The design, synthesis and activity of non-ATP competitive inhibitors of pp60(c-src) tyrosine kinase. Part 1: Hydroxynaphthalene derivatives

Article abstract of DOI:10.1016/S0960-894X(00)00039-1

A series of hydroxynaphthalene pp60(c-src) non-peptide inhibitors was designed, using the crystal structure of the insulin receptor tyrosine kinase as a qualitative model, to target the peptide substrate binding site. Representative inhibitors were shown to bind non-competitively with respect to ATP. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00039-1

Bioorganic & Medicinal Chemistry Letters 10 (2000) 477±481
The Design, Synthesis and Activity of Non-ATP Competitive
Inhibitors of pp60c-src Tyrosine Kinase.
Part 1: Hydroxynaphthalene Derivatives
Thomas H. Marsilje,y Karen L. Milkiewicz and David G. Hangauer*
Department of Medicinal Chemistry, School of Pharmacy, State University of New York at Bu€alo, Bu€alo, NY, 14260-1200, USA
Received 10 November 1999; accepted 7 January 2000
AbstractÐA series of hydroxynaphthalene pp60c-src non-peptide inhibitors was designed, using the crystal structure of the insulin
receptor tyrosine kinase as a qualitative model, to target the peptide substrate binding site. Representative inhibitors were shown to
bind non-competitively with respect to ATP. # 2000 Elsevier Science Ltd. All rights reserved.
Protein tyrosine kinases (PTKs) are signal transduction
enzymes that have been shown to be extensively
involved in the formation and maintenance of cancer.
Consequently, an intense e€ort is currently underway to
develop non-peptide inhibitors for individual PTKs,1
some of which have advanced to clinical trials. pp60c-src
is a prototype for the nonreceptor PTK's and is an
emerging target for drugs that may be useful in treating
a wide range of cancers.2,3 A knock-out of the `src' gene
in mice led to only one defect; osteoclasts that do not
resorb bone. However, this lone defect can be prevented
by inserting a src gene that produces mutant pp60c-src
wherein the enzymatic kinase activity has been lost.4
These and other reports suggest that inhibition of pp60c-src
could provide broad anti-cancer activities without
inducing serious toxicity.
inhibitors for the more unique peptide substrate binding
sites can provide a greater level of con®dence that
acceptable selectivity has been achieved. A second
potential advantage of this alternate approach is that
inhibitors binding ca. 1000-fold weaker than ATP-com-
petitive inhibitors could be equally potent in the intra-
cellular environment wherein they may be competing
with mM concentrations of external protein substrates
rather than mM levels of ATP.1
The utilization of naphthalene-based peptide substrate
mimics as non-ATP competitive PTK inhibitors was
®rst introduced by Saperstein et al.6 in their rational
design of non-peptide insulin receptor (IRTK) and
epidermal growth factor receptor (EGFRTK) PTK
inhibitors.6 This concept was later extended into the
analogous quinoline, isoquinoline, and iminochromene
rings by Burke et al.7 and Huang et al.8 providing non-
ATP competitive p56lck and pp60c-src tyrosine kinase
inhibitors.
The vast majority of known PTK non-peptide inhibitors
bind in the highly conserved ATP substrate site.1
Nevertheless, some of these ATP competitive PTK
inhibitors have been shown to have good selectivity
when tested against a small panel of isolated protein
kinases. However, it is estimated that there are
approximately 2000 di€erent protein kinases (in addi-
tion to other ATP-utilizing proteins) in humans,5 all of
which utilize ATP as the second substrate. Testing a
given inhibitor against a full panel of isolated kinases is
currently not feasible. Consequently, targeting PTK
In the current study, the crystal structure of the auto-
inhibited human IRTK catalytic domain9 was used to
carry out qualitative molecular modeling studies
(SYBYLTM, 6.4, Tripos Inc., St. Louis) wherein a
naphthalene ring was superimposed upon the IRTK Tyr
1,162. The IRTK region containing Tyr 1,162 folds
back into the active site, with Tyr 1,162 positioned
analogous to a phosphorylatable Tyr in a peptide sub-
strate, thereby autoinhibiting the tyrosine kinase. This
superimposition indicated that an amide carbonyl
should be placed at the 2-position (Scheme 1) of the
naphthalene ring to mimic the Tyr 1,162 carbonyl and a
*Corresponding author. Tel.: +1-716-645-6299; fax: +1-716-645-
2393; e-mail: hangauer@acsu.bu€alo.edu
yCurrent aliation: The Scripps Research Institute, Dept. of Chem-
istry, BCC-483, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, USA.
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00039-1

478
T. H. Marsilje et al. / Bioorg. Med. Chem. Lett. 10 (2000) 477±481
Scheme 1.
hydroxyl group should be positioned at the 6-position
to mimic the Tyr 1,162 hydroxyl group. These modeling
studies also indicated that a hydroxyl group at the 3-
position could mimic the Tyr 1,162 NH.
N-phenyl (X=0) and N-benzyl (X=1) amides were
chosen based upon the increase in pp60c-src inhibitor
potency observed with iminochromene analogues con-
taining appended hydroxy N-phenyl amide side-chains.8
Analogues wherein the 6-hydroxyl group was either
deleted or moved were also prepared to determine if a
drop in potency occurs as predicted from the modeling
studies.
In order to test these design concepts experimentally,
the 2-position carbonyl group was appended as either a
methyl ester or as a series of amides (Table 1). The hydroxy
Table 1. pp60c-src Inhibitory activity of hydroxynaphthalene derivatives and select published inhibitorsa±c
Compound
R1
R2
R3
R4
R5
R6
R7
X
% Inhibition at 100 mM (std. dev.)
IC50 (mM)
1a
1b
1c
1d
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
2r
2s
2t
OH
OH
OH
NH2
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
OH
H
H
H
OH
H
H
H
H
H
H
OH
OH
OH
OH
H
H
H
H
H
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
N/A
N/A
N/A
N/A
OH
H
N/A
N/A
N/A
N/A
H
OH
H
H
H
OH
H
H
OMe
H
H
H
H
H
H
H
H
OH
H
N/A
N/A
N/A
N/A
H
N/A
N/A
N/A
N/A
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
0
1
5 (‹2)
47 (‹3)
19 (‹6)
Inactive
12 (‹4)
51 (‹1)
60 (‹7)
14 (‹2)
39 (‹5)
89 (‹1)
23 (‹5)
56 (‹1)
33 (‹5)
35 (‹8)
13 (‹3)
14 (‹2)
Inactive
4 (‹7)
41 (‹2)
49 (‹4)
42 (‹2)
55 (‹3)
42 (‹3)
68 (‹5)
40 (‹3)
45 (‹5)
30 (‹15)
41 (‹2)
37 (‹5)
22 (‹3)
43 (‹3)
n.t.
n.t.
n.t.
n.t.
n.t.
150
n.t.
n.t.
n.t.
16
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
H
OH
OH
OH
OH
H
H
H
H
OH
OH
OH
OH
H
H
H
OH
OH
H
OH
OH
H
H
OH
H
H
OMe
H
OMe
H
OH
H
H
OH
OH
H
OMe
H
OMe
H
OMe
H
OH
H
H
OH
H
H
OH
OH
H
H
OH
OH
H
H
H
H
H
H
H
H
H
H
H
OH
OH
OH
H
H
OH
OH
H
H
H
H
H
OH
OH
H
OH
OH
OH
2u
2v
n.t.
n.t.
H
H
0
Iminochromene 9TA
Piceatannol
ST-638
Emodind
Lit8: 0.118
Lit13: 66 (lck)
Lit14: 18
Lit15: 38
Tyrophostin A47
aThe previously described assay procedure11 was used with the following assay components, ®nal concentrations and conditions: 50.0 mM MOPS,
4.02 mM MgCl2, 6.00 mM K3 citrate (used as a Mg2+ bu€er to stabilize the free Mg2+ at 0.5 mM), 99.0 mM KCl, 10.0 mM 2-mercaptoethanol, 198
mM ATP, 19.8 mM ADP, 10 U full length human puri®ed recombinant pp60c-src (Upstate Biotechnology Inc.), 2.00 mM RR-SRC, 4.0 % DMSO,
pH 7.2, 37 ꢀC. These overall assay conditions have been shown12 to reproduce the intracellular conditions of pH, temp., free Mg2+ (0.5 mM), ionic
strength, osmolality, ATP/ADP and reduction potential.
bAll new compounds were characterized by proton NMR, EI or FAB(+) MS and are pure by TLC.
cN/A=Not applicable, n.t.=not tested.
dATP-competitive.

T. H. Marsilje et al. / Bioorg. Med. Chem. Lett. 10 (2000) 477±481
479
The series of 2-carbonyl-3,5-dihydroxy naphthalene
inhibitors (1a, 2a±2d, 2i±2l, 2o±2p) and 2-carbonyl-3,7-
dihydroxy naphthalene inhibitors (1c, 2t±2u) were syn-
thesized from commercially available (Aldrich) 3,5-
dihydroxy-2-naphthoic acid and 3,7-dihydroxy-2-naph-
thoic acid, respectively. The methyl esters 1a and 1c
were obtained by re¯uxing the respective acid starting
materials for 48 h in methanol pre-saturated with HCl
gas. The amides (2a±2d, 2i±2l, 2o±2p, 2t±2u) were syn-
thesized by coupling the respective carboxylic acid with
commercially available (Aldrich or Lancaster) amines
using one of two methods. The ®rst method utilized the
NBS/PPh3 methodology as described by Froyen.10 The
second method utilized IIDQ (Aldrich) as the coupling
reagent. The carboxylic acid was ®rst reacted with
1.0 equiv IIDQ in anhydrous DMF at rt for 24 h. The
respective amine (2.0 equiv) was then added neat and
the reaction was heated to 80 ꢀC for 2±6 h. After aqu-
eous work up, puri®cation was achieved by silica gel
chromatography and precipitation from CH2Cl2/hex-
ane, followed by preparative C-18 RP-HPLC (CH3CN/
H2O), if necessary. The benzyl amines were commer-
cially available only as their corresponding hydroxyl
protected methyl ethers. Consequently, after amide
formation, the hydroxyl groups were deprotected by
treatment with 6 eq. BBr3 in DCM for 1 min at 78 ꢀC
followed by 1 h at rt.
order to accurately determine the relative potency of
our newly designed class of pp60c-src inhibitors, we
also tested the inhibitory activity of four previously
published, non-ATP competitive PTK inhibitors.
Piceatannol, ST-638, and Tyrphostin A47 were chosen
because they are commercially available (Sigma or Cal-
biochem), and are representative of the spectrum of
known non-ATP competitive PTK inhibitors. Emodin
(Calbiochem) is ATP-competitive when analyzed with
the tyrosine kinase p56lck. Prior to this communication,
iminochromene 9TA was the most potent non-ATP
competitive pp60c-src inhibitor reported.8 Since imino-
chromene 9TA was not commercially available, it was
synthesized using a novel route,16 and included in this
study.
The inhibitory activities shown in Table 1 for com-
pounds 1a±d and 2a±2v were determined using puri®ed,
full length, human recombinant pp60c-src. Due to the
number of compounds tested, and the associated cost,
their rank order potencies were ®rst determined at a
constant inhibitor concentration (100 mM). As predicted
by the modeling studies, based upon analogy to the
IRTK Tyr 1,162 hydroxyl group, a preference for posi-
tioning the naphthalene hydroxyl group on carbon 6
versus 5 or 7 was observed in both the ester (1b, 47%
versus 1a, 5% & 1c, 19%) and amide (e.g. 2f, 89%
versus 2b, 51% and 2t, 68%) series. The prediction that
attaching a hydroxyl group at naphthalene carbon 3
(mimicking the Tyr 1,162 NH) would improve potency
was also con®rmed (2f, 89% versus 2v, 45%). Finally,
the prediction that extending the inhibitor as an amide
at the 2 position (mimicking the peptide bond) could
further improve potency was con®rmed as well (e.g. 2f,
89% versus 1b, 47%).
The series of 2-carbonyl, 3,6-dihydroxy naphthalene
inhibitors (1b, 2e±2h, 2m±2n, 2q±2s) was synthesized
from 3,6-dihydroxy-2-naphthoic acid 6 using the meth-
ods described above. The synthesis of intermediate 6
that was developed is shown in Scheme 2 beginning with
commercially available 2,7-dihydroxynaphthalene
(Aldrich).
3
Compound 1d was synthesized from 3-amino-2-naph-
thoic acid (Aldrich) by reaction with TMS-diazo-
methane in DCM at room temperature. Compound 2v
was synthesized from 6-hydroxy-2-naphthoic acid
(Aldrich) using the amidation method described by
Froyen.10
The data provided in Table 1 demonstrate that moving
the hydroxyl group from the optimal 6 position to the
adjacent naphthalene carbon 5 results in a di€erent
structure activity pro®le with regard to the optimal
concurrent positioning of the hydroxyl group(s) in the
amide side chain (e.g. 2f/2g versus 2b/2c). Also of note is
the replacement of the amide side chain hydroxyl group
with a corresponding methoxy group in compounds 2i±
2n. In the case of the N-phenyl amides (2i±2j), their
Kinase assay conditions have been shown to in¯uence
the measured inhibitory activity.1 Consequently, in
Scheme 2.

480
T. H. Marsilje et al. / Bioorg. Med. Chem. Lett. 10 (2000) 477±481
activity, relative to the corresponding hydroxy amides
(2b±2c), was not reduced as signi®cantly as in the case of
the N-benzyl amides (2k±2n versus 2o±2q, 2s). This
suggests that in the benzyl derivatives, the amide side
chain hydroxyl groups either interact with the enzyme
as hydrogen bond donors, or the methoxy groups are
too large to ®t in the binding site.
References and Notes
1. Lawrence, D. S.; Nui, J. Pharmacol. Ther. 1998, 77, 81.
2. Levitzki, A. Anti-Cancer Drug Des. 1996, 11, 175.
3. Milkiewicz, K. L.; Marsilje, T. H.; Woodworth, R. P. Jr;
Bifulco, N., Jr; Hangauer, M. J., Hangauer, D. G., Bioorg.
Med. Chem. Lett., 2000, 10, 483.
4. Schwartzberg, P. L.; Xing, L.; Ho€man, O.; Lowell, C. A.;
Garrett, L.; Boyce, B. F.; Varmus, H. E. Genes Dev. 1997, 11,
2835.
A more quantitative analysis of the selectivity for
positioning a hydroxyl group on carbon 6 versus 5 is
provided by comparing the IC50's of 2f (16 mM) versus
2b (150 mM), respectively. These results also con®rm
that a drop in % inhibition from ca. 90 to 50% repre-
sents an order of magnitude di€erence in potency, as
expected. Similarly, a drop in % inhibition from ca. 50
to 10% would represent another order of magnitude
di€erence in potency.
5. Hunter, T. Semin. Cell. Biol. 1994, 5, 367.
6. Saperstein, R.; Vicario, P. P.; Strout, H. V.; Brady, E.; Sla-
ter, E. E.; Greenlee, W. J.; Ondeyka, D. L.; Patchett, A. A.;
Hangauer, D. G. Biochemistry 1989, 28, 5694.
7. Burke, T. R.; Lim, B.; Marquez, V. E.; Li, Z-H.; Bolen, J.
B.; Stefanova, I.; Horak, I. D. J. Med. Chem. 1993, 36, 425.
8. Huang, C.-K.; Feng-Ying, W.; You-Xi, A. Bioorg. Med.
Chem. Lett. 1995, 5, 2423.
9. Hubbard, S. R.; Wei, L.; Ellis, L.; Hendrickson, W. A.
Nature 1994, 372, 746.
A direct comparison of the most potent inhibitor from
this series, compound 2f, with the ®ve previously repor-
ted PTK inhibitors shown in Table 1 demonstrates that,
under these assay conditions, 2f is more potent by one
to two orders of magnitude. Interestingly, iminochro-
mene 9TA was previously reported8 to have an IC50 of
118 nM against pp60c-src, and was the most potent
known non-ATP competitive pp60c-src inhibitor, but
under the current assay conditions only a 30% inhibi-
tion at 100 mM was observed. These results re-empha-
size1 the importance of comparing protein kinase
inhibitors under identical assay conditions.
10. Froyen, P. Tetrahedron Lett. 1997, 38 (30), 5359.
11. Lai, J. H.; Marsilje, T. H.; Choi, S.; Nair, S. A.; Hangauer,
D. G. J. Peptide Res. 1998, 51, 271.
12. Choi, S. Ph.D. Thesis, SUNY at Bu€alo, Bu€alo, NY,
1995, and Warren, S. D.; Choi, S.; Hangauer, D. G. manu-
script in preparation.
13. Thakkar, K.; Geahlen, R. L.; Cushman, M. J. Med. Chem.
1993, 36, 2950.
14. Shiraishi, T.; Owada, M. K.; Tatsuka, M.; Yamashita, T.;
Watanabe, K.; Kakunaga, T. Cancer Res. 1989, 49, 2374.
15. Fredenhagen, A.; Mett, H.; Meyer, T.; Buchdunger, E.;
Regenass, U.; Roggo, B. E.; Petersen, F. J. Antibiotics 1995,
48, 1355.
16. 3-Aminophenol was converted to the corresponding
TBDMS ether (1.1 equiv TBDMS-Cl, 1.1 equiv DIEA, 5 mol%
DMAP, DMF, 24 h, rt, 71%). The resulting aniline was
coupled using 2.0 equiv of cyanoacetic acid (1.1 equiv EDCI,
1.1 equiv TEA, DMF, 18 h, 75 ꢀC, 70%). Condensation of the
resulting amide with 1.2 equiv. of 2,3-dihydroxybenzaldehyde
(cat. piperidine, abs. EtOH, 2 h, 60 ꢀC) followed by deprotec-
tion (1.1 equiv. TBAF, THF, 15 m, 43% overall) gave imino-
chromene 9TA with satisfactory elemental, FAB(+)MS and
1H NMR analysis after puri®cation by ¯ash chromatography
(10:1, DCM:MeOH).
17. The % inhibition was measured using ATP concentrations
of 200, 500 and 1000 mM while holding the inhibitor con-
centration constant. If the inhibitor is directly competing with
ATP, then this 5-fold overall increase in [ATP] is equivalent to
decreasing the inhibitor concentration 5-fold in terms of the
e€ect on % inhibition. Consequently the % inhibition should
decrease to the value observed in the IC50 dose±response curve
(obtained with 200 mM ATP) for 1/5 of the set inhibitor con-
centration used in this experiment if direct competition with
ATP is occurring. For inhibitor 2f (set at 25 mM) a 62% (‹5),
54% (‹3) and 50% (‹1) inhibition at 200, 500 and 1000 mM
ATP, respectively, was obtained whereas the level of inhibition
should have dropped to ca. 20% at 1000 mM ATP if direct
competition with ATP were occurring. Similarly, for inhibitor
2b (set at 300 mM) an 84% (‹1), 81% (‹1) and 77% (‹2)
inhibition at 200, 500 and 1000 mM ATP, respectively, was
obtained.
A goal of these studies was to obtain non-peptide pp60c-src
inhibitors which do not compete with ATP. Conse-
quently the % inhibition of pp60c-src by 2f and 2b at
constant inhibitor concentrations was monitored as a
function of increasing [ATP] up to a cellular mimetic 1
mM level. Since the [ATP] had little e€ect on the %
inhibition, both 2f and 2b are non-competitive inhibi-
tors with respect to ATP under these assay conditions.17
The high cost of many kinases has stimulated other
researchers to monitor inhibitor potency as a function
of increasing [ATP] for the same purpose.6,7,18±21
In summary, structure-based design has produced a
series of hydroxynaphthalene pp60c-src non-peptide
inhibitors which do not compete with ATP. Results
with compounds from this series in cell-based assays, as
well as detailed kinetic studies under various assay con-
ditions, will be reported in due course. An extension of
these design concepts from the naphthalene sca€old to
an indole sca€old is reported in the following paper.
Acknowledgements
We gratefully acknowledge the Kapoor Foundation for
providing the ®nancial support (to D.G.H.) for this
work. T.H.M. and K.L.M gratefully acknowledge the
American Foundation for Pharmaceutical Education
for support as AFPE Fellows. Compound 1c was syn-
thesized by Donald E. Higgs, SUNY at Bu€alo.
18. Davis, P. D.; Hill, C. H.; Keech, E.; Lawton, G.; Nixon, J.
S.; Sedgwick, A. D.; Wadsworth, J.; Westmacott, D.; Wilk-
inson, S. E. FEBS Lett. 1989, 259 (1), 61.
19. Davis, P. D.; Elliott, L. H.; Harris, W.; Hill, C. H.; Hurst,
S. A.; Keech, E.; Kumar, M. K. H.; Lawton, G.; Nixon, J. S.;
Wilkinson, S. E. J. Med. Chem. 1992, 35, 994.

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20. Faltynek, C. R.; Wang, S.; Miller, D.; Mauvais, P.; Gau-
vin, B.; Reid, J.; Xie, W.; Hoekstra, S.; Juniewicz, P.; Sarup,
J.; Lehr, R.; Sawutz, D. G.; Murphy, D. J. Enzyme Inhibition
1995, 9, 111.
21. Sawutz, D. G.; Bode, D. C.; Briggs, G. M.; Reid, J. R.;
Canni€, P.; Caldwell, L.; Faltynek, C. R.; Miller, D.; Dunn, J.
A.; Garavilla, L.; Guiles, J. W.; Weigelt, C.; Michne, W.; Treas-
urywala, A. M.; Silver, P. J. Biochem. Pharmacol. 1996, 51, 1631.