Design, synthesis, and biological evaluation of analogues of the antitumor agent, 2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid (XK469)

1758

J . Med. Chem. 2001, 44, 1758-1776

Design , Syn th esis, a n d Biologica l Eva lu a tion of An a logu es of th e An titu m or

Agen t, 2-{4-[(7-Ch lor o-2-qu in oxa lin yl)oxy]p h en oxy}p r op ion ic Acid (XK469)

Stuart T. Hazeldine,^†Lisa Polin,^†J uiwanna Kushner,^†J ennifer Paluch,^†Kathryn White,^†Matthew Edelstein,^†

Eduardo Palomino,^‡Thomas H. Corbett,^†and J erome P. Horwitz*^,†,§

Department of Internal Medicine, Division of Hematology and Oncology, Wayne State University School of Medicine,

Detroit, Michigan 48201, Walker Cancer Research Institute, Detroit, Michigan 48201, and Barbara Ann Karmanos Cancer

Institute, Detroit, Michigan 48201

Received December 1, 2000

2-{4-[(7-Chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid (XK469) is among the most highly

and broadly active antitumor agents to have been evaluated in our laboratories and is currently

scheduled to enter clinical trials in 2001. The mechanism or mechanisms of action of XK469

remain to be elaborated. Accordingly, an effort was initiated to establish a pharmacophore

hypothesis to delineate the requirements of the active site, via a comprehensive program of

synthesis of analogues of XK469 and evaluation of the effects of structural modification(s) on

solid tumor activity. The strategy formulated chose to dissect the two-dimensional parent

structure into three regionssI, ring A of quinoxaline; II, the hydroquinone connector linkage;

and III, the lactic acid moietysto determine the resultant in vitro and in vivo effects of chemical

alterations in each region. Neither the A-ring unsubstituted nor the B-ring 3-chloro-regioisomer

of XK469 showed antitumor activity. The modulating antitumor effect(s) of substituents of

differing electronegativities, located at the several sites comprising the A-ring of region I, were

next ascertained. Thus, a halogen substituent, located at the 7-position of a 2-{4-[(2-quin-

oxalinyl)oxy]phenoxy}propionic acid, generated the most highly and broadly active antitumor

agents. A methyl, methoxy, or an azido substituent at this site generated a much less active

structure, whereas 5-, 6-, 8-chloro-, 6-, 7-nitro, and 7-amino derivatives all proved to be

essentially inactive. When the connector linkage (region II) of 1 was changed from that of a

hydroquinone to either a resorcinol or a catechol derivative, all antitumor activity was lost. Of

the carboxylic acid derivatives of XK469 (region III), i.e., CONH₂, CONHCH₃, CON(CH₃)₂,

CONHOH, CONHNH₂, CN, or CN₄H (tetrazole), only the monomethyl- and N,N-dimethylamides

proved to be active.

In tr od u ction

2-{4-[(7-Chloro-2-quinoxalinyl)oxy]phenoxy}propion-

ic acid (XK469, 1, Figure 1) is among the most highly

and broadly active antitumor agents to have been

evaluated in our laboratories^1-4(Table 1). The breadth

of activity manifested by 1 against transplanted mouse

tumors equals or surpasses that of any standard agent

against these preclinical models (Table 2). These data

prompted the selection of 1 for development as a

collaborative effort between the National Cancer Insti-

tute (NCI), the DuPont Pharmaceuticals Company

(DPC), and our laboratories, the result of which is that

this agent is currently scheduled to enter clinical trials

in mid-2001.

The discovery and development of the series of

compounds is of note. The methyl ester of 1 (Figure 1,

R,S-2) was found to have substantial antileukemic

activity at DPC, but it was not developed because of

scant human tumor activity in athymic nude mice, poor

activity against B16 melanoma, and modest cytotoxicity

in culture with short exposure times (1 to 4 h). In 1990/

F igu r e 1.

1991, investigators at DPC revived the series and

solid tumor selectivity in our in vitro, disk diffusion soft

agar colony formation assay²for 2 and broad solid tumor

activity in tumor-bearing mice.^1-4However, the water-

insoluble ester produced both highly variable toxicity

and efficacy with consequent variable oral absorption.²

Although we were able to improve absorption with

provided us with several analogues. We found excellent

* To whom all correspondence should be addressed. 110 E. Warren,

Detroit, MI 48201-1379. E-mail: horwitz@kci.wayne.edu.

^†Wayne State University School of Medicine.

^‡Walker Cancer Research Institute.

^§Barbara Ann Karmanos Cancer Institute.

Published on Web 04/28/2001

Synthesis of XK469 Analogues

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11 1759

Ta ble 1. Treatment of Transplanted Tumors of Mouse and Human Origin with XK-469

XK-469

injection route

no. of

injections

total dose,

mg/kg

% body wt.

loss at nadir

activity

rating

tumor-sc

Colon-38

T/C, %

log₁₀kill

cures

iv

po

iv

po

iv

9

4

8

7

8

546

328

400

525

480

400

620

980

300

600

165

300

332

175

-3.5

-4

0

14

0

10

2

>50% cures

3/5

4/6

5/5

1/5

0/5

1/5

0/5

0/6

0/5

0/6

++++

+++

Panc-03

>50% cures

Squam-lung-LC12^a

Mam-16/C

-3

all cures

5.2

-9

Mam-16/C/Taxol

Mam-16/C/Adr

Mam-17/0

Mam-17/Adr

Colon-51

Colon-26

B16 melanoma

Panc-02

-11

-9

2.7

4.1

3.4

3.9

2.7

1.3

2.6

2.8

2.0

6.4

0.8

1.6

8

++++

+++

13

14

7

6

8

4

5

8

9

-12

-4

-5

0

14

4

-12

-11

-3

++

+++

9

30% ILS

160% ILS

30

+++

AML1498 (iv)

L1210 (iv)

MX-1

-7

-2

-16

-8

+++

+++++

+

DMS273 sm cell

14

++

a

Upstaged tumors (median of 288 mg size tumors at first treatment). For conversion of log kill to activity rating, see Biology Methods

in text.

Ta ble 2. In Vivo Activity of XK-469 in Comparison with Standard Agents against Transplanted Tumors of Mice

Mam

-17/

Adr

Squam- iv AML

iv

in vivo

agent

Colon

-38

Mam

-16/C

-16/C/ -16/C/ Colon Panc

Adr

Panc

-03

Mam

-17

Colon

-26

Mel

B16

lung-

LC12

Leuk

1498

Leuk

Taxol

-51

-02

L1210^b

Adriamycin

Taxol

Camp/CPT

VP-16

Vinbl/Vinc

5-FU

Ara-C

Gemzar

Cytoxan

CisDDPt

BCNU

++

++++

(

-

+++

-

(

+

-

+++ ++++

++++ ++++

-

(

+

(

++++ ++++

+

-

++++ ++++

-

+

++

+++

++

+++

++

+++

++

NA

-

NA

+

-

++++

++

-

NA

++

-

NA

-

NA

-

+

-

NA

(

-

++

-

NA

++

++++

+++++

a

-

+++

NA

+++

-

++

-

+

a

+++

NA

-

+

++++

-

++

NA

+++

++++ +++++

+++ ++++

NA

++

NA

-

NA

++

-

+++

++

++++ ++++

++ +++

NA

+++++

(

-

+++ ++++

+

-

++++ ++++ ++++

++++

+

(

-

+++ ++++

NA

++

+++++

-

XK469

++++ ++++ ++++ +++ +++ ++++ ++++ ++++ ++++

++++

+++ +++++

a

b

SRI/NCI data. The L1210 activity rating was expanded because of this tumor’s higher sensitivity to a variety of agents (including

XK469). This expanded rating for L1210 (up to +++++) was used in this (comparison) table and also in Table 1 (see Biology Methods in

Experimental Section).

better formulations,⁴it was apparent that a suitable

clinical formulation of 2 would be difficult.²In search

of a more suitable development candidate, DPC pro-

vided 337 analogues from their inventory, which com-

prised, in the main, congeners of the potent herbicidal

agent, ASSURE (ethyl 2-{4-[(6-chloro-2-quinoxalinyl)-

oxy]phenoxy}propionate), Figure 1, 3a . Of these ana-

logues, 1 proved to be equally solid-tumor-selective in

the in vitro disk assay and even more effective than

either 2 or the corresponding ethyl ester in mice.^2-4Of

interest was the comparison of 1 with 3a . Interestingly,

3a manifested no antitumor activity, whereas 1 showed

no herbicidal activity but instead exhibited impressive

It is of historic interest and, indeed, importance to

review the reasons why the series was originally aban-

doned. Only modest antitumor activity was observed for

2 or 1 in immune-deficient mice.⁴The reason can be

ascribed to the intolerance of immune-deficient mice to

the granulocyte toxicity of the series, which is also seen

with Adriamycin.^2,4Thus, only 35% of a usual conven-

tional mouse dose of Adriamycin or 1 can be adminis-

tered without encountering lethality.^2,4This should not

be a concern in most cancer treatment conditions, with

the exception of AIDS patients. The poor activity against

B16 was a formulation problem, since IV administration

of 1 produced excellent activity (see Table 1). However,

the agent does require a long exposure time (>12 h) to

produce substantial cell killing in culture. This does not

pose a problem in either mice or humans, since the half-

life of 1 is in excess of 8 h in both species (8 to 18 h

half-lives, depending on the assaysNCI data).

1-4

antitumor activity.

In cell culture, normal fibroblasts and GI epithelial

cells have been compared with leukemic cells and solid

tumor cells, with GI epithelial cells being the least

sensitive to 1.^3,4For in vivo treatment and highly active

analogues, the most sensitive vital normal cells were

the GI epithelial cells, with bone marrow cells being the

next most sensitive.^2,4Historically, a 1.3 to 2.0 log kill

of vital normal cells produces lethality in the mouse.

Since 6.4 logs of leukemic cells were killed at dosages

of 1 that were nonlethal in DBA/2 mice (Table 1) and

many solid tumors were as sensitive or more sensitive

than the leukemia (Table 1), general tumor selectivity

is implied.

A review of other XK469 analogues in the DPC

inventory, which were tested in mice, has been pub-

lished.²Most changes resulted in a marked reduction

in efficacy. The 3-chloro-regioisomer (4), much like

ASSURE, as well as the unsubstituted analogue were

without antileukemic activity. Replacement of the lactic

acid moiety in 1 by 2-hydroxyisobutyric acid leads to a

marked reduction in activity, and the corresponding

glycolic acid ethyl ester analogue proved to be without

1760 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11

Hazeldine et al.

by changes in the biological activities as a consequence

of the conversion of the lactic acid moiety (as indicated

in Figure 2) to a series of primary, secondary, and

tertiary amides, represented by i, and hydrazide (j),

hydroxamide (k ), nitrile (l), and tetrazole (m ) deriva-

tives.

Ch em istr y

A diverse spectrum of analogues of 1 was prepared,

as racemic mixtures, using standard synthetic method-

ology for a study of structure-activity relationships.

Region I: Qu in oxa lin es. The several synthetic

routes to the precursory quinoxalines are illustrated in

Scheme 1. The initial objective, that of preparing the

5- and 8-regioisomers of XK469, required the corre-

sponding 2,5- and 2,8-dichloroquinoxalines (Scheme 1,

10 and 13, respectively).

F igu r e 2.

The synthesis of 10 proceeded from 2-nitroaniline (8),

which was converted to 2-chloro-6-nitroaniline on suc-

cessive reactions with a mixture of fuming and concen-

trated sulfuric acids at 160 °C, chlorination, and fol-

lowed by hydrolysis.⁷Cyanomethylation of the latter,

according to Harvey,⁸was followed by acid hydrolysis

of the intermediate nitrile to the corresponding car-

boxylic acid (9). Catalytic reduction of 9 to an aniline

intermediate effected cyclization of the latter to a 3,4-

dihydro-2-quinoxalinol, which on oxidation with alkaline

H₂O₂provided 5-chloro-2-quinoxalinol. Treatment of the

intermediate with POCl₃gave 10 in high yield. The

preparation of 13 utilized commercially available 3-chlo-

ro-2-nitrobenzoic acid (11) as starting material, which

was transformed in 91% yield to 3-chloro-2-nitroaniline,

via a Schmidt reaction.⁹The preparation of N-(3-chloro-

2-nitrophenyl)glycine (12) was achieved, though in

disappointing (6%) yield, by fusion of the weakly basic

3-chloro-2-nitroaniline and bromoacetic acid. The con-

version of 12 to 13 followed the same procedure

described above for the preparation of 10. The N-(4-

chloro-,^10a4-methyl-,^10b4-methoxy-,^10aand 4-iodo-2-ni-

trophenyl)glycines (15a -d ), each derived by fusion of

the corresponding nitroanilines (14a -d ) and bromoace-

tic acid, followed by reductive cyclization and oxidation

in the usual manner, gave the corresponding 7-substi-

tuted 2-quinoxalinol. The latter were then converted,

on treatment with POCl₃to the 2-chloro derivatives,

16a -d . Treatment of 2-quinoxalinol (17) with KNO₃in

H₂SO₄provided the 6-nitro derivative (18a ) in high

yield,¹¹whereas the interaction of 17 with HNO₃in

AcOH gave the 7-regioisomer (18c) in equally high

yield.¹¹Each was converted, without further purifica-

tion, to 2-chloronitroquinoxalines (18b and d ). Reduc-

tion of readily accessible 19 with tin and HCl, followed

by oxidation, as described by Atkinson,¹²provided a

facile preparation of 7-amino-2-quinoxalinol (20). Al-

ternatively, the latter was also obtained by direct

reduction of 18c. Conversion of 20 to a diazonium salt

and the reaction of the latter with sodium azide,

followed by treatment with POCl₃, gave 7-azido-2-

chloroquinoxaline (21). 3-Chloro-2-methoxy-6-nitro-

aniline (22a) and 2-chloro-3-methoxy-6-nitroaniline (22b),

previously reported by Mallory,^13,14were obtained on

treatment of the correspondingly substituted benzo-

furoxans with copper powder and HCl in CH₃OH.

Amidation of 22a and 22b with cyanoacetic acid^15,16was

activity (structures and data not shown). Overall, 1

proved to be the most desirable agent of the series.

The mechanism of action of 1 remains to be elabo-

rated, though common mechanisms of anticancer drug

action, including DNA binding, alkylation, scission,

tubulin binding, and inhibition of pyrimidine/purine

metabolism, have all been previously excluded. In

addition, the possibility of 1 being a selective inhibitor

of cycloxygenase II was only recently eliminated.⁵By

contrast, Snapka and co-workers report^6athat 1 and,

in particular, its R(+)-isomer induce protein-DNA

cross-links in mammalian cells. They suggested that the

primary target of 1 is topoisomerase-IIâ,^6bwhich may

explain its solid tumor selectivity, but further study of

the proposed mechanism is required. In any case, the

detailed structure of the putative receptor is presently

unknown, which precludes consideration of the option

of receptor-based design of structures with enhanced

solid tumor selectivity.

Accordingly, an effort was initiated to establish a

pharmacophore hypothesis, via a comprehensive pro-

gram of synthesis of congeners and bioisoteres of 1. The

approach sought not only to delineate the minimal

requirements of the active site, by stepwise structural

modification and antitumor evaluation, but in addition

to optimize solid tumor selectivity and minimize unto-

ward pharmacological effects, e.g., toxicity, of the lead

compound.

The strategy formulated in the pursuit of these

objectives arbitrarily chose to dissect the two-dimen-

sional parent structure into three regions, as shown in

Figure 2, and to determine the resultant in vitro and

in vivo effects of chemical alterations in each region.

Thus, the modulating antitumor effect(s) of substituents

of differing electronegativities, to be located at the

several sites comprising the A-ring of region I, would

first be ascertained. The importance of both the nature

and length of the linker relative to the biological

response in region II [i.e., a, (1,4)-hydroquinone; b, (1,3)-

resorcinol; c, (1,2)-catechol bridge; d, 4-(4-hydroxyphen-

yl)phenol; e and f, (1,4- and 4,1-) aminophenol; and g

and h, (1,4- and 4,1-) thiophenol] between the 2-hetero-

aryl and R-substituted propionic acid moieties compris-

ing region II would then be examined. Last, the contri-

bution of the carboxylic acid in XK469 would be evaluated

Synthesis of XK469 Analogues

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11 1761

Sch em e 1^a

a

Reagents: (a) (i) H₂SO₄; (ii) Cl₂/AcOH; (iii) H₂O; (iv) (CH₂O)_n, KCN, ZnCl₂/AcOH; (v) H₂SO₄/AcOH; (b) H₂, 10% Pd/C/CH₃OH; (c) (i)

H₂O₂, aq. NaOH; (ii) aq. HCl; (d) POCl₃; (e) NaN₃, H₂SO₄; (f) BrCH₂CO₂H; (g) Sn, HCl/EtOH; (h) Fe, HCl/EtOH; (i) KNO₃, H₂SO₄; (j)

HNO₃/AcOH; (k) (i) NaNO₂, aq. HCl, 0 °C; (ii) NaN₃; (l) (i) NCCH₂CO₂H, PCl₅/PhCH₃; (ii) NaOH/pyridine; (iii) HCl; (iv) Na₂S₂O₄/aq.

EtOH.

followed by cyclization of the intermediate R-cyano-

ortho-nitroacetanilides to 2-cyano-3-quinoxalinol-1-

oxides. Reduction of the latter with Na₂S₂O₄in aqueous

ethanol provided 23a and 23b. The conversions of the

latter to 2-chloro derivatives, 24a and 24b, were readily

effected, on treatment with POCl₃.

The etherification of (R,S)-methyl 2-(4-hydroxyphe-

noxy)propionate (25) with each of the 2-chloroquinoxa-

line derivatives (10, 13, 16b-d , 18b,d , 21, 24a ,b), as

shown in Scheme 2, was achieved by method A: (i) K₂-

CO₃in acetonitrile; (ii) saponification of the intermedi-

ate esters; and (iii) acidification to provide the corre-

sponding 2-{4-[(2-quinoxalinyl)oxy]phenoxy}propionic

acids (26a -j). Reduction of the methyl ester of 26g with

iron powder in acetic acid, followed by hydrolysis of the

resulting amino ester, gave 26k .

7-chloro-2-quinoxalinol (Scheme 3a, 27) with methyl

2-bromopropionoate in acetone in the presence of K₂-

CO₃(method B) provided methyl 2-[(7-chloro-2-quinox-

alinyl)oxy]propionate (28a ) along with the N-alkylated

product (29) in a 4:1 ratio. Chromatographic separation

and saponification of 28a yielded the 2-O-propionic acid

derivative (28b). The etherification of resorcinol (30)

with methyl 2-bromopropionate was achieved with

NaOCH₃in CH₃OH¹⁷(method C). Reaction of the

product, 31, and 16a (method A) followed by hydrolysis

gave the 1,3-linked product, 36. (2-Benzyloxy)phenol

(32) was converted to methyl 2-(2-hydroxyphenoxy)-

propionate (33) by method B, followed by debenzylation

(H₂/10% Pd/C in CH₃OH). Substitution of a 2-oxyphe-

noxy linkage for the hydroquinone bridge in XK469 was

achieved by reaction of 16a with 33 by method D (NaH

in DMF) followed by hydrolysis of the intermediate ester

to give the catechol analogue (37) of XK469. Similarly,

the reaction of 16a and methyl 2-[(4′-hydroxyphenyl)-

4-phenoxy]propionate (35) (prepared by method C),

followed by hydrolysis, yielded a biphenyl analogue (38)

Region II: Con n ector Lin k a ges. The direct linkage

of the 7-chloro-2-quinoxalinyloxy-moiety to the 2-posi-

tion of propionic acid was first undertaken in order to

evaluate the contribution of the hydroquinone bridge

in XK469 to its antitumor activity. The reaction of

1762 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11

Hazeldine et al.

Sch em e 2^a

a

Reagents: (a) K₂CO₃/CH₃CN (method A); (b) (i) aq. NaOH/THF; (ii) aq. HCl; For 26f, (ii) aq. K₂CO₃/THF; (c) Fe/AcOH.

of XK469. N-Alkylation of 4-aminophenol¹⁸(39a, Scheme

3b) with a mixture of ethyl 2-bromopropionate and Na₂-

SO₃, afforded 40a as a crystalline solid. S-Alkylation of

4-mercaptophenol¹⁹(39b) with methyl 2-bromopropi-

onate in DMF in the presence of KHCO₃provided the

mercapto derivative, 40b. Etherification of 40a and 40b

with 16a (method A), followed by hydrolysis of the

intermediate esters, gave the desired nitrogen and

sulfur derivatives, 41a and b, respectively. N-Alkyla-

tion²⁰of 39a with 16a was achieved with HCl in

aqueous acetonitrile, whereas S-alkylation of 39b and

16a was effected with KHCO₃in DMF. The O-alkylation

of both 39a and b and hydrolysis of the intermediate

esters led to the respective carboxylic acids, 42a and b,

utilizing method B.

were evaluated against a second tumor as well, if

supplies permitted.

In Vivo Effica cy Eva lu a tion s in Tu m or -Bea r in g

Mice. Region I: The exceptionally high activity of

racemic XK469 is evident in Tables 1-3. The enantio-

mers of 1 were compared for efficacy and toxicity in C3H

mice bearing Mammary Adenocarcinoma-17/Adr tu-

mors. The schedule was daily × 7, beginning the day

after tumor implant (QD1-6), with intravenous (iv)

injections in a volume of 0.2 mL/injection. The top dose

(62 mg/kg/injection) was planned for 7 days, but we

stopped at six injections (372 mg/kg total) because of

two of five drug deaths. An explanation of this rationale

has been provided by one us (T.H.C.) in an earlier

work.²⁷Additional information to explain dose schedules

and routes are incorporated in the legends to tables.

Region III: Ca r boxylic Acid Der iva tives. Ami-

nolysis of the methyl ester (2) of XK 469 provided the

amide (Scheme 4, 43). Similarly, reaction of 2 with

hydrazine in EtOH gave the hydrazide (44) in good

yield. Conversion of 1 to an acid chloride, followed by

reaction of the product with either methyl- or dimethyl-

amine, provided amides, 45a and b, respectively. The

acid chloride of 1, on treatment with (TMS)₂NOTMS²¹

in toluene, followed by hydrolysis, gave the hydroxamide

derivative, 45c. Reaction of 46 with 2-bromopropioni-

trile, followed by debenzylation of the intermediate with

BBr₃‚S(CH₃)₂,²²gave 2-(4-hydroxyphenoxy)propionitrile

(47). Etherification of the latter with 16a (method A)

gave the nitrile derivative (48) of XK469. Conversion

of the latter to a tetrazole derivative (49) was readily

effected by treatment with NaN₃and NH₄Cl in DMF.

The R-form produced a -18% body weight loss,

whereas the S-form effected a -14% body weight loss

in these mice (20.8 g average at the start of the

posttreatment). The next lower dose (301 mg/kg total

on a QD1-7 schedule IV) for both enantiomers was not

toxic, and the efficacies were essentially identical: 2.9

log kill for the R- and 2.7 log kill for the S-form against

Mammary Adenocarcinoma-17/Adr, a p-glycoprotein

positive multidrug resistant tumor. The equal activity

of the R- and S-forms is not surprising, since the two

are interconverted in the liver of the mouse, with 90%

R (the clinical agent) produced at equilibrium (personal

communication, Drs. B. J asti and Ralph Parchment,

Wayne State University/Karmanos Cancer Institute).

The methyl ester of the R-form (2) was also highly active

(Table 3), but had variable absorption and variable

efficacy due to poor formulation properties, as previously

discussed. The 6-chloro carboxylic acid (3c), like Assure

(3a ), proved to be inactive. Moreover, the 5- and 8-regio-

isomers of XK469, i.e., structures 26a and b, respec-

tively, were also without antitumor activity, though 26b

produced severe neurotoxicity, limiting the total dose

that could be administered to 351 mg/kg (Table 3). The

readily soluble (pH 8) 7-fluoro analogue (6a ) was well

tolerated by the mice and highly active, producing cures

against Colon Adenocarcinoma-38 (Table 3). The draw-

back was a markedly higher dose requirement than that

of the chloro analogue. The 7-fluoro methyl ester (6b)

could only be administered orally as a suspension, which

undoubtedly gave rise to absorption problems and

compromised its activity (Table 3). The 6-fluoro car-

Biologica l Resu lts a n d Discu ssion

In Vitr o Testin g of An a logu es. All newly synthe-

sized analogues of XK469 were initially evaluated in our

in vitro, disk diffusion soft agar colony formation assay,

to determine cytotoxicity against leukemias, solid tu-

mors, and normal cells. Many of the analogues of 1 that

were tested in mice (Tables 3-5) exhibited only modest

cytotoxicity and unimpressive tumor selectivity in tissue

culture. These analogues, based on historic results with

other classes of agents, had only a low probability of

being active, though one, obviously, cannot be certain

of this. On the other hand, analogues without meaning-

ful cytotoxicity were excluded from testing in tumor-

bearing mice. Those manifesting activity in a first tumor

Synthesis of XK469 Analogues

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11 1763

Sch em e 3^{a ,b}

a

Reagents for Scheme 3a: (a) BrCHCH₃CO₂CH₃, K₂CO₃/acetone (method B); (b) (i) aq. NaOH/THF; (ii) aq. HCl; (c) BrCHCH₃CO₂CH₃,

b

NaOCH₃/CH₃OH (method C); (d) H₂, 10% Pd/C/CH₃OH; (e) method A; (f) NaH/DMF (method D). Reagents for Scheme 3b: (a)

BrCHCH₃CO₂Et, Na₂SO₃; (b) BrCHCH₃CO₂CH₃, KHCO₃/DMF; (c) method A; (d) (i) aq. NaOH/THF; (ii) aq. HCl; (e) aq. HCl/CH₃CN; (f)

method B; (g) KHCO₃/DMF.

boxylic acid (5), rather surprisingly, showed activity,

albeit marginal (0.9 log kill, see Table 3). However, this

agent, provided by DPC, was not derived via a regiospe-

cific synthesis, which suggested possible contamination

with the active 7-fluoro derivative (6a ). However, com-

parison of ¹H NMR spectra of 5 and 6a failed to confirm

the presence of the suspected contaminant, though >1%

of an as yet unidentified impurity was seen in the

spectrum of 5. The 7-bromo analogue (7) of 1 was also

highly active and curative. Furthermore, the dose

requirement was no higher than that of XK469. The

limitation was the substantially reduced water solubility

of 7. The 7-iodo derivative (26e) was active (1.8 log kill)

and not toxic at a total dose of 530 mg/kg, iv. However,

26e required higher doses than 1 and had substantially

reduced activity. Likewise, the 7-azido analogue (26h )

was active (2.0 log kill Panc-03 and 1.0 log kill Mam-

17/Adr) but obviously inferior to 1. It is also worthy of

1764 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11

Hazeldine et al.

Sch em e 4^a

a

Reagents: (a) NH₃/CH₃OH; (b) NH₂NH₂/EtOH; (c) (i) (COCl)₂/THF; (ii) RR′NH; (d) (i) SOCl₂; (ii) TMS₂NOTMS/toluene; (iii) H₂O; (e)

BrCHCH₃CN, K₂CO₃/acetone; (f) BBr₃‚S(CH₃)₂/CH₂Cl₂; (g) method A; (h) NaN₃, NH₄Cl/DMF.

note that, whereas the dose requirement for 26h was

low (144 to 216 mg/kg), higher dosages produced deaths

from marrow toxicity. The 7-nitro derivative (26g) was

inactive (note the large dosage used; 1310 mg/kg total,

Table 3). The 6-nitro derivative (26f) was similarly

inactive. The 7-amino analogue (26k ) was not cytotoxic

in tissue culture and, therefore, not tested in mice. The

7-methyl analogue (26c) of XK469 was only marginally

active and had a large dose requirement (750 to 1250

mg/kg, total). The 7-methoxy derivative (26d ) was well

tolerated in mice and highly active against multidrug

resistant tumors, Mam-17/Adr (p-glycoprotein positive)

and Mam-16/C/Adr (p-glycoprotein negative) (2.7 to 3.0

log kill). Thus, 26d was essentially as active as the

chloro analogue against these tumors, but it exhibited

a slightly higher dose requirement. It was also active

against Colon Adenocarcinoma-38 (1.7 log kill), but at

a level substantially lower than that of 1 against the

same tumor. Higher dosages of 26d produced lethality

from GI epithelial damage, essentially identical to that

seen with lethal dosages of XK469. The 7-chloro-8-

methoxyquinoxaline compound (26i) exhibited signifi-

cant activity against Panc-03 (3.0 log kill), Mam-17/Adr

(1.5 log kill), and Mam-16/C/Adr (1.7 log kill). Moreover,

the dose requirement for 26i was low (162 to 324 mg/

kg total), which is about half that of the requirement

for 1. However, the activities of 26i were less than that

elicited by 1, and the dose-response relationship was

very steep (see Panc-03 data in Table 3). In contrast,

the 7-methoxy-8-chloroquinoxaline derivative (26j) was

inactive.

substitution in the 8-position also produced high anti-

tumor activity together with a lower dose requirement,

though the dose-response relationship was excessively

steep.

Region II: The importance of the hydroquinone

linkage to the activity of XK469 is indicated by the

changes to the connector linkage summarized in Table

4. Thus, the direct linkage of the propionic acid moiety,

via oxygen, at the 2 position of 7-chloroquinoxaline (28b)

results in a complete loss of antitumor activity. A similar

effect is observed on substitution of catechol (37),

resorcinol (36), or 4-(4-hydroxyphenyl)phenol (38) link-

ages for hydroquinone. Last, structures bearing the

bioisoteric substitutions, i.e., 41a and b, as well as 42a

and b, all proved to be inactive.

Region III: The doses selected for all analogues listed

in Table 5 were derived from multiple dose-level testing

(two to three dosages per agent). Dosages were escalated

daily until some evidence of toxicity was noted or a very

high total dose was obtained.

Of the derivatives presented in Table 5, only the

amides 43, 45a , and 45b showed interesting activities.

Thus, 43 is clearly active (1.9 log kill) but showed a high

dosage requirement. Dosages over 600 mg/kg total

within a 10 day time period are considered to be a high

dosage,^23,28which has discouraged the development of

agents that show this requirement.

The -NHCH₃derivative (45a ) appears to be more

active than 43, though both the weight loss and the dose

(2300 mg/kg) were higher in the former. The evaluation

of 45b, as indicated in Table 5, was performed on a prior

occasion with the tumor Colon-38 and utilized a sample

of the agent derived from the DPC inventory. A com-

parison of the physical properties of the latter with those

of a sample of 45b, prepared in the present study,

showed them to be identical.

In summary, it is apparent that there is clear re-

quirement for a group in the 7-position for significant

antitumor activity, with Cl ) Br ) F > OCH₃> N₃> I.

In addition, a Cl substituent in the 7-position and OCH₃

Synthesis of XK469 Analogues

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11 1765

Ta ble 3. Structure-In Vivo Activity Relationships for Some 2-{4-[(2-Quinoxalinyl)oxy]phenoxy}propionic Acids and Derivatives

against Murine Tumors

compd

no.^a

no. of

injections

total dose,

mg/kg

% body

wt. loss

T/C,

%

log₁₀

kill

activity

rating

W

H

X

Y

Z

R^a

tumor

Panc-03

1

H

Cl

H

4

328

-4.0

0

2.1^b

++++

4/6 cures

++++

3/5 cures

-

1

H

Cl

H

CH₃

Mam-17/ADR

Colon-38

13

7

620

301

495

-4.0

-8.0

-3.0

0

2

0

3.9

2.9

2.7

3.5^b

1 (R)

1 (S)

2 (R)

30

3a

3b

3c

5

H

Cl

F

H

F

H

C₂H₅

CH₃

H

Colon-38

Panc-03

19

15

14

7

2686

2770

1590

769

0

-4

+1.0

-13

61

>100

34

0.42

none

0.9

-

+

6a

H

12

1085

0

3.7^b

++++

3/5 cures

+

6b^c

7

H

F

H

CH₃

H

Colon-38

10

8

1433

753

466

+1.0

-12.0

-7.0

34

0

0.8

Br

3.0^b

2.1^b

++++

2/5 cures

+++

3/5 cures

-

26a

26b

26c

26d

Cl

H

Cl

H

Panc-03

9

7

10

7

6

7

9

13

7

14

6

1010

561

351

287

1250

750

679

407.4

582

455

585

195

530

700

1310

144

216

324

180

164

162

99

-2.0

+2.0

-4.0

-3.9

+7.1

+4.3

-4.6

0.0

59

>100

74

0.27

none

0

-

H

Panc-03

-

69

21

47

4.6

6.4

0

8

0.1

0.63

0.38

3.0

1.7

2.7

2.1

1.73

0.12

1.8

0.42

0.6

2.0

1.0

3.0

0.64

1.7

1.5

1.2

0

-

CH₃

OCH₃

Mam-17/ADR

Mam-16c/ADR

Colon-38

(

-

++++

++

-13.6

-1.2

0

+++

++

23

0

90

15

41

52

17

30

0

33

7.5

7

13

76

-

26e

26f

26g

26h

26i

H

NO₂

H

I

H

Panc-03

Mam-17/ADR

Panc-03

++

-1.4

- 3.5

+2.9

+4.3

-15.0

-5.0

-6.2

-16.4

-1.5

+11.1

-

NO₂

N₃

Cl

+++

+

8

H

OCH₃

12

7

6

++++

(

26i

H

Cl

OCH₃

H

Mam-16c/ADR

Mam-17/ADR

++

+

-

26j

H

OCH₃

NH₂

Cl

H

Panc-03

14

939

26k ^d

a

In cases in which R ) H, these analogues were water-soluble in aqueous NaHCO₃and were injected iv. In cases in which R ) CH₃,

these analogues were water-insoluble and were injected orally (po). A dose-escalation schedule was followed as presented in ref 27.

Treatment was stopped when weight loss was substantial or drug supply was exhausted. Dosages of >1100 mg/kg indicate an agent with

no clinical promise.^23,28Treatment of Colon-38 or Panc-03 began 3 days post-implant of the tumors. The other tumors were faster growing

b

(1. 0 to 1. 2 day doubling), and treatment began the day after tumor implant. The log kill values were based on tumors that grew (cures

excluded from the calculation). The cures represent >4.0 log kill for these tumors. ^cMade a poor suspension and gave poor oral absorption

d

which may account for the limited activity. Inactive in vitro, not tested in vivo.

An evaluation of 45b against Panc-03 is currently in

progress. Moreover, the activity of 45b against Colon-

38 has also prompted (an in-progress) synthesis of the

R- and S-forms of this agent to ascertain whether the

anticipated activity against the tumors, Panc-03 as well

as Colon-38, is a property of one or both enantiomers.

ring A of region I; (b) alterations in hydroquinone

moiety, the connector linkage comprising region II; and

(c) to derivatives of the carboxylic acid function. The

order of the relative activities of the 7-halogen deriva-

tives was found to be F ≈ Cl ≈ Br > I. On the other

hand, the 3,5,6- and 8-regioisomers of 1 were essentially

all inactive. Changes in the hydroquinone (1,4) linkage

to that of either a resorcinol (1,3) or a catechol (1,2)

derivative all resulted in inactive structures, as did

replacement of either the 1- or 4-oxygen atom compris-

ing the hydroquinone bridge by sulfur or nitrogen. By

contrast, simple alkyl ester and amide derivatives of 1b

showed relatively minor changes in activities relative

to 1.

Su m m a r y

Without knowledge of a target structure for 1, we

elected to delineate the putative requirements of the

active site via a comprehensive structure-activity study

of analogues of 1. The investigation focused on relation-

ships of in vitro and in vivo antitumor activities to (a)

changes in the nature and location of substituents in

1766 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11

Hazeldine et al.

Ta ble 4. Importance of the Connector Linkage to the

Antitumor Activity of XK469

2-Ch lor oqu in oxa lin e Der iva tives. N-(2-Ch lor o-6-n itr o-

p h en yl)glycin e (9). 2-Chloro-6-nitroaniline⁷(1.73 g, 10.0

mmol), paraformaldehyde (0.90 g, 30 mmol), KCN (1.96 g, 30.0

mmol), ZnCl₂(13.63 g, 100.0 mmol), AcOH (25 mL), and

concentrated H₂SO₄(1 drop) were heated at 50 °C overnight.

After cooling, the mixture was poured into ice-water (100 mL)

and extracted with AcOEt (4 × 50 mL). The extracts were

washed with saturated NaHCO₃(50 mL portions), followed

by saturated NaCl (50 mL), then dried (MgSO₄), and concen-

trated to give a yellow solid, purified by chromatography (2:1

hexanes:AcOEt) to give 2-chloro-N-cyanomethyl-6-nitroaniline

1

as a yellow solid (0.54 g, 25% yield): mp 82-83 °C; H NMR

(300 MHz, CDCl₃) δ 7.95 (dd, J ) 8.4, 1.2 Hz, 1 H), 7.56 (dd,

J ) 8.1, 1.2 Hz, 1 H), 6.97 (t, J ) 8.1 Hz, 1 H), 6.89 (bt, J )

7.2 Hz, 1 H), 4.38 (d, J ) 7.2 Hz, 2 H).

2-Chloro-N-cyanomethyl-6-nitroaniline (0.54 g, 2.6 mmol),

AcOH (10 mL), and 50% H₂SO₄(25 mL) were heated at 100

°C for 2 h. After cooling, the mixture was added to ice (100

mL), filtered, washed with ice-water, and dried to give 9 as a

yellow solid (0.47 g): mp 134-137 °C. Additional product was

obtained by extracting the filtrate with AcOEt (2 × 50 mL)

and washing the extracts with water (4 × 100 mL) and then

saturated NaCl (50 mL). The extracts were dried (MgSO₄) and

concentrated under high vacuum to remove the remaining

AcOH to give 0.14 g of product (∼100% total yield): ¹H NMR

(300 MHz, DMSO-d₆) δ 12.93 (bs, 1 H), 7.87 (dd, J ) 8.4, 1.2

Hz, 1 H), 7.56 (dd, J ) 7.8, 1.2 Hz, 1 H), 7.00 (bt, J ) 5.7 Hz,

1 H), 6.89 (t, J ) 8.4 Hz, 1 H), 4.02 (d, J ) 5.7 Hz, 2 H).

2,5-Dich lor oqu in oxa lin e (10). The precursory intermedi-

ate 9 (0.61 g, 2.6 mmol), 10% Pd/C (0.03 g) in CH₃OH (50 mL),

was shaken with H₂(30 psi) in a Parr apparatus for 6 h. The

mixture was then filtered and concentrated, to which 1 N

NaOH (6 mL) and 3% H₂O₂(6 mL) were added and heated for

1 h. After cooling, the solution was acidified to pH 5 with HCl,

filtered, washed with ice-water, and dried to give 5-chloro-

2-quinoxalinol as a light tan solid (0.31 g, 65% yield): mp ∼295

°C (dec); ¹H NMR (300 MHz, DMSO-d₆) δ 12.59 (bs, 1 H), 8.22

(s, 1 H), 7.50 (t, J ) 8.1 Hz, 1 H), 7.41 (dd, J ) 7.8, 0.9 Hz, 1

H), 7.24 (dd, J ) 8.1, 0.9 Hz, 1 H).

5-Chloro-2-quinoxalinol (0.31 g, 1.7 mmol) and POCl₃(2.0

mL, 3.3 g, 21 mmol) were refluxed for 1 h and then concen-

trated, and water (25 mL) and NaHCO₃were added until pH

7. The mixture was then extracted with AcOEt (4 × 25 mL),

and the extracts were washed with saturated NaCl (25 mL),

dried (MgSO₄), and evaporated to dryness. After the residue

was dissolved in CHCl₃, the solution was filtered through silica

gel and the filtrate concentrated to give 10 as a pale yellow

solid (0.24 g, 70% yield): mp 131-134 °C; ¹H NMR (300 MHz,

CDCl₃) δ 8.88 (s, 1 H), 7.97 (dd, J ) 8.7, 0.9 Hz, 1 H), 7.89

(dd, J ) 7.5, 0.9 Hz, 1 H), 7.74 (t, J ) 8.1 Hz, 1 H).

a

b

Inactive in culture; not tested in mice. Total iv: 330mg/kg;

T/C ) 100%; no log kill.

Clearly, the components and topology of 1 combine

to give the optimum solid tumor selectivity, relative to

the majority of analogues prepared and evaluated in this

study. Our ongoing work, which will be the subject of

later reports, includes a study of changes in the anti-

tumor activity of 1 as a consequence of the bioisosteric

replacement of the C₃) N₄comprising ring B of the

quinoxaline ring by (1) quinazoline (N₃) C₄), (2) 1,2,4-

trazine (N₃) N₄), and (3) quinoline (C₃) C₄) ring

systems.

Exp er im en ta l Section

N-(3-Ch lor o-2-n itr op h en yl)glycin e (12). 3-Chloro-2-ni-

troaniline⁹(5.12 g, 29.7 mmol) was heated with bromoacetic

acid (4.25 g, 29.7 mmol) at 125 °C for 1 h with air passing

over the mixture to remove the HBr. Methanol was then added

and the mixture heated until all the solid dissolved. Dilute

NH₄OH was added in portions to maintain the pH > 8 while

the mixture was concentrated by heating. The cooled mixture

was filtered and then washed with dilute NH₄OH, and the

filtrate was washed with ether (50 mL). The aqueous layer

was then concentrated to a small volume and acidified while

hot to pH 3 with concentrated HCl. After cooling, the product

was collected, washed with ice-water, and dried to give 12 as

a yellow-brown solid: mp 195-197 °C (dec). Additional product

was obtained by extracting the filtrate with AcOEt (2 × 25

mL) and washing the extracts with saturated NaCl (25 mL).

After the extract was dried (MgSO₄), it was concentrated to

give a yellow solid (total 0.42 g, 6% total yield): ¹H NMR (300

MHz, DMSO-d₆) δ 12.83 (bs, 1 H), 7.30 (t, J ) 8.4 Hz, 1 H),

6.82 (d, J ) 7.8 Hz, 1 H), 6.73 (d, J ) 8.7 Hz, 1 H), 6.54 (bt,

J ) 6.0 Hz, 1 H), 3.92 (d, J ) 6.0 Hz, 2 H).

Ch em istr y. All commercially available solvents and re-

agents were used without further purification. Melting points

were determined on a Thomas-Hoover melting point apparatus

and are uncorrected. Infrared spectra were measured on a

Perkin-Elmer 1330 spectrometer in KBr pellets, Nujol mull,

or as a thin film. Magnetic resonance (¹H and ¹³C NMR)

spectra were recorded at room temperature on either a Varian

Unity 300, Varian Mercury 400, or GE Q300 instruments in

the chemistry department at Wayne State University, Detroit,

MI, and referenced to a residual solvent signal. Chemical shifts

are reported in ppm downfield from TMS. The splitting pattern

abbreviations are as follows: s, singlet; d, doublet; t, triplet;

q, quartet; b, broad; m, unresolved multiplet. Mass spectra

were recorded on a MS80RFA instrument and other instru-

ments in the chemistry department at Wayne State University,

Detroit, MI. Flash column chromatography was carried out

unless otherwise indicated with silica gel 200-400 mesh, 60

Å (Aldrich), and the crude product was introduced onto the

column as a CHCl₃solution. Thin-layer chromatography was

performed on Whatman PE SIL G/UV (250 µm) plates.

Compounds were visualized by use of 254 or 366 nm light and

I₂vapor. Elemental analyses were performed by M-H-W

Laboratories, Phoenix, AZ. All the compounds are racemic

mixtures.

2,8-Dich lor oqu in oxalin e (13): prepared as described above

for 10 from 12 (0.27 g, 1.2 mmol), using 10% Pd/C (0.01 g)

and CH₃OH (50 mL) followed by 1 N NaOH (3 mL) and 3%

H₂O₂(3 mL) to give 8-chloro-2-quinoxalinol as a light tan solid

Synthesis of XK469 Analogues

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11 1767

Ta ble 5. Some Carboxylic Acid Derivatives of 2-{4-[(7-Chloro-2-quinoxalinyl)oxy]phenoxy}propionic Acid (XK469) against Murine

Tumors

compd

no.^a

no. of

injections

total dosage,

mg/kg

% body

wt. loss

log₁₀

kill^b

activity

rating

R

tumor

T/ C, %

43

44

45a

CONH₂

CONHNH₂

CONHCH₃

Panc-03

13

4

10

4

1320

800

2300

480

-3.6

-6.5

7.3

27

4

39

16

1.9

++

0.75

4.2**

1.2

+

-14.5

-9.3

++++ 1/5 cures

+

+++

45b

45c

48

CON(CH₃)₂

CONHOH

CN

Panc-03

c

9

1530

- 3. 8

2. 2

d

49

CHN₄

Panc-03

7

477

+8.7

>100

none

-

a

These agents were all water-insoluble and were injected orally (po) beginning 3 days after tumor implantation. Two to three dosages

were tested, with the highest nontoxic dosage shown. A dose-escalation schedule was administered as per ref 27. Treatment was stopped

b

when weight loss was substantial (as with 45a) or the drug supply was exhausted. The log kill values were based on tumors that grew

d

(cures excluded from the calculation). The cures represent >4.0 log kill for these tumors. ^cInactive in vitro, not tested in vivo. Tetrazole.

(0.12 g, 57% yield); mp 190-195 °C; ¹H NMR (300 MHz,

DMSO-d₆) δ 12.00 (bs, 1 H), 8.18 (s, 1 H), 7.30 (dd, J ) 8.1,

0.9 Hz, 1 H), 7.66 (dd, J ) 7.8, 0.9 Hz, 1 H), 7.27 (t, J ) 8.1

Hz, 1 H).

8-Chloro-2-quinoxalinol (0.12 g, 0.66 mmol) on treatment

with POCl₃(1.0 mL, 1.6 g, 10 mmol) gave 13 as a white solid

(0.12 g, 92% yield): mp 120-122 °C; ¹H NMR (300 MHz,

CDCl₃) δ 8.83 (s, 1 H), 8.05 (dd, J ) 8.4, 0.9 Hz, 1 H), 7.90

(dd, J ) 7.5, 0.9 Hz, 1 H), 7.71 (t, J ) 8.1 Hz, 1 H).

2,7-Dich lor oqu in oxa lin e^10a(16a ): ¹H NMR (300 MHz,

CDCl₃) δ 8.78 (s, 1 H), 8.07 (d, J ) 9.0 Hz, 1 H), 8.03 (d, J )

2.1 Hz, 1 H), 7.75 (dd, J ) 9.0, 2.1 Hz, 1 H).

2-Ch lor o-7-m eth ylqu in oxa lin e^10b(16b): (reductive cy-

clization of 16b performed using H₂, 10% Pd/C, CH₃OH instead

of tin, HCl, EtOH); ¹H NMR (300 MHz, CDCl₃) δ 8.71 (s, 1 H),

8.00 (d, J ) 8.4 Hz, 1 H), 7.79 (s, 1 H), 7.28 (dd, J ) 8.7, 1.8

Hz, 1 H), 2.60 (s, 3 H).

2-Ch lor o-7-m eth oxyqu in oxa lin e^10a(16c): (reductive cy-

clization of 16c performed using H₂, 10% Pd/C, CH₃OH instead

of tin, HCl, EtOH); ¹H NMR (300 MHz, CDCl₃) δ 8.62 (s, 1 H),

7.96 (d, J ) 9.3 Hz, 1 H), 7.40 (dd, J ) 9.3, 3.0 Hz, 1 H), 7.28

(d, J ) 2.7 Hz, 1 H), 3.95 (s, 3 H).

(EI) m/z (%) 290 (M⁺, 100), 255 (14), 163 (42), 128 (22), 101

(24), 75 (27), 50 (18).

2-Ch lor o-6-n itr oqu in oxa lin e¹¹(18b): (PCl₅was not re-

quired for conversion of 2-OH to 2-Cl); ¹H NMR (400 MHz,

CDCl₃) δ 9.02 (d, J ) 2.4 Hz, 1 H), 8.94 (s, 1 H), 8.59 (dd, J )

9.2, 2.8 Hz, 1 H), 8.19 (d, J ) 9.2 Hz, 1 H).

2-Ch lor o-7-n itr oqu in oxa lin e¹¹(18d ): (PCl₅was not re-

quired for conversion of 2-OH to 2-Cl); ¹H NMR (300 MHz,

CDCl₃) δ 8.94 (s, 1 H), 8.92 (d, J ) 2.4 Hz, 1 H), 8.56 (dd, J )

9.0, 2.4 Hz, 1 H), 8.30 (d, J ) 9.0 Hz, 1 H).

7-Azid o-2-ch lor oqu in oxa lin e (21). A mixture of 20¹²(0.16

g, 1.0 mmol) and HCl (3 mL) was stirred together overnight

before cooling to 0 °C. A cold solution of NaNO₂(0.08 g, 1.2

mmol) in water (2 mL) was added dropwise, and the mixture

was stirred an additional 1 h. NaN₃(0.07 g, 1.1 mmol) in water

(2 mL) was added, and the mixture was allowed to warm to

room temperature. The solid was dissolved in 1 N NaOH; the

solution was filtered, acidified to pH 5 with HCl, and cooled;

and the product was collected, washed with ice-water, and

dried to give 7-azido-2-quinoxalinol as a light brown solid (0.13

1

g, 70% yield): mp >300 °C; H NMR (300 MHz, DMSO-d₆) δ

12.34 (bs, 1 H), 8.04 (s, 1 H), 7.73 (d, J ) 8.4 Hz, 1 H), 6.99 (d,

J ) 7.8 Hz, 1 H), 6.87 (s, 1 H); IR (Nujol) 2130 (N₃) cm^-1

.

N-(4-Iod o-2-n itr op h en yl)glycin e (15d ): prepared as de-

scribed above for 12 from 14a (34.87 g, 132.1 mmol) and

bromoacetic acid (18.92 g, 132.1 mmol) to give 15d as a brown-

red solid (2.90 g, 7% yield); mp 189-190 °C (dec); ¹H NMR

(400 MHz, DMSO-d₆) δ 13.07 (bs, 1 H), 8.37 (bt, J ) 5.6 Hz, 1

H), 8.29 (d, J ) 2.4 Hz, 1 H), 7.74 (dd, J ) 9.2, 1.6 Hz, 1 H),

6.76 (t, J ) 10.0 Hz, 1 H), 4.14 (d, J ) 5.6 Hz, 2 H).

7-Azido-2-quinoxalinol (0.12 g, 0.64 mmol) was treated with

POCl₃(0.60 mL, 0.99 g, 6.4 mmol) (as described for 10) to give

21 as a light brown solid (0.09 g, 69% yield): mp 144-147 °C

1

(dec); H NMR (300 MHz, CDCl₃) δ 8.70 (s, 1 H), 8.07 (d, J )

9.0 Hz, 1 H), 7.61 (d, J ) 2.1 Hz, 1 H), 7.41 (dd, J ) 9.0, 2.4

Hz, 1 H); IR (Nujol) 2140 (N₃) cm^-1

.

7-Ch lor o-8-m eth oxy-2-qu in oxa lin ol (23a ): To a mixture

of 22a ¹³(2.03 g, 10.0 mmol), cyanoacetic acid (1.72 g, 20.0

mmol), and toluene (100 mL) was added PCl₅(4.39 g, 20.0

mmol) in small portions, and the mixture was refluxed while

passing air over the mixture. After 2 h, additional cyanoacetic

acid (0.21 g, 2.5 mmol) and PCl₅(0.55 g, 2.5 mmol) were added,

and the refluxing continued until the yellow color disappeared.

After concentration, the solid was dissolved in AcOEt (200 mL)

and washed with saturated NaCl (50 mL), a mixture of

saturated NaCl (50 mL), and saturated NaHCO₃(25 mL)

followed by saturated NaCl (50 mL). The dried solution

(MgSO₄) was then concentrated and recrystallized from EtOH-

heptane to give R-cyano-3-chloro-2-methoxy-6-nitroacetanilide

2-Ch lor o-7-iod oqu in oxa lin e (16d ). To a refluxing solution

of 15d (2.29 g, 7.11 mmol), concentrated HCl (1.8 mL), and

CH₃OH (75 mL) was added iron powder (2.08 g, 35.6 mmol)

in portions over 2 h, and the mixture was refluxed for an

additional 2 h. The mixture was filtered immediately and

washed with hot CH₃OH. To the filtrate, 1 N NaOH (50 mL)

and 3% H₂O₂(20 mL) were added, and the mixture was heated

for 1 h before filtering hot and washing with dilute NaOH and

CH₃OH. The mixture was then refiltered through Celite and

concentrated to ∼100 mL before acidifying pH 5 with concen-

trated HCl. After cooling, the mixture was filtered, washed

with ice-water, and dried to give crude 7-iodo-2-quinoxalinol

as a brown solid (1.67 g, 86% crude yield). A sample was heated

with CH₃OH, the insoluble material was removed, and the

filtrate was concentrated to give a brown solid: ¹H NMR (400

MHz, DMSO-d₆) δ 12.40 (bs, 1 H), 8.15 (s, 1 H), 7.62 (d, J )

1.6 Hz, 1 H), 7.58 (d, J ) 9.2 Hz, 1 H), 7.50 (d, J ) 8.4 Hz,

1 H).

1

as off-white crystals (2.55 g, 94% yield): mp 170-172 °C; H

NMR (300 MHz, CDCl₃) δ 8.37 (bs, 1 H), 7.77 (d, J ) 9.0 Hz,

1 H), 7.46 (d, J ) 9.3 Hz, 1 H), 3.99 (s, 3 H), 3.63 (s, 2 H).

To this intermediate (2.55 g, 9.45 mmol), dissolved in

pyridine (10 mL), was added 1 N NaOH (9.5 mL, 9.5 mmol),

and the mixture was stirred overnight. Water (75 mL) was

added, and the mixture was filtered and washed with water.

The filtrate was acidified to pH 6 with concentrated HCl. After

cooling, the product was collected, washed with ice-water, and

dried to give 6-chloro-2-cyano-5-methoxy-3-quinoxalinol-1-

oxide as a yellow solid (2.38 g, ∼100% yield): mp ∼280 °C (dec);

Crude 7-iodo-2-quinoxalinol (1.67 g, 6.1 mmol) and POCl₃

(2.8 mL, 4.6 g, 30 mmol) (as described above for 10) gave 16d

as a yellow solid (0.64 g, 36% yield): mp 148-150 °C; ¹H NMR

(300 MHz, CDCl₃) δ 8.77 (s, 1 H), 8.43 (d, J ) 1.8 Hz, 1 H),

8.03 (dd, J ) 8.4, 1.8 Hz, 1 H), 7.81 (d, J ) 8.7 Hz, 1 H); MS

1768 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11

Hazeldine et al.

¹H NMR (300 MHz, DMSO-d₆) δ 12.81 (bs, 1 H), 7.86 (d, J )

9.3 Hz, 1 H), 7.47 (d, J ) 9.3 Hz, 1 H), 3.83 (s, 3 H).

A mixture of this second intermediate (1.06 g, 4.21 mmol),

sodium dithionite (2.59 g, 12.6 mmol), EtOH (25 mL), and

water (50 mL) was refluxed for 2 h. After cooling, concentrated

HCl was added to pH 1 and the mixture was concentrated prior

to the addition of NaOH to pH > 12. The hot solution was

filtered, the residue washed with 1 N NaOH, and the filtrate

acidified to pH 6 with concentrated HCl. After cooling, the solid

was collected, washed with ice-water, and dried to give 23a

as a brown-yellow solid (0.70 g, 79% yield): mp ∼225 °C (dec);

¹H NMR (300 MHz, DMSO-d₆) δ 12.29 (bs, 1 H), 8.15 (s, 1 H),

7.50 (d, J ) 9.0 Hz, 1 H), 7.34 (d, J ) 8.7 Hz, 1 H), 3.81 (s,

3 H).

2,7-Dich lor o-8-m eth oxyqu in oxa lin e (24a ). Compound

23a (0.69 g, 3.3 mmol) was reacted with POCl₃(1.6 mL, 2.6 g,

17 mmol) (as described above for 10) to give 24a as an off-

white solid (0.55 g, 73% yield): mp 136-138 °C; ¹H NMR (300

MHz, CDCl₃) δ 8.76 (s, 1 H), 7.83 (d, J ) 9.0 Hz, 1 H), 7.76 (d,

J ) 9.0 Hz, 1 H), 4.21 (s, 3 H).

8-Ch lor o-7-m eth oxy-2-qu in oxa lin ol (23b). The method

described above for the preparation of 23a was applied. Thus

22b¹⁴(2.06 g, 10.2 mmol), cyanoacetic acid (1.75 g, 20.4 mmol),

toluene (100 mL), and PCl₅(4.47 g, 20.4 mmol) gave R-cyano-

3-chloro-2-methoxy-6-nitroacetanilide as off-white crystals fol-

lowing recrystallization from EtOH-heptane (2.48 g, 91%

yield): mp 226-228 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 10.63

(bs, 1 H), 8.04 (d, J ) 9.3 Hz, 1 H), 7.27 (d, J ) 9.3 Hz, 1 H),

3.97 (s, 3 H), 3.96 (s, 2 H).

This intermediate derivative (2.48 g, 9.20 mmol) was

dissolved in pyridine (30 mL), and treated with 1 N NaOH

(9.2 mL, 9.2 mmol) to give a mixture of 6-chloro-2-cyano-5-

methoxy-3-quinoxalinol-1-oxidaend6-chloro-2-cyano-5-methoxy-3-

quinoxalinol as a yellow solid (2.32 g).

H), 4.79 (q, J ) 6.9 Hz, 1 H), 3.79 (s, 3 H), 1.65 (d, J ) 6.6 Hz,

3 H); ¹³C NMR (75 MHz, CDCl₃) δ 172.4, 157.5, 155.1, 146.6,

141.2, 139.4, 136.2, 132.9, 130.1, 127.5, 126.7, 122.4, 116.1,

73.1, 52.3, 18.6; IR (KBr) 1740 (CdO) cm^-1; MS (EI) m/z (%)

358 (M⁺, 27), 299 (19), 243 (12), 163 (10), 149 (16), 137 (18),

123 (9), 121 (9), 109 (9), 107 (6), 97 (9), 95 (15), 93 (8), 85 (7),

83 (12), 81 (51), 73 (9), 71 (13), 69 (100), 67 (11), 60 (7). Anal.

(C₁₈H₁₅N₂ClO₄) C, H, N.

The methyl ester of 26a (0.26 g, 0.72 mmol), dissolved in

THF (10 mL), was hydrolyzed with 0.1 N NaOH (14.5 mL, 1.45

mmol) to give 26a (0.24 g, 96% yield) as off-white crystals after

recrystallization from EtOH-water: mp 144-145 °C; ¹H NMR

(300 MHz, DMSO-d₆) δ 13.08 (bs, 1 H), 8.90 (s, 1 H), 7.81 (dt,

J ) 4.5, 0.9 Hz, 1 H), 7.66 (d, J ) 4.2 Hz, 2 H), 7.27-7.19 (m,

2 H), 6.97-6.89 (m, 2 H), 4.83 (q, J ) 6.6 Hz, 1 H), 1.50 (d,

J ) 6.6 Hz, 3 H); ¹³C NMR (75 MHz, DMSO-d₆) δ 173.5, 157.9,

155.4, 146.3, 141.0, 140.6, 135.8, 132.4, 131.1, 127.9, 126.9,

122.9, 116.1, 72.4, 18.7; IR (KBr) 3400 (OH), 1685 (CdO) cm^-1

;

MS (EI) m/z (%) 344 (M⁺, 100), 299 (16), 285 (12), 272 (31),

255 (7), 243 (82), 215 (6), 208 (8), 193 (12), 181 (6), 163 (61),

152 (5), 136 (26), 127 (22), 124 (8), 116 (10), 109 (18), 100 (40),

85 (11), 81 (17), 75 (13), 69 (11), 63 (13), 57 (14), 55 (24),

45 (26), 43 (24), 41 (17), 39 (15); HRMS (EI) m/z 344.0558

(M⁺, calcd for C₁₇H₁₃N₂ClO₄344.0564). Anal. (C₁₇H₁₃N₂ClO₄)

C, H, N.

2-{4-[(8-Ch lor o-2-qu in oxa lin yl)oxy]p h en oxy}p r op ion -

ic Acid (26b). The methyl ester of 26b was prepared from 13

(0.16 g, 0.80 mmol), 25 (0.17 g, 0.87 mmol), anhydrous K₂CO₃

(0.15 g, 1.1 mmol), and CH₃CN (5 mL) for 6 h (method A). Pure

material (0.19 g, 66% yield) was obtained after chromatogra-

phy (4:1 hexanes:AcOEt) and recrystallization from EtOH-

1

hexanes to give pale yellow crystals: mp 86-88 °C; H NMR

(300 MHz, CDCl₃) δ 8.69 (s, 1 H), 7.98 (dd, J ) 8.4, 1.2 Hz, 1

H), 7.77 (dd, J ) 7.8, 0.9 Hz, 1 H), 7.52 (dd, J ) 8.1, 7.8 Hz,

1 H), 7.39-7.32 (m, 2 H), 7.01-6.93 (m, 2 H), 4.80 (q, J ) 6.6

Hz, 1 H), 3.79 (s, 3 H), 1.66 (d, J ) 6.6 Hz, 3 H); ¹³C NMR (75

MHz, CDCl₃) δ 172.4, 156.9, 155.0, 146.9, 140.3, 139.7, 137.0,

131.2, 130.3, 127.8, 126.9, 122.1, 116.1, 73.3, 52.2, 18.5; IR

(KBr) 1740 (CdO) cm^-1; MS (EI) m/z (%) 358 (M⁺, 100), 299

(51), 271 (20), 255 (11), 243 (50), 215 (4), 208 (3), 192 (6), 179

(3), 163 (36), 149 (6), 136 (17), 127 (11), 110 (6), 100 (13),

91 (5), 75 (7), 63 (7), 59 (12), 55 (7). Anal. (C₁₈H₁₅N₂ClO₄)

C, H, N.

The mixture (0.25 g) was treated with sodium dithionite

(0.51 g, 2.5 mmol), EtOH (5 mL), and water (10 mL) to give

23b as a brown solid (0.11 g): mp ∼220 °C (dec); ¹H NMR

(300 MHz, DMSO-d₆) δ 11.83 (bs, 1 H), 8.00 (s, 1 H), 7.72 (d,

J ) 9.0 Hz, 1 H), 7.15 (d, J ) 9.0 Hz, 1 H), 3.93 (s, 3 H).

2,7-Dich lor o-8-m eth oxyqu in oxa lin e (24b). A mixture of

crude 23b (1.4 g, ∼6.8 mmol) and POCl₃(3.0 mL, 4.9 g, 32

mmol) (as described above for 10) gave 24b as a pale yellow

1

solid (0.69 g): mp 164-167 °C; H NMR (300 MHz, CDCl₃) δ

8.68 (s, 1 H), 8.06 (d, J ) 9.3 Hz, 1 H), 7.58 (d, J ) 9.3 Hz, 1

H), 4.12 (s, 1 H).

The methyl ester of 26b (0.17 g, 0.47 mmol), dissolved in

THF (5 mL), was hydrolyzed with 0.1 N NaOH (9.5 mL, 0.95

mmol) to give 26b (0.13 g, 81% yield) as off-white-yellow

crystals after recrystallization from EtOH-water: mp 133-

136 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 13 (bs, 1 H), 8.77 (s,

1 H), 7.93 (dd, J ) 8.1, 0.9 Hz, 1 H), 7.82 (dd, J ) 7.5, 0.9 Hz,

1 H), 7.57 (t, J ) 8.1 Hz, 1 H), 7.35-7.27 (m, 2 H), 6.99-6.91

(m, 2 H), 4.82 (q, J ) 6.6 Hz, 1 H), 1.50 (d, J ) 6.9 Hz, 3 H);

¹³C NMR (75 MHz, DMSO-d₆) δ 173.5, 157.3, 155.3, 146.4,

140.9, 140.2, 136.5, 130.8, 130.2, 128.3, 127.8, 122.6, 116.0,

72.6, 18.7; IR (KBr) 3495 (OH), 1725 (CdO) cm^-1; MS (EI) m/z

(%) 344 (M⁺, 100), 299 (17), 272 (42), 255 (12), 243 (92), 236

(10), 215 (7), 208 (8), 192 (7), 179 (6), 163 (53), 152 (9), 136

(27), 127 (21), 124 (12), 110 (24), 100 (22), 97 (37), 85 (22), 83

(45), 81 (31), 73 (29), 71 (34), 69 (53), 67 (23), 60 (20), 57 (59),

55 (69); HRMS (EI) m/z 344.0563 (M⁺, calcd for C₁₇H₁₃N₂ClO₄

344.0564). Anal. (C₁₈H₁₅N₂ClO₄) C, N; H: calcd, 3.80; found,

4.45.

Gen er a l P r ep a r a tion of Qu in oxa lin e Ester s (Meth od

A). A mixture of the 2-chloroquinoxaline, the phenol, anhy-

drous K₂CO₃, and CH₃CN was refluxed until the reaction was

complete. The mixture was filtered hot, the residue washed

with hot acetone, and the filtrate evaporated to dryness. The

residue was dissolved in CH₂Cl₂, filtered through silica gel,

and washed with ether. The filtrate was shaken with saturated

NaCl, containing a small amount of 1 N NaOH, followed by

saturated NaCl. The dried solution (MgSO₄) was evaporated

and the crude residue purified by flash column chromatogra-

phy. If the product was a solid, it was recrystallized; if it was

an oil, it was hydrolyzed.

Gen er a l P r ep a r a tion of Qu in oxa lin e Ca r boxylic Ac-

id s. To a solution of the ester dissolved in THF was added 0.1

N base in portions, and the mixture was stirred overnight at

room temperature. After the mixture was concentrated and

filtered to remove insoluble material, the filtrate was cooled

and acidified to pH 3-4 with 0.25 N HCl. After recooling, the

solid was collected, washed with ice-water, and recrystallized,

if required.

2-{4-[(5-Ch lor o-2-qu in oxa lin yl)oxy]p h en oxy}p r op ion -

ic Acid (26a ). The methyl ester of 26a was prepared from 10

(0.25 g, 1.3 mmol), 25 (0.27 g, 1.4 mmol), anhydrous K₂CO₃

(0.24 g, 1.7 mmol), and CH₃CN (10 mL) for 6 h (method A).

Pure material (0.31 g, 69% yield) was obtained after chroma-

tography (4:1 hexanes:AcOEt) and recrystallization from

EtOH-heptane to give white crystals: mp 111-112 °C; ¹H

NMR (300 MHz, CDCl₃) δ 8.76 (s, 1 H), 7.73-7.65 (m, 2 H),

7.56 (t, J ) 7.8 Hz, 1 H), 7.23-7.16 (m, 2 H), 6.99-6.91 (m, 2

2-{4-[(7-Meth yl-2-qu in oxa lin yl)oxy]p h en oxy}p r op ion -

ic Acid (26c). The methyl ester of 26c was prepared from 16b

(0.89 g, 5.0 mmol), 25 (1.08 g, 5.5 mmol), anhydrous K₂CO₃

(0.95 g, 6.9 mmol), and CH₃CN (25 mL) for 24 h (method A).

Pure material (1.38 g, 82% yield) was obtained after chroma-

tography (4:1 hexanes:AcOEt) and recrystallization from EtOH

1

to give light yellow crystals: mp 138-140 °C; H NMR (300

MHz, CDCl₃) δ 8.59 (s, 1 H), 7.93 (d, J ) 8.4 Hz, 1 H), 7.55 (s,

1 H), 7.43 (dd, J ) 8.4, 1.5 Hz, 1 H), 7.23-7.15 (m, 2 H), 7.00-

6.92 (m, 2 H), 4.79 (q, J ) 6.9 Hz, 1 H), 3.80 (s, 3 H), 2.51 (s,

3 H), 1.66 (d, J ) 6.9 Hz, 3 H); ¹³C NMR (75 MHz, CDCl₃)

δ 172.4, 157.2, 154.9, 147.1, 140.8, 140.0, 138.0, 129.3, 128.4,

126.8, 122.4, 116.1, 73.2, 52.2, 21.6, 18.6; IR (KBr) 1765 (CdO)

Synthesis of XK469 Analogues

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11 1769

cm^-1; MS (EI) m/z (%) 338 (M⁺, 100), 310 (2), 279 (53), 251

(19), 235 (13), 223 (96), 195 (10), 143 (95), 116 (43), 110 (11),

105 (10), 89 (60), 87 (10), 77 (19), 63 (24), 59 (36), 55 (17), 51

(12), 43 (14). Anal. (C₁₉H₁₈N₂O₄) C, H, N.

J ) 1.6 Hz, 1 H), 7.91 (dd, J ) 8.0, 1.6 Hz, 1 H), 7.76 (d, J )

8.8 Hz, 1 H), 7.22 (d, J ) 8.8 Hz, 2 H), 6.94 (d, J ) 8.8 Hz, 2

H), 4.83 (q, J ) 6.8 Hz, 1 H), 1.52 (d, J ) 6.4 Hz, 3 H); ¹³C

NMR (100 MHz, DMSO-d₆) δ 173.8, 157.9, 155.6, 146.5, 141.1,

140.9, 138.8, 136.8, 136.2, 130.8, 123.2, 116.3, 98.1, 72.5, 19.0;

IR (KBr) 3430 (OH), 1720 (CdO) cm^-1; MS (EI) m/z (%) 436

(M⁺, 100), 391 (17), 377 (8), 364 (28), 347 (5), 335 (53), 255

(28), 228 (10), 208 (13), 182 (6), 128 (35), 110 (9), 101 (36), 75

(9), 63 (9), 50 (11); HRMS (EI) m/z 435.9928 (calcd for C₁₇H₁₃N₂-

IO₄435.9920). Anal. (C₁₇H₁₃N₂IO₄) C, H, N.

The methyl ester of 26c (0.55 g, 1.6 mmol), dissolved in THF

(15 mL), was hydrolyzed with 0.1 N NaOH (32 mL, 3.2 mmol)

to give 26c (0.50 g, 94% yield) as off-white crystals after

recrystallization from EtOH-water: mp 178-180 °C; ¹H NMR

(300 MHz, DMSO-d₆) δ 13.07 (bs, 1 H), 8.72 (s, 1 H), 7.91 (d,

J ) 8.4 Hz, 1 H), 7.51 (s, 1 H), 7.49 (dd, J ) 8.4, 1.5 Hz, 1 H),

7.25-7.16 (m, 2 H), 6.98-6.89 (m, 2 H), 4.83 (q, J ) 6.6 Hz, 1

H), 2.44 (s, 3 H), 1.50 (d, J ) 6.6 Hz, 3 H); ¹³C NMR (75 MHz,

DMSO-d₆) δ 173.4, 157.6, 155.3, 146.7, 141.3, 139.8, 138.7,

137.9, 129.8, 128.6, 122.9, 116.2, 72.6, 21.5, 18.7; IR (KBr) 3495

(OH), 1730 (CdO) cm^-1; MS (EI) m/z (%) 324 (M⁺, 99), 279

(13), 265 (10), 252 (21), 235 (8), 223 (100), 143 (68), 116 (23),

110 (9), 89 (27), 63 (9); HRMS (EI) m/z 324.1110 (M⁺, calcd

for C₁₈H₁₆N₂O₄324.1110). Anal. (C₁₈H₁₆N₂O₄) C, H, N.

2-{4-[(6-Nit r o-2-q u in oxa lin yl)oxy]p h en oxy}p r op ion -

ic Acid (26f). The methyl ester of 26f was prepared from 18b

(0.42 g, 2.0 mmol), 25 (0.43 g, 2.2 mmol), anhydrous K₂CO₃

(0.38 g, 2.8 mmol), and CH₃CN (10 mL) for 2 h (method A).

Pure material (0.69 g, 93% yield) was obtained after chroma-

tography (3:1 hexanes:AcOEt) and recrystallization from EtOH

1

to give light yellow crystals: mp 124-125 °C; H NMR (400

MHz, CDCl₃) δ 8.92 (d, J ) 2.4 Hz, 1 H), 8.80 (s, 1 H), 8.42

(dd, J ) 9.2, 2.4 Hz, 1 H), 7.83 (d, J ) 9.6 Hz, 1 H), 7.22-7.16

(m, 2 H), 6.99-6.93 (m, 2 H), 4.79 (q, J ) 6.4 Hz, 1 H), 3.80 (s,

3 H), 1.66 (d, J ) 7.6 Hz, 3 H); ¹³C NMR (100 MHz, CDCl₃) δ

172.7, 158.9, 155.6, 146.3, 143.9, 142.2, 138.4, 130.2, 129.1,

125.5, 124.2, 122.7, 116.3, 73.3, 52.7, 18.9; IR (KBr) 1735 (Cd

O) cm^-1; MS (EI) m/z (%) 369 (M⁺, 100), 339 (19), 310 (94),

283 (15), 266 (6), 254 (25), 236 (7), 225 (9), 220 (13), 208 (12),

174 (8), 144 (10), 132 (5), 128 (23), 117 (6), 110 (6), 101 (20),

91 (9), 75 (6), 69 (5), 63 (6), 59 (11), 57 (7), 55 (7), 50 (7). Anal.

(C₁₈H₁₅N₃O₆) C, H, N.

2-{4-[(7-Met h oxy-2-q u in oxa lin yl)oxy]p h en oxy}p r op i-

on ic Acid (26d ). The methyl ester of 26d was prepared from

16c (0.97 g, 5.0 mmol), 25 (1.08 g, 5.5 mmol), anhydrous K₂-

CO₃(0.95 g, 6.9 mmol), and CH₃CN (25 mL) for 24 h (method

A). Pure material (1.48 g, 84% yield) was obtained after

chromatography (4:1 hexanes:AcOEt) and recrystallization

1

from EtOH to give peach crystals: mp 107-109 °C; H NMR

(300 MHz, CDCl₃) δ 8.50 (s, 1 H), 7.91 (d, J ) 9.6 Hz, 1 H),

7.24 (dd, J ) 9.3, 2.7 Hz, 1 H), 7.23-7.16 (m, 2 H), 7.08 (d,

J ) 2.7 Hz, 1 H), 6.99-6.92 (m, 2 H), 4.79 (q, J ) 6.6 Hz, 1

H), 3.89 (s, 3 H), 3.80 (s, 3 H), 1.66 (d, J ) 6.6 Hz, 3 H); ¹³C

NMR (75 MHz, CDCl₃) δ 172.4, 161.2, 157.6, 154.9, 147.1,

141.8, 135.8, 135.4, 129.7, 122.4, 119.7, 116.1, 106.2, 73.1, 55.6,

52.2, 18.5; IR (KBr) 1755 (CdO) cm^-1; MS (EI) m/z (%) 354

(M⁺, 100), 326 (4), 295 (36), 267 (8), 251 (10), 239 (48), 225

(10), 208 (4), 159 (45), 148 (8), 117 (14), 110 (4), 102 (5), 89

(5), 77 (8), 59 (7). Anal. (C₁₉H₁₈N₂O₅) C, H, N.

The methyl ester of 26f (0.33 g, 0.89 mmol), dissolved in

THF (15 mL), was hydrolyzed with 0.1 N K₂CO₃(22.5 mL, 2.25

mmol) to give 26f (0.28 g, 88% yield) as a yellow solid: mp

1

167-169 °C; H NMR (400 MHz, DMSO-d₆) δ 9.01 (s, 1 H),

8.80 (d, J ) 3.6 Hz, 1 H), 8.39 (dd, J ) 8.8, 2.4 Hz, 1 H), 7.88

(d, J ) 10.0 Hz, 1 H), 7.29-7.24 (m, 2 H), 6.99-6.93 (m, 2 H),

4.85 (q, J ) 6.4 Hz, 1 H), 1.52 (d, J ) 6.4 Hz, 3 H); ¹³C NMR

(100 MHz, DMSO-d₆) δ 173.8, 159.4, 155.9, 146.1, 143.8, 143.5,

138.1, 129.4, 125.0, 124.6, 123.2, 116.3, 72.5, 19.0; IR (KBr)

3400 (OH), 1725 (CdO) cm^-1; MS (EI) m/z (%) 355 (M⁺, 100),

339 (5), 325 (10), 310 (38), 296 (8), 283 (48), 266 (5), 254 (56),

236 (7), 225 (16), 220 (11), 208 (22), 192 (5), 182 (9), 174 (16),

144 (9), 128 (42), 109 (15), 101 (37), 91 (10), 83 (10), 81 (10),

75 (12), 69 (14), 65 (11), 63 (13), 57 (17), 55 (18), 50 (14); HRMS

(EI) m/z 355.0804 (calcd for C₁₇H₁₃N₃O₆355.0804). Anal.

(C₁₇H₁₃N₃O₆) C, H, N.

The methyl ester of 26d (0.50 g, 1.4 mmol), dissolved in THF

(15 mL), was hydrolyzed with 0.1 N NaOH (28 mL, 2.8 mmol)

to give 26d (0.49 g, 97% yield) as off-white crystals after

recrystallization from EtOH-water: mp ∼110-140 °C; ¹H

NMR (300 MHz, DMSO-d₆) δ 13.07 (bs, 1 H), 8.61 (s, 1 H),

7.90 (d, J ) 9.3 Hz, 1 H), 7.25 (dd, J ) 9.3, 2.7 Hz, 1 H), 7.23-

7.16 (m, 2 H), 7.07 (d, J ) 2.7 Hz, 1 H), 6.98-6.89 (m, 2 H),

4.83 (q, J ) 6.6 Hz, 1 H), 3.83 (s, 3 H), 1.50 (d, J ) 6.9 Hz, 3

H); ¹³C NMR (75 MHz, DMSO-d₆) δ 173.4, 161.4, 158.0, 155.3,

146.7, 141.7, 136.5, 135.2, 130.0, 123.0, 119.9, 116.2, 106.7,

72.5, 56.3, 18.7; IR (KBr) 3505 (OH), 1745 (CdO) cm^-1; MS

(EI) m/z (%) 340 (M⁺, 90), 312 (8), 295 (10), 281 (13), 268 (21),

251 (9), 239 (85), 225 (22), 211 (6), 208 (7), 159 (100), 148 (9),

144 (12), 117 (58), 109 (22), 102 (22), 91 (24), 89 (29), 81 (26),

77 (50), 75 (19), 65 (26), 63 (33), 55 (32), 45 (47); HRMS (EI)

m/z 340.1060, (M⁺, calcd for C₁₈H₁₆N₂O₅340.1059). Anal.

(C₁₈H₁₆N₂O₅‚H₂O) C, H, N.

2-{4-[(7-Nit r o-2-q u in oxa lin yl)oxy]p h en oxy}p r op ion -

ic Acid (26g). The methyl ester of 26g was prepared from

18d (1.05 g, 5.0 mmol), 25 (1.08 g, 5.5 mmol), anhydrous K₂-

CO₃(0.95 g, 6.9 mmol), and CH₃CN (25 mL) for 2 h (method

A). Pure material (1.68 g, 91% yield) was obtained after

chromatography (3:1 hexanes:AcOEt) to give a yellow liquid,

which solidified very slowly: ¹H NMR (300 MHz, CDCl₃) δ 8.81

(s, 1 H), 8.62 (d, J ) 2.4 Hz, 1 H), 8.36 (dd, J ) 9.3, 2.4 Hz, 1

H), 8.18 (d, J ) 9.3 Hz, 1 H), 7.25-7.16 (m, 2 H), 7.02-6.93

(m, 2 H), 4.80 (q, J ) 6.9 Hz, 1 H), 3.81 (s, 3 H), 1.66 (d, J )

6.9 Hz, 3 H); ¹³C NMR (75 MHz, CDCl₃) δ 172.3, 158.2, 155.4,

148.3, 146.2, 142.6, 142.0, 139.4, 130.3, 123.6, 122.4, 120.8,

116.2, 73.2, 52.3, 18.5; IR (film) 1755 (CdO) cm^-1; MS (EI)

m/z (%) 369 (M⁺, 80), 310 (100), 283 (16), 266 (7), 254 (25),

236 (8), 225 (7), 220 (13), 208 (13), 182 (5), 174 (9), 132 (4),

128 (27), 116 (4), 109 (5), 101 (18), 91 (8), 75 (8), 63 (7), 59

(17), 55 (6), 50 (7); HRMS (EI) m/z 369.0962 (M⁺, calcd for

2-{4-[(7-Iod o-2-q u in oxa lin yl)oxy]p h en oxy}p r op ion ic

Acid (26e). The methyl ester of 26e was prepared from 16d

(0.29 g, 1.0 mmol), 25 (0.21 g, 1.1 mmol), anhydrous K₂CO₃

(0.18 g, 1.3 mmol), and CH₃CN (10 mL) for 4 h (method A).

Pure material (0.37 g, 82% yield) was obtained after chroma-

tography (4:1 hexanes:AcOEt) and recrystallization from EtOH

1

to give pale yellow crystals: mp 134-135 °C; H NMR (400

MHz, CDCl₃) δ 8.65 (s, 1 H), 8.15 (d, J ) 1.6 Hz, 1 H), 7.83

(dd, J ) 8.8, 1.6 Hz, 1 H), 7.72 (d, J ) 8.8 Hz, 1 H), 7.19-7.13

(m, 2 H), 6.97-6.91 (m, 2 H), 4.78 (q, J ) 6.8 Hz, 1 H), 3.79 (s,

3 H), 1.65 (d, J ) 6.8 Hz, 3 H); ¹³C NMR (100 MHz, CDCl₃) δ

172.8, 157.5, 155.3, 146.7, 141.0, 140.0, 138.9, 136.8, 136.5,

130.3, 122.7, 116.3, 96.6, 73.3, 52.7, 18.9; IR (KBr) 1750 (Cd

O) cm^-1; MS (EI) m/z (%) 450 (M⁺, 100), 404 (4), 402 (4), 391

(48), 364 (8), 347 (6), 335 (28), 255 (19), 228 (7), 220 (5), 208

(8), 128 (23), 101 (23), 81 (6), 75 (7), 63 (7), 59 (11), 55 (6), 50

(8), 45 (5). Anal. (C₁₈H₁₅N₂IO₄) C, H, N.

C

₁₈H₁₅N₃O₆369.0961).

The methyl ester of 26g (0.52 g, 1.41 mmol), dissolved in

THF (20 mL), was hydrolyzed with 0.1 N NaOH (28.5 mL, 2.85

mmol) to give 26g (0.30 g, 60% yield) as a dark brown solid:

1

mp ∼85 °C; H NMR (300 MHz, DMSO-d₆) δ 13.06 (bs, 1 H),

9.00 (s, 1 H), 8.41 (d, J ) 2.4 Hz, 1 H), 8.36 (dd, J ) 9.3, 2.4

Hz, 1 H), 8.25 (d, J ) 9.0 Hz, 1 H), 7.33-7.23 (m, 2 H), 7.01-

6.91 (m, 2 H), 4.84 (q, J ) 6.6 Hz, 1 H), 1.51 (d, J ) 6.6 Hz, 3

H); ¹³C NMR (75 MHz, DMSO-d₆) δ 173.3, 158.6, 155.6, 148.3,

146.2, 144.0, 142.0, 139.2, 130.8, 123.1, 122.8, 121.3, 116.3,

72.6, 18.7; IR (KBr) 3415 (OH), 1725 (CdO) cm^-1; MS (EI) m/z

(%) 355 (M⁺, 100), 339 (2), 325 (3), 310 (45), 296 (7), 283 (41),

266 (5), 254 (55), 236 (8), 225 (10), 220 (10), 208 (22), 182 (9),

The methyl ester of 26e (0.23 g, 0.51 mmol), dissolved in

THF (10 mL), was hydrolyzed with 0.1 N NaOH (10.2 mL, 1.02

mmol) to give 26e (0.21 g, 95% yield) as cream crystals after

recrystallization from EtOH-water: mp 164-166 °C; ¹H NMR

(400 MHz, DMSO-d₆) δ 13.00 (bs, 1 H), 8.81 (s, 1 H), 8.07 (d,

1770 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11

Hazeldine et al.

174 (15), 128 (43), 116 (7), 109 (14), 101 (30), 91 (6), 81 (10),

75 (12), 63 (12); HRMS (EI) m/z 355.0802 (M⁺, calcd for

C₁₇H₁₃N₃O₆355.0804). Anal. (C₁₇H₁₃N₃O₆) C, H, N.

prepared from 24b (0.69 g, 3.0 mmol), 25 (0.65 g, 3.3 mmol),

anhydrous K₂CO₃(0.57 g, 4.1 mmol), and CH₃CN (15 mL) for

4 h (method A). Mostly pure material (0.84 g, 72% crude yield)

was obtained after chromatography (2:1 hexanes:AcOEt) to

give a yellow liquid, which gradually solidified. A sample was

recrystallized from acetone-hexanes (2×) to give off-white

2-{4-[(7-Azid o-2-q u in oxa lin yl)oxy]p h en oxy}p r op ion -

ic Acid (26h ). The methyl ester of 26h was prepared from 21

(0.09 g, 0.4 mmol), 25 (0.09 g, 0.5 mmol), anhydrous K₂CO₃

(0.08 g, 0.6 mmol), and CH₃CN (5 mL) for 4 h (method A). Pure

material (0.15 g, 94% yield) was obtained after chromatogra-

phy (4:1 hexanes:AcOEt) to give a yellow liquid: ¹H NMR (300

MHz, CDCl₃) δ 8.57 (s, 1 H), 7.98 (d, J ) 8.7 Hz, 1 H), 7.37 (d,

J ) 2.4 Hz, 1 H), 7.21 (dd, J ) 8.7, 2.4 Hz, 1 H), 7.20-7.13

(m, 2 H), 6.98-6.90 (m, 2 H), 4.77 (q, J ) 6.9 Hz, 1 H), 3.79 (s,

3 H), 1.65 (d, J ) 6.6 Hz, 3 H); ¹³C NMR (75 MHz, CDCl₃) δ

172.4, 157.7, 155.1, 146.7, 142.4, 141.0, 138.2, 137.2, 130.4,

122.4, 120.0, 116.1, 115.6, 73.2, 52.2, 18.5; IR (Film) 2110 (N₃),

1755 (CdO) cm^-1; MS (EI) m/z (%) 365 (M⁺, 76), 337 (40), 312

(6), 309 (9), 306 (12), 277 (58), 250 (46), 233 (23), 222 (55), 205

(7), 194 (32), 179 (8), 167 (9), 152 (6), 144 (15), 142 (25), 139

(30), 131 (10), 120 (10), 115 (100), 110 (14), 103 (14), 91 (22),

88 (44), 81 (13), 76 (34), 64 (68), 59 (56), 55 (25), 52 (14), 50

(16), 43 (12), 39 (10); HRMS (EI) m/z 365.1121 (M⁺, calcd for

1

crystals: mp 116-118 °C; H NMR (300 MHz, CDCl₃) δ 8.51

(s, 1 H), 7.94 (d, J ) 9.0 Hz, 1 H), 7.36 (d, J ) 9.0 Hz, 1 H),

7.37-7.31 (m, 2 H), 6.98-6.92 (m, 2 H), 4.80 (q, J ) 6.9 Hz, 1

H), 4.04 (s, 3 H), 3.78 (s, 3 H), 1.65 (d, J ) 6.9 Hz, 3 H); ¹³C

NMR (100 MHz, CDCl₃) δ 172.8, 157.4, 156.5, 155.0, 147.0,

138.2, 137.2, 135.3, 128.0, 122.4, 116.3, 116.0, 113.4, 73.2, 57.0,

52.6, 18.8; IR (KBr) 1730 (CdO) cm^-1; MS (EI) m/z (%) 388

(M⁺, 100), 329 (39), 301 (13), 293 (5), 285 (11), 273 (58), 267

(5), 259 (21), 193 (30), 178 (14), 164 (8), 151 (10), 102 (8), 88

(8). Anal. (C₁₉H₁₇N₂ClO₅) C, H, N.

The crude methyl ester of 26j (0.80 g, ∼2.0 mmol), dissolved

in THF (25 mL), was hydrolyzed with 0.1 N NaOH (40 mL,

4.0 mmol) to give 26j (0.70 g, 91% yield) as light yellow crystals

after recrystallization from EtOH-water: mp 148-150 °C; ¹H

NMR (400 MHz, DMSO-d₆) δ 13.00 (bs, 1 H), 8.62 (s, 1 H),

7.95 (d, J ) 9.6 Hz, 1 H), 7.58 (d, J ) 9.6 Hz, 1 H), 7.31 (d,

J ) 9.2 Hz, 2 H), 6.95 (d, J ) 8.8 Hz, 2 H), 4.85 (q, J ) 6.4 Hz,

1 H), 3.98 (s, 3 H), 1.51 (d, J ) 6.4 Hz, 3 H); ¹³C NMR (100

MHz, DMSO-d₆) δ 173.8, 157.9, 156.8, 155.4, 146.6, 138.1,

137.9, 135.2, 128.7, 123.0, 116.1, 115.1, 114.7, 72.5, 57.5, 19.0;

IR (KBr) 3440 (OH), 1740 (CdO) cm^-1; MS (EI) m/z (%) 374

(M⁺, 100), 329 (13), 302 (24), 293 (5), 285 (8), 273 (69), 267

(8), 259 (28), 236 (7), 193 (29), 178 (14), 164 (5), 151 (10), 110

(6), 102 (6), 97 (9), 83 (9), 69 (9), 55 (6); HRMS (EI) m/z

C

₁₈H₁₅N₅O₄365.1124).

The methyl ester of 26h (0.15 g, 0.41 mmol), dissolved in

THF (5 mL), was hydrolyzed with 0.1 N NaOH (8.2 mL, 0.82

mmol) to give 26h (0.13 g, 93% yield) as a tan solid: mp 140-

142 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 13.06 (bs, 1 H), 8.72

(s, 1 H), 8.01 (d, J ) 8.4 Hz, 1 H), 7.39-7.30 (m, 2 H), 7.25-

7.16 (m, 2 H), 6.97-6.87 (m, 2 H), 4.82 (q, J ) 6.6 Hz, 1 H),

1.50 (d, J ) 6.6 Hz, 3 H); ¹³C NMR (75 MHz, DMSO-d₆) δ 173.4,

158.0, 155.4, 146.4, 142.3, 140.7, 139.1, 137.1, 130.7, 122.9,

120.5, 116.2, 115.6, 72.5, 18.7; IR (KBr) 3440 (OH), 2230 (N₃),

1730 (CdO) cm^-1; MS (EI) m/z (%) 351 (M⁺, 3), 323 (2), 279

(4), 251 (7), 222 (5), 195 (4), 182 (42), 137 (16), 115 (10), 110

(100), 93 (8), 81 (37), 74 (21), 65 (19), 55 (11), 53 (14), 44 (15),

39 (11); HRMS (EI) m/z 351.0974 (M⁺, calcd for C₁₇H₁₃N₅O₄

351.0968). Anal. (C₁₇H₁₃N₅O₄) C, H, N.

2-{4-[(7-Ch lor o-8-m e t h oxy-2-q u in oxa lin yl)oxy]p h e -

n oxy}p r op ion ic Acid (26i). The methyl ester of 26i was

prepared from 24a (0.17 g, 0.74 mmol), 25 (0.16 g, 0.82 mmol),

anhydrous K₂CO₃(0.14 g, 1.0 mmol), and CH₃CN (10 mL) for

4 h (method A). Pure material (0.22 g, 76% yield) was obtained

after chromatography (4:1 hexanes:AcOEt) and recrystalliza-

tion from EtOH-heptane to give white crystals: mp 115-116

°C; ¹H NMR (300 MHz, CDCl₃) δ 8.65 (s, 1 H), 7.72 (d, J ) 9.0

Hz, 1 H), 7.55 (d, J ) 9.3 Hz, 1 H), 7.23-7.16 (m, 2 H), 6.99-

6.91 (m, 2 H), 4.79 (q, J ) 6.9 Hz, 1 H), 3.90 (s, 3 H), 3.78 (s,

3 H), 1.65 (d, J ) 6.9 Hz, 3 H); ¹³C NMR (75 MHz, CDCl₃) δ

172.4, 156.4, 155.1, 150.3, 146.9, 139.3, 138.7, 134.7, 128.3,

127.6, 124.2, 122.5, 116.1, 73.2, 62.1, 52.3, 18.5; IR (KBr) 1760

(CdO) cm^-1; MS (EI) m/z (%) 388 (M⁺, 100), 359 (4), 329 (41),

313 (10), 311 (10), 301 (23), 285 (14), 273 (36), 270 (8), 255 (7),

209 (7), 193 (22), 180 (11), 163 (13), 151 (13), 136 (9), 121 (18),

110 (8), 102 (11), 88 (17), 76 (12), 63 (9), 59 (24), 55 (10), 50

(6). Anal. (C₁₉H₁₇N₂ClO₅) C, H, N.

374.0672 (M⁺, calcd for C₁₈H₁₅N₂ClO₅374.0670). Anal. (C₁₈

₁₅N₂ClO₅) C, H, N.

2-{4-[(7-Am in o-2-qu in oxa lin yl)oxy]p h en oxy}p r op ion -

-

H

ic Acid (26k ). To the methyl ester of 26g (1.29 g, 3.49 mmol),

dissolved in AcOH (35 mL), was added iron powder (1.02 g,

17.4 mmol), and the mixture was stirred overnight at room

temperature. After the mixture was concentrated, water and

saturated NaHCO₃were added until pH 7. Hot AcOEt (50 mL)

was added, and the mixture was filtered and washed with

AcOEt. The aqueous layer was extracted with AcOEt (50 mL),

and the combined AcOEt was washed with saturated NaCl

(25 mL), dried (MgSO₄), and concentrated. Pure material (0.93

g, 78% yield) was obtained after chromatography (1:1 hexanes:

AcOEt) to give a yellow solid: mp 165-168 °C; ¹H NMR (300

MHz, CDCl₃) δ 8.34 (s, 1 H), 7.80 (d, J ) 8.7 Hz, 1 H), 7.20-

7.12 (m, 2 H), 6.98 (dd, J ) 8.7, 2.4 Hz, 1 H), 6.97-6.90 (m, 2

H), 6.84 (d, J ) 2.4 Hz, 1 H), 4.77 (q, J ) 6.6 Hz, 1 H), 3.79 (s,

3 H), 3.75 (vbs, 2 H), 1.65 (d, J ) 6.6 Hz, 3 H); ¹³C NMR (75

MHz, CDCl₃) δ 172.5, 157.7, 154.8, 148.7, 147.3, 142.1, 134.3,

134.1, 129.8, 122.4, 118.6, 116.1, 107.6, 73.2, 52.3, 18.6; IR

(KBr) 3360 (NH), 3250 (NH), 1735 (CdO) cm^-1; MS (EI) m/z

(%) 339 (M⁺, 100), 311 (10), 280 (20), 252 (7), 236 (12), 224

(54), 196 (8), 161 (11), 144 (55), 140 (5), 133 (6), 117 (37), 105

(10), 90 (28), 81 (12), 74 (10), 69 (15), 63 (21), 59 (26), 57 (18),

55 (21), 43 (22), 41 (26), 39 (16). Anal. (C₁₈H₁₇N₃O₄) C, H, N.

The methyl ester of 26k (0.16 g, 0.47 mmol), dissolved in

THF (10 mL), was hydrolyzed with 0.1 N NaOH (9.4 mL, 0.94

mmol) to give 26k (0.05 g, 33% yield) as a yellow solid: mp

156-158 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 13.00 (bs, 1 H),

8.24 (s, 1 H), 7.63 (d, J ) 9.0 Hz, 1 H), 7.14 (d, J ) 9.0 Hz, 2

H), 6.98 (dd, J ) 9.0, 2.1 Hz, 1 H), 6.90 (d, J ) 9.0, 2 H), 6.54

(d, J ) 2.1 Hz, 1 H), 5.94 (bs, 2 H), 4.81 (q, J ) 6.6 Hz, 1 H),

1.49 (d, J ) 6.6 Hz, 3 H); ¹³C NMR (75 MHz, DMSO-d₆) δ 173.5,

157.8, 155.0, 151.6, 147.0, 142.4, 133.4, 132.4, 129.7, 122.9,

119.2, 116.1, 105.2, 72.5, 18.7; IR (KBr) 3445 (OH), 3350 (NH),

3225 (NH), 1715 (CdO) cm^-1; MS (EI) m/z (%) 325 (M⁺, 100),

297 (8), 280 (7), 266 (9), 253 (13), 236 (7), 224 (57), 196 (7),

182 (4), 144 (60), 140 (6), 117 (31), 110 (13), 105 (5), 90 (24),

81 (6), 63 (15), 55 (6); HRMS (EI) m/z 325.1061 (M⁺, calcd for

C₁₇H₁₅N₃O₄325.1063). Anal. (C₁₇H₁₅N₃O₄) C, H, N.

The methyl ester of 26i (0.18 g, 0.46 mmol), dissolved in

THF (10 mL), was hydrolyzed with 0.1 N NaOH (9.2 mL, 0.92

mmol) to give 26i (0.16 g, 94% yield) as off-white crystals after

recrystallization from EtOH-water: mp 172-173 °C (softens

1

at 120 °C); H NMR (300 MHz, DMSO-d₆) δ 13.02 (bs, 1 H),

8.81 (s, 1 H), 7.75 (d, J ) 9.0 Hz, 1 H), 7.66 (d, J ) 9.0 Hz, 1

H), 7.25 (d, J ) 9.0 Hz, 2 H), 6.95 (d, J ) 9.0, 2 H), 4.84 (q,

J ) 6.6 Hz, 1 H), 3.78 (s, 3 H), 1.50 (d, J ) 6.6 Hz, 3 H); ¹³C

NMR (75 MHz, DMSO-d₆) δ 173.4, 156.7, 155.4, 150.1, 146.4,

140.0, 139.3, 134.5, 128.4, 126.7, 124.7, 123.0, 116.1, 72.5, 62.2,

18.7; IR (KBr) 3440 (OH), 1720 (CdO) cm^-1; MS (EI) m/z (%)

374 (M⁺, 100), 345 (6), 329 (12), 313 (10), 301 (22), 297 (42),

285 (12), 273 (45), 270 (9), 258 (11), 255 (11), 209 (12), 193

(32), 180 (12), 163 (19), 151 (19), 139 (9), 136 (14), 121 (15),

110 (14), 107 (8), 102 (16), 100 (9), 94 (12), 88 (27), 81 (11), 76

(16), 73 (8), 65 (15), 63 (15), 55 (13), 53 (9), 50 (10); HRMS

(EI) m/z 374.0668 (M⁺, calcd for C₁₈H₁₅N₂ClO₅374.0670). Anal.

(C₁₈H₁₅N₂ClO₅) C, H, N.

Met h yl 2-[(7-Ch lor o-2-q u in oxa lin yl)oxy]p r op ion a t e

(28a ). (Meth od B). Compound 27 (0.18 g, 1.0 mmol), methyl

2-bromopropionate (0.12 mL, 0.18 g, 1.05 mmol), anhydrous

K₂CO₃(0.17 g, 1.2 mmol), and acetone (25 mL) were refluxed

2-{4-[(8-Ch lor o-7-m e t h oxy-2-q u in oxa lin yl)oxy]p h e -

n oxy}p r op ion ic Acid (26j). The methyl ester of 26j was

Synthesis of XK469 Analogues

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11 1771

together for 8 h. After being concentrated it was mixed with

AcOEt, filtered through silica gel, and concentrated. Pure 28a

(0.12 g, 44% yield) was obtained after chromatography (10:

1-4:1 hexanes:AcOEt) and recrystallization from a small

volume of hexanes (2×) to give white crystals: mp 70-72 °C;

¹H NMR (300 MHz, CDCl₃) δ 8.56 (s, 1 H), 7.95 (d, J ) 8.7

Hz, 1 H), 7.78 (d, J ) 2.1 Hz, 1 H), 7.53 (dd, J ) 8.7, 2.1, 1 H),

5.50 (q, J ) 6.9 Hz, 1 H), 3.78 (s, 3 H), 1.71 (d, J ) 6.9 Hz, 3

H); ¹³C NMR (75 MHz, CDCl₃) δ 171.5, 156.4, 140.3, 139.3,

137.7, 135.9, 130.0, 127.7, 126.3, 70.5, 52.2, 17.4; IR (KBr) 1760

(CdO) cm^-1; MS (EI) m/z (%) 266 (M⁺, 55), 251 (2), 235 (8),

207 (100), 180 (84), 163 (74), 152 (52), 136 (26), 124 (23), 100

(21), 75 (11), 59 (21). Anal. (C₁₂H₁₁N₂ClO₃) C, H, N.

Meth yl 2-(7-Ch lor o-2-oxo-2H-qu in oxa lin yl)p r op ion a te

(29). Compound 29 was also obtained after 28a following

chromatography of the crude mixture as yellow crystals,

recrystallized from a small volume of hexanes (2×) (0.03 g,

11% yield): mp 122-124 °C; ¹H NMR (300 MHz, CDCl₃) δ 8.28

(s, 1 H), 7.85 (d, J ) 8.4 Hz, 1 H), 7.34 (dd, J ) 8.4, 1.8, 1 H),

7.19 (d, J ) 1.8 Hz, 1 H), 5.62-5.50 (m, 1 H), 3.75 (s, 3 H),

1.73 (d, J ) 7.2 Hz, 3 H); ¹³C NMR (75 MHz, CDCl₃) δ 169.4,

153.8, 150.0, 137.1, 132.5, 132.4, 132.2, 124.3, 113.6, 52.8, 51.2,

13.9; IR (KBr) 1750 (CdO) cm^-1; MS (EI) m/z (%) 266 (M⁺,

44), 234 (24), 207 (38), 179 (100), 163 (7), 152 (18), 144 (9),

124 (12), 117 (12), 111 (12), 89 (10), 75 (21), 63 (9), 59 (9). Anal.

(C₁₂H₁₁N₂ClO₃) C, H, N.

2-[(7-Ch lor o-2-qu in oxa lin yl)oxy]p r op ion ic Acid (28b).

Ester 28a (0.07 g, 0.26 mmol), dissolved in THF (5 mL), was

hydrolyzed with 0.1 N NaOH (5.2 mL, 0.52 mmol) to give 28b

(0.07 g, ∼100% yield) as an off-white solid: mp 152-154 °C;

¹H NMR (400 MHz, DMSO-d₆) δ 13.11 (bs, 1 H), 8.66 (s, 1 H),

7.97 (d, J ) 8.0 Hz, 1 H), 7.73 (d, J ) 2.4 Hz, 1 H), 7.62 (dd,

J ) 9.2, 2.4 Hz, 1 H), 5.38 (q, J ) 7.2 Hz, 1 H), 1.60 (d, J ) 7.6

Hz, 3 H); ¹³C NMR (100 MHz, DMSO-d₆) δ 172.7, 157.2, 140.7,

140.5, 137.8, 135.6, 131.1, 128.2, 126.3, 70.9, 17.9; IR (KBr)

1720 (CdO) cm^-1; MS (EI) m/z (%) 252 (M⁺, 32), 207 (19), 193

(63), 180 (81), 163 (54), 152 (100), 136 (24), 124 (29), 117 (12),

105 (18), 100 (20), 90 (10), 77 (12), 75 (10), 69 (20), 63 (12);

HRMS (EI) m/z 252.0301 (M⁺, calcd for C₁₁H₉N₂ClO₃252.0302).

Anal. (C₁₁H₉N₂ClO₃) C, H, N.

Meth yl 2-(3-Hydr oxyph en oxy)pr opion ate (31). (Meth od

C). To a solution of sodium methoxide, prepared from sodium

(2.30 g, 100 mmol) and CH₃OH (50 mL), was added 30 (5.50

g, 50 mmol), under an Ar atmosphere. Methyl 2-bromopropi-

onate (3.8 mL, 5.7 g, 33 mmol) was added over 0.5 h at reflux.

The mixture was refluxed overnight, and the CH₃OH was then

removed by distillation. After drying, toluene (50 mL) was

added, and the mixture was allowed to stand overnight. The

toluene was decanted and the washing procedure repeated.

Toluene (50 mL), AcOH (4 mL), and water (50 mL) were added,

and the mixture was stirred until all the solid dissolved. The

toluene layer was separated and the aqueous layer extracted

with additional toluene (50 mL). The organic extract was

washed with water (2 × 50 mL), dried (MgSO₄), and concen-

trated before purifying by chromatography (4:1 hexanes:

AcOEt) to give 31 as a colorless liquid. ¹H NMR (300 MHz,

CDCl₃) δ 7.12 (t, J ) 8.1 Hz, 1 H), 6.55-6.38 (m, 3 H), 4.95 (s,

1 H), 4.73 (q, J ) 6.9 Hz, 1 H), 3.77 (s, 3 H), 1.62 (d, J ) 6.9

Hz, 3 H).

with water (2 × 10 mL). After drying and concentrating, the

residue was purified by chromatography (5:1 hexanes:AcOEt)

to give 33 as a colorless liquid (1.03 g, 72% yield): ¹H NMR

(300 MHz, CDCl3) δ 7.00-6.78 (m, 5 H), 4.69 (q, J ) 6.9 Hz,

1 H), 3.78 (s, 3 H), 1.69 (d, J ) 6.9 Hz, 3 H).

Meth yl 2-(4,4′-Hydr oxyph en ylph en oxy)pr opion ate (35).

Sodium (0.46 g, 20 mmol), CH₃OH (25 mL), 34 (1.92 g, 10.0

mmol), and methyl 2-bromopropionate (1.14 mL, 1.71 g, 10.0

mmol) (method C). After being washed with toluene (2 × 25

mL) and mixed with AcOEt (50 mL), AcOH (1 mL), and water

(25 mL). Extracted with AcOEt (2 × 25 mL), washed with

saturated NaCl (25 mL), dried (MgSO₄), and concentrated

before purifying by chromatography (aluminum oxide 150

mesh, 58 Å, Neutral, Brockmann 1 (Aldrich), AcOEt) to give

35 as an off-white solid: mp 115-120 °C; ¹H NMR (400 MHz,

CDCl₃) δ 7.42 (d, J ) 6.4 Hz, 2 H), 7.38 (d, J ) 7.2 Hz, 2 H),

6.90 (d, J ) 6.4 Hz, 2 H), 6.85 (d, J ) 6.4 Hz, 2 H), 5.20 (s, 1

H), 4.80 (bq, J ) 6.4 Hz, 1 H), 3.78 (s, 3 H), 1.65 (d, J ) 6.4

Hz, 3 H).

2-{3-[(7-Ch lor o-2-qu in oxa lin yl)oxy]p h en oxy}p r op ion -

ic Acid (36). The methyl ester of 36 was prepared from 16a

(0.50 g, 2.5 mmol), 31 (0.61 g, 3.1 mmol), anhydrous K₂CO₃

(0.54 g, 3.9 mmol), and CH₃CN (15 mL) for 24 h (method A).

Pure material (0.55 g, 61% yield) was obtained after chroma-

tography (3:1 hexanes:AcOEt) and recrystallization from EtOH

to give peach colored crystals: mp 167-169 °C; ¹H NMR (300

MHz, CDCl₃) δ 8.66 (s, 1 H), 7.99 (d, J ) 8.7 Hz, 1 H), 7.79 (d,

J ) 2.1 Hz, 1 H), 7.56 (dd, J ) 8.7, 2.1 Hz, 1 H), 7.35 (t, J )

8.1 Hz, 1 H), 6.90 (dd, J ) 7.8, 1.5 Hz, 1 H), 6.83 (d, J ) 2.7

Hz, 1 H), 6.82 (dd, J ) 13.8, 2.1 Hz, 1 H), 4.79 (q, J ) 6.9 Hz,

1 H), 3.77 (s, 3 H), 1.65 (d, J ) 6.6 Hz, 3 H); ¹³C NMR (75

MHz, CDCl₃) δ 172.2, 158.7, 157.2, 153.6, 140.6, 139.3, 138.2,

136.3, 130.1, 130.0, 128.3, 126.8, 114.4, 112.3, 108.9, 72.9, 52.3,

18.4; IR (KBr) 1755 (CdO) cm^-1; MS (EI) m/z (%) 358 (M⁺,

48), 299 (100), 271 (14), 255 (8), 244 (14), 192 (9), 163 (22),

136 (15), 119 (9), 100 (11), 92 (10), 64 (13), 59 (22). Anal.

(C₁₈H₁₅N₂ClO₄) C, H, N.

The methyl ester of 36 (0.55 g, 1.53 mmol), dissolved in THF

(25 mL), was hydrolyzed with 0.1 N NaOH (30.7 mL, 3.07

mmol) to give 36 (0.50 g, 94% yield) as a light tan solid: mp

132-135 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 12.75 (bs, 1 H),

8.76 (s, 1 H), 7.98 (d, J ) 8.7 Hz, 1 H), 7.72 (s, 1 H), 7.61 (d,

J ) 9.0 Hz, 1 H), 7.40-7.30 (m, 1 H), 6.95-6.85 (m, 3 H), 4.83

(q, J ) 6.6 Hz, 1 H), 1.49 (d, J ) 6.6 Hz, 3 H); ¹³C NMR (75

MHz, DMSO-d₆) δ 173.3, 159.0, 157.5, 153.7, 140.5, 140.3,

138.1, 135.5, 130.7, 128.5, 126.5, 114.4, 112.5, 108.9, 72.3, 18.6;

IR (KBr) 3525 (OH), 1725 (CdO) cm^-1; MS (EI) m/z (%) 344

(M⁺, 0.1), 304 (21), 299 (2), 285 (4), 199 (32), 185 (18), 149 (16),

137 (16), 129 (9), 121 (11), 105 (82), 97 (10), 95 (15), 93 (10),

91 (11), 83 (14), 81 (48), 79 (11), 77 (48), 73 (12), 71 (12), 69

(100), 67 (14), 57 (21), 55 (28); HRMS (EI) m/z 299.0585 (M -

CO₂H, calcd for C₁₆H₁₂N₂ClO₂299.0587). Anal. (C₁₇H₁₃N₂ClO₄‚

H₂O) C, H, N.

2-{2-[(7-Ch lor o-2-qu in oxa lin yl)oxy]p h en oxy}p r op ion -

ic Acid (37). Meth od D. Compound 16a (0.20 g, 1.0 mmol),

compound 33 (0.22 g, 1.1 mmol), 60% NaH (0.06 g, 1.5 mmol),

and DMF (5 mL) were stirred together overnight at room

temperature. The mixture was poured into saturated NaCl (25

mL) containing AcOH (0.1 mL), extracted with AcOEt (2 × 25

mL), washed with saturated NaCl (25 mL), dried (MgSO₄), and

concentrated. Pure material (0.28 g, 78% yield) was obtained

after chromatography (4:1 hexanes:AcOEt) to give a light

yellow liquid: ¹H NMR (300 MHz, CDCl₃) δ 8.72 (s, 1 H), 8.00

(d, J ) 9.0 Hz, 1 H), 7.74 (d, J ) 2.4 Hz, 1 H), 7.55 (dd, J )

8.7, 2.4 Hz, 1 H), 7.30-7.22 (m, 2 H), 7.10 (dt, J ) 7.8, 1.2 Hz,

1 H), 6.94 (dd, J ) 7.8, 1.2 Hz, 1 H), 4.69 (q, J ) 6.9 Hz, 1 H),

3.62 (s, 3 H), 1.29 (d, J ) 6.9 Hz, 3 H); ¹³C NMR (75 MHz,

CDCl₃) δ 171.7, 157.4, 149.5, 142.3, 140.7, 139.0, 138.1, 135.9,

130.0, 128.0, 126.6, 123.3, 122.3, 73.8, 52.0, 18.3; IR (Film)

1760 (CdO) cm^-1; MS (EI) m/z (%) 358 (M⁺, 14), 299 (16), 255

(100), 228 (5), 215 (6), 208 (6), 163 (19), 136 (13), 121 (10), 110

(5), 100 (8), 91 (13), 81 (5), 69 (5), 65 (6), 59 (11), 55 (7), 52 (6);

HRMS (EI) m/z 358.0720 (M⁺, calcd for C₁₈H₁₅N₂ClO₄358.0720).

Meth yl 2-(2-Hyd r oxyp h en oxy)p r op ion a te (33). Phenol

32 (1.83 mL, 2.09 g, 10.0 mmol), methyl 2-bromopropionate

(1.25 mL, 1.87 g, 11.0 mmol), anhydrous K₂CO₃(1.73 g, 12.5

mmol), and acetone (25 mL) were refluxed overnight (method

B). The mixture was filtered hot and washed with hot acetone,

and the filtrate was filtered through silica gel and concentrated

to give methyl 2-(2-benzyloxyphenoxy)propionate as a brown

liquid (2.08 g, 73% crude yield): ¹H NMR (300 MHz, CDCl₃) δ

7.50-7.30 (m, 5 H), 7.00-6.82 (m, 4 H), 5.20-5.10 (m, 2 H),

4.80 (q, J ) 6.9 Hz, 1 H), 3.72 (s, 3 H), 1.62 (d, J ) 7.2 Hz, 3

H).

The intermediate (2.08 g, 7.2 mmol), 10% Pd/C (0.10 g), and

CH₃OH (50 mL) were debenzylated by shaking with H₂(30

psi) in a Parr apparatus for 8 h. The mixture was filtered,

concentrated, and dissolved in toluene (50 mL) then washed

1772 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11

Hazeldine et al.

The methyl ester of 37 (0.28 g, 0.78 mmol), dissolved in THF

(10 mL), was hydrolyzed with 0.1 N NaOH (16 mL, 1.6 mmol)

to give 37 (0.24 g, 89% yield) as off-white crystals after

recrystallization from EtOH-water: mp 129-133 °C; ¹H NMR

(300 MHz, DMSO-d₆) δ 12.92 (bs, 1 H), 8.84 (s, 1 H), 8.00 (d,

J ) 8.7 Hz, 1 H), 7.69 (d, J ) 2.1 Hz, 1 H), 7.60 (dd, J ) 8.7,

2.1 Hz, 1 H), 7.34-7.27 (m, 1 H), 7.27-7.18 (m, 1 H), 7.08-

6.98 (m, 2 H), 4.73 (q, J ) 6.6 Hz, 1 H), 1.10 (d, J ) 6.6 Hz, 3

H); ¹³C NMR (75 MHz, DMSO-d₆) δ 172.8, 157.6, 149.6, 141.9,

140.5, 140.0, 138.1, 135.5, 130.7, 128.4, 127.1, 126.4, 123.5,

(5 mL), followed by saturated NaCl (25 mL). After the extracts

were dried (MgSO₄) and concentrated, the residue was purified

by chromatography (3:1 hexanes:AcOEt) to give 40b (2.10 g,

99% yield) as a colorless liquid, which solidified on cooling:

mp 55-60 °C; H NMR (300 MHz, CDCl₃) δ 7.40-7.32 (m, 2

H), 6.80-6.72 (m, 2 H), 5.61 (s, 1 H), 3.69 (s, 3 H), 3.65 (q,

J ) 6.9 Hz, 1 H), 1.43 (d, J ) 6.9 Hz, 3 H).

2-{4-N-[(7-Ch lor o-2-q u in oxa lin yl)oxy]p h en yla m in o}-

p r op ion ic Acid (41a ). The ethyl ester of 41a was prepared

from 16a (0.30 g, 1.5 mmol), 40a (0.35 g, 1.7 mmol), anhydrous

K₂CO₃(0.29 g, 2.1 mmol), and CH₃CN (10 mL) for 6 h (method

A). Pure material (0.47 g, 84% yield) was obtained after

chromatography (3:1 hexanes:AcOEt) to give a yellow liquid:

¹H NMR (300 MHz, CDCl₃) δ 8.60 (s, 1 H), 7.94 (d, J ) 9.3

Hz, 1 H), 7.73 (d, J ) 2.4 Hz, 1 H), 7.94 (dd, J ) 9.0, 2.4 Hz,

1 H), 7.10-7.03 (m, 2 H), 6.69-6.62 (m, 2 H), 4.28 (bs, 1 H),

4.21 (q, J ) 7.2 Hz, 2 H), 4.13 (q, J ) 6.6 Hz, 1 H), 1.49 (d,

J ) 6.6 Hz, 3 H), 1.27 (t, J ) 7.2 Hz, 3 H); ¹³C NMR (75 MHz,

CDCl₃) δ 174.7, 158.2, 144.7, 144.4, 140.9, 139.6, 138.1, 136.3,

130.2, 128.2, 127.0, 122.5, 114.2, 61.5, 52.6, 19.2, 14.5; IR (NaCl

film) 3375 (NH), 1730 (CdO) cm^-1; MS (EI) m/z (%) 371 (M⁺,

16), 298 (100), 163 (3), 149 (4), 119 (7), 118 (7), 81 (5), 69 (10),

57 (7), 55 (6); HRMS (EI) m/z 371.1033 (calcd for C₁₉H₁₈N₃-

ClO₃371.1037).

1

122.1, 115.4, 73.0, 18.3; IR (KBr) 3485 (OH), 1715 (CdO) cm^-1

;

MS (EI) m/z (%) 344 (M⁺, 1), 300 (5), 285 (3), 272 (14), 255

(100), 228 (6), 215 (11), 208 (7), 192 (31), 180 (4), 163 (23), 136

(18), 121 (7), 110 (13), 100 (10), 81 (5), 55 (5), 43 (5); HRMS

(EI) m/z 344.0563 (M⁺, calcd for C₁₇H₁₃N₂ClO₄344.0564). Anal.

(C₁₇H₁₃N₂ClO₄) C, H, N.

2-{4-[4-[(7-Ch lor o-2-qu in oxalin yl)oxy]ph en yl]ph en oxy}-

p r op ion ic Acid (38). The methyl ester of 38 was prepared

from 16a (0.25 g, 1.26 mmol), 35 (0.37 g, 1.36 mmol),

anhydrous K₂CO₃(0.24 g, 1.7 mmol), and CH₃CN (10 mL) for

4 h (method A). Pure material (0.42 g, 76% yield) was obtained

after chromatography (4:1 hexanes:AcOEt) and recrystalliza-

tion from CHCl₃-EtOH to give white crystals: mp 149-151

°C; ¹H NMR (300 MHz, CDCl₃) δ 8.70 (s, 1 H), 7.99 (d, J ) 8.7

Hz, 1 H), 7.79 (d, J ) 2.4 Hz, 1 H), 7.65-7.51 (m, 5 H), 7.35-

7.28 (m, 2 H), 7.01-6.93 (m, 2 H), 4.84 (q, J ) 6.9 Hz, 1 H),

3.80 (s, 3 H), 1.67 (d, J ) 6.9 Hz, 3 H); ¹³C NMR (75 MHz,

CDCl₃) δ 172.5, 157.4, 157.2, 151.6, 140.5, 139.4, 138.1, 136.2,

133.8, 130.0, 128.2, 127.9, 126.8, 121.6, 115.5, 72.7, 52.3, 18.6;

IR (KBr) 1755 (CdO) cm^-1; MS (EI) m/z (%) 434 (M⁺, 37), 375

(12), 347 (24), 323 (5), 320 (11), 295 (18), 293 (6), 291 (5), 265

(6), 247 (5), 234 (6), 223 (9), 217 (18), 207 (5), 199 (100), 197

(9), 187 (10), 181 (8), 163 (6), 157 (6), 152 (7), 139 (18), 137

(7), 135 (11), 128 (5), 95 (5), 93 (5), 91 (7), 85 (76), 81 (19), 77

(12), 69 (20), 67 (10). Anal. (C₂₄H₁₉N₂ClO₄) C, H, N.

The methyl ester of 38 (0.38 g, 0.87 mmol), dissolved in THF

(15 mL), was hydrolyzed with 0.1 N NaOH (17.5 mL, 1.75

mmol) to give 38 (0.31 g, 84% yield) as off-white crystals after

recrystallization from EtOH-water: mp 183-185 °C; ¹H NMR

(300 MHz, DMSO-d₆) δ 13.05 (bs, 1 H), 8.86 (s, 1 H), 8.04 (d,

J ) 9.0 Hz, 1 H), 7.75 (d, J ) 2.1 Hz, 1 H), 7.72-7.64 (m, 3

H), 7.60 (d, J ) 8.7 Hz, 2 H), 7.36 (d, J ) 8.7 Hz, 2 H), 6.94 (d,

J ) 8.7 Hz, 2 H), 4.86 (q, J ) 6.6 Hz, 1 H), 1.50 (d, J ) 6.9 Hz,

3 H); ¹³C NMR (75 MHz, DMSO-d₆) δ 173.4, 157.8, 157.6,

151.8, 140.6, 140.4, 138.1, 137.7, 135.5, 132.7, 130.7, 128.5,

128.2, 127.9, 126.4, 122.3, 115.7, 72.2, 18.7; IR (KBr) 3440

(OH), 1715 (CdO) cm^-1; MS (EI negative ion) m/z (%) 839

(2M - H, 7), 419 (M - H, 100), 347 (79), 189 (7), 81 (25). Anal.

(C₂₃H₁₇N₂ClO₄) C, H, N.

The ethyl ester of 41a (0.47 g, 1.3 mmol), dissolved in THF

(25 mL), was hydrolyzed with 0.1 N NaOH (38 mL, 3.8 mmol)

to give 41a (0.14 g, 32% yield) as a brown solid after being

washed with ether before being filtered and acidified: mp ∼135

°C; ¹H NMR (400 MHz, DMSO-d₆) δ 8.76 (s, 1 H), 8.02 (d, J )

8.8 Hz, 1 H), 7.78 (d, J ) 2.4 Hz, 1 H), 7.65 (dd, J ) 8.8, 2.4

Hz, 1 H), 7.05-6.99 (m, 2 H), 6.63-6.57 (m, 2 H), 3.94 (q, J )

7.2 Hz, 1 H), 1.38 (d, J ) 7.2 Hz, 3 H); ¹³C NMR (100 MHz,

DMSO-d₆) δ 176.6, 158.7, 146.3, 143.3, 140.9, 140.8, 138.1,

135.6, 130.9, 128.4, 126.6, 122.6, 113.5, 51.9, 18.9; IR (KBr)

3400 (NH), 1725 (CdO) cm^-1; MS (EI) m/z (%) 299 (84), 284

(100), 271 (7), 256 (27), 243 (4), 228 (5), 192 (4), 163 (11), 136

(27), 108 (12), 100 (10), 80 (15), 65 (7), 63 (5), 53 (6), 50 (5).

Anal. (C₁₇H₁₄N₃ClO₃) C, H, N.

2-{4-[(7-Ch lor o-2-qu in oxa lin yl)oxy]p h en ylth io}p r op i-

on ic Acid (41b). The methyl ester of 41b was prepared from

16a (0.50 g, 2.5 mmol), 40b (0.58 g, 2.7 mmol), anhydrous K₂-

CO₃(0.47 g, 3.4 mmol), and CH₃CN (15 mL) for 6 h (method

A). Pure material (0.94 g, 100% yield) was obtained after

chromatography (4:1 hexanes:AcOEt) to give a yellow liquid:

¹H NMR (300 MHz, CDCl₃) δ 8.68 (s, 1 H), 8.00 (d, J ) 9.0

Hz, 1 H), 7.76 (d, J ) 2.1 Hz, 1 H), 7.61-7.53 (m, 3 H), 7.28-

7.21 (m, 2 H), 3.82 (q, J ) 7.2 Hz, 1 H), 3.72 (s, 3 H), 1.53 (d,

J ) 7.2 Hz, 3 H); ¹³C NMR (75 MHz, CDCl₃) δ 172.5, 156.8,

152.4, 140.0, 139.1, 137.9, 135.9, 134.7, 129.9, 128.1, 126.4,

121.9, 52.0, 45.3, 17.3; IR (film) 1740 (CdO) cm^-1; MS (EI)

m/z (%) 374 (M⁺, 72), 315 (100), 298 (12), 287 (7), 259 (17),

239 (6), 211 (9), 163 (25), 153 (9), 136 (22), 125 (15), 123 (8),

100 (12), 97 (9), 91 (9), 81 (8), 73 (7), 69 (13), 59 (22), 57 (10),

55 (14); HRMS (EI) m/z 374.0492 (M⁺, calcd for C₁₈H₁₅N₂ClO₃S

374.0492).

E t h yl 2-(4-H yd r oxyp h en yla m in o)p r op ion a t e (40a ).

Aniline 39a (2.78 g, 25.0 mmol), methyl 2-bromopropionate

(3.60 mL, 5.02 g, 27.5 mmol), and Na₂SO₃(6.43 g, 50.0 mmol)

were heated together under Ar at 125 °C for 8 h. After cooling,

AcOEt (50 mL), water (25 mL), and saturated NaHCO₃were

added until pH 8. The layers were separated, and the aqueous

layer was extracted with AcOEt (50 mL). The AcOEt layers

were washed with saturated NaCl (25 mL), dried (MgSO₄), and

concentrated. The residue was mixed with 1:2 hexanes:AcOEt,

and the insoluble, unreacted 39a was removed. After recon-

centrating, the residue was purified by chromatography (3×)

(1:2-2:1-3:1 hexanes:AcOEt) and recrystallized from AcOEt-

The methyl ester of 41b (0.94 g, 2.5 mmol), dissolved in THF

(35 mL), was hydrolyzed with 0.1 N NaOH (50 mL, 5.0 mmol)

to give 41b (0.81 g, 90% yield) as off-white crystals after

recrystallization from EtOH-water: mp 127-129 °C; ¹H NMR

(300 MHz, DMSO-d₆) δ 12.75 (bs, 1 H), 8.77 (s, 1 H), 7.97 (d,

J ) 8.7 Hz, 1 H), 7.67 (d, J ) 1.8 Hz, 1 H), 7.60 (dd, J ) 8.7,

2.1 Hz, 1 H), 7.50 (d, J ) 8.7 Hz, 2 H), 7.29 (d, J ) 8.4 Hz, 2

H), 3.89 (q, J ) 6.9 Hz, 1 H), 1.37 (d, J ) 7.2 Hz, 3 H); ¹³C

NMR (75 MHz, DMSO-d₆) δ 173.7, 157.5, 152.2, 140.5, 140.2,

138.1, 135.6, 133.5, 131.0, 130.7, 128.6, 126.5, 122.5, 45.0, 17.9;

IR (KBr) 3485 (OH), 1710 (CdO) cm^-1; MS (EI) m/z (%) 360

(M⁺, 85), 315 (87), 301 (17), 287 (28), 283 (52), 278 (11), 259

(94), 256 (40), 228 (14), 226 (12), 224 (13), 192 (16), 181 (9),

163 (76), 136 (100), 125 (27), 109 (16), 100 (39), 97 (28), 91

(20), 85 (10), 83 (15), 81 (14), 75 (13), 73 (16), 71 (14), 69 (27),

63 (12), 59 (14), 57 (21), 55 (26), 45 (22), 43 (25), 41 (17), 39

(10); HRMS (EI) m/z 360.0333 (M⁺, calcd for C₁₇H₁₃N₂ClO₃S

360.0335). Anal. (C₁₇H₁₃N₂ClO₃S) C, H, N.

1

hexanes to give 40a as off-white crystals: mp 85-87 °C; H

NMR (400 MHz, CDCl₃) δ 6.63-6.57 (m, 2 H), 6.52-6.46 (m,

2 H), 4.14 (q, J ) 7.2 Hz, 2 H), 4.02 (q, J ) 6.9 Hz, 1 H), 1.42

(d, J ) 7.5 Hz, 3 H), 1.21 (d, J ) 6.9 Hz, 3 H); ¹³C NMR (75

MHz, CDCl₃) δ 175.8, 149.2, 140.3, 116.5, 115.9, 61.5, 53.7,

19.1, 14.4.

Meth yl 2-(4-Hyd r oxyp h en ylth io)p r op ion a te (40b). Thio

39a (1.26 g, 10.0 mmol), methyl 2-bromopropionate (1.20 mL,

1.80 g, 10.5 mmol), anhydrous KHCO₃(1.10 g, 11.0 mmol),

and DMF (5 mL) were stirred together overnight at room

temperature. Water (25 mL) was added, and the mixture was

extracted with AcOEt (2 × 25 mL). The extracts were washed

with saturated NaCl (20 mL) containing saturated NaHCO₃

Synthesis of XK469 Analogues

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11 1773

2-{4-[(N-7-Ch lor o-2-qu in oxa lin yl)a m in o]p h en oxy}p r o-

p ion ic Acid (42a ). To 16a (0.40 g, 2.0 mmol) and 39a (0.22

g, 2.0 mmol), dissolved in CH₃CN (15 mL), was added 0.2 N

HCl (10 mL, 2.0 mmol), and the mixture was refluxed

overnight. The volume of CH₃CN was reduced by distillation

and, after cooling, diluted with water (50 mL), and saturated

NaHCO₃was added to adjust to pH 7. The mixture was

extracted with AcOEt (2 × 50 mL), washed with saturated

NaCl (25 mL), dried (MgSO₄), concentrated, and purified by

chromatography (1:1 hexanes:AcOEt) to give 4-[(N-7-chloro-

2-quinoxalinyl)amino]phenol as a yellow solid (0.36 g, 66%

yield): mp 195-200 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 9.79

(s, 1 H), 9.23 (bs, 1 H), 8.43 (s, 1 H), 7.78-7.70 (m, 3 H), 7.60

(d, J ) 1.6 Hz, 1 H), 7.30 (dd, J ) 8.8, 1.6 Hz, 1 H), 6.80 (d,

J ) 8.0 Hz, 2 H); ¹³C NMR (100 MHz, DMSO-d₆) δ 153.7, 150.8,

142.5, 141.8, 135.8, 134.7, 132.3, 130.5, 125.5, 125.0, 121.5,

115.9; IR (KBr) 3350 (OH), 3220 (NH) cm^-1; MS (EI) m/z (%)

270 (M - H, 100), 180 (4), 163 (3), 152 (17), 136 (4), 125 (4),

117 (4), 100 (4), 90 (3), 81 (4), 69 (4), 65 (5), 57 (5), 55 (6);

HRMS (EI) m/z 270.0431 (M - H, calcd for C₁₄H₉N₃ClO

270.0434).

The intermediate (0.41 g, 1.5 mmol), methyl 2-bromopropi-

onate (0.22 mL, 0.33 g, 1.9 mmol), anhydrous K₂CO₃(0.26 g,

1.9 mmol), and acetone (15 mL) were refluxed overnight

(method B). The residue was purified by chromatography (1:1

hexanes:AcOEt) to give the methyl ester of 42a as a brown-

yellow liquid, which slowly solidified (0.48 g, 89% yield): ¹H

NMR (400 MHz, CDCl₃) δ 8.26 (s, 1 H), 7.76 (d, J ) 8.0 Hz, 1

H), 7.68 (d, J ) 2.4 Hz, 1 H), 7.59-7.54 (m, 2 H), 7.32 (dd,

J ) 8.8, 2.4 Hz, 1 H), 7.30 (bs, 1 H), 6.89-6.83 (m, 2 H), 4.76

(q, J ) 6.8 Hz, 1 H), 3.78 (s, 3 H), 1.63 (d, J ) 6.4 Hz, 3 H);

¹³C NMR (100 MHz, CDCl₃) δ 173.4, 154.0, 150.2, 142.1, 139.1,

136.0, 133.3, 129.8, 126.0 (2C), 122.0, 115.9, 73.0, 52.8, 18.9;

IR (NaCl film) 3360 (NH), 1735 (CdO) cm^-1; MS (EI) m/z (%)

356 (M - H, 40), 297 (4), 269 (100), 253 (3), 163 (12), 152 (4),

136 (5); HRMS (EI) m/z 356.0800 (M - H, calcd for C₁₈H₁₅N₃-

ClO₃356.0802).

3.1 mmol), and acetone (15 mL) were refluxed together for 21

h (method B). The residue was purified by chromatography

(4:1 hexanes:AcOEt) and recrystallized from EtOH-heptane

(2×) to give the methyl ester of 42b as yellow crystals (0.56 g,

60% yield): mp 112-114 °C; ¹H NMR (300 MHz, CDCl₃) δ 8.39

(s, 1 H), 7.91 (d, J ) 8.7 Hz, 1 H), 7.85 (d, J ) 2.4 Hz, 1 H),

7.63-7.54 (m, 2 H), 7.57 (dd, J ) 9.0, 2.4 Hz, 1 H), 7.02-6.95

(m, 2 H), 4.84 (q, J ) 6.9 Hz, 1 H), 3.80 (s, 3 H), 1.68 (d, J )

6.6 Hz, 3 H); ¹³C NMR (75 MHz, CDCl₃) δ 172.0, 159.1, 159.0,

143.1, 142.4, 138.3, 137.1, 136.1, 130.3, 129.3, 127.1, 119.6,

116.4, 72.7, 52.4, 18.5; IR (KBr) 1735 (CdO) cm^-1; MS (EI)

m/z (%) 374 (M⁺, 100), 315 (26), 287 (99), 271 (13), 259 (13),

163 (45), 136 (22), 125 (10), 100 (13), 59 (17). Anal. (C₁₈H₁₅N₂-

ClO₃S) C, H, N.

The methyl ester of 42b (0.19 g, 0.51 g, 1.3 mmol), dissolved

in THF (5 mL), was hydrolyzed with 0.1 N NaOH (10.2 mL,

1.02 mmol) to give 42b (0.12 g, 66% yield) as yellow crystals

after recrystallization from EtOH-water: mp 159-163 °C; ¹H

NMR (300 MHz, DMSO-d₆) δ 13.15 (bs, 1 H), 8.42 (s, 1 H),

7.90 (d, J ) 8.7 Hz, 1 H), 7.74 (d, J ) 2.1 Hz, 1 H), 7.62 (dd,

J ) 8.7, 2.1 Hz, 1 H), 7.56 (d, J ) 8.7 Hz, 2 H), 7.00 (d, J ) 8.7

Hz, 2 H), 4.91 (q, J ) 6.6 Hz, 1 H), 1.52 (d, J ) 6.9 Hz, 3 H);

¹³C NMR (75 MHz, DMSO-d₆) δ 173.1, 159.4, 159.0, 143.7,

142.1, 138.3, 137.2, 135.6, 131.0, 129.7, 126.8, 118.4, 116.8,

72.2, 18.6; IR (KBr) 3485 (OH), 1735 (CdO) cm^-1; MS (EI) m/z

(%) 360 (M⁺, 78), 315 (7), 301 (11), 287 (100), 271 (8), 259 (14),

163 (50), 136 (24), 125 (15), 100 (14), 97 (9), 75 (7), 69 (8), 63

(6), 55 (6), 50 (5); HRMS (EI) m/z 360.0333 (M⁺, calcd for

C

₁₇H₁₃N₂ClO₃S 360.0335). Anal. (C₁₇H₁₃N₂ClO₃S) C, H, N.

2-{4-[(7-Ch lor o-2-qu in oxa lin yl)oxy]p h en oxy}p r op ion -

a m id e (43). To a solution of 2 (0.18 g, 0.50 mmol) in CH₃OH

(20 mL) was added NH₃gas for 1 h, and the mixture was

stirred overnight. The solvent was removed, and the crude

material was recrystallized from AcOEt to give 43 as white

1

crystals (0.12 g, 70% yield): mp 210-211 °C; H NMR (300

MHz, DMSO-d₆) δ 8.82 (s, 1 H), 8.04 (d, J ) 9.0 Hz, 1 H), 7.77

(d, J ) 2.1 Hz, 1 H), 7.67 (dd, J ) 8.7, 2.1 Hz, 1 H), 7.56 (s, 1

H), 7.27 (s, 1 H), 7.27-7.19 (m, 2 H), 7.02-6.94 (m, 2 H), 4.62

(q, J ) 6.6 Hz, 1 H), 1.44 (d, J ) 6.6 Hz, 3 H); ¹³C NMR (75

MHz, DMSO-d₆) δ 173.8, 158.0, 155.4, 146.4, 140.6, 140.4,

138.0, 135.5, 130.7, 128.4, 126.4, 122.9, 116.6, 74.5, 19.1; IR

(KBr) 3375 (NH), 3185 (NH), 1665 (CdO) cm^-1; MS (EI) m/z

(%) 343 (M⁺, 69), 325 (9), 299 (100), 272 (34), 255 (18), 244

(46), 192 (8), 163 (45), 136 (28), 110 (9), 100 (16), 91 (6), 75

(7), 63 (7), 55 (7), 44 (29). Anal. (C₁₇H₁₄N₃ClO₃) C, H, N.

The methyl ester of 42a (0.48 g, 1.3 mmol), dissolved in THF

(15 mL), was hydrolyzed with 0.1 N NaOH (27 mL, 2.7 mmol)

to give 42a (0.38 g, 83% yield) as yellow crystals after

recrystallization from EtOH-water: mp 244-246 °C; ¹H NMR

(400 MHz, DMSO-d₆) δ 12.85 (bs, 1 H), 9.93 (s, 1 H), 8.46 (s,

1 H), 7.87-7.81 (m, 2 H), 7.78 (d, J ) 8.8 Hz, 1 H), 7.68 (d,

J ) 2.4 Hz, 1 H), 7.39 (dd, J ) 8.8, 2.6 Hz, 1 H), 6.92-6.86

(m, 2 H), 4.78 (q, J ) 6.4 Hz, 1 H), 1.49 (d, J ) 6.8 Hz, 3 H);

¹³C NMR (100 MHz, DMSO-d₆) δ 174.0, 153.6, 150.7, 142.3,

141.7, 135.9, 134.8, 134.2, 130.6, 125.7, 125.3, 121.1, 115.7,

2-{4-[(7-Ch lor o-2-qu in oxa lin yl)oxy]p h en oxy}p r op ion -

h yd r a zid e (44). To a solution of 3 (0.36 g, 1.0 mmol) in EtOH

(25 mL) was added hydrazine (0.66 mL, 0.67 g, 20 mmol)

dropwise, and the mixture was stirred overnight at room

temperature. After cooling, the mixture was filtered and

washed with cold EtOH to give 44 as white crystals (0.31 g,

86% yield): mp 201-203 °C; ¹H NMR (300 MHz, DMSO-d₆) δ

9.39 (s, 1 H), 8.80 (s, 1 H), 8.01 (d, J ) 8.7 Hz, 1 H), 7.72 (d,

J ) 2.4 Hz, 1 H), 7.65 (dd, J ) 8.7, 2.1 Hz, 1 H), 7.21 (d, J )

9.0 Hz, 2 H), 6.97 (d, J ) 9.0 Hz, 2 H), 4.71 (q, J ) 6.6 Hz, 1

H), 4.30 (s, 2 H), 1.43 (d, J ) 6.6 Hz, 3 H); ¹³C NMR (75 MHz,

DMSO-d₆) δ 170.5, 158.0, 155.2, 146.4, 140.6, 140.4, 138.0,

135.5, 130.7, 128.4, 126.4, 122.9, 116.6, 73.6, 19.2; IR (KBr)

3290 (NH), 1660 (CdO) cm^-1; MS (EI) m/z (%) 358 (M⁺, 22),

299 (33), 272 (100), 255 (20), 244 (40), 207 (6), 192 (10), 163

(34), 136 (24), 124 (5), 110 (11), 100 (16), 91 (7), 87 (22), 81

(6), 75 (9), 65 (9), 59 (30), 55 (10), 50 (7). Anal. (C₁₇H₁₅N₄ClO₃)

C, H, N.

72.5, 19.0; IR (KBr) 3400 (OH), 3290 (NH), 1710 (CdO) cm^-1

;

MS (EI negative ion) m/z (%) 685 (2M - H, 3), 342 (M - H,

86), 298 (9), 270 (100), 265 (4), 214 (3), 166 (2), 150 (2), 134

(6), 121 (3), 111 (6), 97 (11), 62 (46). Anal. (C₁₇H₁₄N₃ClO₃)

C, H, N.

2-{4-[(7-Ch lor o-2-q u in oxa lin yl)t h io]p h en oxy}p r op i-

on ic Acid (42b). Compound 16a (0.50 g, 2.5 mmol), 39b (0.32

g, 2.5 mmol), anhydrous KHCO₃(0.28 g, 2.8 mmol), and DMF

(5 mL) were stirred together overnight at room temperature.

Water (25 mL) was added, and the mixture was extracted with

AcOEt (2 × 25 mL). The extracts were washed with saturated

NaCl (20 mL) containing saturated NaHCO₃(5 mL), followed

by saturated NaCl (25 mL). After drying (MgSO₄), the extract

was concentrated to give 4-[(7-chloro-2-quinoxalinyl)thio]phe-

nol as an orange solid (0.74 g, ∼100% crude yield). A sample

was recrystallized from EtOH-heptane (2 ×) to give yellow-

orange crystals: mp 188-190 °C; ¹H NMR (300 MHz, DMSO-

d₆) δ 10.09 (bs, 1 H), 8.44 (s, 1 H), 7.98 (d, J ) 8.7 Hz, 1 H),

7.86 (d, J ) 2.1 Hz, 1 H), 7.72 (dd, J ) 8.7, 2.1, 1 H), 7.52-

7.45 (m, 2 H), 6.97-6.87 (m, 2 H); ¹³C NMR (75 MHz, DMSO-

d₆) δ 159.8, 159.6, 143.5, 142.1, 138.2, 137.5, 135.5, 131.0,

129.6, 126.8, 117.5, 115.5; IR (KBr) 3485 (OH) cm^-1; MS (EI)

m/z (%) 288 (M⁺, 100), 271 (4), 253 (6), 163 (23), 136 (27), 126

(25), 100 (25), 97 (32), 81 (14), 75 (16), 69 (11), 63 (12), 53 (14),

50 (10), 45 (10), 39 (13). Anal. (C₁₄H₉N₂ClOS) C, H, N.

Gen er a l P r oced u r e for Meth yl a n d Dim eth yl Am id es.

To the acid dissolved in THF containing catalytic quantity of

pyridine was added (COCl)₂(2 N in CH₂Cl₂) dropwise at 0 °C,

and the mixture was stirred until the evolution of gas ceased

and then stirred overnight at room temperature. The amine

(2 N in THF) was added until the mixture was basic, and it

was concentrated. Water and saturated NaHCO₃were added

until pH 8, and the mixture was extracted with AcOEt. The

extract was washed with saturated NaCl, dried (MgSO₄), and

concentrated.

The intermediate (0.74 g, 2.5 mmol), methyl 2-bromopropi-

onate (0.35 mL, 0.52 g, 3.1 mmol), anhydrous K₂CO₃(0.43 g,

1774 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11

Hazeldine et al.

2-{4-[(7-Ch lor o-2-qu in oxa lin yl)oxy]p h en oxy}p r op ion -

m eth yla m id e (45a ). Acid 1 (0.52 g, 1.5 mmol), THF (5 mL),

pyridine (12 µL), (COCl)₂(1.88 mL, 3.76 mmol), and methyl-

amine were reacted to give 45a as white crystals after

recrystallizing from AcOEt (0.48 g, 89% yield): mp 181-182

°C; ¹H NMR (300 MHz, CDCl₃) δ 8.65 (s, 1 H), 7.97 (d, J ) 8.7

Hz, 1 H), 7.74 (d, J ) 2.1 Hz, 1 H), 7.54 (dd, J ) 9.0, 2.4 Hz,

1 H), 7.23-7.15 (m, 2 H), 7.00-6.92 (m, 2 H), 6.53 (bs, 1 H),

4.69 (q, J ) 6.6 Hz, 1 H), 2.88 (d, J ) 4.8 Hz, 3 H), 1.61 (d,

J ) 6.6 Hz, 3 H); ¹³C NMR (75 MHz, CDCl₃) δ 172.5, (165.6),

157.5, 154.5, 146.9, 140.5, 139.2, 138.1, 136.2, 130.0, 128.2,

126.7, 122.6, 116.4, 75.8, (29.6), 25.8, 18.7; IR (KBr) 3370 (NH),

1650 (CdO) cm^-1; MS (EI) m/z (%) 357 (M⁺, 85), 299 (91), 285

(8), 272 (27), 255 (24), 244 (41), 192 (13), 163 (50), 136 (34),

124 (7), 110 (12), 100 (25), 91 (12), 86 (94), 81 (15), 75 (14), 69

(28), 63 (16), 58 (100), 55 (23), 50 (13). Anal. (C₁₈H₁₆N₃ClO₃)

C, H, N.

AcOEt) to give 47 as a light yellow liquid, which solidified on

cooling (1.30 g, 84% yield): ¹H NMR (300 MHz, CDCl₃) δ 6.96-

6.86 (m, 2 H), 6.86-6.77 (m, 2 H), 5.01 (s, 1 H), 4.78 (q, J )

6.6 Hz, 1 H), 1.76 (d, J ) 6.9 Hz, 3 H).

2-{4-[(7-Ch lor o-2-qu in oxa lin yl)oxy]p h en oxy}p r op ion i-

tr ile (48). Compound 16a (0.40 g, 2.0 mmol), compound 47

(0.36 g, 2.2 mmol), anhydrous K₂CO₃(0.38 g, 2.7 mmol), and

CH₃CN (10 mL) were reacted for 4 h (method A), and the

product was purified by chromatography (4:1 hexanes:AcOEt)

and recrystallized from heptane to give 48 as off-white crystals

(0.57 g, 87% yield): mp 135-137 °C; ¹H NMR (400 MHz,

CDCl₃) δ 8.66 (s, 1 H), 7.97 (d, J ) 9.2 Hz, 1 H), 7.75 (d, J )

2.4 Hz, 1 H), 7.54 (dd, J ) 8.8, 2.4 Hz, 1 H), 7.27-7.22 (m, 2

H), 7.12-7.07 (m, 2 H), 4.91 (q, J ) 6.4 Hz, 1 H), 1.83 (d, J )

6.4 Hz, 3 H); ¹³C NMR (100 MHz, CDCl₃) δ 157.7, 154.0, 148.0,

140.7, 139.5, 138.3, 136.5, 130.3, 128.6, 127.0, 123.1, 118.4,

117.3, 63.3, 20.2; MS (EI) m/z (%) 325 (M⁺, 41), 271 (26), 243

(100), 215 (7), 208 (11), 179 (5), 163 (78), 136 (55), 124 (12),

109 (19), 100 (52), 81 (28), 75 (25), 69 (15), 63 (38), 54 (43), 52

(26), 50 (24). Anal. (C₁₇H₁₂N₃ClO₂) C, H, N.

2-{4-[(7-Ch lor o-2-qu in oxa lin yl)oxy]p h en oxy}p r op ion -

d im eth yla m id e (45b). Acid 1 (0.52 g, 1.5 mmol), in THF (5

mL), pyridine (12 µL), (COCl)₂(1.8 mL, 3.6 mmol), and

dimethylamine gave 45b. This was purified by chromatogra-

phy (1:2 hexanes:AcOEt) and recrystallization from EtOH-

hexanes to give pale yellow crystals (0.45 g, 82% yield): mp

7-Ch lor o-2-{4-[1-(1H-tetazol-5-yl)eth oxy]ph en oxy}qu in -

oxa lin e (49). Nitrile 48 (0.48 g, 1.5 mmol), NH₄Cl (0.10 g, 1.9

mmol), NaN₃(0.12 g, 1.8 mmol), and DMF (5 mL) were heated

at 80 °C overnight. The mixture was concentrated, and dilute

NH₄OH (10 mL) was added. The mixture was heated, adding

more dilute NH₄OH to maintain pH 8. The filtered mixture

was washed with water, and the filtrate was acidified to pH

3-4 with 0.25 N HCl. After cooling, the solid was collected,

washed with ice-water, dried, and recrystallized from AcOEt-

heptane to give 49 as brown crystals (0.38 g, 70% yield): mp

1

122-124 °C; H NMR (400 MHz, CDCl₃) δ 8.62 (s, 1 H), 7.95

(d, J ) 8.8 Hz, 1 H), 7.73 (d, J ) 1.6 Hz, 1 H), 7.52 (dd, J )

8.8, 2.4 Hz, 1 H), 7.19-7.13 (m, 2 H), 6.97-6.92 (m, 2 H), 4.97

(q, J ) 6.4 Hz, 1 H), 3.15 (s, 3 H), 2.97 (s, 3 H), 1.63 (d, J ) 6.4

Hz, 3 H); ¹³C NMR (75 MHz, CDCl₃) δ 170.8, 157.6, 154.8,

146.4, 140.5, 139.3, 137.9, 136.1, 130.0, 128.1, 126.7, 122.5,

115.7, 74.1, 36.5, 36.3, 17.6; IR (KBr) 1635 (CdO) cm^-1; MS

(EI) m/z (%) 371 (M⁺, 28), 299 (30), 272 (6), 255 (11), 244 (7),

192 (6), 163 (15), 136 (11), 100 (100), 91 (6), 81 (11), 72 (50),

69 (22), 55 (11), 45 (8). Anal. (C₁₉H₁₈N₃ClO₃) C, H, N.

1

198-199 °C (dec); H NMR (400 MHz, DMSO-d₆) δ 8.81 (s, 1

H), 8.03 (d, J ) 8.0 Hz, 1 H), 7.73 (d, J ) 2.4 Hz, 1 H), 7.67

(dd, J ) 9.2, 2.4 Hz, 1 H), 7.27-7.22 (m, 2 H), 7.12-7.07 (m,

2 H), 5.98 (q, J ) 6.4 Hz, 1 H), 1.72 (d, J ) 6.4 Hz, 3 H); ¹³C

NMR (100 MHz, DMSO-d₆) δ 158.2, 154.8, 147.0, 140.9, 140.6,

138.2, 135.7, 131.0, 128.7, 126.6, 123.4, 117.4, 67.8, 20.8; IR

(KBr) 2620 (OH) cm^-1; MS (EI negative ion with NH₄OH) m/z

(%) 735 (2M - H,5), 367 (M - H,100), 271 (2), 95 (14). Anal.

(C₁₇H₁₃N₆ClO₂) C, H, N.

2-{4-[(7-Ch lor o-2-qu in oxa lin yl)oxy]p h en oxy}p r op ion -

h yd r oxa m ic Acid (45c). Acid 1 (0.17 g, 0.5 mmol) and SOCl₂

(0.35 mL, 0.57 g, 4.8 mmol) were refluxed for 1 h. After cooling,

the mixture was concentrated in vacuo and the acid chloride

dissolved in toluene (10 mL). (TMS)₂NOTMS (0.14 g, 0.56

mmol) was added, and the mixture was stirred for 0.25 h before

it was concentrated. The residue was dissolved in toluene (10

mL) and stirred overnight in the open. The resulting solid was

washed with AcOEt and dried to give 45c as an off-white solid

(0.14 g, 79% yield): mp 190 °C (dec); ¹H NMR (300 MHz,

DMSO-d₆) δ 10.88 (s, 1 H), 8.96 (bs, 1 H), 8.81 (s, 1 H), 8.02

(d, J ) 8.7 Hz, 1 H), 7.74 (d, J ) 2.1 Hz, 1 H), 7.66 (dd, J )

8.7, 2.1 Hz, 1 H), 7.22 (d, J ) 8.7 Hz, 2 H), 6.98 (d, J ) 8.7 Hz,

2 H), 4.67 (q, J ) 6.6 Hz, 1 H), 1.44 (d, J ) 6.6 Hz, 3 H); ¹³C

NMR (75 MHz, DMSO-d₆) δ 168.1, 158.0, 155.2, 146.4, 140.6,

140.4, 138.0, 135.5, 130.7, 128.4, 126.4, 122.9, 116.6, 73.0, 19.2;

IR (KBr) 3415 (OH), 3165 (NH), 1650 (CdO) cm^-1; MS (EI)

m/z (%) 359 (M⁺, 1), 344 (18), 299 (9), 272 (100), 255 (6), 244

(100), 237 (10), 216 (19), 209 (18), 181 (16), 163 (57), 152 (19),

136 (61), 124 (19), 110 (59), 100 (54), 88 (10), 81 (47), 75 (27),

70 (21), 65 (24), 63 (33), 59 (19), 53 (19), 50 (21). Anal.

(C₁₇H₁₄N₃ClO₄) C, H, N.

Biology. Meth od s. In Vitr o. Our “disk diffusion soft agar

colony formation assay”²³is designed to compare the relative

cytotoxicity of an agent against leukemia cells, solid tumor

cells (including mutlidrug resistant solid tumors), and normal

cells.^2,4,23The inhibition is expressed in zone units, with 200

units ) 6.5 mm. On average, a 10-fold dilution of a cytotoxic

agent produces a 330-zone unit change. Activity against a

drug-sensitive leukemia (L1210 or P388) provides the reference

point. The leukemic cell can represent antiproliferative leu-

kemic active agents of past discoveries. The agent needs

greater activity against the drug-insensitive solid tumors than

against the leukemia cells or normal cells in the soft agar assay

to be of interest. Normal fibroblasts were used in current

studies. For the operation of the assay, the tumor cells are

isolated from live tissue, i.e., a tumor growing in a mouse. The

cells are then seeded in the soft agar. The drug is placed on a

filter paper disk (standard hole punch of Whatman #1), which

is then placed on top of the soft agar (60 mm plate). The drug

diffuses off the disk as the tumor cells are replicating, creating

a zone of inhibition of colony formation.

In Vivo. Treatment was carried out against early stage

Pancreatic Ductal Adenocarcinoma-03²⁴and/or Mammary

Adenocarcinoma-17/Adr.^25,26The general methods of tumor

transplantation, drug treatment, endpoint determination, and

interpretation of data have been published.^2-4,24-28A brief

summary follows:

a . Tu m or a n d An im a l Ma in ten a n ce. Mouse tumors are

maintained in the mouse strain of origin and are transplanted

into the appropriate F₁hybrid (or the inbred mouse of origin)

for therapy trials. Individual mouse body weights for each

experiment are within 5 g, and all mice are over 17 g at the

start of therapy. The mice are supplied food and water ad

libitum.

2-(4-Hyd r oxyp h en oxy)p r op ion itr ile (47). A mixture of

phenol 46 (2.04 g, 10.0 mmol), 2-bromopropionitrile (1.33 mL,

2.06 g, 15.0 mmol), anhydrous K₂CO₃(1.73 g, 12.5 mmol), and

acetone (25 mL) was refluxed overnight. The reaction mixture

was filtered hot and washed with hot acetone, and the filtrate

was filtered through silica gel and then concentrated in vacuo

to give 2-(4-benzyloxyphenoxy)propionitrile as a pale brown

liquid, which solidified (2.41 g, 95% crude yield): mp 86-87

1

°C; H NMR (300 MHz, CDCl₃) δ 7.47-7.30 (m, 5 H), 7.00-

6.90 (m, 4 H), 5.04 (s, 2 H), 4.79 (q, J ) 6.9 Hz, 1 H), 1.77 (d,

J ) 6.9 Hz, 3 H).

To the intermediate (2.41 g, 9.51 mmol) in CH₂Cl₂(10 mL)

was added BBr₃‚S(CH₃)₂(3.11 g, 10.0 mmol), and the solution

was stirred for 2 h at room temperature. The solution was

diluted with AcOEt (25 mL), saturated NaHCO₃(15 mL), and

water (10 mL). The layers were separated, and the aqueous

layer was extracted with AcOEt (25 mL). The extracts were

washed with saturated NaCl (10 mL), dried (MgSO₄), and

concentrated before purifying by chromatography (4:1 hexanes:

b. Ch em oth er a p y of Solid Tu m or s. The animals were

pooled, implanted subcutaneously with 30 to 60 mg tumor

Synthesis of XK469 Analogues

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11 1775

fragments by a 12-gauge trocar, and again pooled before

unselective distribution to the various treatment and control

groups (5 mice or 6 mice per group). For early stage treatment,

chemotherapy was started 1 to 3 days after tumor implanta-

tion while the number of cells is relatively small (10⁷to 5 ×

10⁷cells). Tumors were measured with a caliper twice weekly,

or three times weekly for the more rapidly growing tumors).

Mice were sacrificed when their tumors reached 1500 mg (i.e.,

before they could cause the animal discomfort). Tumor weights

were estimated from two-dimensional measurements.

c. Tu m or Weigh t (in mg) ) (a × b²)/2, where a and b are

the tumor length and width in (mm), respectively.

weight loss nadir (mean of group) of greater than 20% or

greater than 20% drug deaths is considered to indicate an

excessively toxic dosage in a single course trial.

Ack n ow led gm en t. Thanks are due to the Resource

Laboratory of the Chemistry Department, Wayne State

University, Detroit, MI, wherein all instrumental analy-

ses were performed. The authors are grateful for the

support of this research through grants from the

National Institutes of Health (CA 82341), the Virtual

Discovery Program of the Barbara Ann Karmanos

Cancer Institute, and the J ack and Miriam Shenkman

Cancer Research Fund.

d . Qu a n tified En d P oin ts for Assessin g An titu m or

Activity for Solid Tu m or s. The following quantified end

points are used to assess antitumor activity.

1. Tu m or Gr ow th Dela y (T - C Va lu e). T is the median

time (in days) required for the treatment group tumors to reach

a predetermined size (e.g., 1000 mg), and C is the median time

(in days) for the control group tumors to reach the same size.

Tumor-free survivors are excluded from these calculations

(cures are tabulated separately). In our judgment, this value

is the single most important criterion of antitumor effective-

ness because it allows the quantification of tumor cell kill.

2. Ca lcu la tion of Tu m or Cell Kill. For subcutaneously

(sc) growing tumors, the log cell kill is calculated from the

following formula: log₁₀cell kill total (gross) ) T - C value in

days/3.32T_d, where T - C is the tumor growth delay as

described above and T_dis the tumor volume doubling time in

days, estimated from the best fit straight line from a log-linear

growth plot of the control group tumors in exponential growth

(100-800 mg range). The conversion of the T - C values to

log cell kill is possible because the T_dof tumors regrowing

posttreatment (Rx) approximates the T_dvalues of the tumors

in untreated control mice.

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J M0005149

Article Doi

Design, synthesis, and biological evaluation of analogues of the antitumor agent, 2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid (XK469)

DOI: 10.1021/jm0005149

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Article abstract of DOI:10.1021/jm0005149

Full text of DOI:10.1021/jm0005149

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