Optimization of 6,7-disubstituted-4-(arylamino)quinoline-3-carbonitriles as orally active, irreversible inhibitors of human epidermal growth factor receptor-2 kinase activity

J. Med. Chem. 2005, 48, 1107-1131

1107

Optimization of 6,7-Disubstituted-4-(arylamino)quinoline-3-carbonitriles as

Orally Active, Irreversible Inhibitors of Human Epidermal Growth Factor

Receptor-2 Kinase Activity

Hwei-Ru Tsou,*^,†Elsebe G. Overbeek-Klumpers,^†William A. Hallett,^†Marvin F. Reich,^†M. Brawner Floyd,^†

Bernard D. Johnson,^†Ronald S. Michalak,^‡Ramaswamy Nilakantan,^†Carolyn Discafani,^§Jonathan Golas,^§

Sridhar K. Rabindran,^§Ru Shen,^§Xiaoqing Shi,^§Yu-Fen Wang,^§Janis Upeslacis,^†and Allan Wissner^†

Chemical and Screening Sciences, Chemical Development, and Oncology, Wyeth Research, 401 North Middletown Road,

Pearl River, New York 10965

Received August 26, 2004

A series of new 6,7-disubstituted-4-(arylamino)quinoline-3-carbonitrile derivatives that function

as irreversible inhibitors of human epidermal growth factor receptor-2 (HER-2) and epidermal

growth factor receptor (EGFR) kinases have been prepared. These compounds demonstrated

enhanced activities for inhibiting HER-2 kinase and the growth of HER-2 positive cells compared

to our EGFR kinase inhibitor 86 (EKB-569). Three synthetic routes were used to prepare these

compounds. They were prepared mostly by acylation of 6-amino-4-(arylamino)quinoline-3-

carbonitriles with unsaturated acid chlorides or by amination of 4-chloro-6-(crotonamido)-

quinoline-3-carbonitriles with monocyclic or bicyclic anilines. The third route was developed

to prepare a key intermediate, 6-acetamido-4-chloroquinoline-3-carbonitrile, that involved a

safer cyclization step. We show that attaching a large lipophilic group at the para position of

the 4-(arylamino) ring results in improved potency for inhibiting HER-2 kinase. We also show

the importance of a basic dialkylamino group at the end of the Michael acceptor for activity,

due to intramolecular catalysis of the Michael addition. This, along with improved water

solubility, resulted in compounds with enhanced biological properties. We present molecular

modeling results consistent with the proposed mechanism of inhibition. Binding studies of one

compound, 25o (C-14 radiolabeled), showed that it binds irreversibly to HER-2 protein in BT474

cells. Furthermore, it demonstrated excellent oral activity, especially in HER-2 overexpressing

xenografts. Compound 25o (HKI-272) was selected for further studies and is currently in phase

I clinical trials for the treatment of cancer.

Introduction

domain. These phosphorylated residues serve as docking

sites for recruitment of downstream signaling proteins,

Protein tyrosine kinases play a key role in signal

transduction pathways that regulate numerous cellular

functions including proliferation, differentiation, migra-

tion, and angiogenesis. A large number of receptor and

nonreceptor tyrosine kinases have been identified.

Among them, the epidermal growth factor receptor

(EGFR) tyrosine kinase (known as EGFR, HER-1, or

ErbB-1) and human epidermal growth factor receptor-2

(known as HER-2, ErbB-2 or neu) have been widely

studied as drug discovery targets. They are two of the

four members of the EGF receptor family that also

includes HER-3 (ErbB-3) and HER-4 (ErbB-4). These

four closely related proteins share common structural

elements such as an extracellular ligand binding domain

and an intracellular tyrosine kinase domain. At least

12 different ligands that bind to the EGFR, HER-3, and

HER-4 receptors have been identified. However, there

is no known ligand for HER-2. Upon ligand binding,

these receptors dimerize with themselves or with other

family members and undergo autophosphorylation of

tyrosine residues within the C-terminal cytoplasmic

leading to subsequent signal transduction cascades.¹

Hyperactivation of the EGFR family of receptors

leading to uncontrolled cell proliferation is seen in a

variety of cancers.²Activation of EGFR may be due to

overexpression of the receptors (head and neck, lung,

breast, bladder, prostate, and kidney tumors), mutations

resulting in constitutive activation (brain tumors), or

overexpression of the ligands (EGF or TGFR in pancre-

atic, prostate, lung, ovary, and colon cancers).^3,4In

contrast, activation of HER-2 occurs mainly via over-

expression in breast, ovarian, lung, prostate, gastric,

and oral cancers^2,5leading to spontaneous homodimer-

ization and activation of downstream signaling events

in a ligand-independent manner. In particular, overex-

pression of HER-2 was found in 25-30% of breast

cancer cases and has been correlated with poor prog-

nosis in patients.⁶

Since deregulation of the EGFR and HER-2 tyrosine

kinases has been associated with a variety of cancers,

small-molecule inhibitors that could control the tyrosine

phosphorylation event could be of therapeutic value as

potential antitumor agents. Indeed, there are several

ATP-competitive EGFR tyrosine kinase inhibitors cur-

rently in clinical trials for the treatment of cancer.^7,8

Among them, a 6,7-disubstituted 4-anilinoquinoline-3-

* To whom correspondence should be addressed. Phone: (845) 602-

4712. Fax: (845) 602-5561. E-mail: tsouh@wyeth.com.

^†Chemical and Screening Sciences.

^‡Chemical Development.

^§Oncology.

Published on Web 01/27/2005

1108 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4

Tsou et al.

carbonitrile 86 (EKB-569), developed by us,^9,10is an

orally active, irreversible inhibitor of the EGFR kinase.

This compound carries a Michael acceptor at the 6-posi-

tion, which forms a covalent bond to Cys 773 located in

the ATP binding pocket of the EGFR enzyme. The

water-solubilizing dimethylamino group at the end of

the Michael acceptor serves as an intramolecular base

catalyst operating via a cyclic five-membered ring

mechanism to catalyze the Michael addition process.⁹

This enhanced reactivity with the EGFR enzyme along

with improved bioavailability resulted in 86 displaying

potent antitumor activity in EGFR-dependent tumor

models. However, since 86 shows less efficacy in HER-2

dependent tumor models, we continued with our efforts

to develop irreversible inhibitors with improved activity

toward HER-2 expressing tumors. These efforts are the

subject of this communication.

Figure 1.

EGFR and HER-2 share an 82% sequence identity in

the kinase domains; the identity is even higher in the

active site.¹¹We constructed a homology model of HER-2

kinase for this study using the crystal structure of

EGFR kinase in complex with erlotinib, a quinazoline-

based inhibitor (also known as Tarceva, or CP-358774).¹²

Since Cys 773 in EGFR is conserved as Cys 805 in HER-

2, the development of an irreversible inhibitor of the

latter kinase appeared to be feasible. The most notable

difference in the ATP binding region between these

kinases is a single amino acid residue, i.e., Ser 783 in

HER-2 vs Cys 775 in EGFR. The high level of identity

between the ATP binding regions of these two enzymes

suggested that it would be difficult to design molecules

that would be selective for one over the other. Designing

an inhibitor that showed improved efficacy in HER-2

models (compared to 86) appeared to be a more realistic

undertaking. The binding models of our inhibitors

indicated that the aniline portion fits into a long

lipophilic pocket. Since this is the region of the binding

pocket with the most difference between the two kinases

and since there is room to introduce large lipophilic

groups at the para position of the aniline, we explored

various modifications to the 4-arylamino portion of the

inhibitors in an empirical search to identify molecules

with improved HER-2 activity. Interestingly, several

other groups^13-15have independently discovered, for

quinazoline-based inhibitors, that large lipophilic sub-

stituents lead to improved HER-2 activity and, in some

cases, improved dual HER-2/EGFR activity.^16,17Here,

we describe the synthesis and SAR of a series of 6,7-

disubstituted 4-(arylamino)quinoline-3-carbonitriles modi-

fied with a variety of lipophilic substituents on the

arylamino ring. Compared to our EGFR kinase inhibitor

86, several of these compounds, as exemplified by 25o

(Figure 1), show improved activity toward HER-2 kinase

while retaining good potency for EGFR kinase. Most

importantly, this dual HER-2 and EGFR inhibitor, 25o,

also demonstrated improved in vivo efficacy in HER-2

dependent tumor models.

5-amino-1-benzylindole and 5-amino-1-benzylindazole)

and then reduced with iron and aqueous ammonium

chloride to yield the corresponding 6-amino-4-anilino-

quinoline-3-carbonitriles 4, which were converted to the

final products by two methods. In method 1A, the

amines 4 were reacted with 4-bromocrotonyl chloride 5

and then the bromo group was displaced with secondary

amines to yield the final compounds 7. In method 1B,

the final compounds 8 were prepared in one step by

reacting the amines 4 with 4-(N,N-disubstituted amino)-

crotonyl chlorides 6. Both methods shown in Scheme 1

were used successfully in our early work⁹to prepare 86

and similar 4-anilinoquinoline-3-carbonitriles as inhibi-

tors of EGFR kinase. Our original preparation of 1a,b

involved a high-temperature cyclization of a nitro-

containing intermediate.⁹Although we never experi-

enced any difficulty with this cyclization, we did have

some concerns about performing the cyclization on a

large scale. Therefore, we developed an alternative

synthesis where the cyclization was carried out on an

acetoamido intermediate 14 as shown in Scheme 2

(method 2). The commercially available 2-amino-5-

nitrophenol 9 was protected as acetamide 10. It was

then ethylated and reduced to yield 4-acetamido-3-

ethoxyaniline 12, which upon condensation with ethyl

(ethoxymethylene)cyano acetate 13 yielded the acet-

amido intermediate 14. Cyclization of 14 was carried

out in Dowtherm at 250-255 °C to yield 6-acetamido-

4-hydroxyquinoline-3-carbonitrile 15. Chlorination of 15

generated the 4-chloro intermediate 16. The subsequent

reactions are similar to those already described (Scheme

1) with the addition that the acetamido group of 18 was

deacetylated to amine 19 before reacting with the

desired crotonyl chloride 20 to yield the final compounds

21.

A third synthetic method was developed where the

6-crotonamide side chain was first attached to the

quinoline-3-carbonitrile ring, followed by introduction

of the anilines at the C-4 position, as shown in Scheme

3 (method 3). 4-Chloro-6-nitro compound 1b was re-

duced to the 4-chloro-6-amino compound 22, which was

then condensed with 4-(N,N-dimethylamino)crotonyl

chloride to yield 23. This was then converted to the final

products 25 by heating with a variety of anilines 24 in

methoxyethanol or 2-propanol using a catalytic amount

of pyridine hydrochloride. This method was proved

Chemistry

Most of the 6,7-disubstituted 4-anilinoquinoline-3-

carbonitriles (7, 8, 21, 25) described here were prepared

by one of the three methods shown in Schemes 1-3. In

Scheme 1, 4-chloro-6-nitroquinoline-3-carbonitriles 1a,b⁹

were reacted with a substituted aniline 2 (including

Carbonitriles as Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1109

Scheme 1. Methods 1A and 1B^a

1110 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4

Scheme 1. (Continued)

Tsou et al.

^a(i) i-PrOH, pyridine hydrochloride, reflux; (ii) Fe, NH₄Cl, MeOH, H₂O, reflux; (iii) THF, Hunig base, 4-bromocrotonyl chloride, 0 °C;

(iv) DMF, NaI, R′′R′NH, room temperature; (v) N-methyl-2-pyrrolidinone or DMF, from 0 °C to room temperature.

useful when optimizing the 4-anilino groups for HER-2

activity while keeping the 6-crotonamide side chain

constant.

pared by alkylation of imidazole with 2-chloro-4-ni-

trobenzyl bromide to yield 41, followed by reduction of

the nitro group. The 4-benzyoxyaniline 44 was readily

prepared by reacting phenol 43 with benzyl bromide

under basic conditions, followed by reduction of the nitro

group as shown. The synthesis of 3-chloro-4-(4-phen-

ylthiazol-2-ylmethyl)phenylamine, 51, where the thia-

zole group is attached via a carbon atom instead of a

heteroatom, is shown in Scheme 7. The toluene deriva-

tive 45 was heated with tert-butoxy-N,N,N′,N′-tetra-

methylmethanediamine, 46, to yield the enamine 47,¹⁸

which was then converted to the phenylacetonitrile 48

by reacting with hydroxylamine-O-sulfonic acid in warm

water.¹⁹Heating the nitrile 48 with H₂S in triethyl-

amine and DMF in a steel bomb gave the thioacetamide

49, which was then condensed with phenacyl bromide

50 to yield the desired aniline 51. Scheme 8 shows the

synthesis of several bicyclic anilines. 5-Amino-1-ben-

zylindole 54 was prepared by benzylation of the 5-nitro-

1H-indole 52, followed by reduction of the nitro group

to the amine. 5-Amino-1-benzylindazole 55 and 5-amino-

2,3-dihydro-1-benzylindole 57 were prepared in a simi-

lar manner. 5-Amino-1-phenylsulfonylindole 59 was

synthesized similarly except that phenylsulfonyl chlo-

ride was used.

For some compounds (31, 32, 36-38) where the

4-aniline ring bears nitrogen-containing para substit-

uents, the synthetic pathways are shown in Schemes 4

and 5. The amino group of 26 was first protected with

BOC anhydride, the nitro group was reduced to yield

the aniline 28, and this was condensed with chloride

23 followed by deprotection of the BOC group to yield

29. Reductive amination of the amino group of 29 with

aldehydes 30 using sodium triacetoxyborohydride in

acetic acid gave 31 and 32. An alternative approach was

used to prepare the 7-methoxy intermediate 35 as

shown in Scheme 5. The 4-chloro compound 1a was first

condensed with aniline 28, then the nitro group was

reduced followed by the reaction of 34 with 4-(N,N-

dimethylamino)crotonyl chloride in a manner similar to

that shown in Scheme 1 (method 1B). After deprotection

of the BOC group with TFA, the key intermediate 35

was obtained. Condensing 35 with acid anhydride, acid

chloride, or phenyl isocyanate yielded amides 36 and

37 or urea 38.

The preparations of the substituted anilines that were

needed to prepare some of the inhibitors are shown in

Schemes 6 and 7. 3-Chloro-4-fluoronitrobenzene was

reacted with 5-phenylthiazole-2-thiol under basic condi-

tions to yield 39, which was then reduced with iron and

acetic acid in methanol to yield the aniline 40. A similar

synthetic sequence was used in the reaction of 3-chloro-

4-fluoronitrobenzene with phenols, alcohols, and amines.

4-(1H-Imidazole-1-ylmethyl)-3-chloroaniline 42 was pre-

Preparations of some representative Michael acceptor

side chains are shown in Schemes 9 and 10. tert-Butyl

4-bromocrotonamide 60²⁰was converted into the corre-

sponding iodide 61 by refluxing with sodium iodide in

acetone. The iodide 61 was reacted with a variety of

secondary amines such as pyrrolidine at 0 °C to yield

amino ester 62. After removal of the tert-butyl group

Carbonitriles as Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1111

Scheme 2. Method 2^a

^a(i) Ac₂O, HOAc, 60 °C; (ii) EtBr, DMF, K₂CO₃, 60 °C; (iii) H₂, Pd/C, THF; (iv) toluene, 90 °C; (v) Dowtherm, 250 °C; (vi) POCl₃,

diglyme, 100 °C; (vii) pyridine hydrochloride, i-PrOH or methoxyethanol, reflux; (viii) aqueous HCl, reflux; (xi) CH₃CN, N-methyl-2-

pyrrolidinone or DMF, from 0 °C to room temperature.

under acidic conditions, a suspension of amino acid HCl

salt 63 in CH₃CN containing a catalytic amount of

N-methyl-2-pyrrolidinone (or DMF) was heated with

oxalyl chloride to afford the acid chloride 64 as a

hydrochloride salt, which was used immediately in

subsequent reactions. 4-(1H-Imidazol-4-yl)-but-2-enoic

acid hydrochloride, 67, was prepared by reacting 1-tri-

tyl-1H-imidazole-4-acetate 65²¹with triethyl phospho-

noacetate and n-BuLi in THF at -78 °C and then with

DIBAL²²to yield 66 (Scheme 10). Ester hydrolysis and

removal of the trityl group from 66 were followed by

acid chloride formation to give 68. 3-(2-Dimethylami-

nophenyl)acrylic acid 70 was prepared by heating

2-(dimethylamino)benzaldehyde 69²³in pyridine with

malonic acid using a catalytic amount of piperidine. Acid

70 was then converted to acid chloride HCl salt 71 using

oxalyl chloride. Acid chlorides 73 and 74 were prepared

in a similar manner (Scheme 11).

Compounds with other types of Michael acceptors

(82-85) were prepared as shown in Scheme 12. The

6-amino derivative 72 was treated with 2-chloro-1-

ethanesulfonyl chloride 78 in triethylamine at 0 °C,

resulting in the vinylsulfonamide 82. Preparation of acid

chlorides 79 and 80 were reported earlier;²⁴these were

reacted with 72 to yield 83 and 84, respectively. Finally,

commercially available 2-(bromomethyl)acrylic acid 81

was reacted with 72 to afford 85.

highly homologous EGFR kinase in complex with erlo-

tinib¹²was used as the template. Appropriate side chain

substitutions were made, a large sphere of water

molecules were added, and the whole structure was

minimized using Quanta/CHARMm.²⁵

By use of this model, ligands were hand-docked in

accordance with our previous studies of compounds in

the quinazoline/cyanoquinoline class,^9,10,26and the EG-

FR-erlotinib complex.¹²The complex was again opti-

mized using Quanta/CHARMm. Because EGFR and

HER-2 are closely related in sequence, the construction

of the homology model presented no significant chal-

lenges.

In the final model of the HER-2-25o complex (see

Figure 2), the N1 atom of the quinoline is hydrogen-

bonded to the backbone NH of Met 801 at a distance of

3.32 Å. The cyano nitrogen is 2.76 Å from the OH of

Ser 783. The chloroaniline portion of the inhibitor is

surrounded by residues Thr 798, Lys 753, Leu 133, Thr

143, and Asp 144, while the pyridylmethoxy group lies

in a pocket surrounded by Val 773, Met 774, Arg 784,

Ser 783, Val 777, Phe 864, and Thr 862.

The sulfur of Cys 805 is 3.43 Å from the reactive

Michael acceptor carbon. The dimethylamino group,

which we have shown earlier⁹can function as an

intramolecular catalyst for Michael additions, is also

close to the sulfhydryl hydrogen of Cys 805.

A paper describing a study of the binding modes of

6,7-disubstituted 4-anilinoquinoline-3-carbonitriles to

the closely related EGFR kinase appeared recently.²⁷

In it, the authors contend that they were unable to

Molecular Modeling

A homology model for the catalytic domain of HER-2

kinase was built as follows. The crystal structure of the

1112 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4

Scheme 3. Method 3^a

Tsou et al.

^a(i) Fe, HOAc, NaOAc, MeOH, reflux; (ii) Me₂NCH₂CHdCHCOCl in CH₃CN, N-methyl-2-pyrrolidinone or DMF, from 0 °C to room

temperature; (iii) pyridine hydrochloride, methoxyethanol, or i-PrOH, reflux.

reproduce a binding mode such as ours where the

headpiece is buried deep into the ATP binding pocket

and the Michael acceptor is positioned suitably to

interact with Cys 773 (analogous to Cys 805 of HER-2

kinase). They reported a distance of 5.97 Å between the

â-carbon and the sulfur of Cys 773 in their model. We

point out that the ligand and the protein have consider-

able flexibility even when complexed. Thermal motion

of the protein and ligand atoms should, in our opinion,

be adequate to bring the â-carbon and the sulfur of Cys

773 close enough to form a covalent interaction. A close

examination of the EGFR-erlotinib crystal structure¹²

leads us to the same conclusion.

sisting of the purified cytoplasmic domain of HER-2

(amino acids 676-1255) or EGFR (amino acids 645-

1186). Concentrations of compounds to inhibit auto-

phosphorylation of these proteins by 50% were mea-

sured to generate the IC₅₀values. As we indicated in

our earlier publications, the IC₅₀values generated by

our method are routinely higher than the IC₅₀values

produced by other researchers using the full-length

enzymes and exogenous peptide substrates, but the

relative potencies remain consistent.

In an earlier report, we showed that 86 functions as

an irreversible inhibitor of EGFR kinase.²⁸We will show

herein that compounds, as exemplified by 25o, bind

irreversibly to HER-2 kinase. As we have cautioned in

our earlier publications,^9,24care should be taken in

interpreting the IC₅₀values of compounds that function

as irreversible inhibitors. The IC₅₀values for such

inhibitors should depend on the extent to which the

covalent interaction has occurred. In addition, we have

suggested, and it has been recapitulated by others,²⁷

that the IC₅₀values could arise from two components,

one reflecting reversible binding and another reflecting

Results and Discussion

Kinase and Cellular Inhibitory Activities. The

compounds shown in Tables 1-5 were evaluated for

their ability to inhibit HER-2 and EGFR kinases. While

some other researchers may determine IC₅₀values of

inhibitors in soluble formats using the entire HER-2 and

EGFR enzymes along with exogenous peptide sub-

strates, we use a solid-phase ELISA-based assay con-

Carbonitriles as Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1113

Scheme 4^a

Figure 3. Scatter plot comparing the activities of compounds

against the EGFR and HER-2 kinase enzymes. Note that mean

values of log IC₅₀are plotted. The error bars (horizontal for

EGFR, vertical for HER-2) represent the standard error of the

respective mean values. Note that the number of observations

for each compound varies from 1 to 30. The single-point

observations show no error bars. The least-squares regression

line is also shown.

levels of expression of HER-2 and/or EGFR (Tables

1-5). Three human carcinoma cell lines were used:

A431 (epidermoid), which overexpresses EGFR; SKBR3

(breast), which overexpresses HER-2 and, to a lesser

extent, EGFR; SW620 (colon), which serves as a control

line not expressing either EGFR or HER-2 to a signifi-

cant extent.

^a(i) (Boc)₂O, DMAP, THF, 0 °C to room temperature; (ii) Fe,

HOAc, MeOH, reflux; (iii) pyridine hydrochloride, methoxyethanol,

reflux, then CH₂Cl₂, TFA, from 0 to 25 °C; (iv) NaBH(OAc)₃,

dichloroethane, HOAc, room temperature.

To get a global view of these data (Tables 1-5), a

matrix of correlation coefficients was constructed and

is shown in Table 6. It is evident that there is a

reasonable correlation between the EGFR and HER-2

inhibitory activities. This is shown in detail in the

scatter plot of the HER-2 and EGFR activities (Figure

3). This correlation is expected in view of the high

sequence homology of the catalytic domains of these two

kinases. In addition, we find a good correlation between

the activities of the compounds in inhibiting the growth

of the A431 and SKBR3 cell lines. Again, this is expected

given that both these cell lines are known to express,

to a large degree, and to depend on these growth factor

receptors. The correlation between the A431 and SKBR3

cell lines data is significantly higher than the correlation

between either of these data and the SW620 cell line

results.

On the other hand, we see a low correlation between

the kinase activities and the ability of these compounds

to inhibit the growth of the A431 and SKBR3 cell lines.

Since factors other than the ability of a compound to

inhibit an enzyme, such as the degree of cell perme-

ability, play a role here, this might be expected. Like-

wise, the kinase data do not correlate well with the

SW620 cell activities.

Figure 2. Model of the HER-2-25o complex. The H-bond

between the quinoline N1 and Met 801 is shown. Also shown

are distances between the sulfur of Cys 805 and the Michael

acceptor â-carbon, and the sulfhydryl hydrogen and the

nitrogen of the dimethylamino group, the suggested intramo-

lecular catalyst.

the subsequent covalent binding. One would therefore

expect that the IC₅₀values would be time-dependent

insofar as an inhibitor that reacts more slowly with the

enzyme, or does not react at all, would be expected to

have a higher IC₅₀value. However, an absolute depen-

dence of the IC₅₀value with reactivity of the inhibitors

cannot be counted on because of the component of the

IC₅₀that reflects reversible (noncovalent) binding.

As mentioned in the Introduction, we adopted the

same chemical scaffold used with our irreversible EGFR

inhibitor 86 and focused our synthetic efforts on intro-

ducing large lipophilic groups at the para position of the

3-chloroaniline ring because this portion of the molecule,

when bound, resides in the region where the enzymes

are most different. Since our efforts were directed

The compounds were also evaluated for their ability

to inhibit the growth of several cell lines with varying

1114 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4

Tsou et al.

Scheme 5^a

^a(i) i-PrOH, reflux; (ii) Fe, HOAc, MeOH, reflux; (iii) Me₂NCH₂CHdCHCOCl in CH₃CN, N-methyl-2-pyrrolidinone or DMF, from 0 °C

to room temperature; (iv) CH₂Cl₂, TFA; (v) (PhCO)₂O, DMA, 60 °C for 36, nicotinyl chloride hydrochloride, (i-Pr)₂NEt, DMA, 25 °C for 37;

(vi) Ph-NdCdO, DMA, 25 °C.

Scheme 6^a

Scheme 7^a

^a(i) Heat and distill at 160 °C; (ii) H₂NOSO₃H, hot H₂O, 55 °C;

(iii) Et₃N, DMF, H₂S, heat at 80 °C in a steel bomb; (iv) EtOH,

reflux.

86 for EGFR kinase. The compound also demonstrated

better activity for the HER-2 overexpressing cell line

SKBR3. To further increase lipophilicity and steric bulk,

a phenyl group was introduced at the C-5 (8d) and C-4

(8e) positions of the thiazole ring. While 8d is a

significantly less potent inhibitor of HER-2 kinase

compared to 7d, the regioisomer 8e is 6-fold more potent

than 7d in the HER-2 assay. Also, when the thioether

linkage of 8e was replaced with a methylene bridge, the

resulting compound 8f remained as potent as 8e. In

addition, other compounds with large lipophilic substit-

uents were prepared showing enhanced activity toward

HER-2 kinase compared to 86. It is evident that for

HER-2 inhibition, the benzyloxy compound 8b and the

phenoxy compound 25a are significantly more potent

^a(i) NaH, DMF, room temperature, or NaOMe, DMF; (ii) Fe,

NH₄Cl, MeOH, H₂O, reflux, or Fe, HOAc, MeOH, reflux; (iii) THF,

reflux; (iv) Cs₂CO₃, DMF, 25 °C.

toward finding compounds with improved HER-2 activ-

ity relative to 86 (EKB-569), we discuss the data using

this compound as a reference.

The compounds listed in Table 1 represent our initial

exploration into the effects of placing large lipophilic

groups at the para position of the 4-anilino substituent.

It is evident that 7d, bearing a 2-thiazolsulfanyl sub-

stituent, is 15-fold more potent than 86 in inhibiting

the HER-2 kinase while retaining similar potency as

Carbonitriles as Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1115

Scheme 8^a

Scheme 11^a

^a(i) N-Methyl-2-pyrrolidinone, from 0 °C to room temperature.

Scheme 12^a

^a(i) K₂CO₃, DMF, heat; (ii) Fe, HOAc, MeOH, H₂O, reflux; (iii)

NaH,benzene; (iv) Raney Ni, hydrazine, EtOH.

Scheme 9^a

^a(i) NaI, acetone, reflux; (ii) THF, 0 °C; (iii) 1 N HCl, reflux;

(iv) CH₃CN, (COCl)₂, N-methyl-2-pyrrolidinone or DMF (cat.), 55

°C.

than 86 (16-fold and 7-fold, respectively) while the

corresponding 4-(pyridinyl)oxy compound 7b is signifi-

cantly less potent. Furthermore, 8b has about the same

potency as 86 against the EGFR kinase. These results

are similar to those found by other researchers in the

quinazoline-based series, where enhancement of HER-2

activity was observed for compounds bearing bulky

substituents on the 4-aniline ring.^14,16,17

We also looked at several compounds that have

bicyclic aniline substituents such as 21b, 21c, 8g, and

8h in Table 2. These compounds are significantly more

potent than 86 in inhibiting HER-2 kinase, and they

show good activity in inhibiting the growth of the

SKBR3 cell line.

^a(i) THF, Et₃N, 0 °C; (ii) N-methyl-2-pyrrolidinone, from -15

°C to room temperature; (iii) N-methyl-2-pyrrolidinone, from 0 °C

to room temperature; (iv) CH₃CN, Hunig’s base, reflux.

Throughout this work, we used either a methoxy or

ethoxy substituent at the 7-position. There is no clear

advantage of either substituent with respect to potency

(for example, compare 8b with 8i and compare 25a with

8j in Table 3). However, from our early work, we found

that a number of the inhibitors that have a 7-methoxy

substituent showed a very weak positive response in an

Ames test after microsomal incubation while the cor-

Scheme 10^a

^a(i) Triethyl phosphonoacetate, n-BuLi, THF, then DIBAL, CH₂Cl₂, -78 °C to room temperature; (ii) MeOH, NaOH, then HCl; (iii)

CH₂Cl₂, (COCl)₂, cat. DMF, reflux; (iv) malonic acid, pyridine, piperidine (cat.), 110 °C; (v) CH₂Cl₂, (COCl)₂, DMF (cat.), reflux.

1116 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4

Tsou et al.

Table 1. Initial Exploration of the Para Substituents for HER-2 Activity

kinase assays

cell proliferation assays

IC₅₀(µM)

synthetic

method

compd

R

Y

HER-2^a

EGFR^b

A431^c

SKBR3^c

SW620^c

7a

7b

7c

7d

7e

8a

8b

8c

8d

8e

8f

21a

25a

86

Me

Et

1-imidazolyl

1A

1B

2

0.80

0.77

0.65

0.041

0.042

0.519

12.7

0.053

0.403

0.032

0.125

0.214

0.500

0.445

0.08

1.2

0.40

2.7

>9

O-(4-pyridinyl)

8.1

6.0

2.8

Me

Et

S-[2-(3-methylimidazolyl)]

S-(2-thiazolyl)

0.155

0.083

1.055

2.6

0.076

0.348

1.007

0.013

0.074

0.625

0.184

1.23

0.132

0.516

2.839

0.43

0.108

0.209

0.574

0.374

0.082

0.129

0.052

0.08

0.0013

0.0015

0.1094

1.2

0.0027

0.0156

0.0462

0.0094

0.0049

0.0021

0.0039

0.01

1.093

1.592

5.48

CH₂-(1-imidazolyl)

4-morpholinyl

Et

2.1

OCH₂Ph

0.486

1.300

0.961

0.842

0.328

0.993

0.683

0.68

Me

Et

S-(2-pyrimidyl)

S-[2-(5-phenylthiazolyl)]

S-[2-(4-phenylthiazolyl)]

CH₂-[2-(4-phenylthiazolyl)]

O-CH₂-[2-(3-methylimidazolyl)]

O-Ph

Me

Et

3

Et

F

^aConcentration needed to inhibit the autophosphorylation of the cytoplasmic domain of HER-2 by 50% as determined from the dose-

response curve. ^bConcentration needed to inhibit the autophosphorylation of the cytoplasmic domain of EGFR by 50% as determined

from the dose-response curve. ^cDose-response curves were determined at five concentrations. The IC₅₀(µM) values are the concentrations

needed to inhibit cell growth by 50% as determined from these curves.

Table 2. 2,3-Dihydro-1H-indole, 1-H-indole, and 1H-Indazole Inhibitors

kinase assays

cell proliferation assays

IC₅₀(µM)

synthetic

method

compd

series

R

X

HER-2^a

EGFR^b

A431^c

SKBR3^c

SW620^c

8g

II

III

I

Et

Me

Et

CH₂Ph

SO₂Ph

1B

2

0.015

0.075

0.23

0.10

0.13

0.34

0.082

0.084

0.1

0.23

0.17

0.0014

0.0034

0.028

0.74

0.87

2.1

8h

21b

21c

II

2

0.025

0.015

1.1

^aConcentration needed to inhibit the autophosphorylation of the cytoplasmic domain of HER-2 by 50% as determined from the dose-

response curve. ^bConcentration needed to inhibit the autophosphorylation of the cytoplasmic domain of EGFR by 50% as determined

from the dose-response curve. ^cDose-response curves were determined at five concentrations. The IC₅₀(µM) values are the concentrations

needed to inhibit cell growth by 50% as determined from these curves.

responding ethoxy derivatives did not. Because of this

observation, the majority of the compounds prepared

possessed the 7-ethoxy substituent.

It is interesting to note the difference in the kinase

activities between the pair of isomers where a methyl

group was introduced at the methylene bridge of the

benzyloxy group. While the R isomer (25v) showed

potent kinase activity, the corresponding S isomer (25w)

was 340-fold less potent for the HER-2 enzyme, sug-

gesting a space requirement in the lipophilic pocket.

Conformationally restricting the benzyloxy group by

using 2,3-dihydro-1H-inden-1-yloxy substituents having

both R and S isomer configurations (8p and 8q, respec-

tively) results in inhibitors with poor HER-2 kinase and

to a lesser extent for EGFR activity relative to 8b.

Additional analogues of 8b were prepared wherein the

benzyloxy group was replaced with benzylamine (31),

N-methylbenzylamine (8s), and 1-naphthylmethylamine

(32). The benzylamine analogue 31 is as potent as 8b,

whereas 8s shows much reduced activity. Poor activity

was also observed for compounds where the benzyl-

amine group in 31 was replaced with benzamide (36),

nicotinamide (37), and phenylurea (38).

Preliminary in vivo experiments indicated that of the

compounds we initially evaluated (Tables 1 and 2), the

benzyl-substituted derivative 8b looked the most prom-

ising for further modifications. Replacement of the

benzyloxy group of 8b with the slightly longer phenyl-

ethoxy group results in a compound, 25c, that is slightly

less potent in both kinase and cell proliferation assays

(Table 3). By comparision of the activity of 25a, 8b, and

25c, it is clear that the chain length of the para

substituent contributes to potency in inhibiting HER-2

kinase in the order of benzyloxy > phenoxy > phenyl-

ethoxy. The position of the benzyloxy substituent is also

important for activity, with a preference for the 4-posi-

tion of the 3-chloroaniline ring. Switching the benzyloxy

substitutent from the 4- to the 3-position by replacing

the 3-chloro group gives 8r, which is clearly an inferior

inhibitor of both HER-2 and EGFR kinases.

Carbonitriles as Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1117

Table 3. Optimization of the Aniline Substituents for HER-2 Activity

kinase assays

cell proliferation assays

IC₅₀(µM)

synthetic

compd series

R

X

Y

A

Z

method HER-2^aEGFR^bA431^cSKBR3^cSW620^c

7f

IV

V

Et

Me

Et

H

Cl

H

Cl

O

3-F-Ph

1A

1B

2

0.033

0.076

0.039

0.363

0.081

0.008

0.044

3.775

0.131

2.1

0.034 0.120

0.053 0.108

0.024 0.083

0.185 0.108

0.110 0.211

0.007 0.148

0.031 0.307

0.574 0.680

0.277 0.077

0.0025

0.0027

0.0038

0.0098

0.0033

0.0012

0.0022

0.0800

0.0033

0.0023

0.0063

0.04

0.511

0.486

1.120

1.019

0.549

0.965

0.580

1.404

0.307

0.29

8b

Ph

8i

Ph

8j

8k

2,4-di-F-Ph

3-Cl-Ph

2-OMe-Ph

4-(OCH₂Ph)Ph

2-thienyl

8l

8m

8n

8o

8p

(1R)-2,3-dihydro-1H-inden-1-yloxy

0.38

0.40

0.069

0.11

0.53

0.37

8q

V

(1S)-2,3-dihydro-1H-inden-1-yloxy

9.8

0.3

8r

V

OCH₂Ph

H

13.2

2.1

13

0.25

1.43

-

8s

IV

V

H

Cl

H

NMe Ph

O

0.034

21d

25a

25b

25c

25d

25e

25f

25g

25h

25i

25j

25k

25l

25m

25n

25o

25p

25q

25r

25s

25t

25u

25v

25w

31

2-Cl-Ph

0.010

0.184

0.498

0.281

0.406

1.134

0.732

0.078

1.331

0.388

0.024

0.161

1.477

0.132

0.028

0.059

0.269

0.062

0.064

1.184

0.020

0.018

0.076

0.011 0.120

0.445 0.052

0.345 0.136

0.165 0.254

0.163 0.423

0.768 0.342

0.401 0.285

0.064 0.102

0.362 0.970

0.140 0.262

0.051 0.069

0.195 0.071

0.894 0.114

0.111 0.052

0.285 0.151

0.092 0.086

0.341 0.056

0.023 0.089

0.188 0.864

0.238 0.708

0.010 0.564

0.015 0.569

0.0029

0.0039

0.0016

0.0319

0.0110

0.0059

0.0028

0.0082

0.0378

0.0033

0.0093

0.0019

0.0078

0.0127

0.0011

0.0018

0.0004

0.0037

0.0110

0.0631

0.0127

0.0280

0.0013

0.013

0.948

0.683

0.924

0.500

1.219

0.540

1.472

0.584

1.200

1.073

0.593

2.220

0.410

0.357

0.551

0.730

0.197

0.366

2.147

3.261

0.973

0.775

0.14

OPh

3

IV

V

H

O

Ph

3

Cl

CH₂Ph

3

Et Cl Cl

Ph

3

Et

H

CN

Cl

Ph

3

2-F-Ph

3

4-F-Ph

3

Et Cl Cl

3-F-Ph

3

Et

Me

H

Cl

3,5-di-F-Ph

4-Cl-Ph

3

3-CN-Ph

(CH₂)₃CH₃

3-Me-Ph

3

cyclohexyl

2-pyridinyl (HKI-272)

3-pyridinyl

2-furyl

3

2-benzothiazolyl

4-quinolinyl

1-naphthyl

2-naphthyl

[(1R)-1-phenylethyl]oxy

[(1S)-1-phenylethyl]oxy

Ph

1-naphthyl

Ph

3-pyridyl

NHPh

3

0.10

0.77

0.053

0.15

V

3

26

0.193.8

0.578

0.93

IV

VI

H

NH

0.103

0.083

0.045 0.048

0.034 0.17

0.0020

0.023

32

36

13

3.9

1.1

0.40

0.072 0.1

0.21 2.6

0.81

>5

0.62

37

0.086

0.50

2.7

38

>5

^aConcentration needed to inhibit the autophosphorylation of the cytoplasmic domain of HER-2 by 50% as determined from the dose-

response curve. ^bConcentration needed to inhibit the autophosphorylation of the cytoplasmic domain of EGFR by 50% as determined

from the dose-response curve. ^cDose-response curves were determined at five concentrations. The IC₅₀(µM) values are the concentrations

needed to inhibit cell growth by 50% as determined from these curves.

In our early work on the EGFR kinase inhibitors, we

found that a small lipophilic substituent (such as Cl) in

the 3-position of the aniline ring was important for

EGFR inhibitory activity. A similar observation was

made in this work; namely, the 3-chloroaniline com-

pounds (8b, 8i) are more potent than the corresponding

des-chloroaniline derivatives (25b, 8j) in kinase assays,

but this is not evident in the cell proliferation assays.

However, the benefit of the 3-chloro group was abolished

when an additional chloro substituent was introduced

at the 5-position, namely, a 3,5-dichloro-4-benzyloxy-

aniline derivative (25d vs 8b; 25h vs 7f). Replacement

of the 3-chloro group of the aniline in 8b with a 3-nitrile

resulted in a much less potent compound 25e in the

kinase assays, but this effect is not as evident in the

cell proliferation assays.

With respect to the substituent on the benzyloxy ring

of 8b, the 3-chloro (8l) and 2-chloro (21d) derivatives

are more potent than 8b in the kinase assays; however,

their cellular activities remained similar to that of 8b.

This effect on the kinase and cellular activities parallels

that seen with 7f, where a fluoro atom is at the

3-position. What was not expected is that, unlike the

potent 2-chloro analogue 21d, the 2-fluoro counterpart

25f was much less active in the kinase assays but

remained potent in the cell proliferation assays. While

the 3-fluoro derivative 7f showed good activity, the

corresponding 3,5-difluoro compound 25i was a less

effective inhibitor of both the HER-2 and EGFR en-

zymes. Compounds bearing 3-cyano (25k) or 3-methyl

(25m) are inferior to the corresponding 3-fluoro (7f) and

3-chloro (8l) compounds. Introduction of a 4-benzyloxy

1118 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4

Table 4. Optimization of the C-6 Crotonamide Side Chain

Tsou et al.

kinase assays

cell proliferation assays

IC₅₀(µM)

synthetic

method

compd

R

HER-2^a

EGFR^b

A431^c

SKBR3^c

SW620^c

7g

7h

7i

N(Me)(CH₂)₂OMe

N(Me)(CH₂)₂OH

1A

1B

0.183

0.027

0.966

0.028

0.104

0.171

0.025

0.169

0.058

0.412

0.059

0.029

0.540

0.040

0.196

0.603

0.076

0.059

0.069

0.068

0.020

0.719

0.030

0.098

0.151

0.031

0.221

0.282

0.247

0.020

0.016

0.360

0.026

0.343

0.321

0.053

0.078

0.024

0.043

0.072

3.115

0.258

0.156

0.167

0.244

0.092

0.063

0.737

0.037

0.127

0.085

0.158

0.229

0.132

0.108

0.125

0.829

0.0057

0.0048

0.1161

0.0043

0.0027

0.0034

0.0042

0.0017

0.0032

0.0200

0.0099

0.0456

0.0327

0.0157

0.0196

0.0032

0.0027

0.0053

0.1485

0.817

0.648

21.099

0.461

1.638

0.807

0.397

0.792

4.107

0.657

2.842

5.862

1.162

0.951

2.451

0.306

0.486

0.309

1.727

N(Me)-^D-glucamine

N(i-Pr)₂

7j

7k

7l

N[CH₂CH(OH)CH₃]₂

2-methoxymethyl-1-pyrrolidinyl

N-piperidinyl

4-OH-1-piperidinyl

4,4-dihydroxy-1-piperidinyl

2,6-dimethyl-1-piperidinyl

N-morpholinyl

N-thiomorpholinyl

N-(4-methylpiperazinyl)

N-azetidinyl

2-carbomethoxy-1-aziridinyl

1-aza-12-crown-4

NMe₂

N-pyrrolidinyl

4-imidazolyl

7m

7n

7o

7p

7q

7r

7s

7t

7u

7v

8b

8t

8u

^aConcentration needed to inhibit the autophosphorylation of the cytoplasmic domain of HER-2 by 50% as determined from the dose-

response curve. ^bConcentration needed to inhibit the autophosphorylation of the cytoplasmic domain of EGFR by 50% as determined

from the dose-response curve. ^cDose-response curves were determined at five concentrations. The IC₅₀(µM) values are the concentrations

needed to inhibit cell growth by 50% as determined from these curves.

Table 5. Inhibitors Bearing C-6 Michael Acceptor Groups Other Than Crotonamide

kinase assays

IC₅₀(µM)

cell proliferation assays

IC₅₀(µM)

compd

series

R

HER-2^a

EGFR^b

A431^c

SKBR3^c

SW620^c

75

76

77

82

83

84

85

VII

VIII

XI

8-quinolinyl

(2-NMe₂)Ph

4-imidazolyl

1.134

1.391

0.227

0.061

0.246

0.089

3.781

0.990

1.569

0.255

0.045

0.223

0.075

1.191

13.576

14.398

>8.85

0.617

0.360

0.150

6.522

4.1526

7.6035

0.6371

0.0673

0.1295

0.0069

2.2685

28.749

32.841

>8.85

1.495

NMe₂

morpholinyl

0.360

XI

0.401

15.501

X

^aConcentration needed to inhibit the autophosphorylation of the cytoplasmic domain of HER-2 by 50% as determined from the dose-

response curve. ^bConcentration needed to inhibit the autophosphorylation of the cytoplasmic domain of EGFR by 50% as determined

from the dose-response curve. ^cDose-response curves were determined at five concentrations. The IC₅₀(µM) values are the concentrations

needed to inhibit cell growth by 50% as determined from these curves.

substituent on the benzyloxy ring of 8b (as shown in

8n) is detrimental to activity.

series, the 2-pyridinyl (25o), 2-furyl (25q), and 2-thienyl

(8o) analogues each showed similar activities compared

to 8b.

In our earlier work,⁹we found that adding a basic

dialkyamino group to the terminus of the crotonamide

side chain at the 6-position of 4-anilinoquinoline-3-

carbonitrile significantly increased the activity in both

kinase and cell proliferation assays. On the basis of our

binding model of 25o complexed with HER-2, the water-

solubilizing dimethylamino group points out of the ATP

We then replaced the phenyl ring of the benzyloxy

group of 8b with cylcohexyl (25n), bicyclic, or hetero-

cyclic groups. The cyclohexyl derivative 25n was as

potent an inhibitor as 8b for HER-2 kinase but showed

10-fold less activity against EGFR kinase. The 1-naph-

thyl compound (25t) and 2-naphthyl compound (25u)

were slightly more potent in the kinase assays but less

potent in cell proliferation assays. In the heterocyclic

Carbonitriles as Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1119

Table 6. Correlations between log(IC₅₀) in EGFR and HER-2

Kinase Assays and Cell Proliferation Assays on Cell Lines

A431, SKBR3, and SW620^a

log IC₅₀

EGFR HER-2 A431 SKBR3 SW620

EGFR (log IC₅₀

HER-2 (log IC₅₀

A431 (log IC₅₀

SKBR3 (log IC₅₀

SW620 (log IC₅₀

)

1.000

0.815

0.365

0.335

0.489

)

1.000

0.281

0.255

0.370

)

1.000

0.934

0.478

)

1.000

0.411

1.000

^aThe Pearson product moment correlation coefficients are

tabulated and are calculated using the formula

n( XY) - ( X)( Y)

∑

∑ ∑

R )

[n X²- ( X)²][n Y²- ( Y)²]

∑ ∑ ∑ ∑

x

binding pocket toward the aqueous environment. Also,

the â-carbon of the crotonamide group is 3.43 Å away

from the sulfur atom of Cys 805, and the dimethylamino

moiety is situated 4.46 Å from the hydrogen of the

sulfhydryl group. We have proposed that the dialkyl-

amino group of compounds such as 25o could serve as

an intramolecular base catalyst for Michael additions

by extracting the proton from the nucleophilic sulfhydryl

group (Cys 805 for HER-2 and Cys 773 for EGFR)

through a cyclic five-membered ring mechanism.

Table 4 shows compounds where the terminal dialkyl-

amino group of the crotonamide Michael acceptor was

varied. With a few exceptions, it is evident that for most

of these analogues, the kinase and cellular activities are

minimally affected by the choice of the dialkylamino

group. One exception is compound 7p, which carries a

2,6-dimethyl-1-piperidinyl group. It is much less active

than the corresponding compound 7m carrying an

N-piperidinyl group, suggesting that the steric bulk

around the basic nitrogen may hinder the intramolecu-

lar base catalysis of the Michael addition of Cys 805 to

form a covalent adduct. Likewise, the loss of kinase

potency of 7v may be due to the steric bulk of 1-aza-

12-crown-4. However, 7v was as potent as 8b in the cell

proliferation assays. A highly water-soluble analogue

7i carrying an N-methyl-D-glucamine group was also

less active in the kinase and cell proliferation assays.

Compound 77, where the Michael acceptor is terminated

with an imidazole ring, showed reduced activity in both

kinase and cell proliferation assays (Table 5) compared

to compounds with the terminal dimethylamino group.

This loss of activity is likely due to the reduced basicity

of the imidazole ring of 77 compared to the imidazole

in 8u. Also, the proposed intramolecular base catalysis

of the Michael addition would not be expected to occur

with 77 but could occur with 8u. Similarly, quinolino

and dimethylaminophenyl substituted derivatives 75

and 76, respectively, show reduced activities most likely

due to reduced basicity. There is no major difference in

activities between compounds in the crotonamide series

such as 8b and 7q compared to the coreresponding

compounds 83 and 84 in the methacrylamide series. As

expected, compound 85, lacking a basic amino substitu-

ent but carrying a carboxylic group, is a poor inhibitor

of both HER-2 and EGFR. Finally, compound 82 with

an ethylenesulfonamide group is as potent as 8b. From

these in vitro studies, it is clear that among the best

Figure 4. Inhibition of HER-2 phosphorylation in the 3T3/

neu tumor model and BT474 xenograft. 3T3/neu (A) and BT474

(B) tumor-bearing mice were treated with vehicle or a single

dose of compound (three to five animals per group). Tumors

from control and treated mice were dissected at various times

after compound administration. Protein extracts from each

time point were pooled and analyzed by SDS-PAGE and

immunoblotting. Extracts from individual tumors were also

analyzed to determine the variability between animals. Phos-

phorylation of HER-2 was detected using phosphotyrosine

antibodies and normalized to HER-2 levels, detected with anti

HER-2 antibodies. Blots were quantified by scanning densi-

tometry. Data for 25o in part B are from ref 29.

dual inhibitors of HER-2 and EGFR kinases are 7f, 7m,

8b, 8o, 8t, and 25o. These compounds were selected for

further in vivo studies.

Selectivity and Irreversible Binding to HER-2.

Compound 25o was evaluated in two additional tyrosine

kinases assays, namely, KDR and Src. The IC₅₀values

(0.8 µM for KDR and 1.4 µM for Src) were 14- and 24-

fold greater for these kinases compared to HER-2.

Compound 25o was also evaluated against a number

of serine-threonine kinases among which were Akt,

cyclin D1/CDK4, cyclin E/CDK2, cyclin B1/CDK1, IKK-

2, MK-2, PDK1, c-Raf, and Tpl-2, and no significant

inhibitory activity was observed.²⁹It is clear that 25o

is a selective inhibitor of members of the ErbB family

of kinases.

In our earlier work,⁹EGFR kinase inhibitor 86 was

shown to form a covalent adduct with EGFR kinase. We

proposed that this is the result of a covalent interaction

between the Michael acceptor group of 86 with Cys 773

located within the ATP binding pocket of EGFR. Since

25o and 86 have identical Michael acceptor functional-

ity and since the target cysteine residue is conserved

in HER-2 (Cys 805), it is expected that 25o will

1120 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4

Tsou et al.

Table 7. Comparison of in Vivo Effects (% T/C) of HER-2 Inhibitor Leads and EKB-569 (86) in Tumor Models Sensitive to HER-2 or

EGFR^a

% T/C (p value)

3T3/neu

BT474

SKOV3

SUM190

40 mg/kg

A431

compd

80 mg/kg

40 mg/kg

10 mg/kg

41 (0.06)

40 mg/kg

7f

1 (<0.01)

27 (<0.01)

3 (<0.01)

44 (<0.01)

13 (<0.01)

46 (<0.01)

36 (<0.01)

5 (<0.01)

44 (<0.01)

4 (<0.01)

12 (<0.01)

6 (<0.01)

34 (0.01)

7m

8b

8o

8t

13 (<0.01)

27 (<0.01)

26 (<0.01)

29 (0.06)

47 (0.07)

25o

86

2 (<0.01)

5 (<0.01)

12 (<0.01)

18 (<0.01)

34 (<0.01)

70 (0.16)

14 (<0.01)

24 (<0.01)

20 (0.01)

32 (0.11)

56 (0.09)

6 (<0.01)

^aTumor cells (3T3/neu, SKOV3, SUM-190, A431) or tumor fragments (BT474) were implanted subcutaneously in female athymic (nude)

mice. For hormone-dependent tumors (BT474, SKOV3), estrogen pellets were implanted 1 week prior to tumor implantation. When tumors

reached a size of 90-200 mg, they were randomly assigned to treatment groups (5-10 animals per group) (staging, day 0). Compounds

were formulated in 0.5% methylcellulose and 2.0% polysorbate-80 (Tween-80) and adminstered orally once daily for 20 days (10 days for

3T3/neu and A431 xenograft). For 3T3/neu tumors, dosing was started the day after tumor cell implantation (day 0) because of rapid

growth. Growth of tumors was determined once a week. Tumor mass was estimated using the formula (length)(width²)/2 and was expressed

as relative tumor growth (RTG), the ratio of the tumor mass to the mass on the staging day (except 3T3/neu). The effect of the compounds

on tumor growth is shown as % T/C, the RTG or mean tumor weight of treated group/RTG or mean tumor weight of placebo group,

expressed as a percentage. Data were analyzed using a one-tailed Student’s t-test (equal variance) after log transformation of the data.

A p value of <0.05 indicates statistical significance of tumor inhibition. The data shown are from day 21 (3T3/neu, BT474, and A431

xenografts), day 28 (SUM-190 xenograft), and day 35 (SKOV3 xenograft), with the p value in parentheses. No compound-related adverse

effects (death, weight loss) were observed in these studies.

irreversibly bind to HER-2. Washout experiments were

designed to determine if 25o functions as an irreversible

binding inhibitor in intact BT474 cells by measuring the

ability of the compound to inhibit the autophosphory-

lation of the receptor after removal of 25o from the

media. We found that after preincubation of cells with

25o (5 h) followed by extensive washing to remove free

drug from the media, autophosphorylation of HER-2

was still inhibited.²⁹

A more direct indication of a covalent interaction with

the enzyme was obtained using C-14 labeled 25o.²⁹

Labeled compound was incubated with the purified

cytoplasmic domain of HER-2. After denaturation, the

sample was analyzed by SDS-PAGE and fluorography.

A single ∼95 kDa labeled band, corresponding to the

HER-2 cytoplasmic domain, was observed. This radio-

labeled signal was greatly diminished if the protein was

preincubated with unlabeled 25o prior to addition of

radiolabeled 25o. Similar experiments were performed

by incubating C-14 labeled 25o with intact BT474 cells.

A prominent band corresponding to ∼185 kDa incorpo-

rated labeled drug. Additionally, incorporation of labeled

drug could be inhibited when the cells were pretreated

with a 5-fold excess of cold compound prior to exposure

to the labeled drug. Finally, the labeled protein could

be immunoprecipitated with an anti-HER-2 antibody

suggesting that the ∼185 kDa species is in fact HER-2.

These data strongly support our claim that 25o func-

tions as an irreversible binding inhibitor of HER-2 in

solution and in intact cells.

HER-2 Phosphorylation in in Vivo Tumor Mod-

els. To determine whether inhibition of tumor growth

in vivo was associated with a block in the target receptor

function, the phosphorylation of HER-2 was evaluated

in xenografts after a single oral dose of 40 mg/kg of 7f

or 25o (Figure 4). Compound 7f reduced HER-2 phos-

phorylation in 3T3/neu tumor model by 30% within 3 h

as shown in Figure 4A. Inhibition reached a maximum

of 71% at 6 h and was partially reversed by 24 h (59%).

In BT474 xenografts, 7f reduced HER-2 phosphorylation

by 52% in 3 h as shown in Figure 4B. Maximum

inhibition was observed at 6 h (87%), which was

sustained at 24 h (79%) and partially reversed by 48 h

(58%). Similar effects were observed with 25o,²⁹al-

though substantial reduction of phosphorylation (84%

inhibition) was observed earlier, within 1 h of compound

dosing, and was partly reversed (57% inhibition) by 24

h. The sustained inhibition of phosporylation of HER-2

seen in the in vivo studies of both the 3T3/neu tumor

model and BT474 xenograft is consistent with irrevers-

ible inhibition of the target enzyme HER-2.

In Vivo Efficacy. The best compounds 7f, 7m, 8b,

8o, 8t, and 25o based on the in vitro assays were

evaluated in a number of tumor models in nude mice

(Table 7). Our EGFR clinical lead 86 was also included

in the testing for comparison. One model used for this

evaluation involved murine fibroblast 3T3 cells trans-

fected with HER-2/neu. Treatment was initiated the day

after cell implantation in this model because of the rapid

growth of the transplant. The compounds were dosed

orally once a day for 10 days. After day 10, dosing was

discontinued. It is evident that compounds 7f, 8b, and

25o are more effective in reducing tumor growth than

86 when administered orally at 40 mg/kg. This cor-

relates well with the higher potency of 7f, 8b, and 25o

against HER-2 enzyme compared to 86. However, at a

higher dose of 80 mg/kg, similar efficacy (95-99%

inhibition) was observed for most of these analogues.

Compounds were evaluated in three traditional tumor

models that overexpress HER-2; among these were

xenografts of BT-474, SKOV3, and SUM190 cells. In

BT474 and SUM190 tumor models, 7f, 8b, and 25o

showed better efficacy and activity than 86. For ex-

ample, 86 inhibited the growth of BT474 tumors by 30%

at a 10 mg/kg dose, whereas 7f, 8b, and 25o showed

higher inhibition (59%, 73%, and 66%, respectively) at

the same dose. Since 7f, 8b, and 25o inhibit EGFR

kinase and proliferation of EGFR-overexpressing cells

in vitro, the effect of these compounds in A431 xeno-

grafts was evaluated. Although all these compounds

suppressed the growth of A431 tumor (44-66% inhibi-

tion), they were less potent than 86 (94% inhibition) in

this tumor model.

Carbonitriles as Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1121

crystallized from ethyl acetate to give 3.3 g (84%) of light-

Conclusions

yellow crystals (39): mp ) 126-128 °C; HRMS (ESI) m/z

We have described three different synthetic routes

and resultant SAR of a series of 6,7-disubstituted

4-anilinoquinoline-3-carbonitriles. These compounds func-

tion as irreversible binding inhibitors of HER-2 and

EGFR kinases consistent with our binding models for

these compounds. Significantly, some of these com-

pounds showed better activity in HER-2 kinase and

HER-2 overexpressing cell proliferation assays com-

pared to our EGFR kinase inhibitor 86. They also

demonstrated improved potency and efficacy in HER-2

dependent in vivo models compared to 86. On the basis

of the results described herein and on extensive pharmo-

kinetic and toxicological studies, one member of this

series, 25o, has entered clinical trials for further

development as an antitumor agent in breast and other

HER-2 dependent cancers.

1

348.986 43 (M + H)⁺¹, ∆ ) -0.25 mmu; H NMR (DMSO-d₆)

δ 8.52 (s, 1H), 8.43 (d, 1H, J ) 2.4 Hz), 8.17 (dd, 1H, J ) 8.7,

2.4 Hz), 7.72 (d, 2H, J ) 5.2 Hz), 7.52-7.40 (m, 4H). Anal.

(C₁₅H₉ClN₂O₂S) C, H, N, Cl, S.

3-Chloro-4-(5-phenylthiazol-2-ylsulfanyl)phenyl-

amine (40). A mixture of 3.13 g (8.97 mmol) of 2-(2-chloro-4-

nitrophenylsulfanyl)-5-phenyl-1,3-thiazol 39 and 3.5 g (62.6

mmol) of iron powder in 37 mL of MeOH and 3.1 mL of HOAc

was mechanically stirred and refluxed for 2.5 h. The hot

reaction mixture was filtered, and the solvents were evapo-

rated in vacuo. The residue was partitioned between saturated

NaHCO₃and EtOAc. The organic layer was separated and

dried to a solid. After it was washed with ether, 2.3 g (80%) of

the product (40) as a light-gray solid was obtained: mp ) 121-

123 °C; HRMS (ESI) m/z 319.012 51 (M + H)⁺¹, ∆ ) -0.02

1

mmu; H NMR (DMSO-d₆) δ 8.05 (s, 1H), 7.53-7.48 (m, 3H),

7.40-7.27 (m, 3H), 6.85 (d, 1H, J ) 1.9 Hz), 6.62 (dd, 1H, J )

8.5, 1.9 Hz), 6.11 (bs, 2H). Anal. (C₁₅H₁₁ClN₂S₂) C, H, N, S,

Cl.

Experimental Section

2-(2-Chloro-4-nitrophenoxymethyl)-1-methyl-1H-im-

idazole. The title compound was prepared from 4.60 g (0.0410

mol) of 1-methyl-1H-imidazol-2-yl)methanol,³¹7.92 g (0.0454

mol) of 3-chloro-4-fluoronitrobenzene, and 1.64 g (0.0410 mol)

of 60% sodium hydride in oil using the method described above

for 39 to yield 7.98 g (72%) of the title compound: mp ) 182-

Biology. The preparation of the enzymes, the details of our

kinase autophosphorylation assays, the cell proliferation as-

says, and the in vivo studies have been described in detail in

our earlier publications.^24,29

HER-2 Phosphorylation in Xenografts. Athymic female

nude mice (Charles River Laboratories, Wilmington, MA; five

animals/group) were implanted subcutaneously with 2 × 10⁶

3T3/neu cells or a single BT474 tumor fragment (∼30 mm³).

When tumors reached 200-300 mg in size, animals were given

a single oral dose (40 mg/kg) of 25o or 7f. Tumors from control

and treated animals were excised at various times up to 48 h

and minced. Tumor fragments were suspended in 10 mM Tris,

pH 7.5, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 1%

sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 10 µg/mL

pepstatin, 10 µg/mL leupeptin, 10 µg/mL aprotinin, 2 mM

sodium vanadate, and 100 mM sodium fluoride (all chemical

reagents were obtained from Sigma-Aldrich, St Louis, MO) and

lysed by homogenization on ice with a polytron. After clarifica-

tion by centrifugation, protein concentration in lysates was

184 °C; MS (ES+) m/z 268.0 (M + H)⁺¹

.

2-(2-Chloro-4-nitrophenylsulfanyl)thiazole. The title

compound was prepared from 10.01 g (85.45 mmol) of thiazole-

2-thiol and 15 g (85.45 mmol) of 2-chloro-1-fluoro-4-nitroben-

zene using the method described above for 39 to yield 22 g

(95%) of the title compound as a yellow solid: MS (ESI) m/z

273.0 (M + H)⁺¹

.

2-(2-Chloro-4-nitrophenylsulfanyl)-4-phenylthiazole.

To a mixture of 5.8 g (0.03 mol) of 2-mercapto-4-phenylthiazole

and 1.78 g (0.033 mol) of NaOMe in 50 mL of DMF was added

5.79 g (0.033 mol) of 3-chloro-4-fluoronitrobenzene. The reac-

tion mixture was stirred at room temperature for about 5 h.

Then it was poured into about 100 mL of ice-water. The

resulting solid was filtered and recrystallized from 95% EtOH

to give 7.64 g (73%) of the title compound as a light-yellow

solid: mp ) 120-124 °C; MS (ESI) m/z 349.3 (M + H)⁺.

1-(4-Amino-2-chlorobenzyl)-1H-imidazole (42). A solu-

tion of 10 g (39.9 mmol) of 2-chloro-4-nitrobenzyl bromide³²

and 5.4 g (79.8 mmol) of imidazole in 125 mL of THF was

refluxed for 45 min. The solvent was removed, and the residue

was dissolved in EtOAc. The solution was washed with H₂O

and dried with MgSO₄. The solvent was removed, and the

residue was extracted with ether. The ether solution was

diluted with 2 volumes of hexane. The resulting precipitate

was collected, giving 4.3 g of 1-(2-chloro-4-nitrobenzyl)-1H-

imidazole (41) as a white solid. This material was mechanically

stirred at reflux in 180 mL of MeOH and 52 mL of H₂O

containing 8.1 g of NH₄Cl and 6.6 g of iron powder for 2 h.

The mixture was filtered, and solvent was removed. The

residue was dissolved in EtOAc and washed with saturated

NaHCO₃solution. The solution was treated with activated

charcoal and dried with MgSO₄. Solvent was removed, giving

3.9 g of the product 42 as an oil that was used without

additional purification.

2-(Benzyloxy)-1,3-dichloro-5-nitrobenzene. A solution

of 2.74 g (13.17 mmol) of 2,6-dichloro-4-nitrobenzene, 2.35 mL

(3.38 g, 19.76 mmol) of benzyl bromide, and 10.73 g (32.92

mmol) of cesium carbonate in 67 mL of DMF was stirred at

room temperature overnight. After the solvent was removed

by rotary evaporation, the residue was partitioned between

EtOAc and water. The organic layer was dried (MgSO₄) and

evaporated to give a brown semisolid, which was recrystallized

from EtOAc to give 1.088 g (28%) of the starting material as

bright-yellow crystals: mp ) 164-166 °C. The mother liquor

was evaporated, and the residue was recrystallized from ether

to give 2.06 g (52%) of the title compound as beige crystals:

mp ) 78-80 °C; HRMS (ESI+) m/z 298.0026, 4 ) 1.1 mmu.

estimated using the BioRad

protein assay (BioRad, Her-

DC

cules, CA). An amount of 60 µg of lysate pooled from each

group was analyzed by SDS-PAGE and immunoblotting with

phosphotyrosine-specific antibodies (BD Biosciences Phar-

Mingen, San Diego, CA). HER-2 specific antibodies were used

as controls for protein loading. Blots were quantified by

scanning densitometry (FluorS MultiImage analyzer, BioRad).

Extracts from individual tumors were analyzed to determine

variability between animals.

Chemistry. ¹H NMR spectra were determined with a NT-

300 WB spectrometer at 300 MHz. Chemical shifts (δ) are

expressed in parts per million relative to the internal standard

tetramethylsilane. Electrospray mass spectra were recorded

in positive mode on a Micromass Platform spectrometer.

Electron impact and high-resolution mass spectra were ob-

tained on a Finnigan MAT-90 spectrometer. Some high-

resolution electrospray mass spectra with higher precision

were obtained on a Brucker 9.4T FTMS spectrometer. Chro-

matographic purifications were by flash chromatography using

Baker 40 µm silica gel. Melting points were determined in open

capillary tubes on a Meltemp melting point apparatus and are

uncorrected.

Preparation of Substituted Anilines. 2-(2-Chloro-4-

nitrophenylsulfanyl)-5-phenyl-1,3-thiazole (39). A solu-

tion of 2.2 g (11.4 mmol) of 2-mercapto-5-phenyl-1,3-thiazole³⁰

in 35 mL of DMF was added dropwise to a stirring mixture of

546 mg (13.6 mmol) of 60% sodium hydride in oil suspended

in 7 mL of DMF, under nitrogen. After 30 min, a solution of 2

g (11.4 mmol) of 3-chloro-4-fluorobenzene in 14 mL of DMF

was added dropwise. This was stirred for 16 h at room

temperature. The DMF was evaporated, and the brown residue

was stirred with 100 mL of hexane and filtered. The residue

was washed with three portions of water and ether and

1122 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4

Tsou et al.

4-Benzyloxy-3,5-dichlorophenylamine (44). A mixture

of 1.77 g (5.94 mmol) of 2-(benzyloxy)-1,3-dichloro-5-nitroben-

zene, 2.33 g of iron powder, 2.2 mL of HOAc, and 92 mL of

MeOH was refluxed for 3 h. After filtration, the filtrate was

evaporated to a residue that was partitioned between EtOAc

and saturated KHCO₃solution. The organic layer was evapo-

rated to give a light-brown oil, which was triturated with

ether-hexane (1:1) to give 1.41 g (88%) of the titile compound

as a beige solid: mp ) 75-77 °C; HRMS (ESI+) m/z 268.628 45,

4 ) -0.62 mmu.

H₂O. The organic layer was washed successively with aqueous

NaHSO₃, H₂O, aqueous NaHCO₃, and H₂O, dried, and con-

centrated to give an amber liquid: ¹H NMR (CDCl₃) δ 6.90

(dt, 1H, J ) 8.3, 14.8 Hz), 5.86 (d, 1H, J ) 14.8 Hz), 3.91 (d,

2H, J ) 8.3 Hz), 1.48 (s, 9H).

(E)-4-(Pyrrolidin-1-yl)but-2-enoic Acid tert-Butyl Ester

(62). To a stirred solution of 2.08 g (7.75 mmol) of (E)-4-

iodobut-2-enoic acid tert-butyl ester (61) in 15.5 mL of THF at

0 °C was added 1.29 mL (15.5 mmol) of pyrrolidine. The

solution was warmed to 25 °C, stirred 15 min, and partitioned

between toluene and aqueous KHCO₃. The organic layer was

washed with water, dried, and concentrated to give 1.50 g

2-(4-Amino-2-chlorophenyl)ethanethioamide (49). A

solution of 15 g (76.3 mmol) of (2-chloro-4-nitrophenyl)-

acetonitrile (48)¹⁹in 100 mL of DMF was stirred at -78 °C as

H₂S was introduced to saturation. To the solution was added

7.7 g (76.3 mmol) of Et₃N. The cooled mixture was placed in a

steel bomb that was maintained at 80 °C for 16 h. The bomb

was cooled and vented. The solution was treated with activated

charcoal and filtered. The residue was diluted with ice/water,

and the solid was collected and washed with H₂O and hexane.

The solid was air-dried and dissolved in 150 mL of MeOH.

Elemental sulfur was removed by filtration. The solvent was

removed, and the product was recrystallized from EtOAc-

hexane to give 8.5 g (56%) of the title compound as tan

(92%) of amber oil: MS (ES+) m/z 212.0 (M + H)⁺¹

.

(E)-4-(Pyrrolidin-1-yl)but-2-enoic Acid Hydrochloride

(63). A solution of 1.49 g (7.05 mmol) of (E)-4-(pyrrolidin-1-

yl)but-2-enoic acid tert-butyl ester (62) in 14.1 mL of 1 N HCl

was refluxed for 15 min, diluted with toluene, and concentrated

1

to give an amber solid: MS (ES+) m/z 155.8 (M + H)⁺¹; H

NMR (DMSO-d₆) δ 12.7 (bs, 1H), 11.4 (bs, 1H), 6.83 (dt, 1H, J

) 6.8, 14.4 Hz), 6.18 (d, 1H, J ) 14.4 Hz), 3.95 (d, 2H, J ) 6.8

Hz), 3.05 (m, 2H), 1.92 (m, 6H).

4-(1H-Imidazol-4-yl)-but-2-enoic Acid Hydrochloride

(67). 1H-Imidazole-4-acetic acid, 1-(triphenylmethyl) methyl

ester (65)²¹was converted to the title compound using the

method reported by Takacs.²²A solution of 6.45 g (28.8 mmol)

of triethyl phosphonoacetate in 100 mL of THF was cooled to

-78 °C with stirring under an inert atmosphere. A solution

of 12 mL of n-BuLi in hexanes (2.5 M, 30 mmol) was added

dropwise. After 10 min, a solution of 1H-imidazole-4-acetic

acid, 1-(triphenylmethyl) methyl ester in 60 mL of THF was

added. After the mixture was stirred for 20 min, a solution of

27.5 mL of DIBAL in CH₂Cl₂(1 M, 27.5 mmol) was added.

The mixture was stirred for 1.5 h and then allowed to slowly

warm to room temperature over 2 h. Solid Na₂SO₄(H₂O)₁₀was

added followed by 3 mL of H₂O. The mixture was filtered, and

the solvent was removed. The residue was dissolved in ethyl

acetate, washed with saturated Na₂CO₃, and dried (MgSO₄).

The product, which is a mixture of cis and trans isomers, was

treated with NaOCH₃in CH₃OH to convert most of the

material to the trans isomer, which was purified by silica gel

chromatography giving 5.9 g of intermediate (66). A 4.77 g

portion of this ester (66) was hydrolyzed by dissolving it in 48

mL of CH₃OH containing 9 mL of 5 M NaOH. After being

stirred for 2 h, the mixture was neutralized to pH 4-5 and

extracted with ethyl acetate. The solution was dried (MgSO₄),

and the solvent was removed. The trityl group was removed

by refluxing this material in a mixture of 40 mL of H₂O, 40

mL of EtOH, and 5 mL of concentrated HCl for 1.5 h. The

mixture was cooled, and solid trityl alcohol was filtered off.

Solvent was removed from the filtrate and the remaining H₂O

was coevaporated with toluene several times, resulting in 1.3

g of crude product HCl salt (67), which was used in the next

step without additional purification.

3-(2-Dimethylaminophenyl)acrylic Acid (70). A solution

of 18 g (120.7 mmol) of 2-(dimethylamino)benzaldehyde (69),²³

12.6 g (120.7 mmol) of malonic acid, and 6 drops of piperidine

in 107 mL of pyridine was heated to 110 °C for 3 h. The

mixture was diluted with 600 mL of H₂O, and 30 mL of 5 N

NaOH was added. The solution was washed twice with ether

(washing discarded). The solution was acidified to pH 5-6,

saturated with NaCl, and extracted with ethyl acetate. The

extract was dried (MgSO₄), and the solvents were removed.

The solid was dissolved in ether, activated charcoal was added,

and the mixture was filtered. The solution was concentrated,

and 5.8 g (25%) of solid 70 was obtained by filtration. MS

(ES+) m/z 192.1 (M + H)⁺¹. Anal. (C₁₁H₁₃NO₂) C, H, N.

crystals: MS (ESI+) m/z 201.0 (M + H)⁺¹

.

3-Chloro-4-[(4-phenyl-1,3-thiazol-2-yl)methyl]aniline

(51). A solution of 4.56 g (22.92 mmol) of phenacyl bromide

(50) and 4.6 g (22.92 mmol) of 2-(4-amino-2-chlorophenyl)-

ethanethioamide (49) in 47 mL of EtOH was refluxed for 3 h.

The mixture was poured into saturated NaHCO₃and extracted

with ether. The solution was dried (MgSO₄). Solvent was

removed and the residue was chromatographed on silica gel,

eluting with CH₂Cl₂-ether mixtures to yield 8.3 g (95%) of

the HBr salt of the titile compound as a light-tan solid: MS

(ES+) m/z 301.1 (M + H)⁺¹

.

1-Benzyl-1H-indazol-5-amine (55). A mixture of 5 g

(30.65 mmol) of 5-nitroindazole (53), 5.76 g (33.68 mmol) of

benzyl bromide, and 4.66 g (33.72 mmol) of K₂CO₃in 50 mL

of DMF was heated at 75 °C for 4 h. After cooling, the reaction

mixture was poured into 50 mL of H₂O. The solid was collected

and washed with H₂O to give a mixture of two isomers: 1-N-

benzyl-5-nitro-1H-indazole (major) and 2-N-benzyl-5-nitro-1H-

indazole (minor). The crude mixture was dissolved in 35 mL

of acetone. After filtration, 10.8 mL of H₂O was added slowly

to the filtrate with stirring. After 1 h, 2.9 g (37%) 1-N-benzyl-

5-nitro-1H-indazole was collected as yellow crystals. A mixture

of 1-N-benzyl-5-nitro-1H-indazole (1.4 g, 5.53 mmol) and iron

powder (2.25 g) in 84 mL of MeOH and 33 mL of HOAc was

refluxed for 2 h. The hot reaction mixture was filtered, and

the filtrate was concentrated. The residue was partitioned

between EtOAc and NaHCO₃solution. The organic layer was

concentrated to give 1.3 g (100%) of the title compound as

orange crystals (55): HRMS (EI) m/z 223.1094, 4 ) 1.6 mmu.

1-Benzyl-2,3-dihydro-1H-indol-5-ylamine (57). A solu-

tion of 3.11 g (0.0184 mmol) of 5-nitroindoline (56) and 2.48

mL (3.56 g, 0.0208 mol) of benzyl bromide in 35 mL of CH₃CN

was refluxed for 2 h. The solvent was removed in vacuo, and

the solid was stirred with saturated aqueous NaHCO₃. The

resulting solid was chromatographed on silica gel and eluted

with 1:9 EtOAc-hexanes to yield 2.83 g (58%) of 1-benzyl-5-

nitro-2,3-dihydro-1H-indole: mp ) 87-88 °C; MS (ES+) m/z

255.0 (M + H)⁺¹. A mixture of 2.54 g (0.01 mol) of 1-benzyl-

5-nitro-2,3-dihydro-1H-indole, 2.23 g (0.04 mol) of iron powder,

and 2.14 g (0.04 mol) of NH₄Cl in 65 mL of EtOH and 10 mL

of H₂O was vigorously stirred and refluxed for 3 h. The reaction

mixture was filtered through Celite; then the solvents were

removed from the filtrate in vacuo. The residue was extracted

with CH₂Cl₂, and these extracts were passed through Mag-

nesol. The CH₂Cl₂was removed in vacuo to give 2.04 g (91%)

of 57 as an oil.

Preparation of Representative Intermediates. 4-(4-

Benzyloxy-3-chlorophenylamino)-7-ethoxy-6-nitroquino-

line-3-carbonitrile Hydrochloride (3g). A suspension of

2.60 g (11.15 mmol) of 4-benzyloxy-3-chloroaniline, 3.1 g (11.15

mmol) of 4-chloro-7-methoxy-6-nitroquinoline-3-carbonitrile,⁹

and 0.3 g of pyridine hydrochloride in 180 mL of i-PrOH under

nitrogen was refluxed with stirring for 8 h. When the mixture

was coooled, a yellow solid was obtained, which was washed

Preparation of the Michael Acceptor Groups. (E)-4-

Iodobut-2-enoic Acid tert-Butyl Ester (61). A mixture of

4.42 g (20 mmol) of (E)-tert-butyl 4-bromobut-2-enoate (60),

6.0 g (40 mmol) of NaI, and 40 mL of acetone was refluxed for

1 h. The cooled mixture was partitioned between CH₂Cl₂and

Carbonitriles as Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1123

with i-PrOH and ether to give 4.9 g (85%) of yellow solid: mp

) 251-255 °C; HRMS (ESI) m/z 475.113 95 (M + H)⁺¹, ∆ )

-2.81 mmu; ¹H NMR (DMSO-d₆) δ 10.98 (bs, 1H), 9.33 (s, 1H),

8.88 (s, 1H), 7.67 (s, 1H), 7.58 (d, 1H, J ) 2.0 Hz), 7.51-7.35

(m, 7H), 5.27 (s, 2H), 4.36 (q, 2H, J ) 6.8 Hz), 1.43 (t, 3H, J )

6.8 Hz). Anal. (C₂₅H₁₉N₄O₄Cl‚0.5HCl) H, N. C: calcd, 60.89;

found, 59.38.

using the method described above for 7h: MS (ES+) m/z 551.1

(M + H)⁺¹, 276.2 (M + 2H)⁺²; ¹H NMR (DMSO-d₆) δδ 9.97 (s,

1H), 9.83 (s, 1H), 9.04 (s, 1H), 8.76 (s, 1H), 7.71 (m, 3H), 7.53

(s, 1H), 7.40 (s, 1H), 7.16 (d, 1H, J ) 8.4 Hz), 6.76 (m, 2H),

4.07 (s, 3H), 3.49 (m, 2H), 2.47 (s, 6H). Anal. (C₂₆H₂₃ClN₆O₂S₂‚

1.4H₂O) C, H, N.

(E)-N-{4-[3-Chloro-4-(3-fluorobenzyloxy)-phenylamino]-

3-cyano-7-ethoxy-6-quinolinyl}-(dimethylamino)-2-buten-

amide (7f). The title compound was prepared using the

method described above for 7h to yield the product (29%) as a

yellow glass after silica gel chromatography: HRMS (ESI) m/z

6-Amino-4-(4-benzyloxy-3-chlorophenylamino)-7-ethoxy-

quinoline-3-carbonitrile (4g). A 4.6 g (9 mmol) sample of

3g in 138 mL of methanol was brought to reflux with stirring.

To this was added 3.52 g of iron powder suspended in 55 mL

of acetic acid, and reflux was continued for 2 h. The reaction

mixture was filtered hot and left to crystallize, producing 4.9

g of yellow solid as the acetate salt. This was suspended into

700 mL of ethyl acetate and 300 mL of saturated KHCO₃and

stirred overnight. The aqueous phase was extracted twice with

500 mL portions of ethyl acetate, and the organic phases were

combined, dried, and evaporated to a volume of about 100 mL.

This yielded 2.73 g (68%) of product in three crops as off-white

crystals: mp ) 237-239 °C; HRMS (ESI) m/z 445.141 15 (M

1

574.199 79 (M + H)⁺¹, ∆ ) -1.79 mmu; H NMR (DMSO-d₆)

δ 9.62 (s, 1H), 9.50 (s, 1H), 8.97 (s, 1H), 8.47 (s, 1H), 7.51-

7.43 (m, 1H), 7.39-7.30 (m, 4H), 7.26-7.16 (m, 3H), 6.82-

6.73 (m, 1H), 6.62-6.57 (m, 1H), 5.25 (s, 2H), 4.31 (q, 2H, J )

7.0 Hz), 3.07 (d, 2H, J ) 5.6 Hz), 2.18 (s, 6H), 1.47 (t, 3H, J )

7.0 Hz). Anal. (C₃₁H₂₉ClFN₅O₃‚1H₂O) C, H, N.

(E)-N-{4-[4-(Benzyloxy)-3-chloroanilino]-3-cyano-7-

ethoxy-6-quinolinyl}-4-[methyl(2,3,4,5,6-pentahydroxy-

hexyl)amino]-2-butenamide (7i). The title compound was

prepared using the method described above for 7h. The crude

product was purified by preparative TLC developed with

EtOAc-MeOH-NH₄OH ) 40:4:2. The resulting material was

dissolved in acetone and filtered through Celite to obtained

125 mg (20%) of light-yellow solid: mp ) 72 °C; HRMS (ESI)

m/z 705.256 68 (M + H)⁺¹, ∆ ) -0.28 mmu; ¹H NMR (DMSO-

d₆) δ 9.59 (s, 1H), 9.53 (s, 1H), 8.84 (s, 1H), 8.46 (s, 1H), 7.49

(d, 2H, J ) 6.9 Hz), 7.44-7.35 (m, 7H), 7.27-7.19 (m, 2H),

5.21 (s, 2H), 4.45 (s, 1H), 4.29 (q, 4H, J ) 6.9 Hz), 4.0 (m, 2H),

3.84 (bs, 1H), 3.56 (m, 2H) 3.48 (bs, 1H), 3.25 (d, 2H, J ) 7.4

Hz), 3.11 (m, 1H), 2.77 (m, 2H), 2.34 (s, 1H), 2.13 (s, 3H), 1.46

(t, 3H, J ) 6.8 Hz). Anal. (C₃₆H₄₀ClN₅O₈‚2.5H₂O) C, H, N.

1

+ H)⁺¹, ∆ ) -1.43 mmu; H NMR (DMSO-d₆) δ 9.10 (s, 1H),

8.30 (s, 1H), 7.52-7.48 (m, 2H), 7.46-7.32 (m, 3H), 7.28-7.19

(m, 4H), 5.44 (bs, 2H), 5.19 (s, 2H), 4.23 (q, 2H, J ) 6.9 Hz),

1.45 (t, 3H, J ) 6.9 Hz). Anal. (C₂₅H₂₁N₄O₂Cl) C, H, N.

Synthesis Using Method 1A. (E)-N-{4-[4-(Benzyloxy)-

3-chloroanilino]-3-cyano-7-ethoxy-6-quinolinyl}-4-[(2-hy-

droxyethyl)(methyl)amino]-2-butenamide (7h). To a sus-

pension of 6-amino-4-(4-benzyloxy-3-chlorophenylamino)-7-

methoxyquinoline-3-carbonitrile (1 g, 2.25 mmol) (4) in 12 mL

of THF at 0 °C, was added Hunig’s base (0.78 mL, 4.5 mmol),

followed by dropwise addition of 4-bromocrotonyl chloride (0.33

mL, 2.9 mmol) in 35 min. After the mixture was stirred for 2

h at 0 °C, the solvent was evaporated. The residue was

partitioned between EtOAc and saturated sodium bicarbonate

solution. The organic layer was dried (Na₂SO₄), filtered, and

evaporated. The crude product was purified by column chro-

matography using a gradient of ethyl acetate in methylene

chloride to yield 0.79 g (62% yield) of a yellow solid as a 1:1

mixture of (E)-N-{4-[4-(benzyloxy)-3-chloroanilino]-3-cyano-7-

ethoxy-6-quinolinyl}-4-bromo-2-butenamide and (E)-N-{4-[4-

(benzyloxy)-3-chloroanilino]-3-cyano-7-ethoxy-6-quinolinyl}-4-

chloro-2-butenamide. MS (ES+) m/z 593 (M + H)⁺¹, 547 (M +

(E)-N-{4-[4-(Benzyloxy)-3-chloroanilino]-3-cyano-7-

ethoxy-6-quinolinyl}-4-(1-piperidinyl)-2-butenamide (7m).

The title compound was prepared using the method described

above for 7h except NaI was replaced by 0.153 mL (0.88 mmol)

of Hunig’s base. Chromatography of the crude residue on a

short column, eluting with 10% MeOH/EtOAc, yielded 273 mg

(52%) of yellow glass: mp ) 105-107°C; HRMS (ESI) m/z

1

595.235 15 (M + H)⁺¹, ∆ ) -0.54 mmu; H NMR (DMSO-d₆)

δ 9.61 (s, 1H), 9.51 (s, 1H), 8.94 (s, 1H), 8.47 (s, 1H), 7.49 (d,

2H, J ) 6.9 Hz), 7.44-7.35 (m, 5H), 7.27-7.18 (m, 2H), 6.8-

6.73 (m, 1H), 6.58 (d, 1H, J ) 15.3 Hz), 5.21 (s, 2H), 4.31 (q,

2H, J ) 6.9 Hz), 3.11 (bs, 2H), 2.36 (bs, 4H), 1.51 (d, 4H, J )

5 Hz), 1.46 (t, 3H, J ) 6.9 Hz), 1.40 (bs, 2H). Anal. (C₃₄H₃₄-

ClN₅O₃‚2.3H₂O) C, H, N.

2H)⁺¹

.

A 0.18 g (0.316 mmol) portion of the bromo/chloro mixture

prepared above was dissolved in 1.8 mL of DMF and cooled to

0 °C. Sodium iodide (24 mg, 0.158 mmol) was added, follwed

by methoxyethylmethylamine (0.28 mL, 3.16 mmol). The

reaction mixture was stirred overnight at room temperature.

The reaction solution was evaporated, and the residue was

partitioned between EtOAc and saturated NaHCO₃. The

organic layer was dried (Na₂SO₄), filtered, and evaporated to

an oil. Chromatography of the crude residue on a short column,

eluting with EtOAc, then 5% MeOH/EtOAc, 10% MeOH/

EtOAc, and 15% MeOH/EtOAc yielded 135 mg (71%) of yellow

(E)-N-{4-[4-(Benzyloxy)-3-chloroanilino]-3-cyano-7-

ethoxy-6-quinolinyl}-4-[(2R,6S)-2,6-dimethylpiperidinyl]-

2-butenamide (7p). The title compound was prepared using

the method described above for 7h except NaI was replaced

by Hunig’s base. Chromatography of the crude residue was

done on a short column, eluting with EtOAc, 5% MeOH/

EtOAc,EtOAc/MeOH/NH₄OH ) 40:4:1. The resulting material

was purified in preparative plates, developing in EtOAc/

MeOH/NH₄OH ) 40:4:1 to yield a light-yellow solid (43%

1

solid: mp ) 55-70 °C; H NMR (DMSO-d₆) δ 9.62 (bs, 1H),

9.49 (s, 1H), 8.96 (s, 1H), 8.47 (s, 1H), 7.49 (d, 2H, J ) 6.9

Hz), 7.44-7.32 (m, 5H), 7.27-7.18 (m, 2H), 6.8-6.73 (m, 1H),

6.58 (d, 1H, J ) 15.4 Hz), 5.21 (s, 2H), 4.31 (q, 2H, J ) 6.9

Hz), 3.43 (t, 2H, J ) 5.8 Hz), 3.33 (s, 2H), 3.23 (s, 3H), 3.19 (d,

2H, J ) 5.5 Hz), 2.2 (s, 3H), 1.46 (t, 3H, J ) 6.9 Hz). Anal.

(C₃₃H₃₄ClN₅O₄‚0.3H₂O) C, H, N.

1

yield): mp ) 98-99 °C; H NMR (DMSO-d₆) δ 9.61 (s, 1H),

9.49 (s, 1H), 8.93 (s, 1H), 8.47 (s, 1H), 7.49 (d, 2H, J ) 6.9

Hz), 7.44-7.35 (m, 5H), 7.27-7.18 (m, 2H), 6.98-6.91 (m, 1H),

6.61 (d, 1H, J ) 15.3 Hz), 5.21 (s, 2H), 4.31 (q, 2H, J ) 6.9

Hz), 3.45 (d, 2H, J ) 5.8 Hz), 2.41 (bs, 2H), 1.54 (m, 3H), 1.46

(t, 3H, J ) 6.9 Hz), 1.28-1.14 (m, 3H), 1.05 (d, 6H, J ) 6.1

Hz). Anal. (C₃₆H₃₈ClN₅O₃‚1.05H₂O) C, H, N.

(E)-N-{4-[3-Chloro-4-(4-pyridinyloxy)anilino]-3-cyano-

7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-buten-

amide (7b). The title compound was prepared using the

method described above for 7h: MS (ES+) m/z 543.4 (M +

(E)-N-{4-[4-(Benzyloxy)-3-chloroanilino]-3-cyano-7-

ethoxy-6-quinolinyl}-4-(4-morpholinyl)-2-butenamide (7q).

The title compound was prepared using the method described

above for 7h. Chromatography of the crude residue on a short

column, eluting with EtOAc and then 5% MeOH/EtOAc and

10% MeOH/EtOAc, yielded a yellow solid (75% yield): HRMS

(ESI) m/z 597.2144 (M + H)⁺¹, ∆ ) -0.95 mmu; ¹H NMR

(DMSO-d₆) δ 9.61 (bs, 1H), 9.52 (s, 1H), 8.95 (s, 1H), 8.47 (s,

1H), 7.49 (d, 2H, J ) 6.9 Hz), 7.44-7.32 (m, 5H), 7.24-7.18

(m, 2H), 6.79-6.72 (m, 1H), 6.59 (d, 1H, J ) 15.4 Hz), 5.21 (s,

2H), 4.32 (q, 2H, J ) 6.9 Hz), 3.59 (t, 4H, J ) 4.4 Hz), 3.14 (d,

1

H)⁺¹, 272.2 (M + 2H)⁺²; H NMR (DMSO-d₆) δ 10.08 (s, 1H),

9.77 (s, 1H), 8.95 (s, 1H), 8.76 (s, 1H), 7.66 (m, 2H), 7.51 (m,

2H), 7.40 (d, 1H, J ) 2.1 Hz), 7.22 (dd, 1H, J ) 8.4 Hz, 2.1

Hz), 6.84 (m, 2H), 6.23 (m, 2H), 4.36 (q, 2H, J ) 6.9 Hz), 3.85

(m, 2H), 2.52 (s, 6H), 1.44 (t, 3H, J ) 6.9 Hz). Anal. C₂₉H₂₇-

ClN₆O₃‚4H₂O) C, N. H: calcd, 5.74; found, 5.20.

(E)-N-(4-{[3-Chloro-4-(1,3-thiazol-2-ylsulfanyl)phenyl]-

amino}-3-cyano-7-methoxy-6-quinolinyl)-4-(dimethylami-

no)-2-butenamide (7d). The title compound was prepared

1124 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4

Tsou et al.

2H, J ) 5.5 Hz), 2.38 (s, 4H), 1.46 (t, 3H, J ) 6.9 Hz). Anal.

(C₃₃H₃₂ClN₅O₄‚0.35H₂O) C, H, N.

4.49 (s, 2H), 4.01 (s, 3H), 2.20 (s, 6H), 3.10 (d, 2H, J ) 5.6

Hz). Anal. (C₃₃H₂₉ClN₆O₂S‚1.25 H₂O) C, H, N.

(E)-N-{4-[(1-Benzyl-1H-indol-5-yl)amino]-3-cyano-7-

ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamide

(8g). The titile compound was prepared using the method

described above for 8d. The crude product was chromato-

graphed on silica gel. The column was eluted with 1:49

MeOH-CH₂Cl₂to give 175 mg (23%) of 8g: mp ) 209-212

(E)-N-{4-[4-(Benzyloxy)-3-chloroanilino]-3-cyano-7-

ethoxy-6-quinolinyl}-4-(1,4,7-trioxa-10-azacyclododecan-

10-yl)-2-butenamide (7v). The title compound was prepared

using the method described above for 7h except NaI was

replaced by Hunig’s base. Chromatography of the crude residue

on a short column, eluting with 5% MeOH/EtOAc, 10% MeOH/

EtOAc, EtOAc/MeOH/NH₄OH ) 40:4:1, yielded a yellow glass

°C; MS (ES+) m/z 545.0 (M + H)⁺¹, 272.9, 273.2 (M + 2H)⁺²

;

¹H NMR (DMSO-d₆) δ 9.65 (s, 1H), 9.50 (s, 1H), 8.97 (s, 1H),

8.35 (s, 1H) 7,49 (m, 3H), 7.28 (m, 4H), 7.18 (m, 2H), 7.04 (dd,

1H, J ) 1.83 Hz, J ) 8.67 Hz), 6.77 (dt, 1H, J ) 5.91 Hz, J )

15.36 Hz), 6.54 (m, 2H), 5.45 (s, 2H), 4.29 (q, 2H, J ) 6.9),

3.08 (d, 2H, J ) 5.5 Hz), 2.18 (s, 6H), 1.46 (t, 3H, J ) 6.9 Hz).

Anal. (C₃₃H₃₂N₆O₂‚0.5H₂O) C, H, N.

1

(82% yield): mp ) 82-83 °C; H NMR (DMSO-d₆) δ 9.62 (s,

1H), 9.38 (s, 1H), 8.98 (s, 1H), 8.46 (s, 1H), 7.49 (d, 2H, J )

6.9 Hz), 7.44-7.32 (m, 5H), 7.27-7.18 (m, 2H), 6.82-6.75 (m,

1H), 6.66 (d, 1H, J ) 15.3 Hz), 5.21 (s, 2H), 4.31 (q, 2H, J )

6.9 Hz), 3.56 (t, 12H, J ) 4.2 Hz), 3.28 (d, 2H, J ) 5.3 Hz),

2.65 (t, 4H, J ) 4.5 Hz), 1.46 (t, 3H, J ) 6.9 Hz). Anal. (C₃₇H₄₀-

ClN₅O₆‚0.5H₂O) C, H, N.

(E)-N-{4-[(1-Benzyl-1H-indazol-5-yl)amino]-3-cyano-7-

methoxy-6-quinolinyl}-4-(dimethylamino)-2-buten-

amide (8h). The title compound was prepared using the

method described above for 8d: MS (ES+) m/z 532.1 (M +

Synthesis Using Method 1B. (E)-N-{4-[3-Chloro-4-(5-

phenylthiazol-2-ylsulfanyl)phenylamino]-3-cyano-7-meth-

oxy-6-quinolinyl}-4-(dimethylamino)-2-butenamide (8d).

A 0.044 mL (0.505 mmol) portion of oxalyl chloride was added

to 93 mg (0.562 mmol) of 4-(dimethylamino)-1-ylbut-2-enoic

acid hydrochloride in 1.2 mL of CH₃CN. This was followed by

the addition of a trace amount of DMF. This solution was

heated in an oil bath at 60 °C for 30 min after gas evolution

began. Then the orange reaction solution was concentrated to

about ¹/₄of its volume in vacuo without the application of heat.

This solution was then cooled in an ice bath, and 145 mg (0.281

mmol) of 6-amino-4-[3-chloro-4-(5-phenylthiazol-2-ylsulfanyl)-

phenylamino]-7-methoxyquinoline-3-carbonitrile in 1.2 mL of

N-methylpyrrolidinone was added dropwise. Cooling and stir-

ring were continued for 2 h, then the reaction mixture was

poured into saturated aqueous NaHCO₃solution. The resulting

brown precipitate was collected and chromatographed on silica

gel. The column was eluted with a gradient of EtOAc to 1:9:

0.2 MeOH-CH₂Cl₂-Et₃N. The product was washed with ether

to give 88 mg (50%) of 8d: HRMS (ESI) m/z 627.138 10 (M +

1

H)⁺¹; H NMR (DMSO-d₆) δ 9.75 (s, 1H), 9.70 (s, 1H), 9.5 (s,

1H), 8.45 (s, 1H), 8.1 (s, 1H), 7.7 (m, 2H), 7.4 (s, 1H), 7.3 (m,

4H), 7.2 (m, 2H), 6.6 (m, 2H), 5.7 (s, 2H), 4.02 (s, 3H), 3.08 (d,

2H, J ) 5.0 Hz), 2.18 (s, 6H), 2.2 (s, 6H). Anal. (C₃₁H₂₉N₇O₂‚

1.5H₂O) C, H, N.

(E)-N-[4-(4-Benzyloxy)anilino)-3-cyano-7-ethoxy-6-quin-

olinyl]-4-(dimethylamino)-2-butenamide (8i). The title

compound was prepared using the method described above for

8d. The crude product was recrystallized from EtOAc-1%

MeOH to produce 587 mg (54%) of beige crystals: mp ) 131-

1

133 °C; HRMS (EI) m/z 541.1891, ∆ ) -1.0 mmu; H NMR

(DMSO-d₆) δ 9.64 (bs, 2H), 8.97 (s, 1H), 8.45 (s, 1H), 7.51-

7.44 (m, 2H), 7.42-7.32 (m, 5H), 7.26-7.16 (m, 2H), 6.8-6.56

(m, 2H), 5.21 (s, 2H), 4.02 (s, 3H), 3.06 (d, 2H, J ) 5.4 Hz),

2.17 (s, 6H). Anal. (C₃₀H₂₈ClN₅O₃‚3H₂O) C, H, N.

(E)-N-{4-[4-(Benzyloxy)anilino]-3-cyano-7-methoxy-6-

quinolinyl}-4-(dimethylamino)-2-butenamide (8j). The

title compound was prepared using the method described above

for 8d. Chromatography of the crude residue on a column,

eluting with 10% MeOH-EtOAc, 15% MeOH-EtOAc, and

EtOAc-MeOH-Et₃N ) 40:4:1 yielded a yellow solid (38%

yield): mp ) 193-195 °C; ¹H NMR (DMSO-d₆) δ 9.64 (bs, 1H),

9.56 (s, 1H), 8.95 (s, 1H), 8.41 (s, 1H), 7.49 (d, 2H, J ) 6.8

Hz), 7.43-7.33 (m, 4H), 7.21 (d, 2H, J ) 6.9 Hz), 7.04 (dd, 2H,

J ) 2.1 Hz, J ) 6.8 Hz), 6.8-6.71 (m, 1H), 6.57 (d, 1H, J )

15.4 Hz), 5.11 (s, 2H), 4.01 (s, 3H), 3.04 (d, 2H, J ) 7.4 Hz),

2.17 (s, 6 H); HRMS (ESI) m/z 508.234 32 (M + H)⁺¹, ∆ )

-0.33 mmu. Anal. (C₃₀H₂₉N₅O₃‚1.5H₂O) C, H, N.

(E)-N-{4-[3-Chloro-4-(3-chlorobenzyloxy)phenylamino]-

3-cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-bu-

tenamide (8l). The title compound was prepared using the

method described above for 8d. The crude product was

chromatographed to yield a yellow glassy solid: HRMS (ESI)

m/z 590.169 20 (M + H)⁺¹, ∆ ) -2.83 mmu; ¹H NMR (DMSO-

d₆) δ 9.62 (s, 1H), 9.50 (s, 1H), 8.97 (s, 1H), 8.47 (s, 1H), 7.56

(s, 1H), 7.47-7.39 (m, 5H), 7.26-7.17 (m, 2H), 6.82-6.57 (m,

2H), 5.24 (s, 2H), 4.31 (q, 2H, J ) 5.8 Hz), 3.08 (d, 2H, J ) 3.6

Hz), 2.18 (s, 6H), 1.47 (t, 3H, J ) 7.0 Hz). Anal. (C₃₁H₂₉Cl₂N₅O₃‚

2H₂O‚0.1EtOAc) C, H, N.

(E)-N-[4-(4-{[4-(Benzyloxy)benzyl]oxy}-3-chloroanilino)-

3-cyano-7-ethoxy-6-quinolinyl]-4-(dimethylamino)-2-bu-

tenamide (8n). This compound was prepared as a light-yellow

solid (61%) using the method described for 8d: MS (ES+) m/z

661.8 (M + H)⁺¹, 331.5 (M + 2H)⁺²; ¹H NMR (DMSO-d₆) δ 9.60

(s,1H), 9.51 (s,1H), 8.96 (s,1H), 8.47 (s,1H), 7.48-7.33 (m, 9H),

7.27-7.18 (m, 2H), 7.05 (d, 2H, J ) 8.7 Hz), 6.82-6.73 (m,

1H), 6.59 (d, 1H, J ) 14.7 Hz), 5.12 (s, 4H), 4.30 (q, 2H, J )

6.9 Hz), 3.07 (d, 2H, J ) 6 Hz), 2.18 (s, 6H), 1.46 (t, 3H, J )

6 Hz). Anal. (C₃₈H₃₆ClN₅O₄‚H₂O) C, H, N.

(2E)-N-{4-[3-Chloro-4-(2-thienylmethoxy)anilino]-3-cy-

ano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-buten-

amide (8o). This compound was prepared using the method

described for 8d. Chromatography of the crude material on a

silica gel pack, eluting with 5% MeOH-CHCl₃, yielded a tan

foam (51% yield): HRMS (ESI) m/z 561.160 24 (M + H)⁺¹, ∆

1

H)⁺¹, ∆ ) -2.74 mmu; H NMR (DMSO-d₆) δ 9.99 (bs, 1H),

9.72 (s, 1H), 9.09 (s, 1H), 8.75 (s, 1H), 8.15 (s, 1H), 7.78 (d,

1H, J ) 8.5 Hz), 7.57-7.52 (m, 3H), 7.44-7.30 (m, 4H), 7.20

(dd, 1H, J ) 8.5 Hz, J ) 2.3 Hz), 6.83-6.62 (m, 2H), 4.07 (s,

3H), 3.07 (d, 2H, J ) 5.5 Hz), 2.19 (s, 6H). Anal. (C₃₂H₂₇-

ClN₆O₂S₂‚3.2H₂O‚0.5EtOAc) C, H, N.

(E)-N-[4-(4-Benzyloxy-3-chlorophenylamino)-3-cyano-

7-ethoxy-6-quinolinyl]-4-(dimethylamino)-2-buten-

amide (8b). The title compound was prepared using the

method described above for 8d: pale-yellow crystals (62%

yield); mp ) 108-112 °C; HRMS (ESI) m/z 1111.414 43 (2M

1

+ H)⁺¹, ∆ ) -0.29 mmu; H NMR (DMSO-d₆) δ 9.61 (s, 1H),

9.49 (s, 1H), 8.97 (s, 1H), 8.47 (s, 1H), 7.53-7.48 (m, 2H), 7.45-

7.35 (m, 5H), 7.27-7.18 (m, 2H), 6.81-6.57 (m, 2H), 5.22 (s,

2H), 4.31 (q, 2H, J ) 5.2 Hz), 3.07 (d, 2H, J ) 3.7 Hz), 2.18 (s,

6H), 1.47 (t, 3H, J ) 5.2 Hz). Anal. (C₃₁H₃₀ClN₅O₃‚1.25H₂O)

C, H, N.

(E)-N-(4-{3-Chloro-4-[(4-phenyl-1,3-thiazol-2-yl)sulfanyl]-

anilino}-3-cyano-7-ethoxy-6-quinolinyl)-4-(dimethylami-

no)-2-butenamide (8e). The title compound was prepared

using the method described above for 8d: 55% yield: MS

(ES+) m/z 641.3 (M + H)⁺¹, 321.2 (M + 2H)^{+2 1}H NMR

;

(DMSO-d₆) δ 9.98 (s, 1H), 9.51 (s, 1H), 9.06 (s, 1H), 8.75 (s,

1H), 8.00 (s, 1H), 7.92 (s, 1H), 7.89 (s, 1H), 7.79 (d, 1H), 7.46

(m, 3H), 7.36 (d, 2H, J ) 7.23 Hz), 7.18 (dd, 1H, J ) 8.52 Hz,

J ) 2.31 Hz), 6.80 (dt, 1H, J ) 15.33 Hz, J ) 5.79 Hz), 6.64

(d, 1H, J ) 15.45 Hz), 4.36 (q, 2H, J ) 6.93 Hz), 3.09 (d, 2H,

J ) 5.55 Hz), 2.19 (s, 6H), 1.49 (t, 3H, J ) 6.93 Hz). Anal.

(C₃₃H₂₉ClN₆O₂S₂‚H₂O) C, H, N.

(E)-N-(4-{3-Chloro-4-[(4-phenyl-1,3-thiazol-2-yl)-

methyl]anilino}-3-cyano-7-methoxy-6-quinolinyl)-4-

(dimethylamino)-2-butenamide (8f). The title compound

was prepared using the method described above for 8d: MS

(ES+) m/z 609.2 (M + H)⁺¹, 305.2 (M + 2H)^{+2 1}H NMR

;

(DMSO-d₆) δ 9.75 (s, 1H), 9.69 (s, 1H), 9.03 (s, 1H), 8.63 (s,

1H), 7.94 (m, 3H), 7.49 (m, 4H), 7.35 (m, 2H), 7.19 (dd, 1H, J

) 2.2 Hz, J ) 8.3 Hz), 6.79 (m, 1H), 6.63 (d, 1H, J ) 15.4 Hz),

Carbonitriles as Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1125

1

) -0.54 mmu; H NMR (DMSO-d₆) δ 9.61 (bs, 1H), 9.49 (s,

9.45 (s, 1H), 8.87 (s, 1H), 8.46 (s, 1H), 7.60 (s, 1H), 7.50-7.32

(m, 8H), 7.23 (m, 2H), 7.04 (s, 1H), 6.48 (d, 1H, J ) 15.8 Hz),

5.21 (s, 2H), 4.27 (q, 2H, J ) 5.7 Hz), 3.36 (s, 2H), 1.39 (t, 3H,

J ) 5.7). Anal. (C₃₂H₂₇ClN₆O₃‚1.4H₂O) C, H. N: calcd, 13.91;

found, 12.78.

1H), 8.97 (s, 1H), 8.47 (s, 1H), 7.58 (dd, 1H, J ) 1.1 Hz, J ) 5

Hz), 7.37 (d, 2H, J ) 10 Hz), 7.29 (d, 1H, J ) 8.8 Hz), 7.24-

7.17 (m, 2H), 7.06-7.04 (m, 1H), 6.81-6.74 (m, 1H), 6.59 (d,

1H, J ) 15.4 Hz), 5.38 (s, 2H), 4.30 (q, 2H, J ) 6.9 Hz), 3.07

(d, 2H, J ) 5.1 Hz), 2.17 (s, 6H), 1.47 (t, 3H, J ) 6.9 Hz). Anal.

(C₂₉H₂₈ClN₅O₃S) C, H, N.

(E)-N-{4-[4-(Benzyloxy)-3-chloroanilino]-3-cyano-7-

ethoxy-6-quinolinyl}-3-(8-quinolinyl)-2-propenamide (75).

The acid chloride hydrochloride salt of 3-quinolin-8-ylacrylic

acid³³was prepared by refluxing 1.61 g (8.09 mmol) of the acid

in 20.6 mL of SOCl₂. After removal of the excess reagent, the

resulting solid was washed several times with dry ether. This

material was then used to prepare the title compound by the

method described above for 8d: MS (ES+) m/z 627.40 (M +

(E)-N-(4-{3-Chloro-4-[(1R)-2,3-dihydro-1H-inden-1-yloxy]-

anilino}-3-cyano-7-ethoxy-6-quinolinyl)-4-(dimethylami-

no)-2-butenamide (8p). This compound was prepared using

the method described for 8d: MS (ES+) m/z 581.9 (M + H)⁺¹

,

291.5 (M + 2H)⁺²; ¹H NMR (DMSO-d₆) δ 9.64 (s, 1H), 9.50 (s,

1H), 8.99 (s, 1H), 8.49 (s, 1H), 7.43-7.35 (m, 6H), 7.25 (m, 2H),

6.79 (m, 1H), 6.60 (d, 1H, J ) 14.5 Hz), 5.92 (m, 1H), 4.31 (q,

2H, J ) 5.2 Hz), 3.11 (m, 1H), 3.08 (d, 2H, J ) 6 Hz), 2.86 (m,

1H), 2.54 (m, 1H), 2.18 (s, 6H), 2.09 (m, 1H), 1.47 (t, 3H, J )

5.2 Hz). Anal. (C₃₃H₃₂ClN₅O₃‚1.2H₂O) C, H, N.

1

H)⁺¹, 313.7 (M + 2H)⁺²; H NMR (DMSO-d₆) δ 9.67 (s, 1H),

9.66 (s, 1H), 9.09 (s, 1H), 9.00 (m, 1H), 8.49 (s, 1H), 8.44 (dd,

1H, J ) 1.7 Hz, J ) 8.3 Hz), 8.20 (d, 1H, J ) 7.20 Hz), 8.08 (d,

1H, J ) 7.2 Hz), 7.73 (t, 1H, J ) 7.7 Hz), 7.64 (m, 1H), 7.50-

7.22 (m, 11H), 5.23 (s, 2H), 4.36 (q, 2H, J ) 5.7 Hz), 1.51 (t,

3H, J ) 5.7 Hz). Anal. (C₃₇H₂₈ClN₅O₃‚0.66H₂O) C, H, N.

(E)-N-(4-{3-Chloro-4-[(1S)-2,3-dihydro-1H-inden-1-yloxy]-

anilino}-3-cyano-7-ethoxy-6-quinolinyl)-4-(dimethylami-

no)-2-butenamide (8q). This compound was prepared using

(E)-N-{4-[4-(Benzyloxy)-3-chloroanilino]-3-cyano-7-

ethoxy-6-quinolinyl}-3-[2-(dimethylamino)phenyl]-2-pro-

penamide (76). The acid chloride of 3-[2-(dimethylamino)-

phenyl]acrylic acid (70) was prepared using (COCl)₂as described

above and was utilized by the method described above for 8d

the method described for 8d: MS (ES+) m/z 581.9 (M + H)⁺¹

,

291.5 (M + 2H)⁺²; ¹H NMR (DMSO-d₆) δ 9.64 (s, 1H), 9.50 (s,

1H), 8.98 (s, 1H), 8.49 (s, 1H), 7.35 (m, 6H), 7.25 (m, 2H), 6.79

(m, 1H), 6.60 (d, 1H, J ) 14.5 Hz), 5.92 (m, 1H), 4.31 (q, 2H,

J ) 5.2 Hz), 3.11 (m, 1H), 3.08 (d, 2H, J ) 6.9 Hz), 2.86 (m,

1H), 2.54 (m, 1H), 2.18 (s, 6H), 2.09 (m, 1H), 1.47 (t, 3H, J )

5.2 Hz). Anal. (C₃₃H₃₂ClN₅O₃‚1.2H₂O) C, H, N.

to prepare the title compound: MS (ES+) m/z 618.0 (M + H)⁺¹

,

309.6 (M + 2H)⁺²; ¹H NMR (DMSO-d₆) δ 9.64 (s, 1H), 9.53 (s,

1H), 9.12 (s, 1H), 8.48 (s, 1H), 7.93 (d, 1H, J ) 15.8 Hz), 7. 61

(m, 1H), 7.52-7.08 (m, 13H), 5.22 (s, 2H), 4.35 (q, 2H, J ) 6.9

Hz), 2.69 (s, 6H), 1.50 (t, 3H, J ) 6.9 Hz). Anal. (C₃₆H₃₂ClN₅O₃‚

5H₂O) C, H, N.

(E)-N-{4-[3-(Benzyloxy)anilino]-3-cyano-7-ethoxy-6-

quinolinyl}-4-(dimethylamino)-2-butenamide (8r). This

compound was prepared using the method described for 8d:

MS (ES+) m/z 522.1 (M + H)⁺¹, 261.8 (M + 2H)⁺²; H NMR

(E)-N-{4-[4-(Benzyloxy)-3-chloroanilino]-3-cyano-7-

ethoxy-6-quinolinyl}-3-(1H-4-imidazolyl)acrylamide (77).

This was prepared from 0.44 g (1.0 mmol) of 6-amino-4-[4-

(benzyloxy)-3-chlorophenylamino]-7-ethoxyquinoline-3-carbo-

nitrile (4g) and 3-(1H-imidazol-4-yl)acryloyl chloride hydro-

chloride as described above for 8d. The acid chloride was

prepared from 0.235 g (1.7 mmol) of the corresponding acid

and 8.5 mL of SOCl₂at reflux temperature followed by

evaporation. The crude product was triturated with hot MeOH

to give 376 mg (67%) of light-yellow solid: mp ) 196-201 °C

(dec); MS (ES+) m/z 283.1, 284.1 (M + 2H)⁺²; ¹H NMR (DMSO-

d₆) δ 12.3 (bs, 1H), 9.62 (bs, 1H), 9.50 (s, 1H), 9.02 (s, 1H),

8.47 (s, 1H), 7.76 (s, 1H), 7.45-7.30 (m, 10H), 7.23 (m, 1H),

7.08 (d, 1H, J ) 15.1 Hz), 5.22 (s, 2H), 4.31 (q, 2H, J ) 6.8

Hz), 1.49 (t, 3H, J ) 6.8 Hz). Anal. (C₃₁H₂₅ClN₆O₃‚2H₂O‚H₂-

CO₃) C, H, N.

1

(DMSO-d₆) δ 9.60 (s, 1H), 9.80 (s, 1H), 9.00 (s, 1H), 8.45 (s,

1H), 7.41(m, 6H), 7.30 (t, 1H, J ) 4.7 Hz), 6.82 (m, 4H), 6.60

(d, 1H, J ) 15.5 Hz), 5.10 (s, 2H), 4.34 (q, 2H, J ) 6.8 Hz),

2.17 (s, 6H), 3.12 (d, 2H, J ) 6.8 Hz), 1.47 (t, 3H, J ) 6.8 Hz).

Anal. (C₃₁H₃₁N₅O₃‚H₂O) C, H, N.

(E)-N-(4-{4-[Benzyl(methyl)amino]-3-chloroanilino}-3-

cyano-7-ethoxy-6-quinolinyl)-4-(dimethylamino)-2-bu-

tenamide (8s). This compound was prepared using the

method described for 8d: MS (ES+) m/z 569.1 (M + H)⁺¹; ¹H

NMR (DMSO-d₆) δ 11.35-11.19 (m, 2H), 10.0 (s, 1H), 9.17 (s,

1H), 9.05 (s, 1H), 7.67 (s, 1H), 7.60 (s, 1H), 7.35-7.25 (m, 6H),

7.18 (d, 1H, J ) 7.8 Hz), 6.96-6.87 (m, 1H), 6.8 (d, 1H, J )

16.5 Hz), 4.33 (q, 2H, J ) 7.5 Hz), 4.26 (s, 1H), 3.95 (t, 2H, J

) 3.9 Hz), 2.74 (s, 6H), 2.65 (s, 3H), 1.50 (t, 3H, J ) 3.9 Hz).

Anal. (C₃₂H₃₃ClN₆O₂‚3HCl‚1.3H₂O) C, H, N.

Synthesis Using Method 2. 2-Acetamido-5-nitrophenol

(10). To a stirred suspension of 400 g (2.6 mol) of 2-amino-5-

nitrophenol (9) in 1.6 L of HOAc at 60 °C was added 368 mL

(3.9 mol) of Ac₂O over 1.5 h. The reaction mixture was stirred

at 60 °C for 2 h. More Ac₂O was added until less than 1% of

2-amino-5-nitrophenol (9) was present, as determined by

HPLC. An amount of 2 L of H₂O was added to the reaction

mixture after it was cooled to 25 °C. The suspension was

stirred in H₂O for 1 h, filtered, washed with 2 × 640 mL of

H₂O, washed with 2 × 640 mL of heptane, and dried in an

oven to yield 486.7 g (96%) product (10): mp > 205 °C.

4-Acetamido-3-ethoxynitrobenzene (11). To a stirred

suspension of 400 g (2.04 mol) of 2-acetamido-5-nitrophenol

(10) and 790 g (5.72 mol) of K₂CO₃in 2.0 L of DMF at 60-62

°C was added 294 g (2.70 mol) of EtBr over 1 h. The reaction

mixture was stirred at 60 °C for 2 h. More EtBr was added

until less than 1% of 2- acetamido-5-nitrophenol (10) was

present, as determined by TLC. An amount of 4 L of H₂O was

added to the reaction mixture after it was cooled to 25 °C. The

suspension was stirred in H₂O for 30 min, filtered, washed

with 4 × 1 L of warm (50 °C) H₂O (maintain pH < 8) and 1 L

of heptane, and dried at 60-65 °C in an oven to yield 447.8 g

(98%) of product 11: mp ) 164-165 °C.

(E)-N-{4-[4-(Benzyloxy)-3-chloroanilino]-3-cyano-7-

ethoxy-6-quinolinyl}-4-(pyrrolidin-1-yl)-2-butenamide (8t).

To a stirred suspension of 1.20 g (6.26 mmol) of (E)-4-

(pyrrolidin-1-yl)but-2-enoic acid hydrochloride (63) in 14 mL

of MeCN at 25 °C was added 0.01 mL of N-methyl-2-

pyrrolidinone (NMP, catalyst) followed by 0.55 mL (∼6.26

mmol) of oxalyl chloride. The resulting mixture was stirred

at 55 °C for 10 min, concentrated to a volume of ∼5 mL at

<15 °C, and cooled at 0 °C as 7.0 mL of NMP was added. To

this solution was added a solution of 1.56 g (3.5 mmol) of 4g

in 7.0 mL of NMP. The solution was warmed to 25 °C, stirred

for 1 h, and treated with aqueous K₂CO₃. The resulting

precipitate was filtered, washed with H₂O, and dried. The

product was purified by short column chromatography on silica

gel with final elution by 25:25:2:1 CH₂Cl₂-EtOAc-MeOH-

Et₃N to give 1.63 g (80%) of amber glass: MS (ES+) m/z 582.2,

1

584.2 (M + H)⁺¹; H NMR (DMSO-d₆) δ 9.62 (s, 1H), 9.52 (s,

1H), 8.96 (s, 1H), 8.47 (s, 1H), 7.51 (d, 1H, J ) 1.6 Hz), 7.49

(s, 1H), 7.45-7.15(m, 7H), 6.83 (dt, 1H, J ) 5.6, J ) 15.6 Hz),

6.62 (d, 1H, J ) 15.6 Hz), 5.22 (s, 2H), 4.31 (q, 2H, J ) 7.0

Hz), 3.33 (d, 2H, J ) 5.6 Hz), 2.56 (m, 4H), 1.74 (m, 4H), 1.47

(t, 3H, J ) 7.0 Hz). Anal. (C₃₃H₃₂ClN₅O₃‚0.75MeOH) C, H, N.

(E)-N-{4-[4-(Benzyloxy)-3-chloroanilino]-3-cyano-7-

ethoxy-6-quinolinyl}-4-(1H-imidazol-4-yl)-2-buten-

amide (8u). This compound was prepared by the method

described above for 8d using 4-(1H-imidazol-4-yl)but-2-enoic

acid hydrochloride (67): MS (ES+) m/z 579.3 (M + H)⁺¹, 296.2

(M + 2H)⁺²; ¹H NMR (DMSO-d₆) δ 12.06 (bs, 1H), 9.61 (s, 1H),

3-(4-Acetamido-3-ethoxyaniline)-2-cyanopropenoic Acid

Ethyl Ester (14). An amount of 410 g (1.83 mol) of 4-acet-

amido-3-ethoxynitrobenzene (11) in 4.2 L THF was reduced

to 3-(4-acetamido-3-ethoxyaniline)-2-cyanopropenoic acid ethyl

ester under hydrogenation (at 28-30 °C, 50 psi) with 35 g of

1126 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4

Tsou et al.

10% Pd-C for 3 h. The reaction mixture was filtered through

a 0.2 µm cartridge and rinsed with THF. The filtrate was

concentrated to give the aniline, which was stirred in 1.3 L of

toluene containing 50 g of anhydrous Na₂SO₄at 25 °C for 20

min. The suspension was filtered, and the cake was washed

with 200 mL of toluene. To the filtrate containing 12 in 1.5 L

of toluene was added 475 g (2.8 mol) of ethyl (ethoxymethyl-

ene)cyanoacetate 13 and 3.5 L of toluene. The suspension was

stirred and refluxed at 90 °C for 16 h. The reaction mixture

was cooled to room temperature over 1 h and stirred at room

temperature for 30 min. The light-yellow solid was filtered,

washed with toluene (2 × 400 mL), and dried in a vacuum

oven at 60-65 °C for 24 h to give 524 g (90.3%) of 14 as a

mixture of E and Z isomers.

3-Cyano-7-ethoxy-4-hydroxy-6-N-acetylquinoline (15).

An amount of 210 g (0.664 mol) of 14 was stirred in 12 L of

Dowtherm A at 250-255 °C for 20 h. After the mixture was

cooled to room temperature, the solid was filtered and washed

with toluene (4 × 210 mL). The solid crude material was

stirred in THF at reflux temperature (66 °C) for 30 min and

cooled. The dark-brown solid was filtered, washed with THF

(4 × 100 mL), and dried in a 60-65 °C vacuum oven to yield

75.3 g (42%) of 15: mp > 250 °C.

4-Chloro-3-cyano-7-ethoxy-6-N-acetylquinoline (16). To

a dark-brown suspension of 80.0 g (0.296 mol) of 15 in 1.6 L

of diethylene glycol dimethyl ether was added 96.0 mL (1.04

mol) of phosphorus oxychloride in one portion. The reaction

mixture was stirred at 100-102 °C for 45 min. The reaction

mixture was cooled to 80-85 °C, and 25 g of Celite was added.

The reaction mixture was filtered through a Celite pad with 3

× 100 mL and 1 × 50 mL of diethylene glycol dimethyl ether.

The filtrate was concentrated to a volume of 875 mL and

stirred in an aqueous solution of 75.0 g (0.543 mol) of K₂CO₃

(maintaining temperature less than 50 °C). The yellow solid

was filtered, washed with 3 × 100 g of warm H₂O and 200

mL of toluene, then dried at 60-65 °C in an oven to yield 55.45

g (65%) of 16: mp ) 250 °C.

bath at 133 °C for 1 h. When the mixture was cooled, a solid

formed. This was filtered and stirred with 100 mL of saturated

aqueous NaHCO₃. The solid was filtered to give 2.45 g (94%)

of the product (19c): mp ) 263-267 °C; MS (ES+) m/z 641.3

(M + H)⁺¹

.

(E)-N-(4-{3-Chloro-4-[(1-methyl-1H-imidazol-2-yl)-

methoxy]anilino}-3-cyano-7-ethoxy-6-quinolinyl)-4-(di-

methylamino)-2-butenamide (21a). The title compound was

prepared from 1.18 g (7.13 mmol) of 4-(dimethylamino)-1-yl-

but-2-enoic acid, 0.56 mL (820 mg, 6.46 mmol) of oxalyl

chloride, and 1.6 g (3.56 mmol) of 6-amino-4-[3-chloro-4-(1-

methyl-1H-imidazol-2-ylmethoxy)phenylamino]-7-ethoxyquin-

oline-3-carbonitrile (19a) using the method described above

for 8d to yield 1.031 g (52%) of 21a as a yellow solid: mp )

203-206 °C; MS (ES+) m/z 560.1 (M + H)⁺¹, 281.3 (M + H)⁺²

;

¹H NMR (DMSO-d₆) δ 9.61 (s, 1H,), 9.49 (s, 1H), 8.97 (s, 1H),

8.48 (s, 1H), 7.37 (m, 3H), 7.21 (m, 2H), 6.89 (s, 1H), 6.78 (dt,

1H, J ) 15.4 Hz, J ) 5.9 Hz), 6.38 (d, 1H, J ) 15.4 Hz), 5.24

(s, 2H), 4.31 (q, 2H, J ) 6.96 Hz), 3.73 (s, 3H), 3.08 (d, 2H, J

) 5.58 Hz), 2.18 (s, 6H), 1.47 (t, 3H, J ) 6.96 Hz). Anal. (C₂₉H₃₀-

ClN₇O₃‚0.5H₂O) C, H, N.

(2E)-N-{4-[(1-Benzyl-2,3-dihydro-1H-indol-5-yl)amino]-

3-cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-bu-

tenamide (21b). The title compound was prepared from 662

mg (4.25 mmol) of 4-(dimethylamino)-1-yl-but-2-enoic acid,

0.35 mL (508 mg, 4.0 mmol) of oxalyl chloride, and 871 mg

(2.83 mmol) of 6-amino-4-(1-benzyl-2,3-dihydro-1H-indol-5-

ylamino)-7-ethoxyquinoline-3-carbonitrile (19b) using the

method described above for 8d to yield 585 mg (53%) of a tan

solid (21b): mp ) 199-201 °C; MS (ES+) m/z 546.9 (M + H)⁺¹

,

274.0 (M + 2H)⁺²; ¹H NMR (DMSO-d₆) δ 9.49 (s, 1Η), 9.46 (s,

1Η), 9.19 (s, 1H), 9.46 (s, 1H), 8.91 (s, 1H), 8.35 (s, 1H), 7.32

(m, 7H), 6.97 (s, 1H), 6.92 (dd, 1H, J ) 8.28 Hz, J ) 1.88 Hz),

6.77 (dt, 1H, J ) 5.96 Hz, J ) 15.36 Hz), 6.58 (m, 2H), 4.48,

(s, 1H), 4.28 (q, 2H, J ) 7.04 Hz), 3.29, (t, 2H, J ) 6.04 Hz),

2.92 (t, 2H, J ) 8.20 Hz) 2.18 (s, 6H), 1.46 (t, 3H, J ) 6.92

Hz). Anal. (C₃₃H₃₄N₆O₂) C, H, N.

N-{4-[3-Chloro-4-(1-methyl-1H-imidazol-2-ylmethoxy)-

phenylamino]-3-cyano-7-ethoxyquinolin-6-yl}acetamide

Hydrochloride (18a). A mixture of 6.10 g (0.0211 mol) of

N-(4-chloro-3-cyano-7-ethoxyquinolin-6-yl)acetamide (16), 4.55

g (0.0191 mol) of 3-chloro-4-(1-methyl-1H-imidazol-2-ylmeth-

oxy)phenylamine (17a), and 2.21 g (0.0191 mol) of pyridine

hydrochloride in 50 mL of i-PrOH was refluxed for 16 h. The

solid was filtered and washed with H₂O and ether to give 2.86

(E)-N-(3-Cyano-7-ethoxy-4-{[1-(phenylsulfonyl)-1H-in-

dol-5-yl]amino}-6-quinolinyl)-4-(dimethylamino)-2-bu-

tenamide (21c). The title compound was prepared from 662

mg (4 mmol) of 4-(dimethylamino)-1-yl-but-2-enoic acid, 0.31

mL (457 mg, 3.6 mmol) of oxalyl chloride, and 1.971 g (2 mmol)

of 6-amino-4-(1-benzenesulfonyl-1H-indol-5-ylamino)-7-eth-

oxyquinoline-3-carbonitrile (19c) using the method described

above for 8d to yield 517 mg (43%) of a tan solid (21c): mp )

g of 18a: mp ) 251-252 °C; MS (ES+) m/z 491.1 (M + H)⁺¹

.

243-245 °C; MS (ES+) m/z 595.0 (M + H)⁺¹, 298.2 (M + H)⁺²

;

¹H NMR (DMSO-d₆) δ 9.69 (s, 1H), 9.48 (s, 1H), 8.98 (s, 1H),

8.45 (s, 1H), 7.95 (m, 3H), 7.76 (d, 1H, J ) 3.66 Hz), 7.69 (m,

1H), 7.58 (m, 2H), 7.45 (d, 1H, J ) 1.92 Hz), 7.39 (s, 1H), 7.27

(dd, 1H, J ) 8.85 Hz, J ) 2.04 Hz), 6.84 (d, 1H, J ) 3.66 Hz),

6.75 (dt, 1H, J ) 15.39 Hz, J ) 5.88 Hz), 6.29 (m, 1H), 4.31 (q,

2H, J ) 6.84 Hz), 3.07, d, 2H, J ) 5.61 Hz), 2.17 (s, 6H), 1.47

(t, 3H, J ) 6.93 Hz). Anal. (C₃₂H₃₀N₆O₄S‚0.25H₂O) C, H, N.

(E)-N-{4-[3-Chloro-4-(2-chlorobenzyloxy)-phenylamino]-

3-cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-bu-

tenamide (21d). A 958 mg (2 mmol) sample of 19d was

reacted with 4-(dimethylamino)-but-2-enoic acid chloride (20)

as described above for 8d to yield 780 mg (66%) of product as

a yellow glass after silica gel chromatography: HRMS (ESI)

m/z 590.171 17 (M + H)⁺¹, ∆ ) -0.86 mmu; ¹H NMR (DMSO-

d₆) δ 9.62 (s, 1H), 9.49 (s, 1H), 8.97 (s, 1H), 8.48 (s, 1H), 7.68-

7.65 (m, 1H), 7.55-7.53 (m, 1H), 7.44-7.38 (m, 4H), 7.30-

7.27 (m, 1H), 7.22 (dd, 1H, J ) 6.6, 1.9 Hz), 6.81-6.74 (m,

1H), 6.61-6.57 (m, 1H), 5.27 (s, 2H), 4.31 (q, 2H, J ) 5.2 Hz),

3.07 (d, 2H, J ) 3.8 Hz), 2.18 (s, 6H), 1.47 (t, 3H, J ) 5.2 Hz).

Anal. (C₃₁H₂₉Cl₂N₅O₃‚1H₂O) C, H, N, Cl.

N-[4-(1-Benzenesulfonyl-1H-indol-5-ylamino)-3-cyano-

7-ethoxyquinolin-6-yl]acetamide (18c). A solution of 3.29

g (0.0113 mol) of N-(4-chloro-3-cyano-7-ethoxyquinolin-6-yl)-

acetamide (16), 3.09 g (0.0113 mol) of 1-benzenesulfonyl-1H-

indol-5-ylamine (17c),³⁴and 1.31 g (0.0113 mol) of pyridine

hydrochloride in 50 mL of 2-methoxyethanol was refluxed for

2 h. The reaction mixture was poured into H₂O. The resulting

mixture was extracted with 3 × 250 mL portions of EtOAc.

The combined extracts were concentrated in vacuo until a solid

formed. This was filtered to give 3.04 g (51%) of the product

(18c): mp ) 255-257 °C; MS (ES+) m/z 526.0 (M + H)⁺¹

.

6-Amino-4-[3-chloro-4-(1-methyl-1H-imidazol-2-yl-

methoxy)phenylamino]-7-ethoxyquinoline-3-carboni-

trile (19a). A mixture of 3.27 g of N-{4-[3-chloro-4-(1-methyl-

1H-imidazol-2-ylmethoxy)phenylamino]-3-cyano-7-ethoxy-

quinolin-6-yl}acetamide hydrochloride (18a) in 10 mL of H₂O

and 20 mL of 12 N HCl was stirred and heated in a oil bath

for 2 h. The solvents were evaporated in vacuo, and the solid

was treated with 50 mL of saturated aqueous NaHCO₃, and

then filtered. This solid was taken up in EtOAc and passed

through Magnesol. This filtrate was evaporated to a solid in

vacuo and digested with EtOAc to give 1.77 g (59%) of product

Synthesis Using Method 3. 4-Chloro-6-(5-(dimethyl-

amino)-2-oxopent-3-enyl)-7-ethoxyquinoline-3-carboni-

trile (23). A solution of 4-dimethylaminocrotonyl chloride

freshly prepared from 9 g (54.3 mmol) of 4-dimethylamino-

crotonic acid hydrochloride was slurried in 20 mL of acetoni-

trile, and 6.69 g (27 mmol) of 6-amino-4-chloro-7-ethoxyquin-

oline-3-carbonitrile (22) as a slurry in 150 mL of 1-methyl-2-

pyrrolidinone was added under nitrogen. This was cooled in

(19a): mp ) 222-225 °C; MS (ES+) m/z 449.0 (M + H)⁺¹

.

6-Amino-4-(1-benzenesulfonyl-1H-indol-5-ylamino)-7-

ethoxyquinoline-3-carbonitrile (19c). A mixture of 2.82 g

(5.27 mmol) of N-[4-(1-benzenesulfonyl-1H-indol-5-ylamino)-

3-cyano-7-ethoxyquinolin-6-yl]acetamide (18c) in 20 mL of 12

N HCl and 10 mL of H₂O was stirred and heated in an oil

Carbonitriles as Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1127

an ice bath and stirred for 2.5 h. The solution was poured into

1.3 L of a 1:1 mixture of saturated KHCO₃and water and was

stirred for 20 min, and the precipitate was filtered off, washed

with water, and dried. Crystallization from acetonitrile yielded

7.97 g (82%) of brown solid in two crops, ESMS m/z 358.8 (M

+ H)⁺¹. The product was used without further purification.

-0.66 mmu; ¹H NMR (DMSO-d₆) δ 9.74 (bs, 1H), 9.50 (s, 1H),

9.00 (s, 1H), 8.62 (s, 1H), 7.55-7.52 (m, 2H), 7.48-7.33 (m,

6H), 6.81-6.60 (m, 2H), 5.01 (s, 2H), 4.33 (q, 2H, J ) 5.2 Hz),

3.08 (d, 2H, J ) 5.1 Hz), 2.17 (s, 6H), 1.48 (t, 3H, J ) 5.2 Hz).

Anal. (C₃₁H₂₉Cl₂N₅O₃‚0.5H₂O‚0.25EtOAc) C, H, N.

(E)-N-{4-[4-(Benzyloxy)-3-cyanoanilino]-3-cyano-7-

ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamide (25e).

The title compound was prepared from a mixture of 0.500 g

(1.39 mmol) of 23, 0.374 g (1.67 mmol) of 5-amino-2-(benzyl-

oxy)benzonitrile, and 0.322 g (2.78 mmol) of pyridine hydro-

chloride in 35 mL of i-PrOH using the same procedure as

described for 25g. A 680 mg sample of crude product was

purified by flash chromatography (silica gel, 7% MeOH in

CHCl₃) to give 0.560 g (74%) of off-white solid. ¹H NMR

(DMSO-d₆) δ 9.68 (s, 1H), 9.51 (s, 1H), 8.97 (s, 1H), 8.49 (s,

1H), 7.69 (m, 1H), 7.46 (m, 8H), 6.77 (m, 1H), 6.59 (m, 1H),

5.31 (s, 2H), 4.30 (q, 2H, J ) 6.9 Hz), 3.07 (d, 2H, J ) 5.55

Hz), 2.18 (s, 6H), 1.48 (t, 3H, J ) 6.9 Hz); HRMS (ESI) m/z

547.244 18 (M + H)⁺¹, ∆ ) -1.04 mmu. Anal. (C₃₂H₃₀N₆O₃‚

1.4H₂O) C, H, N.

(E)-N-{4-[3-Chloro-4-(2-fluorobenzyloxy)anilino]-3-cy-

ano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-buten-

amide (25f). The title compound was prepared from a mixture

of 0.500 g (1.39 mmol) of 23, 0.421 g (1.67 mmol) of 3-chloro-

4-[(2-fluorobenzyl)oxy]aniline, and 0.322 g (2.78 mmol) of

pyridine hydrochloride in 45 mL of i-PrOH using the same

procedure described for 25g. A 0.740 g sample of crude product

was purified by flash chromatography (silica gel, 7% MeOH

in CHCl₃) to give 0.590 g (74%) of yellow solid; HRMS (ESI)

m/z 574.20070 (M + H)⁺¹, ∆ ) - 0.88 mmu; ¹H NMR (DMSO-

d₆) δ 9.62 (s, 1H), 9.50 (s, 1H), 8.97 (s, 1H), 8.48 (s, 1H), 7.61

(m, 1H), 7.38 (m, 2H), 7.35 (m, 2H), 7.25 (m, 4H), 6.78 (m,

1H), 6.58 (m, 1H), 5.25 (s, 2H), 4.33 (m, 2H), 3.17 (d, 2H, J )

3.96 Hz), 3.07 (d, 2H, J ) 3.84 Hz), 2.18 (s, 6H), 1.47 (t, 3H, J

) 5.19 Hz). Anal. (C₃₁H₂₉ClFN₅O₃‚H₂O) C, H, N.

(E)-N-(4-{Chloro-4-[(4-fluorobenzyl)oxy]anilino}-3-cy-

ano-7-ethoxy-6-quinolinyl)-4-(dimethylamino)-2-butene-

amide (25g). A mixture of 0.350 g (0.975 mmol) of (E)-N-(4-

chloro-3-cyano-7-ethoxy-6-quinolinyl)-4-(dimethylamino)-2-

butenamide (23), 0.295 g (1.17 mmol) of 3-chloro-4-(4-

fluorobenzyloxy)aniline, and 0.226 g (1.95 mmol) of pyridine

hydrochloride in 20 mL of i-PrOH was refluxed under N₂for

10.5 h. The cooled reaction mixture was poured into 100 mL

of H₂O and basified with K₂CO₃. Addition of EtOAc caused

the gummy mixture to solidify, and this material was collected,

washed with EtOAc, and dried. A 0.545 g sample of crude

product was purified by flash chromatography (silica, 8%

MeOH in CHCl₃) to give 0.510 g (53%) of yellow-orange foam:

1

HRMS (ESI) m/z 574.199 63 (M + H)⁺¹, ∆ ) -0.69 mmu; H

NMR (DMSO-d₆) δ 9.61 (s, 1H), 9.49 (s, 1H), 8.97 (s, 1H), 8.47

(s, 1H), 7.54 (m, 2H), 7.37 (s, 2H), 7.25 (m, 4H), 6.78 (m, 1H),

6.59 (m, 1H), 5.20 (s, 2H), 4.30 (q, 2H, J ) 6.96 Hz), 3.07 (d,

2H, J ) 5.52 Hz), 2.18 (s, 6H), 1.47 (t, 3H, J ) 6.96 Hz). Anal.

(C₃₁H₂₉ClFN₅O₃‚0.4H₂O) C, H, N.

(E)-N-[4-(3-Chloro-4-phenoxyanilino)-3-cyano-7-ethoxy-

6-quinolinyl]-4-(dimethylamino)-2-butenamid (25a). This

compound was prepared as a yellow solid (0.57 g, 75%) by

method 3 described for the preparation of 25g using 23 and

0.366 g (3.62 mmol) of 3-chloro-4-phenoxyphenylamine: HRMS

(ES+) m/z 542.195 57 (M + H)⁺¹, ∆ ) 0.22 mmu; ¹H NMR

(DMSO-d₆) δ 9.78 (s, 1H), 9.51 (s, 1H), 9.02 (s, 1H), 8.55 (s,

1H), 7.46 (s, 1H), 7.42 (s, 1H), 7.35 (t, 2H, J ) 6.3 Hz), 7.24 (d,

1H, J ) 3.3 Hz), 7.18 (d, 1H, J ) 6.6 Hz), 7.09 (t, 1H, J ) 6.6

Hz), 6.96 (d, 2H, J ) 6.6 Hz), 6.82-6.76 (m, 1H), 6.62 (d, 1H,

J ) 9.9 Hz), 4.32 (q, 2H, J ) 6.6 Hz), 3.08 (d, 2H, J ) 3 Hz),

2.17 (s, 6H), 1.47 (t, 3H, J ) 3 Hz). Anal. (C₃₀H₂₈ClN₅O₃‚H₂O)

C, H, N.

(E)-N-{4-[3,5-Dichloro-4-(3-fluorobenzyloxy)anilino]-3-

cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-bu-

tenamide (25h). A 720 mg (2 mmol) sample of 23 was reacted

with 647 mg (2.4 mmol) of 3,5-dichloro-4-[(3-fluorobenzyl)oxy]-

aniline as described above for 25g to yield 457 mg (38%) of

beige crystals in two crops from CH₃CN: mp ) 128-131 °C;

(E)-N-{4-[4-(Benzyloxy)anilino]-3-cyano-7-ethoxy-6-

quinolinyl}-4-(dimethylamino)-2-butenamide (25b). The

product was prepared by method 3 described for the prepara-

tion of 25g using 0.36 g (1.0 mmol) of 23, 0.28 g (1.2 mmol) of

4-benzyloxyaniline hydrochloride, 92 mg (0.8 mmol) of pyridine

hydrochloride, and 3.0 mL of methoxyethanol at reflux for 30

min. The product was purified by short column chromatogra-

phy on silica gel with 25:25:2:1 CH₂Cl₂-EtOAc-MeOH-Et₃N

to give 487 mg (94%) of amber glass: MS (ES+) m/z 521.9 (M

1

HRMS (ESI) m/z 608.162 27 (M + H)⁺¹, ∆ ) -0.33 mmu; H

NMR (DMSO-d₆) δ 9.75 (s, 1H), 9.50 (s, 1H), 8.99 (s, 1H), 8.62

(s, 1H), 7.50-7.41 (m, 2H), 7.38-7.31 (m, 4H), 7.25-7.19 (m,

1H), 6.81-6.74 (m, 1H), 6.64-6.59 (m, 1H), 5.04 (s, 2H), 4.33

(q, 2H, J ) 5.1 Hz), 3.08 (bs, 2H), 2.17 (s, 6H), 1.48 (t, 3H, J

) 5.1 Hz). Anal. (C₃₁H₂₈Cl₂FN₅O₃‚H₂O) C, H, N.

1

+ H)⁺¹; H NMR (DMSO-d₆) δ 9.56 (s, 1H), 9.49 (s, 1H), 8.96

(s, 1H), 8.41 (s, 1H), 7.48 (m, 2H), 7.38 (m, 3H,), 7.22 (d, 2H,

J ) 6.9 Hz), 7.05 (d, 2H, J ) 6.9 Hz), 6.77 (dt, 1H, J ) 5.3, J

) 15.4 Hz), 6.58 (d, J ) 15.4 Hz), 5.12 (s, 2H), 4.29 (q, 2H, J

) 6.9 Hz), 3.08 (d, 2H, J ) 5.3 Hz), 2.18 (s, 6H), 1.46 (t, 3H, J

) 6.9 Hz). Anal. (C₃₁H₃₁N₅O₃‚MeOH) C, H, N.

(E)-N-{4-[3-Chloro-4-(3,5-difluorobenzyloxy)anilino]-3-

cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-bu-

tenamide (25i). As described in the preparation of 25g, 720

mg (2 mmol) of 23 and 644 mg (2.4 mmol) of 3-chloro-4-(3,5-

difluorobenzyloxy)aniline were reacted to yield 432 mg (37%)

of golden brown crystals from CH₃CN: mp ) 101-104 °C;

(E)-N-{4-[3-Chloro-4-(2-phenylethoxy)anilino]-3-cyano-

7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-buten-

amide (25c). 25c was prepared from a mixture of 0.250 g

(0.696 mmol) of 23, 0.207 g (0.835 mmol) of 3-chloro-4-

phenylethoxyaniline, and 0.161 g (1.39 mmol) of pyridine

hydrochloride in 12 mL of i-PrOH using the same procedure

described for 25g. A 1.00 g sample of crude product was

purified by flash chromatography (silica gel, 8% MeOH in

CHCl₃) to give 0.700 g (74%) of tan foam: ¹H NMR (DMSO-

d₆) δ 9.59 (s, 1H), 9.48 (s, 1H), 8.97 (s, 1H), 8.45 (s, 1H), 7.28

(m, 10H), 6.78 (m, 1H), 6.58 (m, 1H), 4.28 (m, 4H), 3.09 (m,

4H), 2.17 (s, 6H), 1.47 (t, 3H, J ) 6.96 Hz); HRMS (ESI) m/z

570.226 63 (M + H)⁺¹, ∆ ) -0.17 mmu. Anal. (C₃₂H₃₂ClN₅O₃‚

0.5H₂O) C, H, N.

(E)-N-[4-(4-Benzyloxy-3,5-dichlorophenylamino)-3-cy-

ano-7-ethoxy-6-quinolinyl]-4-(dimethylamino)-buten-

amide (25d). As described in the preparation of 25g, 448 mg

(1.25 mmol) of 23 and 402 mg (1.5 mmol) of 4-benzyloxy-3,5-

dichloroaniline in 35 mL of i-PrOH were reacted to yield 203

mg (27%) of beige crystals in two crops from CH₃CN: mp )

106-109 °C; HRMS (ESI) m/z 590.171 37 (M + H)⁺¹, ∆ )

1

HRMS (CI) m/z 592.1913 (M + H)⁺¹, ∆ ) 1.4 mmu; H NMR

(DMSO-d₆) δ 9.73 (s, 1H), 9.63 (s, 1H), 8.97 (s, 1H), 8.48 (s,

1H), 7.39-7.37 (m, 2H), 7.25-7.20 (m, 5H), 6.81-6.58 (m, 2H),

5.27 (s, 2H), 4.31 (q, 2H, J ) 5.2 Hz), 3.08 (d, 2H, J ) 3.9 Hz),

2.18 (s, 6H), 1.47 (t, 3H, J ) 5.2 Hz). Anal. (C₃₁H₂₈ClF₂N₅O₃‚

1.12H₂O) C, H, N.

(E)-N-{4-[3-Chloro-4-(3-cyanobenzyloxy)anilino]-3-cy-

ano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-buten-

amide (25k). The title compound was prepared from a

mixture of 0.500 g (1.39 mmol) of 23, 0.431 g (1.67 mmol) of

3-[(4-amino-2-chlorophenoxy)methyl]benzonitrile, and 0.322 g

(2.78 mmol) of pyridine hydrochloride in 36 mL of i-PrOH

using the same procedure described for 25g. A 0.740 g sample

of crude product was purified by flash chromatography (silica

gel, 7% MeOH in CHCl₃) to give 0.600 g (74%) of yellow solid.

¹H NMR (DMSO-d₆) δ 9.63 (s, 1H), 9.06 (s, 1H), 8.97 (s, 1H),

8.47 (s, 1H), 7.94 (s, 1H), 7.83 (m, 2H), 7.65 (m, 2H), 7.39 (s,

2H), 7.23 (m, 2H), 6.77 (m, 1H), 6.59 (m, 1H), 5.29 (s, 2H),

4.30 (q, 2H, J ) 5.16 Hz), 3.07 (d, 2H, J ) 3.9 Hz), 2.17 (s, J

1128 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4

Tsou et al.

) 6 Hz), 1.47 (t, 3H, J ) 5.16 Hz); HRMS (ESI) m/z 291.106 65

(d, 1H, J ) 5.91 Hz), 7.98 (m, 2H), 7.74 (d, 1H, J ) 5.01 Hz),

7.56 (m, 3H), 7.46 (d, 2H, J ) 6.69 Hz), 7.38 (m, 2H), 7.25 (dd,

1H, J ) 6.6 Hz, J ) 1.89 Hz), 6.78 (m, 1H), 6.60 (m, 1H), 5.67

(s, 2H), 4.31 (q, 2H, J ) 5.16 Hz), 3.07 (d, 2H J ) 3.81 Hz),

2.17 (s, 6H), 1.47 (t, 3H, J ) 5.16 Hz). Anal. (C₃₅H₃₂ClN₅O₃‚

0.5H₂O) C, H, N.

(M + 2H)⁺², ∆ ) 0.68 mmu. Anal. (C₃₂H₂₉ClN₆O₃‚H₂O) C, H,

N.

(E)-N-{4-[3-Chloro-4-(3-methylbenzyloxy)anilino]-3-cy-

ano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-buten-

amide (25m). The title compound was prepared from a

mixture of 0.500 g (1.39 mmol) of 23, 0.414 g (1.67 mmol) of

3-chloro-4-(3-methylbenzyloxy)aniline, and 0.322 g (2.78 mmol)

of pyridine hydrochloride in 25 mL of i-PrOH using the same

procedure as for 25g. A 0.700 g sample of crude product was

purified by flash chromatography (silica, 5% MeOH in CHCl₃)

to give 430 mg (54%) of tan foam: HRMS (ESI) m/z 570.225 10

(M + H)⁺¹, ∆ ) -0.27 mmu; ¹H NMR (DMSO-d₆) δ 9.61 (s,

1H), 9.49 (s, 1H), 8.97 (s, 1H), 8.47 (s, 1H), 7.29 (m, 7H), 6.78

(m, 1H), 6.59 (M, 1H), 5.17 (s, 2H), 4.30 (q, 2H, J ) 5.19 Hz),

3.07 (d, 2H, J ) 3.99 Hz), 2.33 (s, 3H), 2.18 (s, 6H), 1.47 (t,

3H, J ) 5.19 Hz). Anal. (C₃₂H₃₂ClN₅O₃‚0.6H₂O) C, H, N.

(E)-N-{4-[3-Chloro-4-(2-naphthylmethoxy)anilino[-3-

cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-bu-

tenamide (25u). The title compound was prepared from a

mixture of 0.500 g (1.39 mmol) of 23, 0.474 g (1.67 mmol) of

3-chloro-4-(2-naphthylmethoxy)aniline, and 0.322 g (2.78 mmol)

of pyridine hydrochloride in 25 mL of i-PrOH using the same

procedure as described for 25g. A 0.900 g sample of crude

product was purified by flash chromatography (silica gel, 7%

MeOH in CHCl₃) to give 0.590 g (70%) of yellow foam: HRMS

(ESI) m/z 606.225 88 (M + H)⁺¹, ∆ ) -0.77 mmu; ¹H NMR

(DMSO-d₆) δ 9.63 (s, 1H), 9.51 (s, 1H), 8.98 (s, 1H), 8.47 (s,

1H), 7.96 (m, 4H), 7.62 (d, 1H, J ) 7.53 Hz), 7.54 (m, 2H),

7.39 (m, 2H), 7.30 (m, 1H), 7.23 (m, 1H), 6.78 (m, 1H), 6.59

(m, 1H), 5.39 (s, 2H), 4.30 (q, 2H, J ) 5.19 Hz), 3.06 (d, 2H, J

) 3.75 Hz), 2.17 (s, 6H), 1.47 (t, 3H, J ) 5.19 Hz). Anal. (C₃₅H₃₂-

ClN₅O₃) C, H, N.

(E)-N-{4-[3-Chloro-4-(cyclohexylmethoxy)anilino]-3-

cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-bu-

tenamide (25n). The title compound was prepared from a

mixture of 0.650 g (1.81 mmol) of 23, 0.521 g (2.17 mmol) of

3-chloro-4-(cyclohexylmethoxy)aniline, and 0.420 g (3.62 mmol)

of pyridine hydrochloride in 30 mL of i-PrOH using the same

procedure as described for 25g. A 1.00 g sample of crude

product was purified by flash chromatography (silica gel, 7%

MeOH in CHCl₃) to give 0.860 g (84%) of yellow foam: HRMS

(E)-N-[4-(3-Chloro-4-{[(1R)-1-phenylethyl]oxy}anilino)-

3-cyano-7-ethoxy-6-quinolinyl]-4-(dimethylamino)-2-bu-

tenamide (25v). This compound was prepared as a yellow

solid (0.6 g, 74%) by the same method for the preparation of

25g, using 0.5 g (1.39 mmol) of 23 and 0.414 g (1.67 mmol) of

3-chloro-4-{[(1R)-1-phenylethyl]oxy}aniline: MS(ESI+) m/z

1

(ESI) m/z 281.632 08 (M + 2H)⁺², ∆ ) -0.38 mmu; H NMR

(DMSO-d₆) δ 9.59 (s, 1H), 9.49 (s, 1H), 8.96 (s, 1H), 8.46 (s,

1H), 7.38 (s, 1H), 7.33 (d, 1H, J ) 1.86 Hz), 7.17 (m, 2H), 6.78

(m, 1H), 5.59 (m, 1H), 4.33 (q, 2H, J ) 3.84 Hz), 3.87 (d, 2H,

J ) 4.62 Hz), 3.07 (d, 2H, J ) 3.87 Hz), 2.18 (s, 6H), 1.80 (m,

6H), 1.47 (t, 3H, J ) 5.22 Hz), 1.16 (m, 5H). Anal. (C₃₁H₃₆-

ClN₅O₃‚0.25H₂O) C, H, N.

1

570.2 (M + H)⁺¹; H NMR (DMSO-d₆) δ 9.54 (s, 1H), 9.47 (s,

1H), 8.93 (s, 1H), 8.44 (s, 1H), 7.43 (d, 2H, J ) 6 Hz), 7.36-

7.33 (m, 4H), 7.27 (t, 1H, J ) 5.1 Hz), 7.05 (t, 2H, J ) 3.3 Hz),

6.78-6.72 (m, 1H), 6.58 (d, 1H, J ) 9.3 Hz), 5.61 (q, 1H, J )

6 Hz), 4.29 (q, 2H, J ) 6.3 Hz), 3.06 (d, 2H, J ) 6.3 Hz), 2.16

(s, 6H), 1.58 (d, 3H, J ) 3.3 Hz), 1.45 (t, 3H, J ) 6.3 Hz). Anal.

(C₃₂H₃₂ClN₅O₃‚0.5H₂O) C, H, N.

(E)-N-{4-[3-Chloro-4-(2-pyridinylmethoxy)anilino]-3-

cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-bu-

tenamide (25o). This compound was prepared as a yellow

solid (0.86 g, 85%) by the method described for 25g using 0.65

g (1.81 mmol) of 23 and 0.42 g (3.62 mmol) of 3-chloro-4-(2-

pyridinylmethoxy)aniline: HRMS (ES+) m/z 557.205 89 (M +

H)⁺¹, ∆ ) -0.36 mmu; ¹H NMR (DMSO-d₆) δ 9.62 (s, 1H), 9.49

(s, 1H), 8.96 (s, 1H), 8.60 (d, 1H, J ) 3.9 Hz), 8.47 (s, 1H),

7.88 (t, 1H, J ) 3.9 Hz), 7.58 (d, 1H, J ) 3.9 Hz), 7.39-7.35

(m, 3H), 7.26 (d, 1H, J ) 7.8 Hz), 7.19 (d, 1H, J ) 8.1 Hz),

6.81-6.73 (m, 1H), 6.59 (d, 1H, J ) 7.8 Hz), 5.28 (s, 2H), 4.30

(q, 2H, J ) 6.9 Hz), 3.07 (d, 2H, J ) 3.9 Hz), 2.17 (s, 6H), 1.46

(t, 3H, J ) 3.9 Hz). Anal. (C₃₀H₂₉ClN₆O₃‚1.1H₂O) C, H, N.

(E)-N-[4-(3-Chloro-4-{[(1S)-1-phenylethyl]oxy}anilino)-

3-cyano-7-ethoxy-6-quinolinyl]-4-(dimethylamino)-2-bu-

tenamide (25w). This compound was prepared as a yellow

solid (0.56 g, 70%) by the same method described for 25g using

0.5 g (1.39 mmol) of 23 and 0.414 g of (1.67 mmol) 3-chloro-

4-{[(1S)-1-phenylethyl]oxy}aniline: HRMS (ESI) m/z 570.225 71

(M + H)⁺¹, ∆ ) -0.94 mmu; ¹H NMR (DMSO-d₆) δ 9.55 (s,

1H), 9.48 (s, 1H), 8.93 (s, 1H), 8.45 (s, 1H), 7.43 (d, 2H, J )

6.3 Hz), 7.38-7.24 (m, 5H), 7.06 (d, 2H, J ) 1.5 Hz), 6.80-

6.71 (m, 1H), 6.58 (d, 1H, J ) 15.6 Hz), 5.61 (q, 1H, J ) 6.3

Hz), 4.29 (q, 2H, J ) 6.3 Hz), 3.06 (d, 2H, J ) 6.3 Hz), 2.16 (s,

6H), 1.58 (d, 3H, J ) 3.3 Hz), 1.45 (t, 3H, J ) 6.3 Hz). Anal.

(C₃₂H₃₂ClN₅O₃‚H₂O) C, H, N.

N,N-Bis(tert-butoxycarbonyl)-2-chloro-4-nitroani-

line (27). To a stirred solution of 8.63 g (50 mmol) of 2-chloro-

4-nitroaniline and 0.30 g (2.5 mmol) of DMAP in 50 mL of THF

at 0 °C was added 22.9 g (105 mmol) of (Boc)₂O. The resulting

purple solution was warmed to 25 °C, stirred for 3 h, and

concentrated. The residue was partitioned between EtOAc and

1 M citric acid. The EtOAc layer was washed well with brine,

dried, and concentrated to give 19.2 g (100%) of off-white

solid: mp ) 103-110 °C. Anal. (C₁₆H₂₁ClN₂O₆) C, H, N.

N¹,N¹-Bis(tert-butoxycarbonyl)-2-chloro-p-phenylene-

diamine (28). A stirred mixture of 13.5 g (36.2 mmol) of 27,

7.09 g (127 mg atom) of iron powder, 14.5 mL (253 mmol) of

HOAc, and 181 mL of MeOH was refluxed for 30 min and

concentrated to remove MeOH. The residue was stirred with

EtOAc and H₂O for 15 min and filtered. The EtOAc layer was

washed with aqueous NaHCO₃and H₂O, dried, and concen-

trated to give 12.4 g (100%) of off-white solid: ¹H NMR

(DMSO-d₆) δ 7.00 (d, 1H, J ) 8.6 Hz), 6.77 (bs, 1H), 6.61 (d,

1H, J ) 8.6 Hz), 4.46 (bs, 2H), 1.36 (s, 18H).

(E)-N-[4-(4-Amino-3-chloroanilino)-3-cyano-7-ethoxy-

6-quinolinyl]-4-(dimethylamino)-2-butenamide (29). A

stirred mixture of 3.99 g (11.1 mmol) of 23, 4.56 g (13.3 mmol)

of 28, 2.56 g (22.2 mmol) of pyridine hydrochloride, and 67

mL of methoxyethanol was refluxed for 1 h. The cooled mixture

was stirred with dilute aqueous K₂CO₃(pH ∼9). The resulting

(E)-N-{4-[3-Chloro-4-(2-furylmethoxy)anilino]-3-cyano-

7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-buten-

amide (25q). The title compound was prepared from a mixture

of 0.500 g (1.39 mmol) of 23, 0.374 g (1.67 mmol) of 3-chloro-

4-(2-furylmethoxy)aniline, and 0.322 g (2.78 mmol) of pyridine

hydrochloride in 25 mL of i-PrOH using the same procedure

as for 25g. A 0.600 g sample of the crude product was purified

by flash chromatography (silica gel, 5% MeOH in CHCl₃) to

give 0.360 mg (47%) of brown foam: HRMS (ESI) m/z

1

546.192 59 (M + H)⁺¹, ∆ ) 2.33 mmu; H NMR (DMSO-d₆) δ

9.61 (s, 1H), 9.49 (s, 1H), 8.97 (s, 1H), 8.47 (s, 1H), 7.73 (s,

1H), 7.34 (m, 3H), 7.21 (m, 1H), 6.77 (m, 1H), 6.62 (m, 2H),

6.49 (m, 1H), 5.16 (s, 2H), 4.30 (q, 2H, J ) 5.19 Hz), 3.07 (d,

2H, J ) 3.69 Hz), 2.18 (s, 6H), 1.47 (t, 3H, J ) 5.19 Hz). Anal.

(C₂₉H₂₈ClN₅O₄‚0.3H₂O) C, H, N.

(E)-N-{4-[3-Chloro-4-(1-naphthylmethoxy)anilino[-3-

cyano-7-ethoxy-6-quinolinyl}-4-(dimethylamino)-2-bu-

tenamide (25t). The title compound was prepared from a

mixture of 0.500 g (1.39 mmol) of 23, 0.474 g (1.67 mmol) of

3-chloro-4-(1-naphthylmethoxy)aniline, and 0.322 g (2.78 mmol)

of pyridine hydrochloride in 23 mL of i-PrOH using the same

procedure as described for 25g. The crude product was

dissolved in CHCl₃-MeOH, neutralized with concentrated

NH₄OH, and evaporated. A 0.960 mg sample of this material

was purified by flash chromatography (silica gel, 7% MeOH

in CHCl₃) to give 0.720 mg (86%) of brown foam: HRMS (ESI)

1

m/z 606.226 84 (M + H)⁺¹, ∆ ) 0.19 mmu; H NMR (DMSO-

d₆) δ 9.64 (s, 1H), 9.51 (s, 1H), 8.98 (s, 1H), 8.48 (s, 1H), 8.14

Carbonitriles as Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1129

precipitate was filtered, washed well with water, and dried to

give 5.84 g brown solid.

acylation of 7.99 g (14.8 mmol) of 34 with 4.90 g (29.6 mmol)

of 4-dimethylaminobut-2-enoic acid hydrochloride in 65 mL of

MeCN was carried out using the same procedure described

for the preparation of 23 to provide 9.06 g of the intermediate

N,N-bis(tert-butoxycarbonyl) derivative. The crude product was

subjected to short column chromatography on silica gel with

final elution by 25:5:1 EtOAc-MeOH-Et₃N to provide 7.30 g

(75%) of amber solid, which was sufficiently pure for conversion

to the title compound.

The solid was stirred with 111 mL of CH₂Cl₂at 0 °C and

treated with 55 mL of TFA. The resulting solution was warmed

to 25 °C, stirred for 15 min, diluted with toluene, and

concentrated. The residue was partitioned between 20:1

EtOAc-MeOH and aqueous K₂CO₃. The organic layer was

washed with brine, dried, and concentrated. Short column

chromatography of the residue on silica gel with final elution

by 25:5:1 EtOAc-MeOH-Et₃N gave 3.05 g (60%) of amber

solid: mp ) 222-232 °C (dec); MS (ES+) m/z 465.2, 467.2 (M

The solid was stirred with 112 mL of CH₂Cl₂at 0 °C and

treated with 56 mL of TFA. The resulting solution was warmed

to 25 °C, stirred for 5 min, diluted with toluene, and concen-

trated. The residue was partitioned between 20:1 EtOAc-

MeOH and aqueous K₂CO₃. The organic layer was washed with

brine, dried, and concentrated to give 4.89 g (97%) of light-

yellow solid that was sufficiently pure for further reactions.

Short column chromatography of a portion of the product on

SG with final elution by 25:5:1 EtOAc-MeOH-Et₃N gave a

yellow solid: mp ) 190-200 °C (dec); MS (ES+) m/z 451.2,

1

+ H)⁺¹; H NMR (DMSO-d₆) δ 9.49 (s, 1H), 9.47 (s, 1H), 8.91

(s, 1H), 8.38 (s, 1H), 7.33 (s, 1H), 7.14 (d, 1H, J ) 2.3 Hz),

6.98 (dd, 1H, J ) 2.3 Hz, J ) 8.6 Hz), 6.80 (d, 1H, J ) 8.6 Hz),

6.77 (dt, 1H, J ) 5.6 Hz, J ) 15.4 Hz), 6.57 (d, 1H, J ) 15.4

Hz), 5.41 (bs, 2H), 4.28 (q, 2H, J ) 7.0 Hz), 3.09 (d, 2H, J )

5.6 Hz), 2.19 (s, 6H), 1.46 (t, 3H, J ) 7.0 Hz) Anal. (C₂₄H₂₅-

ClN₆O₂‚0.5H₂O) C, H, N.

(E)-N-{4-[4-(Benzylamino)-3-chloroanilino]-3-cyano-7-

ethoxy-6-quinolinyl}-4-(dimethylamino)-2-butenamide

(31). To a stirred suspension of 465 mg (1.0 mmol) of 29 in

4.0 mL of DCE were added successively 0.21 g (2.0 mmol) of

benzaldehyde, 0.59 g (2.8 mmol) of NaBH(OAc)₃, and 0.23 mL

(4.0 mmol) of HOAc at 25 °C. After 18 h the reaction mixture

was partitioned between CH₂Cl₂and aqueous NaHCO₃. The

organic layer was washed with water, dried, and concentrated.

Short column chromatography of the residue on silica gel with

final elution by 25:25:2:1 CH₂Cl₂-EtOAc-MeOH-Et₃N gave

375 mg (68%) of an amber foam: MS (ES+) m/z 555.1, 557.1

1

453.2 (M + H)⁺¹; H NMR (DMSO-d₆) δ 9.67 (s, 1H), 9.48 (s,

1H), 8.92 (s, 1H), 8.39 (s, 1H), 7.36 (s, 1H), 7.14 (d, 1H, J )

2.3 Hz), 6.98 (dd, 1H, J ) 2.3 Hz, J ) 8.6 Hz), 6.80 (d, 1H, J

) 8.6 Hz), 6.75 (dt, 1H, J ) 5.8 Hz, J ) 15.4 Hz), 6.58 (d, 1H,

J ) 15.4 Hz), 5.41 (bs, 2H), 4.01 (s, 3H), 3.15 (d, 2H, J ) 5.8

Hz), 2.24 (s, 6H). Anal. (C₂₃H₂₃ClN₆O₂) C, H, N.

(E)-N-(4-{4-[(Benzoyl)amino]-3-chloroanilino}-3-cyano-

7-methoxy-6-quinolinyl)-4-(dimethylamino)-2-buten-

amide (36). A stirred solution of 226 mg (0.50 mmol) of 35,

0.34 g (1.5 mmol) of benzoic anhydride, and 1.5 mL of

dimethylacetamide (DMA) was heated at 60 °C for 20 h. The

reaction mixture was partitioned between 20:1 CH₂Cl₂-MeOH

and aqueous K₂CO₃. The organic layer was washed with H₂O,

dried, and concentrated. Short column chromatography of the

residue on silica gel with final elution by 25:5:1 EtOAc-

MeOH-Et₃N gave 0.20 g (72%) of amber solid: mp ) 170-

1

(M + H)⁺¹; H NMR (DMSO-d₆) δ 9.49 (s, 1H), 9.46 (s, 1H),

8.89 (s, 1H), 8.37 (s, 1H), 7.33 (s, 1H) 7.40-7.15 (m, 5H), 7.22

(d, 1H, J ) 2.3 Hz), 6.98 (dd, 1H, J ) 2.3, J ) 8.7 Hz), 6.77

(dt, 1H, J ) 5.6 Hz, J ) 15.4 Hz), 6.59 (d, J ) 15.4 Hz), 6.56

(d, 1H, J ) 8.7 Hz), 6.16 (t, J ) 6.0 Hz), 4.44 (d, 2H, J ) 6.0

Hz), 4.30 (q, 2H, J ) 6.9 Hz), 3.12 (d, 2H, J ) 5.6 Hz), 2.21 (s,

6H), 1.45 (t, 3H, J ) 6.9 Hz). Anal. (C₃₁H₃₁ClN₆O₂‚0.5H₂CO₃‚

0.5MeOH) C, H, N.

180 °C; MS (ES+) m/z 555.3, 557.3 (M + H)⁺^{1 1}H NMR

;

(DMSO-d₆) δ 10.0 (s, 1H), 9.74 (s, 1H), 9.70 (s, 1H), 9.04 (s,

1H), 8.63 (s, 1H), 8.03 (s, 1H), 7.95 (m, 2H), 7.65-7.40 (m, 6H),

6.79 (dt, 1H, J ) 5.7 Hz, J ) 15.3 Hz), 6.63 (d, 1H, J ) 15.3

Hz), 4.06 (s, 3H), 3.02 (d, 2H, J ) 5.7 Hz), 2.20 (s, 6H). Anal.

(C₃₀H₂₇ClN₆O₃) C, H, N.

(E)-N-(4-{3-Chloro-4-[(1-naphthylmethyl)amino]anili-

no}-3-cyano-7-ethoxy-6-quinolinyl)-4-(dimethylamino)-

2-butenamide (32). The product was prepared from 465 mg

(1.0 mmol) of 29 and 0.31 g (2.0 mmol) of 1-naphthaldehyde

as described above for 31 to give 546 mg (91%) of yellow

foam: MS (ES+) m/z 604.8, 606.7 (M + H)⁺¹; ¹H NMR (DMSO-

d₆) δ 9.65 (s, 1H), 9.50 (s, 1H), 8.88 (s, 1H), 8.38 (s, 1H), 8.22

(d, 1H, J ) 8.2 Hz), 7.97 (dd, 1H, J ) 1.6 Hz, J ) 8.2 Hz), 7.82

(m, 1H), 7.56 (m, 2H), 7.44 (d, 1H, J ) 1.6 Hz), 7.42 (s, 1H),

7.35 (s, 1H), 7.27 (d, 1H, J ) 2.4 Hz), 6.99 (dd, 1H, J ) 2.4

Hz, J ) 8.7 Hz), 6.78 (dt, 1H, J ) 5.6 Hz, J ) 15.3 Hz), 6.67

(d, 1H, J ) 15.3 Hz), 6.58 (d, 1H, J ) 8.7 Hz), 6.18 (t, 1H, J )

5.8 Hz), 4.92 (d, 2H, J ) 5.8 Hz), 4.29 (q, 2H, J ) 6.9 Hz),

3.08 (d, 2H, J ) 5.6 Hz), 2.18 (s, 6H), 1.45 (t, 3H, J ) 6.9 Hz).

Anal. (C₃₅H₃₃ClN₆O₂‚2.5H₂O) C, H, N.

(E)-N-(4-{3-Chloro-4-[(nicotinoyl)amino]anilino}-3-cy-

ano-7-methoxy-6-quinolinyl)-4-(dimethylamino)-2-bu-

tenamide (37). A solution of 226 mg (0.50 mmol) of 35 in 1.5

mL of DMA was treated with 164 mg (0.92 mmol) of nicotinyl

chloride hydrochloride and 0.16 mL (0.92 mmol) of disopro-

pylethylamine. The resulting mixture was stirred at 25 °C for

16 h and at 40 °C for 1 h, treated with aqueous K₂CO₃at 25

°C, stirred for 30 min, and filtered. The yellow solid was

washed with a large volume of H₂O and dried to give 0.26 g

(94%) of product: mp ) 208-218 ) °C (dec); MS (ES+) m/z

556.3,558.3 (M + H)⁺^{1 1}H NMR (DMSO- d₆) δ 10.3 (s, 3H),

;

9.76 (s, 1H), 9.70 (s, 1H), 9.16 (s, 1H), 9.05 (s, 1H), 8.78 (m,

1H), 8.63 (s, 1H), 8.34 (m, 1H), 7.60-7.10 (m, 5H), 6.79 (dt,

1H, J ) 5.6, J ) 15.6 Hz), 6.63 (d, 1H, J ) 15.6 Hz), 4.05 (s,

3H), 3.07 (d, 2H, J ) 5.6 Hz), 2.18 (s, 6H) Anal. (C₂₉H₂₆ClN₇O₃)

C, H, N.

Di(tert-butyl)-2-chloro-4-[(3-cyano-7-methoxy-6-nitro-

4-quinolinyl)amino]phenylimidodicarbonate (33). A stirred

mixture of 5.27 g (20 mmol) of 4-chloro-7-methoxy-6-nitro-

quinoline-3-carbonitrile (1a), 8.22 g (24 mmol) of 28, and 100

mL of i-PrOH was refluxed for 1 h, cooled, and partitioned

between EtOAc and aqueous K₂CO₃. The organic layer was

washed with water, dried, and concentrated. The crude product

was recrystallized from EtOAc-hexane to give 10.3 g (90%)

of yellow solid: mp ) 235-240 °C (dec); MS (ES+) m/z 570.1,

(E)-N-(4-{4-[(Anilinocarbonyl)amino]-3-chloroanilino}-

3-cyano-7-methoxy-6-quinolinyl)-4-(dimethylamino)-2-

butenamide (38). A solution of 226 mg (0.50 mmol) of 35 and

0.18 g (1.5 mmol) of phenyl isocyanate in 1.5 mL of DMA was

stirred at 25 °C for 60 min, diluted with 20 mL of Et₂O and a

few drops of H₂O, and stirred for 30 m. The resulting yellow

solid was filtered off, washed with boiling Et₂O, and dried to

give 220 mg (77%) of product: mp ) 200-210 °C (dec); MS

(ES+) m/z 570.2, 572.2 (M + H)⁺¹; ¹H NMR (DMSO-d₆) δ 9.67

(s, 3H), 9.39 (s, 1H), 8.99 (s, 1H), 8.53 (s, 1H), 8.33 (s, 1H),

8.18 (d, 1H, J ) 8.9 Hz), 7.50-7.15 (m, 7H), 6.78 (dt, 1H, J )

5.7 Hz, J ) 15.3 Hz), 6.64 (d, 1H, J ) 15.3 Hz), 4.04 (s, 3H),

3.08 (d, 2H, J ) 5.7 Hz), 2.18 (s, 6H). Anal. (C₃₀H₂₈ClN₇O₃) C,

H, N.

572.1 (M + H)⁺¹

.

Di(tert-butyl)4-[(6-amino-3-cyano-7-methoxy-4-quino-

linyl)amino]-2-chlorophenylimidodicarbonate (34). A

stirred mixture of 10.2 g (17.9 mmol) of 33, 3.5 g (62.6 mg

atom) of iron powder, 7.2 mL (125 mmol) of HOAc, and 179

mL of MeOH was refluxed for 2 h, cooled, and concentrated to

remove MeOH. The residue was stirred in EtOAc-H₂O and

filtered to remove iron salts. The organic layer was washed

with H₂O, NaHCO₃, and H₂O, dried, and concentrated to give

9.08 g of tan solid, which was sufficiently pure for the next

step: MS (ES+) m/z 540.1, 542.1 (M + H)⁺¹

.

N-{4-[4-(Benzyloxy)-3-chloroanilino]-3-cyano-7-ethoxy-

6-quinolinyl}ethylenesulfonamide (82). To a solution of

300 mg (0.68 mmol) of 72 and 0.34 mL (2.43 mmol) of

(E)-N-[4-(4-Amino-3-chloroanilino)-3-cyano-7-methoxy-

6-quinolinyl]-4-(dimethylamino)-2-butenamide (35). The

1130 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4

Tsou et al.

triethylamine in 34 mL of THF at 0 °C was added 0.212 mL

(2.03 mmol) of 2-chloro-1-ethanesulfonyl chloride (78) over 10

min. The reaction mixture was stirred at 0 °C for 40 min.

Solvent was evaporated. EtOAc, NaOH, and brine were added,

and the solid was filtered. After the filtrate was evaporated,

acetone was added to dissolve the product. It was filtered, and

the acetone solution was evaporated to the crude product as a

yellow gum. It was treated with EtOAc and filtered. The EtOAc

layer was evaporated and the product was precipitated out

with hexane and EtOAc to yield 109 mg (30%) of yellow glass:

mp ) 96-97 °C; HRMS (ESI) m/z 534.119 23, ∆ ) 0.68 mmu

1H), 5.57 (s, 1H), 5.18 (s, 1H), 5.11 (s, 2H), 5.00 (s, 2H), 4.10

(q, 2H, J ) 5.2 Hz), 3.33 (bs, 2H), 1.37 (t, 3H, J ) 5.2 Hz).

Anal. (C₂₉H₂₅ClN₄O₄‚1.2H₂O) C, H, N.

Acknowledgment. The authors thank Drs. Tarek

Mansour, Philip Frost, and Semiramis Ayral-Kaloustian

for their support and encouragement. We also thank

members of the Wyeth Discovery Biology group for in

vivo evaluations and members of the Wyeth Discovery

Analytical Chemistry group for analytical and spectral

determinations.

1

(M + H)⁺¹; H NMR (DMSO-d₆) δ 9.69 (s, 1H), 9.5 (bs, 1H),

8.48 (s, 1H), 8.24 (s, 1H), 7.49 (d, 2H, J ) 7 Hz), 7.44-7.32

(m, 5H), 7.28-7.21 (m, 2H), 6.83 (dd, 1H, J ) 9.9 Hz, J ) 16.4

Hz), 6.01-5.91 (m, 2H), 5.22 (s, 2H), 4.23 (q, 2H, J ) 6.9 Hz),

1.44 (t, 3H, J ) 6.8 Hz). Anal. (C₂₇H₂₃ClN₄O₄S‚1.2 H₂O) C, H,

N.

Supporting Information Available: Experimental pro-

cedures for compounds 2d,k, 7a,c,e,g,j-l,n,o,r-u, 8a,c,k,m,

17a, and 25j,l,p,r,s and elementary analysis data for com-

pounds 7a-v, 8a-u, 21a-d, 25a-w, 31, 32, 36-38, 75-77,

and 82-86. This material is available free of charge via the

Internet at http://pubs.acs.org.

N-{4-[4-(Benzyloxy)-3-chloroanilino]-3-cyano-7-ethoxy-

6-quinolinyl}-2-[(dimethylamino)methyl]acrylamide (83).

An amount of 400 mg (0.9 mmol) of 72 in 4 mL (0.22 mmol) of

NMP was cooled to -15 °C. To a solution of 0.298 g (1.80 mmol)

of 2-dimethylaminomethylacrylic acid hydrochloride³⁵in 15 mL

of CH₃CN at 60 °C was added 0.285 mL (3.24 mmol) of (COCl)₂

for 30 min. The resulting 2-dimethylaminomethylacrylic acid

chloride solution was evaporated to a yellow oil. It was

suspended in 15 mL of CH₃CN and added dropwise to an NMP

solution (4 mL) containing 400 mg (0.90 mmol) of 72 at -15

°C. The yellow suspension was stirred overnight from -15 °C

to room temperature. The reaction mixture was poured into

EtOAc-1 N NaOH solution and stirred for 30 min. The

resulting yellow precipitate was collected and purified on

preparative TLC plates, eluting with 10% MeOH-EtOAc in

1% HOAc to yield 50.5 mg (12%) of light-yellow solid: mp )

References

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1

110-111 °C; H NMR (DMSO-d₆) δ 11.9 (s, 1H), 9.61 (s, 1H),

9.25 (s, 1H), 8.47 (s, 1H), 7.49 (d, 2H, J ) 7.1 Hz), 7.44-7.35

(m, 6H), 7.26-7.18 (m, 3H), 6.19 (s, 1H), 5.65 (s, 1H), 5.21 (s,

2H), 4.31 (q, 2H, J ) 6.9 Hz), 2.3 (s, 6H), 1.48 (t, 3H, J ) 6.9

Hz). Anal. (C₃₁H₃₀ClN₅O₃‚1.3 H₂O) C, H, N.

N-{4-[4-(Benzyloxy)-3-chloroanilino]-3-cyano-7-ethoxy-

6-quinolinyl}-2-(morpholinylmethyl)acrylamide (84). To

a solution of 281 mg (1.35 mmol) of 2-(morpholin-4yl)methyl-

acrylic acid hydrochloride³⁵in 3 mL of CH₃CN at 60 °C was

added 0.106 mL (1.22 mmol) of (COCl)₂. This was stirred at

60 °C for 15 min, evaporated, and cooled to 0 °C. It was added

to a solution of 300 mg (0.68 mmol) of 72 in 3 mL of NMP at

0 °C, and the mixture was stirred overnight from 0 °C to room

temperature. The reaction mixture was stirred with saturated

NaHCO₃. The bright-yellow suspension was filtered and

purified by preparative TLC plates developing with 5%

MeOH-EtOAc to yield 119 mg (30%) of light-yellow solid: mp

) 95-97 °C; HRMS (ESI) m/z 597.2144, ∆ ) 1.37 mmu (M +

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Nilakantan, R.; Shen, R.; Wang, Y.-F.; Greenberger, L. M.; Tsou,

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H)⁺^{1 1}H NMR (DMSO-d₆) δ 11.1 (s, 1H), 9.63 (s, 1H), 9.17 (s,

;

1H), 9.06 (s, 1H), 8.48 (s, 1H), 7.5-7.35 (m, 7H), 7.26-7.17

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JM040159C

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Optimization of 6,7-disubstituted-4-(arylamino)quinoline-3-carbonitriles as orally active, irreversible inhibitors of human epidermal growth factor receptor-2 kinase activity

DOI: 10.1021/jm040159c

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