Design, synthesis, and biological evaluation of a series of lavendustin A analogues that inhibit EGFR and Syk tyrosine kinases, as well as tubulin polymerization

Article abstract of DOI:10.1021/jm000387g

A series of N-alkylamide analogues of the lavendustin A pharmacophore were synthesized and tested for inhibitiofi of the epidermal growth factor receptor (EGFR) protein tyrosine kinase and the nonreceptor protein tyrosine kinase Syk. Although several comp

Full text of DOI:10.1021/jm000387g

J . Med. Chem. 2001, 44, 441-452
441
Design , Syn th esis, a n d Biologica l Eva lu a tion of a Ser ies of La ven d u stin A
An a logu es Th a t In h ibit EGF R a n d Syk Tyr osin e Kin a ses, a s Well a s Tu bu lin
P olym er iza tion
Fanrong Mu,† Stephanie L. Coffing,† David J . Riese II,† Robert L. Geahlen,† Pascal Verdier-Pinard,‡
Ernest Hamel,‡ J ill J ohnson,§ and Mark Cushman*,†
Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmacal Sciences, Purdue
University, West Lafayette, Indiana 47907, Screening Technologies Branch, Developmental Therapeutics Program, Division of
Cancer Treatment and Diagnosis, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick,
Maryland 21702, and Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer
Institute, National Institutes of Health, Rockville, Maryland 20852
Received September 5, 2000
A series of N-alkylamide analogues of the lavendustin A pharmacophore were synthesized and
tested for inhibition of the epidermal growth factor receptor (EGFR) protein tyrosine kinase
and the nonreceptor protein tyrosine kinase Syk. Although several compounds in the series
were effective inhibitors of both kinases, it seemed questionable whether their inhibitory effects
on these kinases were responsible for the cytotoxic properties observed in a variety of human
cancer cell cultures. Accordingly, a COMPARE analysis of the cytotoxicity profile of the most
cytotoxic member of the series was performed, and the results indicated that its cytotoxicity
profile was similar to that of antitubulin agents. This mechanism of action was supported by
demonstrating that most compounds in the series were moderately effective as inhibitors of
tubulin polymerization. This suggests that the lavendustin A analogues reported here, as well
as some of the previously reported lavendustin A analogues, may be acting as cytotoxic agents
by a mechanism involving the inhibition of tubulin polymerization.
In tr od u ction
The protein tyrosine kinases (PTKs) play critical roles
in many of the signal transduction processes that control
cell growth, differentiation, mitosis, and death. They are
therefore important targets for the development of
therapeutic agents for the treatment of diseases that
are characterized by uncontrolled cell proliferation, such
as cancer and psoriasis.1-3 Fractionation of a butyl
acetate culture extract from Streptomyces griseolaven-
analogues.6-15 The ester derivative 3 and the amide 4
dus led to the isolation of the novel PTK inhibitor
lavendustin A.4 Structure 1 was proposed for lavendus-
were both found to compare favorably with the laven-
dustin A pharmacophore 2. In particular, the ester 3
was as potent as 2 versus the EGFR tyrosine kinase in
a cell-free assay, but 3 was more potent than 2 as an
inhibitor of EGF-stimulated DNA synthesis in ER 22
cells.10 Furthermore, the amide 4 was reported to be
more potent than 2 as an inhibitor of EGFR tyrosine
kinase in a cell-free system.13 In view of these results,
and as an extension of our earlier work on the solid-
phase synthesis of lavendustin A and derivatives,15 we
decided to synthesize and evaluate a series of amide
derivatives of lavendustin A having the general struc-
ture 5. These compounds have been tested versus both
the receptor PTK EGFR in BaF3 mouse lymphoid cells
as well as the nonreceptor PTK Syk in a cell-free system.
In addition, the new compounds in this series were
evaluated as cytotoxic agents using a variety of cultured
human cancer cell lines, and the cytotoxicity profile of
at least one agent, 13b, was found to be similar to that
of antitubulin drugs when analyzed using the COM-
PARE algorithm.16-18 Since the COMPARE program
was developed as a predictor of mechanism of action,
these results led to the hypothesis that the lavendustin
1
tin A on the basis of H and 13C NMR data, and this
tentative assignment was confirmed by total synthesis.4
Lineweaver-Burke analysis carried out in the presence
of varying concentrations of ATP and the substrate
indicated that the inhibition was competitive with
respect to ATP and noncompetitive with respect to the
substrate when tested on the epidermal growth factor
receptor (EGFR) tyrosine kinase,4 although subsequent
studies showed that lavendustin A can function as a
hyperbolic mixed-type inhibitor with respect to both
ATP and substrate.5 It was also determined that the
lavendustin A fragment 2 was as potent as the parent
compound 1, suggesting that 2 is the biologically active
“pharmacophore” of lavendustin A.
These initial reports have stimulated work on the
synthesis of lavendustin A itself and the synthesis and
biological investigation of a variety of lavendustin
* To whom correspondence should be addressed. Tel: 765-494-1465.
Fax: 765-494-1414. E-mail: cushman@pharmacy.purdue.edu.
† Purdue University.
‡ Frederick Cancer Research and Development Center.
§ National Institutes of Health.
10.1021/jm000387g CCC: $20.00 © 2001 American Chemical Society
Published on Web 01/03/2001

442 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3
Mu et al.
mented or against a single compound. The result of the
analysis is a list of compounds in rank order and
Pearson correlation coefficients, which provide an in-
dication of the similarity of the patterns of cell line
responses. Correlation coefficients of g0.60 are generally
considered to be meaningful. For example, when a
COMPARE is performed using paclitaxel as the seed,
the correlation coefficient for vinblastine sulfate (an-
other tubulin-interactive agent) is 0.88, whereas the
correlation coefficient for phyllanthoside (a topoi-
somerase II inhibitor) is 0.422. The hypothetical mech-
anism of action suggested by COMPARE analysis can
then be confirmed by laboratory testing. The reason that
COMPARE analysis is a predictor of mechanism of
action is that biological targets are expressed to a
different extent in different cell lines, so that compounds
which interact with a single biological target will have
similar cytotoxicity patterns when tested in these cell
lines.26
A COMPARE analysis was performed using 13b as
a seed. The top 20 compounds in the COMPARE
analysis included 9 taxanes, 3 colchicine analogues, 2
combretastatins, and vinblastine, all of which interact
with tubulin. Representative correlation coefficients
between 13b and various tubulin-interactive drugs were
docetaxel, 0.790; vinblastine sulfate, 0.761; paclitaxel,
0.703; and maytansine, 0.701.
A analogues in our series were functioning as inhibitors
of tubulin polymerization, and the appropriate assays
were performed to test this hypothesis.
Th e COMP ARE Algor ith m
The COMPARE program was developed at the De-
velopmental Therapeutics Program, National Cancer
Institute (NCI), to interpret the emerging data from the
60-cell line in vitro human cancer cell cytotoxicity screen
and display it in way that would facilitate comparison
of the different patterns of dose-response curves pro-
duced by different cytotoxic agents. Paull and colleagues
developed the “mean graph” representation of the
screening data in which the mean concentration affect-
Syn th esis
The synthesis of the series of lavendustin A analogues
13a -m is a modification of a previously published route
used to prepare a series of lavendustin A hydroxamic
acid derivatives (Scheme 1).10 Treatment of commer-
cially available 5-aminosalicylic acid (7) with di-tert-
butyl dicarbonate and triethylamine in aqueous dioxane
afforded the Boc-protected intermediate 8. Reaction of
8 with the appropriate primary amines 9a -m in the
presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodi-
imide hydrochloride (EDCI), 1-hydroxybenzyltriazole
hydrate (HOBt), and triethylamine in dry DMF yielded
the series of amides 10a -m . Deprotection of the inter-
mediates 10a -m with a mixture of dichloromethane
and trifluoroacetic acid resulted in the formation of the
primary amines 11a -m . Reaction of 11a -m with 2,5-
dihydroxybenzaldehyde (12) afforded the corresponding
Schiff bases, which were reduced with sodium cy-
anoborohydride to provide the desired lavendustin A
analogues 13a -m .
As outlined in Scheme 2, a variation of this route was
executed in order to obtain the congeners 18a -c, in
which the aniline part of the molecule is substituted in
the para position with various amides. Similarly, the
lavendustin A analogue 23, having a â-phenethylamide
substituent in the meta position relative to the aniline
nitrogen but lacking an adjacent phenolic hydroxyl
group, was prepared as shown in Scheme 3.
ing all 60 cell lines at three levels of effect (GI50
)
concentration causing 50% inhibition of growth; TGI )
concentration causing total inhibition of growth; LC50
) concentration causing 50% cell kill) is plotted in the
midline of the graph and the behavior of each individual
cell line is represented as a deflection to the left for cells
more resistant than the mean and to the right for cells
more sensitive than the mean.17 An important applica-
tion of this manner of presentation is provided by the
richly informative patterns of activity that emerge.
Correlations of activity patterns can be quantified using
a pattern recognition algorithm named COMPARE.16
Use of the COMPARE program led to the realization
that compounds with the same or similar mechanisms
of action often result in cytotoxicity patterns that are
similar.16 This approach has resulted in identification
of new tubulin polymerization inhibitors,19 topoi-
somerase I and II inhibitors,20,21 and dihydroorotate
dehydrogenase inhibitors.22 In another example, the use
of COMPARE linked the effects of cucurbitacin23 and
jasplakinolide24 to the actin cytoskeleton. Compounds
with unique cytotoxicity profiles, suggesting modes of
action not shared with the known clinically active
classes of chemotherapeutic agents, have also been
identified.25
Biologica l Resu lts a n d Discu ssion
The lavendustin A analogues were examined for
antiproliferative activity against the human cancer cell
lines in the NCI cytotoxicity screen, in which the activity
of each compound was evaluated using approximately
55 different cancer cell lines of diverse tumor origins.
The mean-graph midpoint values (MGMs) listed in
A COMPARE analysis for a test agent can be run
against the entire database of more than 77 000 com-
pounds that have been tested in the NCI cell line
cytotoxicity assay or against a database of standard
agents whose mechanism of cytotoxicity is well-docu-

Lavendustin A Analogues
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3 443
Sch em e 1
Sch em e 2
Sch em e 3
Table 1 are based on a calculation of the average GI50
values for all of the cancer cell lines tested (ap-
proximately 55) in which GI50 values below and above
the test range (10-4-10-8 M) are taken as the minimum
(10-8 M) and maximum (10-4 M) drug concentrations
used in the screening test.18 A more detailed listing of
the cytotoxicities of each compound in eight representa-
tive human cancer cell lines is presented in Table 2.
With the exception of the cholestane derivative 13m ,
all of the compounds in the series were found to be
cytotoxic in human cancer cell cultures, with MGM
values ranging from 0.35 to 20.4 µM. Starting with the
basic â-phenylethylamine 13a , which had an MGM of
14.8 µM, various substituents were introduced into the
para position in order to determine how they affected
cytotoxicity. The substituent that resulted in the great-
est cytotoxicity was fluorine (13b, MGM 0.35 µM),
followed by bromine (13c, MGM 7.6 µM), chlorine (13f,
MGM 10.0 µM), methoxy (13d , MGM 15.4 µM), and
hydroxy (13g, MGM 20.4 µM). Lengthening the chain
of 13a by one methylene unit had no appreciable effect
on cytotoxicity (13j, MGM 13.2 µM), and shortening it
by one methylene group also had little effect on cyto-
toxicity (13e, MGM 15.5 µM). The replacement of the
benzene ring by a 2-pyridyl substituent (13i, MGM 16.0
µM) or a 2-tetralinyl substituent (13l, MGM 11.2 µM)
did not result in any appreciable change in activity,
while replacement of the benzene ring by a morpholine
ring caused a slight decrease in cytotoxicity (13h , MGM
19.5 µM). The appendage of a long hydrocarbon chain
to the amide in 13k resulted in one of the more cytotoxic
compounds (MGM 2.7 µM), but it was not an inhibitor
of tubulin polymerization or Syk PTK (see below).

444 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3
Ta ble 1. Inhibitory Activities of Lavendustin A Analogues
Mu et al.
IC50 (µM)
MCF-7
DNAe
MCF-10A
compd
MGMa
tubulinb
Sykc
EGFRd
DNAf
4
8.7 ( 0.4
14.8 ( 1.4
0.35 ( 0.05
7.6 ( 2.4
15.4
15.5
10.0 ( 0.95
20.4
3.6 ( 0.7
3.6 ( 1
NTg
5
5
45
50
25
25
5
14 ( 10
10 ( 4
4 ( 2
11 ( 4
9 ( 3
14 ( 3
11 ( 3
10 ( 1
6 ( 1
11 ( 2
17 ( 4
9 ( 1
15 ( 2
16 ( 5
7 ( 0.0
2 ( 0.0
3 ( 0.5
6 ( 0.7
NTg
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
13k
13l
13m
18a
18b
18c
23
4.0 ( 0.4
4.2 ( 0.5
4.2 ( 0.8
3.2 ( 0.6
3.2 ( 0.7
4.5 ( 1
12 ( 4
7 ( 0
35 ( 11
>100
14 ( 0
17 ( 4
13 ( 2
16 ( 3
14 ( 3
11 ( 5
2 ( 0.2
1 ( 0.3
10 ( 3
>50
16 ( 3
3 ( 1
0.4 ( 0.1
5 ( 1
8 ( 4
46 ( 13
∼300
>50
15 ( 6
19.5
2.1 ( 0.4
3.6 ( 0.3
5.0 ( 1
1.5
16.0 ( 0.55
13.2 ( 0.3
2.7 ( 0.1
11.2 ( 1.4
>100
8.8 ( 1.7
6.4 ( 1.2
5.3 ( 1.3
12.1 ( 0.8
NTg
28
1 ( 0.4
35 ( 7
4 ( 1
*
>40
50
5.3 ( 0.9
>40
8
NTg
18
6.2 ( 1
>100
>200
33 ( 5
∼500
34 ( 9
4.9 ( 0.9
5.7 ( 1
5
35
>100
7 ( 2
3 ( 1
6.9 ( 1
6 ( 1
a
b
Mean graph midpoint for growth inhibition of all human cancer cell lines (approximately 55) successfully tested. IC50 values for
inhibition of tubulin polymerization. c IC50 values for in vitro inhibition of Syk PTK. dIC50 values for inhibition of EGFR phosphorylation
in BaF3 mouse lymphoid cells. e IC50 values for inhibition of DNA synthesis in MCF-7 cells. f IC50 values for inhibition of DNA synthesis
in MCF-10A cells. NT, not tested. * Stimulates EGFR tyrosine phosphorylation.
g
Ta ble 2. Cytotoxicities of Lavendustin A Analogues in Human Cancer Cell Cultures
GI50 (µM)a
leukemia
CCRF-CEM
lung
HOP-62
colon
HCT-116
CNS
SF-539
melanoma
UACC-62
ovarian
OVCAR-3
renal
SN12C
prostate
DU-145
breast
MDA-MB-435
compd
MGMb
4
0.56
2.7
0.29
NTc
3.2
19
20
12
18
12
23
17
2.5
NTc
20
22
3.8
17
20
13
17
18
9.2
7.6
23
17
15
16
20
17
24
2.1
16
10
7.4
6.4
16
2.3
17
9.2
15.8
16
12
14
7.8
8.6
14
15
13
18
17
17
14
3.2
14
12
9.4
7.9
13
4.0
7.7
11
4.2
5.5
21
5.9
62
87
6.1
18
7.7
6.3
72
12
9.8
19
22
19
15
2.6
18
15
16
18
16
21
16
16
17
17
17
8.4
11
17
15
14
13
17
15
19
13
10
16
30
20
23
13
13
18
3.2
9.2
12
5.1
5.8
15
8.7
14.8
0.35
7.6
15.4
15.5
10.0
20.4
19.5
16.0
13.2
2.7
11.2
8.8
6.4
5.3
12.1
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
13k
13l
18a
18b
18c
23
3.2
21
1.6
8.4
2.5
19
29
16
NTc
1.8
1.0
2.4
0.34
1.4
2.7
0.59
0.83
4.4
17
6.5
1.9
7.9
6.8
2.7
3.0
2.8
20
2.7
9.7
8.5
3.4
15
9.7
7.4
3.2
13
16
3.8
a
b
The cytotoxicity GI50 values are the concentrations (µM) corresponding to 50% growth inhibition. Mean graph midpoint for growth
inhibition of all human cancer cell lines successfully tested. c NT, not tested.
Because the hydrocarbon chain in 13k might be ex-
pected to be membrane-interactive, it was replaced by
the cholestanyl moiety in 13m , since that would be
expected to bind strongly to biological membranes.
However, the resulting compound 13m proved to be
inactive.
Inhibition of EGF-stimulated EGFR phosphorylation
by a number of the lavendustin A analogues was tested
in BaF3 cells. After addition of inhibitor to the cells,
they were stimulated by EGF under conditions which
induce abundant receptor phosphorylation but not
receptor downregulation or internalization.27 Immuno-
precipitates from the lysed cells were resolved by
polyacrylamide gel electrophoresis and electrotrans-
ferred onto nitrocellulose. The resulting Western blots
were probed with a mouse monoclonal antiphosphoty-
rosine antibody, and the bound antibody was detected
by probing the blot with a horseradish peroxidase-
coupled goat anti-mouse antibody. The antibody com-
plexes were visualized by enhanced chemiluminescence.
The resulting IC50 values for inhibition of EGFR phos-
phorylation are listed in Table 1. A total of 18 com-
pounds were tested, of which 5 showed appreciable
activity (IC50 10 µM or less). The three most potent
compounds were 13j (IC50 1 µM), having a γ-phenyl-
A series of analogues 18a -c was designed by moving
the amide to the para position and eliminating the
phenolic hydroxyl group. The extension of the amide
substituent by consecutive addition of methylene groups
led to small increases in cytotoxicity (18a , MGM 8.8 µM;
18b, MGM 6.4 µM; 18c, MGM 5.3 µM). However, these
small differences in cytotoxicity may not be significant
based on the standard deviations reported. A final
modification in this series involved the elimination of
the phenolic hydroxyl group from 13a (MGM 14.8 µM),
resulting in 23 (MGM 12.1). Comparison of 13a and 23
indicates that the phenolic hydroxyl group of 13a is not
a requirement for cytotoxicity.

Lavendustin A Analogues
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3 445
propyl substituent on the amide nitrogen, 13b (IC50
4
tors of the polymerization reaction. In previous studies
under the same reaction conditions, known potent
antimitotic agents such as combretastatin A-4 and
dolastatin 10 reproducibly yield IC50 values in the 0.5-
1.0 µM range. Compounds 4 and 13b,h were also shown
to be weak inhibitors of the binding of [3H]colchicine to
tubulin. In an assay with tubulin at 1 µM, [3H]colchicine
at 5 µM, and the three agents at 50 µM, the percent
inhibitions for these compounds were 13b, 38 ( 10%;
13h , 34 ( 20%; and 4, 45 ( 20%; as compared to 98 (
2% with 5 µM combretastatin A-4 (25) (data not
presented).
µM), having a p-fluoro-â-phenylethyl substituent, and
13l (IC50 4 µM), having a â-tetralinyl substituent. These
three compounds were followed by the benzyl analogue
13e (IC50 8 µM) and the â-phenylethyl congener 13a
(IC50 10 µM). Each of these values was relatively close
to the IC50 values for inhibition of DNA synthesis in
MCF-7 cells and MCF-10A cells (Table 1). Since MCF-
10A cells are EGF-dependent, while MCF-7 cells are not,
the fact that the two cell types exhibit similar responses
to the lavendustin A analogues is evidence that inhibi-
tion of EGFR tyrosine kinase activity is not relevant to
the cytotoxic activities of the lavendustin
A
analogues.28-31 Compounds 13c,d ,f,g, 18c, and 23 did
not show appreciable activity versus EGFR tyrosine
kinase at the concentrations tested.
To assay the new lavendustin A congeners versus a
representative nonreceptor PTK, Syk was obtained from
lysates of Sf9 cells infected with a baculovirus directing
the expression of the full-length enzyme as a fusion
protein with glutathione S-transferase. GST-Syk was
isolated by affinity chromatography on glutathione-
agarose. The activities of the inhibitors were assayed
by monitoring the transfer of 32P from [γ-32P]ATP to
tyrosyl residues on the immobilized kinase. Reactions
were terminated by the addition of EDTA. The im-
mobilized kinase was separated from unreacted [γ-32P]-
ATP by centrifugation. The extent of kinase autophos-
phorylation was determined by liquid scintillation
spectrometry. The resulting IC50 values are listed in
Table 1. In general, IC50 values for the inhibition of Syk
were comparable to those obtained for the inhibition of
EGFR. Four compounds (13g,h and 18a ,b), however,
did display considerable selectivity for Syk as compared
to the EGFR. These IC50 values are comparable to that
of the Syk-selective inhibitor, piceatannol (24), which
had an IC50 value of 5 µg/mL in this assay.
Whether or not the cytotoxicities of the lavendustin
A analogues reported here are due to PTK inhibition is
questionable. Since almost all of the EGFR tyrosine
kinase activity must be inhibited before effects are seen
on cell growth,27 it is unlikely that the potencies of
EGFR inhibitors seen here could possibly be responsible
for the effects seen on inhibition of cancer cell growth,
since the IC50 values for EGFR inhibition are close to
the MGMs observed for growth inhibition. The fact that
lavendustin A did not inhibit the PTK activity of the
mutant protein pp60srcF527, but nevertheless did exhibit
antiproliferative activity, previously led other investiga-
tors to the conclusion that the antiproliferative effects
of lavendustin A could be due to actions on cellular
targets downstream of pp60srcF527 or receptors unre-
lated to the kinase.11 We therefore also considered other
possible targets for the new lavendustin analogues that
might be responsible for their inhibitory effects on
cancer cell growth.
The activity of these compounds as inhibitors of
tubulin polymerization was unexpected on the basis of
prior literature reports, which have indicated that
polyhydroxylated trans-stilbenes, benzylanilines, and
related compounds are inhibitors of the PTKs, while
their polymethoxylated cis-stilbene analogues and re-
lated compounds are inhibitors of tubulin polymeriza-
tion. For example, in addition to lavendustin A and its
analogues, other polyhydroxylated stilbene PTK inhibi-
tors include piceatannol (24) and related polyhydroxy-
lated trans-stilbenes,7,32,33 while the polymethoxylated
cis-stilbene inhibitors of tubulin polymerization include
combretastatin A-4 (25) and its analogues.34-37 Poly-
methoxylated trans-stilbenes are much less active as
inhibitors of tubulin polymerization and as cytotoxic
agents than their cis counterparts.33 Neussbaumer et
al. previously reported that the methylated lavendustin
A analogues 26 and 27 have antiproliferative effects and
that 27 acts “by blocking the cell cycle at mitosis by
perturbing the microtubules of the mitotic spindle
apparatus”.12,38 This would basically be in agreement
with our prior study which had indicated that the
benzylamine 28 and analogous compounds are antimi-
totic agents that act by inhibition of tubulin polymeri-
zation.37 Furthermore, the methylated lavendustin A
analogue 27 did not inhibit EGFR tyrosine kinase in a
cell-free system.38
However, when we examined human Burkitt lym-
phoma cells treated with several members (4, 13b,c,e,f,k,
and 18a -c) of this series of compounds for evidence of
mitotic arrest (increase in G2/M cells), only 13b caused
such an effect. We thus have evidence for an antitubulin
effect at the cellular level only with the most cytotoxic
member of the series.
The abilities of the present polyhydroxylated benzy-
laniline derivatives of lavendustin A to inhibit both
receptor and nonreceptor PTKs, as well as tubulin
polymerization, is a novel observation which raises the
possibility that the antiproliferative effects observed
both in this series and in the other series of lavendustin
A analogues reported in the literature may actually be
due in part to inhibition of tubulin polymerization,
rather than any effect on PTK inhibition. The laven-
dustin A derivatives reported are also unusual tubulin
As stated previously, the COMPARE analysis of 13b
suggested that the lavendustin A analogues in this
series might be interacting with tubulin. As shown in
Table 1, this turned out to be the case. All of the
compounds except 13k ,m inhibited tubulin polymeri-
zation with IC50 values ranging from 2.1 to 6.9 µM.
Under the reaction conditions used here, these values
indicate the compounds are moderately active as inhibi-

446 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3
Mu et al.
its culture conditions have been described previously.42,43
Briefly, these cells were propagated in RPMI supplemented
with 10% heat-inactivated fetal bovine serum and 300 µg/mL
G418. The BaF3 mouse lymphoid cell lines engineered to
express either EGFR (BaF3/EGFR) or ErbB2 and ErbB3
together (BaF3/2+3) and the culture conditions for these cell
lines have been described earlier.27 These cells were propa-
gated in RPMI supplemented with 10% fetal bovine serum,
300 µg/mL G418, and 10% medium conditioned by WeHI cells.
This conditioned medium serves as a source for interleukin 3.
MCF-10A human mammary epithelial cells and MCF-7,
MDA-MB-231, and MDA-MB-453 human mammary tumor cell
lines were obtained from the American Type Culture Collection
(ATCC). These lines were propagated according to ATCC
recommendations.
In h ibition of Recep tor Tyr osin e P h osp h or yla tion As-
sa y. The assay for inhibition of ErbB family receptor tyrosine
phosphorylation was adapted from a previously described
protocol.27,43 Briefly, 200-mL cultures of CEM/4, BaF3/EGFR,
or BaF3/2+3 cells were grown to saturation density (∼106 cells/
mL) and were incubated for 24 h at 37 °C in serum-free
medium to reduce basal levels of receptor tyrosine phospho-
rylation. The cells were collected by centrifugation and resus-
pended in serum-free medium at a final concentration of ∼107
cells/mL (∼20 mL of cells). Cells were transferred to micro-
centrifuge tubes in 1-mL aliquots and putative kinase inhibi-
tors were added to the cells. Each tyrosine kinase inhibitor
was tested at 3-5 different concentrations. The inhibitors were
dissolved in 5 µL of DMSO; hence, cells treated with 5 µL of
DMSO were used as a solvent control. Cells were incubated
in the presence of inhibitor for 2 h at 37 °C, then were
incubated on ice for 20 min. Chilling the cells reduces the
amount of ligand-induced receptor downregulation.27
Ligand was then added to the appropriate samples at a final
concentration of 100 ng/mL and the samples were mixed and
incubated on ice for 7 min. Recombinant human EGF (Sigma)
was used as the ligand for EGFR, while neuregulin1â (NRG1â;
R&D Systems) was used as the ligand for ErbB3 and ErbB4.
Note that because ErbB3 lacks kinase activity, ligand-induced
ErbB2 and ErbB3 phosphorylation in the BaF3/2+3 cells is
the result of ErbB2 kinase activity.27 Following incubation with
ligand, the cells were collected by centrifugation, the super-
natant was removed by aspiration, and the cells were resus-
pended in an isotonic lysis buffer containing 0.5% NP40/Igepal
CA-630 (nonionic detergent; Sigma).
The cells were incubated for 20 min on ice to permit lysis.
The samples were centrifuged for 10 min at 4 °C to collect the
nuclei and cellular debris. The supernatants (cell lysates) were
transferred to fresh tubes. Concanavalin A Sepharose (Amer-
sham/Pharmacia) beads were added to each sample (35 µL of
a 50% v/v slurry) and the samples were incubated at 4 °C for
30 min. Concanavalin A Sepharose precipitates the cellular
glycoproteins, which include ErbB family receptors. The
precipitated glycoproteins were washed 3 times with 500 µL
of ice-cold lysis buffer, then were eluted by boiling the beads
for 5 min in 80 µL of reducing SDS protein sample buffer. The
beads were collected by centrifugation and one-half of the
eluted glycoproteins (40 µL) were recovered and resolved by
SDS/PAGE on a 7.5% acrylamide gel.
polymerization inhibitors, since they are not poly-
methoxylated cis-stilbene or benzylaniline analogues.
Exp er im en ta l Section
Gen er a l. Melting points are uncorrected. Nuclear magnetic
resonance spectra for proton (1H NMR) were recorded on a
300-MHz spectrometer. The chemical shift values are ex-
pressed in ppm (parts per million) relative to tetramethylsilane
as internal standard; s ) singlet, d ) doublet, m ) multiplet,
bs ) broad singlet. Microanalyses were performed at the
Purdue Microanalysis Laboratory, and all values were within
(0.4% of the calculated compositions. Column chromatography
was carried out using Merck silica gel (230-400 mesh).
Analytical thin-layer chromatography (TLC) was performed
on silica gel GF (Analtech) glass-coated plates (2.5 × 10 cm
with 250 µm layer and prescored), and spots were visualized
with UV light at 254 nm. Most chemicals and solvents were
analytical grade and used without further purification. Com-
mercial reagents were purchased from Aldrich Chemical Co.
(Milwaukee, WI).
Tu bu lin Assa ys. Electrophoretically homogeneous tubulin
was purified from bovine brain as described previously.39 The
tubulin polymerization and colchicine binding assays were
performed as described previously,40 except that Beckman
DU7400/7500 spectrophotometers equipped with “high-per-
formance” temperature controllers were used in the former
assay. Unlike the manual control possible with the previously
used Gilford spectrophotometers, the polymerization assays
required use of programs provided by MDB Analytical Associ-
ates, South Plainfield, NJ , since the Beckman instruments are
microprocessor-controlled. The Beckman instruments were
unable to maintain 0 °C, and the lower temperature in the
assays fluctuated between 2 and 4 °C. Temperature changes
were, however, more rapid than in the Gilford instruments
with the jump from the lower temperature to 30 °C taking
about 20 s and the reverse jump about 100 s.
Syk Assa ys. Preparation of GST-Syk was as described
previously.41 Autophosphorylation reactions contained 50 mM
Tris/HCl, pH 7.4, 5 mM MnCl2, 5 µM ATP, 5 µCi [γ-32P]ATP,
1 mM sodium orthovanadate, 5 mM p-nitrophenyl phosphate
and 1.5% DMSO, which was used as a carrier for the inhibi-
tors. Reactions were terminated by the addition of EDTA to a
final concentration of 10 mM. Beads were washed 2 times in
50 mM Tris/HCl, pH 7.4, 1 mM sodium orthovanadate and 10
mM EDTA and counted by liquid scintillation spectrometry.
IC50 values were determined graphically and represent the
concentration of inhibitor that gives half-maximal inhibition
as compared to control assays carried out in the absence of
inhibitor but in the presence of DMSO carrier.
The resolved glycoproteins were electroblotted onto nitrocel-
lulose (BiotraceNT; Gelman Sciences). The resulting blot was
blocked by incubation for 45 min at room temperature in a
solution consisting of 5% bovine serum albumin (Sigma)
dissolved in Tris-buffered normal saline (TBS) supplemented
with 0.05% Tween-20 (TBS-T). The blot was then probed with
a mouse monoclonal antiphosphotyrosine antibody (4G10;
Upstate Biotechnology). The blot was washed with TBS-T 5
times for 6 min each, and primary antibody binding was
detected by probing the blot with a goat anti-mouse antibody
conjugated to horseradish perioxidase (HRP; Pierce). The blot
was washed with TBS-T 12 times for 10 min each, after which
HRP activity was visualized by enhanced chemiluminescence
(ECL; Amersham Pharmacia Biotech). The resulting chemi-
lumigrams were digitized using a Linotype-Hell J ade flatbed
Cell Lin es a n d Cell Cu ltu r e. The CEM human T lym-
phocyte cell line engineered to express ErbB4 (CEM/4) and

Lavendustin A Analogues
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3 447
scanner and the amount of receptor tyrosine phosphorylation
was quantified using NIH Image software. The amount of
receptor tyrosine phosphorylation in samples from cells treated
with a putative receptor tyrosine kinase inhibitor were com-
pared to a standard curve generated using samples from cells
treated with DMSO solvent control. This enabled us to
determine the concentration of a given tyrosine kinase inhibi-
tor that was necessary to cause a 50% reduction in receptor
tyrosine phosphorylation. This value is reported as the receptor
tyrosine phosphorylation IC50 value.
(m, 6 H), 7.00 (dd, J ) 8.82, 2.14 Hz, 1 H), 6.90 (d, J ) 8.8 Hz,
1 H), 6.60 (bs, 1 H), 6.34 (s, 1 H), 3.68 (q, J ) 6.859 Hz, 2 H),
2.93 (t, J ) 7.1 Hz, 2 H), 1.51 (s, 9 H).
5-[N-(ter t-Bu toxyca r bon yl)a m in o]-N-(4-flu or o-â-p h en -
eth yl)sa licyla m id e (10b). From compound 8 (0.75 g, 2.9
mmol), EDCI (0.85 g, 4.5 mmol), HOBt (0.6 g, 4.5 mmol),
triethylamine (1.65 mL, 11.8 mmol) and 4-fluorophenethy-
lamine (9b) (1.2 g, 9.0 mmol), a similar procedure as that
described for 10a gave pure 10b (0.58 g, 53%) as a white
crystalline solid: mp 173-174 °C; 1H NMR (300 MHz, DMSO-
d6) δ 11.68 (s, 1 H), 9.12 (bs, 1 H), 8.70 (t, J ) 5.75 Hz, 1 H),
7.87 (s, 1 H), 7.28 (m, 3 H), 7.12 (t, J ) 8.79 Hz, 2 H), 6.81 (d,
J ) 8.83 Hz, 1 H), 3.49 (q, J ) 6.92 Hz, 2 H), 2.84 (t, J ) 7.35
Hz, 2 H), 1.46 (s, 9 H).
5-[N-(ter t-Bu toxyca r bon yl)a m in o]-N-(4-br om o-â-p h en -
eth yl)sa licyla m id e (10c). From compound 8 (0.66 g, 2.6
mmol), EDCI (0.75 g, 3.9 mmol), HOBt (0.53 g, 3.9 mmol),
triethylamine (1.45 mL, 10.4 mmol) and 4-bromophenethy-
lamine (9c) (1.0 g, 5.0 mmol), a similar procedure as that
described for 10a gave pure 10c (0.55 g, 49%) as a white
crystalline solid: mp 196-197 °C; 1H NMR (300 MHz, DMSO-
d6) δ 11.66 (s, 1 H), 9.13 (bs, 1 H), 8.71 (t, J ) 4.99 Hz, 1 H),
7.81 (s, 1 H), 7.49 (dd, J ) 8.20, 1.81 Hz, 1 H), 7.30 (d, J )
8.20 Hz, 2 H), 7.22 (d, J ) 8.11 Hz, 2 H), 6.81 (d, J ) 9.11 Hz,
1 H), 3.51 (q, J ) 6.84 Hz, 2 H), 2.83 (t, J ) 7.29 Hz, 2 H),
1.47 (s, 9 H).
5-[N -(t er t -Bu t oxyca r b on yl)a m in o]-N -(4-m e t h oxy-â-
p h en eth yl)sa licyla m id e (10d ). From compound 8 (0.74 g,
2.9 mmol), EDCI (0.85 g, 4.5 mmol), HOBt (0.6 g, 4.5 mmol),
triethylamine (1.65 mL, 11.8 mmol) and 4-methoxyphenethy-
lamine (9d ) (1.32 g, 9.0 mmol), a similar procedure as that
described for 10a gave pure 10d (0.64 g, 55%) as a white
crystalline solid: mp 159-160 °C; 1H NMR (300 MHz, DMSO-
d6) δ 11.72 (s, 1 H), 9.12 (bs, 1 H), 8.70 (t, J ) 5.01 Hz, 1 H),
7.88 (s, 1 H), 7.29 (dd, J ) 8.65, 1.50 Hz, 1 H), 7.16 (d, J )
7.74 Hz, 2 H), 6.86 (d, J ) 7.29 Hz, 2 H), 6.81 (d, J ) 8.65 Hz,
1 H), 3.72 (s, 3 H), 3.47 (q, J ) 6.38 Hz, 2 H), 2.77 (t, J ) 8.20
Hz, 2 H), 1.46 (s, 9 H).
5-[N-(ter t-Bu t oxyca r b on yl)a m in o]-N-(b en zyl)sa licyl-
a m id e (10e). From compound 8 (0.77 g, 3.0 mmol), EDCI (0.85
g, 4.5 mmol), HOBt (0.6 g, 4.5 mmol), triethylamine (1.65 mL,
11.8 mmol) and benzylamine (9e) (0.65 mL, 6.0 mmol), a
similar procedure as that described for 10a gave pure 10e (0.54
g, 53%) as a white crystalline solid: mp 184-185 °C; 1H NMR
(300 MHz, DMSO-d6) δ 11.74 (s, 1 H), 9.15 (bs, 2 H), 7.94 (s,
1 H), 7.41-7.21 (m, 6 H), 6.84 (d, J ) 9.11 Hz, 1 H), 4.51 (d,
J ) 4.10 Hz, 2 H), 1.46 (s, 9 H).
In h ibition of Cellu la r DNA Syn th esis Assa y. The assay
for inhibition of cellular DNA synthesis was adapted from a
previously described protocol.44 Briefly, human mammary
(tumor) cells were seeded in 1-mL aliquots into 24-well culture
dishes at a density of 105 cells/well. Cells were incubated for
24 h at 37 °C, and a tyrosine kinase inhibitor dissolved in
DMSO was added to each well in a volume of 10 µL. Each
tyrosine kinase inhibitor was assayed at 3-5 different con-
centrations and each concentration was assayed using 3-4
wells of cells. Cells treated with 10 µL of DMSO served as the
solvent control. Cells were then incubated for 48 h at 37 °C.
[3H]Thymidine (1.5 µCi; Amersham Pharmacia Biotech) dis-
solved in a 1.5 µL of an aqueous solution was added to each
well and the cells were incubated for an additional 2 h at 37
°C. The culture medium was aspirated from the wells, and the
cells were rinsed once with 1 mL of ice-cold phosphate-buffered
saline (PBS) and once with 1 mL of ice-cold 10% trichloroacetic
acid (TCA). Incorporated [3H]thymidine was precipitated by
incubating the cells for at least 30 min at 4 °C in 1 mL of 10%
TCA. Following incubation, the TCA solution was aspirated
from each well and the precipitated (incorporated) [3H]thymi-
dine was solubilized by incubating the cells for 30 min at 95
°C in 500 µL of 3% perchloric acid. The perchloric acid samples
were transferred to scintillation vials containing 10 mL of
Cytoscint scintillation cocktail (ICN). The incorporated [3H]-
thymidine was assayed by scintillation counting on a Packard
Tricarb scintillation counter. The amount of [3H]thymidine
incorporation observed in the cells treated with the solvent
control was divided by 2 (two) to determine the amount of half-
maximal [3H]thymidine incorporation. Dose-response curves
for each combination of putative tyrosine kinase inhibitor and
cell line were then constructed using the [3H]thymidine
incorporation data. The dose-response curves and the half-
maximal [3H]thymidine values were used to calculate the
concentration of each inhibitor required to inhibit [3H]thymi-
dine incorporation by 50% in a given cell line. This value is
reported as the DNA synthesis IC50 value.
5-[N-(ter t-Bu toxyca r bon yl)a m in o]sa licylic Acid (8). To
a mixture of 5-aminosalicylic acid (7) (3.0 g, 19.6 mmol) in
dioxane (50 mL) and water (25 mL) were added triethylamine
(4.0 mL, 29.3 mmol) followed by di-tert-butyl dicarbonate (6.4
g, 29.3 mmol). The reaction mixture was stirred at room
temperature for 24 h. Solvent was removed by rotary evapora-
tion, and 3 N aqueous hydrochloric acid (30 mL) was added
dropwise to the residue. A precipitate was obtained, collected,
washed with water, and dried to provide 8 (4.76 g, 96%) as a
5-[N-(ter t-Bu toxyca r bon yl)a m in o]-N-(4-ch lor o-â-p h en -
eth yl)sa licyla m id e (10f). From compound 8 (0.61 g, 2.4
mmol), EDCI (0.69 g, 3.6 mmol), HOBt (0.49 g, 3.6 mmol),
triethylamine (1.3 mL, 9.2 mmol) and 4-chlorophenethylamine
(9f) (1.12 g, 7.2 mmol), a similar procedure as that described
for 10a gave pure 10f (0.55 g, 58%) as a white crystalline
solid: mp 192-193 °C; 1H NMR (300 MHz, CDCl3) δ 11.66 (s,
1 H), 9.13 (bs, 1 H), 8.71 (t, J ) 5.42 Hz, 1 H), 7.87 (s, 1 H),
7.31 (d, J ) 8.39 Hz, 2 H), 7.29 (d, J ) 8.21 Hz, 2 H), 7.27 (dd,
J ) 8.39, 2.04 Hz, 1 H), 6.80 (d, J ) 8.77 Hz, 1 H), 3.52 (q, J
) 6.17 Hz, 2 H), 2.84 (t, J ) 7.06 Hz, 2 H), 1.47 (s, 9 H).
5-[N-(ter t-Bu toxycar bon yl)am in o]-N-(4-h ydr oxy-â-ph en -
eth yl)sa licyla m id e (10g). From compound 8 (0.61 g, 2.4
mmol), EDCI (0.69 g, 3.6 mmol), HOBt (0.49 g, 3.6 mmol),
triethylamine (1.3 mL, 9.3 mmol) and 4-hydroxyphenethy-
lamine (9g) (1.2 g, 8.7 mmol), a similar procedure as that
described for 10a gave pure 10g (0.37 g, 42%) as a white
crystalline solid: mp 194-195 °C; 1H NMR (300 MHz, DMSO-
d6) δ 11.73 (s, 1 H), 9.19 (s, 1 H), 9.12 (s, 1 H), 8.69 (t, J )
4.51 Hz, 1 H), 7.88 (s, 1 H), 7.31 (d, J ) 9.3 Hz, 1 H), 7.05 (d,
J ) 8.22 Hz, 2 H), 6.82 (d, J ) 8.68 Hz, 1 H), 6.68 (d, J ) 7.67
Hz, 2 H), 3.43 (q, J ) 8.03 Hz, 2 H), 2.72 (t, J ) 7.20 Hz, 2 H),
1.46 (s, 9 H).
1
solid: mp 279-280 °C; H NMR (300 MHz, DMSO-d6) δ 9.29
(s, 1 H), 7.96 (d, J ) 1.81 Hz, 1 H), 7.47 (dd, J ) 8.93, 2.69 Hz,
1 H), 6.85 (d, J ) 8.88 Hz, 1 H), 1.44 (s, 9 H).
5-[N-(ter t-Bu toxyca r bon yl)a m in o]-N-(â-p h en eth yl)sa l-
icyla m id e (10a ). To a solution of 8 (1.5 g, 5.93 mmol) in dry
DMF (15 mL) were added EDCI (1.71 g, 8.92 mmol), HOBt
(1.2 g, 8.92 mmol) and triethylamine (1.65 mL, 11.86 mmol).
After stirring at room temperature for 24 h, â-phenethylamine
(9a ) (3.7 mL, 29.5 mmol) was added dropwise and the reaction
continued for 48 h at room temperature under argon. Water
(300 mL) was then added and the mixture stirred for 5 min.
The product was then extracted with ethyl acetate (5 × 50
mL). The combined organic extracts were washed with brine
(1 × 40 mL), dried over sodium sulfate, filtered, and the solvent
removed. Purification was achieved by flash chromatography
(silica gel 75 g, ethyl acetate/hexane 1:4 by volume) to yield
pure 10a (1.09 g, 52%) as a white crystalline solid: 1H NMR
(300 MHz, CDCl3) δ 12.13 (s, 1 H), 7.74 (s, 1 H), 7.36-7.24
5-[N-(ter t-Bu t oxyca r b on yl)a m in o]-N-(2-m or p h olin o-
eth yl)sa licyla m id e (10h ). From compound 8 (0.38 g, 1.5
mmol), EDCI (0.43 g, 2.2 mmol), HOBt (0.30 g, 2.2 mmol),
triethylamine (0.87 mL, 6.0 mmol) and 4-(2-aminoethyl)-

448 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3
Mu et al.
morpholine (9h ) (0.39 mL, 3.0 mmol), a similar procedure as
that described for 10a gave pure 10h (0.33 g, 56%) as a white
crystalline solid: mp 154-155 °C; 1H NMR (300 MHz, CDCl3)
δ 11.02 (s, 1 H, OH), 7.83 (bs, 1 H, NH), 7.01 (dd, J ) 9.82,
2.14 Hz, 1 H), 6.91 (d, J ) 9.9 Hz, 1 H), 6.68 (bs, 1 H), 6.50 (d,
J ) 2.01 Hz, 1 H), 3.56 (t, J ) 4.40 Hz, 4 H), 3.47 (m, 4 H),
2.39 (m, 4 H), 1.43 (s, 9 H).
1 H), 7.67 (d, J ) 2.74 Hz, 1 H), 7.35-7.20 (m, 6 H), 7.03 (d,
J ) 8.77 Hz, 1 H), 3.54 (q, J ) 6.47 Hz, 2 H), 2.86 (t, J ) 7.12
Hz, 2 H).
5-Am in o-N-(4-flu or o-â-p h en eth yl)sa licyla m id e (11b).
From compound 10b (0.56 g, 1.5 mmol), a similar procedure
as that described for 11a provided white solid 11b (0.43 g,
104%): mp 200-202 °C dec; 1H NMR (300 MHz, DMSO-d6) δ
11.9 (bs, 1 H), 9.83 (bs, 1 H), 8.73 (t, J ) 5.50 Hz, 1 H), 7.71
(d, J ) 2.54 Hz, 1 H), 7.31-7.26 (m, 2 H), 7.23 (d, J ) 2.67
Hz, 1 H), 7.12 (t, J ) 8.93 Hz, 1 H), 7.02 (d, J ) 8.77 Hz, 1 H),
3.54 (q, J ) 6.90 Hz, 2 H), 2.84 (t, J ) 7.15 Hz, 2 H).
5-Am in o-N-(4-br om o-â-p h en eth yl)sa licyla m id e (11c).
From compound 10c (0.54 g, 1.2 mmol), a similar procedure
as that described for 11a provided white solid 11c (0.41 g,
101%): mp 201-202 °C dec; 1H NMR (300 MHz, DMSO-d6) δ
12.48 (bs, 1 H), 9.27 (bs, 2 H), 8.73 (t, J ) 5.78 Hz, 1 H), 7.77
(d, J ) 2.90 Hz, 1 H), 7.49 (d, J ) 8.35 Hz, 2 H), 7.27 (dd, J )
9.08, 2.54 Hz, 1 H), 7.23 (d, J ) 8.36 Hz, 2 H), 7.00 (d, J )
8.72 Hz, 1 H), 3.55 (q, J ) 6.18 Hz, 2 H), 2.84 (t, J ) 7.26 Hz,
2 H).
5-Am in o-N-(4-m eth oxyp h en eth yl)sa licyla m id e (11d ).
From compound 10d (0.64 g, 1.65 mmol), a similar procedure
as that described for 11a provided white solid 11d (0.48 g,
100%): mp 196-198 °C dec; 1H NMR (300 MHz, DMSO-d6) δ
12.03 (bs, 1 H), 9.40 (bs, 2 H), 8.74 (t, J ) 5.73 Hz, 1 H), 7.74
(d, J ) 2.86 Hz, 1 H), 7.28 (dd, J ) 9.06, 2.87 Hz, 1 H), 7.18
(d, J ) 8.58 Hz, 2 H), 7.00 (d, J ) 8.59 Hz, 1 H), 6.87 (d, J )
8.58 Hz, 2 H), 3.73 (s, 3 H), 3.52 (q, J ) 6.19 Hz, 2 H), 2.79 (t,
J ) 7.63 Hz, 2 H).
5-[N-(ter t-Bu t oxyca r b on yl)a m in o]-N-(2-p yr id in -2-yl-
eth yl)sa licyla m id e (10i). From compound 8 (0.25 g, 0.99
mmol), EDCI (0.28 g, 1.5 mmol), HOBt (0.26 g, 1.5 mmol),
triethylamine (0.3 mL, 2.1 mmol) and 2-(2-aminoethyl)pyridine
(9i) (0.20 mL, 1.7 mmol), a similar procedure as that described
for 10a gave pure 10i (0.30 g, 85%) as a white crystalline
1
solid: mp 182-184 °C dec; H NMR (300 MHz, DMSO-d6) δ
11.68 (s, 1 H), 9.13 (s, 1 H), 8.53 (d, J ) 4.57 Hz, 1 H), 7.88 (s,
1 H), 7.72 (td, J ) 7.67, 1.80 Hz, 1 H), 7.30 (d, J ) 7.76 Hz, 1
H), 7.23 (m, 2 H), 6.80 (d, J ) 8.78 Hz, 1 H), 3.65 (t, J ) 7.17
Hz, 2 H), 3.00 (t, J ) 7.10 Hz, 2 H), 1.46 (s, 9 H).
5-[N-(ter t-Bu toxyca r bon yl)a m in o]-N-(3-p h en yl-1-p r o-
p yl)sa licyla m id e (10j). From compound 8 (0.38 g, 1.5 mmol),
EDCI (0.43 g, 2.2 mmol), HOBt (0.30 g, 2.2 mmol), triethy-
lamine (0.87 mL, 6 mmol) and 3-phenyl-1-propylamine (9j)
(0.43 mL, 3.0 mmol), a similar procedure as that described for
10a gave pure 10j (0.45 g, 41%) as a white crystalline solid:
1
mp 111-112 °C; H NMR (300 MHz, DMSO-d6) δ 11.85 (s, 1
H), 9.11 (s, 1 H), 8.72 (s, 1 H), 7.75 (s, 1 H), 7.34-7.14 (m, 6
H), 6.82 (d, J ) 8.8 Hz, 1 H), 3.27 (t, J ) 6.98 Hz, 2 H), 2.59
(t, J ) 7.65 Hz, 2 H), 1.80 (quintet, J ) 7.15 Hz, 2 H), 1.43 (s,
9 H).
5-Am in o-N-(ben zyl)sa licyla m id e (11e). From compound
10e (0.53 g, 1.55 mmol), a similar procedure as that described
for 11a provided white solid 11e (0.38 g, 101%): mp 202-204
5-[N-(ter t-Bu toxyca r bon yl)a m in o]-N-(d od ecyl)sa licyl-
a m id e (10k ). From compound 8 (0.38 g, 1.5 mmol), EDCI (0.43
g, 2.2 mmol), HOBt (0.30 g, 2.2 mmol), triethylamine (0.87 mL,
6 mmol) and dodecylamine (9k ) (0.55 g, 3.0 mmol), a similar
procedure as that described for 10a gave pure 10k (0.35 g,
1
°C dec; H NMR (300 MHz, DMSO-d6) δ 12.27 (bs, 1 H), 9.40
(m, 2 H), 9.18 (t, J ) 5.80 Hz, 1 H), 7.73 (s, 1 H), 7.35 (d, J )
1.78 Hz, 1 H), 7.37-7.20 (m, 5 H), 7.00 (d, J ) 8.46 Hz, 1 H),
4.54 (d, J ) 5.79 Hz, 2 H).
1
56%) as a white crystalline solid: mp 104-105 °C; H NMR
(300 MHz, DMSO-d6) δ 11.89 (s, 1 H), 9.09 (s, 1 H), 7.86 (s, 1
H), 7.28 (d, J ) 7.36 Hz, 1 H), 6.81 (d, J ) 8.8 Hz, 1 H), 3.25
(t, J ) 6.81 Hz, 2 H), 1.46 (s, 9 H), 1.22 (m, 20 H), 0.84 (t, J )
6.73 Hz, 3 H).
5-Am in o-N-(4-ch lor o-â-p h en eth yl)sa licyla m id e (11f).
From compound 10f (0.46 g, 1.18 mmol), a similar procedure
as that described for 11a provided white solid 11f (0.34 g,
100%): mp 199-200 °C dec; 1H NMR (300 MHz, DMSO-d6) δ
12.48 (bs, 1 H), 9.33 (bs, 2 H), 8.73 (t, J ) 5.01 Hz, 1 H), 7.70
(d, J ) 2.80 Hz, 1 H), 7.36 (d, J ) 8.19 Hz, 2 H), 7.28 (d, J )
8.09 Hz, 2 H), 7.26 (dd, J ) 9.21, 2.51 Hz, 1 H), 6.98 (d, J )
8.68 Hz, 1 H), 3.54 (q, J ) 6.10 Hz, 2 H), 2.85 (t, J ) 7.00 Hz,
2 H).
5-Am in o-N-(4-h yd r oxy-â-p h en eth yl)sa licyla m id e (11g).
From compound 10g (0.35 g, 0.94 mmol), a similar procedure
as that described for 11a provided white solid 11g (0.25 g,
100%): mp 139-140 °C dec; 1H NMR (300 MHz, CD3OD) δ
7.82 (d, J ) 2.65 Hz, 1 H), 7.34 (dd, J ) 8.71, 2.69 Hz, 1 H),
7.08 (d, J ) 8.02 Hz, 2 H), 7.02 (d, J ) 8.76 Hz, 1 H), 3.60 (t,
J ) 7.13 Hz, 2 H), 2.81 (t, J ) 7.02 Hz, 2 H).
5-Am in o-N-(2-m or ph olin oeth yl)salicylam ide (11h ). From
compound 10h (0.30 g, 0.82 mmol), a similar procedure as that
described for 11a provided white solid 11h (0.21 g, 100%): 1H
NMR (300 MHz, CDCl3) δ 10.82 (s, 1 H, OH), 7.83 (bs, 1 H,
NH), 7.01 (dd, J ) 8.90, 2.14 Hz, 1 H), 6.98 (d, J ) 9.01 Hz, 1
H), 6.50 (d, J ) 2.01 Hz, 1 H), 3.56 (t, J ) 4.20 Hz, 4 H), 3.47
(m, 4 H), 2.39 (m, 4 H).
5-[N-[(ter t-Bu toxyca r bon yl)m eth yl]a m in o]-N-(1,2,3,4-
tetr a h yd r on a p h th a len -2-yl)sa licyla m id e (10l). From com-
pound 8 (0.40 g, 1.6 mmol), EDCI (0.46 g, 2.4 mmol), HOBt
(0.32 g, 2.4 mmol), triethylamine (0.90 mL, 6.3 mmol) and
1,2,3,4-tetrahydronaphthalen-2-amine hydrochloride (9l) (0.43
g, 2.3 mmol), a similar procedure as that described for 10a
gave pure 10l (0.36 g, 59%) as a white crystalline solid: mp
1
211-212 °C; H NMR (300 MHz, DMSO-d6) δ 11.62 (s, 1 H,
OH), 9.11 (s, 1 H, NH), 8.64 (t, J ) 7.14 Hz, 1 H), 7.91 (s, 1
H), 7.34 (d, J ) 8.71 Hz, 1 H), 7.10 (m, 4 H), 6.82 (d, J ) 8.75
Hz, 1 H), 4.23 (m, 1 H), 3.06 (dd, J ) 16.19, 4.99 Hz, 1 H),
2.89-2.77 (m, 3 H), 2.06-1.98 (m, 1 H), 1.88-1.75 (m, 1 H),
1.46 (s, 9 H).
4-[N-(ter t-Bu toxyca r bon yl)a m in o]-N-(3â-ch olesta n yl)-
sa licyla m id e (10m ). From compound 8 (0.20 g, 0.8 mmol),
EDCI (0.23 g, 1.2 mmol), HOBt (0.16 g, 1.2 mmol), triethy-
lamine (0.44 mL, 3.2 mmol) and 3â-aminocholestane hydro-
chloride (9m ) (0.35 g, 0.82 mmol), a similar procedure as that
described for 10a gave pure 10m (0.31 g, 62%) as a white
crystalline solid: mp 230-231 °C dec; 1H NMR (300 MHz,
CDCl3) δ 12.36 (s, 1 H, OH), 7.77 (bs, 1 H, NH), 7.26 (s, 1 H),
7.01 (dd, J ) 8.88, 2.92 Hz, 1 H), 6.90 (d, J ) 8.75 Hz, 1 H),
6.36 (bs, 1 H, NH), 3.95 (m, 1 H), 1.99-1.67 (m, 3 H), 1.63 (d,
J ) 2.78 Hz, 4 H), 1.52 (s, 9 H), 1.47-0.99 (m, 9 H), 0.91 (d,
J ) 6.47 Hz, 3 H), 0.87 (dd, J ) 6.59, 1.26 Hz, 6 H), 0.81 (s, 3
H), 0.65 (s, 3 H).
5-Am in o-N-(2-pyr idin -2-yleth yl)salicylam ide (11i). From
compound 10i (0.29 g, 0.81 mmol), a similar procedure as that
described for 11a provided white solid 11i (0.21 g, 100%): mp
1
163-165 °C; H NMR (300 MHz, DMSO-d6) δ 12.08 (s, 1 H),
8.85 (t, J ) 5.57 Hz, 1 H), 8.69 (d, J ) 5.13 Hz, 1 H), 8.11 (td,
J ) 7.66, 1.67 Hz, 1 H), 7.75 (d, J ) 2.67 Hz, 1 H), 7.63 (d, J
) 7.92, 1 H), 7.58 (t, J ) 6.41 Hz, 1 H), 7.33 (dd, J ) 8.71,
2.76 Hz, 1 H), 7.03 (d, J ) 8.72 Hz, 1 H), 3.73 (q, J ) 5.82 Hz,
2 H), 3.15 (t, J ) 6.66 Hz, 2 H).
5-Am in o-N-(â-p h en eth yl)sa licyla m id e (11a ). A solution
of 10a (1.0 g, 2.8 mmol) in 6:1 dichloromethane:trifluoroacetic
acid (9 mL) was stirred at room temperature for 2 h. The
solvent was evaporated in vacuo, and diethyl ether (20 mL)
was added. The precipitate was collected, washed with ether
and dried to provide white solid 11a (0.72 g, 100%): mp 190-
5-Am in o-N-(3-p h en yl-1-p r op yl)sa licyla m id e (11j). From
compound 10j (0.35 g, 0.95 mmol), a similar procedure as that
described for 11a provided white solid 11j (0.23 g, 90%): mp
162-164 °C; 1H NMR (300 MHz, DMSO-d6) δ 12.10 (bs, 1 H),
8.76 (t, J ) 5.52 Hz, 1 H), 7.74 (d, J ) 2.69 Hz, 1 H), 7.31-
7.02 (m, 6 H), 7.00 (d, J ) 8.71 Hz, 1 H), 3.70 (bs, 1 H), 3.32
1
191 °C; H NMR (300 MHz, DMSO-d6) δ 8.73 (t, J ) 1.8 Hz,

Lavendustin A Analogues
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3 449
(q, J ) 5.71 Hz, 2 H), 2.63 (t, J ) 7.35 Hz, 2 H), 1.84 (quintet,
J ) 7.37 Hz, 2 H).
(s, 1 H), 6.61 (d, J ) 9.76 Hz, 1 H), 6.44 (dd, J ) 8.40, 2.65 Hz,
1 H), 5.43 (bs, 1 H, NH), 4.09 (d, J ) 4.68 Hz, 2 H), 3.49 (q, J
) 7.22 Hz, 2 H), 2.81 (t, J ) 7.15 Hz, 2 H); ESMS m/z 458 (M+
+ 2), 457 (M+ + 1), 456 (M+). Anal. (C22H21BrN2O4) C, H, N,
Br.
5-Am in o-N-(d od ecyl)sa licyla m id e (11k ). From com-
pound 10k (0.35 g, 0.83 mmol), a similar procedure as that
described for 11a provided white solid 11k (0.27 g, 101%): mp
1
158-160 °C; H NMR (300 MHz, DMSO-d6) δ 8.70 (s, 1 H),
5-[N-[(2,5-Dih yd r oxyp h en yl)m eth yl]a m in o]-N-(4-m eth -
oxy-â-p h en eth yl)sa licyla m id e (13d ). From compounds 11d
(0.60 g, 2.0 mmol), 12 (0.35 g, 2.5 mmol) and NaBH3CN (0.25
g, 4.0 mmol), a similar procedure as that described for 13a
gave pure 13d (0.50 g, 61%) as a white crystalline solid: mp
7.76 (d, J ) 2.43 Hz, 1 H), 7.35 (dd, J ) 8.72, 2.65 Hz, 1 H),
7.06 (d, J ) 8.74 Hz, 1 H), 3.30 (t, J ) 6.8 Hz, 2 H), 1.25 (m,
20 H), 0.85 (t, J ) 6.64 Hz, 3 H).
5-Am in o-N-(1,2,3,4-tetr a h yd r on a p h th a len -2-yl)sa licyl-
a m id e (11l). From compound 10l (0.35 g, 0.91 mmol), a similar
procedure as that described for 11a provided white solid 11l
(0.25 g, 98%): mp 208-209 °C dec; 1H NMR (300 MHz, DMSO-
d6) δ 8.67 (d, J ) 7.54 Hz, 1 H), 7.75 (d, J ) 2.51 Hz, 1 H),
7.25 (dd, J ) 8.69, 2.56 Hz, 1 H), 7.10 (m, 4 H), 6.98 (d, J )
8.72 Hz, 1 H), 4.25 (m, 1 H), 3.09 (dd, J ) 16.46, 5.29 Hz, 1
H), 2.89-2.76 (m, 3 H), 2.03 (m, 1 H), 1.89-1.79 (m, 1 H).
1
173-174 °C dec; H NMR (300 MHz, DMSO-d6) δ 11.42 (s, 1
H, OH), 8.78 (s, 1 H, OH), 8.71 (t, J ) 5.42 Hz, 1 H, NH), 8.57
(s, 1 H, OH), 7.15 (d, J ) 8.60, 2 H), 7.02 (d, J ) 2.41 Hz, 1
H), 6.85 (d, J ) 8.64 Hz, 2 H), 6.72 (dd, J ) 8.76, 2.57 Hz, 1
H), 6.65 (d, J ) 8.88 Hz, 1 H), 6.63 (d, J ) 3.03 Hz, 1 H), 6.60
(d, J ) 8.53 Hz, 1 H), 6.44 (dd, J ) 8.48, 2.88 Hz, 1 H), 5.42
(t, J ) 5.68 Hz, 1 H, NH), 4.10 (d, J ) 4.82 Hz, 2 H), 3.72 (s,
3 H), 3.46 (q, J ) 6.91 Hz, 2 H), 2.76 (t, J ) 7.02 Hz, 2 H);
ESMS m/z 431 (MNa+), 409 (MH+). Anal. (C23H24N2O5) C, H,
N.
5-Am in o-N-(3â-ch olesta n yl)sa licyla m id e (11m ). From
compound 10m (0.29 g, 0.46 mmol), a similar procedure as
that described for 11a provided white solid 11m (0.21 g,
1
5-[N-[(2,5-Dih yd r oxyp h en yl)m et h yl]a m in o]-N-(b en z-
yl)sa licyla m id e (13e). From compounds 12 (0.38 g, 2.6
mmol), 11e (0.54 g, 2.2 mmol) and NaBH3CN (0.14 g, 2.2
mmol), a similar procedure as that described for 13a gave pure
13e (0.55 g, 69%) as a white crystalline solid: mp 195-196
87%): mp 200-202 °C dec; H NMR (300 MHz, DMSO-d6) δ
12.18 (bs, 1 H, OH), 8.51 (d, J ) 7.73 Hz, 1 H), 7.73 (d, J )
2.46 Hz, 1 H), 7.26 (dd, J ) 8.72, 2.56 Hz, 1 H), 6.98 (d, J )
8.74 Hz, 1 H), 1.94-0.98 (m, 20 H), 0.88 (d, J ) 6.35 Hz, 3 H),
0.83 (d, J ) 6.65 Hz, 6 H), 0.80 (s, 3 H), 0.62 (s, 3 H).
1
°C; H NMR (300 MHz, DMSO-d6) δ 11.43 (s, 1 H), 9.18 (t, J
5-[N-[(2,5-Dih yd r oxyp h en yl)m et h yl]a m in o]-N-(p h en -
eth yl)sa licyla m id e (13a ). 2,5-Dihydroxybenzaldehyde (12)
(0.21 g, 1.40 mmol) was added to 11a (0.35 g, 1.40 mmol) in
benzene (40 mL), and the mixture was heated to reflux under
argon for 24 h, using a Dean-Stark trap. The reaction mixture
was then concentrated to remove the benzene completely, and
the residue was redissolved in methanol (15 mL). While
stirring, sodium cyanoborohydride NaBH3CN (0.18 g, 2.81
mmol) was added in three portions during 30 min, and the
reaction mixture was stirred at room temperature for an
additional 1 h. To the reaction mixture was then added a
saturated solution of NaCl (100 mL) containing 37% HCl (0.28
g, 2.8 mmol). The reaction mixture was extracted with ethyl
acetate (3 × 50 mL). The combined organic layers were washed
with brine (10 mL), dried over sodium sulfate and concentrated
to furnish the crude product, which was further purified by
flash chromatography (silica gel 40 g, ethyl acetate:hexane 1:1).
The product 13a (0.37 g, 71.4%) was isolated as a light yellow
solid: mp 175-177 °C; 1H NMR (300 MHz, DMSO-d6) δ 11.43
(s, 1 H, OH), 8.76 (m, 2 H, OH, NH), 8.58 (s, 1 H, OH), 7.35-
7.20 (m, 5 H), 7.05 (d, J ) 2.51, 1 H), 6.74 (dd, J ) 2.28, 8.79
Hz, 1 H), 6.68 (d, J ) 8.97 Hz, 1 H), 6.66 (d, J ) 3.53 Hz, 1 H),
6.63 (d, J ) 8.56 Hz, 1 H), 6.46 (dd, J ) 2.75, 8.58 Hz, 1 H),
5.43 (bs, 1 H, NH), 4.11 (s, 2 H), 3.52 (q, J ) 6.52 Hz, 2 H),
2.86 (t, J ) 7.19 Hz, 2 H); FABMS (Gly) m/z 379 (MH+). Anal.
(C22H22N2O4) C, H, N.
) 6.24 Hz, 1 H), 8.75 (s, 1 H), 8.56 (s, 1 H), 7.38-7.20 (m, 5
H), 7.09 (d, J ) 2.69 Hz, 1 H), 6.76 (dd, J ) 8.46, 2.66 Hz, 1
H), 6.68 (d, J ) 8.91 Hz, 1 H), 6.65 (d, J ) 3.11 Hz, 1 H), 6.60
(d, J ) 8.46 Hz, 1 H), 6.43 (dd, J ) 8.46, 3.12 Hz, 1 H), 5.41
(t, J ) 5.41 Hz, 1 H), 4.49 (d, J ) 5.79, 2 H), 4.09 (d, J ) 5.35
Hz, 2 H); CIMS m/z 379 (MH+). Anal. (C21H20N2O4) C, H, N.
5-[N-[(2,5-Dih ydr oxyph en yl)m eth yl]am in o]-N-(4-ch lor o-
â-p h en eth yl)sa licyla m id e (13f). From compounds 11f (0.34
g, 1.2 mmol), 12 (0.16 g, 1.2 mmol) and NaBH3CN (0.15 g, 2.4
mmol), a similar procedure as that described for 13a gave pure
13f (0.34 g, 69%) as a light yellow crystalline solid: mp 158-
1
160 °C; H NMR (300 MHz, DMSO-d6) δ 11.35 (s, 1 H, OH),
8.77 (s, 1 H, OH), 8.71 (t, J ) 5.31 Hz, 1 H, NH), 8.56 (s, 1 H,
OH), 7.48 (d, J ) 8.20 Hz, 2 H), 7.21 (d, J ) 8.28 Hz, 2 H),
7.00 (d, J ) 2.87, 1 H), 6.72 (dd, J ) 8.55, 2.40 Hz, 1 H), 6.67
(d, J ) 9.37 Hz, 1 H), 6.60 (s, 1 H), 6.61 (d, J ) 9.76 Hz, 1 H),
6.44 (dd, J ) 8.40, 2.65 Hz, 1 H), 5.44 (t, J ) 5.29 Hz, 1 H,
NH), 4.09 (d, J ) 4.68 Hz, 2 H), 3.49 (q, J ) 7.22 Hz, 2 H),
2.81 (t, J ) 7.15 Hz, 2 H); ESMS m/z 414 (M+ + 2), 412 (M+).
Anal. (C22H21ClN2O4) C, H, N, Cl.
5-[N-[(2,5-Dih yd r oxyp h en yl)m et h yl]a m in o]-N-(4-h y-
d r oxyp h en eth yl)sa licyla m id e (13g). From compounds 11g
(0.22 g, 0.81 mmol), 12 (0.14 g, 0.97 mmol) and NaBH3CN (0.15
g, 2.3 mmol), a similar procedure as that described for 13a
gave pure 13g (0.18 g, 55%) as a slightly yellow crystalline
solid: mp 175-176 °C; 1H NMR (300 MHz, DMSO-d6) δ 11.38
(s, 1 H, OH), 9.87 (s, 1 H, OH), 8.76 (s, 1 H, OH), 8.68 (t, J )
5.33 Hz, 1 H, NH), 8.56 (s, 1 H, OH), 7.30 (t, J ) 7.07 Hz, 2
H), 7.10 (t, J ) 8.92 Hz, 1 H), 7.00 (d, J ) 2.78 Hz, 1 H), 6.73
(dd, J ) 8.68, 2.61 Hz, 1 H), 6.67 (d, J ) 9.09 Hz, 2 H), 6.62
(d, J ) 2.50 Hz, 1 H), 6.59 (d, J ) 8.62 Hz, 1 H), 6.42 (dd, J )
8.44, 2.85 Hz, 1 H), 5.40 (bs, 1 H, NH), 4.11 (d, J ) 3.88 Hz,
2 H), 3.47 (q, J ) 5.92 Hz, 2 H), 2.82 (t, J ) 7.08 Hz, 2 H);
ESMS m/z 417 (MNa+), 395 (MH+). Anal. (C22H22N2O5) C, H,
N.
5-[N-[(2,5-Dih yd r oxyp h en yl)m eth yl]a m in o]-N-(2-m or -
p h olin oeth yl)sa licyla m id e (13h ). From compounds 12 (0.12
g, 0.80 mmol), 11h (0.20 g, 0.75 mmol) and NaBH3CN (0.19 g,
3.0 mmol), a similar procedure as that described for 13a gave
pure 13h (0.18 g, 55%) as a yellow crystalline solid: mp 184-
186 °C dec; 1H NMR (300 MHz, DMSO-d6) δ 11.25 (s, 1 H,
OH), 8.77 (s, 1 H, OH), 8.66 (t, J ) 4.39 Hz, 1 H, NH), 8.55 (s,
1 H, OH), 7.02 (d, J ) 2.21 Hz, 1 H), 6.74 (dd, J ) 8.80, 2.55
Hz, 1 H), 6.67 (d, J ) 8.00 Hz, 1 H), 6.63 (d, J ) 2.87 Hz, 1 H),
6.60 (d, J ) 8.66 Hz,1 H), 6.43 (dd, J ) 8.42, 2.85 Hz, 1 H),
5.45 (s, 1 H, NH), 4.08 (s, 2 H), 3.56 (t, J ) 4.42 Hz, 4 H), 3.38
(m, 4 H), 2.43 (m, 4 H); ESMS m/z 388 (MH+). Anal.
(C20H25N5O5) C, H, N.
5-[N -[(2,5-D ih y d r o x y p h e n y l)m e t h y l]a m in o ]-N -(4-
flu or op h en eth yl)sa licyla m id e (13b). From compounds 11b
(0.56 g, 2.0 mmol), 12 (0.35 g, 2.5 mmol), and NaBH3CN (0.26
g, 4.1 mmol), a similar procedure as that described for 13a
gave pure 13b (0.34 g, 42%) as a slightly yellow crystalline
solid: mp 154-156 °C; 1H NMR (300 MHz, CDCl3) δ 11.37 (s,
1 H), 8.77 (s, 1 H), 8.72 (t, J ) 5.33 Hz, 1 H), 8.56 (s, 1 H),
7.27 (t, J ) 7.07 Hz, 2 H), 7.10 (t, J ) 8.92 Hz, 2 H), 7.00 (d,
J ) 2.78 Hz, 1 H), 6.74 (dd, J ) 8.68, 2.61 Hz, 1 H), 6.67 (d,
J ) 9.09 Hz, 1 H), 6.62 (s, 1 H), 6.59 (d, J ) 8.62 Hz, 1 H),
6.42 (dd, J ) 8.44, 2.85 Hz, 1 H), 5.42 (bs, 1 H, NH), 4.09 (d,
J ) 3.88 Hz. 2 H), 3.47 (q, J ) 5.92 Hz, 2 H), 2.82 (t, J ) 7.08
Hz, 2 H); ESMS m/z 419 (MNa+), 397 (MH+). Anal. (C22H21
-
FN2O4) C, H, N, F.
5-[N-[(2,5-Dih ydr oxyph en yl)m eth yl]am in o]-N-(4-br om o-
â-p h en eth yl)sa licyla m id e (13c). From compounds 11c (0.48
g, 1.4 mmol), 12 (0.24 g, 1.4 mmol) and NaBH3CN (0.20 g, 2.9
mmol), a similar procedure as that described for 13a gave pure
13c (0.42 g, 64%) as a white crystalline solid: mp 179-180
1
°C; H NMR (300 MHz, CDCl3) δ 11.35 (s, 1 H), 8.77 (s, 1 H),
8.71 (t, J ) 2.44 Hz, 1 H), 8.56 (s, 1 H), 7.48 (d, J ) 8.20 Hz,
2 H), 7.21 (d, J ) 8.28 Hz, 2 H), 7.00 (d, J ) 2.87, 1 H), 6.72
(dd, J ) 8.55, 2.40 Hz, 1 H), 6.67 (d, J ) 9.37 Hz, 1 H), 6.60

450 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3
Mu et al.
5-[N-[(2,5-Dih yd r oxyp h en yl)m eth yl]a m in o]-N-(2-p yr i-
d in -2-yleth yl)sa licyla m id e (13i). From compounds 11i (0.24
g, 0.93 mmol), 12 (0.17 g, 1.2 mmol) and NaBH3CN (0.25 g,
4.0 mmol), a similar procedure as that described for 13a gave
pure 13i (0.18 g, 51%) as a white solid: mp 114-116 °C dec;
1H NMR (300 MHz, DMSO-d6) δ 11.68 (s, 1 H, OH), 9.13 (s, 1
H, OH), 8.79 (m, 2 H), 8.62 (t, J ) 2.12 Hz, 1 H, NH), 8.59 (s,
1 H, OH), 8.53 (d, J ) 4.67 Hz, 1 H), 7.72 (td, J ) 7.67, 1.80
Hz, 1 H), 7.01 (d, J ) 2.51 Hz, 1 H), 6.74 (dd, J ) 8.87, 2.31
Hz, 1 H), 6.69 (d, J ) 8.83 Hz, 1 H), 6.65 (d, J ) 2.53 Hz, 1 H),
6.61 (d, J ) 8.94 Hz, 1 H), 6.46 (dd, J ) 8.61, 2.77 Hz, 1 H),
5.39 (t, J ) 1.98 Hz, 1 H, NH), 4.08 (s, 2 H), 3.65 (t, J ) 7.20
Hz, 2 H), 3.05 (t, J ) 7.31 Hz, 2 H); ESMS m/z 380 (MH+).
Anal. (C21H21N3O4) C, H, N.
NMR (300 MHz, DMSO-d6) δ 9.71 (s, 1 H), 7.82 (d, J ) 8.64
Hz, 2 H), 7.54 (d, J ) 8.68 Hz, 2 H), 1.47 (s, 9 H).
4-[N -(t er t -Bu t oxyca r b on yl)a m in o]-N -(b e n zyl)b e n z-
a m id e (16a ). From compound 15 (0.38 g, 1.5 mmol), EDCI
(0.43 g, 2.2 mmol), HOBt (0.30 g, 2.2 mmol), triethylamine
(0.87 mL, 6.0 mmol) and benzylamine (9e) (0.33 mL, 3.0
mmol), a similar procedure as that described for 10a gave pure
16a (0.34 g, 69%) as a white crystalline solid: mp 198-199
1
°C; H NMR (300 MHz, CDCl3) δ 7.74 (d, J ) 8.58 Hz, 2 H),
7.43 (d, J ) 8.55 Hz, 2 H), 7.37-7.26 (m, 5 H), 4.64 (d, J )
5.63 Hz, 2 H), 1.52 (s, 9 H).
4-[N-(ter t-Bu toxycar bon yl)am in o]-N-(â-ph en eth yl)ben z-
a m id e (16b). From compound 15 (0.278 g, 1.16 mmol), EDCI
(0.45 g, 2.34 mmol), HOBt (0.32 g, 2.34 mmol), triethylamine
(0.36 mL, 4.68 mmol) and â-phenethylamine (9a ) (1.0 mL, 11.7
mmol), a similar procedure as that described for 10a gave pure
16b (0.23 g, 58%) as a white crystalline solid: mp 210-211
5-[N-[(2,5-Dih yd r oxyp h en yl)m eth yl]a m in o]-N-(3-p h en -
yl-1-p r op yl)sa licyla m id e (13j). From compounds 12 (0.13
g, 0.98 mmol) 11j (0.22 g, 0.81 mmol) and NaBH3CN (0.2 g,
3.1 mmol), a similar procedure as that described for 13a gave
pure 13j (0.19 g, 60%) as a white crystalline solid: mp 152-
1
°C; H NMR (300 MHz, CDCl3) δ 7.64 (d, J ) 8.69 Hz, 2 H),
7.40 (d, J ) 8.65 Hz, 2 H), 7.36-7.22 (m, 5 H), 6.62 (s, 1 H),
6.05 (t, J ) 6.5 Hz, 1 H), 3.71 (q, J ) 6.8 Hz, 2 H), 2.93 (t, J
) 6.9 Hz, 2 H), 1.52 (s, 9 H).
1
154 °C; H NMR (300 MHz, DMSO-d6) δ 11.56 (s, 1 H, OH),
8.77 (s, 1 H, OH), 8.69 (t, J ) 4.58 Hz, 1 H, NH), 8.56 (s, 1 H,
OH), 7.32-7.16 (m, 5 H), 7.03 (d, J ) 2.42 Hz, 1 H), 6.75 (dd,
J ) 8.82, 2.53 Hz, 1 H), 6.68 (d, J ) 8.80 Hz, 1 H), 6.64 (d, J
) 2.90 Hz, 1 H), 6.62 (d, J ) 8.59 Hz, 1 H), 6.45 (dd, J ) 8.53,
2.94 Hz, 1 H), 5.40 (t, J ) 5.78 Hz, 1 H, NH), 4.10 (d, J ) 4.51
Hz, 2 H), 3.29 (q, J ) 4.87 Hz, 2 H), 2.62 (t, J ) 7.53 Hz, 2 H),
1.83 (quintet, J ) 7.26 Hz, 2 H); CIMS m/z 393 (MH+). Anal.
(C23H24N2O4) C, H, N.
4-[N-(ter t-Bu toxyca r bon yl)a m in o]-N-(3-p h en yl-1-p r o-
p yl)ben za m id e (16c). From compound 15 (0.38 g, 1.5 mmol),
EDCI (0.43 g, 2.2 mmol), HOBt (0.30 g, 2.2 mmol), triethy-
lamine (0.87 mL, 6.0 mmol) and 3-phenyl-1-propylamine (9j)
(0.43 mL, 3.0 mmol), a similar procedure as that described for
10a gave pure 16c (0.43 g, 81%) as a white crystalline solid:
mp 178-180 °C; 1H NMR (300 MHz, CDCl3) δ 7.61 (d, J )
8.69 Hz, 2 H), 7.42 (d, J ) 8.70 Hz, 2 H), 7.33-7.19 (m, 5 H),
4.69 (q, J ) 6.19 Hz, 2 H), 2.73 (t, J ) 7.28 Hz, 2 H), 1.97
(quint, J ) 7.47 Hz, 2 H), 1.53 (s, 9 H).
5-[N-[(2,5-Dih ydr oxyph en yl)m eth yl]am in o]-N-(dodecyl)-
sa licyla m id e (13k ). From compounds 11k (0.30 g, 0.93
mmol), 12 (0.14 g, 1.0 mmol) and NaBH3CN (0.13 g, 2.0 mmol),
a similar procedure as that described for 13a gave pure 13k
(0.23 g, 56%) as a slightly yellow crystalline solid: mp 126-
4-Am in o-N-(b en zyl)b en za m id e (17a ). From compound
16a (0.30 g, 0.92 mmol), a similar procedure as that described
for 11a provided white solid 17a (0.20 g, 100%): mp 199-200
1
128 °C; H NMR (300 MHz, DMSO-d6) δ 11.59 (s, 1 H, OH),
1
°C dec; H NMR (300 MHz, DMSO-d6) δ 8.67 (t, J ) 5.66 Hz,
8.77 (s, 1 H, OH), 8.62 (t, J ) 2.12 Hz, 1 H, NH), 8.56 (s, 1 H,
OH), 7.01 (d, J ) 2.51 Hz, 1 H), 6.74 (dd, J ) 8.87, 2.31 Hz, 1
H), 6.69 (d, J ) 8.83 Hz, 1 H), 6.65 (d, J ) 2.53 Hz, 1 H), 6.61
(d, J ) 8.94 Hz, 1 H), 6.46 (dd, J ) 8.61, 2.77 Hz, 1 H), 5.39
(t, J ) 1.98 Hz, 1 H, NH), 4.09 (s, 2 H), 3.25 (t, J ) 7.90 Hz,
2 H), 1.50 (m, 2 H), 1.20 (s, 18 H), 0.88 (t, J ) 6.87 Hz, 3 H);
CIMS m/z 443 (MH+). Anal. (C26H38N2O4) C, H, N.
1 H, NH), 7.70 (d, J ) 8.57 Hz, 2 H), 7.34-7.20 (m, 5 H), 6.71
(d, J ) 8.52 Hz, 2 H), 5.11 (bs, 2 H, NH2), 4.44 (d, J ) 4.22
Hz, 2 H).
4-Am in o-N-(â-p h en eth yl)ben za m id e (17b). From com-
pound 16b (0.066 g, 0.19 mmol), a similar procedure as that
described for 11a provided 17b (0.045 g, 98.6%) as a white
solid: 1H NMR (300 MHz, CDCl3) δ 7.54 (d, J ) 8.51 Hz, 2
H), 7.35-7.24 (m, 5 H), 6.65 (d, J ) 8.44 Hz, 2 H), 6.15 (bs, 1
H), 4.46 (bs, 2 H), 3.68 (q, J ) 6.32 Hz, 2 H), 2.92 (t, J ) 6.80
Hz, 2 H).
5-[N-[(2,5-Dih yd r oxyp h en yl)m eth yl]a m in o]-N-(1,2,3,4-
tetr a h yd r on a p h th a len -2-yl)sa licyla m id e (13l). From com-
pounds 11l (0.32 g, 1.13 mmol), 12 (0.19 g, 1.36 mmol) and
NaBH3CN (0.3 g, 4.7 mmol), a similar procedure as that
described for 13a gave pure 13l (0.28 g, 61%) as a yellow
crystalline solid: mp 159-161 °C; 1H NMR (300 MHz, DMSO-
d6) δ 11.30 (s, 1 H, OH), 8.79 (s, 1 H, OH), 8.62 (t, J ) 4.58, 1
H, NH), 8.58 (s, 1 H, OH), 7.11 (m, 5 H), 6.74 (dd, J ) 8.83,
2.41 Hz, 1 H), 6.67 (d, J ) 8.87 Hz, 1 H), 6.65 (d, J ) 2.82 Hz,
1 H), 6.60 (d, J ) 8.49 Hz, 1 H), 6.45 (dd, J ) 8.44, 2.84 Hz,
1 H), 5.45 (t, J ) 5.30 Hz, 1 H, NH), 4.22 (m, 1 H), 4.11 (d, J
) 4.51 Hz, 2 H), 3.07 (dd, J ) 16.34, 5.10 Hz, 1 H), 2.89-2.76
(m, 3 H), 2.03 (m, 1 H), 1.80 (m, 1 H); CIMS m/z 405 (MH+).
Anal. (C24H24N2O4) C, H, N.
4-Am in o-N-(3-p h en yl-1-p r op yl)ben za m id e (17c). From
compound 16c (0.43 g, 0.92 mmol), a similar procedure as that
described for 11a provided 17c (0.23 g, 100%) as a white
1
solid: mp 126-128 °C; H NMR (300 MHz, DMSO-d6) δ 8.11
(t, J ) 5.25 Hz, 1 H, NH), 7.64 (d, J ) 8.59 Hz, 2 H), 7.30-
7.17 (m, 5 H), 6.71 (d, J ) 8.55 Hz, 2 H), 5.51 (bs, 2 H, NH2),
3.23 (q, J ) 7.04 Hz, 2 H), 2.60 (t, J ) 7.39 Hz, 2 H), 1.81
(quint, J ) 7.21 Hz, 2 H).
4-[N-[(2,5-Dih yd r oxyp h en yl)m et h yl]a m in o]-N-(b en z-
yl)ben za m id e (18a ). From compounds 12 (0.17 g, 1.25 mmol),
17a (0.34 g, 1.04 mmol) and NaBH3CN (0.26 g, 4.2 mmol), a
similar procedure as that described for 13a gave pure 18a (0.28
g, 77%) as a slightly yellow crystalline solid: mp 204-205 °C;
1H NMR (300 MHz, DMSO-d6) δ 8.81 (s, 1 H, OH), 8.57 (t, J
) 3.79 Hz, 1 H, NH), 8.57 (s, 1 H, OH), 7.65 (d, J ) 8.72 Hz,
2 H), 7.32-7.18 (m, 6 H), 6.63 (d, J ) 8.37 Hz, 1 H), 6.58 (d,
J ) 2.76 Hz, 1 H), 6.55 (d, J ) 8.72 Hz, 2 H), 6.44 (dd, J )
8.46, 2.81 Hz, 1 H), 4.43 (d, J ) 5.88 Hz, 2 H), 4.16 (s, 2 H);
CIMS m/ z 349 (MH+). Anal. (C21H20ClN2O3) C, H, N.
4-[N-[(2,5-Dih yd r oxyp h en yl)m eth yl]a m in o]-N-(â-p h en -
eth yl)ben za m id e (18b). From compounds 12 (0.031 g, 0.22
mmol), 17b (0.045 g, 0.22 mmol) and NaBH3CN (0.03 g, 0.47
mmol), a similar procedure as that described for 13a gave pure
18b (0.040 g, 59%) as a yellow solid: mp 154-156 °C; 1H NMR
(300 MHz, DMSO-d6) δ 8.81 (s, 1 H, OH), 8.57 (s, 1 H, OH),
8.10 (t, J ) 5.38 Hz, 1 H, NH), 7.58 (d, J ) 8.41 Hz, 2 H),
7.33-7.17 (m, 6 H), 6.63 (d, J ) 8.47 Hz, 1 H), 6.58 (d, J )
2.74 Hz, 1 H), 6.54 (d, J ) 8.48 Hz, 2 H), 6.45 (dd, J ) 8.47,
2.83 Hz, 1 H), 4.17 (d, J ) 4.56 Hz, 2 H), 3.41 (q, J ) 8.15 Hz,
5-[N -[(2,5-Dih yd r oxyp h e n yl)m e t h yl]a m in o]-N -(3â-
ch olesta n yl)sa licyla m id e (13m ). From compounds 11m
(0.15 g, 0.28 mmol), 12 (0.05 g, 0.35 mmol) and NaBH3CN (0.14
g, 2.2 mmol), a similar procedure as that described for 13a
gave pure 13m (0.08 g, 44%) as a yellow solid: mp 202-204
1
°C dec; H NMR (300 MHz, DMSO-d6) δ 11.65 (s, 1 H, OH),
8.79 (s, 1 H, OH), 8.58 (s, 1 H, OH), 8.38 (d, 1 H, NH), 7.04 (d,
J ) 2.16 Hz, 1 H), 6.72 (dd, J ) 8.81, 2.18 Hz, 1 H), 6.66-6.64
(m, 2 H), 6.59 (d, J ) 8.56 Hz, 1 H), 6.45 (dd, J ) 8.50, 2.70
Hz, 1 H), 5.35 (bs, 1 H, NH), 4.09 (s, 2 H), 3.81 (m, 1 H), 1.95-
1.00 (m, 21 H), 0.90 (d, J ) 6.34 Hz, 3 H), 0.86 (dd, J ) 6.66,
1.17 Hz, 6 H), 0.82 (s, 3 H), 0.63 (s, 3 H); CIMS m/z 646 (MH+).
Anal. (C41H60N2O4) C, H, N.
4-[(ter t-Bu toxycar bon yl)am in o]ben zoic Acid (15). From
4-aminobenzoic acid (14) (0.5 g, 3.65 mmol), triethylamine
(1.02 mL, 7.30 mmol) and di-tert-butyl dicarbonate (1.59 g, 7.30
mmol), a similar procedure as that described for 8 provided
1
pure 15 (0.86 g, 100%) as a white solid: mp 191-192 °C; H

Lavendustin A Analogues
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3 451
2 H), 2.80 (t, J ) 7.89 Hz, 2 H); CIMS m/z 363 (MH+). Anal.
(C22H22N2O3) C, H, N.
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Hydroxylated 2-(5′-Salicyl)naphthalenes as Protein-Tyrosine
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4-[N-[(2,5-Dih yd r oxyp h en yl)m eth yl]a m in o]-N-(3-p h en -
yl-1-p r op yl)ben za m id e (18c). From compounds 12 (0.17 g,
0.85 mmol), 17c (0.20 g, 0.8 mmol) and NaBH3CN (0.21 g, 3.2
mmol), a similar procedure as that described for 13a gave pure
18c (0.22 g, 73%) as a slightly yellow crystalline solid: mp
133-134 °C; 1H NMR (300 MHz, DMSO-d6) δ 8.80 (s, 1 H,
OH), 8.57 (s, 1 H, OH), 8.00 (t, J ) 5.49 Hz, 1 H, NH), 7.58 (d,
J ) 8.69 Hz, 2 H), 7.30-7.14 (m, 6 H), 6.62 (d, J ) 8.54 Hz, 1
H), 6.57 (d, J ) 2.76 Hz, 1 H), 6.53 (d, J ) 8.70 Hz, 2 H), 6.42
(dd, J ) 8.50, 2.90 Hz, 1 H), 4.15 (s, 2 H), 3.21 (q, J ) 6.60 Hz,
2 H), 2.59 (t, J ) 7.84 Hz, 2 H), 1.79 (quint, J ) 7.55 Hz, 2 H);
CIMS m/z 377 (MH+). Anal. (C23H24N2O3) C, H, N.
3-[N-(ter t-Bu toxyca r bon yl)a m in o]ben zoic Acid (20).
From 3-aminobenzoic acid (14) (0.75 g, 5.47 mmol), triethy-
lamine (1.5 mL, 8.20 mmol) and di-tert-butyl dicarbonate (1.78
g, 8.20 mmol), a similar procedure as that described for 8
provided pure 20 (1.3 g, 100%) as a white solid: mp 189-190
1
°C; H NMR (DMSO-d6) δ 9.53 (s, 1 H), 8.13 (s, 1 H), 7.61 (d,
J ) 7.66 Hz, 1 H), 7.53 (d, J ) 7.52 Hz, 1 H), 7.35 (t, J ) 7.91
Hz, 1 H), 1.47 (s, 9 H).
3-[N-(ter t-Bu toxycar bon yl)am in o]-N-(â-ph en eth yl)ben -
zoyla m id e (21). From compound 20 (0.48 g, 2.03 mmol), EDCI
(0.45 g, 2.34 mmol), HOBt (0.33 g, 2.34 mmol), triethylamine
(0.37 mL, 4.68 mmol) and â-phenethylamine (9a ), a similar
procedure as that described for 10a gave pure 21 (0.35 g, 51%)
as a white solid: mp 186-188 °C; 1H NMR (300 MHz, CDCl3)
δ 7.51 (t, J ) 4.86 Hz, 1 H), 7.36-7.31 (m, 4 H), 7.24 (d, J )
4.08 Hz, 1 H), 7.22 (d, J ) 2.3 Hz, 1 H), 6.61 (s, 1 H), 6.22 (bs,
1 H), 3.71 (q, J ) 6.9 Hz, 2 H), 2.93 (t, J ) 7.0 Hz, 2 H), 1.53
(s, 9 H).
3-Am in o-N-(â-p h en eth yl)ben za m id e (22). From com-
pound 21 (0.23 g, 0.66 mmol), a similar procedure as that
described for 11a provided white solid 22 (0.16 g, 100%): mp
137-139 °C; 1H NMR (300 MHz, DMSO-d6) δ 8.47 (t, 1 H),
7.35-7.18 (m, 7 H), 6.99 (s, 2 H), 3.46 (q, J ) 7.00 Hz, 2 H),
2.83 (t, J ) 7.06 Hz, 2 H).
3-[N-[(2,5-Dih yd r oxyp h en yl)m eth yl]a m in o]-N-(â-p h en -
eth yl)ben za m id e (23). From compounds 12 (0.25 g, 1.77
mmol), 22 (0.38 g, 1.58 mmol) and NaBH3CN (0.2 g, 3.16
mmol), a similar procedure as that described for 13a gave pure
1
23 (0.42 g, 73%) as a light yellow solid: mp 124-126 °C; H
NMR (300 MHz, DMSO-d6) δ 8.81 (s, 1 H, OH), 8.58 (s, 1 H,
OH), 8.35 (t, J ) 4.93 Hz, 1 H, NH), 7.33-7.19 (m, 5 H), 7.11
(t, J ) 7.74 Hz, 1 H), 7.03 (d, J ) 2.11 Hz, 1 H), 6.95 (d, J )
7.61 Hz, 1 H), 6.69 (dd, J ) 2.24, 8.24 Hz, 1 H), 6.64 (d, J )
8.40 Hz, 1 H), 6.24 (s, 1 H), 6.45 (dd, J ) 2.91, 8.28 Hz, 1 H),
6.20 (t, J ) 4.56 Hz, 1 H, NH), 4.17 (d, J ) 5.46 Hz, 2 H), 3.48
(q, J ) 6.55 Hz, 2 H), 2.83 (t, J ) 7.67 Hz, 2 H); CIMS m/z 363
(MH+). Anal. (C22H22N2O3) C, H, N.
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Ack n ow led gm en t. This research was made possible
by grants from the Showalter Trust, Purdue Research
Foundation, National Institutes of Health (CA37372 and
CA80770), and American Chemical Society (IRG 58-006-
40).
Su p p or tin g In for m a tion Ava ila ble: Elemental analyses
for compounds 13a -m , 18a -c, and 23. This material is
available free of charge via the Internet at http://pubs.acs.org.
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