Design and synthesis of benzoic acid derivatives as influenza neuraminidase inhibitors using structure-based drug design

4030

J . Med. Chem. 1997, 40, 4030-4052

Design a n d Syn th esis of Ben zoic Acid Der iva tives a s In flu en za Neu r a m in id a se

In h ibitor s Usin g Str u ctu r e-Ba sed Dr u g Design ¹

Pooran Chand, Yarlagadda S. Babu, Shanta Bantia, Naiming Chu, L. Brent Cole, Pravin L. Kotian,

W. Graeme Laver,^†J ohn A. Montgomery, Ved P. Pathak,^‡Sandra L. Petty, David P. Shrout,

David A. Walsh,* and Gerald M. Walsh

BioCryst Pharmaceuticals, Inc., 2190 Parkway Lake Drive, Birmingham, Alabama 35244

Received J uly 23, 1997^X

A series of 94 benzoic acid derivatives was synthesized and tested for its ability to inhibit

influenza neuraminidase. The enzyme-inhibitor complex structure was determined by X-ray

crystallographic analysis for compounds which inhibited the enzyme. The most potent

compound tested in vitro, 5 (4-(acetylamino)-3-guanidinobenzoic acid), had an IC₅₀) 2.5 ×

10^-6M against N9 neuraminidase. Compound 5 was oriented in the active site of the

neuraminidase in a manner that was not predicted from the reported active site binding of

GANA (4) with neuraminidase. In a mouse model of influenza, 5 did not protect the mice

from weight loss due to the influenza virus when dosed intranasally.

Ch a r t 1. Sialic Acid Derivatives

In tr od u ction

Influenza is an acute respiratory disease that causes

annual epidemics in humans afflicting between 20 and

40 million people in the United States each year. It is

particularly dangerous to the very young, the elderly

and/or debilitated patients, and those who have sup-

pressed immune systems. Approximately 300 000 people

are hospitalized, and about 15 000-20 000 persons die.

This mortality figure can be as high as 40 000-70 000

during a more severe outbreak. Four times this cen-

tury, a particularly virulent new strain of flu virus has

emerged resulting in far more disease and deaths than

usual. The pandemics of 1957 and 1968 together cost

some $32 billion (in 1995 dollars) in lost productivity

and medical expenses.²

Ch a r t 2. Inhibitors of Influenza Neuraminidase

Neuraminidase (EC 3.2.1.18) is one of two major

surface glycoproteins of both type A and B influenza

viruses and is essential for viral replication in vitro.³

The enzyme active site of viral neuraminidase is con-

served despite up to 75% sequence variation in amino

acid sequence between the neuraminidases of influenza

A and B, which makes it an attractive target for

structure-based drug design (SBDD). Inhibition of

neuraminidase by complexation with the active site

could lead to drugs which have a broad spectrum of

activity against various flu strains.

Sialic acid (1) has relatively weak (IC₅₀∼ 10^-3M) in

vitro viral neuraminidase inhibitory activity, but recent

reports^4,5have shown that deoxysialic acid derivatives

2-4 (Chart 1) are potent neuraminidase inhibitors.

Compound 4, which has been described as a transition-

state inhibitor,⁶has shown activity against influenza

in the clinic both prophylactically and therapeutically

in experimentally infected humans.⁷The ring system

in 2-4 assumes a more planar conformation than the

ring system in 1, and this observation has led to other

reports^8,9which have indicated that aromatic deriva-

tives related to 5 (Chart 2) where the ring system is

completely planar also bond to the neuraminidase active

site and possess in vitro activity. However, one of the

same reports⁹also indicated that 7 (Chart 2), an

aromatic derivative closely related to 4, was inactive in

vitro in the H3N2 strain of influenza.

Aromatic derivatives related to 5 are chemically more

readily accessible than the more difficult-to-prepare and

costly analogues of 4. Our goals for this project were

to prepare orally active, potent, and selective viral

neuraminidase inhibitors which could be easily and

economically prepared. Our concurrent studies with 5

in the H1N9 viral strain of influenza gave us similar

results to those previously reported^8,9for the B/Lee/40

and H3N2 strains; in addition, we observed cyclization

in 5 (Chart 2) which resulted in loss of neuraminidase

inhibitory potency. By studying the interactions of 5

and other derivatives with the active site of neuramini-

dase, we believed we could design analogues which

contained substituents which would maximize the in-

teractions with the active site and also impart chemical

* Author to whom correspondence should be addressed.

^†The Australian National University, Canberra 260, Australia.

^‡Present address: Alanex Corp., 3550 General Atomics Ct., San

Diego, CA 92121.

^XAbstract published in Advance ACS Abstracts, November 15, 1997.

Benzoic Acids as Influenza Inhibitors

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 25 4031

Sch em e 1^a

Sch em e 2^a

a

Reagents: (a) fuming HNO₃; (b) 1. H₂, 10% Pd/C, MeOH, 2.

HCl, NaNO₂, 3. CuCl, NaCN; (c) 1. 10% NaOH, EtOH, ∆, 4 h, 2.

1 N HCl.

stability to these planar molecules. Hopefully, we could

also explain any anomalous results such as the inactiv-

ity of 7 in vitro. This report describes our initial work

to determine suitable replacements for each of the

substituents on 5. Our approach was to initially test

the synthesized compound at a concentration of 7 mM

in an in vitro assay¹⁰for the inhibition of N9 neuramini-

dase. If the compound showed good activity, an IC₅₀

was determined. A solution of certain interesting

compounds was also soaked (see Experimental Section)

into crystals¹¹of neuraminidase. X-ray diffraction data

were collected for the enzyme-inhibitor complex and

compared with the refined N9 native structure. The

difference Fourier maps were calculated to identify the

inhibitor in the active site of the enzyme. The inhibitory

activity of the compound coupled with its mode of

bonding in the active site was used to design other

derivatives for synthesis.

a

Reagents: (a) NaSCH₃, DMF; (b) mClPBA, CH₂Cl₂; (c) 1. 1 N

NaOH, 2. HCl; (d) KNO₃, H₂SO₄; (e) H₂, PtO₂, MeOH; (f)

SdC[NHCO₂(CH₃)₃]₂, Et₃N, DMF, HgCl₂; (g) 1. TFA, 2. 1 N NaOH,

3. HCl; (h) H₂O₂, HOAc; (i) 90% fuming HNO₃.

pounds which contain several complimentary polar

groups contiguously placed around a planar benzene

ring a daunting chemical challenge. Replacement of the

ring hydroxyl in 2 with the guanidine substituent in 4

increases the in vitro potency of this series of molecules

by approximately 5 orders of magnitude, so it was

desirable to use the guanidino group as a key substitu-

ent for our series of benzoic acid inhibitors. The

chemistry associated with the synthesis of guanidine

derivatives has been reviewed recently.⁴⁰In most

instances, our guanidino substituents were synthesized

by three general methods: reaction of an amine with

cyanamide; reaction of an amine with aminoimi-

nomethanesulfonic acid;³⁴and reaction of an amine with

N,N′-bis(tert-butoxycarbonyl)thiourea⁴¹facilitated by

HgCl₂.⁴²The latter method allowed for protected guani-

dine derivatives to be prepared which could readily be

purified by column chromatography before the charged

groups such as carboxyl and/or guanidino were un-

masked. Purification of the highly polar molecules after

the final synthetic step proved troublesome. The tert-

butoxy protecting groups could be readily removed by

TFA,⁴³or both tert-butoxy protecting group removal and

hydrolysis of an ester could be accomplished in one step

using 6 N HCl.

Table 2 lists the disubstituted benzoic acid derivatives

which were tested for in vitro neuraminidase inhibitory

activity. Most of the compounds listed in Table 2 are

novel, and their preparations are described in the

Experimental Section. Scheme 3 illustrates the syn-

thesis for several targets from the key intermediates 92

and 139. These intermediates were prepared using

Gassman’s method⁴⁵for aldehyde synthesis. Aldehyde

92 was converted to the alcohol 94, the iminoguanidine

derivative 121, and the aminoethyl analogue 131 by

standard methods. Both aldehydes 92 and 139 were

converted to their respective oximes 119 and 144, while

139 was converted to the aminohydroxyethyl derivative

142.

Ch em istr y

Table 1 lists monosubstituted benzoic acid derivatives

and isosteres that were tested for in vitro neuramini-

dase inhibitory activity. Most of these compounds could

be purchased or prepared by literature methods. The

synthesis of diol 46 is outlined in Scheme 1. Nitration

of 2-phenyl-1,3-propanediol (41)²⁹with fuming nitric

acid gives predominantly the para isomer 42 which is

relatively stable and can be characterized. Hydrogena-

tion of 42 over palladium gives the amine 44 which was

then diazotized and converted to the nitrile 45. Hy-

drolysis of 45 gave 46. Scheme 2 depicts the synthesis

of sulfoxide 57 and sulfone 58. Methyl 4-(bromomethyl)-

benzoate (53) was converted to the key intermediate 54

with sodium thiomethoxide in DMF. The methylthio

derivative was reacted with m-chloroperbenzoic acid to

give sulfoxide 55 or reacted with H₂O₂in acetic acid to

give sulfone 56. Hydrolysis of these esters gave 57 and

58, respectively.

The three-dimensional structure of the active site of

N9 neuraminidase and the structure of 2 bonded to the

active site have been determined.³⁹The active site has

12 amino acids in contact with 2, while another 6 amino

acids stabilize this site. The bonding environment is a

very polar and charged environment. There is only one

small hydrophobic pocket where the methyl substituent

of the -NHCOCH₃group resides. This polar require-

ment makes the synthesis and purification of com-

Scheme 4 depicts the preparation of various deriva-

tives containing a two-carbon substituent ortho to the

4032 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 25

Chand et al.

Sch em e 3^a

Sch em e 4^a

a

Reagents: (a) t-BuOCl, CH₃SCH₂CO₂C₂H₅, Et₃N, CH₂Cl₂, -70

°C; (b) CH₃COCl, Et₃N; (c) Raney Ni, THF; (d) 1 N NaOH, 16 h;

(e) t-BuOCl, C₆H₅SCH₂CONH₂, Et₃N, CH₂Cl₂, -70 °C; (f) 1 N

NaOH, 1 h; (g) 1. ClCO₂C₂H₅, Et₃N, THF, 2. NaBH₄, MeOH, 3.

HCl.

Sch em e 5^a

a

Reagents: (a) MsCl, pyridine, CH₂Cl₂; (b) CuO, CuCl₂, acetone,

DMF; (c) NaBH₄, MeOH; (d) 1. 1 N NaOH, 2. 1 N HCl; (e)

aminoguanidine bicarbonate, HCl, EtOH; (f) hydroxylamine hy-

drochloride, EtOH; (g) NaH, MeNO₂, DMF; (h) H₂, PtO₂, EtOH;

(i) DMAP, acetic anhydride, CH₂Cl₂.

amino function. These were again prepared utilizing a

Gassman⁵²procedure. Starting with ester 95, the

diester 98 was prepared by standard methods. The

diester could be hydrolyzed to the diacid 122, or the

aliphatic ester could be selectively hydrolyzed to the

monoacid 99 in the presence of an aromatic ester by

using shorter reaction times. The selective reduction

of an acid in the presence of an ester to give 100 was

accomplished by preparing the mixed anhydride of the

acid and reduction with NaBH₄. The amide 126 could

be prepared from 95 in a similar manner.

a

Reagents: (a) 1. Pd(OAc)₂, toluene, 2. CH₂dCHCH₂I, HOAc;

(b) AD mix-R for isomer A, AD mix-â for isomer B, t-BuOH, H₂O;

(c) 1. 1 N NaOH, 2. HCl; (d) 1. Pd(OAc)₂, toluene, 2. CH₂dCHCH-

(OC₂H₅)₂, Et₃N; (e) NaBH₄, MeOH.

A terephthalic acid derivative (160) was prepared by

the procedure given in Scheme 6. Selective esterifica-

tion of one carboxyl group gave 156 which in turn was

converted through standard methods to target 160.

The syntheses of compounds which have substituents

designed to mimic the glycerol substituent in 2 are

outlined in Scheme 5. The reaction of 107 with pal-

ladium acetate and an appropriate alkene gave adducts

108 and 113. Intermediate 108 was converted to the

dihydroxy esters 109 and 110 using commercially avail-

able AD mix-R or -â. In this instance, the acids obtained

after hydrolysis, 111 and 112, had no optical rotation

at the sodium D-line and were chemically, physically,

and spectroscopically indistinguishable from each other.

Aldehyde 113 was reduced with NaBH₄to yield alcohol

115 after hydrolysis.

Scheme 7 depicts the synthesis of several 3,5-disub-

stituted benzoic acid derivatives. The acid group in

185⁵¹could be selectively reduced in the presence of an

ester using lithium tri-tert-butoxyaluminohydride to

give both the aldehydo- (186) and the hydroxymethyl-

(187) substituted compounds. The aldehyde was con-

verted to the oxime (188), and both the nitro and hy-

droxyimino groups were reduced catalytically over

palladium to give the diamine 189. The aliphatic amino

group in 189 could be preferentially substituted in the

presence of the aromatic amino group to yield 191 via

Benzoic Acids as Influenza Inhibitors

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 25 4033

Sch em e 6^a

the carboxyl and acetylamino substituents bonded to the

active site in a similar manner and position as did the

same substituents in 2.³⁹Compounds 9-58 listed in

Table 1 were tested to determine suitable replacements

for the acetylamino substituent located in a para posi-

tion to the carboxyl group in the benzoic acid derivative.

Derivatives 11, 12, 14-17, 31-34, 39, 46, 49, and 57

showed a measurable degree of inhibitory activity at a

concentration of 7 mM with 14, 16, and 31 being the

most potent. Interestingly, 14 (C₆H₅CONH) had an IC₅₀

) 1 mM, but no electon density corresponding to this

inhibitor was present in the active site of the enzyme.

One explanation for the results with 14 is that it

interacts with the neuraminidase at a position other

than the catalytic site. Derivatives with one (48) or two

(51) -CH₂- spacer groups between the acetylamino

substituent and the aromatic ring were inactive at 7 mM

in vitro.

a

Reagents: (a) MeOH, H₂SO₄, ∆, 1 h; (b) 1. SOCl₂, DMF, 2.

MeNH₂, H₂O, CH₂Cl₂; (c) SnCl₂, EtOH; (d) 1. 1 N NaOH, 2. HCl;

(e) cyanamide, HCl, EtOAc, 37-38 °C, 16 h.

intermediate 190, or both amino groups could be

substituted simultaneously to give the disubstituted 193

via 192.

Compounds 59-82 contain substituents in a meta

position to the carboxyl group in the benzoic acid

derivatives. These substituents may substitute for the

glycerol side chain in 2³⁹or for the guanidino substitu-

ent in 4.⁴Of these derivatives tested, 60, 64, and 67

had the highest degree of activity at 7 mM; however,

67 could not be located in the active site. The -NHCN

substituent in 60 and the guanidino substituent in 64

were positioned in the same site as the glycerol sub-

stituent in 2 and 4 and not in the same site as the

guanidino substituent of 4. This observation is in

agreement with the results reported by Singh et al.⁸for

5 (Table 2) in B/Lee neuraminidase. The activity for

the derivative 72, where one -CH₂- spacer is placed

between the guanidino substituent and the aromatic

ring, or 76, where the guanidino group is replaced by

an amidino substituent, is reduced and for the deriva-

The nitro substituent in 188 could be reduced over

platinum oxide preferentially in the presence of the

hydroxyimino group to give the monoamine 194, which

is then converted to 197. The hydroxymethyl-substi-

tuted 187 was converted to the aminoethyl-substituted

204 through a series of standard reactions.

Resu lts a n d Discu ssion

Initial results from the testing of 11 (Table 1) were

encouraging since this relatively simple monosubsti-

tuted benzoic acid derivative showed some in vitro

inhibitory activity against the H1N9 strain of influenza

neuraminidase. Furthermore, X-ray data showed that

this weak inhibitor soaked into the N9 crystal and that

Sch em e 7^a

a

Reagents: (a) 1. (ClCO)₂, DMF, CH₂Cl₂, 2. CuI, LiAlH[OC(CH₃)₃]₃, THF; (b) NH₂OH‚HCl, EtOH; (c) H₂, 10% Pd/C, EtOH; (d)

SdC[NHCO₂C(CH₃)₃]₂, Et₃N, DMF, HgCl₂; (e) 6 N HCl; (f) H₂, PtO₂, EtOH; (g) 1. TFA, 2. 1 N NaOH, 3. 1 N HCl; (h) pyridinium

chlorochromate, CH₂Cl₂; (i) NaH, CH₃NO₂, DMF; (j) 1. NaBH₄, EtOH, 2. HOAc.

4034 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 25

Chand et al.

Benzoic Acids as Influenza Inhibitors

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 25 4035

F igu r e 1. Comparison of the binding of 5 (green) and 119

(white) in the active site of H1N9 neuraminidase.

tive 81, where two -CH₂- spacer groups are between

the guanidino substituent and the aromatic ring, is

abolished.

In compounds 83-90, the p-acetylamino substituent

was held constant while various isosteres were substi-

tuted for the carboxyl group. Of these, the derivatives

containing the -SO₃H (86) and -PO₃H₂(87) groups

retained activity.

On the basis of the results obtained for the compounds

listed in Table 1, the derivatives in Table 2 were

prepared to determine the effect on potency when a

second substituent for interaction with the active site

was added to the benzoic acid derivatives. The propen-

sity for substituents located in the meta and para

positions of the benzoic acid derivatives to undergo

chemical reactions with each other was also assessed.

In compounds 91-115, the p-acetylamino substituent

was held constant while various hydroxylated substit-

uents were placed in the meta position to mimic the

glycerol side chain of 2. The in vitro activity of these

compounds was disappointing since none of the com-

pounds had increased activity over 11 which has a

hydrogen in the meta position.

Compounds 116-131 in Table 2 were tested to

determine if other substituents would bond in the pocket

where the glycerol substituent of 2 bonds. Clearly, 5

was the most potent compound tested in vitro. Al-

though 122 and 126 possessed some in vitro activity,

they could not be located in the difference electron

density maps. Of the compounds which did bond to the

active site (5, 117, 119, 130, and 131), only the hydroxy-

imino substituent of 119 did not point to the glycerol

pocket. The hydroxyimino of 119 was positioned in the

pocket in which the guanidino substituent of 4 bonds,

and the acetylamino group had different interactions

with the active site than in 5 (Figure 1).

Replacement of the acetylamino substituent was

studied with compounds 136-178. Of these, the (meth-

ylsulfonyl)amino (136), (methylamino)carbonyl (160),

aminosulfonyl (164), and (methylsulfoxyl)methyl (173)

all bonded in the same site as did the acetylamino group

of 5 (Figures 2 and 3) albeit with different interactions.

Only the o-(methylamino)carbonyl substituent of 160

appeared to undergo cyclization with the guanidino

4036 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 25

Chand et al.

Benzoic Acids as Influenza Inhibitors

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 25 4037

F igu r e 2. Comparison of the binding of 5 (green, HNCOCH₃),

136 (yellow, NHSO₂CH₃), and 160 (white, CONHCH₃) of the

active site of H1N9 neuraminidase.

F igu r e 4. Comparison of the binding of 5 (green, 4-NH-

COCH₃), 184 (yellow, 5-NHC(dNH)NH₂), and 196 (blue,

5-CHdNOH).

F igu r e 5. Effects of daily intranasal administration of 5, 10

mg/kg, twice per day, on the time course of body weight loss

in mice after infection with influenza virus. Values are means

( SE, n ) 5/group.

F igu r e 3. Comparison of the binding of 5 (green, NHCOCH₃),

164 (white, SO₂NH₂), and 173 (blue, CH₂SOCH₃) in the active

site of H1N9 neuraminidase.

effects of 5 intranasal treatment in influenza-infected

mice are shown in Figure 5. Compound 5 failed to

prevent weight loss and was not different from the

vehicle controls. The effects of 2 (DANA) intranasal

treatment are shown in Figure 6. Compound 2 pro-

duced a significant attenuation of weight loss compared

to the vehicle control but did not completely prevent

weight loss in response to infection compared to the

uninfected, untreated control. The results in Figure 7

show that active immunization by infection with the

influenza virus is completely effective in preventing

weight loss in response to a second infection.

group at higher temperatures. Compounds 136, 164,

and 173 appeared to be stable up to their melting points.

Again, the hydroxyimino substituent of 144 bonded to

the same pocket where the guanidino substituent in 4

bonded.

Examples of meta-substituted benzoic acids are com-

pounds 179-204. These compounds without the p-

acetylamino group were tested to determine a combi-

nation of substituents which would bond in each of the

guanidino and glycerol bonding sites of 4. Figure 4

depicts the comparison of 5, 184, and 196 in the active

site of the neuraminidase enzyme. The loss of the

interactions of the acetylamino-substituent interaction

with the active site reduced the in vitro potency

compared with 5 for all of these (179-204) derivatives.

The most potent compound in this report against

neuraminidase activity, 5, was tested in vivo in an

influenza infection weight loss model in the mouse

according to the method of J ohansson et al.⁵³The

Con clu sion s

The planar benzene ring provides a scaffold to support

substituents which will interact with the active site of

influenza neuraminidase. Based upon the data pre-

sented herein, a direct correlation between the interac-

tions observed for the substituents of DANA (2) and

GANA (4) and the active site cannot be extended to the

4038 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 25

Chand et al.

in a different manner. The -NHSO₂CH₃, -SO₂NH₂,

and -CH₂SOCH₃groups appear to be stable at elevated

temperatures when located contiguous to a guanidino

group.

Although 2 (DANA), a relatively weak inhibitor of

influenza neuraminidase, was moderately, but signifi-

cantly, effective in preventing weight loss in influenza-

infected mice, the most potent inhibitor, 5, was not. It

is not clear why 5 was not active in vivo, but it is

possible that it may be cleared rapidly from the lung

by metabolism and/or absorption. Active immunization

demonstrated that weight loss can be prevented by this

stimulus for antibody production against influenza, in

agreement with the findings of J ohansson et al.,⁵³who

further demonstrated that immunization with purified

influenza neuraminidase was also completely effective

in preventing weight loss in the influenza-infected

mouse. Thus, it appears that 5 may not have properties

suitable for in vivo efficacy. Similar results to those

seen for 5 were obtained with 160.

This present study lays the foundation to select

appropriate substituents for arrangement around a

benzene ring that would lead to compounds possessing

inhibitory potency in the same range as 4. Future

reports will discuss compounds with more than three

substituents about a benzene ring and the exploitation

of a lipophilic pocket in the active site of neuraminidase

recently reported.^54,55

F igu r e 6. Effects of daily intranasal administration of 2

(DANA), 10 mg/kg, twice per day, on the time course of body

weight loss in mice after infection with influenza virus.

*Significantly different from vehicle-treated mice, p < 0.05.

Values are means ( SE, n ) 5/group.

Exp er im en ta l Section

Bioch em istr y. The in vitro assay is based on the method

reported by von Itzstein et al.¹⁰The neuraminidase from the

H1N9 strain of influenza was obtained by the method de-

scribed by Laver et al.¹¹Values for the IC₅₀were measured

via a spectrofluorometric technique that uses 2′-(4-methylum-

belliferyl)-R-^D-acetylneuraminic acid as substrate. This sub-

strate was cleaved by neuraminidase to yield a fluorescent

product which can be quantified. The assay mixture contained

inhibitors at various concentrations (4-6 points) and enzyme

in 32.5 mM MES ((2-(N-morpholino)ethanesulfonic acid) buffer,

4 mM CaCl₂at pH 6.5 (total volume ) 80 µL). The reaction

was started by the addition of 20 µL of the substrate to a final

concentration of 75 µM. After 10 min at 37 °C, 2.4 mL of 0.1

M glycine/NaOH (pH 10.2) was added to 0.1 mL of the reaction

mixture to terminate the reaction. A blank was run with the

same substrate solution with no enzyme. Fluorescence was

read using an Aminco-Bowman fluorescence spectrophotom-

eter (excitation, 360 nm; emission, 450 nm), and substrate

blanks were subtracted from the sample readings. The IC₅₀

was calculated by plotting percent inhibition versus the

inhibitor concentration, and determination of each point was

performed in duplicate.

Biology. Mice (Balb/c females, 31-53 days of age) were

infected intranasally under light ether anesthesia with A/Tur-

key/Mass/76A/Beijing/32/92[R] (H6N2). The IC₅₀of 5 against

this strain was 2.25 µM (n ) 2). The infection was at 200 times

the MID₅₀(mouse infectious dose, 50%). Compound 5 was

dissolved in phosphate-buffered saline and administered in-

tranasally 5-10 min before infection at 10 mg/kg. The mice

were treated thereafter at 10 mg/kg/day intranasally, twice a

day. Body weight was measured daily. DANA (2) was tested

for comparison under similar conditions at 10 mg/kg/day

intranasally, twice a day. The IC₅₀of DANA against this

strain was 6 µM (n ) 3). A vehicle-treated group and an

untreated/uninfected group were also included. There were

5 mice/group. In a second experiment mice were actively

immunized with virus and then infected again to demonstrate

that active immunity prevents weight loss in the mouse in

response to intranasal influenza infection. Differences among

groups were analyzed by analysis of variance with a least

significant difference test.

F igu r e 7. Effects of active immunization on weight loss in

mice after infection with influenza. Values are means ( SE,

n ) 10/group.

benzene ring. From the enzyme-inhibitor complex of

5, it can be seen that the interaction of the guanidino

substituent is more favorable with the active site pocket

where the glycerol substituent of 4 resides, rather than

in the pocket where the guanidino substituent of 4

bonds. Considering this observation, it is not surprising

that Williams et al.⁹did not see any in vitro inhibitory

activity for 7.

Interactions for individual substituents on the ben-

zene ring with the active site cannot be considered to

be additive. Each combination of substituents has

unique steric and electronic interactions with each other

which influences the overall interaction of the compound

with the active site. This makes it difficult to predict a

substituent arrangement for this series that will give

an enhanced bonding to the active site.

Moieties which can be substituted for the -NH-

COCH₃group are -NHSO₂CH₃, -CONHCH₃, -SO₂-

NH₂, and -CH₂SOCH₃. These groups all bond to the

same pocket as does the -NHCOCH₃group albeit each

Benzoic Acids as Influenza Inhibitors

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 25 4039

Cr ysta llogr a p h y. Influenza neuraminidase has an N-

terminal hydrophobic sequence which anchors the glycoprotein

to the viral membrane. This sequence is followed by a highly

variable “stalk” which raises the globular neuraminidase

“heads” away from the membrane. Neuraminidase heads

released with protease contain all the enzymatic and antigenic

activity and are used for crystallization. Purified N9 neuramini-

dase¹¹was crystallized using the hanging drop technique in

which droplets of protein solution on siliconized cover slips are

inverted on a linbro plate. The droplet consisted of equal

volumes of protein solution (10-15 mg/mL in water) and

potassium phosphate buffer (1.7 M, pH 6.6). This mixture was

equilibrated through vapor phase with a reservoir of 1.9 M

potassium phosphate buffer at pH 6.8. Large rhombic dodeca-

hedral crystals grew in a few days. The space group has been

identified as cubic, I432 with cell dimension a ) 183.8 Å. The

crystals are stable at room temperature indefinitely in the

artificial mother liquor, and they diffract strongly to at least

2-Å resolution on a rotating anode source.

Inhibitor complexes with neuraminidase were prepared by

diffusing the compound into N9 crystals that were transferred

into a 2-mL solution of the stabilization buffer which was the

same as the reservoir solution used in crystallization. The

final concentration of the inhibitor in the solution was 1-2

mM. The N9 crystals were allowed to equilibrate with the

inhibitor compound for at least 24 h before data collection.

All X-ray intensity measurements were recorded with a

Nicolet/Siemens X-100 multiwire area detector. The detector

is mounted on a Rigaku RU-300 rotating anode X-ray genera-

tor operating at 100 mA and 50 kV with a 0.3 × 0.3 focus and

a Cu anode. The data collection parameters were crystal to

detector distance of 16 cm, swing angle of 28°, frame width of

0.1°, and exposure time of 240-300 s. Each crystal provided

about 800-900 frames of data before radiation damage

deteriorated the diffraction quality.

The X-ray intensity data were processed using the XENGEN

suite of programs. The integrated intensities were then scaled

and merged to produce a final data set containing only unique

reflections. The completeness of data to 2.1 Å is generally

about 90%. The refinement of the inhibitor complexes was

performed using X-PLOR simulated annealing protocol. Dif-

fraction data in 10-2.1-Å resolution range were used in the

refinement with a 2σ cutoff on F_o’s. The starting model for

the refinement of the inhibitor complexes is the 1.9-Å refined

uncomplexed neuraminidase native structure. The water

molecules in the active site of the native structure were

removed prior to the refinement of the complex structure. A

full cycle of X-PLOR simulated annealing protocol was carried

out on this model. F_o- F_cand 2(F_o- F_c) difference Fourier

maps were calculated, and a model of the inhibitor molecule

was fitted into the electron density in the active site. Water

molecules were also placed in the active site based on the

difference electron density maps, and these positions were

compared to the active site water structure in the native

neuraminidase model. At the end of the simulated annealing

protocols and positional and temperature factor refinement,

the crystallographic R-factor converged to 17-19% at 2-Å

resolution for the inhibitor-complex structures.

and the residue was purified by column chromatography on

silica gel (6:1:0.5 EtOAc-MeOH-H₂O). The appropriate

fractions (R_f) 0.45) were combined and concentrated to give

0.14 g (12%) of 6 as a brown powder: mp 195-198 °C dec; ¹H

NMR (400 MHz, DMSO-d₆) δ 2.52 (s, 3H), 5.41 (br s, 1H), 6.21

(br s, 1H), 6.57 (br s, 1H), 7.50 (d, J ) 7.8 Hz, 1H), 7.75 (dd,

J ) 7.8, 1.4 Hz, 1H), 8.05 (s, 1H), 12.50 (s, 1H); IR (KBr) 3336,

1635, 1558, 1384, 1363 cm^-l; MS (ES⁺) 177 (M + 1). Anal.

(C₁₀H₁₀N₄O₂‚0.6H₂O) C, H; N: calcd, 24.46; found, 23.96.

4-[(Meth oxyca r bon yl)a m in o]ben zoic Acid (22). A solu-

tion of 5.0 g (0.033 mol) of methyl p-aminobenzoate (20;

Aldrich) in 100 mL of CH₂Cl₂was cooled to 0-5 °C, and 3.2

mL (0.04 mol) of pyridine, 2.8 mL (0.04 mol) of methyl

chloroformate, and 0.5 g of DMAP were added. The reaction

mixture was stirred at ambient temperature for 16 h and then

diluted with 100 mL of H₂O and 50 mL of CH₂Cl₂. The

mixture was vigorously shaken, and the layers were separated.

The organic layer was dried (Na₂SO₄) and concentrated to give

5 g (72%) of methyl 4-[(methoxycarbonyl)amino]benzoate

(21): mp 177-178 °C.

A mixture of 1.5 g (0.006 mol) of 21, 10 mL of deionized

H₂O, and 7 mL of 1 N NaOH was heated at 60-70 °C for 8 h.

The reaction mixture was filtered, and the filtrate was made

acidic with 6 N HCl. The precipitate was collected by filtra-

tion, washed with H₂O, and dried to yield 0.75 g (54%) of 22

as a white solid: mp 195-196 °C; ¹H NMR (400 MHz, DMSO-

d₆) δ 3.69 (s, 3H), 7.56 (d, J ) 8.7 Hz, 2H), 7.87 (d, J ) 8.7 Hz,

2H), 10.04 (s, 1H), 12.62 (s, 1H); IR (KBr) 3330, 1716, 1687,

1610, 1552, 1420 cm^-l; MS (ES^-) 194 (M - 1). Anal. (C₁₀H₉-

NO₅‚0.25H₂O) C, H, N.

4-[[(Meth yla m in o)ca r bon yl]oxy]ben zoic Acid (30). To

0.24 g (0.0018 mol) of 4-hydroxybenzoic acid (29; Aldrich) in

10 mL of THF was added 460 µL (0.0035 mol) of Et₃N, and

the mixture stirred for 15 min. A 230-µL (0.0018-mol) portion

of trimethylsilyl bromide was added, and stirring continued

for another 30 min followed by the addition of 114 µL (0.0019

mL) of methyl isocyanate. The reaction was quenched by the

addition of 1 mL of H₂O, and the solvent was evaporated. To

the residue were added 15 mL of H₂O and 2 mL of 6 N NaOH,

and the suspended impurities were removed through filtration.

The filtrate was acidified using 6 N HCl, and the precipitate

was collected by filtration. The solid was dried to give 0.097

g (28%) of 30 as a white solid: mp 223-225 °C; ¹H NMR (400

MHz, DMSO-d₆) δ 2.67 (d, J ) 4.6 Hz, 3H), 7.21 (d, J ) 8.7

Hz, 2H), 7.77 (m, 1H), 7.95 (d, J ) 8.7 Hz, 2H), 12.90 (br s,

1H); IR (KBr) 3361, 1709, 1685, 1533, 1293, 1219 cm^-1; MS

(CI⁺) 196 (M + 1). Anal. (C₉H₉NO₄) C, H, N.

4-[2-[[(Am in oim in om e t h yl)a m in o]im in o]e t h yl]b e n -

zoic Acid (37). A mixture of 0.82 g (0.005 mol) of 4-acetyl-

benzoic acid (36; Aldrich), 0.68 g (0.005 mol) of aminoguanidine

bicarbonate, and 0.084 g (0.01 mol) of concentrated HCl in 30

mL of EtOH was heated at reflux for 16 h. Upon cooling, a

light brown precipitate formed, which was collected by filtra-

tion, washed with cold EtOH, and dried to give 0.9 g (79%) of

37 as a light brown powder: mp 328 °C dec; ¹H NMR (400

MHz, CF₃CO₂D) δ 2.45 (m, 3H), 7.94 (dd, J ) 6.9, 1.7 Hz, 2H),

8.25 (dd, J ) 6.9, 1.7 Hz, 2H); IR (KBr) 3180, 2382, 2350, 1679,

1635, 1594, 1384 cm^-1; MS (ES⁺) 221 (M + 1). Anal.

(C₁₀H₁₂N₄O₂‚0.25H₂O) C, H, N.

Ch em istr y. Gen er a l P r oced u r es. Melting points were

determined in open capillary tubes in a Melt-Temp II melting

2-(4-Nitr op h en yl)-1,3-O-d in itr op r op a n ed iol (42). To 10

mL of cooled 90% fuming HNO₃at 0-5 °C was added

portionwise 1.0 g (0.0066 mol) of 2-phenyl-1,3-propanediol

(41)²⁹over a 10-min period. The reaction mixture was stirred

for 30 min at 0-5 °C and for 30 min at ambient temperature

and then poured into 100 mL of cold H₂O. The mixture was

extracted thrice with 25 mL of EtOAc. The combined organic

layers were washed with 20 mL of a saturated Na₂CO₃solution

and 20 mL of brine, dried (MgSO₄), and concentrated to give

a yellow oil. This oil was purified by flash chromatography

on silica gel (10-50% Et₂O in C₆H₁₄). The appropriate

fractions were combined and concentrated to give 1.3 g (67%)

of 42 as a light yellow solid: mp 53-55 °C. The 2-nitro isomer

43 was also obtained in a 20% yield as a light yellow oil. On

a large scale, 42 can be obtained by crystallization of the crude

oil from Et₂O: ¹H NMR (400 MHz, CDCl₃) δ 3.63 (m, 1H), 4.77

(dd, J ) 6.4, 4.5 Hz, 4H), 7.48 (m, 2H), 8.26 (m, 2H); IR (KBr)

1

point apparatus and are uncorrected; H NMR spectra were

obtained in CDCl₃, Me₂SO-d₆, or TFA-d with Me₄Si as internal

standard or in D₂O or ND₄OD with DSS as internal standard

on a Bruker AM400 or Bruker AM360 spectrometer; IR spectra

were run as KBr pellets on a BioRad FTS-7 FTIR spectrom-

eter; mass spectra were determined on a Fisons Trio 2000

quadrupole mass spectrometer. Elemental analyses were

obtained from Atlanta Microlab, Inc. (Norcross, GA) and are

within 0.4% of the calculated values unless otherwise noted.

1-(Am in oim in om eth yl)-2-m eth ylben zim id a zole-6-ca r -

boxylic Acid (6). To a mixture of 0.97 g (0.005 mol) of

4-(acetylamino)-3-aminobenzoic acid (8)¹²in 40 mL of EtOH

were added 0.44 mL (0.005 mol) of concentrated HCl and 1.5

g (0.036 mol) of cyanamide. The reaction mixture was stirred

at ambient temperature for 48 h. Compound 5 precipitated

and was removed by filtration. The filtrate was concentrated,

4040 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 25

Chand et al.

3076, 2883, 1593, 1522, 1359, 1285 cm^-1; MS (ES^-) 286.5 (M

- 1). Anal. (C₉H₉N₃O₈) C, H, N.

was separated, washed successively with 10 mL of a saturated

NaHCO₃solution and 10 mL of brine, dried (Na₂SO₄), and

concentrated to give 0.68 g of oily residue. The crude was

purified by flash column chromatography on silica gel (40-

100% EtOAc in C₆H₁₄followed by 20% MeOH in EtOAc) to

give 0.17 g (30%) ofthe sulfone 56 and 0.35 g (66%) of 55 as a

white solid: mp 69-72 °C; ¹H NMR (400 MHz, CDCl₃) δ 2.48

(s, 3H), 3.93 (s, 3H), 4.02 (d, J ) 7.1 Hz, 2H), 7.37 (d, J ) 8.3

Hz, 2H), 8.06 (d, J ) 8.3 Hz, 2H); IR (KBr) 3417, 3002, 2950,

1719, 1610, 1435, 1280, 1101, 1032 cm^-1; MS (ES⁺) 213.3 (M

+ 1). Anal. (C₁₀H₁₂O₃S) C, H.

2-(4-Cya n op h en yl)-1,3-p r op a n ed iol (45). To 3.0 g (0.01

mol) of 42 in 25 mL of MeOH was added 0.3 g of 10% Pd/C

under N₂. The resulting slurry was hydrogenated for 30 min

at 50 psi, evacuated to remove ammonia, and then further

hydrogenated for an additional 30 min at 50 psi. The catalyst

was removed by filtration through Celite, and the solvent was

removed in vacuo to furnish 1.8 g of 2-(4-aminophenyl)-1,3-

propanediol (44).

To 1.8 g (0.01 mol) of 44 in 5 mL of ice-concentrated HCl

(2:3) cooled to 0 °C was added dropwise with stirring 0.7 g

(0.01 mol) of sodium nitrite in 5 mL of H₂O. The resulting

diazonium solution was neutralized carefully with a saturated

sodium carbonate solution to neutral pH; 10 mL of toluene

was added, and the reaction mixture was cooled to 0 °C. To

this cold solution was added slowly a solution containing 0.6

g (0.003 mol) of CuCl and 0.37 g (0.007 mol) of NaCN in 2 mL

of H₂O; the mixture stirred sequentially in the cold for 10 min,

at room temperature for 1 h, and at 50 °C for 1 h. The reaction

mixture was cooled, the organic layer was separated, and the

aqueous layer was extracted with four 20-mL portions of

EtOAc. The combined organic layers were washed with 20

mL of brine, dried (Na₂SO₄), and concentrated to furnish 1.6

g of crude product. The crude was purified by flash column

chromatography (EtOAc) to furnish 1.0 g (57%) of 45 which

was recrystallized from Et₂O to furnish 45 as a tan solid: mp

77-79 °C; ¹H NMR (400 MHz, CDCl₃) δ 2.29 (s, 2H), 3.13 (m,

1H), 3.98 (m, 4H), 7.39 (d, J ) 8.2 Hz, 2H), 7.62 (d, J ) 8.2

Hz, 2H); IR (KBr) 3474, 3291, 2239, 1609, 1507, 1472, 1418;

MS (ES^-) 176.1 (M - 1). Anal. (C₁₀H₁₁NO₂) C, H, N.

2-(4-Ca r boxyp h en yl)-1,3-p r op a n ed iol (46). To a solution

of 0.3 g (0.0017 mol) of 45 in 2.5 mL of EtOH was added 7.5

mL of 10% NaOH solution. The solution was heated at reflux

for 4 h and then cooled. The reaction mixture was acidified

with 1 N HCl to pH 2 and extracted thrice with 20 mL of

EtOAc. The combined organic layers were dried (Na₂SO₄) and

concentrated to give 0.2 g (60%) of brown residue. The residue

was recrystallized from EtOAc-Et₂O to obtain 0.08 g of 46 as

4-[(Meth ylsu lfoxy)m eth yl]ben zoic Acid (57). A mixture

of 1.2 g (0.0055 mol) of 55 and 17 mL (0.017 mol) of 1 N NaOH

was stirred at ambient temperature for 1 h. The reaction

mixture was filtered and the filtrate pH adjusted to 5 with

concentrated HCl. The precipitate was collected by filtration

and washed with H₂O and Et₂O to give 0.7 g (64%) of 57 as a

white, crystalline solid: mp 177-178 °C; ¹H NMR (400 MHz,

DMSO-d₆) δ 2.49 (s, 3H), 4.02 (d, J ) 12.65 Hz, 1H), 4.23 (d,

J ) 12.65 Hz, 1H), 7.45 (d, J ) 8.2 Hz, 2H), 7.94 (d, J ) 8.2

Hz, 2H), 12.99 (br s, 1H); IR (KBr) 2919, 2778, 2618, 2496,

1701, 1262, 1005 cm^-1; MS (ES⁺) 1992 (M + 1). Anal.

(C₉H₁₀O₃S‚0.25H₂O) C, H.

Meth yl 4-[(Meth ylsu lfon yl)m eth yl]ben zoa te (56). To

a solution of 1.8 g (0.009 mol) of 54 in 10 mL of glacial HOAc

was added 5.2 mL (0.046 mol) of 30% H₂O₂. The mixture was

heated at reflux for 1 h and cooled, and the resulting

precipitate was collected by filtration and dried to give 1.6 g

(89%) of 56 as a white solid. An analytical sample, mp 162-

164 °C, was recrystallized from EtOAc: ¹H NMR (400 MHz,

DMSO-d₆) δ 2.94 (s, 3H), 3.87 (s, 3H), 4.62 (s, 2H), 7.56 (d, J

) 8.4 Hz, 2H), 7.99 (d,J ) 8.4 Hz, 2H); IR (KBr) 3005, 2982,

1720, 1614, 1440, 1300, 1277, 1125, 1101 cm^-1; MS (ES⁺) 229.8

(M + 1). Anal. (C₁₀H₁₂O₄S) C, H.

4-[(Meth ylsu lfon yl)m eth yl]ben zoic Acid (58). A mix-

ture of 0.26 g (0.001 mol) of 56 and 4 mL (0.004 mol) of 1 N

NaOH was stirred at ambient temperature for 2 h. The

mixture was filtered, and the filtrate pH was adjusted to 4

using 1 N HCl. The precipitate was collected by filtration,

washed with H₂O, and recrystallized from EtOH to give 0.16

g (65%) of 58 as a white, crystalline solid: mp 249-253 °C;

¹H NMR (400 MHz, DMSO-d₆) δ 2.93 (s, 3H), 4.60 (s, 2H), 7.53

(d, J ) 8.2 Hz, 2H), 7.96 (d, J ) 8.2 Hz, 2H), 13.05 (br s, 1H);

1

a brown solid: mp 128-131 °C; H NMR (400 MHz, DMSO-

d₆) δ 2.88 (m, 1H), 3.68 (m, 4H), 4.59 (s, 2H), 7.40 (m, 2H),

7.81 (m, 2H), 12.79 (s, 1H); IR (KBr) 3339, 2875, 1690, 1320,

1296, 1037 cm^-1; MS (ES^-) 194.9 (M - 1). Anal. (C₁₀H₁₂O₄)

C, H.

IR (KBr) 3017, 2945, 2829, 2682, 2551, 1686, 1286, 1131 cm^-1

;

MS (ES^-) 212.9 (M - 1). Anal. (C₉H₁₀O₄S) C, H.

4-[(Acetyla m in o)m eth yl]ben zoic Acid (48). A mixture

of 1.51 g (0.01 mol) of 4-(aminomethyl)benzoic acid (47;

Aldrich) and 1.5 g (0.018 mol) of anhydrous NaOAc in 5 mL of

glacial HOAc was heated at reflux for 18 h. Upon cooling, the

mixture was poured into 50 mL of cold H₂O. The precipitate

was separated by filtration and washed several times with cold

H₂O. The cake was recrystallized from H₂O to give 1.4 g (73%)

of 48 as a white powder: mp 195 °C; ¹H NMR (400 MHz,

DMSO-d₆) δ 1.89 (s, 3H), 4.31 (d, J ) 5.9 Hz, 2H), 7.35 (d, J

) 8.3 Hz, 2H), 7.89 (d, J ) 8.3 Hz, 2H), 8.42 (t, J ) 5.9 Hz,

1H), 12.87 (br s, 1H); IR (KBr) 3393, 1690, 1606, 1543, 1264,

1178 cm^-1; MS (ES⁺) 194 (M + 1). Anal. (C₁₀H₁₁NO₃) C, H,

N.

Meth yl 4-[(Meth ylth io)m eth yl]ben zoa te (54). To 1.8 g

(0.025 mol) of NaSCH₃in 25 mL of DMF cooled to 0 °C was

added 5.5 g (0.024 mol) of methyl 4-(bromomethyl)benzoate

(53; Aldrich), and the mixture stirred under N₂at ambient

temperature for 16 h. The reaction mixture was poured into

150 mL of H₂O and extracted thrice with 50-mL portions of

EtOAc. The combined organic layers were washed with 50

mL of brine, dried (MgSO₄), and concentrated to give 5.3 g of

colorless liquid. The oil was vacuum-distilled to furnish 3.9 g

(85%) of 54 as a colorless liquid, bp 122-124 °C/0.6 mmHg;

¹H NMR (600 MHz, CDCl₃) δ 2.0 (s, 3H), 3.7 (s, 2H), 3.9 (s,

3H), 7.4 (d, J ) 8.2 Hz, 2H), 8.0 (d, J ) 8.2 Hz, 2H); IR (KBr)

2951, 2916, 1720, 1610, 1435, 1280; MS (ES⁺) 197 (M + 1).

Anal. (C₁₀H₁₂O₂S) C, H.

3-(Cya n oa m in o)ben zoic Acid (60). To 9.1 g (0.066 mol)

of 3-aminobenzoic acid (59; Aldrich) in 70 mL of HOAc-H₂O

(1:1) was added 8.1 g (0.099 mol) of NaOAc, and the mixture

was cooled in an ice-water bath. A total of 8.4 g (0.079 mol)

of cyanogen bromide was added in two batches over a 10-min

period. The mixture was stirred at ambient temperature for

18 h and then poured into 350 g of an ice-water mixture with

vigorous stirring. The precipitate was collected by filtration,

washed with 70 mL of cold H₂O, air-dried, and recrystallized

from MeOH-H₂O to give 10.4 g (95%) of 60 as a white solid:

1

mp 238-240 °C; H NMR (400 MHz, DMSO-d₆) δ 7.19-7.22

(m, 1H), 7.46-7.53 (m, 2H), 7.59-7.62 (m, 1H); IR (KBr) 3177,

2228, 1683, 1595, 1448, 1316 cm^-1; MS (ES^-) 161 (M - 1).

Anal. (C₈H₆N₂O₂‚0.2H₂O) C, H, N.

Meth yl 3-[[Am in o(cyan oim in o)m eth yl]am in o]ben zoate

(66). A solution of 0.8 g (0.0086 mol) of sodium dicyanamide

in 5 mL of H₂O was treated dropwise with a solution of 1.3 g

(0.0086 mol) of methyl 3-aminobenzoate (65; Aldrich) in 15 mL

of 1 N HCl. The resulting clear solution was heated at 80-90

°C for 2 h, and a precipitate formed upon cooling. The solid

was collected by filtration and recrystallized from CHCl₃-

MeOH to give 1.8 g (quantitative) of 66 as a white solid: mp

203-204 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 3.17 (d, J ) 5.1

Hz, 0.6H), 3.34 (s, 3H), 4.10 (q, J ) 5.1 Hz, 0.2H), 7.14 (s, 2H),

7.43-7.47 (m, 1H), 7.60-7.66 (m, 2H), 8.00-8.01 (m, 1H), 9.32

(s, 1H); IR (KBr) 3420, 3329, 2179, 1720, 1562 cm^-1; MS (ES⁺)

219.0 (M + 1). Anal. (C₁₀H₁₀N₄O₂‚0.2MeOH) C, H, N.

3-[[Am in o(cyan oim in o)m eth yl]am in o]ben zoic Acid (67).

A mixture of 7.3 g (0.033 mol) of 66 in 20 mL of H₂O and 34

mL of 1 N NaOH was heated at reflux for 2 h. The cooled

mixture was filtered, and the filtrate pH was adjusted to 4.0-

Meth yl 4-[(Meth ylsu lfoxy)m eth yl]ben zoa te (55). To 1.0

g (0.003 mol) of m-chloroperbenzoic acid (50% in 10 mL of CH₂-

Cl₂) at 0 °C was added 0.5 g (0.0025 mol) of 54. The mixture

was stirred at ambient temperature overnight and then poured

into 10 mL of a saturated Na₂CO₃solution. The organic layer

Benzoic Acids as Influenza Inhibitors

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 25 4041

4.5 with 6 N HCl. The precipitate was collected by filtration,

washed with 30 mL of H₂O, and dried to give 1.3 g (18%) of

67 as a white solid: mp >300 °C. Further acidification of the

filtrate gave an additional 4 g of slightly impure 67: ¹H NMR

(400 MHz, DMSO-d₆) δ 7.12 (s, 2H), 7.42 (m, 1H), 7.6 (m, 2H),

7.98 (m, 1H), 9.31 (s, 1H), 12.95 (br s, 1H); IR (KBr) 3434,

3361, 2196, 1702, 1658, 1565 cm^-1; MS (ES^-) 203 (M - 1).

Anal. (C₉H₈N₄O₂‚0.25H₂O) C, H, N.

MHz, DMSO-d₆) δ 3.42 (s, 2H), 6.92 (s, 1H), 7.33-7.94 (m, 8H);

IR (KBr) 3354, 3161, 1670, 1636, 1402 cm^-1; MS (ES⁺) 179 (M

+ 1). Anal. (C₉H₁₀N₂O₂‚0.2H₂O) C, H, N.

Meth yl 3-(2-Am in oeth yl)ben zoa te (78). To a suspension

of 0.095 g (0.0025 mol) of NaBH₄in 2.5 mL of THF was added

dropwise over a 10-min period at ambient temperature a

solution of 0.19 mL (0.0025 mol) of TFA in 2.5 mL of THF. A

solution of 0.18 g (0.001 mol) of 74 in 2.5 mL of THF was then

added, and the mixture was stirred at ambient temperature

for 4 h. The mixture was cooled, and H₂O was added dropwise

while keeping the temperature below 10 °C. The mixture was

concentrated, and 5 mL of H₂O was added to the residue. The

mixture was made basic with 1 N NaOH and extracted thrice

with 10-mL portions of CH₂Cl₂. The combined extracts were

dried (MgSO₄) and concentrated to give an oil as a residue.

The oil was converted to the hydrochloride and the resulting

solid recrystallized from EtOH to furnish 0.12 g (52%) of 78

as a white solid: mp 120-122 °C; ¹H NMR (400 MHz, DMSO-

d₆) δ 3.00-3.05 (m, 4H), 3.85 (s, 3H), 7.47-7.51 (m, 1H), 7.57

(d, J ) 7.7 Hz, 1H), 7.83-7.85 (m, 2H), 8.23 (br s, 2H); IR

(KBr) 3423, 3003, 1724, 1591, 1285 cm^-1; MS (ES⁺) 180.4 (M

+ 1). Anal. (C₁₀H₁₃NO₂‚HCl‚0.75H₂O) C, H, N.

3-[(Acetyla m in o)m eth yl]ben zoic Acid (71). To a stirred

mixture of 0.23 g (0.0012 mol) of 70,³³0.037 g (0.003 mol) of

DMAP, and 0.86 mL (0.006 mol) of Et₃N in 12 mL of CH₂Cl₂

was added dropwise 0.23 mL (0.0023 mol) of acetic anhydride.

The mixture was stirred at ambient temperature for 4 h and

then concentrated. The white residue was treated with 2 mL

of H₂O, and the pH was adjusted to 8 with 1 N NaOH. The

mixture was extracted thrice with 2 mL of EtOAc, and the

combined extracts were dried (MgSO₄) and concentrated. The

solid residue was recrystallized from EtOAc to yield 0.14 g

1

(59%) of 71 as a white solid: mp 165-166 °C; H NMR (400

MHz, DMSO-d₆) δ 1.88 (s, 3H), 4.29 (d, J ) 5.9 Hz, 2H), 7.45

(m, 2H), 7.82 (m, 2H), 8.42 (s, 1H), 12.95 (br s, 1H); IR (KBr)

3306, 2835, 1691, 1642, 1352, 1292 cm^-1; MS (ES^-) 192.2 (M

- 1). Anal. (C₁₀H₁₁NO₃) C, H, N.

Meth yl 3-[2-(Acetyla m in o)eth yl]ben zoa te (79). To a

mixture of 2.2 g (0.01 mol) of 78, 7 mL (0.05 mol) of Et₃N, and

0.25 g (0.002 mol) of DMAP in 25 mL of CH₂Cl₂was added

dropwise with stirring at 0 °C 1.9 mL (0.02 mol) of acetic

anhydride. The mixture was stirred at ambient temperature

overnight and then poured into 20 mL of H₂O. The organic

layer was separated, and the aqueous layer was extracted

twice with 10-mL portions of CH₂Cl₂. The combined organic

layers were dried (MgSO₄) and concentrated. The residue was

purified by flash column chromatography on silica gel (75-

80% EtOAc in C₆H₁₄) to yield 0.96 g (44%) of 79 as a colorless

oil: ¹H NMR (400 MHz, CDCl₃) δ 1.95 (s, 3H), 2.88 (t, J ) 7.0

Hz, 2H), 3.53 (m, 2H), 3.92 (s, 3H), 5.62 (br s, 1H), 7.39 (m,

2H), 7.91 (m, 2H); IR (KBr) 3297, 3082, 2951, 1722, 1655, 1554,

3-[[(Am in oim in om eth yl)a m in o]m eth yl]ben zoic Acid

(72). A mixture of 0.1 g (0.0064 mol) of 70 in 7 mL of a

saturated NaHCO₃solution was treated dropwise with 6 N

NaOH until all solids dissolved (2 drops). To this solution was

added 0.08 g (0.0064 mol) of aminoiminomethanesulfonic

acid;³⁴the mixture was stirred at ambient temperature for 16

h and concentrated. The residue was recrystallized from H₂O

to give 0.072 g (58%) of 72 as a white solid: mp 308-311 °C;

¹H NMR (400 MHz, CF₃CO₂D) δ 4.53 (s, 2H), 7.55-7.61 (m,

2H), 8.09 (s, 1H), 8.14 (d, J ) 7.7 Hz, 1H); IR (KBr) 3399, 3248,

3079, 2363, 2337, 1670, 1628, 1550, 1396 cm^-1; MS (ES⁺) 194.5

(M + 1). Anal. (C₉H₁₁N₃O₂) C, H, N.

Meth yl 3-(Cya n om eth yl)ben zoa te (74). A mixture of 5.0

g (0.022 mol) of methyl 3-(bromomethyl)benzoate (73; May-

bridge), 2.9 g (0.044 mol) of KCN, and 0.5 g (0.0018 mol) of

18-crown-6 in 50 mL of CH₃CN was vigorously stirred for 24

h at ambient temperature. The mixture was filtered, the

filtrate was concentrated to ∼30 mL, and the volume was

adjusted to 100 mL with H₂O. The yellow mixture was

extracted with portions of CH₂Cl₂until the aqueous layer was

almost colorless. The combined organic layers were dried

(MgSO₄) and concentrated to give an oil. This oil was purified

by column chromatography on silica gel eluted with Et₂O. The

appropriate fractions were combined and concentrated. The

residue was vacuum-distilled to give 3.5 g (92%) of 74 as a

light yellow oil: bp 101.5 °C/0.05 mmHg; ¹H NMR (400 MHz,

DMSO-d₆) δ 3.82 (s, 2H), 3.89 (s, 3H), 7.42 (m, 1H), 7.51 (m,

1H), 7.96 (m, 2H); IR (KBr) 2955, 2249, 1718, 1436, 1286, 1200

cm^-1; MS (no ionization). Anal. (C₁₀H₉NO₂) C, H, N

1286, 1203 cm^-1; MS (ES⁺) 222.4 (M + 1). Anal. (C₁₂H₁₅

NO₃‚0.25H₂O) C, H, N.

-

3-[2-(Acetyla m in o)eth yl]ben zoic Acid (80). A mixture

of 0.35 g (0.0016 mol) of 79 and 3.2 mL (0.0032 mol) of 1 N

NaOH was stirred at ambient temperature for 2 h. The

mixture was filtered, and the filtrate pH was adjusted to 6

using concentrated HCl. The mixture was concentrated, 2 mL

of H₂O was added, and the precipitate was collected by

filtration to yield 0.1 g (31%) of 80 as a white solid: mp 146-

1

148 °C; H NMR (400 MHz, DMSO-d₆) δ 1.77 (s, 3H), 2.76 (t,

J ) 7.2 Hz, 2H), 3.26 (m, 2H), 7.42 (m, 2H), 7.79 (s, 2H), 7.91

(s, 1H), 12.89 (s, 1H); IR (KBr) 3333, 3130, 1688, 1628, 1591,

1567, 1313, 1267, 1199 cm^-1; MS (ES^-) 206.2 (M - 1). Anal.

(C₁₃H₁₃NO₃) C, H, N.

3-[2-[(Am in oim in om et h yl)a m in o]et h yl]ben zoic Acid

(81). A solution of 0.23 g (0.0011 mol) of 77 and 0.32 g (0.0023

mol) of Na₂CO₃in 2.2 mL of H₂O was treated portionwise with

0.15 g (0.0011 mol) of aminoiminomethanesulfonic acid³⁴over

a 10-min period. The reaction mixture was stirred at ambient

temperature for 1 h, and the solid which precipitated was

collected by filtration and dried in vacuo at toluene reflux

temperature to yield 0.15 g (64%) of 81 as a white solid: mp

Meth yl 3-(2-Am in o-2-im in oeth yl)ben zoa te (75). A solu-

tion of 1.9 g (0.011 mol) of 74 in 100 mL of dry CH₂Cl₂and 15

mL of absolute EtOH was cooled to 0 °C and treated with a

stream of HCl gas for 10 min. The mixture was refrigerated

for 6 days and then concentrated in vacuo with the bath

temperature at 0 °C. The white solid residue was dissolved

in 200 mL of anhydrous MeOH, and the solution was treated

with a stream of NH₃gas for 20 min. The mixture was then

warmed at 50 °C for 18 h. The mixture was cooled and filtered,

and the filtrate was concentrated to give 2.5 g (quantitative)

of white foam as a residue. The foam was pulverized and dried

in vacuo at toluene reflux temperature to give 75 as a white

1

>310 °C; H NMR (400 MHz, CF₃CO₂D) δ 3.1 (t, 2H), 3.7 (t,

2H), 7.6 (m, 2H), 8.1 (s, 1H), 8.2 (d, 1H); IR (KBr) 3354, 3149,

1658, 1519, 1386 cm^-1; MS (ES⁺) 208.4 (M + 1). Anal.

(C₁₀H₁₃N₃O₂) C, H, N.

5-[(3-Ca r boxyp h en yl)a m in o]-1H-tetr a zole (82). A mix-

ture of 5.0 g (0.031 mol) of 60, 2.4 g (0.037 mol) of NaN₃, and

2.0 g (0.037 mol) of NH₄Cl in 20 mL of dry DMF was heated

at 160 °C for 16 h. The mixture was concentrated, and the

residue was diluted with 150 mL of H₂O. The mixture was

made basic with dilute NaOH and filtered, and the filtrate

was acidified with 6 N HCl. The precipitate was collected by

filtration, air-dried, and recrystallized from MeOH-EtOAc to

give 2.3 g (36%) of 82 as a cream-colored solid: mp 243-245

1

solid: mp 139-140 °C; H NMR (400 MHz, DMSO-d₆) δ 3.82

(s, 2H), 3.87 (s, 3H), 7.55 (m, 1H), 7.78 (m, 1H), 7.91 (m, 1H),

8.08 (m, 1H), 8.89-9.34 (m, 4H); IR (KBr) 3362, 3030, 1718,

1688, 1313, 1285 cm^-1; MS (ES⁺) 193 (M + 1). Anal.

(C₁₀H₁₂N₂O₂‚HCl) C, H, N.

3-(2-Am in o-2-im in oeth yl)ben zoic Acid (76). A mixture

of 0.2 g (0.0009 mol) of 75 and 5 mL of NH₄OH was stirred at

ambient temperature for 24 h. The mixture was concentrated,

the residue diluted with 5 mL of H₂O, and the resulting solid

collected by filtration. The cake was washed with H₂O and

dried in vacuo at acetone reflux temperature to give 0.14 g

(88%) of 76 as a white powder: mp 219-221 °C; ¹H NMR (400

1

°C; H NMR (400 MHz, DMSO-d₆) δ 7.44 (dd, J ) 7.86, 8.0

Hz, 1H), 7.54 (ddd, J ) 7.65, 1.17, 1.15 Hz, 1H), 7.77 (ddd, J

) 8.16, 2.12, 1.12 Hz, 1H), 8.21 (m, 1H), 10.05 (s, 1H); IR (KBr)

3277, 3080, 1688, 1629, 1600, 1047, 1463, 1311, 1242, 1056

cm^-1; MS (ES⁺) 206.3 (M + 1). Anal. (C₈H₇N₅O₂) C, H, N.

4042 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 25

Chand et al.

5-[4-(Acetyla m in o)p h en yl]-1H-tetr a zole (90). A stirred

mixture of 10 g (0.062 mol) of 4′-cyanoacetanilide (89; Aldrich),

4.6 g (0.07 mol) of NaN₃, and 3.75 g (0.07 mol) of NH₄Cl in

100 mL of DMF was heated at 120 °C for 20 h and then poured

into 800 mL of ice-water with vigorous stirring. The resulting

precipitate was collected by filtration and slurried with 200

mL of H₂O with vigorous stirring. The solid was collected by

filtration and dried to yield 3.4 g (27%) of 90 as a white solid:

mp 287 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 2.07 (s, 3H), 7.78

(d, J ) 8.8 Hz, 2H), 7.95 (d, J ) 8.8 Hz, 2H), 10.24 (s, 1H); IR

(KBr) 3312, 3267, 1678, 1603, 1549, 1502, 1321 cm^-1; MS (ES^-)

202 (M - 1). Anal. (C₉H₉N₅O) C, H, N.

Meth yl 4-(Acetyla m in o)-3-(h yd r oxym eth yl)ben zoa te

(93). To a mixture of 3.3 g (0.015 mol) of methyl 4-(acetyl-

amino)-3-formylbenzoate (92)⁴⁵in 30 mL of MeOH was added

0.3 g (0.0078 mol) of NaBH₄at 0 °C in three portions at 10-

min intervals. The mixture was stirred at 0 °C for 0.75 h after

the final addition was complete. The mixture was neutralized

with H⁺resin and filtered, and the filtrate was concentrated.

The residue was recrystallized from EtOAc-hexane to give

2.6 g (78%) of 93 as yellow needles: mp 131-132 °C; ¹H NMR

(400 MHz, DMSO-d₆) δ 2.10 (s, 3H), 3.84 (s, 3H), 4.56 (d, J )

5.5 Hz, 2H), 5.49 (t, J ) 5.5 Hz, 1H), 7.82 (d, J ) 1.2 Hz, 2H),

8.04 (s, 1H), 9.44 (s, 1H); IR (KBr) 3197, 1721, 1667, 1590,

1540, 1436, 1287, 1123 cm^-1; MS (ES⁺) 206 (M - H₂O). Anal.

(C₁₁H₁₃NO₄) C, H, N.

filtered through Celite, and the filtrate was concentrated. The

residue was recrystallized from EtOAc-hexane to give 5.2 g

1

(93%) of 98 as a white solid: mp 119 °C; H NMR (400 MHz,

DMSO-d₆) δ 1.17 (t, J ) 7.0 Hz, 3H), 2.06 (s, 3H), 3.83 (s, 2H),

3.84 (s, 3H), 4.07 (q, J ) 7.0 Hz, 2H), 7.67 (d, J ) 8.4 Hz, 1H),

7.85 (dd, J ) 8.4, 2.0 Hz, 1H), 7.87 (d, J ) 2.0 Hz, 1H), 9.56

(s, 1H); IR (KBr) 3350, 2993, 1712, 1691, 1589, 1521, 1279,

1213 cm^-1; MS (ES⁺) 280.6 (M + 1). Anal. (C₁₄H₁₇NO₅) C, H,

N.

r-[2-(Acet yla m in o)-5-(m et h oxyca r b on yl)p h en yl]a ce-

tic Acid (99). To a mixture of 4.18 g (0.015 mol) of 98 in 60

mL of MeOH was added 15 mL of 1 N NaOH over a 10-min

period. The mixture was stirred at ambient temperature for

1 h and diluted with 50 mL of H₂O. The mixture was filtered,

and the filtrate was neutralized with concentrated HCl. The

precipitate was collected by filtration and washed with H₂O,

and the cake was recrystallized from EtOAc to give 2.3 g (61%)

of 99 as a white solid: mp 194 °C; ¹H NMR (400 MHz, DMSO-

d₆) δ 2.07 (s, 3H), 3.74 (s, 2H), 3.84 (s, 3H), 7.72 (d, J ) 8.3

Hz, 1H), 7.82 (dd, J ) 8.4, 2.0 Hz, 1H), 7.84 (d, J ) 2 Hz, 1H),

9.55 (s, 1H), 12.40 (s, 1H); IR (KBr) 3476, 3327, 3008, 2958,

1723, 1709, 1693, 1676, 1590, 1531, 1439, 1289, 1205 cm^-1

;

MS (ES⁺) 252.5 (M + 1). Anal. (C₁₂H₁₃NO₅) C, H, N.

Meth yl 4-(Acetyla m in o)-3-(2-h yd r oxyeth yl)ben zoa te

(100). To a mixture of 1 g (0.004 mol) of 99, 0.56 mL (0.004

mol) of Et₃N, and 10 mL of THF at 0 °C was added 0.38 mL of

ethyl chloroformate over a 5-min period. The mixture was

stirred at 0 °C for 20 min and then filtered. The filtrate

containing the mixed anhydride was treated with 0.48 g (0.013

mol) of NaBH₄followed by the dropwise addition of 2.6 mL of

MeOH over a 1-h period at 10 °C. After addition was complete,

the mixture was stirred for 30 min and then carefully

quenched with 1 N HCl (pH ∼7). The mixture was treated

with 25 mL of H₂O and 25 mL of Et₂O. The aqueous layer

was extracted thrice with 10 mL of CH₂Cl₂. The organic layers

were combined, dried (Na₂SO₄), and concentrated. The residue

was purified by flash column chromatography in silica gel (75-

100% EtOAc in hexane) to give 0.55 g (58%) of 100 as a white

solid: mp 134-136 °C. The ¹H NMR indicated that this

sample was contaminated with ∼15% of the ethyl ester: ¹H

NMR (400 MHz, CDCl₃) δ 1.38 (t, J ) 7.1 Hz, 3 × 0.15H),

2.16 (s, 3H), 2.52 (s, 1H), 2.89 (t, J ) 5.3 Hz, 2H), 3.89 (s, 3 ×

0.85H), 4.00 (m, 2H), 4.36 (q, J ) 14.3, 7.1 Hz, 2 × 0.15H),

7.85 (d, J ) 1.8 Hz, 1H), 7.90 (dd, J ) 8.5, 2.0 Hz, 1H), 8.04

(d, J ) 8.5 Hz, 1H), 9.33 (br s, 1H); IR (KBr) 3251, 1726, 1678,

1542, 1286 cm^-1; MS (ES^-) 236.1 (M - 1). Anal. (C₁₂H₁₅NO₅)

C, H, N.

4-(Acetyla m in o)-3-(h yd r oxym eth yl)ben zoic Acid (94).

A mixture of 0.84 g (0.0038 mol) of 93 in 4.5 mL (0.0045 mol)

of 1 N NaOH was stirred at ambient temperature for 8 h. The

mixture was filtered, the filtrate was made acidic with

concentrated HCl, the resulting precipitate was collected by

filtration, and the cake was recrystallized from H₂O to give

0.48 (61%) of 94 as a white powder: mp 180-181 °C; ¹H NMR

(400 MHz, DMSO-d₆) δ 2.09 (s, 3H), 4.55 (s, 2H), 5.46 (br s,

1H), 7.76-7.81 (m, 2H), 8.00 (s, 1H), 9.42 (s, 1H), 12.75 (br s,

1H); IR (KBr) 3261, 1696, 1662, 1527, 1286, 1241, 1015 cm^-1

;

MS (ES^-) 208 (M - 1). Anal. (C₁₀H₁₁NO₄) C, H, N.

Eth yl r-[2-(Acetylam in o)-5-(m eth oxycar bon yl)ph en yl]-

r-(m eth ylth io)a ceta te (97). To a mixture of 15.1 g (0.1 mol)

of methyl 4-aminobenzoate (95; Aldrich) in 400 mL of CH₂Cl₂

at -70 °C was added a solution of 12.0 g of tert-butyl

hypochlorite in 50 mL of CH₂Cl₂dropwise over a 10-min

period. The mixture was stirred for 10 min and then treated

dropwise with a solution of 13.4 g (0.1 mol) of ethyl (meth-

ylthio)acetate in 50 mL of CH₂Cl₂over a 10-min period. The

stirring was continued for 1 h, and then the solution was

treated dropwise with a solution of 11.2 g (0.11 mol) of

triethylamine in 40 mL of CH₂Cl₂over a 10-min period. The

reaction mixture was allowed to warm to ambient temperature

and then diluted with 120 mL of H₂O. The organic layer was

separated, dried (Na₂SO₄), and concentrated to give crude 96

as an oil.

A mixture of the above 96, 40 mL of triethylamine, and 200

mL of Et₂O was cooled to 0-5 °C in an ice bath. A solution of

8.6 g (0.11 mol) of acetyl chloride in 50 mL of Et₂O was added

dropwise over a 15-min period. The reaction mixture was

stirred for 1 h and then diluted with 200 mL of H₂O. The

organic layer was separated, washed with H₂O, dried (Na₂-

SO₄), and concentrated to give a syrup as a residue. This syrup

was purified by column chromatography on silica gel (EtOAc-

hexane, 1:2). The appropriate fractions were combined and

concentrated to give a solid residue. The solid was recrystal-

lized from EtOAc-hexane to give 9.5 g (29%) of 97 as a white

solid: mp 95-96 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 1.17 (t,

J ) 7.0 Hz, 3H), 2.08 (s, 3H), 2.10 (s, 3H), 3.85 (s, 3H), 4.12-

4.17 (m, 2H), 5.10 (s, 1H), 7.67 (d, J ) 8.5 Hz, 1H), 7.88 (dd,

J ) 8.5, 2.0 Hz, 1H), 8.14 (d, J ) 2.0 Hz, 1H), 9.66 (s, 1H); IR

(KBr) 3224, 1717, 1645, 1534, 1299 cm^-1; MS (ES⁺) 326.4 (M

+ 1). Anal. (C₁₅H₁₉NO₅S) C, H, N.

4-(Acetyla m in o)-3-(2-h yd r oxyeth yl)ben zoic Acid (101).

A mixture of 0.71 g (0.003 mol) of 100 and 6 mL of 1 N NaOH

was stirred at ambient temperature for 1 h. The mixture was

filtered and the filtrate pH adjusted to 2-3 with 1 N HCl. The

mixture was allowed to stand for 1 h, and the precipitate was

collected by filtration, washed with H₂O and then Et₂O, and

dried to give 0.58 g (87%) of 101 as a light brown solid: mp

206-208 °C dec; ¹H NMR (400 MHz, DMSO-d₆) δ 2.09 (s, 3H),

2.82 (t, J ) 6.3 Hz, 2H), 3.64 (t, J ) 6.3 Hz, 2H), 5.10 (br s,

1H), 7.75 (s, 2H), 7.82 (s, 1H), 9.63 (s, 1H), 12.75 (s, 1H); IR

(KBr) 3346, 1683, 1590, 1532, 1431 cm^-1; MS (ES^-) 222 (M -

1). Anal. (C₁₁H₁₃NO₄) C, H, N.

Meth yl 3-(2-Hyd r oxyeth oxy)-4-n itr oben zoa te (103). A

mixture of 5.3 g (0.027 mol) of methyl 3-hydroxy-4-nitroben-

zoate (102; Lancaster), 9.15 g (0.065 mol) of K₂CO₃, 0.8 g

(0.0053 mol) of NaI, and 7.3 g (0.059 mol) of 2-bromoethanol

in 50 mL of acetone was heated at reflux for 48 h. A TLC

analysis (silica gel, 3:2 hexane-EtOAc) indicated that the

reaction was not complete. An additional 11 g (0.089 mol) of

2-bromoethanol and 100 mL of acetone were added, and the

mixture was heated at reflux for 60 h. The mixture was

poured into 400 mL of H₂O and extracted thrice with 200-mL

portions of EtOAc. The combined organic layers were con-

centrated, and the residue was recrystallized from EtOAc-

hexane to give 3.3 g (50%) of 103 as an orange solid: mp 97-

98 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 3.70-3.74 (m, 2H),

3.90 (s, 3H), 4.26 (t, J ) 4.8 Hz, 2H), 4.94 (t, J ) 5.8 Hz, 1H),

7.66 (dd, J ) 8.4, 1.5 Hz, 1H), 7.81 (d, J ) 1.5 Hz, 1H), 8.3 (d,

Eth yl r-[2-(Acetyla m in o)-5-(m eth oxyca r bon yl)p h en yl]-

a ceta te (98). A slurry of Raney nickel in THF was prepared

by washing a 20-g sample of a commercial preparation with

several portions of H₂O and then washing with three portions

of THF. A stirred mixture of 6.5 g (0.02 mol) of 97 in 150 mL

of THF was treated portionwise with the washed Raney nickel

slurry over a 30-min period. The mixture was cautiously

Benzoic Acids as Influenza Inhibitors

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 25 4043

J ) 8.4 Hz, 1H); IR (KBr) 3556, 3452, 1723, 1612, 1527, 1295

cm^-1; MS (did not ionize). Anal. (C₁₀H₁₁NO₆) C, H, N.

4-(Acetylam in o)-3-(2-h ydr oxyeth exy)ben zoic Acid (106).

A mixture of 3.3 g (0.0136 mol) of 103 and 0.05 g of PtO₂in 50

mL of EtOH was hydrogenated for 40 min at 35 psi. The

mixture was filtered and concentrated to give 2.9 g (100%) of

methyl 4-amino-3-(2-hydroxyethoxy)benzoate (104) as an off-

white solid.

To 3.0 g (0.014 mol) of 104 dissolved in 200 mL of CH₂Cl₂

were added 1.5 mL (0.016 mol) of acetic anhydride and 1.34

mL (0.0156 mol) of pyridine. After stirring for 3 h at room

temperature, the reaction mixure was poured into 70 mL of

H₂O and the organic layer washed thrice with 50 mL of H₂O.

The organic layer was concentrated, treated with 50 mL of

MeOH and a catalytic amount of NaOCH₃, and stirred for 1

h. The solution was neutralized with H⁺resin, filtered, and

then concentrated. The residue was recrystallized from EtOAc

to give 1.9 g (53%) of 105 as an off-white solid.

2H), 3.91-3.98 (m, 1H), 4.21 (t, J ) 4.7 Hz, 1H), 4.31 (q, J )

7.1 Hz, 2H), 4.85 (d, J ) 3.8 Hz, 1H), 7.83-7.88 (m, 2H), 8.15

(m, 1H), 9.81 (br s, 1H); IR 3508, 3260, 1684, 1591, 1541, 1290

cm^-1; MS (ES⁺) 282 (M + 1). Anal. (C₁₄H₁₉NO₅) C, H, N.

4-(Acetyla m in o)-3-(2,3-d ih yd r oxyp r op yl)ben zoic Acid ,

Isom er A (111). A mixture of 0.12 g (0.0004 mol) of 109 and

2 mL of 1 N NaOH was stirred at ambient temperature for 15

h. The mixture was filtered and the filtrate made acidic with

concentrated HCl. The mixture was concentrated; the residue

was dissolved in 5 mL of MeOH and filtered. The filtrate was

concentrated, and the residue was purified by column chro-

matography on silica gel (85:15:1 EtOAc-MeOH-HOAc) to

give 0.06 g (54%) of 111 as an off-white powder: mp 181-183

1

°C; H NMR (400 MHz, CD₃OD) δ 2.17 (s, 3H), 2.82 (dd, J )

7.1, 7.7 Hz, 1H), 2.92 (dd, J ) 7.0, 4.0 Hz, 1H), 3.48 (m, 2H),

3.84 (m, 1H), 7.82-7.88 (m, 2H), 7.93 (d, J ) 1.5 Hz, 1H); IR

(KBr) 3369, 3289, 1699, 1514, 1286 cm^-1; MS (ES⁺) 254 (M +

1). Anal. (C₁₂H₁₅NO₅‚0.2H₂O) C, H, N.

4-(Acetyla m in o)-3-(2,3-d ih yd r oxyp r op yl)ben zoic Acid ,

Isom er B (112). Isomer B was prepared by the above

procedure using 0.09 g (0.0003 mol) of 110 and 2 mL of 1 N

NaOH. This method gave 0.055 g (67%) of 112 as an off-white

powder: mp 180-182 °C; ¹H NMR (400 MHz, CD₃OD) δ 2.17

(s, 3H), 2.82 (dd, J ) 7.1, 7.7 Hz, 1H), 2.92 (dd, J ) 9.3, 4.0

Hz, 1H), 3.48 (m, 2H), 3.84 (m, 1H), 7.82-7.88 (m, 2H), 7.93

(d, J ) 1.3 Hz, 1H); IR (KBr) 3389, 3256, 1699, 1651, 1518,

1284 cm^-1; MS (ES^-) 252 (M - 1). Anal. (C₁₂H₁₅NO₅‚0.75H₂O)

C, H, N.

Eth yl 4-(Acetyla m in o)-3-(3-oxo-1-p r op en yl)ben zoa te

(113). Compound 113 was synthesized according to the

procedure used to prepare 108. A mixture of 8.3 g (0.04 mol)

of 107, 4.5 g (0.02 mol) of Pd(OAc)₂, 17.5 g (0.135 mol) of

acrolein diethyl acetal, and 11.2 g (0.11 mol) of Et₃N gave 1.1

g (21%) of 113 as an off-white, flocculent solid: mp 171 °C; ¹H

NMR (400 MHz, DMSO-d₆) δ 1.34 (t, J ) 7.0 Hz, 3H), 2.16 (s,

3H), 4.34 (q, J ) 7.0 Hz, 2H), 6.82 (dd, J ) 7.7, 15.7 Hz, 1H),

7.80-7.87 (m, 1H), 7.91 (d, J ) 1 5.7 Hz, 1H), 7.98 (dd, J )

8.3, 2.0 Hz, 1H), 8.28 (d, J ) 2.0 Hz, 1H), 9.73 (d, J ) 7.7 Hz,

1H), 10.05 (s, 1H); IR (KBr) 3293, 1718, 1672, 1522, 1285, 1234

cm^-1; MS (ES^-) 260 (M - 1). Anal. (C₁₄H₁₅NO₄‚0.2H₂O) C,

H, N.

Eth yl 4-(Acetyla m in o)-3-(3-h yd r oxy-1-p r op en yl)ben -

zoa te (114). A mixture of 0.52 g (0.002 mol) of 113 in 20 mL

of MeOH at 0 °C was treated with a mixture of 0.09 g (0.0024

mol) of NaBH₄in 2 mL of MeOH and then stirred for 15 min.

The mixture was neutralized with H⁺resin (Dowex-50W) and

filtered. The filtrate was concentrated, and the residue was

purified by column chromatography on silica gel (19:1 CHCl₃-

MeOH) to give 0.41 g (76%) of 114 as a pale yellow powder:

mp 118-119 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 1.32 (t, J )

7.0 Hz, 3H), 2.10 (s, 3H), 4.17 (ddd, J ) 5.3, 5.1, 1.8 Hz, 2H),

4.31 (q, J ) 7.0 Hz, 2H), 4.93 (t, J ) 5.3 Hz, 1H), 6.34 (dt, J

) 15.8, 5.1 Hz, 1H), 6.81 (d, J ) 15.8 Hz, 1H), 7.70 (d, J ) 8.4

Hz, 1H), 7.78 (dd, J ) 8.4, 2.0 Hz, 1H), 8.06 (d, J ) 2.0 Hz,

1H), 9.65 (s, 1H); IR (KBr) 3273, 1720, 1662, 1527, 1287, 1257

cm^-1; MS (ES^-) 262 (M - 1). Anal. (C₁₄H₁₇NO₄‚0.5H₂O) C,

H, N.

A mixture of 1.5 g (0.006 mol) of 105 and 12 mL (0.012 mol)

of 1 N NaOH was stirred at ambient temperature for 1 h. The

mixture was filtered and the filtrate neutralized with concen-

trated HCl. The resulting precipitate was collected by filtra-

tion, washed with H₂O, and dried to give 0.47 g (34%) of 106

as a tan solid: mp 207.5-208.5 °C; ¹H NMR (400 MHz, DMSO-

d₆) δ 2.16 (s, 3H), 3.77 (t, J ) 4.5 Hz, 2H), 4.08 (t, J ) 4.5 Hz,

2H), 5.13 (br s, 1H), 7.51 (d, J ) 1.7 Hz, 1H), 7.54 (dd, J )

1.7, 8.4 Hz, 1H), 8.26 (d, J ) 8.4 Hz, 1H), 9.20 (s, 1H), 12.79

(br s, 1H); IR (KBr) 3351, 1710, 1660, 1604, 1531, 1270 cm^-1

;

MS (ES⁺) 240 (M + 1). Anal. (C₁₁H₁₃NO₅) C, H, N.

Eth yl 4-(Acetyla m in o)-3-(2-p r op en yl)ben zoa te (108). A

solution of 2.1 g (0.01 mol) of ethyl 4-(acetylamino)benzoate

(107)⁴⁶and 1.1 g (0.005 mol) of Pd(OAc)₂in 17 mL of dry

toluene was heated at reflux for 0.75 h under a N₂atmosphere.

The solution was cooled to 60 °C, and the toluene was decanted

from the residue. The residue was washed with fresh toluene,

and the combined toluene layers were heated at reflux for 3

h. The solution was cooled, and the resulting precipitate was

collected by filtration. The filter cake was combined with the

residue, and the mixture was dried under vacuum for 3 h. A

mixture of this palladium adduct, 8.4 g (0.05 mol) of allyl

iodide, and 40 mL of glacial HOAc was stirred at ambient

temperature for 16 h. The mixture was filtered through Celite,

the filtrate was concentrated, and the residue was purified by

column chromatography on silica gel (1:1 EtOAc-hexane) to

give 0.85 g (69%) of 108 as a white powder: mp 127-128 °C;

¹H NMR (400 MHz, CDCl₃) δ 1.39 (t, J ) 7.1 Hz, 3H), 2.17 (s,

3H), 3.44 (d, J ) 6.0 Hz, 2H), 4.36 (q, J ) 7.1 Hz, 2H), 5.14 (d,

J ) 16.8 Hz, 1H), 5.24 (d, J ) 10.0 Hz, 1H), 5.98 (m, 1H), 7.43

(br s, 1H), 7.87 (s, 1H), 7.95 (dd, J ) 4.0, 2.0 Hz, 1H), 8.14 (br

s, 1H); IR (KBr) 3298, 1716, 1658, 1525, 1280; MS (ES⁺) 248

(M + 1). Anal. (C₁₄H₁₇NO₃) C, H, N.

E t h yl 4-(Acet yla m in o)-3-(2,3-d ih yd r oxyp r op yl)b en -

zoa te, Isom er A (109). A mixture of 5 mL of t-BuOH, 5 mL

of H₂O, and 1.4 g of AD mix-R (Aldrich) was stirred for 5 min.

To this mixture was added 0.1 g (0.0004 mol) of 108, and the

mixture stirred at ambient temperature for 48 h. The mixture

was cooled to 0 °C, 1.5 g (0.012 mol) of Na₂SO₃was added,

and the reaction mixture stirred at 0 °C for 20 min and then

at ambient temperature for 30 min. The mixture was ex-

tracted thrice with 15-mL portions of EtOAc. The combined

extracts were dried (Na₂SO₄) and concentrated, and the

residue was purified by column chromatography on silica gel

(50-100% EtOAc in hexane) to give 0.075 g (66%) of 109 as a

white powder: mp 114-116 °C; ¹H NMR (400 MHz, CD₃-

COCD₃) δ 1.35 (t, J ) 7.0 Hz, 3H), 2.10 (s, 3H), 2.83-3.00 (m,

2H), 3.47-3.60 (m, 2H), 3.91-3.97 (m, 1H), 4.21 (t, J ) 5.0

Hz, 1H), 4.31 (q, J ) 7.0 Hz, 2H), 4.85 (d, J ) 3.5 Hz, 1H),

7.83-7.88 (m, 2H), 8.15 (m, 1H), 9.81 (br s, 1H); IR 3506, 3260,

1684, 1593, 1541, 1290 cm^-1; MS (ES^-) 280 (M - 1). Anal.

(C₁₄H₁₉NO₅) C, H, N.

4-(Acetyla m in o)-3-(3-h yd r oxy-1-p r op en yl)ben zoic Acid

(115). A mixture of 0.27 g (0.001 mol) of 114 and 5 mL of 0.5

N NaOH was stirred at ambient temperature for 8 h. The

mixture was neutralized with H⁺resin and quickly filtered.

Upon cooling of the filtrate, a pale yellow solid precipitated.

The solid was collected by filtration, washed with cold H₂O,

1

and dried to give 0.065 g (28%) of 115: mp 214-215 °C; H

NMR (400 MHz, DMSO-d₆) δ 2.08 and 2.09 (2 s, 3H), 4.16 (d,

J ) 3.7 Hz, 2H), 4.92 (br s, 1H), 6.32 (dt, J ) 15.8, 5.0 Hz,

1H), 6.80 (dt, J ) 15.8, 1.6 Hz, 1H), 7.65 (d, J ) 8.4 Hz, 1H),

7.77 (dd, J ) 8.4, 2.0 Hz, 1H), 8.06 (d, J ) 2.0 Hz, 1H) 9.63 (s,

1H), 12.86 (br s, 1H); IR (KBr) 3314, 2886, 1675, 1524, 1229

cm^-1; MS (ES⁺) 218 (M + 1). Anal. (C₁₂H₁₃NO₄) C, H, N.

Meth yl 4-(Acetylam in o)-3-[(h ydr oxyim in o)m eth yl]ben -

zoa te (118). A mixture of 0.44 g (0.002 mol) of 92⁴⁵and 0.17

g (0.0025 mol) of HONH₂‚HCl in 10 mL of EtOAc was heated

at reflux for 16 h. The mixture was filtered, and the filtrate

was concentrated. The residue was recrystallized from EtOAc-

hexane to give 0.25 g (53%) of 118 as a pale yellow powder:

E t h yl 4-(Acet yla m in o)-3-(2,3-d ih yd r oxyp r op yl)b en -

zoa te, Isom er B (110). Isomer B was prepared by the above

procedure using 0.09 g of 108 and 1.4 g of AD mix-â (Aldrich).

The method gave 0.068 g (75%) of 110 as a white powder: mp

1

116-117 °C; H NMR (400 MHz, CD₃COCD₃) δ 1.35 (t, J )

7.1 Hz, 3H), 2.10 (s, 3H), 2.84-3.00 (m, 2H), 2.47-3.60 (m,

4044 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 25

Chand et al.

mp 200-203 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 2.15 (s, 3H),

3.85 (s, 3H), 7.92 (dd, J ) 8.67, 2.0 Hz, 1H), 8.17 (d, J ) 2.0

Hz, 1H), 8.33 (d, J ) 8.6 Hz, 1H), 8.43 (s, 1H), 10.76 (s, 1H),

11.69 (s, 1H); IR (KBr) 3214, 3100, 3010, 1720, 1598, 1550,

1282 cm^-1; MS (ES⁺) 237.1 (M + 1). Anal. (C₁₁H₁₂N₂O₄) C,

H, N.

4-(Acetylam in o)-3-[(h ydr oxyim in o)m eth yl]ben zoic Acid

(119). A mixture of 0.14 g (0.0006 mol) of 118 and 1 mL of 1

N NaOH was stirred at ambient temperature for 4 h. The

mixture was filtered, and the filtrate was neutralized with 1

N HCl. The resulting precipitate was collected by filtration,

washed with H₂O, and dried to give 0.095 g (71%) of 119 as a

white powder: mp 215-218 °C dec; ¹H NMR (400 MHz,

DMSO-d₆) δ 2.15 (s, 3H), 7.90 (dd, J ) 8.6, 2.0 Hz, 1H), 8.15

(d, J ) 2.0 Hz, 1H), 8.31 (d, J ) 8.6 Hz, 1H), 8.42 (s, 1H),

10.74 (s, 1H), 11.66 (s, 1H), 12.93 (s, 1H); IR (KBr) 3597, 3288,

3087, 2995, 2916, 1693, 1589, 1543, 1195 cm^-1; MS (ES^-) 220.4

(M - 1). Anal. (C₁₀H₁₀N₂O₄‚0.25H₂O) C, H, N.

Met h yl 4-(Acet yla m in o)-3-[[(a m in oim in om et h yl)h y-

d r a zin o]m eth yl]ben zoa te (120). A mixture of 2.2 g (0.01

mol) of 92,⁴⁵1.4 g (0.01 mol) of aminoguanidine bicarbonate,

1.7 mL (0.02 mol) of concentrated HCl, and 50 mL of EtOH

was heated at reflux for 4 h. The reaction mixture was

concentrated to ∼0.5 times the original volume and cooled. The

precipitate was collected by filtration and dried to give 2.1 g

(69%) of 120 as a fluffy white solid: mp 278 °C; ¹H NMR (400

MHz, DMSO-d₆) δ 2.15 (s, 3H), 3.87 (s, 3H), 7.90 (m, 6H), 8.33

(s, 1H), 8.45 (d, J ) 2.0 Hz, 1H), 10.47 (s, 1H); IR (KBr) 3449,

3271, 1724, 1670, 1631, 1291 cm^-1; MS (ES⁺) 278 (M + 1).

Anal. (C₁₂H₁₅N₅O₃‚HCl) C, H, N.

2-[2-(Ace t yla m in o)-5-(m e t h oxyca r b on yl)p h e n yl]-2-

(p h en ylth io)a ceta m id e (124). A mixture of 13 g (0.041 mol)

of 123 and 16.4 mL (0.12 mol) of Et₃N in 400 mL of THF at 0

°C under a N₂atmosphere was treated with 3.2 mL (0.045 mol)

of acetyl chloride, and the mixture stirred at ambient tem-

perature for 1 h. The reaction progress was monitored by TLC

(silica gel, 3:1 EtOAc-hexene). An additional 1.2 mL (0.016

mol) of acetyl chloride was added, and the mixture stirred at

ambient temperature overnight. The reaction was not com-

plete so an additional 1.2 mL (0.010 mol) of acetyl chloride

was added and the mixture stirred for 4 h. The mixture was

diluted with 300 mL of EtOAc and 200 mL of H₂O. The

organic layer was separated, washed twice with 100-mL

portions of H₂O, and concentrated. The residue was chro-

matographed twice on silica gel (50-67% EtOAc in hexene).

The appropriate fractions were combined and concentrated,

and the residue was recrystallized twice from CHCl₃-hexane

to give 1.5 g (10%) of 124 as a white solid: mp 173-174 °C;

¹H NMR (400 MHz, DMSO-d₆) δ 2.13 (s, 3H), 3.92 (s, 3H), 5.34

(s, 1H), 7.29 (m, 5H), 7.63 (s, 1H), 7.84 (dd, J ) 8.5, 2.0 Hz,

1H), 7.94 (m, 3H), 10.47 (s, 1H); IR (KBr) 3382, 3273, 3185,

1727, 1681, 1670, 1527, 1291, 1278, 1224 cm^-1; MS (ES⁺) 359.2

(M + 1). Anal. (C₁₈H₁₈N₂O₄S‚0.25H₂O) C, H, N.

2-[2-(Acetyla m in o)-5-(m eth oxyca r bon yl)p h en yl]a cet-

a m id e (125). An activated sample of Raney Ni was prepared

by washing 20 g of a commercial preparation thrice with H₂O

and thrice with THF. This THF slurry was added portionwise

over a 1-h period to a stirred solution of 1.25 g (0.0035 mol) of

124 in 70 mL of THF. The mixture was filtered through Celite,

the filtrate concentrated, and the residue dried to give 0.68

(78%) of 125 as a white solid. An analytical sample, mp 235

°C, was recrystallized from MeOH: ¹H NMR (400 MHz,

DMSO-d₆) δ 2.11 (s, 3H), 3.55 (s, 2H), 3.84 (s, 3H), 7.28 (s,

1H), 7.82 (dd, J ) 8.5, 2.0 Hz, 1H), 7.85 (s, 1H), 7.89 (d, J )

2.0 Hz, 1H), 7.96 (dd, J ) 8.2, 5 Hz, 1H), 10.39 (s, 1H); IR

(KBr) 3408, 3276, 3211, 1712, 1665, 1522, 1273 cm^-1; MS (ES⁺)

251.3 (M + 1). Anal. (C₁₂H₁₄N₂O₄) C, H, N.

4-(Acet yla m in o)-3-[[(a m in oim in om et h yl)h yd r a zin o]-

m eth yl]ben zoic Acid (121). A mixture of 0.54 g (0.0017 mol)

of 120 and 5 mL of 1 N NaOH was stirred at ambient

temperature for 24 h. The mixture was filtered and the filtrate

neutralized with concentrated HCl. The precipitate was

collected by filtration, washed with H₂O, and dried to give 0.41

1

g (80%) of 121 as a white powder: mp 326-327 °C; H NMR

2-[2-(Acetyla m in o)-5-ca r boxyp h en yl]a ceta m id e (126).

A mixture of 0.48 g (0.0019 mL) of 125, 2.1 mL of 1 N NaOH,

and 3 mL of H₂O was stirred at ambient temperature for 3 h

followed by 1 h at 35 °C. The mixture was filtered, the filtrate

pH was adjusted to 4.4 with 0.5 N HCl, and the volume of the

filtrate was reduced by ∼50%. The precipitate was collected

by filtration and then recrystallized twice from H₂O to give

0.1 g (22%) of 126 as a cream-colored solid: mp 324-326 °C;

¹H NMR (400 MHz, DMSO-d₆) δ 2.10 (s, 3H), 3.53 (s, 2H), 7.26

(s, 1H), 7.79 (dd, J ) 8.4, 2.0 Hz, 1H), 7.85 (s, 1H), 7.87 (d, J

) 2.0 Hz, 1H), 7.91 (dd, J ) 8.2, 5.2 Hz, 1H), 10.35 (s, 1H),

12.77 (s, 1H); IR (KBr) 3360, 3180, 1705, 1685, 1668, 1596,

1525, 1323, 1280 cm^-1; MS (ES⁺) 237.3 (M + 1). Anal.

(C₁₁H₁₂N₂O₄‚0.15H₂O) C, H; N: calcd, 11.72; found, 11.27.

Met h yl 4-(Acet yla m in o)-3-(1-h yd r oxy-2-n it r oet h yl)-

ben zoa te (127). A suspension of 0.06 g (0.0023 mol) of 95%

NaH in 2.5 mL of DMF at 0 °C under an N₂atmosphere was

treated with 0.9 mL (0.015 mol) of MeNO₂and then stirred

for 20 min. A 0.33-g (0.0015 mol) sample of 92⁴⁵was added,

and the mixture stirred at 0 °C for 30 min. The reaction

mixture was acidified with HOAc and poured into 100 mL of

H₂O. The mixture was extracted thrice with 25-mL portions

of EtOAc, and the combined extracts were washed with 25 mL

of brine, dried (MgSO₄), and concentrated. The residue was

purified by flash column chromatography on silica gel (90%

Et₂O in hexene) to give 0.24 g (57%) of 127 as a white solid:

mp 127-129 °C; ¹H NMR (400 MHz, CDCl₃) δ 2.23 (s, 3H),

3.91 (s, 3H), 4.14 (d, J ) 4.2 Hz, 1H), 4.53 (dd, J ) 13.8, 3.1

Hz, 1H), 4.81 (dd, J ) 13.8, 10.3 Hz, 1H), 5.60 (d, t, J ) 10.3,

3.6 Hz, 1H), 7.86 (d, J ) 2.0 Hz, 1H), 8.0 (dd, J ) 8.6, 2.0 Hz,

1H), 8.23 (d, J ) 8.6 Hz, 1H), 9.14 (s, 1H); IR (KBr) 3233, 1705,

1678, 1552, 1515, 1298 cm^-1; MS (ES⁺) 283.1 (M + 1). Anal.

(C₁₂H₁₄N₂O₆) C, H, N.

(400 MHz, CF₃COOD) δ 2.50 (s, 3H), 8.03 (d, J ) 8.5 Hz, 1H),

8.33 (m, 2H), 8.64 (br s, 1H); IR (KBr) 3379, 3129, 1668, 1637,

1386, 1294 cm^-1; MS (ES⁺) 264 (M + 1). Anal. (C₁₁H₁₃N₅O₃‚

2H₂O) C, H, N.

2-[2-(Acetyla m in o)-5-ca r boxyp h en yl]a cetic Acid (122).

A mixture of 0.3 g (0.001 mol) of 98 and 3 mL of 1 N NaOH

was stirred at ambient temperature for 16 h. The mixture

was filtered, and the filtrate was neutralized with 1 N HCl.

The precipitate was collected by filtration, washed with H₂O,

and recrystallized from MeOH-H₂O to give 0.14 g (59%) of

122 as cream-colored needles: mp 240-241 °C; ¹H NMR (400

MHz, DMSO-d₆) δ 2.07 (s, 3H), 3.72 (s, 2H), 7.66 (d, J ) 8.3

Hz, 1H), 7.79 (dd, J ) 8.4, 2 Hz, 1H), 7.84 (d, J ) 2.0 Hz, 1H),

9.53 (s, 1H), 12.61 (br s, 1H); IR (KBr) 3289, 3013, 1693, 1620,

1388, 1286 cm^-1; MS (ES^-) 236.5 (M - 1). Anal. (C₁₁H₁₁NO₅)

C, H, N.

2-[2-Am in o-5-(m e t h oxyca r b on yl)p h e n yl]-2-(p h e n yl-

th io)a ceta m id e (123). A solution of 10 g (0.066 mol) of

methyl 4-aminobenzoate (95; Aldrich) in 400 mL of CH₂Cl₂at

-70 °C under a N₂atmosphere was treated with a solution of

8.6 g (0.079 mol) of tert-butyl hypochlorite in 20 mL of CH₂Cl₂

over a 5-min period. The reaction mixture was stirred for 10

min and then treated in one portion with a solution of 11 g

(0.066 mol) of 1-(phenylthio)acetamide (Parish) in 300 mL of

CH₂Cl₂. The solution was stirred at -70 °C for 5 h, at -40 °C

for 1 h, and at -10 °C for 1 h and then cooled again to -70

°C. The mixture was treated in one portion with 11 mL (0.079

mol) of Et₃N, stirred for 30 min at -70 °C, and then allowed

to warm to room temperature. A suspended solid was collected

by filtration, and the filter cake was suspended in ∼500 mL

of hexane to remove color. The solid was collected by filtration

and dried to give 16.9 g (81%) of 123. An analytical sample,

mp 181-182 °C, was recrystallized from EtOAc-hexene to

yield a white solid: ¹H NMR (400 MHz, DMSO-d₆) δ 3.74 (s,

3H), 5.16 (s, 1H), 6.25 (s, 2H), 6.67 (d, J ) 8.5 Hz, 1H), 7.22

(m, 1H), 7.32 (m, 5H), 7.57 (dd, J ) 8.5, 2.1 Hz, 1H), 7.67 (s,

1H), 7.93 (d, J ) 2.1 Hz, 1H); IR (KBr) 3369, 3259, 3183, 1687,

1661, 1602, 1509, 1290, 1236 cm^-1; MS (ES⁺) 317.2 (M + 1,

40), 207.2 (M - C₆H₅SH, 10). Anal. (C₁₆H₁₆N₂O₃S) C, H, N.

Meth yl 4-(Acetyla m in o)-3-(2-n itr ovin yl)ben zoa te (128).

A mixture of 2.1 g (0.0075 mol) of 127, 0.17 g (0.0014 mol) of

DMAP, 1 mL (0.011 mol) of acetic anhydride, and 15 mL of

CH₂Cl₂was stirred at ambient temperature for 1.5 h. The

precipitate was collected by filtration and recrystallized from

EtOH to give 1.1 g (45%) of 128 as a yellow solid: mp 200-

Benzoic Acids as Influenza Inhibitors

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 25 4045

203 °C dec; ¹H NMR (400 MHz, DMSO-d₆) δ 2.16 (s, 3H), 3.87

(s, 3H), 7.8 (dd, J ) 8.6, 5.0 Hz, 1H), 8.04 (dd, J ) 8.6, 2.0 Hz,

1H), 8.18 (d, J ) 13.4 Hz, 1H), 8.24 (d, J ) 13.4 Hz, 1H), 8.36

(d, J ) 2.0 Hz, 1H), 10.18 (s, 1H); IR (KBr) 3455, 3269, 3098,

1716, 1665, 1515, 1340, 1281, 1239 cm^-1; MS (ES^-) 263.2 (M

- 1). Anal. (C₁₂H₁₂N₂O₅) C, H, N.

4 h. The reaction mixture was cooled, and the resulting

precipitate was collected by filtration, washed with cold EtOAc,

and dried to give 3 g (86%) of 135 as a white powder: mp 160-

1

164 °C; H NMR (400 MHz, DMSO-d₆) δ 1.31 (t, J ) 7.1 Hz,

3H), 3.13 (s, 3H), 4.32 (q, J ) 7.1 Hz, 2H), 7.51 (br s, 4H), 7.66

(d, J ) 8.5 Hz, 1H), 7.76 (d, J ) 2.0 Hz, 1H), 7.92 (dd, J ) 8.5,

2.0 Hz, 1H), 9.40 (br s, 1H), 9.98 (br s, 1H); IR (KBr) 3368,

3179, 1680, 1611, 1340, 1296, 1170 cm^-1; MS (ES^-) 299 (M -

1). Anal. (C₁₁H₁₆N₄O₄S‚HCl‚0.5H₂O) C, H, N.

Meth yl 4-(Acetyla m in o)-3-(2-n itr oeth yl)ben zoa te (129).

To a solution of 0.89 g (0.0027 mol) of 128 in 15 mL of EtOH

at 0 °C was added dropwise over a 10-min period 0.31 g (0.008

mol) of NaBH₄in 20 mL of EtOH. The mixture was stirred

for 1 h at 0 °C and acidified to pH 5.6 using 1 N HCl. The

mixture was concentrated, and the residue was diluted with

20 mL of EtOAc and 20 mL of H₂O. The layers were

separated, and the aqueous layer was extracted thrice with

20-mL portions of EtOAc. The combined organic layers were

washed with 25 mL of brine, dried (MgSO₄), and concentrated.

The residue was triturated thrice with 100 mL of boiling Et₂O

and then recrystallized from EtOH to give 0.31 g (43%) of 129

as a white solid: mp 169-170 °C; ¹H NMR (400 MHz, DMSO-

d₆) δ 2.11 (s, 3H), 3.33 (m, 2H), 3.83 (s, 3H), 4.80 (t, J ) 7.2

Hz, 2H), 7.72 (m, 1H), 7.82 (m, 2H), 9.58 (s, 1H); IR (KBr) 3421,

3278, 1717, 1664, 1543, 1293 cm^-1; MS (ES^-) 265.3 (M - 1).

Anal. (C₁₂H₁₄N₂O₅) C, H, N.

3-[(Am in oim in om eth yl)am in o]-4-[(m eth ylsu lfon yl)am i-

n o]ben zoic Acid (136). A mixture of 1.8 g (0.005 mol) of 135

and 15 mL of 1 N NaOH was stirred at ambient temperature

for 4 h. The mixture was filtered, and the filtrate was

neutralized with HCl. The resulting precipitate was collected

by filtration, washed with H₂O, and dried to give 1.2 g (81%)

of 136 as an off-white powder: mp 200 °C; ¹H NMR (400 MHz,

DMSO-d₆) δ 2.75 (s, 3H), 7.32 (d, J ) 9.1 Hz, 1H), 7.51-7.62

(m, 6H); IR (KBr) 3437, 1668, 1601, 1379, 1321, 1157 cm^-1

;

MS (ES^-) 271 (M - 1). Anal. (C₉H₁₂N₄O₂S‚0.5H₂O) C, H, N.

2-[(Meth ylsu lfon yl)a m in o]-5-(m eth oxyca r bon yl)ben z-

a ld eh yd e Tr im eth ylen em er ca p ta l (138). To a solution of

5.0 g (0.019 mol) of 137⁴⁵in 90 mL of CH₂Cl₂and 4.5 mL (0.055

mol) of pyridine was added 4.3 mL (0.055 mol) of methane-

sulfonyl chloride dropwise over a 5-min period, and the

mixture heated at reflux overnight. The reaction mixture was

cooled and washed successively with a 10% HOAc solution

followed by H₂O. The organic layer was dried (Na₂SO₄) and

concentrated, and the residue was purified by column chro-

matography on silica gel (1:1 EtOAc-hexane). The appropri-

ate fractions were combined and concentrated to give 5.0 g

(77%) of 138 as a yellow solid. An analytical sample, mp 168-

168.5 °C, was recrystallized from EtOH: ¹H NMR (400 MHz,

CDCl₃) δ 1.90-2.01 (m, 1H), 2.20-2.25 (m, 1H), 2.93-2.98 (m,

2H), 3.05-3.11 (m, 5H), 3.91 (s, 3H), 5.4 (s, 1H), 7.77 (d, J )

8.6 Hz, 1H), 8.00 (dd, J ) 8.6, 2.0 Hz, 1H), 8.05 (d, J ) 20.0

Hz, 1H), 8.21 (br s, 1H); IR (KBr) 3301, 2905, 1723, 1335, 1162

cm^-1; MS (ES⁺) 348 (M + 1). Anal. (C₁₃H₁₇NO₄S₃) C, H, N.

2-[(Meth ylsu lfon yl)a m in o]-5-(m eth oxyca r bon yl)ben z-

a ld eh yd e (139). A solution of 7.7 g (0.022 mol) of 138 in 4.4

mL of DMF and 39 mL of acetone was added dropwise over a

5-min period to a mixture of 2.1 g (0.026 mol) of CuO and 7.0

g (0.055 mol) of CuCl₂in 174 mL of acetone heated at reflux.

The mixture was heated at reflux for 2 h, cooled, and filtered

through Celite, and the cake was washed with 10% EtOH in

CH₂Cl₂. The filtrate was diluted with 125 mL of H₂O and 150

mL of CH₂Cl₂. The filtrate was shaken, the layers were

separated, and the aqueous layer was extracted twice with

100-mL portions of H₂O, dried (Na₂SO₄), and concentrated. The

residue was recrystallized from EtOH to give 4.5 g (79%) of

139 as a beige solid: mp 161-163 °C; ¹H NMR (400 MHz,

DMSO-d₆) δ 3.31 (s, 3H), 3.89 (s, 3H), 7.69 (d, J ) 8.8 Hz,

1H), 8.23 (dd, J ) 8.8, 2.1 Hz, 1H), 8.51 (d,J ) 2.1 Hz, 1H),

10.10 (s, 1H), 10.68 (s, 1H); IR (KBr) 3138, 1712, 1664, 1612,

4-(Acetyla m in o)-3-(2-n itr oeth yl)ben zoic Acid (130). A

mixture of 0.25 g (0.0009 mol) of 129 in 1.8 mL of 1 N NaOH

was stirred at ambient temperature for 0.75 h. The mixture

was filtered, and the filtrate pH was adjusted to 5-6 with

glacial HOAc. The resulting solid was collected by filtration,

washed with H₂O, and dried to give 0.18 g (78%) of 130 as a

1

white solid: mp >220 °C dec; H NMR (400 MHz, DMSO-d₆)

δ 2.11 (s, 3H), 3.3 (t, J ) 7.2 Hz, 2H), 4.80 (t, J ) 7.2 Hz, 2H),

7.66 (m, 1H), 7.81 (m, 2H), 9.57 (s, 1H), 12.84 (br s, 1H); IR

(KBr) 3461, 3269, 2996, 1695, 1656, 1556, 1281 cm^-1; MS (ES^-)

251.2 (M - 1). Anal. (C₁₁H₁₂N₂O₅) C, H, N.

4-(Acetyla m in o)-3-(2-a m in oeth yl)ben zoic Acid (131). A

mixture of 0.2 g (0.0008 mol) of 130 and 0.04 g of PtO₂in 8

mL of MeOH was hydrogenated at 50 psi for 4 h. The reaction

mixture was filtered through Celite, and the cake was washed

thrice with MeOH. The cake was dissolved in 100 mL of H₂O

and again filtered through Celite to remove catalyst. This

filtrate was concentrated, and the residue was recrystallized

from H₂O-EtOH to give 0.08 g (45%) of 131 as a tan solid:

mp 212-213 °C dec; ¹H NMR (400 MHz, DMSO-d₆) δ 2.09 (s,

3H), 2.94 (m, 4H), 7.58 (d, J ) 7.7 Hz, 1H), 7.74 (m, 2H); IR

(KBr) 3195, 2990, 1668, 1532, 1380 cm^-1; MS (ES⁺) 223 (M +

1). Anal. (C₁₁H₁₄N₂O₃‚0.25H₂O) C, H, N.

Eth yl 3-Nitr o-4-[(m eth ylsu lfon yl)am in o]ben zoate (133).

To 10 mL of fuming HNO₃at 5-10 °C was added portionwise

2.8 g (0.008 mol) of ethyl 4-[(methylsulfonyl)amino]benzoate

(132)¹⁸over a 15-min period. The mixture was stirred at 10-

15 °C for 0.5 h and then poured into 50 mL of cold H₂O. The

precipitate was collected by filtration, washed with H₂O, dried,

and recrystallized from EtOAc to give 1.7 g (72%) of 133 as

yellow crystals: mp 124 °C; ¹H NMR (400 MHz, CDCl₃) δ 1.42

(t, J ) 7.2 Hz, 3H), 3.23 (s, 3H), 4.43 (q, J ) 7.2 Hz, 2H), 7.95

(d, J ) 8.9 Hz, 1H), 8.31 (dd, J ) 8.9, 2.0 Hz, 1H), 8.93 (d, J

) 2.0 Hz, 1H), 10.06 (s, 1H); IR (KBr) 3271, 1719, 1624, 1535,

1342, 1173 cm^-1; MS (ES^-) 287 (M - 1). Anal. (C₁₀H₁₂N₂O₆S)

C, H, N.

1155, 1137 cm^-1

C, H, N.

; MS (ES^-) 256 (M - 1). Anal. (C₁₀H₁₁NO₅S)

Meth yl 3-(1-Hyd r oxy-2-n itr oeth yl)-4-[(m eth ylsu lfon -

yl)a m in o]ben zoa te (140). To a mixture of 2.6 g (0.01 mol)

of 139 and 1.2 g (0.02 mol) of MeNO₂in 40 mL of EtOH at 0

°C (ice-salt bath) was added dropwise a solution of 1.2 g (0.02

mol) of KOH in 18 mL of EtOH and 2 mL of H₂O over a 20-

min period. The mixture was stirred for 0.5 h at 0 °C and

then at ambient temperature for 4 h. The mixture was

neutralized with 2 N HCl and then poured into 100 mL of H₂O.

The mixture was extracted with CHCl₃, and the organic layer

was dried (Na₂SO₄) and concentrated. The residue was

purified by column chromatography on silica gel (1-5% MeOH

in CHCl₃). The appropriate fractions were combined and

concentrated, and the residue was recrystallized from EtOAc-

hexane to give 0.8 g (25%) of 140 as an off-white solid: mp

E t h yl 3-Am in o-4-[(m et h ylsu lfon yl)a m in o]b en zoa t e

(134). A mixture of 1 g (0.0035 mol) of 133 and 0.02 g of PtO₂

in 40 mL of EtOH was hydrogenated at 20 psi overnight. The

mixture was filtered through Celite, and the filtrate volume

was reduced to 20 mL. A precipitate formed which was

collected by filtration, washed with cold EtOH, and dried to

1

give 0.75 g (84%) of 134 as a white powder: mp 151 °C; H

NMR (400 MHz, DMSO-d₆) δ 1.29 (t, J ) 7.2 Hz, 3H), 2.90 (s,

3H), 4.26 (q, J ) 7.2 Hz, 2H), 5.40 (br s, 2H), 7.14 (dd, J )

8.2, 2.0 Hz, 1H), 7.21 (d, J ) 8.2 Hz, 1H), 7.38 (d, J ) 2.0 Hz,

1H), 8.94 (br s, 1H); IR (KBr) 3476, 3368, 3277, 1705, 1620,

1523, 1157 cm^-1; MS (ES^-) 257 (M - 1). Anal. (C₁₀H₁₄N₂OS)

C, H, N.

E t h yl 3-[(Am in oim in om et h yl)a m in o]-4-[(m et h ylsu l-

fon yl)a m in o]ben zoa te (135). A mixture of 2.6 g (0.01 mol)

of 134, 0.84 mL (0.01 mol) of concentrated HCl, 4.2 g (0.1 mol)

of cyanamide, and 100 mL of EtOAc was heated at reflux for

1

148 °C; H NMR (400 MHz, DMSO-d₆) δ 3.16 (s, 3H), 3.86 (s,

3H), 4.56 (dd, J ) 12.7, 9.8 Hz, 1H), 4.82 (dd, J ) 12.7, J )

2.9 Hz, 1H), 5.73 (dd, J ) 7.8, 2.9 Hz, 1H), 6.41 (br s, 1H),

7.51 (d, J ) 8.42 Hz, 1H), 7.92 (dd, J ) 8.4, 2.1 Hz, 1H), 8.16

(d, J ) 2.1 Hz, 1H), 9.65 (s, 1H); IR (KBr) 3398, 3275, 1685,

1563, 1442, 1330 cm^-1; MS (ES^-) 317 (M - 1). Anal.

(C₁₁H₁₄N₂O₇S) C, H, N.

4046 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 25

Chand et al.

Meth yl 3-(2-Am in o-1-h yd r oxyeth yl)-4-[(m eth ylsu lfon -

yl)a m in o]ben zoa te (141). A mixture of 0.2 g (0.0006 mol)

of 140 and 0.02 g of PtO₂in 10 mL of EtOH was hydrogenated

at 50 psi for 8 h. The mixture was filtered through Celite,

and the filtrate was concentrated to 5 mL. The filtrate was

diluted with 5 mL of EtOAc and 10 mL of hexane and cooled

to 0 °C. The precipitate was collected by filtration, washed

with a 1:1 solution of EtOAc-hexane, and dried to give 0.1 g

(55%) of 141 as a white powder: mp 172-173 °C dec; ¹H NMR

(400 MHz, DMSO-d₆) δ 2.68 (s, 3H), 2.82 (dd, J ) 12.2, 7.8

Hz, 1H), 3.09 (dd, J ) 12.2, 4.5 Hz, 1H), 3.75 (s, 3H), 4.92 (dd,

J ) 4.5, 7.8 Hz, 1H), 7.24 (d, J ) 9.3 Hz, 1H), 7.61 (dd, J )

9.3, 2.2 Hz, 1H), 7.81 (d, J ) 2.2 Hz, 1H); IR (KBr) 3141, 3021,

1695, 1605, 1494, 1273 cm^-1; MS (ES⁺) 289 (M + 1). Anal.

(C₁₁H₁₆N₂O₅S) C, H, N.

trated. The residue was recrystallized from EtOAc-hexane

to give 5.0 g (70%) of 147 as a beige solid. An analytical

sample, mp 147-149 °C, was prepared by two additional

recrystallizations from EtOAc-hexane: ¹H NMR (400 MHz,

CDCl₃) δ 1.29 (d, J ) 6.8 Hz, 6H), 1.38 (t, J ) 7.2 Hz, 3H),

2.58 (m, 1H), 3.61 (br s, 2H), 4.34 (q, J ) 7.2 Hz, 2H), 7.26 (br

s, 1H), 7.33-7.53 (m, 3H); IR (KBr) 3428, 3270, 1712, 1653,

1527, 1438 cm^-1; MS (ES⁺) 251 (M + 1). Anal. (C₁₃H₁₈N₂O₃)

C, H, N.

Eth yl 3-[(Am in oim in om eth yl)a m in o]-4-[(2-m eth ylp r o-

p ion yl)a m in o]ben zoa te (148). A mixture of 1.7 g (0.0068

mol) of 147, 5.7 g (0.136 mol) of cyanamide, and 0.6 mL (0.0068

mol) of concentrated HCl in 50 mL of EtOAc was heated at

reflux for 48 h. The mixture was concentrated, and the residue

was purified by column chromatography on silica gel (0-3%

MeOH in EtOAc). The appropriate fractions were combined

and concentrated, and the residue was triturated with EtOAc.

The resulting solid was collected by filtration and dried to give

0.76 g (34%) of 148. An analytical sample, mp 175-177 °C,

was isolated as a white solid by a recrystallization from Me-

OH-Et₂O: ¹H NMR (400 MHz, DMSO-d₆) δ 1.12 (d, J ) 6.8

Hz, 6H), 1.32 (t, J ) 7.1 Hz, 3H), 2.80 (m, 1H), 4.31 (q, J )

7.1 Hz, 2H), 7.51 (br s, 3H), 7.76 (d, J ) 1.9 Hz, 1H), 7.89 (dd,

J ) 1.9, 8.4 Hz, 1H), 7.95 (d, J ) 8.4 Hz, 1H), 9.29 (s, 1H),

3-(2-Am in o-1-h ydr oxyeth yl)-4-[(m eth ylsu lfon yl)am in o]-

ben zoic Acid (142). A mixture of 0.05 g (0.000 17 mol) of

141 and 44 mL (0.000 44 mol) of 1 N NaOH was stirred at

ambient temperature for 4 h. The mixture was filtered, the

filtrate neutralized with 1 N HCl, and the mixture allowed to

stand at ambient temperature overnight. The precipitate was

collected by filtration, washed with H₂O, and dried to give

1

0.038 g (81%) of 142 as a white powder: mp 274-276 °C; H

NMR (400 MHz, ND₄OD) δ 2.90 (m, 5H), 5.08 (dd, J ) 4.6,

7.6 Hz, 1H), 7.23 (d, J ) 8.4 Hz, 1H), 7.71 (dd, J ) 2.2, 8.4 Hz,

1H), 7.80 (d, J ) 2.2 Hz, 1H); IR (KBr) 3264, 1608, 1590, 1530,

1395, 1327 cm^-1; MS (ES⁺) 275 (M + 1). Anal. (C₁₀H₁₄N₂O₅-

S‚0.2H₂O) C, H, N.

9.95 (s, 1H); IR (KBr) 3306, 3153, 1720, 1675, 1587, 1307 cm^-1

;

MS (ES⁺) 293 (M + 1). Anal. (C₁₄H₂₀N₄O₃‚0.5H₂O) C, H, N.

3-[(Am in oim in om et h yl)a m in o]-4-[(2-m et h ylp r op ion -

yl)a m in o]ben zoic Acid (149). A mixture of 0.36 g (0.0012

mol) of 148 and 2.4 mL of 1 N NaOH was stirred at 38 °C for

2 h. The mixture was filtered, and the filtrate pH was adjusted

to 7 with 1 N HCl. The resulting precipitate was collected by

filtration, and then the cake was dissolved in a minimum

amount of 1 N NaOH. The pH of this solution was adjusted

to 7 with 1 N HCl. The resulting precipitate was collected by

filtration, washed with H₂O, and dried to give 0.15 g (52%) of

Meth yl 3-[(Hyd r oxyim in o)m eth yl]-4-(m eth ylsu lfon yl-

a m in o)ben zoa te (143). A mixture of 2.6 g (0.01 mol) of 139

and 0.87 g (0.0125 mol) of HONH₂‚HCl in 40 mL of EtOH was

heated at reflux for 2 h. The mixture was filtered hot, the

filtrate was concentrated, and the residue was triturated with

H₂O. The resulting solid was collected by filtration, dried, and

recrystallized from EtOAc/hexane to give 2.4 g (89%) of 143

as an off-white powder, mp 160 °C; ¹H NMR (400 MHz, DMSO-

d₆) δ 3.25 (s, 3H), 3.85 (s, 3H), 7.62 (d, J ) 8.7 Hz, 1H), 7.96

(dd, J ) 8.7 Hz and J ) 2.1 Hz, 1H), 8.19 (d, J ) 2.1 Hz, 1H),

8.50 (s, 1H), 10.59 (s, 1H), 11.84 (s, 1H); IR (KBr) 3361, 1698,

1621, 1341, 1286, 1159 cm^-1; MS (ES⁺) 273 (M + 1). Anal.

(C₁₀H₁₂N₂O₅S) C, H, N.

1

149 as a white solid: mp 238-239.5 °C; H NMR (400 MHz,

DMSO-d₆and CD₃OD) δ 1.17 (d, J ) 6.8 Hz, 6H), 2.71 (m,

1H), 7.53 (d, J ) 8.3 Hz, 1H), 7.78 (d, J ) 1.6 Hz, 1H), 7.86

(dd, J ) 1.6, 8.3 Hz, 1H); IR (KBr) 3408, 3207, 1683, 1640,

1538, 1383 cm^-1; MS (ES^-) 263 (M - 1). Anal. (C₁₂H₁₆N₄O₃‚

0.5H₂O) C, H, N.

3-[(Hyd r oxyim in o)m eth yl]-4-[(m eth ylsu lfon yl)a m in o]-

ben zoic Acid (144). A mixture of 0.54 g (0.002 mol) of 143,

5.0 mL of 1 N NaOH, and 2.5 mL of H₂O was stirred at 40 °C

for 16 h. The mixture was allowed to cool to ambient

temperature and filtered, and the filtrate was neutralized with

HCl. The resulting precipitate was collected by filtration,

washed with H₂O, and dried to give 0.4 g (81%) of 144 as a

Eth yl 4-(2-F u r oyla m in o)-3-n itr oben zoa te (151). A solu-

tion of 2.3 g (0.011 mol) of ethyl 4-amino-3-nitrobenzoate

(150)⁴⁸in 30 mL of pyridine was treated with 1.1 mL (0.011

mol) of 2-furoyl chloride (Aldrich), and the mixture stirred at

ambient temperature overnight. The mixture was poured into

500 mL of H₂O, and the resulting precipitate was collected by

filtration and recrystallized from EtOAc to give 2.4 g (72%) of

151. An analytical sample, mp 155-156.5 °C, was isolated

as a yellow solid after two additional recrystallizations from

EtOAc: ¹H NMR (400 MHz, DMSO-d₆) δ 1.35 (t, J ) 7.0 Hz,

3H), 4.36 (q, J ) 7.0 Hz, 2H), 6.79 (dd, J ) 1.8, 3.5 Hz, 1H),

7.42 (dd, J ) 3.5, 0.7 Hz, 1H), 8.07 (dd, J ) 1.8, 0.7 Hz, 1H),

8.28 (m, 2H), 8.52 (m, 1H), 11.15 (s, 1H); MS (no ionization);

1

white powder: mp 250 °C; H NMR (400 MHz, DMSO-d₆) δ

3.24 (s, 3H), 7.59 (d, J ) 8.6 Hz, 1H), 7.95 (dd, J ) 8.6, 2.0

Hz, 1H), 8.16 (d, J ) 2.0 Hz, 1H), 8.42 (s, 1H), 10.54 (s, 1H),

11.80 (s, 1H), 13.00 (s, 1H); IR (KBr) 3442, 3033, 1691, 1610,

1343, 1289 cm^-1; MS (ES^-) 257 (M - 1). Anal. (C₉H₁₀N₂O₅S)

C, H, N.

IR (KBr) 3323, 1713, 1687, 1587, 1508, 1270 cm^-1

(C₁₄H₁₂N₂O₆) C, H, N.

. Anal.

Eth yl 4-[(2-Meth ylp r op ion yl)a m in o]-3-n itr oben zoa te

(146). A 29.4-g (0.125-mol) sample of ethyl 4-[(2-methylpro-

pionyl)amino]benzoate (145)⁴⁷was added portionwise to 200

mL of stirred, fuming HNO₃at 10 °C. After the addition was

complete, the mixture was stirred at ambient temperature for

30 min. The reaction mixture was poured into H₂O and

extracted thrice with 500-mL portions of EtOAc. The com-

bined extracts were washed with 500 mL of 1% NaHCO₃and

then 500 mL of H₂O and concentrated. The residue was

purified by column chromatography on silica gel. The ap-

propriate fractions were combined and concentrated to give

17 g (49%) of 146 as a yellow solid. An analytical sample, mp

74-76 °C, was recrystallized from hexane: ¹H NMR (400 MHz,

CDCl₃) δ 1.32 (d, J ) 7.0 Hz, 6H), 1.42 (t, J ) 7.1 Hz, 3H),

2.69 (m, 1H), 4.41 (q, J ) 7.1 Hz, 2H), 8.27 (dd, J ) 1.8, 8.8

Hz, 1H), 8.89 (d, J ) 1.8 Hz, 1H), 8.95 (d, J ) 8.8 Hz, 1H),

10.67 (br s, 1H); IR (KBr) 3344, 2986, 1712, 1620, 1508, 1335

cm^-1; MS (ES⁺) 281 (M + 1). Anal. (C₁₃H₁₆N₂O₅) C, H, N.

Eth yl 3-Am in o-4-[(2-m eth ylp r op ion yl)a m in o]ben zoa te

(147). A mixture of 8.0 g (0.029 mol) of 146 and 0.05 g of PtO₂

in 75 mL of EtOH was hydrogenated at 35 psi for 2 h. The

mixture was filtered through Celite and the filtrate concen-

Eth yl 3-Am in o-4-(2-fu r oylam in o)ben zoate (152). A mix-

ture of 2.0 g (0.0066 mol) of 151 and 0.05 g of PtO₂in 200 mL

of EtOH was hydrogenated at 30 psi for 1 h. The mixture was

filtered through Celite, and the filtrate was concentrated to

give 1.2 g (67%) of 152 as a yellow solid. An analytical sample,

mp 134.5-135.5 °C, was recrystallized from EtOAc-hexane:

¹H NMR (400 MHz, DMSO-d₆) δ 1.31 (t, J ) 7.1 Hz, 3H), 4.28

(q, J ) 7.1 Hz, 2H), 5.26 (s, 2H), 6.70 (dd, J ) 1.8, 3.5 Hz,

1H), 7.20 (dd, J ) 1.9, 8.2 Hz, 1H), 7.33 (dd, J ) 0.8, 3.5 Hz,

1H), 7.39 (d, J ) 8.2 Hz, 1H), 7.44 (d, J ) 1.9 Hz, 1H), 7.94

(dd, J ) 0.8, 1.8 Hz, 1H), 9.62 (s, 1H); IR (KBr) 3360, 1701,

1662, 1432, 1316 cm^-1; MS (ES⁺) 275.1 (M + 1). Anal.

(C₁₄H₁₄N₂O₄) C, H, N.

3-[(Am in oim in om eth yl)a m in o]-4-(2-fu r oyla m in o)ben -

zoic Acid (154). A mixture of 0.52 g (0.0019 mol) of 152, 1.56

g (0.037 mol) of cyanamide, 10 drops of concentrated HCl, and

100 mL of EtOAc was heated at reflux for 14 h. The resulting

precipitate was collected by filtration and dried to give 0.4 g

(58%) of ethyl 3-[(aminoiminomethyl)amino]-4-(2-furoylamino)-

benzoate hydrochloride (153) as an off-white solid.

Benzoic Acids as Influenza Inhibitors

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 25 4047

A mixture of 0.3 g (0.0009 mol) of crude 153 and 2 mL of 1

N NaOH was stirred at ambient temperature for 16 h. An

additional 1 mL of 1 N NaOH was added and the mixture

heated at 50 °C for 6 h. The mixture was filtered, and the

filtrate pH was adjusted to 8 with 1 N HCl. The resulting

precipitate was collected by filtration and then dissolved in a

minimum amount of NH₄OH. The pH was adjusted to 10 with

6 N HCl, and the resulting precipitate was collected by

filtration and dried to yield 0.04 g (20%) of 154 as a tan solid:

mp 227-228 °C; ¹H NMR (400 MHz, ND₄OD) δ 7.20 (dd, J )

1.8, 3.5 Hz, 1H), 7.78 (dd, J ) 3.5, 0.8 Hz, 1H), 8.03 (d, J )

1.8 Hz, 1H), 8.11 (dd, J ) 8.5, 1.8 Hz, 1H), 8.25 (dd, J ) 0.8,

1.8 Hz, 1H), 8.49 (d, J ) 8.5 Hz, 1H); IR (KBr) 3455, 3424,

1693, 1511, 1366 cm^-1; MS (ES⁺) 289.1 (M + 1). Anal.

(C₁₃H₁₂N₄O₄) C, H, N.

°C for 16 h. The precipitate which formed was collected by

filtration, and the cake was mixed with 0.4 g (0.01 mol) of

cyanamide and 10 mL of EtOAc. The mixture was stirred

overnight at 37-38 °C. The resulting precipitate was collected

by filtration, washed with EtOAc, and recrystallized from

MeOH-Et₂O to give 0.12 g (40%) of 160 as a white solid: mp

>300 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 2.80 (d, J ) 4.6 Hz,

3H), 3.17 (s, 0.75H), 7.72-7.97 (m, 7H), 8.76 (q, J ) 4.6 Hz,

1H), 9.87 (s, 1H), 13.38 (br s, 1H); IR (KBr) 3379, 3164, 1719,

1679, 1646, 1544 cm^-1; MS (ES⁺) 237 (M + 1). Anal.

(C₁₀H₁₂N₄O₃‚HCl‚H₂O‚0.25MeOH) C, H, N.

Meth yl 3-Am in o-4-(a m in osu lfon yl)ben zoa te (162).

A

mixture of 0.65 g (0.0025 mol) of methyl 4-(aminosulfonyl)-3-

nitrobenzoate (161)⁴⁹and 0.12 g of PtO₂in 20 mL of EtOAc

was hydrogenated at 50 psi for 1 h. The mixture was filtered

through Celite, and the cake was washed with five 5-mL

portions of MeOH. The combined filtrates were concentrated

to give 0.49 g (85%) of 162. An analytical sample, mp 211-

213 °C, was obtained as a light orange solid by recrystallization

from 95% EtOH: ¹H NMR (360 MHz DMSO-d₆) δ 3.83 (s, 3H),

6.07 (s, 2H), 7.12 (dd, J ) 1.5, 8.3 Hz, 1H), 7.39 (s, 2H), 7.44

(d, J ) 1.5 Hz, 1H), 7.63 (d, J ) 8.3 Hz, 1H); IR (KBr) 3453,

3365, 3269, 1709, 1432, 1329 cm^-1; MS (ES^-) 229 (M - 1).

Anal. (C₈H₁₀N₂O₄S) C, H, N.

3-[(Am in oim in om eth yl)a m in o]-4-(a m in osu lfon yl)ben -

zoic Acid (164). A mixture of 0.4 g (0.0018 mol) of 162, 1.9

g (0.045 mol) of cyanamide, 0.23 mL of concentrated HCl, and

10 mL of EtOAc was heated at reflux for 6 h. The solution

was cooled, the resulting solid was collected by filtration, and

the cake was washed thrice with 10-mL portions of EtOAc and

then dried to give 0.44 g (79%) of methyl 3-[(aminoiminometh-

yl)amino]-4-(aminosulfonyl)benzoate hydrochloride (163) as a

white solid.

A mixture of 0.3 g (0.001 mol) of the above crude ester 163

and 5 mL of 1 N NaOH was stirred at ambient temperature

for 1 h. The mixture was filtered, and the filtrate pH was

adjusted to 6 using 1 N HCl. The resulting precipitate was

collected by filtration and dried to give 0.2 g (78%) of 164

contaminated with urea. An analytical sample, mp >320 °C,

was isolated as a white solid by slurrying the solid thrice with

5-mL portions of fresh MeOH for 15 min and then drying the

solid: ¹H NMR (360 MHz, CF₃CO₂D) δ 8.34 (m, 3H); IR (KBr)

3443, 3668, 1698, 1620, 1381, 1332, 1159 cm^-1; MS (no

ionization). Anal. (C₈H₁₀N₄O₄S‚0.25H₂O) C, H; N: calcd,

21.33; found, 21.88.

3-Am in o-4-(2-h yd r oxyeth yl)ben zoic Acid (166). A mix-

ture of 0.9 g (0.0043 mol) of 4-(2-hydroxyethyl)-3-nitrobenzoic

acid (165)⁵⁰and 0.05 g of PtO₂in 20 mL of EtOH was

hydrogenated at 30 psi for 3 h. The mixture was filtered

through Celite and the filtrate concentrated to give 0.8 g

(quantitative) of 166. An analytical sample, mp 173-174 °C,

was isolated as an off-white solid from a recrystallization from

EtOH: ¹H NMR (400 MHz, DMSO) δ 2.65 (t, J ) 6.8 Hz, 2H),

3.62 (t, J ) 6.8 Hz, 2H), 4.69 (br s, 1H), 5.12 (br s, 2H), 7.03

(d, J ) 7.7 Hz, 1H), 7.09 (dd, J ) 7.7, 1.6 Hz, 1H), 7.25 (d, J

) 1.6 Hz, 1H), 12.5 (br s, 1H); IR (KBr) 3391, 3225, 2878, 1680,

1581, 1444, 1314, 1019 cm^-1; MS (ES⁺) 182.3 (M + 1). Anal.

(C₉H₁₁NO₃) C, H, N.

4-(Meth oxyca r bon yl)-2-n itr oben zoic Acid (156). A mix-

ture of 20 g (0.095 mol) of nitroterephthalic acid (155; Aldrich)

and 10 mL of H₂SO₄in 100 mL of anhydrous MeOH was

heated at reflux for 1 h. The mixture was concentrated, and

the residue was poured into 200 mL of a saturated NaHCO₃

solution. The mixture was washed with CHCl₃, and then the

pH of the aqueous layer was adjusted to 2 with 2 N HCl. The

mixture was extracted twice with approximately 200-mL

portions of EtOAc, and the combined organic layers were

washed with H₂O and brine and dried (Na₂SO₄). The residue

was recrystallized from EtOAc-hexane to give 11.8 g (55%)

1

of 156 as cream-colored crystals: mp 131-132 °C; H NMR

(400 MHz, DMSO-d₆) δ 3.93 (s, 3H), 7.98 (d, J ) 8.2 Hz, 1H),

8.30 (dd, J ) 1.6, 8.0 Hz, 1H), 8.43 (d, J ) 1.6 Hz, 1H), 14.23

(br s, 1H); IR (KBr) 3115, 1720, 1545, 1501, 1438, 1358 cm^-1

;

MS (ES^-) 224 (M - 1). Anal. (C₉H₇NO₆) C, H, N.

Met h yl 4-[(Met h yla m in o)ca r b on yl]-3-n it r ob en zoa t e

(157). A mixture of 3.6 g (0.016 mol) of 156, 2 drops of DMF,

and 32 mL of SOCl₂was heated at reflux for 3 h. The mixture

was concentrated, and the residue was dissolved in 10 mL of

CH₂Cl₂. This solution was added dropwise over a 5-min period

to a stirred solution of 2 g (0.026 mol) of 40% aqueous MeNH₂

and 5 mL of H₂O. The mixture was stirred for 10 min resulting

in the formation of a precipitate. The solid was collected by

filtration, washed with H₂O, dried, and recrystallized from

EtOAc-hexane to give 2.1 g (55%) of 157 as white needles:

1

mp 155 °C; H NMR (400 MHz, DMSO-d₆) δ 2.77 (d, J ) 4.6

Hz, 3H), 3.92 (s, 3H), 7.76 (d, J ) 8.0 Hz, 1H), 8.29 (dd, J )

8.0, 1.5 Hz, 1H), 8.45 (d, J ) 1.5 Hz, 1H), 8.73 (d, J ) 4.6 Hz,

1H); IR (KBr) 3277, 1727, 1653, 1532, 1294, 1247 cm^-1; MS

(ES⁺) 339 (M + 1). Anal. (C₁₀H₁₀N₂O₅) C, H, N.

Meth yl 3-Am in o-4-[(m eth yla m in o)ca r bon yl]ben zoa te

(158). A mixture of 3.8 g (0.01 mol) of 157 and 15.3 g (0.08

mol) of SnCl₂in 80 mL of EtOH was heated at 70 °C for 1 h.

The reaction mixture was diluted with 150 mL of H₂O, and a

saturated NaHCO₃solution was added to adjust the pH to 8.

The mixture was extracted with about 200 mL of EtOAc. The

organic layer was washed with H₂O and brine, dried (Na₂SO₄),

and concentrated. The residue was recrystallized from EtOAc-

hexane to give 2.2 g (64%) of 158 as tan needles: mp 167-

1

168 °C; H NMR (400 MHz, DMSO-d₆) δ 2.74 (d, J ) 4.6 Hz,

3H), 3.82 (s, 3H), 6.59 (s, 2H), 7.04 (dd, J ) 1.7, 8.2 Hz, 1H),

7.35 (d, J ) 1.7 Hz, 1H), 7.53 (d, J ) 8.2 Hz, 1H), 8.35 (m,

1H); IR (KBr) 3464, 3317, 1715, 1637, 1589, 1553 cm^-1; MS

(ES⁺) 209 (M + 1). Anal. (C₁₀H₁₂N₂O₃) C, H, N.

Meth yl 3-Am in o-4-(2-h yd r oxyeth yl)ben zoa te (167). To

a cooled (ice bath) solution of acidic MeOH (prepared from 50

mL of anhydrous MeOH and 5 mL of acetyl chloride) was

added 0.86 g (0.0047 mol) of 166, and the resulting solution

stirred at ambient temperature overnight. A small amount

of the hydrochloride, mp 217 °C, had precipitated so it was

collected by filtration and used to characterize 167 hydrochlo-

ride. The filtrate was concentrated, and the residue was

treated with 5 mL of H₂O and 25 mL of EtOAc. The pH was

adjusted to 7-8 with concentrated NH₄OH. The layers were

separated, and the aqueous layer was extracted thrice with

10-mL portions of EtOAc. The combined organic layers were

dried (Na₂SO₄) and concentrated to give 1.0 g (91%) of 167 as

a gum: ¹H NMR (400 MHz, DMSO-d₆) δ 2.90 (t, J ) 6.4 Hz,

2H), 3.75 (t, J ) 6.4 Hz, 2H), 7.45 (d, J ) 8.0 Hz, 1H), 7.8 (dd,

J ) 8.0, 1.7 Hz, 1H), 7.98 (d, J ) 1.7 Hz, 1H); IR (KBr) 3555,

2956, 2585, 2036, 1723, 1490, 1281 cm^-1; MS (ES⁺) 196.3 (M

+ 1). Anal. (C₁₀H₁₃NO₃‚HCl) C, H, N.

3-Am in o-4-[(m eth ylam in o)car bon yl]ben zoic Acid (159).

A mixture of 0.62 g (0.003 mol) of 158 and 4.5 mL of 1 N NaOH

was stirred at ambient temperature for 4 h. The mixture was

filtered, the filtrate was neutralized with concentrated HCl,

and the pH was then adjusted to 3 with HOAc. The resulting

precipitate was collected by filtration, washed with H₂O, and

dried to give 0.4 g (69%) of 159 as a pale yellow powder: mp

215-217 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 2.74 (d, J ) 4.5

Hz, 3H), 6.54 (s, 2H), 7.02 (dd, J ) 1.6, 8.2 Hz, 1H), 7.32 (d, J

) 1.6 Hz, 1H), 7.51 (d, J ) 8.2 Hz, 1H), 8.32 (m, 1H), 12.86

(br s, 1H); IR (KBr) 3455, 3423, 1697, 1628, 1589, 1538 cm^-1

;

MS (ES⁺) 195 (M + 1). Anal. (C₉H₁₀N₂O₃) C, H, N.

3-[(Am in oim in om et h yl)a m in o]-4-[(m et h yla m in o)ca r -

bon yl]ben zoic Acid (160). A mixture of 0.19 g (0.001 mol)

of 159, 0.59 g (0.014 mol) of cyanamide, 0.08 g (0.001 mol) of

concentrated HCl, and 10 mL of EtOAc was stirred at 37-38

4048 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 25

Chand et al.

Meth yl 3-[[[(ter t-Bu toxyca r bon yl)a m in o][(ter t-bu toxy-

ca r bon yl)im in o]m et h yl]a m in o]-4-(2-h yd r oxyet h yl)b en -

zoa te (168). To a solution of 0.75 g (0.0039 mol) of 167 in 3.5

mL of DMF were added 1.9 mL (0.014 mol) of Et₃N and 1.1 g

(0.004 mol) of N,N-bis(tert-butoxycarbonyl)thiourea.⁴¹The

mixture was cooled to 0 °C and treated with 1.2 g (0.0043 mol)

of HgCl₂. The reaction mixture was stirred at 0 °C for 0.5 h

and then at ambient temperature for 24 h. The mixture was

diluted with 30 mL of EtOAc and filtered through Celite. The

filtrate was washed thrice with 20-mL portions of H₂O, dried

(Na₂SO₄), and concentrated. The residue was purified by flash

column chromatography on silica gel (20-30% EtOAc in

hexane) to give 1 g (64%) of 168 as a white solid: mp 147-

was stirred at 0 °C for 0.5 h and then at ambient temperature

overnight. The mixture was diluted with 50 mL of EtOAc and

filtered through Celite. The filtrate was washed successively

with 10 mL of H₂O and 10 mL of brine, dried (MgSO₄), and

concentrated. The residue was purified by flash column

chromatography on silica gel (30% hexane in EtOAc) to give

0.63 g (54%) of 172 as a white solid: mp 140-141 °C (EtOAc-

1

hexane); H NMR (400 MHz, DMSO-d₆) δ 1.32 (s, 9H), 1.51

(s, 9H), 2.53 (s, 3H), 3.87 (s, 3H), 4.07 (d, J ) 13.0 Hz, 1H),

4.31 (d,J ) 13.0 Hz, 1H), 7.54 (d,J ) 8.0 Hz, 1H), 7.87 (dd, J

) 8.0, 1.7 Hz, 1H), 8.05 (d, J ) 1.7 Hz, 1H), 9.95 (s, 1H), 11.46

(s, 1H); IR (KBr) 3421, 3262, 3122, 1728, 1648, 1591, 1404,

1307, 1232, 1130, 1060 cm^-1; MS (ES⁺) 470.9 (M + 1). Anal.

(C₂₁H₃₁N₃O₇S) C, H, N.

1

150 °C; H NMR (400 MHz, DMSO-d₆) δ 1.35 (s, 9H), 1.55 (s,

9H), 2.76 (t, J ) 6.3 Hz, 2H), 3.65 (m, 2H), 3.9 (s, 3H), 4.83 (t,

J ) 4.5 Hz, 1H), 7.45 (d, J ) 8.0 Hz, 1H), 7.80 (dd, J ) 8.0,

1.6 Hz, 1H), 8.05 (d, J ) 1.6 Hz, 1H), 9.98 (s, 1H), 11.55 (s,

1H); IR (KBr) 3559, 3266, 3127, 3011, 2971, 1724, 1650, 1596,

1459, 1291, 1230, 1163, 1127, 1057, 760 cm^-1; MS (ES⁺) 438.6

(M + 1). Anal. (C₂₁H₃₁N₃O₇) C, H, N.

3-[(Am in oim in om eth yl)am in o]-4-[(m eth ylsu lfoxy)m eth -

yl]ben zoic Acid (173). A solution of 0.4 g (0.0009 mol) of

172 in 8.5 mL of CH₂Cl₂was treated dropwise with 1.6 mL

(0.021 mol) of TFA and stirred at ambient temperature for 32

h. The solution was treated with 1.3 mL (0.017 mol) of TFA

and stirred for 1 h. The mixture was concentrated, and the

residue was chased twice with 10-mL portions of CH₂Cl₂. A

mixture of the residue and 4.3 mL of 1 N NaOH was stirred

at ambient temperature for 2 h. The mixture was filtered,

and the filtrate volume was reduced to 2 mL. The pH was

adjusted to 10-10.5 with 1 N HCl, and the resulting precipi-

tate was collected by filtration and dried to give 0.13 g (59%)

3-[(Am in oim in om eth yl)a m in o]-4-(2-h yd r oxyeth yl)ben -

zoic Acid (169). To a solution of 0.9 g (0.002 mol) of 168 in

20 mL of CH₂Cl₂at 0 °C was added dropwise 3.3 mL (0.04

mol) of TFA, and the mixture stirred at ambient temperature

for 32 h. An additional 0.8 mL (0.011 mol) of TFA was added,

and the mixture stirred for 1 h. The mixture was filtered, the

filtrate concentrated, and the residue chased twice with 10-

mL portions of CH₂Cl₂. The residue was stirred with 18 mL

of 3 N NaOH for 1 h. The mixture was filtered, and the filtrate

pH was adjusted to 5 using concentrated HCl and then

readjusted to 7 using 1 N NaOH. The precipitate was collected

by filtration, successively washed with small amounts of

MeOH and Et₂O, and dried to give 0.29 g (59%) of 169 as a

white solid: mp >270 °C dec; ¹H NMR (400 MHz, NH₄OD)

3.22 (t, J ) 7.0 Hz, 2H), 4.15 (t, J ) 7.0 Hz, 2H), 7.75 (d, J )

8.0 Hz, 1H), 7.81 (d, J ) 1.7 Hz, 1H), 7.98 (dd, J ) 1.7, 8.0 Hz,

1H); IR (KBr) 3413, 3343, 3078, 2870, 1689, 1665, 1533, 1389,

1069 cm^-1; MS (ES^-) 222.4 (M - 1). Anal. (C₁₀H₁₃N₃O₃‚

0.25H₂O) C, H, N.

Met h yl 4-[(Met h ylsu lfoxy)m et h yl]-3-n it r ob en zoa t e

(170). To 100 mL of concentrated H₂SO₄cooled to 0-5 °C was

added 5.3 g (0.025 mol) of 55. This suspension was treated in

five portions over a 30-min period with 12.6 g (0.125 mol) of

KNO₃. The mixture was stirred at 4 °C for 66 h and then

poured in 1 L of ice H₂O. The mixture was extracted with

five 150-mL portions of EtOAc, and the combined extracts were

washed with 100 mL of 10% NH₄OH and 100 mL of brine,

dried (MgSO₄), and concentrated. The residue was purified

by flash column chromatography on silica gel (20-0% hexane

in EtOAc) to give 2.7 g (42%) of 170 as a white solid: mp 129-

130 °C (EtOH); ¹H NMR (400 MHz, CDCl₃) δ 2.67 (s, 3H), 3.99

(s, 3H), 4.13 (d, J ) 12.6 Hz, 1H), 4.64 (d, J ) 12.6 Hz, 1H),

7.63 (d, J ) 8.0 Hz, 1H), 8.29 (dd, J ) 8.0, 1.7 Hz, 1H), 8.77

(d, J ) 1.7 Hz, 1H); IR (KBr) 3411, 3012, 2944, 1714, 1531,

1440, 1270 cm^-1; MS (ES^-) 256.4 (M - 1). Anal. (C₁₀H₁₁NO₅)

C, H, N.

Met h yl 3-Am in o-4-[(m et h ylsu lfoxy)m et h yl]b en zoa t e

(171). A mixture of 0.63 g (0.0025 mol) of 170, 0.12 g of PtO₂,

and 25 mL of MeOH was hydrogenated at 50 psi for 1 h. The

mixture was filtered through Celite, and the filtrate was

concentrated. The residue was purified by flash column

chromatography on silica gel (10-30% CHCl₃-MeOH-NH₄-

OH (80:18:2) in CH₂Cl₂) to give 0.2 g (35%) of 171. An

analytical sample, mp 161-162 °C, was obtained as a yellow

solid by recrystallization from EtOH: ¹H NMR (400 MHz,

CDCl₃) δ 2.55 (s, 3H), 3.89 (d, J ) 13.6 Hz, 1H), 3.90 (s, 3H),

4.19 (d, J ) 13.6 Hz, 1H), 7.04 (d, J ) 8.2 Hz, 1H), 7.40 (d, J

) 1.7 Hz, 1H), 7.42 (s, 1H); IR (KBr) 3441, 3341, 3237, 1700,

1660, 1426; MS (ES⁺) 228.4 (M + 1). Anal. (C₁₀H₁₃NO₃S) C,

H, N.

Meth yl 3-[[[(ter t-Bu toxyca r bon yl)a m in o][(ter t-bu toxy-

ca r bon yl)im in o]m eth yl]a m in o]-4-[(m eth ylsu lfoxy)m eth -

yl]ben zoa te (172). A mixture of 0.57 g (0.0025 mol) of 171,

5 mL of DMF, 1.2 mL (0.009 mol) of Et₃N, and 0.8 g (0.003

mol) of N,N′-bis(tert-butoxycarbonyl)thiourea⁴¹at 0 °C was

treated with 0.8 g (0.003 mol) of HgCl₂. The reaction mixture

1

of 173 as a white solid: mp 211-213 °C; H NMR (400 MHz,

CF₃CO₂D) δ 2.95 (s, 3H), 4.36 (d, J ) 13.8 Hz, 1H), 4.73 (d, J

) 13.8 Hz, 1H), 7.76 (d, J ) 8.1 Hz, 1H), 8.30-8.40 (m, 1H);

IR (KBr) 3502, 3356, 3140, 1684, 1640, 1404, 1382, 1012; MS

(ES⁺) 256.2 (M + 1). Anal. (C₁₀H₁₃N₃O₃S‚H₂O) C, H, N.

Met h yl 4-[(Met h ylsu lfon yl)m et h yl]-3-n it r ob en zoa t e

(174). To 50 mL of 90% fuming HNO₃at 0-5 °C was added

portionwise over a 20-min period 4.2 g (0.019 mol) of 56. The

reaction mixture was stirred at 0-5 °C for 0.5 h and at

ambient temperature overnight and then poured into 1 L of

cold H₂O. The mixture was extracted with four 200-mL

portions of EtOAc, and the combined extracts were washed

successively with 100 mL of a saturated Na₂CO₃solution and

200 mL of brine, dried (MgSO₄), and concentrated. The

residue was purified by flash column chromatography on silica

gel (25-0% hexane in Et₂O) to give 3.8 g (75%) of 174 as a

light yellow solid: mp 114-115 °C; ¹H NMR (400 MHz) δ 3.04

(s, 3H), 3.93 (s, 3H), 5.08 (s, 2H), 7.84 (d, J ) 8.0 Hz, 1H),

8.32 (dd, J ) 8.0, 1.7 Hz, 1H), 8.49 (d, J ) 1.7 Hz, 1H); IR

(KBr) 3103, 3019, 2961, 2928, 1727, 1538, 1298, 1141 cm^-1

;

MS (ES^-) 272.7 (M - 1). Anal. (C₁₀H₁₁NO₆S) C, H, N.

A 1.3-g (25%) sample of the 2-nitro isomer 175 was also

isolated as a light yellow solid: mp 115-117 °C; ¹H NMR (400

MHz, DMSO-d₆) δ 2.99 (s, 3H), 3.33 (s, 3H), 3.87 (s, 2H), 7.86

(dd, J ) 8.0, 1.5 Hz, 1H), 7.92 (d, J ) 8.0 Hz, 1H), 8.13 (d, J

) 1.4 Hz, 1H); IR (KBr) 3010, 1747, 1534, 1306, 1122 cm^-1

;

MS (ES^-) 272.1 (M - 1). Anal. (C₁₀H₁₁NO₆S) C, H, N.

Meth yl 3-Am in o-4-[(m eth ylsu lfon yl)m eth yl]ben zoa te

(176). A mixture of 1.2 g (0.004 mol) of 174, 0.2 g of PtO₂,

and 50 mL of MeOH was hydrogenated at 50 psi for 1 h. The

mixture was filtered through Celite, and the volume of the

filtrate was reduced to 20 mL. The hot solution was slowly

cooled to give 0.5 g (48%) of 176 as a tan solid: mp 151-153

°C; ¹H NMR (400 MHz, DMSO-d₆) δ 2.93 (s, 3H), 3.81 (s, 3H),

4.47 (s, 2H), 5.56 (br s, 2H), 7.14 (dd, J ) 7.9, 1.7 Hz, 1H),

7.22 (d, J ) 7.9 Hz, 1H), 7.35 (d, J ) 1.7 Hz, 1H); IR (KBr)

3447, 3364, 3014, 2929, 1705, 1580, 1435, 1289, 1252, 1133

cm^-1; MS (ES⁺) 244.2 (M + 1). Anal. (C₁₀H₁₃NO₄S) C, H, N.

Meth yl 3-[[[(ter t-Bu toxyca r bon yl)a m in o][(ter t-bu toxy-

car bon yl)im in o]m eth yl]am in o]-4-[(m eth ylsu lfon yl)m eth -

yl]ben zoa te (177). A mixture of 2.3 g (0.0094 mol) of 176,

20 mL of DMF, 4.6 mL of Et₃N (0.033 mol), and 2.9 g (0.01

mol) of N,N′-bis(tert-butoxycarbonyl)thiourea⁴¹stirred at 0 °C

was treated with 2.8 g (0.01 mol) of HgCl₂. The mixture was

stirred at 0 °C for 0.5 h and then at ambient temperature

overnight. The mixture was diluted with 50 mL of EtOAc and

filtered through Celite. The filtrate was washed thrice with

25-mL portions of H₂O and twice with 25-mL portions of brine,

dried (MgSO₄), and concentrated. The residue was purified

by flash column chromatography on silica gel (30-0% hexane

Benzoic Acids as Influenza Inhibitors

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 25 4049

in Et₂O) to give 2.7 g (60%) of 177 as a white solid: mp 145-

148 °C; ¹H NMR (400 MHz, CDCl₃) δ 1.44 (s, 9H), 1.55 (s, 9H),

2.92 (s, 3H), 3.93 (s, 3H), 4.40 (s, 2H), 7.60 (d,J ) 8.0 Hz, 1H),

7.94 (d, J ) 8.0 Hz, 1H), 8.28 (s, 1H), 10.15 (s, 1H), 11.62 (s,

1H); IR (KBr) 3269, 3134, 2983, 1727, 1650, 1403, 1233, 1156,

1132 cm^-1; MS (ES⁺) 486.7 (M + 1). Anal. (C₂₁H₃₁N₃O₈S) C,

H, N.

3,5-Bis[(a m in oim in om eth yl)a m in o]ben zoic Acid (184).

A mixture of 2.3 g (0.01 mol) of 3,5-diaminobenzoic acid

dihydrochloride semihydrate (179; Aldrich) and 2.5 g (0.06 mol)

of cyanamide in 50 mL of absolute EtOH was heated at reflux

for 8 h. The mixture was cooled, the precipitate collected by

filtration, and the cake washed with several portions of EtOH

and dried to give 2.2 g (73%) of 184 as an off-white powder:

mp 280 °C dec; ¹H NMR (400 MHz, D₂O) δ 1.20 (t, J ) 7.0 Hz,

0.6H), 3.67 (q, J ) 7.0 Hz, 0.4H), 7.42 and 7.72 (aromatic

protons, 3H); IR (KBr) 3320, 3160, 1672, 1560, 1379 cm^-1; MS

(ES⁺) 237 (M + 1). Anal. (C₉H₁₂N₆O₂‚1.5HCl‚0.2EtOH) C, H,

N.

3-[(Am in oim in om et h yl)a m in o]-4-[(m et h ylsu lfon yl)-

m eth yl]ben zoic Acid (178). A solution of 0.49 g (0.001 mol)

of 177 in 10 mL of CH₂Cl₂was treated dropwise with 1.9 mL

(0.025 mol) of TFA, and the mixture stirred at ambient

temperature for 16 h. An additional 1.9 mL (0.025 mol) of

TFA was added, and the mixture stirred for 1 h. The mixture

was concentrated, and the residue was chased twice with 10-

mL portions of CH₂Cl₂. A mixture of the residue and 5 mL of

1 N NaOH was stirred at ambient temperature for 1.5 h. The

mixture was filtered, and the filtrate was concentrated to 1

mL. The pH was adjusted to 6 using HOAc. The resulting

precipitate was collected by filtration, washed with a 1:1

solution of 2-PrOH-Et₂O, and dried to give 0.22 g (88%) of

178 as a white solid: mp 261-264 °C; ¹H NMR (400 MHz,

CF₃CO₂D) δ 3.22 (s, 3H), 4.79 (s, 2H), 7.75 (d, J ) 8.0 Hz, 1H),

7.25 (s, 1H), 8.29 (d, J ) 8.0 Hz, 1H); IR (KBr) 3538, 3461,

3365, 3163, 1684, 1621, 1404, 1386, 1291, 1133 cm^-1; MS (ES^-)

269.9 (M - 1). Anal. (C₁₀H₁₃N₃O₄S‚H₂O) C, H, N.

Meth yl 3-F or m yl-5-n itr oben zoa te (186) a n d Meth yl

3-(Hyd r oxym eth yl)-5-n itr oben zoa te (187). To a solution

of 1.5 g (0.02 mol) of DMF in 30 mL of CH₂Cl₂at 0 °C was

added 5 mL (0.057 mol) of oxalyl chloride, and the mixture

stirred at ice bath temperature for 1 h. The solution was

concentrated, and the residue was suspended in 50 mL of THF

and 30 mL of MeCN. To this mixture at -30 °C was added

dropwise a solution of 4.5 g (0.02 mol) of 3-(methoxycarbonyl)-

5-nitrobenzoic acid (185)⁵¹

and 1.6 g (0.02 mol) of pyridine in

10 mL of THF over a 10-min period. The reaction mixture

was stirred at -30 °C for 1 h and then treated with a

suspension of 0.38 g (0.002 mol) of CuI in 10 mL of THF. The

reaction mixture was cooled to -70 °C, and 40 mL (0.04 mol)

of a 1 M solution in THF of lithium tri-tert-butoxyaluminohy-

dride (Aldrich) was added dropwise over a 10-min period. The

mixture was stirred for an additional 10 min at -78 °C and

then quenched with 50 mL of 2 N HCl. The layers were

separated, and the organic layer was washed with a NaHCO₃

solution, dried (Na₂SO₄), and concentrated. The residue was

purified by column chromatography on silica gel (33% EtOAc

in hexane) to give 0.25 g (6%) of 186 as an off-white solid: mp

85 °C; ¹H NMR (400 MHz, CDCl₃) δ 4.05 (s, 3H), 8.86 (d, J )

1.5 Hz, 1H), 8.9 (dd, J ) 2.2, 1.5 Hz, 1H), 9.09 (dd, J ) 2.2,

1.6 Hz, 1H), 8.19 (s, 1H); IR (KBr) 3076, 2962, 2872, 1736,

1703, 1616, 1540, 1355, 1295, 1206, 1184 cm^-1; MS (ES^-) 209.9

(M - 1, 5). Anal. (C₉H₇NO₅) C, H, N.

3,5-Bis(cya n oa m in o)ben zoic Acid (180). To a mixture

of 3.8 g (0.025 mol) of 3,5-diaminobenzoic acid (179; Aldrich)

and 4.1 g (0.05 mol) of NaOAc in 30 mL of a 1:1 solution of

HOAc-H₂O at 0 °C was added 6.5 g (0.06 mol) of cyanogen

bromide in two portions over a 10-min period. The reaction

mixture was stirred at ambient temperature overnight and

then poured onto 130 g of ice. The precipitate was collected

by filtration, washed with H₂O, and dried. One recrystalli-

zation from MeOH gave 2.5 g (50%) of 180 as a gray solid:

1

mp >300 °C dec; H NMR (400 MHz, DMSO-d₆) δ 6.80-7.18

(m, 3H), 10.68 (br s, 2H), 13.0 (br s, 1H); IR (KBr) 3173, 2229,

1737, 1617, 1519, 1453 cm^-1; MS (ES^-) 201 (M - 1). Anal.

(C₉H₆N₄O₂) C, H, N.

Other fractions when combined and concentrated gave 2.3

g (55%) of 187 as an off-white solid: mp 77 °C; ¹H NMR (400

MHz, CDCl₃) δ 2.4 (br s, 1H), 3.99 (s, 3H), 4.88 (s, 2H), 8.35

(m, 1H), 8.44 (m, 1H), 8.76 (m, 1H); IR (KBr) 3497, 3096, 2961,

1722, 1528, 1355, 1300, 1216, 1062, 738 cm^-1; MS (ES^-) (no

ionization). Anal. (C₉H₉NO₅) C, H, N.

Another run using the exact procedure as above except that

only 20 mL (0.02 mol) of a 1 M solution in THF of lithium

tri-tert-butoxyaluminohydride was added gave 1.4 g (33%) of

186 and 0.8 g (19%) of 187.

Eth yl 3-Am in o-5-[[[(ter t-bu toxyca r bon yl)a m in o][(ter t-

bu toxyca r bon yl)im in o]m eth yl]a m in o]ben zoa te (182). To

a mixture of 1.8 g (0.01 mol) of ethyl 3,5-diaminobenzoate (181,

Pfaltz & Bauer), 8 mL of DMF, 4.9 mL (0.035 mol) of Et₃N,

and 2.8 g (0.01 mol) of N,N′-bis(tert-butoxycarbonyl)thiourea⁴¹

stirred at 0 °C was added 3 g (0.011 mol) of HgCl₂. The

mixture was stirred at 0 °C for 0.5 h and at ambient

temperature for 24 h and diluted with 75 mL of EtOAc. The

slurry was filtered through Celite; the filtrate was washed with

four 15-mL portions of H₂O, dried (Na₂SO₄), and concentrated.

The residue was purified by flash column chromatography on

silica gel (10-20% EtOAc in hexane) to give 2.5 g (60%) of

182 as a white solid: mp 126-128 °C; ¹H NMR (400 MHz,

DMSO-d₆) δ 1.30 (t, J ) 7.0 Hz, 3H), 1.40 (s, 9H), 1.50 (s, 9H),

4.25 (q, J ) 7.0 Hz, 2H), 5.51 (s, 2H), 6.89 (s, 1H), 7.00 (s,

1H), 7.35 (s, 1H), 9.92 (s, 1H), 11.35 (s, 1H); IR (KBr) 3577,

3410, 3263, 2981, 1710, 1646 cm^-1; MS (ES⁺) 423.6 (M + 1).

Anal. (C₂₀H₃₀N₄O₆) C, H, N.

Meth yl 3-[(N-Hyd r oxylim in o)m eth yl]-5-n itr oben zoa te

(188). A mixture of 0.42 g (0.002 mol) of 186 and 0.21 g (0.003

mol) of hydroxylamine hydrochloride in 5 mL of EtOH was

heated at reflux for 2 h. The mixture was poured into H₂

O,

and the resulting precipitate was collected by filtration. The

cake was recrystallized from EtOAc-hexane to give 0.22 g

(49%) of 188 as an off-white solid: mp 144-145 °C;

¹H NMR

(400 MHz, DMSO-d₆

) δ 3.94 (s, 3H), 8.43 (s, 1H), 8.56 (m, 2H),

8.65 (m, 1H), 11.84 (s, 1H); IR (KBr) 3406, 3105, 3067, 2960,

1705, 1531, 1305, 1276, 1221, 978 cm^-1; MS (ES^-) 223.4 (M -

1). Anal. (C₉H₈N₂O₅) C, H, N.

Sod iu m 3-Am in o-5-[(a m in oim in om et h yl)a m in o]ben -

zoa te (183). To a solution of 1.8 g (0.0043 mol) of 182 in 20

mL of CH₂Cl₂at 0 °C was added dropwise 3.3 mL (0.043 mol)

of TFA, and the solution stirred at ambient temperature for 6

h. An additional 3.3 mL (0.043 mol) of TFA was added, and

the mixture stirred overnight. The mixture was concentrated,

and the residue was chased twice with 20-mL portions of CH₂-

Cl₂. A mixture of the residue and 22.5 mL (0.068 mol) of 3 N

NaOH was stirred at ambient temperature for 0.75 h, and then

the pH was adjusted to 7-8 with 1 N HCl. The solution was

filtered, and the filtrate was concentrated to one-half its

volume. The filtrate was allowed to stand at ambient tem-

perature for 2 h, and the resulting precipitate was collected

by filtration, washed successively with a small amount of

MeOH and Et₂O, and dried to give 0.66 g (66%) of 183 as a

Meth yl 3-Am in o-5-(a m in om eth yl)ben zoa te (189).

A

mixture of 0.1 g (0.0005 mol) of 188, 0.02 g of 10% Pd/C, 30

mL of EtOH, and 0.5 mL of concentrated HCl was hydroge-

nated at 40 psi for 2 h. The mixture was filtered through

Celite and the filtrate concentrated. The residue was recrys-

tallized from EtOH-toluene-H₂O to give 0.09 g (76%) of 189

as a white solid: mp 224-226 °C; ¹H NMR (600 MHz, DMSO-

d₆) δ 3.85 (s, 3H), 4.01 (m, 2H), 7.30 (s, 1H), 7.58 (s, 1H), 7.69

(s, 1H), 8.47 (br s, 4H); IR (KBr) 3440, 3024, 2577, 1725, 1314,

1228 cm^-1; MS (ES⁺) 181.5 (M + 1). Anal. (C₉H₁₂N₂O₂‚

2HCl‚0.75H₂O) C, H, N.

Met h yl 3-Am in o-5-[[[[(ter t-b u t oxyca r b on yl)a m in o]-

[(ter t-bu toxyca r bon yl)im in o]m eth yl]a m in o]m eth yl]ben -

zoa te (190). A mixture of 0.76 g (0.003 mol) of 189, 6 mL of

DMF, 2.5 mL (0.018 mol) of Et₃N, and 2.1 g (0.008 mol) of

N,N′-bis(tert-butoxycarbonyl)thiourea⁴¹at 0 °C was treated

with 2 g (0.008 mol) of HgCl₂. The reaction mixture was

1

white solid: mp 198-203 °C; H NMR (400 MHz, DMSO-d₆)

δ 5.2 (s, 2H), 6.45 (s, 1H), 6.9 (s, 1H), 7.05 (s, 1H), 8.05 (br s,

4H), 12.1 (br s, 1H); IR (KBr) 3362, 3216, 3006, 1677, 1629,

1599, 1543, 1395, 1201 cm^-1; MS (ES⁺) 195.3 (M + 1). Anal.

(C₈H₉N₄NaO₂‚H₂O) C, H, N.

4050 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 25

Chand et al.

stirred at 0 °C for 0.5 h and at ambient temperature overnight

and then diluted with 50 mL of EtOAc. The slurry was filtered

through Celite and the filtrate washed successively thrice with

25-mL portions of H₂O and twice with 25-mL portions of brine.

The organic layer was dried (MgSO₄) and concentrated, and

the residue was purified by flash column chromatography on

silica gel (30% EtOAc in hexane) to give 0.3 g (25%) of 190 as

a white solid. An analytical sample, mp 150 °C, was recrystal-

lized from Et₂O-hexane: ¹H NMR (400 MHz, DMSO-d₆) δ 1.39

(s, 9H), 1.48 (s, 9H), 3.80 (s, 3H), 4.40 (d, J ) 5.7 Hz, 2H),

5.45 (s, 2H), 6.69 (s, 1H), 7.03 (s, 1H), 7.08 (t, J ) 1.8 Hz, 1H),

8.63 (s, 1H), 11.55 (s, 1H); IR (KBr) 3461, 3335, 2983, 1732,

1713, 1648, 1599, 1309, 1229, 1164, 1140 cm^-1; MS (ES⁺) 423.4

(M + 1). Anal. (C₂₀H₃₀N₄O₆) C, H, N.

3-Am in o-5-[[(a m in oim in om et h yl)a m in o]m et h yl]b en -

zoic Acid (191). A solution of 0.12 g (0.0003 mol) of 190 in

2.8 mL of 6 N HCl was heated at reflux for 1 h. The mixture

was concentrated, and the residue was recrystallized from

EtOH-Et₂O to give 0.04 g (69%) of 191 as a tan solid: mp

245-247 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 4.42 (d, J ) 6.3

Hz, 2H), 7.14 (s, 1H), 7.35 (br s, 5H), 7.51 (d, J ) 6.0 Hz, 1H),

8.19 (t, J ) 6.3 Hz, 1H), 13.0 (br s, 1H); IR (KBr) 8415, 3208,

3162, 3056, 2792, 1685, 1644, 1601, 1237; MS (ES⁺) 209.3 (M

+ 1). Anal. (C₉H₁₂N₄O₂‚2HCl) C, H, N.

160 °C (EtOAc-hexane); ¹H NMR (400 MHz, DMSO-d₆) δ 1.40

(s, 9H), 1.51 (s, 9H), 3.87 (s, 3H), 7.92 (s, 1H), 7.96 (s, 1H),

8.22 (s, 2H), 10.10 (s, 1H), 11.27 (s, 1H), 11.48 (s, 1H); IR (KBr)

3455, 3245, 3134, 2983, 1730, 1650, 1636, 1577, 1397, 1320,

1230, 1151, 1104, 769 cm^-1; MS (ES⁺) 436.9 (M + 1, 40). Anal.

(C₂₀H₂₈N₄O₇) C, H, N.

Other fractions when combined and concentrated gave 0.1

g (11%) of methyl 3-amino-5-cyanobenzoate (196) as a yellow

1

solid byproduct: mp 121 °C (EtOAc-hexane); H NMR (400

MHz, DMSO-d₆) δ 3.84 (s, 3H), 5.97 (s, 2H), 7.09 (dd, J ) 2.3,

1.5 Hz, 1H), 7.35 (dd, J ) 2.0, 1.5 Hz, 1H), 7.45 (dd, J ) 2.3,

1.5 Hz, 1H); IR (KBr) 3456, 3365, 3251, 2235, 1790, 1704, 1600,

1455, 1440, 1265, 1256, 1158, 770 cm^-1; MS (ES^-) 174.9 (M -

1). Anal. (C₉H₈N₂O₂) C, H, N.

3-[(Am in oim in om et h yl)a m in o]-5-[(N-h yd r oxyim in o)-

m eth yl]ben zoic Acid (197). A mixture of 0.36 g (0.0008 mol)

of 195 and 1.5 mL of TFA in 5 mL of CH₂Cl₂was stirred at

ambient temperature for 24 h. The mixture was concentrated,

and the residue was chased repeatedly with CH₂Cl₂.

A

mixture of the residue and 3 mL of 1 N NaOH was stirred at

ambient temperature for 6 h. The mixture was filtered, and

the filtrate pH was adjusted to 8 with 1 N HCl. The resulting

precipitate was collected by filtration, washed with H₂O, and

dried to give 0.125 g (64%) of 197 as an off-white solid: mp

248-253 °C dec; ¹H NMR (400 MHz, DMSO-d₆) δ 7.56 (m, 1H),

7.72 (m, 1H), 7.95 (s, 1H), 8.17 (s, 1H), 8.20 (br s, 4H), 11.29

(s, 1H), 13.01 (s, 1H); IR (KBr) 3339, 3181, 2985, 2364, 2338,

1681, 1653, 1585, 1372, 1328 cm^-1; MS (ES⁺) 222.9 (M + 1).

Anal. (C₉H₁₀N₄O₃‚0.75H₂O) C, H, N.

Meth yl 3-Am in o-5-(h yd r oxym eth yl)ben zoa te (198). A

mixture of 1.9 g (0.009 mol) of 187, 0.1 g of PtO₂, and 100 mL

of MeOH was hydrogenated at 50 psi for 0.5 h. The mixture

was filtered through Celite and the filtrate immediately

acidified with concentrated HCl. The mixture was concen-

trated, and the residue was recrystallized from MeOH-toluene

to give 1.3 g (78%) of 198 as a white solid: mp 203-205 °C;

¹H NMR (400 MHz, DMSO-d₆) δ 3.87 (s, 3H), 4.57 (s, 2H), 7.47

(m, 1H), 7.71 (m, 1H), 7.79 (m, 1H); IR (KBr) 3193, 2860, 2648,

2623, 1710, 1608, 1542, 1328, 1233, 1056 cm^-1; MS (ES⁺) 182.4

(M + 1). Anal. (C₍H₁₁NO₃‚HCl) C, H, N.

Meth yl 3-[[[(ter t-Bu toxyca r bon yl)a m in o][(ter t-bu toxy-

ca r bon yl)im in o]m eth yl]a m in o]-5-(h yd r oxylm eth yl)ben -

zoa te (199). A mixture of 2.9 g (0.013 mol) of 198, 15 mL of

DMF, 13 mL (0. 11 mol) of Et₃N, and 4 g (0.016) of N,N′-bis-

(tert-butoxycarbonyl)thiourea⁴¹at 0-5 °C was treated with 3.6

g (0.013 mol) of HgCl₂. The reaction mixture was stirred at

ambient temperature for 16 h and then diluted with 250 mL

of EtOAc. The mixture was filtered through Celite; the filtrate

was washed with 60 mL of H₂O and concentrated. The residue

was purified by column chromatography on silica gel packed

in CHCl₃(5% MeOH in CHCl₃) to give 4.9 g (86%) of 199 as a

cream-colored solid: mp 80-83 °C; ¹H NMR (400 MHz, CDCl₃)

δ 1.51-1.54 (m, 18H), 3.91 (s, 3H), 4.74 (s, 2H), 7.79 (m, 1H),

8.02 (m, 3H), 10.48 (m, 1H), 11.53 (2s, 1H); IR (KBr) 3434,

2982, 1727, 1646, 1321, 1233, 1152, 1107 cm^-1; MS (ES^-) 422.7

(M - 1). Anal. (C₂₀H₂₉N₃O₇) C, H, N.

Meth yl 3-[[[(ter t-Bu toxyca r bon yl)a m in o][(ter t-bu toxy-

ca r bon yl)im in o]m eth yl]a m in o]-5-[[[[(ter t-bu toxyca r bon -

yl)a m in o][(ter t-b u t oxyca r b on yl)im in o]m et h yl]a m in o]-

m eth yl]ben zoa te (192). A mixture of 1.2 g (0.0045 mol) of

189, 15 mL of DMF, 3.2 mL (0.023 mol) of Et₃N, and 2.5 g

(0.009 mol) of N,N′-bis(tert-butoxycarbonyl)thiourea⁴¹at 0 °C

was treated with 2.4 g (0.009 mol) of HgCl₂. The reaction

mixture was stirred at ambient temperature for 16 h, diluted

with 150 mL of EtOAc, and filtered through Celite. The

filtrate was washed with 60 mL of H₂O, dried (Na₂SO₄), and

concentrated. The residue was dissolved in 150 mL of a 7:3

hexane-EtOAc solution and filtered through a 1′′ × 2′′ silica

gel plug. The filtrate was concentrated, and the residue was

recrystallized from EtOAc-hexane to give 1.9 g (61%) of 192

as a white solid: mp 114-118 °C; ¹H NMR (600 MHz, CDCl₃)

δ 1.51 (m, 36H), 3.91 (s, 3H), 4.67 (d, J ) 5.34, 2H), 7.75 (s,

1H), 7.87 (s, 1H), 8.13 (s, 1H), 8.60 (s, 1H), 10.42 (s, 1H), 11.52

(s, 1H), 11.61 (s, 1H); IR (KBr) 2982, 1726, 1643, 1320, 1233,

1155 cm^-1; MS (ES⁺) 664.9 (M + 1). Anal. (C₃₁H₄₈N₆O₁₀

0.75H₂O) C, H, N.

‚

3-[(Am in oim in om eth yl)am in o]-5-[[(am in oim in om eth yl)-

a m in o]m eth yl]ben zoic Acid (193). A solution of 0.8 g

(0.0012 mol) of 192 in 12 mL of 6 N HCl was heated at reflux

for 1 h. The mixture was concentrated, and the resulting oil

was crystallized from 2-PrOH-H₂O to give 0.09 g (23%) of 193

1

as a white solid: mp >300 °C dec; H NMR (400 MHz, CF₃-

CO₂D) δ 4.72 (s, 2H), 7.73 (s, 1H), 8.15 (s, 1H), 8.24 (s, 1H); IR

(KBr) 3206, 1691, 1938, 1604, 1388 cm^-1; MS (ES⁺) 251.5 (M

+ 1). Anal. (C₁₀H₁₄N₆O₂‚HCl‚0.75H₂O) C, H, N.

Meth yl 3-Am in o-5-[(N-h ydr oxylim in o)m eth yl]ben zoate

(194). A mixture of 2.0 g (0.009 mol) of 188, 0.25 g of PtO₂,

and 150 mL of EtOH was hydrogenated at 30 psi for 0.5 h.

The mixture was filtered through Celite, the filtrate was

concentrated, and the residue was recrystallized from EtOAc-

hexane to give 1.1 g (64%) of 194 as an off-white solid: mp

160-162 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 3.81 (s, 3H), 5.53

(s, 2H), 7.04 (m, 1H), 7.19 (m, 1H), 7.29 (t, J ) 1.4 Hz, 1H),

8.04 (s, 1H), 11.20 (s, 1H); IR (KBr) 3324, 3625, 2957, 3885,

7714, 1603, 1441, 1345, 1239 cm^-1; MS (ES⁺) 194.8 (M + 1).

Anal. (C₉H₁₀N₂O₃) C, H, N.

Meth yl 3-[[[(ter t-Bu toxyca r bon yl)a m in o][(ter t-bu toxy-

ca r b on yl)im in o]m e t h yl]a m in o]-5-[(N -h yd r oxyim in o)-

m eth yl]ben zoa te (195). A mixture of 0.97 g (0.005 mol) of

194, 10 mL of DMF, 1.8 g (0.018 mol) of Et₃N, and 1.5 g (0.0055

mol) of N,N-bis(tert-butoxycarbonyl)thiourea⁴¹at 5 °C was

treated with 1.5 g (0.0055 mol) of HgCl₂. The mixture was

stirred at 5 °C for 0.5 h and at ambient temperature for 18 h

and then diluted with 40 mL of EtOAc. The mixture was

filtered through Celite, and the filtrate was washed with H₂O,

dried (Na₂SO₄), and concentrated. The residue was purified

by column chromatography on silica gel (25-50% EtOAc in

hexane) to give 0.7 g (30%) of 195 as a white solid: mp 158-

Meth yl 3-[[[(ter t-Bu toxyca r bon yl)a m in o][(ter t-bu toxy-

ca r bon yl)im in o]m eth yl]a m in o]-5-for m ylben zoa te (200).

A mixture of 2.5 g (0.006 mol) of 199 and 1.5 g (0.007 mol) of

pyridinium chlorochromate (Aldrich) in 25 mL of CH₂Cl₂was

stirred at ambient temperature for 16 h. The mixture was

filtered through a plug of Florisil, diluted to 150 mL with

CHCl₃, and filtered through a plug of silica gel. The filtrate

was concentrated, and the residue was recrystallized from

EtOH-CHCl₃to give 2.1 g (82%) of 200 as a cream-colored

1

solid: mp 138-141 °C; H NMR (400 MHz, CDCl₃) δ 1.52 (s,

9H), 1.55 (s, 9H), 3.96 (s, 3H), 8.28 (s, 1H), 8.41 (s, 1H), 8.54

(s, 1H), 10.05 (s, 1H), 10.64 (s, 1H), 11.61 (s, 1H); IR (KBr)

2982, 1647, 1318, 1149 cm^-1; MS (ES⁺) 422.3 (M + 1). Anal.

(C₂₀H₂₇N₃O₇) C, H, N.

Meth yl 3-[[[(ter t-Bu toxyca r bon yl)a m in o][(ter t-bu toxy-

ca r bon yl)im in o]m eth yl]a m in o]-5-(1-h yd r oxy-2-n itr oeth -

yl)ben zoa te (201). To a stirred suspension of 0.28 g (0.011

mol) of 95% NaH in 8.0 mL of DMF under a N₂atmosphere at

0 °C was added 5.3 mL (0.088 mol) of MeNO₂. Stirring was

continued at 0 °C followed by the addition of 3.7 g (0.009 mol)

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Wildonger, R. A. Preparation of Peptidic Human Leukocyte

Elastase (HLF) Inhibitors. Eur. Pat. Appl. 291234, 1988; Chem.

Abstr. 1989, 110, 232092z.

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M.; Richard, J .; Bauthier, J .; Charlier, R. Benzofurans. LX.

Potential Antianginal Sulfonylaminobenzoylbenzofurans. Eur.

J Med. Chem. (Chim. Ther.) 1977, 12, 81-86.

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Mesomeric Effects of Nitrogen and Oxygen Substituents. Collect.

Czech. Chem. Commun. 1970, 35, 1371-1386.

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of Antineoplastic Agents. XXXII. N-Nitrosoureas. I. J . Med.

Chem. 1963, 6, 669-683.

of 200. The reaction mixture was stirred at 0 °C for 0.5 h,

acidified with HOAc, and poured into 250 mL of H₂O. The

aqueous layer was extracted thrice with 75-mL portions of

EtOAc. The combined organic layers were washed with 75

mL of brine, dried (MgSO₄), and concentrated to give 4.4 g

(quantitative) of 201 as a white solid. An analytical sample,

mp 146-147 °C, was recrystallized from EtOH: ¹H NMR (400

MHz, CDCl₃) δ 1.51 (s, 9H), 1.55 (s, 9H), 3.28 (br s, 1H), 3.93

(s, 3H), 4.58 (m, 2H), 5.52 (dd, J ) 9.1, 3.3 Hz, 1H), 7.82 (s,

1H), 8.05 (s, 1H), 8.14 (s, 1H), 10.52 (s, 1H), 11.60 (br s, 1H);

IR (KBr) 3534, 3276, 3156, 2990, 1721, 1652, 1560, 1234, 1154

cm^-1; MS (ES^-) 481.5 (M - 1). Anal. (C₂₁H₃₀N₄O₉) C, H, N.

Meth yl 3-[[[(ter t-Bu toxyca r bon yl)a m in o][(ter t-bu toxy-

ca r b on yl)im in o]m et h yl]a m in o]-5-(2-n it r oet h yl)b en zo-

a te (202). To a solution of 3.7 g (0.0086 mol) of 201 in 10 mL

of EtOH at 0 °C was added dropwise over a 10-min period a

solution of 1 g (0.026 mol) of NaBH₄in 20 mL of EtOH. The

solution was stirred at 0 °C for 1 h and then the pH adjusted

to 5-6 with a 20% HOAc solution. The solution was diluted

with 20 mL of H₂O, and then the mixture was saturated with

solid NaCl. The mixture was extracted thrice with 25-mL

portions of EtOAc, and the combined extracts were dried

(MgSO₄) and concentrated. The residue was purified by flash

column chromatography on silica gel (25-35% Et₂O in hex-

ane). The appropriate fractions were combined and concen-

trated to give 1 g (26%) of 202 as a white solid: mp 167-169

1

°C; H NMR (400 MHz, CDCl₃) δ 1.5 (s, 9H), 1.6 (s, 9H), 3.40

(t, J ) 7.0 Hz, 2H), 3.90 (s, 3H), 4.70 (t, J ) 7.0 Hz, 2H), 7.65

(s, 1H), 7.85 (s, 1H), 8.15 (s, 1H), 10.4 (s, 1H), 11.6 (s, 1H); IR

(KBr) 2978, 1726, 1649, 1158, 1111 cm^-1; MS (ES⁺) 467.4 (M

+ 1). Anal. (C₂₁H₃₀N₄O₈) C, H, N.

3-(2-Am in oet h yl)-5-[(a m in oim in om et h yl)a m in o]b en -

zoic Acid (204). A mixture of 0.23 g (0.0005 mol) of 202, 0.06

g of PtO₂, and 10 mL of MeOH was hydrogenated at 50 psi for

2 h. The mixture was filtered through Celite, and the filtrate

was concentrated. The residue was purified by flash column

chromatography on silica gel (10-20% CHCl₃-MeOH-con-

centrated NH₄OH (80:18:2) in CH₂Cl₂) to give 0.11 g (51%) of

methyl 3-(2-aminoethyl)-5-[[[(tert-butoxycarbonyl)amino][(tert-

butoxycarbonyl)imino]methyl]amino]benzoate (203).

A solution of 0.11 g (0.000 25 mol) of the above 203 in 2 mL

of 5 N HCl was heated at reflux for 1 h. The mixture was

concentrated, and the residue was successively triturated with

four portions of Et₂O and four portions of EtOAc and dried to

give 0.03 g (36%) of 204 as a tan, highly hygroscopic solid: ¹H

NMR (400 MHz, DMSO-d₆) δ 2.99 (t, J ) 6.2 Hz, 2H), 3.08 (t,

J ) 6.2 Hz, 2H), 7.43 (s, 1H), 7.6 (s, 1H), 7.72 (br s, 3H), 7.74

(s, 1H), 8.16 (br s, 3H), 10.36 (br s, 1H); IR (KBr) 3435, 1673,

1631, 1595 cm^-1; MS (ES⁺) 223.3 (M + 1). Anal. (C₁₀H₁₄N₄O₂‚

2HCl‚H₂O‚0.25EtOAc) C, H, N.

Ack n ow led gm en t. The authors would like to thank

Dr. Shri Niwas for the preparation of 50-52, Dr. Renata

Hawthorne and her staff for providing the IR and MS

data, and Ms. Sandya Ananth for technical assistance

in obtaining the in vitro data. The authors would also

like to thank Dr. Philip E. Morris for his review of the

manuscript and Ms. Linda K. First and Ms. Rosanne

Firneno for their assistance in the preparation of the

manuscript.

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Compounds, 4th ed.; Oxford University: New York, 1965; Vol.

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J M970479E

Article Doi

Design and synthesis of benzoic acid derivatives as influenza neuraminidase inhibitors using structure-based drug design

DOI: 10.1021/jm970479e

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