct Transfer of One-

Carbon Units in the Transformylations of de novo Purine

Biosynthesis. Biochemistry 1982, 21, 2870-2874.

(3) (a) Taylor, E. C.; Harrington, P. J .; Fletcher, S. R.; Beardsley,

G. P.; Moran, R. G. Synthesis of the Antileukemic Agents 5,10-

Dideazaaminopterin and 5,10-Dideaza-5,6,7,8-tetrahydroami-

nopterin. J . Med. Chem. 1985, 28, 914-921. (b) Beardsley, G.

P.; Moroson, B. A.; Taylor, E. C.; Moran, R. G. A New Folate

Antimetabolite, 5,10-Dideaza-5,6,7,8-tetrahydrofolate Is a Potent

Inhibitor of de novo Purine Synthesis. J . Biol. Chem. 1989, 264,

328-333.

ic acid ethyl ester: [R]₅₈₉+11.0° (c ) 0.58, MeOH); H NMR

(CDCl₃) δ 1.35 (t, 3H, J ) 7.2 Hz), 1.75-1.84 (m, 2H), 2.17 (s,

3H), 2.80-3.00 (m, 2H), 3.29 (dd, 1H, J ) 7.0, 12.3 Hz), 3.42

(dd, 1H, J ) 3.1, 12.3 Hz), 3.75-3.83 (m, 1H), 4.31 (q, 2H, J

) 7.2 Hz), 7.50 (s, 1H). Anal. (C₁₂H₁₇N₃O₃S) C, H, S.

5-[4-[(ter t-Bu toxycar bon yl)am in o]-3(R)-h ydr oxybu tyl]-

4-m eth ylth iop h en e-2-ca r boxylic Acid Eth yl Ester (36d ).

Starting with the azide prepared above, the title compound

was prepared in 96% yield according to the general procedure

described for compound 36a : [R]₅₈₉+22.6° (c ) 0.66, MeOH);

¹H NMR (CDCl₃) δ 1.35 (t, 3H, J ) 7.2 Hz), 1.45 (s, 9H), 1.72-

1.81 (m, 2H), 2.16 (s, 3H), 2.78-2.99 (m, 2H), 3.09 (dd, 1H, J

) 7.4, 14.3 Hz), 3.29 (dd, 1H, J ) 2.9, 14.3 Hz), 3.70-3.78 (m,

1H), 4.31 (q, 2H, J ) 7.2 Hz), 7.50 (s, 1H). Anal. (C₁₇H₂₇

-

(4) Moran, R. G.; Baldwin, S. W.; Taylor, E. C.; Shih, C. The 6S-

and 6R-Diastereomers of 5,10-Dideaza-5,6,7,8-tetrahydrofolate

Are Equiactive Inhibitors of de novo Purine Synthesis. J . Biol.

Chem. 1989, 264, 21047-21051.

NO₅S) C, H, N, S.

5-[4-[(ter t-Bu toxyca r bon yl)a m in o]-3(R)-[(m eth ylsu lfo-

n yl)oxy]bu tyl]-4-m eth ylth ioph en e-2-car boxylic Acid Eth -

yl Ester . Starting with the alcohol prepared above, the title

compound was prepared in 93% yield according to the general

procedure described for compound 5-[4-[(tert-butoxycarbonyl)-

amino]-3(S)-[(methylsulfonyl)oxy]butyl]thiophene-2-carboxyl-

ic acid ethyl ester: [R]₅₈₉-7.8° (c ) 0.98, MeOH); ¹H NMR

(CDCl₃) δ 1.35 (t, 3H, J ) 7.2 Hz), 1.45 (s, 9H), 1.98-2.06 (m,

2H), 2.16 (s, 3H), 2.86-2.92 (m, 2H), 3.06 (s, 3H), 3.34-3.43

(m, 2H), 4.31 (q, 2H, J ) 7.2 Hz), 4.77-4.84 (m, 1H), 4.92

(broad, 1H), 7.50 (s, 1H). Anal. (C₁₈H₂₉NO₇S₂) C, H, N, S.

2-[[1-[(ter t-Bu toxyca r bon yl)a m in o]-4-[5-(eth oxyca r bo-

n yl)-3-m eth ylth ien -2-yl]bu t-2(S)-yl]th io]m a lon ic Acid Di-

eth yl Ester . Starting with the thioacetate prepared above,

the title compound was prepared in 88% yield according to

the general procedure described for compound 2-[[1-[(tert-

butoxycarbonyl)amino]-4-[5-(ethoxycarbonyl)thien-2-yl]but-

2(S)-yl]thio]malonic acid diethyl ester: [R]₅₈₉-23.7° (c ) 0.68,

(5) (a) Ray, M. S.; Muggia, F. M.; Leichman, G.; Grunberg, S. M.;

Nelson, R. L.; Dyke, R. W.; Moran, R. G. Phase I Study of

(6R)-5,10-Dideazatetrahydrofolate: a Folate Antimetabolite In-

hibitory to De Novo Purine Synthesis. J . Natl. Cancer Inst. 1993,

85, 1154-1159. (b) Cole, J . T.; Gralla, R. J .; Kardinal, C. G.;

Rivera, N. P. Lometrexol (DDATHF): Phase I Trial of a Weekly

Schedule of This New Antifolate. Proc. Am. Assoc. Cancer Res.

1992, 33, 2468. (c) Wedge, S. R.; Laohavinij, S.; Taylor, G. A.;

Newell, D. R.; Charlton, C. J .; Proctor, M.; Chapman, F.;

Simmons, D.; Oakey, A.; Gumbrell, L.; Calvert, A. H. Proc. Am.

Assoc. Cancer Res. 1993, 34, 1629.

(6) (a) Taylor, E. C.; Wong, G. S. K.; Fletcher, S. R.; Harrington, P.

J .; Beardsley, G. P.; Shih, C. Synthesis of 5,10-Dideazatetrahy-

drofolic Acid (DDATHF) and Analogues. In Chemistry and

Biology of Pteridines, 1986; Cooper, B. A., Whitehead, V. M.,

Eds.; de Gruyter: Berlin, 1986; pp 61-64. (b) Taylor, E. C.; Yoon,

C. Synthesis of 10-(Hydroxymethyl)-5,10-dideaza-5,6,7,8-tet-

rahydrofolic Acid, a Potent New Analogue of DDATHF (Lom-

etrexol). J . Org. Chem. 1994, 59, 7096-7098. (c) Baldwin, S. W.;

Tse, A.; Gossett, L. S.; Taylor, E. C.; Rosowsky, A.; Shih, C.;

Moran, R. G. Structural Features of 5,10-Dideaza-5,6,7,8-tet-

rahydrofolate That Determine Inhibition of Mammalian Glycin-

amide Ribonucleotide Formyltransferase. Biochemistry 1991, 30,

1997-2006.

(7) (a) Shih, C.; Grindey, G. B.; Moran, R. G.; Taylor, E. C.; Gossett,

L. S.; Harrington, P. M.; Worzalla, J . F.; Rinzel, S. M. New

Analogues of 5,10-Dideazatetrahydrofolic Acid (DDATHF) with

Modification in the Phenyl Region. Proc. Am. Assoc. Cancer Res.

1989, 30, 479. (b) Shih, C.; Grindey, G. B.; Gossett, L. S.; Moran,

R. G. Synthesis and Structure-Activity Relationship Studies of

5,10-Dideazatetrahydrofolic Acid. In Chemistry and Biology of

Pteridines, 1989; Curtius, H.-C., Ghisla, S., Blau, N., Eds.; de

Gruyter: Berlin, 1990; pp 1035-1038.

(8) (a) Taylor, E. C.; Harrington, P. M.; Shih, C. A Facile Route to

“Open Chain” Analogues of DDATHF. Heterocycles 1989, 28,

1169-1178. (b) Kelley, J . L.; McLean, E. W.; Cohn, N. K.;

Edelstein, M. P.; Duch, D. S.; Smith, G. K.; Hanlon, M. H.;

Ferone, R. Synthesis and Biological Activity of an Acyclic

Analogue of 5,6,7,8-Tetrahydrofolic Acid, N-[4-[[3-(2,4-Diamino-

1,6-dihydro-6-oxo-5-pyrimidinyl)propyl]amino]-benzoyl]-L-glutam-

ic Acid. J . Med. Chem. 1990, 33, 561-567. (c) Shih, C.; Gossett,

L. S.; Worzalla, J . F.; Rinzel, S. M.; Grindey, G. B.; Harrington,

P. M.; Taylor, E. C. Synthesis and Biological Activity of Acyclic

Analogues of 5,10-Dideaza-5,6,7,8-tetrahydrofolic Acid. J . Med.

Chem. 1992, 35, 1109-1116. (d) Bigham, E. C.; Hodson, S. J .;

Mallory, R.; Wilson, D.; Duch, D. S.; Smith, G. K.; Ferone, R.

Synthesis and Biological Activity of Open-Chain Analogues of

5,6,7,8-Tetrahydrofolic Acid-Potential Antitumor Agents. J . Med.

Chem. 1992, 35, 1399-1410. (e) Taylor, E. C.; Schrader, T. H.;

Walensky, L. D. Synthesis of 10-Substituted “Open-Chain”

Analogues of 5,10-Dideaza-5,6,7,8-tetrahydrofolic Acid (DDATHF,

Lometrexol). Tetrahedron 1992, 48, 19-32. (f) Hodson, S. J .;

Bigham, E. C.; Duch D. S.; Smith, G. K.; Ferone, R. Thienyl and

Thiazolyl Acyclic Analogues of 5-Deazatetrahydrofolic Acid. J .

Med. Chem. 1994, 37, 2112-2115.

1

MeOH); H NMR (CDCl₃) δ 1.34 (t, 3H, J ) 7.1 Hz), 1.44 (s,

9H), 1.75-1.86 (m, 1H), 1.90-1.99 (m, 1H), 2.15 (s, 3H), 2.85-

2.97 (m, 2H), 3.00-3.07 (m, 1H), 3.23-3.39 (m, 2H), 3.80 (s,

6H), 3.87 (s, 1H), 4.31 (q, 2H, J ) 7.1 Hz), 5.12 (broad, 1H),

7.49 (s, 1H). Anal. (C₂₂H₃₃NO₈S₂) C, H, N, S.

6(S)-[2-[5-(Eth oxyca r bon yl)-3-m eth ylth ien -2-yl]eth yl]-

3-oxo-1,4-th ia zin e-2-ca r boxylic Acid Meth yl Ester (37d ).

Starting with the malonate prepared above, compound 37d

was prepared in 88% yield according to the general procedure

described for compound 37a : [R]₅₈₉-26.2° (c ) 0.81, MeOH);

¹H NMR (CDCl₃) δ 1.35 (t, 3H, J ) 7.1 Hz), 1.83-2.03 (m,

2H), 2.16 (s, 3H), 2.83-2.97 (m, 2H), 3.39-3.67 (m, 3H), 3.80,

3.82 (s, 3H), 4.14, 4.25 (s, 1H), 4.31 (q, 2H, J ) 7.1 Hz), 6.29

(broad, 1H), 7.50 (s, 1H). Anal. (C₁₆H₂₁NO₅S₂) C, H, N, S.

5-[2-(2-Am in o-4(3H )-oxo-5,6,7,8-t et r a h yd r op yr im id o-

[5,6-b][1,4]t h ia zin -6(S)-yl)et h yl]-4-m et h ylt h iop h en e-2-

ca r boxylic Acid Eth yl Ester . Starting with lactam prepared

above, the title compound was prepared in 27% yield according

to the general procedure described for compound 5-[2-(2-amino-

4(3H)-oxo-5,6,7,8-tetrahydropyrimido[5,6-b][1,4]thiazin-6(R)-

yl)ethyl]thiophene-2-carboxylic acid ethyl ester: [R]₅₈₉-41.5°

1

(c ) 0.66, DMSO); H NMR (DMSO-d₆) δ 1.25 (t, 3H, J ) 7.0

Hz), 1.62-1.73 (m, 1H), 1.80-1.91 (m, 1H), 2.13 (s, 3H), 2.79-

2.97 (m, 3H), 3.19-3.26 (m, 1H), 3.47-3.55 (m, 1H), 4.22 (q,

2H, J ) 7.0 Hz), 6.05 (s, 2H), 6.68 (s, 1H), 7.53 (s, 1H), 10.10

(s, 1H); HMS calcd for C₁₆H₂₀N₄O₃S₂(M + Na⁺) 403.0875,

found 403.0859.

Ack n ow led gm en t. We thank the members of the

Crystallography, Computational Chemistry, Pharmacol-

ogy, and Medicinal Chemistry Groups for both their

intellectual and technical assistance throughout this

work. We also thank Bob J ackson for his continued

support and Beverly Standifer for her expert assistance

in preparing the manuscript.

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J M9607459

Article Doi

Protein structure-based design, synthesis, and biological evaluation of 5-thia-2,6-diamino-4(3H)-oxopyrimidines: Potent inhibitors of glycinamide ribonucleotide transformylase with potent cell growth inhibition

DOI: 10.1021/jm9607459

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