Inhibitors of Src tyrosine kinase: The preparation and structure-activity relationship of 4-anilino-3-cyanoquinolines and 4-anilinoquinazolines

Article abstract of DOI:10.1016/S0960-894X(00)00493-5

Src is a nonreceptor tyrosine kinase involved in signaling pathways that control proliferation, migration, and angiogenesis. Increased Src expression and activity are associated with an increase in tumor malignancy and poor prognosis. Several quinolines and quinazolines were identified as potent and selective inhibitors of Src kinase activity. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00493-5

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2477±2480
Inhibitors of Src Tyrosine Kinase: The Preparation and
Structure±Activity Relationship of 4-Anilino-3-cyanoquinolines
and 4-Anilinoquinazolines
Yanong D. Wang,* Karen Miller,y Diane H. Boschelli, Fei Ye, Biqi Wu,
M. Brawner Floyd, Dennis W. Powell, Allan Wissner,
Jennifer M. Weber and Frank Boschelli
Chemical Sciences and Oncology, Wyeth-Ayerst Research, Pearl River, NY 10965, USA
Received 20 July 2000; accepted 22 August 2000
AbstractÐSrc is a nonreceptor tyrosine kinase involved in signaling pathways that control proliferation, migration, and angiogen-
esis. Increased Src expression and activity are associated with an increase in tumor malignancy and poor prognosis. Several qui-
nolines and quinazolines were identi®ed as potent and selective inhibitors of Src kinase activity. # 2000 Elsevier Science Ltd. All
rights reserved.
The nontransmembrane or nonreceptor tyrosine kinases
(TKs) have intrinsic kinase activity, are present in the
cytoplasm and nucleus, and participate in diverse signaling
pathways. A large number of nonreceptor TKs have been
identi®ed, including Src, Abl, Jak, Fak, Syk, and ZAP 70.
The Src family consists of Src, Fyn, Lyn, Yes, Lck, Fgr,
Hck, and Blk.1 Src is involved in signaling pathways con-
trolling proliferation, migration, and angiogenesis.
Experimental evidence suggests that increased Src expres-
sion and activity are associated with an increase in tumor
malignancy and poor prognosis.2 Therefore, inhibitors of
Src kinase activity may prove useful for therapeutic
intervention in cancer as well as other proliferative dis-
eases. Several structural types of compounds have been
reported to be inhibitors of Src family kinases,3 includ-
ing the quinazoline 1,4 reported by Rhone-Poulenc
Rorer to be a 500 nM inhibitor of Lck. A similar qui-
nazoline 2, was reported by Parke-Davis to be an inhi-
bitor of epidermal growth factor receptor (EGFr),5 a
receptor tyrosine kinase. Recently Wyeth-Ayerst dis-
closed that 3, the 3-cyanoquinoline analogue of 2, was
also an e€ective inhibitor of EGFr.6 We found that
while 1 had an IC50 of 280 nM for the inhibition of Src
enzymatic activity,7 the corresponding 3-cyanoquinoline
4 had an IC50 of 35 nM. In this paper, we present the
synthesis and structure±activity relationships toward
Src for additional analogues of 1 and 4.
The 4-anilinoquinazoline 6 or 4-anilinoquinolines 7±9
were obtained by the reaction of anilines with 4-chloro-
quinazoline 5a8a or 4-chloroquinolines 5b±5d8b d in 2-
ethoxyethanol using pyridine±HCl as the catalyst as
shown in Scheme 1.6,9 The 3-CH2OH quinoline 10 was
prepared by the reduction of 9 with DIBAL-H. The
phenoxy derivative 11 and benzylamine derivative 12
were prepared by the reaction of the corresponding phe-
nol or benzylamine with 4-chloro-3-cyanoquinoline 5b.
In order to further explore the SAR of the aniline portion
of 1 and 4, several 4-alkoxy-3,5-dimethoxy anilines 15a±
15e were prepared. While the desired anilines could be
obtained by regioselective demethylation of compound
7 followed by alkylation and reduction as documented
in the literature,10 the yield of the demethylation step was
only 10%. To circumvent this low yield, the para-methoxy
group may be directly replaced by di€erent nucleophiles,
*Corresponding author. Tel.: +1-914-732-5253; fax: +1-914-732-
5561; e-mail: wangd@war.wyeth.com
yIntern student from Dartmouth College.
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00493-5

2478
Y. D. Wang et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2477±2480
Scheme 1.
such as alcohols and amines.11 However, the literature
procedure11 used vigorous reaction conditions and often
resulted in unsatisfactory yields of the desired product.
We found a mild and facile reaction condition12 (NaH/
ROH/DMF/THF, room temperature) that e€ected
complete conversion of 13 to the desired products
regioselectively. Furthermore, this reaction condition
tolerates a wide range of alcohol nucleophiles including
primary, secondary and tertiary alcohols. As shown in
Scheme 2, compounds 14a±14e were all generated in
good to excellent yields and were then converted to
compounds 15a±15e by either catalytic hydrogenation
or Fe/HOAc reduction in good yields.
Scheme 3.
methoxy with isopropoxy gave a 3-fold reduction in
activity. We speculate that this is due to a size-limited
hydrophobic interaction of the aniline with the kinase.
We found that the 3-cyanoquinolines were far more
potent Src inhibitors than the corresponding quinazo-
lines. Compound 4 inhibited Src activity with an IC50 of
35 nM. A similar trend was observed to that seen in the
quinazoline series when a bulkier alkoxy group replaced
the 4-methoxy group in the aniline. Changing 4-meth-
oxy to isopropoxy again caused a 3-fold loss of activity.
It is interesting that when either one of the meta or the
para methoxy groups of 4 was removed, the activity
dropped dramatically (from an IC50 of 35 nM for 4 to
IC50s of 980 nM and 950 nM for 7d and 7e, respec-
tively). This is analogous to what was observed by
Rhone-Poulenc Rorer4 for the analogues of 1. There-
fore, all three methoxy groups in the aniline appear to
be required for an energy favored molecular conforma-
tion that can have an e€ective hydrophobic interaction
with the enzyme. Introduction of a bromo or methyl
group into the 2-postion of the aniline (7f and 7g) may
substantially change the optimal conformation, result-
ing in the observed reduced activity.
3-Cyanoquinolines 19a and 19b, which contain water
solubilizing groups, were prepared as shown in Scheme
3.9 Protection of the hydroxy group of commercially
available methyl vanillate followed by nitration and
reduction gave compound 16, which was converted to
the amidine derivative and cyclized with the anion gen-
erated from acetonitrile to provide compound 17.
Chlorination of 11 with POCl3 and deprotection of the
isopropoxy group with AlCl3 both went smoothly to
give 18. Mitsunobu reaction of the phenol with 3-
chloropropanol proceeded readily to give the dichloride.
Subsequent replacement of the 4-chloro by 3, 4, 5-tri-
methoxyaniline and of the alkyl chloride by 4-hydroxy-
piperidine or piperidine provided 19a and 19b.
The 4-anilinoquinazolines and 4-anilino-3-cyanoquino-
lines were evaluated for their inhibitory activity against
Src kinase using an ELISA assay, and the activities are
reported in Table 1.7 In the quinazoline series, 1 showed
moderate activity with an IC50 of 280 nM. However,
only small alkoxy groups were tolerated at the C-4
position of the aniline. When the 4-methoxy group in
the aniline was replaced with a bulkier alkoxy group,
the activity dropped consistently. Replacement of 4-
To further identify the other structural features of 4
required for the inhibition of Src activity, several ana-
logues of 4 were prepared where the 3,4,5-trimethoxy-
phenyl portion was maintained. Substitution of the NH
linker at C-4 with O (11) resulted in an 8-fold loss of
activity, while inserting one methylene group between
NH and trimethoxybenzene (12) led to an even greater
loss of activity. Replacement of the C-3-cyano group
with CO2Et (9) or CH2OH (10) decreased Src inhibitory
activity while the C-3 unsubstituted quinoline 8 was a
moderate Src inhibitor. These results imply that the NH
linker at C-4 and the cyano group at C-3 are both
required for good Src inhibitory activity.
The 3-cyanoquinolines with water solubilizing groups at
C-7, 19a and 19b, were better Src inhibitors than 4,
having IC50s of 5.5 nM and 5.3 nM, respectively. This
Scheme 2.

Y. D. Wang et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2477±2480
2479
Table 1.
Compounds
X
Y
R
IC50 (nM)
1
N
N
N
N
N
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
O
3,4,5-Trimethoxy
3,5-Dimethoxy-4-ethoxy
3,5-Dimethoxy-4-n-butoxy
3,5-Dimethoxy-4-isopropoxy
3,5-Dimethoxy-4-t-butoxy
3,5-Dimethoxy-4-n-dodecanoxy
3,4,5-Trimethoxy
3,5-Dimethoxy-4-ethoxy
3,5-Dimethoxy-4-isopropoxy
3,5-Dimethoxy-4-n-dodecanoxy
3,5-Dimethoxy
280
340
490
830
2700
5200
35
63
110
180
980
950
470
170
200
6a
6b
6c
6d
6e
4
7a
7b
7c
7d
7e
7f
7g
8
N
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-H
C-CO2Et
C-CH2OH
C-CN
C-CN
3,4-Dimethoxy
2-Methyl-3,4,5-trimethoxy
2-Bromo-3,4,5-trimethoxy
3,4,5-Trimethoxy
9
3,4,5-Trimethoxy
3,4,5-Trimethoxy
3,4,5-Trimethoxy
3,4,5-Trimethoxy
>10,000
>10,000
290
10
11
12
NHCH2
>10,000
increased activity may be a result of an additional
interaction of the heteroatom-rich side chain with the
kinase. In an anchorage independent cellular assay13
measuring the inhibition of Src dependent cell pro-
liferation, 19a was more potent than 19b, with IC50s of
1.3 mM and >10 mM, respectively.
A.; Showalter, H. D. H.; Doherty, A. M. J. Med. Chem. 1998,
41, 3276. (d) Dow, R. L.; Bechle, B. M.; Chou, T. T.; God-
dard, C.; Larson, E. Bioorg. Med. Chem. Lett. 1995, 5, 1007.
(e) Missbach, M.; Altmann, E.; Wilder, L.; Susa, M.; Buch-
dunger, E.; Mett, H.; Meyer, T.; Green, J. Bioorg. Med. Chem.
Lett. 2000, 10, 945.
4. Myers, M. R.; Setzer, N. N.; Spada, A. P.; Zulli, A. L.;
Hsu, C. J.; Zilberstein, A.; Johnson, S. E.; Hook, L. E.;
Jacoski, M. V. Bioorg. Med. Chem. Lett. 1997, 7, 417.
5. Fry, D. W.; Kraker, A. J.; McMichael, A.; Ambroso, L. A.;
Nelson, J. M.; Leopold, W. R.; Conners, R. W.; Bridges, A. J.
Science 1994, 265, 1093.
6. (a) Wissner, A.; Johnson, B. D.; Reich, M. F.; Floyd, M.
B.; Kitchen, D. B.; Tsou, H.-R. US Pat. 6002008 A, 1999;
Chem. Abstr. 1998, 129, 302564. (b) Wissner, A.; Berger, D.
M.; Boschelli, D. H.; Floyd, M. B.; Greenberger, L. M.; Gru-
ber, B. C.; Johnson, B. D.; Mamuya, N.; Nilakatan, R.; Reich,
M. F.; Shen, R.; Tsou, H.-R.; Upeslacis, E.; Wang, Y. F.; Wu,
B.; Ye, F.; Zhang, N. J. Med. Chem. 2000, 43, 3244.
7. Src kinase activity was measured in an ELISA format
(Roche Diagnostics Tyrosine Kinase Assay Kit). Src (human
c-Src protein, 3 units/reaction; Upstate Biotechnologies),
reaction bu€er (50 mM Tris±HCl pH 7.5, 10 mM MgCl2, 0.1
mM EGTA, 0.5 mM Na3VO4) and cdc2 substrate peptide
were added to compound and incubated at 30 ꢀC for 10 min.
The reaction was started by the addition of ATP to a ®nal
concentration of 100 mM, incubated at 30 ꢀC for 1 h and
stopped by addition of EDTA. Instructions from the manu-
facturer were followed for subsequent steps. Compounds were
tested in duplicate and the value given is an average of two
determinations with the exception of 4, which was tested seven
times resulting in values of 15, 90, 21, 36, 20, 27, and 40 nM
(average=35 nM).
In an ELISA study examining the e€ect of increasing the
concentration of ATP (100, 500, 1000, and 5000 mM), the
IC50 values for the inhibition of Src activity by 4 steadily
increased (34, 108, 190, and 650 nM, respectively), sug-
gesting that 4 is an ATP competitive inhibitor. In addi-
tion, 4 was much less active in inhibiting a panel of
other kinases including EGFr6b (IC50=1.5 mM). How-
ever, activity was seen with 19a in an assay measuring
Fyn dependent cell proliferation (IC50=6.7 mM).14
Therefore, while these compounds may be selective for
the Src family kinases over other kinase families, they
appeared to be marginally selective for Src over other
Src family members. Further work towards identifying
more potent and selective Src inhibitors is in progress.
References and Notes
1. Schwartzberg, P. L. Oncogene 1998, 17, 1463.
2. Biscardi, J. S.; Tice, D. A.; Parsons, S. J. Adv. Cancer Res.
1999, 76, 61.
3. (a) Hanke, J. H.; Gardner, J. P.; Dow, R. L.; Changelian,
P. S.; Brissette, W. H.; Weringer, F. J.; Pollok, B. A.; Con-
nelly, P. A. J. Biol. Chem. 1996, 271, 695. (b) Missbach, M.;
Jeschke, M.; Feyen, J.; Muller, K.; Glatt, M.; Green, J.; Susa,
M. Bone (NY) 1999, 24, 437. (c) Klutchko, S. R.; Hamby, J.
M.; Boschelli, D. H.; Wu, Z.; Kraker, A. J.; Amar, A. M.;
Hartl, B. G.; Shen, C.; Klohs, W. D.; Steinkampf, R. W.;
Driscoll, D. L.; Nelson, J. M.; Elliott, W. L.; Roberts, B. J.;
Stoner, C. L.; Vincent, P. W.; Dykes, D. J.; Panek, R. L.; Lu,
G. H.; Major, T. C.; Dahring, T. K.; Hallak, H.; Bradford, L.
8. (a) Preparation of 5a: Bridges, A. J.; Zhou, H.; Cody, D. R.;
Rewcastle, G. W; McMichael, A.; Showalter, H. D. H; Fry, D.
W.; Kraker, A. J.; Denny, W. A. J. Med. Chem. 1996, 39, 267.
(b) Preparation of 5b: see ref 6a. (c) Preparation of 5c: Kubo,
K.; Shimizu, T.; Ohyama, S.; Murooka, H.; Nishitoba, T.;
Kato, S.; Kobayashi, Y.; Yagi, M.; Isoe, T.; Nakamura, K.
Osawa, T.; Izawa, T. Bioorg. Med. Chem. Lett. 1997, 7, 2935.

2480
Y. D. Wang et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2477±2480
(d) Preparation of 5d: Marquez, V. E.; Li, Z.; Bolen, J. B.;
Stefanova, I.; Horake, I. J. Med. Chem. 1993, 36, 425.
9. The ®nal compounds were analytically pure as determined
was separated and the aqueous layer was extracted with ethyl
acetate. The combined organics were dried over Na2SO4, con-
centrated and column chromatographed to give 473 mg (85%
yield) of compound 14c.
1
by elementary analysis, H NMR and MS.
10. Tepe, J. J.; Madelengoitia, J. S.; Miller, K. E.; Werbovetz,
K. W.; Spoors, P. G.; Macdonald, T. L. J. Med. Chem. 1996,
39, 2188.
13. Anchorage independent Src cellular assay: Rat2 ®bro-
blasts were stably transformed with a pCDNA plasmid (Invi-
trogen) containing a v-Src:human c-Src fusion under control of
the CMV promoter. The v-Src/Hu c-Src fusion gene has the
catalytic domain of human c-Src fused with the remainder of v-
Src (the amino terminal half of v-Src and the v-Src C-terminal
tail). Ultra-low cluster plates (Costar No. 3474), which do not
support cell attachment, were seeded with 10,000 cells per well
on day one. Compound was added on day two and MTS
reagent (Promega) was added on day ®ve to measure relative
cell number according to manufacturer speci®cations.
11. (a) Oliverio, G. Gazz. Chim. Ital. 1943, 73, 181. (b) Cer-
vello, J.; Figueredo, M.; Marquet, J.; Moreno-Nanas, M.;
Bertran, J.; Lluch, J. M. Tetrahedron Lett. 1984, 25, 4147.
12. The general procedure of regioselective substitution of 13:
NaH (276 mg, 6.9 mmol) was suspended in THF:DMF (1:1, 5
mL) and isopropanol (625 mg, 10.4 mmol) in THF:DMF (1:1,
5 mL) was added dropwise. The resulting mixture was stirred
at room temperature for 40 min. Compound 13 (500 mg, 2.3
mmol) in DMF (15 mL) was added via syringe. The reaction
mixture was stirred at room temperature for 15 min and
quenched with saturated aqueous NH4Cl. The organic layer
14. This assay is similar to the anchorage independent Src cel-
lular assay, except that a v-Src:human fynB fusion gene was
employed. An IC50 >1 0 mM was obtained for 19b in this assay.