N-Hydroxy-3-phenyl-2-propenamides as novel inhibitors of human histone deacetylase with in vivo antitumor activity: Discovery of (2E)-N-hydroxy-3-[4-[[(2-hydroxyethyl)[2-(1H-indol-3-yl)ethyl] amino]methyl]-phenyl]-2-propenamide (NVP-LAQ824)

Article abstract of DOI:10.1021/jm030235w

A series of N-hydroxy-3-phenyl-2-propenamides were prepared as novel inhibitors of human histone deacetylase (HDAC). These compounds were potent enzyme inhibitors, having IC₅₀s < 400 nM in a partially purified enzyme assay. However, potency in cell growth inhibition assays ranged over 2 orders of magnitude in two human carcinoma cell lines. Selected compounds having cellular IC₅₀ < 750 nM were tested for maximum tolerated dose (MTD) and for efficacy in the HCT116 human colon tumor xenograft assay. Four compounds having an MTD ≥ 100 mg/kg were selected for dose-response studies in the HCT116 xenograft model. One compound, 9 (NVP-LAQ824), had significant dose-related activity in the HCT116 colon and A549 lung tumor models, high MTD, and low gross toxicity. On the basis, in part, of these properties, 9 has entered human clinical trials in 2002.

Full text of DOI:10.1021/jm030235w

J . Med. Chem. 2003, 46, 4609-4624
4609
N-Hyd r oxy-3-p h en yl-2-p r op en a m id es a s Novel In h ibitor s of Hu m a n Histon e
Dea cetyla se w ith in Vivo An titu m or Activity: Discover y of
(2E)-N-Hyd r oxy-3-[4-[[(2-h yd r oxyeth yl)[2-(1H-in d ol-3-yl)eth yl]a m in o]m eth yl]-
p h en yl]-2-p r op en a m id e (NVP -LAQ824)
Stacy W. Remiszewski,*,† Lidia C. Sambucetti,‡ Kenneth W. Bair,§ J ohn Bontempo, David Cesarz,
Nagarajan Chandramouli, Ru Chen, Min Cheung, Susan Cornell-Kennon, Karl Dean, George Diamantidis,
Dennis France, Michael A. Green, Kobporn Lulu Howell, Rina Kashi, Paul Kwon, Peter Lassota,
Mary S. Martin, Yin Mou, Lawrence B. Perez, Sushil Sharma, Troy Smith, Eric Sorensen, Francis Taplin,
Nancy Trogani, Richard Versace, Heather Walker, Susan Weltchek-Engler, Alexander Wood, Arthur Wu, and
Peter Atadja
Oncology Research, Novartis Institute for Biomedical Research, 1 Health Plaza, East Hanover, New J ersey 07936-1080
Received May 15, 2003
A series of N-hydroxy-3-phenyl-2-propenamides were prepared as novel inhibitors of human
histone deacetylase (HDAC). These compounds were potent enzyme inhibitors, having IC50s <
400 nM in a partially purified enzyme assay. However, potency in cell growth inhibition assays
ranged over 2 orders of magnitude in two human carcinoma cell lines. Selected compounds
having cellular IC50 < 750 nM were tested for maximum tolerated dose (MTD) and for efficacy
in the HCT116 human colon tumor xenograft assay. Four compounds having an MTD g 100
mg/kg were selected for dose-response studies in the HCT116 xenograft model. One compound,
9 (NVP-LAQ824), had significant dose-related activity in the HCT116 colon and A549 lung
tumor models, high MTD, and low gross toxicity. On the basis, in part, of these properties, 9
has entered human clinical trials in 2002.
In tr od u ction
initiated based on this general HDAI structure in
parallel with a high throughput screen (HTS) of the
Novartis compound archive. Several structural classes
identified from these efforts were examined as leads.
The most promising compound, 8 (NVP-LAK974), was
obtained from the HTS. While this compound met our
basic structural and in vitro requirements, it had poor
efficacy in the HCT116 human colon tumor xenograft
model. We, therefore, instituted a systematic structural
exploration of N-hydroxy-3-phenyl-2-propenamides with
the goal of synthesizing a novel, well tolerated, effica-
cious compound with acceptable pharmaceutical proper-
ties. Our efforts have led to the discovery of 9 (NVP-
LAQ824), a potent HDAI and antitumor agent currently
undergoing human clinical trials against both solid
tumors and leukemia.
Histone acetylation is one major regulator of gene
expression which may act by changing the accessibility
of transcription factors to DNA.1 Cell specific patterns
of gene expression dependent on histone acetylation
result from a balance of the competing activities of two
classes of enzymes, the histone acetyl transferases2,3 and
the histone deacetylases (HDACs).4,5 Perturbations of
this balance have been linked to cancer,6,7 and inhibition
of HDAC has been shown to have antiproliferative
effects on tumor cell lines, resulting in considerable
interest in this field. Several HDAC inhibitors (HDAIs)
are in clinical trials as anticancer agents, including
SAHA (1), FK-228 (2), and MS-275 (3, Chart 1).8-12
We initiated a program to discover novel HDAIs as
anticancer agents and, in undertaking the development
of a new class of inhibitors, set the following minimum
requirements to select compounds for in vivo evalua-
tion: Novel structural type, enzyme potency < 500 nM,
and cellular potency < 750 nM. On the basis of the
known HDAIs trichostatin A13 (TSA, 4), dimethylami-
nobenzamidylcaprylic hydroxamate14,15 (DBCH, 5), and
trapoxin B16 (TpxB, 6), we, and others,17,18 have pro-
posed a generalized HDAI structure as A-B-C 4 (Chart
1), where C is a Zn2+ ligand, B is a spacer, and A is a
specificity element. A synthetic chemistry effort was
Ch em istr y
The synthesis of the majority of compounds was
accomplished by reductive amination of methyl 4-formyl-
cinnamate (10)19 with either primary or secondary
amines using NaBH3CN (Method A) or NaBH(OAc)3
(Method B, Scheme 1) to afford secondary amines 11 or
tertiary amines 12. The aminoesters 11 and 12 were
converted to the corresponding hydroxamic acids 13 and
14 by one of three methods: reaction with aqueous
hydroxylamine in the presence of NaOH (Method C),
treatment with a solution of hydroxylamine in methanol
(Method D), or by adding methanolic NaOMe to a
mixture of the ester and hydroxylamine hydrochloride
in MeOH (Method E). Tertiary amine hydroxamates 14
were prepared by reacting secondary amine 11 or 13
with an alkyl halide or by reductive amination of 11 or
13 with an aldehyde or ketone.
* Corresponding author: Tel: 973-235-7005; fax: 973-235-2448;
e-mail: remisz@netscape.net.
† Present address: Hoffmann-La Roche, Inc. 340 Kingsland St.,
Nutley, NJ 07110.
‡ Present address: Xenogen Corporation, 869 Atlantic Ave., Alame-
da, CA 94501.
§ Present address: Chiron Corporation, 4560 Horton St., Emeryville,
CA 94608.
10.1021/jm030235w CCC: $25.00 © 2003 American Chemical Society
Published on Web 09/13/2003

4610 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Remiszewski et al.
Ch a r t 1
Sch em e 1a
Sch em e 2a
a
Reagents: (a) primary amine, Method A; (b) (BOC)2O, Et3N,
MeOH; (c) Tr-ONH2, EDCI, HOBT, 1-methylmorpholine (NMM),
DMF; (d) 5% H2O/TFA; (e) 1,2,3,4-tetrahydro-9H-pyrido[3,4-b]in-
dole, Method A.
amine to afford 19. Alkylation of aminoester 20 with
1-(2-bromoethyl)benzimidazole and subsequent treat-
ment with hydroxylamine afforded hydroxamate 21.
Reductive amination of methyl 4-acetylcinnamate (22)
with tryptamine followed by hydroxylamine treatment
afforded 23.
a
Reagents: (a) NaBH(CN)3, MeOH, HOAc (optional) (Method
A); (b) NaBH(OAc)3, THF or dichloroethane (DCE), HOAc (op-
tional) (Method B); (c) alkyl halide, base; (d) aldehyde or ketone,
Method A or Method B; (e) HONH2 (aq), NaOH (aq) (Method C);
(f) MeOH/HONH2 (anhydrous, Method D); (g) NaOMe, HONH3Cl,
MeOH (Method E); (i) alkyl halide, amine base; (j) aldehyde or
ketone, Method A.
In Vitr o Biology
Reductive amination of 4-formylcinnamic acid (15)
with a primary amine followed by BOC protection of the
resulting amino acid afforded acid 16 (Scheme 2). EDCI-
mediated coupling of 16 with O-tritylhydroxylamine
followed by acidic deprotection provided hydroxamic
acid 13. The tricyclic derivative 17 was prepared in a
similar fashion.
Compounds were profiled using partially purified
HDAC enzyme obtained from H1299 human lung car-
cinoma cell lysates22 and in antiproliferative assays
using HCT116 human colon and H1299 human lung
carcinoma cells.23 Most of the compounds tested have
enzyme IC50 values below 100 nM, but no clear struc-
ture-activity relationship (SAR) could be proposed.
Larger differences are observed in the cellular IC50s,
with values ranging from 6 to 600 nM for HCT116
Methyl 4-aminocinnamate (18)20 was acylated with
3-indolepropionic acid (Scheme 3). The resulting amide
was treated with NaBH4/HOAc21 followed by hydroxyl-

Inhibitors of Human Histone Deacetylase
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4611
Sch em e 3a
13g in both cell lines. The benzimidazole 21 is 7-fold
less potent in cell growth inhibition than the indole 13g,
while 13t is 3-fold less potent and 13s is 2- to 3-fold
less potent than 8, suggesting that polarity in this
region is detrimental to cellular activity.
A series of tertiary amine analogues of the indole-
containing inhibitors were prepared, along with a small
number of analogues of our lead compound, 8, and an
analogue of 13j (Table 2). Of the compounds prepared,
no clear SAR was deduced for enzyme inhibition or cell
growth inhibition.
In the indole series, aliphatic, relatively nonpolar
amine substitution generally resulted in compounds
having increased cellular potency. Thus, 14a is 13- and
5-fold more potent than 13g in H1299 and HCT116 cell
growth inhibition, respectively, the N-cyclohexyl deriva-
tive 14b is 10- to 12-fold more potent than 13g, and the
N-tetrahydropyranyl derivative 14c is 10- to 15-fold
more potent than 13g in cell growth inhibition (Table
2). The N-hydroxethyl analogue 9 is ca. 3-fold more
potent in each cell line than 13g, but the bis-hydroxy
derivative 14h is 16-fold less potent in each cell line
than 13g.
Aryl substitution of the amine N results in reduced
cellular potency. The benzylamine 14g is 5- and 4-fold
less potent than 13g in H1299 and HCT116 growth
inhibition, respectively. A similar loss of potency is
observed in the 2- and 3-pyridyl derivatives of 8, 14i,
and 14f.
Conformationally restricted indole derivatives 14j and
17 were prepared. The potency of 14j (Table 3) is
essentially equal to the 3-methylene spacer acyclic
analogue 13q (Table 1), while the potency of 17 (Table
3) is similar to that of the 2-substituted indole 13u
(Table 1), and quite different from the 3-substituted
indole 13g (Table 1).
For comparison, we also tested 1, 3, 4, and 6 in our
in vitro assays. Enzyme and cell growth inhibition for
4 and 6 are below 100 nM, in the range of our most
potent HDAIs. The enzyme IC50 of 194 nM for 1 is
within the range observed for our HDAIs, but the cell
growth inhibition is between 241- to 2.5-fold less potent
in H1299 growth inhibition and 321- to 2.6-fold less
potent in HCT116 growth inhibition than our com-
pounds.
a
Reagents: (a) 1H-3-indolepropanoic acid, i-BuOC(O)Cl, NMM,
THF; (b) NaBH4/HOAc, THF; (c) Method C; (d) 1-(2-bromoethyl)-
benzimidazole, DIEA, DMSO; (e) tryptamine, Method A.
growth inhibition and from 30 to 2900 nM in H1299
growth inhibition, and some trends regarding structure
and activity are observed. Possible reasons for the lack
of correlation between enzyme potency and cellular
potency and for large differences in cellular potency
between homologous compounds may be due to differ-
ences in cellular uptake of the compounds or to isoform
specificity of the compounds. Profiling of these com-
pounds in individual HDAC isoforms is in progress.
Cellular potency in homologous series tended to be
better for compounds with even numbers of atoms
between the basic N of the benzylic amine and the
terminal moiety. For example, 13g and 13i, having two
and four methylene groups between the basic N and the
indole, respectively, are between 2- and 5-fold more
potent in H1299 growth inhibition and 3- and 5-fold
more potent in HCT116 growth inhibition than 13q and
13u , having three and one methylene groups between
the basic N and the indole, respectively (Table 1). More
dramatically, there is a loss of 11- and 16-fold in H1299
and HCT116 potency, respectively, when going from two
methylenes in 13b to three in 13r . However, this not
the case for the 2-substituted indoles 13l, having a
2-methylene spacer, and 13p , having a 1-methylene
spacer, where no change in HCT116 inhibition and a
minor 1.7-fold difference in H1299 potency was ob-
served.
In our hands, 3 does not inhibit HDAC enzyme
activity, while the reported IC50 for 3 is 2 µM24 and 4.8
µM.25 One possible explanation for this discrepancy is
the source of the enzyme. Saito24 and Suzuki25 used
HDACs isolated from K562 human erythroleukemia
cells while we used HDACs isolated from H1299 human
lung carcinoma cells. Alternatively, the substrates used,
nuclear histones isolated from K562 cells vs the histone
H4 N-terminus 24mer peptide, may affect the activity
observed with the differing enzyme preparations. Fo-
cusing on cellular activity, 3 is between 227- to 2.4-fold
less potent in H1299 growth inhibition and 111-fold less
potent to equipotent at HCT116 growth inhibition than
our HDAIs.
For the secondary amines, methyl substitution of the
benzylic carbon, 23 (Table 3), or the chain carbon R to
the benzylamine N, 13h (Table 1), had no effect on
enzyme or cellular activity relative to the unsubstituted
analogue 13g. There is no effect on enzyme or HCT116
potency in either enantiomer of hydroxymethyl com-
pounds 13n and 13o, compared to 13g, and only a minor
2-fold loss of H1299 potency. Substitution of the indole
N with a methyl group resulted in improved cellular
potency, with 13c being 2- to 3-fold more potent than
13g. However, the N-cyclopropylmethyl indole 13e was
not significantly better than 13g, being only 1.6-fold
more potent in H1299 potency, and equipotent in
HCT116 potency. Substitution of the 5-position of the
indole had variable effects on cellular potency. The
5-benzyloxy derivative 13a is 6-fold more potent than
13g in H1299 growth inhibition, but equal to 13g in
HCT116 growth inhibition. The 5-fluoro derivative 13f
and the 5-methoxy compound 13m are equipotent to
In Vivo Biology
Selected compounds meeting the in vitro require-
ments discussed above were tested for maximum toler-
ated dose (MTD), defined as the highest dose that

4612 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Remiszewski et al.
Ta ble 1. HDAC Enzyme and Cell Growth Inhibition Data for Secondary Amines

Inhibitors of Human Histone Deacetylase
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4613
Ta ble 1 (Continued)
a
b
Values are mean ( standard deviation (SD) of a minimum of three separate experiments. Values are the mean of two triplicate
experiments.
resulted in no deaths when the compound was admin-
Maximal efficacy was 9% T/C, tumor regressions were
not observed and body weight change ranged from +3.1
to -9.4%.
istered once daily (qd) intravenously (iv) for five con-
secutive days, up to a maximum of 200 mg/kg/day to a
group of four athymic mice. Compounds were adminis-
tered as the lactate salt using 10% DMSO in 5%
dextrose/water (D5W) as the vehicle. MTDs ranged from
10 mg/kg for 13b to > 200 mg/kg for 9 (Table 4).
Following MTD determination, the compounds were
tested for efficacy in athymic mice bearing HCT116
subcutaneous tumor xenografts at the compound’s MTD,
or a maximum of 100 mg/kg, iv qd, 5×/week for 10 or
15 total doses. Athymic mice were implanted subcuta-
neously with a suspension of ≈106 HCT116 human colon
carcinoma cells. After tumors had reached approxi-
mately 100 mm3, the animals were sorted into groups
of eight balanced with respect to the mean and range
of tumor volume and treatment started. Results are
reported as % T/C, the ratio of the change in tumor
volume of treated animals to the change in tumor
volume of control animals. Statistical significance was
determined using a one-tailed Student’s t-test and
physiologic relevance was defined to be T/C e 50%.
Potency in vitro did not correlate with efficacy,
illustrated by 13a , the most potent secondary amine in
vitro, which had modest efficacy and 13b, the next most
potent, which had no efficacy at its MTD. Compounds
having indole groups had the best efficacy and toler-
ability. Substituents on the indole had some effect on
toxicity, with indole N-cyclopropylmethyl derivative 13e
having the lowest MTD and least efficacy. Comparing
13g and 9, addition of the hydroxyethyl group improved
tolerability while maintaining efficacy. For the quinolyl
compounds 13j and 14e, a smaller increase in toler-
ability was observed for the hydroxyethyl derivative, but
this was insufficient to achieve an efficacious dose.
On the basis of their MTD and efficacy in the
screening study, 9, 13f, 13g, and 13m were chosen for
dose-response studies in the HCT116 xenograft model,
with 5-fluorouracil used as a positive control. Com-
pounds were administered iv as the lactate salt in 10%
DMSO/D5W 5×/week for 15 total doses. The positive

4614 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Remiszewski et al.
Ta ble 2. HDAC Enzyme and Cell Growth Inhibition Data for Tertiary Amines
a
b
Values are mean ( standard deviation (SD) of a minimum of three separate experiments. Values are the mean of two triplicate
experiments.
Ta ble 3. HDAC Enzyme and Cell Growth Inhibition Data for Miscellaneous N-Hydroxy-3-phenyl-2-propenamides, Trichostatin A and
Trapoxin B
a
b
Values are mean ( standard deviation (SD) of a minimum of three separate experiments. Values are the mean of two triplicate
experiments.
control, 5-fluorouracil, was administered in 1% DMSO/
0.9% saline at 100 mg/kg iv 1×/week for three total
doses.
Each compound caused statistically significant growth
inhibition for all doses tested and a good dose-response
was observed (Figures 1-4). For 9, T/C ranged from 55%

Inhibitors of Human Histone Deacetylase
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4615
Ta ble 4. Efficacy, MTD and Body Weight Change of Selected
HDAIs Administered to Athymic Mice Bearing HCT116 Human
Colon Carcinoma Xenograftsa
compdb
dose (mg/kg)
% T/C
MTD
∆% body weight
8
9
45c
100c
50e
86
45
>200
50
-3.1 ( 0.41
-0.9 ( 0.42
-3.0 ( 0.96
-3.7 ( 0.32
-3.2 ( 0.41
+0.5 (0.65
-0.9 ( 0.81
-6.7 ( 0.28
-9.4 ( 0.41
-5.4 ( 0.37
-7.4 ( 0.39
-4.1 ( 2.7
+0.1 ( 1.4
+3.1 ( 1.3
12d
45d
85
13a
13b
13c
13d
13e
13f
13g
13j
13m
14a
14c
14e
10c
10
75
50
20
100
100
30
100
25
75c
15d
49g
81
50f
20c
100c
100c
30c
16d
17d
77
100c
25c
9d
29d
32d
78
40e
40
50
50e
F igu r e 2. Growth curves of HCT116 human colon tumor
xenografts, expressed as mean tumor volume ( standard error
of the mean (SEM), after treatment with 13f, 10% DMSO/
D5W, 5-fluorouracil, and 1% DMSO/0.9% saline. Treatment
started 13 days postimplant when tumors were ≈100 mm3.
13f was administered as the lactate salt in 10% DMSO/D5W
qd iv 5 days/week for 15 total doses. 5-Fluorouracil was
administered at 100 mg/kg in 1% DMSO/0.9% saline 1 day/
week for three total doses. For all treatment groups, at all
measurements, p < 0.01 compared to vehicle controls using a
one-tailed Student’s t-test.
a
b
Eight mice per group. Administered qd 5×/week. c 15 doses.
p < 0.01. e 10 doses. f The study was terminated on day 22 post-
implant after the sixth dose, because of the six deaths in the
treatment group. %T/C was calculated on day 22 predose. p <
0.05.
d
g
F igu r e 1. Growth curves of HCT116 human colon tumor
xenografts, expressed as mean tumor volume ( standard error
of the mean (SEM), after treatment with 9, 10% DMSO/D5W,
5-fluorouracil, and 1% DMSO/0.9% saline. Treatment started
13 days postimplant when tumors were ≈100 mm3. 9 was
administered as the lactate salt in 10% DMSO/D5W qd iv 5
days/week for 15 total doses. 5-Fluorouracil was administered
at 100 mg/kg in 1% DMSO/0.9% saline 1 day/week for three
total doses. For all treatment groups, at all measurements,
p < 0.01 compared to vehicle controls using a one-tailed
Student’s t-test.
F igu r e 3. Growth curves of HCT116 human colon tumor
xenografts, expressed as mean tumor volume ( standard error
of the mean (SEM), after treatment with 13g, 10% DMSO/
D5W, 5-fluorouracil, and 1% DMSO/0.9% saline. Treatment
started 13 days postimplant when tumors were ≈100 mm3.
13g was administered as the lactate salt in 10% DMSO/D5W
qd iv 5 days/week for 15 total doses. 5-Fluorouracil was
administered at 100 mg/kg in 1% DMSO/0.9% saline 1 day/
week for three total doses. For all treatment groups, except
10 mg/kg 13g, p < 0.01 at all measurements, using a one-
tailed Student’s t-test; p < 0.05 at all measurements for 13g
at 10 mg/kg.
to 20%, body weights at the end of the study were equal
to or greater than the weights at the start of dosing and
no deaths occurred in any group. Efficacy for 13f ranged
from 40% T/C to 8% T/C, no treatment-related deaths
occurred and a dose-independent 1.7% to 7.5% body
weight loss was observed (Table 5). For 13g, T/C ranged
from 56% to 13%, with no deaths and a dose-indepen-
dent body weight loss from 7% to 10.2%. Results for 13m
were similar to those of 13g, with T/C from 64 to 22%,
no deaths and a dose dependent 4.6% to 12.0% body
weight loss.
Of the four compounds, 9 was of most interest, since,
in the HCT116 dose-response study, animals treated
with 9 exhibited the least gross toxicity as measured
by weight loss, its MTD was >200 mg/kg and it was 2-
to 3-fold more potent in cell growth inhibition than the
other compounds. Therefore, we tested the efficacy of 9
in a second xenograft model and chose the slower
growing A549 human lung carcinoma cell line (mono-
layer growth inhibition IC50 ) 30 nM in this cell line).
Athymic mice were implanted subcutaneously with a
suspension of ≈107 A549 human lung carcinoma cells,
tumors were permitted to grow to approximately 100
mm3, the animals were sorted into groups of eight,
balanced with respect to the mean and range of tumor
volume, and treatment started. Drug was administered
as the lactate salt using the same doses, vehicle, and
schedule as in the HCT116 xenograft study. Mitomycin
C, the positive control, was administered intraperito-
neally (ip) in 10% DMSO/D5W at 2 mg/kg 3×/week for

4616 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Remiszewski et al.
F igu r e 5. Growth curves of A549 human lung tumor xe-
nografts, expressed as mean tumor volume ( standard error
of the mean (SEM), after treatment with 9, 10% DMSO/D5W
(ip), mitomycin C and 10% DMSO/D5W (ip). Treatment started
20 days post implant when tumors were ≈ 100 mm3. 9 was
administered in 10% DMSO/D5W qd iv 5 days/week for 15
total doses. Mitomycin C was administered at 2 mg/kg in 10%
DMSO/D5W ip 3×/week for nine total doses. For all treatment
groups at all measurements, p < 0.01 using a one-tailed
Student’s t-test.
F igu r e 4. Growth curves of HCT116 human colon tumor
xenografts, expressed as mean tumor volume ( standard error
of the mean (SEM), after treatment with 13m , 10% DMSO/
D5W, 5-fluorouracil, and 1% DMSO/0.9% saline. Treatment
started 13 days postimplant when tumors were ≈100 mm3.
13m was administered as the lactate salt in 10% DMSO/D5W
qd iv 5 days/week for 15 total doses. 5-Fluorouracil was
administered at 100 mg/kg in 1% DMSO/0.9% saline 1 day/
week for three total doses. For all treatment groups, except
10 mg/kg 13m , p < 0.01 at all measurements, using a one-
tailed Student’s t-test; p < 0.05 at all measurements for 13m
at 10 mg/kg.
Ta ble 6. Dose-Response of 9 in the A549 Human Lung
Carcinoma Xenograft Studya
Ta ble 5. Dose-Response of 9, 13f, 13g, and 13m in the
doseb
(mg/kg)
body weight
change (%)
HCT116 Human Colon Carcinoma Xenograft Studya
% T/C
dead/total
dose
(mg/kg)
body weight
change (%)
10
25
50
32c
20c
15c
7c
1.3 ( 0.31
-0.9 ( 0.32
-0.4 ( 0.34
0.4 ( 0.29
0/8
0/8
0/8
0/8
compdb
% T/C
dead/total
9
10
25
50
100
10
25
50
100
10
25
50
100
10
25
50
100
55c
38c
27c
20c
40c
19c
13c
8c
2.7 ( 0.50
0.0 ( 0.49
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
100
2.3 ( 0.33
a
b
Eight mice per group. Administered as the lactate salt qd
5×/week for 15 total doses. c p < 0.01 using a one-tailed Student’s
t-test.
1.4 ( 0.33
13f
-6.4 ( 0.39
-6.6 ( 0.81
-1.7 ( 0.40
-7.5 ( 0.34
-10.2 ( 0.47
-7.0 ( 0.21
-7.3 ( 0.31
-9.4 ( 0.41
-4.6 ( 0.46
-5.5 ( 0.14
-8.1 ( 0.61
-12.0 ( 0.51
were screened for efficacy at their MTD or a maximum
of 100 mg/kg, as the lactate salt, in athymic mice using
the subcutaneous HCT116 human colon carcinoma
xenograft model. Four compounds, 9, 13f, 13g, and 13m ,
were selected for dose-response studies in the HCT116
xenograft model, and good responses were observed for
each compound. One compound, 9, was 2- to 3-fold more
potent in cell growth inhibition, had a greater MTD and
caused the least gross toxicity in the HCT116 xenograft
dose-response study. Thus, 9 was selected for testing
in the A549 human lung carcinoma xenograft model,
where a good dose-response was observed with minimal
body weight loss. Compared to 1, which is currently in
clinical trials, 9 is 6-fold more potent at HDAC inhibi-
tion, 48-fold more potent at H1299 cell growth inhibi-
tion, and 193-fold more potent at HCT116 cell growth
inhibition. Compared to 3, also in clinical trials, 9 is 45-
fold more potent at H1299 cell growth inhibition and
67-fold more potent at HCT116 cell growth inhibition.
On the basis, in part, of these properties, 9 has entered
clinical trials as a novel anticancer agent.
13g
13m
56d
40c
13c
17c
64d
38c
24c
22c
a
b
Eight mice per group. Administered as the lactate salt qd
5×/week for 15 total doses. c p < 0.01 using a one-tailed Student’s
d
t-test. p < 0.05 using a one-tailed Student’s t-test.
nine total doses. As in the HCT116 study, an excellent
dose-response was observed, with statistically signifi-
cant tumor growth inhibition observed at all measure-
ments for all doses of 9 (p < 0.01, Figure 5). The T/C
ranged from 32% to 7%, body weight change ranged
from -0.9% to +1.3% (Table 6), and no deaths were
observed.
Su m m a r y
Based on the lead compound from a HTS of the
Novartis compound archive for novel inhibitors of
HDAC, a series of N-hydroxy-3-phenyl-2-propenamides
were prepared. These compounds were all potent inhibi-
tors of partially purified HDAC isolated from H1299
cellular lysates, with IC50s < 400 nM. Their potency in
cell growth inhibition ranged over 2 orders of magnitude
in both H1299 and HCT116 cells. Selected compounds
Exp er im en ta l Section
All chemicals were obtained from commercial suppliers and
used without further purification. Flash column chromatog-
raphy was performed with silica (Merck EM9385, 230-400
mesh). Preparative reverse phase HPLC (RPHPLC) was done
using a C-18 column with
containing 0.1% TFA as the eluent. Analytical RPHPLC was
a gradient of MeCN in H2O

Inhibitors of Human Histone Deacetylase
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4617
done using a Rainin Dynamax HPLC equipped with a PDA
detector using the following columns and systems: a Waters
Symmetry Shield 4.6 × 250 mm C-18 column (5 µM particle
size), linear gradient from 10% to 100% MeCN/(H2O + 0.1%
TFA), flow rate ) 1.5 mL/min (System 1) or linear gradient
from 10% to 100% MeOH/(H2O + 0.1% TFA), flow rate ) 1.5
mL/min (System 2); Waters XTerra 4.6 × 250 mm C-18 column
(5 µM particle size), linear gradient from 10% to 70% MeCN/
(H2O + 0.1% TFA), flow rate ) 1.5 mL/min (System 3) or linear
gradient from 10% to 70% MeOH/(H2O + 0.1% TFA), flow rate
) 1.0 mL/min (System 4). 1H and 13C NMR spectra were
recorded at 500 or 300 and at 125 or 75 MHz, respectively.
Proton and carbon chemical shifts are expressed in ppm
relative to internal tetramethylsilane, and coupling constants
(J ) are expressed in hertz. High-resolution MS was carried out
using a Micromass model LCT TDF/MS. Cells were obtained
from American Type Culture Collection, 12301 Parklawn
Drive, Rockville, MD 20852. Elemental analyses were per-
formed by Robertson Microanalytical Laboratories, Madison,
NJ . Lactic acid (1 N) was purchased from Alfa and was used
as received.
Meth od A. Red u ctive Am in a tion Usin g Na BH3CN.
NaBH3CN (1.2-2 equiv) was added in portions to a MeOH
solution of an aldehyde or ketone and a primary or secondary
amine. In some cases, 1 equiv of HOAc was added to the
mixture before the addition of NaBH3CN. After the reaction
was complete, the volume was reduced by half, this mixture
extracted with EtOAc or CH2Cl2, and the extracts were washed
with sat. NaHCO3 and brine. The organic solution was dried
and evaporated and the residue purified by chromatography,
trituration, or crystallization.
Meth od B. Red u ctive Am in a tion Usin g Na BH(OAc)3.
NaBH(OAc)3 (1-1.5 equiv) was added in portions to a solution
of an aldehyde or ketone and a primary or secondary amine
in dichlororethane (DCE) or THF. In some cases, 1 equiv of
HOAc was added to the mixture before the addition of NaBH-
(OAc)3. After the reaction was complete, the mixture was
diluted with sat. NaHCO3 and extracted with EtOAc or CH2-
Cl2, and the extracts were washed with brine. The organic
solution was dried and evaporated and the residue purified
by chromatography, trituration or crystallization.
Meth od C. Hyd r oxa m a te F or m a tion Usin g Aqu eou s
NH2OH. To a solution of methyl ester in MeOH or EtOH at
0 °C was added HONH2 (50% aq. solution, 10-15 equiv)
followed by 1 M NaOH (7-10 equiv). The mixture was stirred
at 0 °C for 2-4 h, warmed to room temperature, and stirred
until the reaction was complete (TLC). Acidification with 1 M
HCl to pH 7-8 (pH paper) resulted in product precipitation.
The precipitate was filtered, analyzed by RPHPLC, and
purified by preparative RPHPLC if necessary.
was acidified to pH ∼ 6 (pH paper) with HOAc or 1 M HCl
and diluted with EtOAc or CH2Cl2. The mixture was washed
with sat. NaHCO3 and brine, dried, and evaporated and the
residue purified by crystallization, trituration, or preparative
RPHPLC.
Gen er a l P r oced u r e for Neu tr a liza tion of Tr iflu or oa c-
eta te Sa lts. Trifluoroacetate salts of compounds obtained after
HPLC purification were neutralized by dissolving the salt in
CH2Cl2 or EtOAc and washing the solution with sat. NaHCO3
followed by brine. The solution was dried and evaporated to
afford the free base.
N-Hydr oxy-3-[4-[[[3-ph en ylpr opyl]am in o]m eth yl]ph en -
yl]-(2E)-2-p r op en a m id e 8. Following Method A, 3-phenyl-
propylamine (6.76 g, 50.0 mmol), 15 (8.81 g, 50.0), and
NaBH3CN were reacted in MeOH containing 1 equiv of HOAc
to provide 3-[4-[[[3-phenylpropyl]amino]methyl]phenyl]-(2E)-
2-propenoic acid which precipitated from the reaction mixture.
The precipitate was filtered, washed with MeOH, and sus-
pended in 50% H2O/dioxane. Et3N (7.0 mL, 5.08 g, 50.2 mmol)
and (BOC)2O (10.9 g, 49.9 mmol) were added, and the mixture
was stirred for 2 h and extracted with EtOAc. The combined
extracts were washed with brine, dried, and evaporated to give
an oil which was purified by flash chromatography to afford
3-(4-[(tert-butoxycarbonyl)(3-phenylpropyl)amino]methyl]phen-
yl)-(2E)-2-propenoic acid (11.6 g, 59%): 1H NMR (CDCl3) δ 1.48
(s, 9H), 1.86 (s, 2H), 2.60 (m, 2H), 3.23 (m, 2H), 4.47 (s, 2H),
6.47 (d, 15.8 Hz, 1H), 7.24 (m, 7H), 7.53 (d, 7.9 Hz, 2H), 7.80
(d, 16.2 Hz, 1H); m/z 396 (MH+). The acid (11.6 g, 29.3 mmol),
O-tritylhydroxylamine (9.7 g, 35.3 mmol), EDCI‚HCl (6.8 g,
35.3 mmol), HOBT‚H2O (6.0 g, 44.1 mmol) and 1-methylmor-
pholine (NMM) were dissolved in DMF, and the mixture was
stirred. After 16 h, the mixture was diluted with EtOAc and
washed with H2O and the organic solution dried (MgSO4) and
evaporated to dryness. Purification of the residue by flash
chromatography provided (3-phenylpropyl)-[4-((E)-2-trityloxy-
carbamoylvinyl)benzyl]carbamic acid tert-butyl ester (7.8 g,
40%): 1H NMR (CDCl3) δ 1.49 (s, 9H), 1.84 (s, 2H), 2.60 (m,
2H), 3.24 (m, 2H), 4.42 (m, 2H), 6.11 (d, 15.8 Hz, 1H), 7.20
(m, 5H), 7.34 (m, 15H), 7.48 (m, 5H); m/z 653 (MH+). The
O-tritylhydroxamate (7.8 g, 11.9 mmol) was treated with 5%
H2O/TFA for 2 h, the mixture evaporated to dryness and the
residue purified by RPHPLC to afford 8 as the TFA salt which
was neutralized and treated with 1 equiv of lactic acid to
provide 8 as the lactate salt (2.23 g, 46%): retention time
System 3 ) 16.63 min, retention time System 4 ) 29.84 min;
1H NMR (CD3OD) δ 1.36 (d, 6.8 Hz, 5H, lactate), 2.02 (m, 2H),
2.71 (t, 7.5 Hz, 2H), 3.04 (m, 2H), 4.11 (q, 6.9 Hz, 1.75H,
lactate), 4.19 (s, 2H), 6.51 (d, 15.8 Hz, 1H), 7.26 (m, 5H), 7.56
(m, 5H); 13C NMR (CD3OD) δ 19.72, 27.53, 32.19, 50.47, 67.03,
118.55, 126.07, 128.01, 128.04, 128.28, 130.10, 132.68, 136.10,
139.00, 164.46, 178.63; m/z 312 (MH+); HRMS (MH+) calcd
for C19H23N2O2, 311.1760, found 311.1754; Anal. (C19H22N2O2‚
1.75C3H6O3‚0.65H2O), C 60.56%, H 6.29%, N 6.19%.
(2E)-N-Hyd r oxy-3-[4-[[(2-h yd r oxyeth yl)[2-(1H-in d ol-3-
yl)eth yl]a m in o]m eth yl]p h en yl]-2-p r op en a m id e 9. A mix-
ture of (2E)-3-[4-[[[2-(1H-indol-3-yl)ethyl]amino]methyl]phen-
yl]-2-propenoic acid methyl ester (20.00 g, 59.81 mmol),
2-bromoethanol (21.5 mL, 37.9 g, 303 mmol), and K2CO3 in
MeCN was heated at 60 °C for 18 h, cooled, and filtered and
the filtrate evaporated. The residue was purified by flash
chromatography to afford (2E)-3-[4-([(2-hydroxyethyl)-[2-(1H-
indol-3-yl)ethyl]amino]methyl)phenyl]-2-propenoic acid methyl
ester (13.97 g, 62%): m/z 379 (MH+). Following Method C, the
ester (13.90 g, 36.73 mmol) was converted to the hydroxamate
which was treated with 1 equiv of lactic acid to provide 9 as
the lactate salt (7.44 g, 53%): 1H NMR (DMSO-d6) δ 1.24 (d,
6.8 Hz, 3H, lactate), 2.67 (t, 6.4 Hz, 2H), 2.77 (dd, 9.6, 4.71
Hz, 2H), 2.87 (m, 2H), 3.54 (t, 6.4 Hz, 2H), 3.76 (s, 2H), 4.04
(q, 6.9 Hz, 1H, lactate), 6.46 (d, 15.8 Hz, 1H), 6.91 (t, 7.5 Hz,
1H), 7.04 (t, 7.5 Hz, 1H), 7.10 (d, 1.9 Hz, 1H), 7.37 (m, 5H),
7.50 (m, 2H), 10.75 (s, 1H); 13C NMR (DMSO-d6) δ 20.88, 22.85,
55.14, 56.15, 58.45, 59.55, 66.19, 111.70, 112.76, 118.44,
118.57, 118.89, 121.16, 122.86, 127.57, 127.67, 129.61, 133.82,
Meth od D. Hyd r oxa m a te F or m a tion Usin g Meth a n olic
NH2OH. A stock solution of NH2OH was prepared by adding
a solution of KOH (12.9 g (87%), 0.2 mol) in MeOH (100 mL)
to a solution of NH2OH‚HCl (13.9 g, 0.2 mol) in MeOH (200
mL). After 15 min, the mixture was filtered, the filter cake
washed with a minimum of MeOH, and the filtrate volume
reduced to approximately 75 mL. The mixture was filtered and
the volume adjusted to 100 mL with MeOH. The resulting 2
M solution was stored under N2 at -20 °C for up to 2 weeks.
Ca u tion : Th e 2 M NH2OH solu tion d ecom p oses a t r oom
tem p er a tu r e. NH2OH is exp losive w h en d r y. Use p r op er
p r eca u tion s! The ester (1 equiv) was added to 2 M NH2OH
in MeOH (10 equiv) at 0 °C, followed by a solution of KOH
(1.1-1.5 equiv). The ice bath was removed and the mixture
monitored by TLC. When the reaction was complete, a small
amount of dry ice was added to the mixture and the mixture
evaporated to dryness. The residue was purified by RPHPLC
to give the product.
Meth od E. Hyd r oxa m a te F or m a tion Usin g NH2OH‚
HCl a n d Na OMe. To a mixture of 1 equiv of methyl ester
and 7-10 equiv of NH2OH‚HCl in MeOH was added 10-12
equiv of ∼3.0 M NaOMe/MeOH (freshly prepared). The reac-
tion was monitored by TLC, and additional NH2OH‚HCl and
∼3.0 M NaOMe/MeOH were added if necessary. The mixture

4618 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Remiszewski et al.
136.57, 138.55, 141.67, 163.20, 176.73; m/z 380 (MH+); Anal.
(C22H25N3O3‚C3H6O3‚H2O) C, H, N, mp 100-103 °C.
(DMSO-d6) δ 25.7, 32.58, 49.95, 52.95, 109.88, 112.33, 118.58,
118.91, 121.34, 127.38, 127.69, 127.96, 128.80, 133.52, 136.97,
138.52, 143.09, 163.17; m/z 350 (MH+); HRMS (MH+) calcd
for C21H24N3O2, 350.1869, found 350.1876; mp 115.2-118.1 °C;
Anal. (C21H23N3O2‚0.1H2O‚0.3TFA) C, H, N.
N-Hyd r oxy-3-[4-[[[2-[5-(p h en ylm eth oxy)-1H-in d ol-3-yl]-
eth yl]am in o]m eth yl]ph en yl]-(2E)-2-pr open am ide 13a. Fol-
lowing Method B, 5-benzyloxytryptamine (7.2 g, 27.0 mmol),
10 (5.35 g, 28.1 mmol), and NaBH(OAc)3 (1.4 equiv) were
reacted in DCE to afford 9.8 g (82%) of 3-[4-[[[2-[5-(phenyl-
methoxy)-1H-indol-3-yl]ethyl]amino]methyl]phenyl]-(2E)-2-
propenoic acid methyl ester: 1H NMR (DMSO-d6) δ 2.78 (m,
4H), 3.72 (s, 3H), 3.77 (s, 2H), 5.04 (s, 2H), 6.58 (d, 16.2 Hz,
1H), 6.79 (dd, 8.9, 2.5 Hz, 1H), 7.08 (dd, 6.0, 2.3 Hz, 2H), 7.23
(d, 9.0 Hz, 1H), 7.36 (m, 5H), 7.46 (m, 2H), 7.64 (m, 3H), 10.65
(s, 1 H); 13C NMR (DMSO-d6) δ 25.97, 49.82, 51.81, 52.93,
70.22, 102.19, 111.97, 112.36, 112.79, 117.37, 123.74, 128.04,
128.71, 131.96, 132.67, 138.20, 144.44, 144.89, 152.27, 167.14;
m/z 441 (MH+); Anal. (C28H28N2O3) C, H, N. Following Method
C, the ester (1.53 g, 3.47 mmol) was converted to the hydrox-
amate (0.75 g, 50%) which was treated with 1 equiv of lactic
acid to provide 13a as the lactate salt: 1H NMR (DMSO-d6) δ
1.35 (d, 6.78 Hz, 3 H, lactate), 3.14 (br s, 2H), 3.31 (m, 4H),
4.10 (q, 6.78 Hz, 1H, lactate), 5.05 (s, 2H) 6.50 (d, 15.8 Hz,
1H), 6.88 (d, 8.7 Hz, 1H), 7.12 (m, 2H), 7.32 (m, 5H), 7.46 (m,
5H), 7.59 (m 2H); 13C NMR (DMSO-d6) δ 21.66 (lactate), 23.81,
51.20, 69.05 (lactate), 72.56, 103.34, 110.39, 113.74, 114.16,
120.31, 125.54, 128.85, 129.91, 129.89, 131.94, 134.20, 134.47,
137.83, 139.70, 140.86, 154.76, 166.30, 180.87 (lactate); m/z
442 (MH+); Anal. (C27H27N3O3‚C3H6O3) C, H, N.
3-[4-[[[2-(9H-Car bazol-9-yl)eth yl]am in o]m eth yl]ph en yl]-
N-h yd r oxy-(2E)-2-p r op en a m id e 13b. Following Method B,
9H-carbazole-9-ethanamine hydrobromide26 (919 mg, 3.16
mmol), 1.4 equiv of Et3N, 10 (500 mg, 2.63 mmol), and NaBH-
(OAc)3 (1.4 equiv) were reacted in DCE to afford 555 mg (47%)
of 3-[4-[[[2-(9H-carbazol-9-yl)amino]methyl]phenyl]-(2E)-2-pro-
penoic acid methyl ester: 1H NMR (DMSO-d6) δ 2.90 (t, 6.6
Hz, 2H), 3.72 (s, 5H), 4.48 (t, 6.6 Hz, 2H), 6.59 (d, 15.8 Hz,
1H), 7.19 (t, 7.0 Hz, 2H), 7.29 (d, 8.3 Hz, 2H), 7.44 (t, 7.7 Hz,
2H), 7.62 (m, 5H), 8.14 (d 7.9, 2H); 13C NMR (DMSO-d6) δ
43.00, 47.54, 51.38, 52.73, 109.72, 117.47, 119.03, 120.59,
122.42, 125.97, 128.60, 128.71, 132.77, 140.55, 143.85, 144.84,
176.14; m/z 385 (MH+). Following Method E, the ester (405
mg, 1.05 mmol) was converted to the hydroxamate which was
purified by RPHPLC to afford 13b as the TFA salt which was
neutralized and treated with 1 equiv of lactic acid to provide
13b as the lactate salt (190 mg, 40%): retention time System
3 ) 16.82 min, retention time System 4 ) 28.11 min; 1H NMR
(DMSO-d6) δ 1.26 (d, 6.8 Hz, 3H lactate), 3.26 (br s, 2H), 4.04
(q, 6.8 Hz, 1H lactate), 4.12 (br s, 1H), 4.67 (m, 1H), 6.52 (d,
15.8 Hz, 1H), 7.23 (t, 7.35 Hz, 2H), 7.48 (m 5H), 7.62 (m 4H),
8.16 (d, 7.9 Hz, 2H); 13C NMR (DMSO-d6) δ 20.91 (lactate),
45.65, 51.02, 66.21 (lactate), 109.49, 119.56, 120.78, 122.72,
126.24, 128.02, 130.31, 135.30, 138.06, 140.27, 162.94, 176.86
(lactate); m/z 386 (MH+); HRMS (MH+) calcd for C24H24N3O2,
3-[4-[[[2-(3-Ben zofu r a n yl)eth yl]a m in o]m eth yl]p h en yl]-
N-h yd r oxy-(2E)-2-p r op en a m id e 13d . Following Method B,
3-benzofuranethanamine28 (1.10 g, 6.84 mmol), 10 (1.30 g, 6.84
mmol), and NaBH(OAc)3 (1.1 equiv) were reacted in THF to
afford 900 mg (39%) of 3-[4-[[[2-(3-benzofuranyl)ethyl]amino]-
methyl]phenyl]-(2E)-2-propenic acid methyl ester: 1H NMR
(CDCl3) δ 2.93 (m, 4H), 3.80, (s, 3H), 3.83 (s, 2H), 6.41(d, 16.2
Hz, 1H), 7.27 (m, 4H), 7.45 (m, 4H), 7.54 (d, 7.5 Hz, 1H), 7.68
(d, 16.2 Hz, 1H); m/z 336 (MH+). Following Method E, the ester
(840 mg, 2.51 mmol) was converted to the hydroxamate which
was purified by RPHPLC to afford 13d as the TFA salt, which
was neutralized and treated with 1 equiv of lactic acid to
provide 13d as the lactate salt (588 mg, 55%): 1H NMR (CD3-
OD) δ 1.33 (d, 6.8 Hz, 3H, lactate), 3.13 (m, 2H), 3.36 (m, 2H),
4.07 (q, 6.8 Hz, 1H, lactate), 4.24 (s, 2H), 6.51 (d, 15.8 Hz, 1H),
7.29 (m, 2H), 7.54 (m, 7H), 7.68 (s, 1H); 13C NMR (CD3OD) δ
21.81 (lactate), 22.23, 52.43, 69.36 (lactate), 112.92, 117.15,
120.75, 124.26, 126.24, 128.94, 129.83, 131.89, 135.13, 137.73,
140.91, 144.30, 157.38, 166.33, 181.53 (lactate); m/z 337 (MH+);
Anal. (C20H20N2O3‚C3H6O3‚0.5H2O) C, H, N.
3-[4-[[[2-(1-Cyclopr opylm eth yl-1H-in dol-3-yl)eth yl]am i-
n o]m eth yl]p h en yl]-N-h yd r oxy-(2E)-2-p r op en a m id e 13e.
Following Method B, 1-(cyclopropylmethyl)-1H-indole-3-eth-
anamine29 (954 mg, 4.45 mmol), 10 (799 mg, 4.20 mmol), and
NaBH(OAc)3 (1.5 equiv) were reacted in DCE to afford 911
mg (47%) of 3-[4-[[[2-(1-cyclopropylmethyl-1H-indol-3-yl)ethyl]-
amino]methyl]phenyl]-(2E)-2-propenoic acid methyl ester: 1H
NMR (CDCl3) δ 0.34 (q, 5.2 Hz, 2H), 0.60 (m, 2H), 0.88 (m,
1H), 2.99 (m, 4H), 3.48 (s, 1H), 3.80 (s, 3H), 3.82 (s, 2H), 3.92
(d, 6.8 Hz, 2H), 6.41 (d, 15.8 Hz, 1H), 7.07 (m 2H), 7.27 (m,
4H), 7.45 (d, 8.3 Hz, 2H), 7.59 (d, 7.2 Hz, 1H), 7.67 (d, 15.8
Hz, 1H), m/z 389 (MH+). Following Method D, the ester (875
mg, 2.25 mmol) was converted to the hydroxamate which was
purified by RPHPLC to afford 13e as the TFA salt which was
neutralized and treated with 1 equiv of lactic acid to provide
13e as the lactate salt (525 mg, 48%): retention time System
3 ) 16.63 min, retention time System 4 ) 29.84 min; 1H NMR
(CD3OD) δ 0.38 (t, 5.3 Hz, 2H), 0.57 (m, 2H), 1.25 (m, 1H),
1.35 (d, 7.2 Hz, 3.6H, lactate), 2.14 (m, 2H), 3.35 (m, 2H), 4.00
(d, 6.8 Hz, 2H), 4.12 (q, 6.9 Hz, 1.2H, lactate), 4.24 (s, 2H),
6.52 (d, 15.5 Hz, 1H), 7.05 (t, 7.0 Hz, 1H), 7.18 (t, 7.7 Hz, 1H),
7.22 (s, 1H), 7.41 (d, 8.3 Hz, 1H), 7.56 (m, 6H); 13C NMR (CD3-
OD) δ 4.80, 12.82, 21.52 (lactate), 23.69, 51.73, 52.22, 68.81
(lactate), 110.08, 11.28, 119.67, 120.36, 120.56, 123.27, 127.81,
129.86, 131.95, 140.85, 166.45; m/z 390 (MH+); HRMS (MH+)
calcd for C24H28N3O2, 390.2182, found 390.2205; Anal. (C24
27N3O2‚1.2C3H6O3‚0.15H2O‚0.45TFA) C, H, N.
3-[4-[[[2-(5-F lu or o-1H-in d ol-3-yl)eth yl]a m in o]m eth yl]-
-
H
386.1892, found 386.1869; mp 169.5-185.4 °C; Anal. (C24H23
N3O2‚C3H6O3‚0.7TFA‚0.7H2O) C, H, N.
-
p h en yl]-N-h yd r oxy-(2E)-2-p r op en a m id e 13f. Following
Method B, 5-fluorotryptamine (8.30 g, 46.6 mmol), 10 (8.85 g,
46.6 mmol), and NaBH(OAc)3 (1.1 equiv) were reacted in THF
to afford 10.9 g (67%) of 3-[4-[[[2-(5-fluoro-1H-indol-3-yl)ethyl]-
amino]methyl]phenyl]-(2E)-2-propenoic acid methyl ester: m/z
353 (MH+). Following Method D, the ester was converted to
the hydroxamate which was purified by RPHPLC. Neutraliza-
tion of the resulting TFA salt afforded 13f (7.90 g, 72%): 1H
NMR (DMSO-d6) δ 2.96 (m, 4H), 3.18 (s, 2H), 6.50 (d, 15.8
Hz, 1H), 6.91 (dt, 9.2, 2.6 Hz, 1H), 7.31 (m, 3H), 7.47 (m, 3H),
7.57 (m, 2H), 11.02 (s, 1H); 13C NMR (DMSO-d6) δ 23.85, 48.51,
51.43, 103.16, 103.47, 109.33, 109.68, 111.62, 112.72, 119.59,
125.58, 127.68, 127.91, 129.86, 133.31, 134.69, 138.29, 155.54,
158.61, 163.11; m/z 354 (MH+); Anal. (C20H20FN3O2‚H2O) C,
H, N.
N-Hyd r oxy-3-[4-[[[2-(1-m eth yl-1H-in d ol-3-yl)eth yl]a m i-
n o]m et h yl]p h en yl]-(2E)-2-p r op en a m id e 13c. Following
Method A, 1-methyl-1H-indole-3-ethanamine27 (320 mg, 1.84
mmol), 10 (350 mg, 1.84 mmol), HOAc (1 equiv), and NaBH3-
CN (1.5 equiv) were reacted in MeOH to afford 3-[4-[[[2-(1-
methyl-1H-indol-3-yl)ethyl]amino]methyl]phenyl]-(2E)-2-pro-
penoic acid methyl ester (159 mg, 25%): 1H NMR (CDCl3) δ
2.97 (t, 3.4 Hz, 4H), 3.74 (s, 3H), 3.80 (s, 3H), 3.82 (s, 2H),
6.41 (d, 15.8 Hz, 1H), 6.87 (s, 1H), 7.09 (t, 6.8 Hz, 1H), 7.22
(m, 1H), 7.29 (m, 3H), 7.45 (m 2H), 7.59 (d, 7.9 Hz, 1H), 7.67
(d, 15.8 Hz, 1H); 13C NMR (CDCl3) δ 26.04, 33.01, 49.92, 52.08,
53.89, 109.61, 112.68, 117.71, 119.13, 119.39, 122.02, 127.21,
128.54, 129.00, 133.48, 137.51, 145.07, 167.93; m/z 349 (MH+).
Following Method C, the ester (132 mg, 0.379 mmol) was
converted to the hydroxamate which was purified by RPHPLC.
The resulting TFA salt was neutralized to give 13c (28 mg,
21%): retention time System 3 ) 13.06 min, retention time
N-Hyd r oxy-3-[4-[[[2-(1H-in d ol-3-yl)eth yl]a m in o]m eth -
yl]p h en yl]-(2E)-2-p r op en a m id e 13g. Following Method B,
tryptamine (1.78 g, 11.1 mmol), 10 (2.00 g, 10.5 mmol), NaBH-
(OAc)3 (1.5 equiv), and HOAc (1 equiv) were reacted in DCE
to give 3-[4-[[[2-(1H-indol-3-yl)ethyl]amino]methyl]phenyl]-
(2E)-2-propenoic acid methyl ester (2.20 g, 63%): 1H NMR
1
System 4 ) 22.56 min; H NMR (DMSO-d6) δ 2.80 (m, 4H),
3.37 (br s, 3H), 3.72 (s, 3H), 3.76 (s, 2H), 6.45 (d, 15.8 Hz, 1H),
6.99 (t, 7.2 Hz, 1H), 7.13 (m, 2H), 7.45 (m, 7H); 13C NMR

Inhibitors of Human Histone Deacetylase
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4619
(CDCl3) δ 2.91 (m, 4H), 3.72 (s, 3H), 3.74 (s, 2H), 6.33 (d, 16.2
Hz, 1H), 6.92 (d, 2.6 Hz, 1H) 7.03 (dt, 7.5, 1.1 Hz, 1H) 7.11
(dt, 7.6, 1.3 Hz, 1H) 7.19 (m, 2H) 7.26 (d, 7.9 Hz, 1H) 7.36 (m,
2H) 7.53 (d, 7.9 Hz, 1H) 7.59 (d, 16.2 Hz, 1H); 13C NMR (CDCl3)
δ 26.23, 49.80, 52.17, 53.97, 77.09, 77.51, 77.94, 111.63, 114.27,
117.69, 119.34, 119.72, 122.48, 127.86, 128.60, 128.99, 133.44,
136.85, 143.50, 145.18, 168.04; m/z 335 (MH+). Following
Method C, the ester (7.50 g, 22.5 mmol) was converted to the
hydroxamate (7.23 g, 96%), which was treated with 1 M lactic
acid to provide 13g as the lactate salt: 1H NMR (DMSO-d6) δ
1.21 (d, 6.8 Hz, 3H, lactate), 2.95 (s, 4H), 3.90 (q, 6.9 Hz, 1H,
lactate), 3.95 (s, 2H), 6.48 (d, 15.8 Hz, 1H), 6.97 (t, 7.4 Hz,
1H), 7.06 (t, 7.0 Hz, 1H), 7.34 (d, 7.9 Hz, 1H), 7.48 (m, 6H),
10.86 (s, 1H); 13C NMR (DMSO-d6) δ 21.23 (lactate), 24.35,
48.84, 51.67, 66.61 (lactate), 111.72, 111.78, 118.61, 119.39,
121.33, 123.18, 127.43, 127.81, 129.59, 134.37, 136.63, 138.29,
163.03, 177.46 (lactate); m/z 336 (MH+); Anal. (C20H21N3O2‚
C3H6O3‚0.5H2O) C, H, N.
Hz, 2H), 3.53 (m, 2H), 4.31 (s, 2H), 6.51 (d, 15.82 Hz, 1H),
7.58 (m, 5H), 7.83 (t, 7.72 Hz, 1H), 7.99 (m, 1H), 8.14 (dd, 7.72,
5.46 Hz, 2H), 8.73 (s, 1H), 9.03 (d, 1.88 Hz, 1H); m/z 348 (MH+);
HRMS (MH+) calcd for C21H22N3O2, 348.1712, found 348.1696;
Anal. (C21H21N3O2‚1.5 C3H6O3‚H2O), Calcd: C 61.19%, H
6.44%, N 8.39%; Found: C 60.71%, H 6.00%, N 8.30%.
3-[4-[[[2-(1,3-Ben zod ioxol-5-yl)et h yl]a m in o]m et h yl]-
p h en yl]-N-h yd r oxy-(2E)-2-p r op en a m id e 13k . Following
Method B, 1,3-benzodioxole-5-ethanamine (806 mg, 4.89 mmol),
10 (774 mg, 4.07 mmol), and NaBH(OAc)3 (1.4 equiv) were
reacted to afford 3-[4-[[[2-(1,3-benzodioxol-5-yl)ethyl]amino]-
methyl]phenyl]-(2E)-2-propenoic acid methyl ester (650 mg,
47%) which was used without further purification: m/z 340
(MH+). Following Method C, the ester (640 mg, 1.89 mmol)
was converted to 13k (391 mg, 61%): 1H NMR (DMSO-d6) δ
2.64 (m, 4H), 3.70 (s, 2H), 5.93 (s, 2H), 6.42 (d, 15.9 Hz, 1H),
6.63 (dd, 7.9, 1.53 Hz, 1H), 6.78 (m, 2H), 7.32 (d, 8.2 Hz, 2H),
7.42 (d, 15.6 Hz, 1H), 7.47 (d, 7.9 Hz, 2H); 13C NMR (DMSO-
d6) δ 35.46, 50.57, 52.48, 100.56, 108.00, 109.01, 118.35, 121.34,
127.30, 128.41, 133.14, 134.25, 138.17, 142.57, 145.26, 147.09,
162.78; m/z 341 (MH+); Anal. (C19H20N2O4) C, H, N.
N-Hyd r oxy-3-[4-[[[2-(1H-in d ol-3-yl)-1-m eth yleth yl]a m i-
n o]m eth yl]p h en yl]-(2E)-2-p r op en a m id e 13h . Following
Method A, R-methyltryptamine (770 mg, 4.42 mmol), 10 (700
mg, 3.68 mmol), and NaBH3CN were reacted in MeOH to
afford 3-[4-[[[2-(1H-indol-3-yl)-1-methylethyl]amino]methyl]-
phenyl]-(2E)-2-propenoic acid methyl ester (620 mg, 48%): 1H
NMR (CDCl3) δ 1.02 (d, 6.0 Hz, 3H), 2.64 (m, 1H), 2.98 (m,
2H), 3.72 (s, 3H), 3.89 (m 2H), 6.62 (d, 15.8 Hz, 1H) 6.93 (t,
7.5 Hz, 1H) 7.05 (t, 7.5 Hz, 1H) 7.13 (d, 1.9 Hz, 1H) 7.39 (m,
4H) 7.65 (m, 3H), 10.82 (s, 1H); m/z 349 (MH+). Following
Method C, the ester (620 mg, 1.78 mmol) was converted to the
hydroxamate and purified by RPHPLC which, after neutral-
ization, afforded 13h (200 mg, 32%): 1H NMR (DMSO-d6) δ
1.00 (d, 6.0 Hz, 3H), 2.62 (m, 1H), 2.92 (m, 2H), 3.86 (m, 2H),
6.43 (d, 15.8 Hz, 1H), 6.93 (td, 7.4, 0.9 Hz, 1H), 7.04 (td, 7.4,
0.9 Hz, 1H), 7.13 (d, 1.9 Hz, 1H), 7.41 (m, 7H), 10.82 (s, 1H);
13C NMR (DMSO-d6) δ 20.29, 32.55, 50.31, 53.15, 111.71,
112.01, 118.51, 118.80, 121.15, 123.69, 127.67, 127.89, 128.86,
N-Hyd r oxy-3-[4-[[[2-(1H-in d ol-2-yl)eth yl]a m in o]m eth -
yl]p h en yl]-(2E)-2-p r op en a m id e 13l. Following Method B,
1H-indole-2-ethanamine31 (100 mg, 0.63 mmol), 10 (118 mg,
0.62 mmol), and NaBH(OAc)3 (1.1 equiv) were reacted in THF
to afford 3-[4-[[[2-(1H-indol-2-yl)ethyl]amino]methyl]phenyl]-
(2E)-2-propenoic acid methyl ester (131 mg, 27%): 1H NMR
(DMSO-d6) δ 2.90 (s, 4H), 3.72 (s, 3H), 3.84 (s, 2H), 6.15 (s,
1H), 6.62 (d, 16.2 Hz, 1H), 6.95 (m, 2H), 7.27 (d, 7.9 Hz, 1H),
7.40 (dd, 7.7, 3.6 Hz, 3H), 7.67 (m, 3H), 10.91 (s, 1H); 13C NMR
(DMSO-d6) δ 21.01, 27.84, 47.91, 51.37, 52.00, 98.60, 110.57,
117.24, 118.51, 119.02, 120.01, 128.23, 128.64, 132.62, 135.87,
137.84, 144.30, 166.65, 171.94; m/z 335 (MH+). Following
Method C, the ester was converted to 13l (65 mg, 53%): 1H
NMR (DMSO-d6) δ 2.83 (m, 4H), 3.75 (s, 2H), 6.13 (s, 1H), 6.43
(d, 15.8 Hz, 1H), 6.94 (m, 2H), 7.37 (m, 8H), 10.89 (s, 1H); 13
C
133.63, 136.60, 138.54, 163.17; m/z 350 (MH+); Anal. (C21H23
N3O2‚1.5H2O) C, H, N.
-
NMR (DMSO-d6) 28.87, 48.77, 52.88, 98.92, 110.99, 118.75,
118.91, 119.41, 120.36, 127.70, 128.69, 128.82, 133.55, 136.30,
138.56, 138.86, 142.93, 163.17; m/z 336 (MH+); Anal. (C20H21
N3O2‚0.5H2O) C, H, N.
-
N-Hyd r oxy-3-[4-[[[4-(1H-in d ol-3-yl)bu tyl]a m in o]m eth -
yl]p h en yl]-(2E)-2-p r op en a m id e 13i. Following Method A,
1H-indole-3-butanamine30 (1.91 g, 10.1 mmol), 10 (1.59 g, 8.37
mmol), and NaBH3CN were reacted in MeOH to afford 3-[4-
[[[4-(1H-indol-3-yl)butyl]amino]methyl]phenyl]-(2E)-2-prope-
noic acid methyl ester (2.61 g, 86%) which was used without
further purification: m/z 363 (MH+). Following Method C, the
ester (898 mg, 2.48 mmol) was converted to the hydroxamate
and purified by RPHPLC which, after neutralization, afforded
13i (160 mg, 18%): 1H NMR (DMSO-d6) δ 1.51 (m, 3H), 1.67
(m, 2H), 2.54 (m, 2H), 2.66 (t, 7.4 Hz, 2H), 3.70 (s, 2H), 6.43
(d, 15.8 Hz, 1H), 6.94 (t, 7.4 Hz, 1H), 7.05 (m, 2H), 7.42 (m,
7H), 10.72 (s, 1H); 13C NMR (DMSO-d6) δ 24.52, 27.65, 29.18,
48.43, 52.51, 111.19, 114.50, 117.93, 118.24, 120.65, 122.01,
127.12, 127.22, 128.38, 133.07, 136.19, 138.11, 162.69; m/z 364
(MH+); Anal. (C22H25N3O2‚0.25H2O) C, H, N.
N-Hyd r oxy-3-[4-[[[2-(3-qu in olin yl)eth yl]a m in o]m eth yl]-
p h en yl]-(2E)-2-p r op en a m id e 13j. To a solution of 3-quino-
lineacetonitrile (2.30 g, 13.7 mmol) in 5% TFA/MeOH was
added 10% Pd/C 1.0 g, 0.94 mmol), and the mixture was
shaken under H2 (Paar apparatus). After 3 h the mixture was
filtered through Celite, and the filtrate was evaporated to give
3-quinolinethaneamine which was used immediately in the
next reaction. Following Method B, 3-quinolinethaneamine,
10 (2.49 g, 13.1 mmol), and NaBH(OAc)3 (1 equiv) were reacted
in DCE containing 1 equiv of HOAc to afford 3-[4-[[[2-(3-
quinolinyl)ethyl]amino]methyl]phenyl]-(2E)-2-propenoic acid
methyl ester (2.96 g, 78%): retention time System 1 ) 5.35
min, retention time System 2 ) 7.62 min; 1H NMR (CDCl3) δ
3.01 (s, 4H), 3.81 (s, 3H), 3.84 (s, 2H), 6.42 (d, 16.2 Hz, 1H),
7.29 (s, 1H), 7.32 (s, 1H), 7.45 (s, 1H), 7.48 (s, 1H), 7.53 (t, 6.8
Hz, 1H), 7.67 (m, 2H), 7.76 (d, 8.3 Hz, 1H), 7.95 (d, 2.3 Hz,
1H), 8.09 (d, 8.3 Hz, 1H), 8.81 (d, 2.3 Hz, 1H); m/z 347 (MH+).
Following Method D, the ester (2.89 g, 8.35 mmol) was
converted to the hydroxamate which was purified by RPHPLC
to afford 13j (2.75 g, 57%): 1H NMR (CD3OD) δ 3.39 (m, 7.91
N-H yd r oxy-3-[4-[[[2-[5-m et h oxy-1H -in d ol-3-yl]et h yl]-
a m in o]m eth yl]p h en yl]-(2E)-2-p r op en a m id e 13m . Follow-
ing Method B, 5-methoxytryptamine (15.0 g, 78.8 mmol), 10
(19.0 g, 78.8 mmol), and NaBH(OAc)3 (1.4 equiv) were reacted
in THF (250 mL) containing CaSO4 (10.9 g) to afford 14.5 g
(46%) of 3-[4-[[[2-[5-methoxy-1H-indol-3-yl]ethyl]amino]meth-
yl]phenyl]-(2E)-2-propenoic acid methyl ester hydrochloride:
m/z 365 (MH+). Following Method C, the hydrochloride (14.4
g, 35.9 mmol) was converted to the hydroxamate (13.0 g, 98%)
which was treated with 1 equiv of lactic acid to provide 13m
as the lactate salt: 1H NMR (DMSO-d6) δ 1.21 (d, 6.8 Hz, 3H,
lactate), 3.74 (s, 3H), 3.89 (q, 7.0 Hz, 1H, lactate), 3.98 (s, 2H),
6.49 (d, 15.8 Hz, 1H), 6.71 (dd, 8.8, 2.4 Hz, 1H), 6.98 (d, 2.3
Hz, 1H), 7.12 (d, 1.9 Hz, 1H), 7.23 (d, 9.0 Hz, 1H), 7.45 (m,
3H), 7.55 (m, 2H), 10.71 (s, 1H); 13C NMR (DMSO-d6) δ 18.86,
21.66 (lactate), 46.00, 48.91, 53.24, 64.28 (lactate), 97.96,
108.81, 109.02, 109.97, 117.01, 121.42, 125.22, 125.34, 127.23,
129.29, 132.01, 135.75, 136.41, 150.88, 160.53, 175.31 (lactate);
m/z 366 (MH+); Anal. (C21H23N3O3‚C3H6O3) C, H, N.
N-Hydr oxy-3-[4-[[[(1R)-2-h ydr oxy-1-(1H-in dol-3-ylm eth -
yl)e t h yl]a m in o]m e t h yl]p h e n yl]-(2E )-2-p r op e n a m id e
13n . Following Method A, D-tryptophanol (1.0 g, 5.3 mmol),
10 (1.0 g, 5.3 mmol), and NaBH3CN (10 equiv) were reacted
in MeOH to afford 3-[4-[[[(1R)-2-hydroxy-1-(1H-indol-3-ylmeth-
yl)ethyl]amino]methyl]phenyl]-(2E)-2-propenoic acid methyl
ester (1.4 g, 75%): 1H NMR (DMSO-d6) δ 2.79 (m, 3H), 3.33
(m, 2H), 3.72 (s, 3H), 3.85 (s, 2H), 6.60 (d, 15.8 Hz, 1H), 6.92
(t, 7.0 Hz, 1H), 7.04 (t, 7.0 Hz, 1H), 7.12 (d, 2.3 Hz, 1H), 7.33
(t, 7.9 Hz, 3H), 7.43 (d, 7.9 Hz, 1H), 7.64 (m, 3H), 10.80 (s,
1H); m/z 365 (MH+). Following Method C, the ester (1.0 g, 2.7
mmol) was converted to 13n (250 mg, 25%): 1H NMR (CD3-
OD) δ 2.88 (m, 2H), 3.00 (m, 1H), 3.57 (m, 2H), 3.85 (s, 2H),
6.42 (d, 15.8 Hz, 1H), 6.93 (t, 7.5 Hz, 1H), 7.08 (m, 2H), 7.17
(d, 7.9 Hz, 2H), 7.33 (d, 8.3 Hz, 1H), 7.41 (m, Hz, 3H), 7.52 (d,

4620 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Remiszewski et al.
15.8 Hz, 1H); 13C NMR (CD3OD) δ 28.50, 52.20, 60.19, 65.04,
112.72, 112.76, 118.66, 119.86, 120.10, 122.86, 124.69, 129.17,
129.28, 130.35, 135.54, 141.74, 142.82, 166.77; m/z 366 (MH+);
Anal. (C21H23N3O3‚H2O) C, H, N.
(2E)-N-Hyd r oxy-3-[4-[(2-p h en oxyeth yla m in o)m eth yl]-
p h en yl]-2-p r op en a m id e 13s. Following the procedure de-
scribed for the preparation of 8, 2-phenoxyethylamine (4.12
g, 30.0 mmol) and 10 (5.82 g, 33.0 mmol) was converted to
13s: 1H NMR (CD3OD) δ 1.37 (d, 6.8 Hz, 3.75H), 3.5 (m, 2H),
4.13 (q, 6.9 Hz, 1.25H), 4.31 (m, 2H), 4.34 (s, 2H), 6.55 (d, 15.8
Hz, 1H), 7.01 (m, 3H), 7.32 (m, 2H), 7.62 (m, 5H); 13C NMR
(CD3OD) δ 20.43 (lactate), 46.85, 51.28, 63.70 (lactate), 114.88,
119.07, 121.97, 128.67, 129.90, 130.90, 133.53, 136.63, 139.71,
158.62, 165.13, 179.59 (lactate); m/z 313 (MH+); Anal.
(C18H20N2O3‚1.25 C3H6O3‚1.75H2O), Calcd: C 57.23%, H 6.84%,
N 6.14%; Found: C 57.20%, H 5.99%, N 5.93%.
N-Hydr oxy-3-[4-[[[(1S)-2-h ydr oxy-1-(1H-in dol-3-ylm eth -
yl)eth yl]a m in o]m eth yl]p h en yl]-(2E)-2-p r op en a m id e 13o.
Following Method A, L-tryptophanol (700 mg, 3.7 mmol), 10
(800 mg, 4.2 mmol), and NaBHCN3 (8 equiv) were reacted in
MeOH to afford 3-[4-[[[(1S)-2-hydroxy-1-(1H-indol-3-ylmethyl)-
ethyl]amino]methyl]phenyl]-(2E)-2-propenoic acid methyl ester
(710 mg, 58%): 1H NMR (DMSO-d6) δ 2.78 (m, 3H), 3.35 (m,
2H), 3.72 (s, 3H), 3.86 (s, 2H), 6.61 (d, 16.2 Hz, 1H), 6.92 (t,
6.8 Hz, 1H), 7.04 (t, 7.0 Hz, 1H), 7.33 (m, 3H), 7.44 (d, 7.9 Hz,
1H), 7.64 (m, 3H), 10.80 (s, 1H), m/z 365 (MH+). Following
Method C, the ester (710 mg, 1.9 mmol) was converted to 13o
(220 mg, 32%): 1H NMR (DMSO-d6) δ 2.77 (m, 3H), 3.34 (m,
2H), 3.83 (s, 2H), 6.42 (d, 15.8 Hz, 1H), 6.92 (t, 7.4 Hz, 1H),
7.04 (t, 7.5 Hz, 1H), 7.11 (s, 1H), 7.32 (d, 7.5 Hz, 3H), 7.43 (m,
4H), 10.78 (s, 1H); 13C NMR (DMSO-d6) δ 27.22, 50.74, 59.54,
63.33, 111.66, 111.94, 118.47, 118.73, 118.80, 121.13, 123.74,
127.65, 127.93, 128.75, 133.50, 136.57, 138.57, 163.18; m/z 365
(MH+); Anal. (C21H23N3O3‚0.1H2O) C, H, N.
(2E)-N-H yd r oxy-3-[4-[[(1H -in d ol-2-ylm et h yl)a m in o]-
m eth yl]p h en yl]-2-p r op en a m id e 13p . Following Method A,
1H-indole-2-methanamine32 (600 mg, 3.75 mmol), 10 (700 mg,
3.75 mmol), and NaBH3CN (2 equiv) were reacted in MeOH
to afford (2E)-3-[4-[[(1H-indol-2-ylmethyl)amino]methyl]phen-
yl]-2-propenoic acid methyl ester (500 mg, 42%): 1H NMR
(CDCl3) δ 3.81 (s, 3H), 3.83 (s, 2H), 3.98 (s, 2H), 6.35 (s, 1H),
6.44 (d, 16.20 Hz, 1H), 7.13 (m, 2H), 7.35 (m, 3H), 7.53 (m,
3H), 7.69 (d, 15.83 Hz, 1H), 8.52 (s, 1H). Following Method C,
the ester was converted to the hydroxamate and purified by
RPHPLC which, after neutralization, afforded 13p (350 mg,
64%): 1H NMR (DMSO-d6) δ 3.74 (s, 2H), 3.82 (s, 2H), 6.28 (s,
1H), 6.93 (m, 1H), 7.01 (t, 6.8 Hz, 1H), 7.32 (d, 7.9 Hz, 1H),
7.46 (m, 6H), 9.03 (s, 1H), 10.72 (br. s, 1H), 10.94 (s, 1H); 13C
NMR (DMSO-d6) δ 45.86, 52.11, 99.71, 111.31, 118.83, 118.99,
119.80, 120.74, 127.73, 128.39, 128.93, 133.67, 136.52, 138.57,
138.93, 142.49, 163.18; m/z 322 (MH+); Anal. (C19H19N3O2‚H2O)
C, H, N.
(2E)-N-Hyd r oxy-3-[4-[[[3-(1H-in d ol-3-yl)p r op yl]a m in o]-
m eth yl]p h en yl]-2-p r op en a m id e 13q. Following Method B,
1H-indole-3-propanamine (1.23 g, 7.1 mmol), 10 (1.48 g, 7.8
mmol), and NaBH(OAc)3 (1.4 equiv) were reacted in DCE to
afford (2E)-3-[4-[[[3-(1H-indol-3-yl)propyl]amino]methyl]phen-
yl]-2-propenoic acid methyl ester (0.99 g, 40%): 1H NMR (CD3-
OD) δ 1.92 (m, 2H), 2.62 (t, 7.4 Hz, 2H), 2.77 (t, 7.4 Hz, 2H),
3.68 (s, 2H), 3.77 (s, 3H), 6.49 (d, 15.8 Hz, 1H), 7.02 (m, 3H),
7.28 (m, 3H), 7.51 (m, 3H), 7.66 (d, 16.2 Hz, 1 H); m/z 349
(MH+). Following Method D, the ester (0.95 g, 2.73 mmol) was
converted to the hydroxamate 13q which was purified by
RPHPLC and converted to the lactate salt (405 mg, 34%): 1H
NMR (CD3OD) δ 1.32 (d, 6.8 Hz, 3.9H, lactate), 2.11 (m, 2 H),
2.86 (t, 7.0 Hz, 2H), 3.03 (m, 2H), 4.02 (q, 6.8 Hz, 1.3H, lactate),
4.13 (s, 2H), 6.49 (d, 15.8 Hz, 1H), 7.05 (m, 3H), 7.33 (d, 7.9
Hz, 1H), 7.42 (d, 7.9 Hz, 2H), 7.53 (m, 3H); 13C NMR (CD3OD)
δ 21.64, 23.24, 27.98, 51.74, 69.36, 112.42, 114.28, 119.26,
119.74, 119.81, 122.50, 123.36, 128.49, 129.39, 131.55, 134.33,
137.28, 138.33, 140.53, 165.95, 181.94; m/z 350 (MH+); Anal.
(C21H23N3O2‚1.3C3H6O3‚H2O), C, H, N.
(2E)-3-[4-[(3-Ca r ba zol-9-ylp r op yla m in o)m eth yl]-p h en -
yl]-N-h yd r oxy-2-p r op en a m id e 13r . Following the procedure
described for the preparation of 8, 9H-carbazole-9-propan-
amine (2.80 g, 12.5 mmol), and 15 (2.42 g, 13.8 mmol) was
converted to 13r : 1H NMR (CD3OD) δ 1.39 (d, 7.2 Hz, 4.8H,
lactate), 2.31 (m, 2H), 3.01 (m, 2H), 4.12 (s, 2H), 4.21 (q, 6.9
Hz, 1.6H, lactate), 4.55 (t, 6.4 Hz, 2H), 6.50 (d, 16.2 Hz, 1H),
7.25 (t, 7.4 Hz, 2H), 7.33 (d, 7.9 Hz, 2H), 7.52 (m, 7H), 8.11 (d,
7.5 Hz, 2H); 13C NMR (CD3OD) δ 21.27 (lactate), 27.34, 40.92,
46.57, 52.06, 68.30 (lactate), 110.15, 120.32, 120.78, 121.75,
124.73, 127.43, 129.79, 131.85, 133.89, 137.86, 140.79, 141.96,
166.26, 179.33 (lactate); m/z 400 (MH+); Anal. (C25H25N3O2‚
1.6 C3H6O3‚H2O), Calcd: C 61.75%, H 6.71%, N 7.25%;
Found: C 61.44%, H 6.21%, N 7.41%.
(2E)-N-H yd r oxy-3-[4-[[[2-[(2-m et h oxyp h en yl)a m in o]-
eth yl]a m in o]m eth yl]p h en yl]-2-p r op en a m id e 13t. Fol-
lowing Method B, N-(2-methoxyphenyl)-1,2-ethanediamine33
(770 mg, 4.6 mmol), 10 (1.05 g, 5.5 mmol), and NaBH(OAc)3
(1.5 equiv) were reacted in MeOH to afford (2E)-3-(4-[[2-(2-
methoxyphenylamino)ethylamino]methyl]phenyl)-2-propeno-
ic acid methyl ester (2.2 g) which was used directly in the next
step. Following Method C, the crude ester obtained in the
previous reaction (412 mg) was converted to 13t and purified
by RPHPLC (214 mg): 1H NMR (DMSO-d6) δ 3.12 (t, 6.03 Hz,
2H), 3.41 (t, 6.22 Hz, 2H), 3.77 (s, 3H), 4.22 (s, 2H), 6.58 (m,
3H), 6.80 (m, 2H), 7.50 (m, 3H), 7.63 (m, 2H); 13C NMR
(DMSO-d6) δ 45.86, 50.12, 55.60, 109.55, 110.35, 116.66,
120.45, 121.39, 128.04, 130.85, 133.39, 135.85, 137.56, 147.01,
158.90; m/z 342 ((MH+); Anal. (C19H23N3O3‚1.6TFA‚1.3H2O)
C, H.
(2E)-N-H yd r oxy-3-[4-[[(1H -in d ol-3-ylm et h yl)a m in o]-
m eth yl]p h en yl]-2-p r op en a m id e 13u . Following Method A,
1H-indole-3-methanamine (2.60 g, 17.6 mmol), 10 (3.30 g, 17.6
mmol), HOAc (1 equiv), and NaBHCN3 (1.5 equiv) were reacted
in MeOH to afford (2E)-3-(4-[[(1H-indol-3-ylmethyl)amino]-
methyl]phenyl)propenoic acid methyl ester (950 mg, 17%); 1H
NMR (CDCl3) δ 3.80 (s, 3H), 3.87 (s, 2H), 4.00 (s, 2H), 6.41 (d,
15.8 Hz, 1H), 7.13 (m, 2H), 7.21 (m, 1H), 7.36 (m, 3H), 7.48
(m, 2H), 7.63 (m, 2H), 8.19 (s, 1H), m/z 321 (MH+). Following
Method C, the ester (925 mg, 2.89 mmol) was converted to the
hydroxamate and purified by RPHPLC which, after neutral-
ization, afforded 13u (231 mg, 25%): 1H NMR (DMSO-d6) δ
3.75 (s, 2H), 3.83 (s, 2H), 6.44 (d, 15.8 Hz, 3H), 6.97 (t, 7.00
Hz, 1H), 7.06 (m, 1H), 7.23 (d, 2.64 Hz, 1H), 7.46 (m, 7H), 10.86
(s, 1H); 13C NMR (DMSO-d6) δ 43.67, 51.96, 111.23, 113.42,
118.15, 118.21, 118.72, 120.83, 123.34, 126.88, 127.23, 128.37,
133.01, 136.26, 138.15, 142.69, 162.72; m/z 322 (MH+); Anal.
(C19H19N3O2‚H2O) C, H, N.
(2E)-N-Hyd r oxy-3-[4-[[[2-(1H-in d ol-3-yl)eth yl](1-m eth -
yleth yl)a m in o]m eth yl]p h en yl]-2-p r op en a m id e 14a . A so-
lution of (2E)-3-[4-[[[2-(1H-indol-3-yl)ethyl]amino]methyl]-
phenyl]-2-propenoic acid methyl ester (500 mg, 1.50 mmol),
2-iodopropane (1.01 g, 5.9 mmol), and DIEA (1.50 mL, 1.11 g,
8.61 mmol) in MeCN was heated to 50 °C. After 48 h, the
solution was evaporated and the residue purified by flash
chromatography to afford (2E)-3-[4-([[2-(1H-indol-3-yl)ethyl]-
(1-methylethyl)amino]methyl)phenyl]-2-propenoic acid methyl
ester (455 mg, 80%): 1H NMR (DMSO-d6) δ 1.01 (d, 6.4 Hz,
6H), 2.66 (m, 2H), 2.75 (m, 2H), 3.66 (s, 2H), 3.73 (s, 3H), 6.61
(d, 15.8 Hz, 1H), 6.89 (t, 7.9 Hz, 1H), 7.02 (t, 8.1 Hz, 1H), 7.07
(d, 2.3 Hz, 1H), 7.30 (d, 8.7 Hz, 2H), 7.42 (d, 8.3 Hz, 2H), 7.66
(m, 3.96 Hz, 3H), 10.74 (s, 1H); 13C NMR (DMSO-d6) δ 17.96,
20.69, 50.57, 51.36, 53.26, 59.68, 111.21, 117.95, 118.04,
120.65, 122.37, 127.09, 128.14, 128.64, 136.07, 144.48, 166.70;
m/z 377 (MH+). Following Method C, the ester (420 mg, 1.12
mmol) was converted to the hydroxamate which was purified
by RPHPLC to afford 14a (325 mg, 59%): retention time
System 3 ) 8.07 min, retention time System 4 ) 25.12 min;
1H NMR (DMSO-d6) δ 1.36 (d, 6.8 Hz, 3H), 1.39 (d, 6.8 Hz,
3H), 3.09 (m, 3H), 3.35 (m, 1H), 3.71 (m, 1H), 4.45 (m, 2H),
6.57 (d, 15.8 Hz, 1H), 6.95 (t, 7.4 Hz, 1H), 7.08 (t, 7.2 Hz, 1H),
7.21 (d, 1.9 Hz, 1H), 7.33 (m, 2H), 7.62 (m, 5H), 9.50 (s, 1H),
11.00 (s, 1H); 13C NMR (DMSO-d6) δ 16.58, 21.06, 49.90, 53.39,
55.15, 109.31, 112.00, 118.28, 118.88, 120.82, 121.60, 126.92,
128.27, 132.00, 132.15, 136.31, 136.58, 158.78, 159.24, 162.86;
m/z 377 (MH+); HRMS (MH+) calcd for C23H28N3O2, 378.2182,

Inhibitors of Human Histone Deacetylase
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4621
found 378.2207; Anal. (C23H27N3O2‚2 TFA‚0.25H2O) Calcd: C
53.16%, H 4.87%, N 6.89%; Found: C 52.76%, H 4.88%, N
7.32%.
3.51 (m, 2H), 3.62 (s, 2H), 4.24 (s, 2H), 4.46 (br s, 4H), 6.23 (d,
3.0 Hz, 1H), 6.32 (m, 1H), 6.53 (d, 15.8 Hz, 1H), 6.94 (t, 7.4
Hz, 1H), 7.10 (m, 2H), 7.35 (m, 3H), 7.47 (m, 2H), 7.57 (m,
3H); 13C NMR (CD3OD) δ 21.85, 39.18, 54.91, 55.37, 59.33,
61.00, 108.64, 109.65, 111.82, 113.07, 119.26, 120.65, 123.32,
124.96, 128.25, 130.04, 132.29, 133.06, 138.48, 138.76, 140.70,
143.85, 153.55, 158.68; m/z 503 (MH+); Anal. (C28H30N4O5‚
1.5TFA) C, H, F, N.
(2E)-3-[4-[[Cycloh exyl[2-(1H -in d ol-3-yl)et h yl]a m in o]-
m eth yl]p h en yl]-N-h yd r oxy-2-p r op en a m id e 14b. Following
Method A, a mixture of 13g (500 mg, 1.49 mmol), cyclohex-
anone (0.32 mL, 303 mg, 3.09 mmol), and NaBH3CN (4.3
equiv) in MeOH was heated at reflux. After 4 h, the reaction
was cooled, diluted with EtOAc, and washed with sat. NaHCO3
and brine. The organic solution was dried and evaporated, and
the residue was purified by RPHPLC to afford 14b (484 mg,
50%): 1H NMR (DMSO-d6) δ 1.23 (m, 3H), 1.62 (m, 3H), 1.83
(m, 2H), 2.10 (m, 2H), 3.14 (m, 5H), 4.38 (m, 1H), 4.56 (m,
1H), 6.55 (d, 15.8 Hz, 2H), 6.94 (t, 7.0 Hz, 2H), 7.07 (t, 7.0 Hz,
1H), 7.28 (m, 3H), 7.50 (d, 15.8 Hz, 1H), 7.67 (m, 4H), 9.36 (s,
1H), 10.97 (s, 1H); 13C NMR (DMSO-d6) δ 20.70, 24.53, 25.86,
26.10, 49.97, 62.41, 108.79, 111.53, 117.82, 118.41, 120.35,
121.16, 123.35, 126.40, 127.85, 131.58, 131.72, 135.88, 136.09;
m/z 418 (MH+); Anal. (C26H31N3O2‚1.75TFA) C, H, N.
(2E)-N-H yd r oxy-3-[4-[[[2-(1H -in d ol-3-yl)et h yl](t et r a -
h yd r o-2H -p yr a n -4-yl)a m in o]m et h yl]p h en yl]-2-p r op en -
a m id e 14c. Following Method B, (2E)-3-[4-[[[2-(1H-indol-3-
yl)ethyl]amino]methyl]phenyl]-2-propenoic acid methyl ester
(1.02 g, 3.06 mmol), tetrahydro-4H-pyran-4-one (0.32 mL, 346
mg, 3.45 mmol) and NaBH(OAc)3 (1.5 equiv) were reacted to
afford (E)-3-(4-[[[2-(1H-indol-3-yl)ethyl](tetrahydro-2H-pyran-
4-yl)amino]methyl]phenyl)-2-propenoic acid methyl ester (612
mg, 48%): 1H NMR (CDCl3) δ 1.68 (m, 4H), 2.82 (m, 4H), 3.34
(m, 2H), 3.76 (s, 2H), 3.81 (s, 3H), 4.02 (m, 2H), 6.43 (d, 15.83
Hz, 1H), 6.92 (d, 2.26 Hz, 1H), 7.05 (t, 6.97 Hz, 1H), 7.16 (t,
6.97 Hz, 1H), 7.39 (m, 6H), 7.70 (d, 15.83 Hz, 1H), 7.96 (s, 1H).
Following Method C, the ester (319 mg, 0.763 mmol) was
converted to the hydroxamate 14c which was purified by
RPHPLC and converted to the lactate salt (375 mg, 47%): 1H
NMR (CD3OD) δ 1.35 (d, 6.8 Hz, 3.9H, lactate), 1.84 (m, 4H),
2.96 (m, 2H), 3.12 (m, 2H), 3.38 (m, 3H), 4.08 (m, 5H), 6.51 (d,
15.8 Hz, 1H), 6.92 (t, 7.5 Hz, 1H), 7.06 (m, 2H), 7.24 (d, 7.9
Hz, 1H), 7.31 (d, 8.3 Hz, 1H), 7.55 (m, 5H); 13C NMR (CD3OD)
δ 21.17, 24.01, 29.78, 51.82, 55.36, 60.24, 68.16, 68.48, 112.22,
112.44, 119.07, 119.78, 122.52, 123.69, 128.38, 129.25, 131.56,
136.42, 138.19, 141.02, 166.22, 180.06; m/z 420 (MH+); Anal.
(C25H29N3O3‚1.3C3H6O3) C, H, N.
(2-F u r a n ylm et h yl)ca r b a m ic Acid 2-[[[4-[(1E)-3-(H y-
d r oxya m in o)-3-oxo-1-p r op en yl]p h en yl]m eth yl][2-(1H-in -
d ol-3-yl)eth yl]a m in o]eth yl Ester 14d . To a stirred slurry
of THF (50 mL), ice (50 g), K2CO3 (4.25 g, 30.7 mmol), and
2-furanmethanamine (2.8 g, 25.7 mmol) was added 2-bromo-
ethyl chloroformate (3.60 mL, 5.29 g, 28.2 mmol). After 4 h,
the mixture was acidified (1 M HCl), NaCl was added, and
the solution was extracted with EtOAc. The organic extract
was dried (Na2SO4), filtered, and evaporated to afford furan-
2-ylmethylcarbamic acid 2-bromoethyl ester which was used
without further purification. A solution of the bromide (469
mg, 1.80 mmol), (2E)-3-[4-[[[2-(1H-indol-3-yl)ethyl]amino]-
methyl]phenyl]-2-propenoic acid methyl ester (517 mg, 1.55
mmol), and DIEA (0.33 mL, 1.89 mmol) in DMSO (5 mL) was
heated to 60 °C. After 6 h, the reaction was diluted with H2O
and extracted with EtOAc. The organic extracts were washed
with brine, dried (Na2SO4), and evaporated. The resulting
residue was purified by column chromatography to afford (2E)-
3-[4-([[2-(furan-2-ylmethylcarbamoyloxy)ethyl]-[2-(1H-indol-3-
yl)ethyl]amino]methyl)phenyl]-2-propenic acid methyl ester
(428 mg, 55%): 1H NMR (CD3OD) δ 2.81 (m, 4H), 2.91 (m,
2H), 3.74 (s, 2H), 3.77 (d, 4.5 Hz, 3H), 4.18 (t, 5.7 Hz, 2H),
4.24 (s, 2H), 6.20 (d, 3.0 Hz, 1H), 6.30 (m, 1H), 6.48 (d, 16.2
Hz, 1H), 6.90 (t, 7.0 Hz, 1H), 6.98 (s, 1H), 7.04 (t, 7.0 Hz, 1H),
7.29 (d, 8.3 Hz, 1H), 7.36 (m, 4H), 7.48 (m, 2H), 7.67 (d, 15.8
Hz, 1H); 13C NMR (CD3OD) δ 24.33, 38.70, 52.21, 53.96, 56.32,
59.78, 64.11, 107.79, 111.36, 112.19, 114.25, 118.07, 119.39,
119.44, 122.19, 123.23, 128.86, 129.22, 130.69, 134.46, 138.15,
143.24, 143.94, 146.31, 153.83, 169.30; m/z 502 (MH+). Fol-
lowing Method D, the ester (420 mg, 0.837 mmol) was
converted to the hydroxamate which was purified by RPHPLC
to give 14d (230 mg, 55%): 1H NMR (CD3OD) δ 3.23 (m, 2H),
(2E)-N-Hydr oxy-3-[4-[[(2-h ydr oxyeth yl)[2-(3-qu in olin yl)-
eth yl]a m in o]m eth yl]p h en yl]-2-p r op en a m id e 14e. A solu-
tion of 2-(2-bromoethoxy)tetrahydro-2H-pyran (1.67 mL, 10.6
mmol), 3-[4-[[[2-(3-quinolinyl)ethyl]amino]methyl]phenyl]-(2E)-
2-propenoic acid methyl ester (2.63 g, 7.60 mmol), and DIEA
(2.00 mL, 11.4 mmol) in DMSO (25 mL) was heated to 60 °C
for 17 h. The cooled mixture was diluted with H2O and
extracted with EtOAc. The organic extracts were washed with
brine, dried (MgSO4), and evaporated. The residue was purified
by flash chromatography to afford (2E)-3-[4-([(2-quinolin-3-
ylethyl)-[2-(tetrahydro-2H-pyran-2-yloxy)ethyl]amino]methyl)-
phenyl]-2-propenic acid methyl ester (1.36 g, 38%): m/z 475
(MH+). Following Method C, the ester (1.31 g, 2.76 mmol) was
converted to the hydroxamate which was purified by RPHPLC
to afford 14e as the TFA salt which was neutralized and
treated with 1 M lactic acid to provide 14e as the lactate salt
(1.01 g, 76%): 1H NMR (DMSO-d6) δ 1.23 (d, 6.8 Hz, 3H,
lactate), 2.62 (t, 6.2 Hz, 2H), 2.81 (t, 6.8 Hz, 2H), 2.95 (t, 6.8
Hz, 2H), 3.48 (t, 6.2 Hz, 2H), 3.71 (s, 2H), 3.99 (t, 6.8 Hz, 1H),
6.44 (d, 15.8 Hz, 1H), 7.25 (d, 7.9 Hz, 2H), 7.41 (m, 3H), 7.57
(t, 7.4 Hz, 1H), 7.70 (t, 7.4 Hz, 1H), 7.87 (d, 7.9 Hz, 1H), 8.00
(d, 8.3 Hz, 1H), 8.09 (s, 1H), 8.77 (s, 1H); 13C NMR (DMSO-d6)
δ 21.00, 30.37, 48.97, 55.60, 55.95, 58.39, 59.61, 66.37, 118.90,
126.81, 127.57, 127.90, 128.09, 128.99, 129.39, 133.70, 134.02,
134.74, 138.45, 141.71, 146.63, 152.58, 163.16, 177.01; m/z 392
(MH+); Anal. (C23H25N3O3‚C3H6O3‚2.5H2O) C, H, N, mp 68.2-
74.8 °C.
(2E)-N-H yd r oxy-3-(4-[[(3-p h en ylp r op yl)p yr id in -3-yl-
m eth yla m in o]m eth yl]p h en yl)-2-p r op en a m id e 14f. Fol-
lowing Method A, 2-pyridinecarboxaldehyde (1.18 g, 11.0
mmol), (2E)-3-[4-[(3-phenyl-propylamino)-methyl]-phenyl]-2-
propenoic acid methyl ester (3.09 g, 10.0 mmol), HOAc (1
equiv), and NaBH3CN (1.1 equiv) were reacted in MeOH to
afford (2E)-3-(4-[[(3-phenylpropyl)pyridin-3-ylmethylamino]-
methyl]phenyl)-2-propenoic acid methyl ester (3.76 g, 94%):
1H NMR (CDCl3) δ 1.87 (m, 2H), 2.52 (t, 7.2 Hz, 2H), 2.60 (m,
2H), 3.60 (s, 2H), 3.61 (s, 2H), 3.84 (s, 3H), 6.46 (d, 15.8 Hz,
1H), 7.20 (m, 6H), 7.39 (d, 8.3 Hz, 2H), 7.50 (d, 8.3 Hz, 2H),
7.72 (m, 2H), 8.52 (dd, 4.7, 1.7 Hz, 1H), 8.59 (d, 1.9 Hz, 1H);
13C NMR (CDCl3) δ 29.24, 33.85, 52.16, 56.12, 58.49, 77.69,
117.83, 123.78, 126.21, 128.55, 128.74, 129.72, 133.72, 136.91,
142.47, 145.05, 148.98, 150.69, 167.95; m/z 401; (MH+). Fol-
lowing Method D, the ester (3.70 g, 9.20 mmol) was converted
to the hydroxamate which was purified by RPHPLC to afford
14f as the TFA salt which was neutralized and treated with 1
M lactic acid to provide 14f as the lactate salt (3.34 g, 73%):
1H NMR (CD3OD) δ 1.40 (d, 6.8 Hz, 4.5H, lactate), 1.88 (m,
2H), 2.57 (q, 6.9 Hz, 4H), 3.74 (m, 4H), 4.23 (q, 6.9 Hz, 1.5H,
lactate), 6.49 (d, 15.8 Hz, 1H), 7.14 (m, 5H), 7.41 (m, 3H), 7.56
(m, 3H), 7.84 (d, 7.8 Hz, 1H), 8.46 (d, 4.1 Hz, 1H), 8.52 (s, 1H);
13C NMR (CD3OD) δ 21.21, 29.79, 34.63, 54.31, 57.00, 59.58,
68.16, 118.83, 125.65, 127.25, 129.34, 129.77, 131.30, 135.94,
136.67, 139.76, 141.69, 141.76, 143.46, 149.27, 151.03, 166.75,
179.04; m/z 402 (MH+); Anal. (C25H27N3O2‚1.5C3H6O3‚0.2H2O)
C, H, N.
(2E)-N-Hyd r oxy-3-[4-[[[2-(1H-in d ol-3-yl)eth yl](p h en yl-
m eth yl)a m in o]m eth yl]p h en yl]-2-p r op en a m id e 14g. Fol-
lowing Method B, N-benzyltryptamine (2.05 g, 8.20 mmol), 10
(1.49 g, 7.83 mmol), HOAc (1 equiv), and NaBH(OAc)3 were
reacted in DCE to afford (2E)-3-[4-([benzyl-[2-(1H-indol-3-yl)-
ethyl]amino]methyl)phenyl]-2-propenoic acid methyl ester (1.48
g, 45%): 1H NMR (CDCl3) δ 2.80 (m, 2H), 2.97 (m, 2H), 3.67
(s, 2H), 3.69 (s, 2H), 3.80 (s, 3H), 6.41 (d, 16.2 Hz, 1H), 6.89
(d, 2.3 Hz, 1H), 7.01 (t, 7.3 Hz, 1H), 7.15 (t, 7.5 Hz, 1H), 7.32
(m, 11H), 7.68 (d, 16.2 Hz, 1H), 7.90 (s, 1H); m/z 425 (MH+).
Following Method D, the ester (1.45 g, 3.42 mmol) was

4622 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Remiszewski et al.
converted to the hydroxamate which was purified by RPHPLC
to afford 14g as the TFA salt which was neutralized and
treated with 1 M lactic acid to provide 14g as the lactate salt
(649 mg, 40%): 1H NMR (CD3OD) δ 1.37 (d, 6.8 Hz, 4.8H,
lactate), 3.19 (m, 2H), 3.31 (m, 2H), 4.21 (q, 6.8 Hz, 1.6H,
lactate), 4.44 (m, 4H), 6.54 (d, 15.8 Hz, 1H), 6.90 (t, 7.5 Hz,
1H), 7.11 (m, 3H), 7.33 (d, 7.9 Hz, 1H), 7.50 (s, 8H), 7.64 (m,
2H); 13C NMR (CD3OD) δ 20.79, 21.59, 53.50, 58.45, 58.97,
67.68, 109.91, 112.63, 118.83, 120.11, 120.25, 122.84, 124.14,
129.63, 130.57, 132.20, 132.68, 137.93, 138.31, 140.41, 178.47;
m/z 426 (MH+); Anal. (C25H27N3O2‚1.6 C3H6O3‚4H2O); Calcd:
C 59.52%, H 7.01%, N 6.55%; Found C 59.91%, H 5.48%, N
6.22%.
(1.87 g, 4.99 mmol) was converted to hydroxamate 14j (1.66
g, 88%): 1H NMR (DMSO-d6) δ 1.70 (m, 2H), 1.92 (m, 2H),
2.13 (t, 10.7 Hz, 2H), 2.75 (m, 1H), 2.91 (d, 11.3 Hz, 2H), 3.53
(s, 2H), 6.45 (d, 15.8 Hz, 1H), 6.94 (m, 1H), 7.04 (m, 1H), 7.09
(d, 2.3 Hz, 1H), 7.35 (m, 3H), 7.45 (d, 15.8 Hz, 1H), 7.53 (m,
3H), 10.77 (s, 1H); 13C NMR (DMSO-d6) δ 32.76, 32.97, 53.73,
62.15, 111.32, 117.94, 118.46, 119.51, 120.40, 120.69, 126.22,
127.26, 129.21, 133.37, 136.26, 138.07, 140.32, 162.68; m/z 376
(MH+); Anal. (C23H25N3O3‚0.4H2O) C, H, N.
(2E)-N-H yd r oxy-3-[4-[(1,3,4,9-t et r a h yd r o-2H -p yr id o-
[3,4-b]in d ol-2-yl)m eth yl]p h en yl]-2-p r op en a m id e 17. Fol-
lowing the procedure described for the preparation of 8, 1,2,3,4-
tetrahydro-9H-pyrido[3,4-b]indole (5.01 g, 29.1 mmol) and 12
(5.74 g, 32.6 mmol) were converted to 17: 1H NMR (CD3OD)
δ 2.97 (t, 5.6 Hz, 2H, lactate), 3.24 (t, 5.8 Hz, 2H), 3.99 (s,
2H), 4.14 (m, 3H), 6.54 (d, 16.2 Hz, 1H), 7.06 (m, 2H), 7.31 (d,
7.9 Hz, 1H), 7.44 (d, 7.5 Hz, 1H), 7.59 (m, 5 H); 13C NMR (CD3-
OD) δ 21.49 (lactate), 21.62, 51.26, 52.50, 62.17, 68.78 (lactate),
107.96, 112.44, 119.11, 120.48, 122.93, 129.57, 132.31, 138.56,
141.31, 166.59, 180.51; m/z 348 (MH+); Anal. (C21H21N3O2‚
C3H6O3) C, H, N.
(2E)-N-Hyd r oxy-3-[4-[[[2-h yd r oxy-1-(h yd r oxym eth yl)-
et h yl][2-(1H -in d ol-3-yl)et h yl]a m in o]m et h yl]p h en yl]-2-
p r op en a m id e 14h . Following Method A, 1,3-dihydroxyace-
tone dimer (4.2 g, 23 mmol), (2E)-3-[4-[[[2-(1H-indol-3-
yl)ethyl]amino]methyl]phenyl]-2-propenoic acid methyl ester
(1.5 g, 4.5 mmol), and NaBH3CN (3 equiv) were reacted in
MeOH to afford (2E)-3-[4-([(2-hydroxy-1-hydroxymethylethyl)-
[2-(1H-indol-3-yl)ethyl]amino]methyl)phenyl]-2-propenoic acid
methyl ester (920 mg, 50%): m/z 409 (MH+). Following Method
C, the ester (900 mg, 2.21 mmol) was converted to the
hydroxamate which was purified by RPHPLC and neutralized
to afford 14h (370 mg, 41%): retention time System 3 ) 10.22
min, retention time System 4 ) 22.27 min; 1H NMR (CD3OD)
δ 2.87 (m, 2H), 2.99 (m, 2H), 3.62 (m, 4H), 3.90 (s, 2H), 6.43
(d, 15.8 Hz, 1H), 6.96 (m, 3H), 7.30 (m, 4H), 7.42 (m, 2H), 7.53
(d, 16.2 Hz, 1H); 13C NMR (CD3OD) δ 21.83, 31.37, 52.72,
56.01, 60.93, 64.23, 112.72, 114.11, 118.32, 119.76, 119.96,
122.73, 123.94, 126.60, 129.05, 129.19, 131.00, 135.50, 138.61,
141.75, 166.74; m/z 410 (MH+); HRMS (MH+) calcd for
C23H28N3O4, 410.2080, found 410.2107; Anal. (C23H27N3O4‚
0.65CH2Cl2‚0.25THF) C, H, N.
(2E)-N-Hyd r oxy-3-[4-[[3-(1H-in d ol-3-yl)p r op yl]a m in o]-
p h en yl]-2-p r op en a m id e 19. To a solution of 1H-indole-3-
propanoic acid (5.00 g, 26.4 mmol) and i-BuOCCl (3.50 mL,
3.65 g, 26.7 mmol) in THF was added NMM (5.90 mL, 5.43 g,
53.6 mmol). After 2 h, 18 hydrochloride (5.65 g, 26.4 mmol)
was added and the mixture stirred overnight. The reaction
mixture was diluted with EtOAc and washed with 1 M HCl, 1
M NaOH, and brine. The organic extract was dried (MgSO4),
filtered and the filtrate evaporated to give an oil which was
dissolved in EtOAc. Hexane addition afforded (2E)-3-[4-(3-1H-
Indol-3-ylpropionylamino)phenyl]-2-propenoic acid methyl es-
ter as a solid which, after filtration, was used without
purification in the next step (5.65 g, 61%): m/z 439 (MH+). A
solution of HOAc (8.62 g, 144 mmol) in 25 mL of THF was
added dropwise to a suspension of NaBH4 (5.43 g, 144 mmol)
in 100 mL of THF. After 2 h, a solution of the amide (5.04 g,
14.5 mmol) in 50 mL of THF was added to the NaBH4/HOAc
mixture. After 3 h, the reaction mixture was slowly poured
into brine (250 mL) and the mixture stirred overnight. The
mixture was extracted with EtOAc, and the extracts were
washed with water and brine, dried, and evaporated. The
residue was purified (flash chromatography) to an oil which
was dissolved in 4 M HCl/dioxane. The dioxane solution was
diluted with Et2O to give a precipitate which was filtered and
dried to provide (2E)-3-[4-[3-(1H-indol-3-yl)propylamino]phen-
yl]-2-propenoic acid methyl ester hydrochloride (2.48 g, 51%):
1H NMR (DMSO-d6) δ 2.00 (m, 2H), 2.79 (t, 7.5 Hz, 2H), 3.21
(t, 7.4 Hz, 2H), 3.70 (s, 3H), 6.45 (d, 15.8 Hz, 1H), 6.96 (t, 7.4
Hz, 1H), 7.06 (m, 2H), 7.15 (s, 1H), 7.34 (d, 7.9 Hz, 1H), 7.57
(m, 4H), 9.91 (br s, 2H), 10.85 (s, 1H); 13C NMR (DMSO-d6) δ
22.40, 28.31, 46.07, 51.61, 111.76, 113.94, 118.50, 118.65,
121.25, 122.70, 127.44, 130.35, 136.70, 144.84, 167.37; m/z 335
(MH+); mp 187-190 °C; Anal. (C21H22N2O2‚HCl) C, H, N, Cl.
Following Method C, the amine hydrochloride (1.51 g, 4.07 g)
was converted to 19, which precipitated from the reaction
mixture upon neutralization. The free base was treated with
HCl/dioxane as described above to give 19 as the HCl salt (519
mg, 34%): retention time System 3 ) 15.51 min, retention time
(2E)-N-H yd r oxy-3-(4-[[(3-p h en ylp r op yl)-p yr id in -2-yl-
m eth yla m in o]m eth yl]p h en yl)-2-p r op en a m id e 14i. Fol-
lowing Method B, 2-pyridinecarboxaldehyde (1.33 g, 12.4
mmol), (2E)-3-[4-[(3-phenylpropylamino)methyl]phenyl]-2-pro-
penoic acid methyl ester (3.51 g, 11.3 mmol), and NaBH(OAc)3
(1.5 equiv) were reacted in DCE to afford (2E)-3-(4-[[(3-
phenylpropyl)pyridin-2-ylmethylamino]methyl]phenyl)-2-pro-
penoic acid methyl ester (2.72 g, 60%): 1H NMR (CDCl3) δ
1.85 (m, 2H), 2.57 (m, 4H), 3.65 (s, 2H), 3.78 (s, 2H), 3.80 (s,
3H), 6.42 (d, 16.2 Hz, 1H), 7.17 (m, 6H), 7.42 (m, 4H), 7.55 (d,
7.9 Hz, 1H), 7.68 (m, 2H), 8.54 (d, 4.1 Hz, 1 H), m/z 401 (MH+).
Following Method E, the ester (1.29 g, 3.22 mmol) was
converted to the hydroxamate which was purified by RPHPLC
to afford 14i as the TFA salt which was neutralized and
treated with 1 equiv of lactic acid to provide 14i as the lactate
salt (1.5 g, 83%): retention time System 3 ) 4.72 min,
1
retention time System 4 ) 25.32 min; H NMR (DMSO-d6) δ
1.24 (d, 7.2 Hz, 4.5H, lactate), 1.77 (m, 2H), 2.44 (t, 6.8 Hz,
2H), 2.52 (m, 2H), 3.61 (s, 2H), 3.68 (s, 2H), 4.05 (q, 7.0 Hz,
1.5H lactate), 6.45 (d, 15.8 Hz, 1H), 7.17 (m, 6H), 7.48 (m, 6H),
7.77 (m, 1H), 8.48 (d, 4.1 Hz, 1 H); 13C NMR (DMSO-d6) δ 20.42
(lactate), 28.37, 32.69, 52.68, 57.59, 59.49, 65.70 (lactate),
118.46, 121.99, 122.53, 125.48, 127.32, 128.07, 128.15, 129.10,
133.40, 136.43, 140.90, 141.91, 148.59, 159.45, 176.28 (lactate);
m/z 402 (MH+); HRMS (MH+) calcd for C25H27N3O2, 402.2182,
found 402.2199; Anal. (C25H27N3O2‚1.5 C3H6O3‚3H2O‚0.75 TFA‚
0.75 THF) C, H, N.
1
System 4 ) 26.63 min; H NMR (DMSO-d6) δ 2.01 (m, 2H),
2.79 (t, 7.35 Hz, 2H), 3.23 (m, 2H), 6.38 (d, 15.5 Hz, 1H), 6.96
(t, 7.4 Hz, 1H), 7.06 (t, 7.4 Hz, 1H), 7.15 (m, 3H), 7.34 (d, 7.9
(2E)-N-Hyd r oxy-3-[4-[[4-(1H-in d ol-3-yl)-1-p ip er id in yl]-
m eth yl]p h en yl]-2-p r op en a m id e 14j. Following Method B,
3-(piperidin-4-yl)-1H-indole (2.00 g, 10 mmol), 10 (1.90 g, 10
mmol), and NaBH(OAc)3 (1 equiv) were reacted in DCE to
afford (2E)-3-[4-[4-(1H-indol-3-yl)piperidin-1-ylmethyl]phenyl]-
2-propenoic acid methyl ester (2.90 g, 77%): 1H NMR (CDCl3)
δ 1.90 (m, 2H), 2.07 (m, 3H), 2.24 (t, 11.1 Hz, 2H), 2.88 (m,
1H), 3.06 (d, 11.3 Hz, 2H), 3.64 (s, 2H), 3.84 (s, 3H), 6.47 (d,
16.2 Hz, 1H), 7.00 (d, 1.9 Hz, 1H), 7.13 (m, 1H), 7.21 (m, 1H),
7.40 (m, 3H), 7.54 (m, 2H), 7.71 (m, 2H), 8.06 (s, 1H); 13C NMR
(CDCl3) δ 33.36, 33.83, 52.15, 54.81, 63.57, 111.63, 117.78,
119.52, 120.09, 122.35, 127.09, 128.48, 130.18, 133.67, 136.79,
145.16, 167.99; m/z 375 (MH+). Following Method C, the ester
Hz, 1H), 7.40 (d, 15.8 Hz, 1H), 7.51 (m, 3H), 10.84 (s, 1H); 13
C
NMR (DMSO-d6) δ 22.33, 27.98, 47.14, 111.77, 113.83, 118.53,
118.64, 121.27, 122.73, 127.41, 129.27, 136.70, 138.25, 163.44;
m/z 336 (MH+); HRMS (MH+) calcd for C20H22N3O2, 336.1712,
found 336.1725; mp 148-149 °C decomp.; Anal. (C20H21N3O2‚
HCl‚0.15Et2O‚0.4H2O) C, H, N, Cl.
(2E)-N-Hyd r oxy-3-[4-[[[2-(2-m et h yl-1H -b en zim id a zol-
1-yl)eth yl]a m in o]m eth yl]p h en yl]-2-p r op en a m id e 21. A
mixture of 1-(2-bromoethyl)-2-methyl-1H-benzimidazole34 (2.93
g, 12.3 mmol), 20 hydrochloride (2.79 g, 12.3 mmol), and DIEA
(4.3 mL, 24.7 mmol) in DMSO (50 mL) was heated to 50 °C
for 72 h, cooled, diluted with sat. NaHCO3 (aq.), and extracted

Inhibitors of Human Histone Deacetylase
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4623
with EtOAc. The combined extracts were dried (MgSO4),
filtered, and evaporated. The residue was purifed (flash
chromatography) to give an oil. This was dissolved in MeOH
and treated with 4.0 M HCl/dioxane and the solution diluted
with Et2O to afford (2E)-3-[4-[[[2-(2-methyl-1H-benzimidazol-
1-yl)ethyl]amino]methyl]phenyl]-2-propenoic acid methyl ester
hydrochloride (438 mg, 9%): 1H NMR (DMSO-d6) δ 2.93 (s,
3H), 3.46 (s, 2H), 3.73 (s, 3H), 4.21 (s, 2H), 4.94 (t, 6.22 Hz,
2H), 6.71 (d, 16.20 Hz, 1H), 7.57 (m, 2H), 7.67 (m, 3H), 7.78
(m, 3H), 8.14 (m, 1H), 10.37 (s, 2 H); 13C NMR (DMSO-d6) δ
12.13, 40.81, 44.41, 49.74, 51.46, 112.59, 113.87, 118.55,
125.25, 125.74, 128.38, 130.18, 130.57, 131.62, 133.89, 134.40,
143.70, 152.40, 166.50; m/z 350 (MH+); mp 175-179 °C.
Following Method C, the ester hydrochloride (386 mg, 1.00
mmol) was converted to the hydroxamate and triturated with
Et2O to afford 21 (46 mg, 13%): retention time System 3 )
3.01 min, retention time System 4 ) 18.96 min; 1H NMR
(DMSO-d6) δ 2.54 (s, 3H), 2.83 (t, 6.03 Hz, 2H), 3.35 (s, 2H),
3.70 (s, 2H), 4.24 (t, 6.41 Hz, 2H), 6.41 (d, 15.82 Hz, 1H), 7.13
Mon ola yer Gr ow th In h ibition Assa y. All incubations
were performed at 37 °C. The effect of the HDAIs on monolayer
cell proliferation was measured using an adaptation of a
published procedure.23 Cells were plated in 96-well plates at
initial densities of between 1000 and 3000 cells/well and
incubated at 37 °C. After 24 h, test compounds were added to
test wells, vehicle added to vehicle control (VC) wells and
initial growth control wells (IGC) received 10 µL 3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sul-
fophenyl)-2H-tetrazolium, inner salt (MTS) mixture (prepared
on day of use at a ratio of 10 µL of a 0.92 mg/mL solution of
phenazine methosulfate (PMS) to a 190 µL of a 2 mg/mL
solution of MTS), the plate was incubated for 4 h at 37 °C,
and the OD490 of IGC wells was measured on a Molecular
Devices Thermomax at 490 nm using the Softmax program to
determine initial cell density values. The plates were further
incubated for 72 h at 37 °C, 10 µL/well of MTS mixture was
added to the test and VC wells, and the OD490 was measured.
OD490 values for wells containing cells were corrected for media
absorbance. The following formulas were used to calculate
percent growth: If X > T0, % growth ) 100 × ((X - T0)/
(GC - T0)), if X < T0, % growth ) 100 × (X - T0)/T0); where
(m, 2H), 7.26 (d, 7.91 Hz, 2H), 7.47 (m, 5H), 10.73 (s, 1 H); 13
C
NMR (DMSO-d6) δ 12.26 41.89, 46.17, 50.81, 108.39, 116.67,
117.02, 119.56, 119.79, 125.87, 126.85, 131.80, 133.83, 136.70,
140.93, 150.60; m/z 351 (MH+), HRMS (MH+) calcd for
C20H23N4O2, 351.1821, found 351.1850; mp 147-152 °C de-
comp.; Anal. (C20H22N4O2‚0.2Et2O‚0.4H2O), C, H, N.
T0 ) IGC well OD490 - background, GC ) VC well OD490
-
background, X ) compound-treated well OD490 - background.
Effica cy Exp er im en ts. The studies were performed using
outbred athymic (nu/nu) female mice (“Hsd:Athymic Nude-nu”
from Harlan Sprague Dawley, Indianapolis, IN). Mice were
anesthetized with Metofane (Mallinckrodt Veterinary, Inc.,
Mundelein, IL), and a cell suspension (100 µL), containing
either ≈106 HCT116 cells or ≈107 A549 cells, was injected
subcutaneously into the right axillary (lateral) region of each
animal. Tumors were allowed to grow until a volume of ≈100
mm3 was achieved. At this point, mice bearing tumors with
acceptable morphology and size were sorted into groups of
eight for the study. The sorting process produced groups
balanced with respect to mean and range of tumor size. Solid
compound, as the lactate salt, was dissolved in pure DMSO
to create a stock solution. This solution was diluted with D5W
just prior to dosing so the final DMSO concentration was 10%
and the compound remained in solution. Positive-control
animals in the HCT116 xenograft studies received 5-fluoro-
uracil formulated in 0.9% w/v saline and dosed iv at 100 mg/
kg 1×/week for three doses. Positive-control animals in the
A549 xenograft studies received mitomycin C formulated in
10% DMSO/D5W and dosed ip 3×/week for nine doses. Tumors
were measured, and individual animal body weights were
recorded once weekly. Antitumor activity is expressed as %
T/C, the ratio of the change in tumor volume of treated animals
to the change in tumor volume of vehicle control animals.
N-Hyd r oxy-3-[4-[1-[[2-(1H-in d ol-3-yl)eth yl]a m in o]eth -
yl]p h en yl]-(2E)-2-p r op en a m id e 23. Following Method A,
tryptamine (680 mg, 4.25 mmol), 22 (702 mg, 3.44 mmol), and
NaBH3CN were reacted in MeOH to afford 3-[4-[1-[[2-(1H-
indol-3-yl)ethyl]amino]ethyl]phenyl]-(2E)-2-propenoic acid meth-
yl ester: 1H NMR (CDCl3) δ 1.42 (d, 6.8 Hz, 3H), 2.92 (m, 4H),
3.81 (s, 3H), 3.91 (q, 6.4 Hz, 1H), 6.39 (d, 15.8 Hz, 1H), 7.07
(m, 2H), 7.19 (td, 7.5, 1.1 Hz, 1H), 7.27 (m, 2H), 7.37 (ddd,
8.1, 1.1, 0.9 Hz, 1H), 7.46 (m, 3H), 7.64 (d, 15.8 Hz, 1H), 8.06
(s, 1H); m/z 349 (MH+). Following Method C, the ester (460
mg, 1.61 mmol) was converted to the hydroxamate and purified
by RPHPLC which, after neutralization, afforded 23 (223 mg,
40%): 1H NMR (DMSO-d6) δ 1.26 (d, 6.4 Hz, 3H), 2.71 (m,
4H), 3.84 (m, 6.8 Hz, 1H), 6.43 (d, 15.8 Hz, 1H), 6.92 (t, 7.4
Hz, 1H), 7.05 (m, 2H), 7.40 (m, 7H), 10.76 (s, 1H); 13C NMR
(DMSO-d6) δ 23.77, 25.18, 47.76, 57.05, 111.24, 112.18, 118.01,
118.12, 118.35, 120.74, 122.43, 127.07, 127.11, 127.41, 133.32,
136.12, 138.08, 162.71; m/z 350 (MH+); Anal. (C21H23N3O2‚H2O)
C, H, N.
HDAC En zym e Assa y. HDAC was partially purified from
H1299 human non small cell lung carcinoma cells. Cells were
grown to 70-80% confluence in RPMI media in the presence
of 10% fetal calf serum, harvested, and lysed using sonication.
The lysate was centrifuged at 23 420 × g for 10-15 min and
the supernatant applied to a Hiload 26/10 High Performance
Q-sepharose column (Amersham Pharmacia Biotech), previ-
ously equilibrated with a Buffer A (20mM Tris pH8, 0.1 mM
EDTA, 10 mM NH4Cl2, 1 mM â-mercaptoethanol, 5% glycerol,
2 µg/mL aprotinin, 1 µg/mL leupeptin, and 400 mM phenyl
methyl sulfonyl fluoride (PMSF)) and eluted with a linear
gradient of 0-500 mM NaCl in Buffer A at a flow rate of 2.5
mL/min. Four milliliter fractions were collected, and each was
titrated for HDAC activity using a modification of the pub-
lished procedure35 to determine the optimal amount needed
to obtain a signal-to-noise ratio of at least 5:1. The substrate
used is a peptide of amino acid sequence SGRGKGGKGLGKG-
GAKRHRKVLRD, corresponding to the 24 N-terminal amino
acids of human histone H4, biotinylated at the N-terminus and
peracetylated with 3H-acetate at each lysine residue. The
substrate is diluted in 10 µL of Buffer B (100 mM Tris pH 8.0,
2 mM EDTA), added to the enzyme mixture, and incubated
at 37 °C for 1.5 h. The reaction is stopped by the addition of
20 µL of 0.5 N HCl/0.08 M HOAc and extracted with TBME,
and an aliquot of the organic layer is added to Opti-Phase
Supermix liquid scintillation cocktail (Wallac). The mixture
was read on a 1450 MicroBeta Trilux liquid scintillation and
luminescence counter (Wallac) with a color/chemical quench
and dpm correction and the data corrected for background
luminescence.
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