Development of tetrahydroisoquinoline-based hydroxamic acid derivatives: Potent histone deacetylase inhibitors with marked in vitro and in vivo antitumor activities

Article abstract of DOI:10.1021/jm101605z

Inhibition of histone deacetylase (HDAC) results in growth arrest, differentiation, and apoptosis in nearly all tumor cell lines, promoting HDACs as promising targets for antitumor therapy. In our previous study we developed a novel series of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid derivatives as HDAC inhibitors (HDACi), among which compound 7d exhibited promising HDAC8 inhibitory and antiproliferative activities. Herein, we report the design and development of a new class of tetrahydroisoquinoline-bearing hydroxamic acid analogues as potential HDACi and anticancer agents. In vitro biological evaluation of these compounds showed improved HDAC8 inhibition (compounds 31a and 31b exhibited mid-nM IC₅₀ values against HDAC8) and potent growth inhibition in multiple tumor cell lines. Most importantly, compounds 25e, 34a, and 34b exhibited excellent in vivo anticancer activities in a human breast carcinoma (MDA-MB-231) xenograft model compared with suberoylanilide hydroxamic acid (SAHA), an approved HDACi. Collectively, our results indicate that tetrahydroisoquinoline bearing a hydroxamic acid is an excellent template to develop novel HDACi as potential anticancer agents.

Full text of DOI:10.1021/jm101605z

ARTICLE
pubs.acs.org/jmc
Development of Tetrahydroisoquinoline-Based Hydroxamic Acid
Derivatives: Potent Histone Deacetylase Inhibitors with Marked
in Vitro and in Vivo Antitumor Activities
Yingjie Zhang,† Jinhong Feng,† Yuping Jia,† Xuejian Wang,† Lei Zhang,† Chunxi Liu,§ Hao Fang,*,† and
Wenfang Xu*,†
Departments of †Medicinal Chemistry and §Pharmaceutics, School of Pharmacy, Shandong University, Ji'nan, Shandong,
250012, People's Republic of China
S Supporting Information
b
ABSTRACT: Inhibition of histone deacetylase (HDAC) re-
sults in growth arrest, differentiation, and apoptosis in nearly all
tumor cell lines, promoting HDACs as promising targets for
antitumor therapy. In our previous study we developed a novel
series of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid deri-
vatives as HDAC inhibitors (HDACi), among which com-
pound 7d exhibited promising HDAC8 inhibitory and
antiproliferative activities. Herein, we report the design and
development of a new class of tetrahydroisoquinoline-bearing
hydroxamic acid analogues as potential HDACi and anticancer
agents. In vitro biological evaluation of these compounds showed improved HDAC8 inhibition (compounds 31a and 31b exhibited
mid-nM IC50 values against HDAC8) and potent growth inhibition in multiple tumor cell lines. Most importantly, compounds 25e,
34a, and 34b exhibited excellent in vivo anticancer activities in a human breast carcinoma (MDA-MB-231) xenograft model
compared with suberoylanilide hydroxamic acid (SAHA), an approved HDACi. Collectively, our results indicate that tetrahy-
droisoquinoline bearing a hydroxamic acid is an excellent template to develop novel HDACi as potential anticancer agents.
’ INTRODUCTION
the common pharmacophore of these HDACi consists of three
domains: a zinc-binding group (ZBG), such as hydroxamic acid;
a cap group, generally a hydrophobic and aromatic group; a
saturated or unsaturated linker domain, composed of linear or
cyclic structures that connect the ZBG and the cap group.
Treatment of various transformed cells with HDACi results in
significant growth arrest, differentiation, and apoptosis.17ꢀ19 Up
to now, two HDACi, suberoylanilide hydroxamic acid (1, SAHA,
Vorinostat)20,21 and the depsipeptide FK228 (2, Romidepsin),22
have been approved by the US Food and Drug Administration
(FDA) for the treatment of cutaneous T-cell lymphoma
(CTCL), validating HDACs’ potential as an important target
for anticancer therapy. Other HDACi in clinical trials include
hydroxamic acids, such as panobinostat (3, LBH-589), belinostat
(4, PXD101), givinostat (5, ITF-2357), SB-939 (6), R306465
(7), and CRA024781 (8), benzamides, such as entinostat (9,
MS-275), mocetinostat (10, MGCD-0103), and tacedinaline
(11, CI-994), and aliphatic acids, such as valproic acid (12),
sodium phenylbutyrate (13), and pivanex (14, AN-9).23
Histone deacetylases (HDACsR) are enzymes that catalyze
the deacetylation of lysine residues located at the N-terminus of
various protein substrates, such as nucleosomal histiones. The
hydrolysis of the acetyl group from histones results in condensed
chromosomal DNA and transcriptional repression.1ꢀ3 There are
18 human HDACs grouped into 4 classes4,5 based on their
homology to yeast models: classes I (HDACs 1ꢀ3 and 8), II
(HDACs 4ꢀ7, 9, and 10), and IV (HDAC 11) are zinc-
dependent metallohydrolases, namely “classical HDACs”,4
whereas class III HDACs (sirtuins 1ꢀ7) are NADþ dependent.6
All the zinc-dependent HDACs bear a highly conserved
catalytic site.
Over the past few years, multiple nonhistone proteins involved
in cell growth and survival pathways have been identified as
substrates of HDACs, including transcription factors, cytoskele-
tal proteins, molecular chaperones and nuclear import factors.7
In this context, dysregulation of HDACs has been linked to the
pathogenesis of many diseases, of which cancer is the most
intractable. It has been revealed that overexpression of classes I
and II HDACs, especially class I isozymes, is associated with
various human cancers.8ꢀ15 Therefore, there has been a high
level of interest in developing HDAC inhibitors (HDACi), and
numerous structurally diverse HDACi have been developed as
potential anticancer agents (Figure 1).16 As shown in Figure 1,
Recently, we have embarked on the development of tetra-
hydroisoquinoline-based hydroxamic acid as a novel HDACi
scaffold.24 Biological evaluation showed that HDACi with rigid
Received: December 17, 2010
Published: April 05, 2011
r
2011 American Chemical Society
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Figure 1. Pharmacophore model and structures of representative HDACi.
tetrahydroisoquinoline scaffold as the linker domain have com-
parable activities as SAHA (e.g., compound 7d, Figure 1). In the
present study, we report the design, synthesis, and biological
characterization of a new series of tetrahydroisoquinoline deri-
vatives based on 7d to conduct extensive structureꢀactivity
relationship (SAR) studies and to optimize their anticancer
activities. The tert-butoxycarbonyl-protected isoleucine deriva-
tive 25e and its analogues 34a, 34b showed similar, or in some
cases even more potent, in vitro and in vivo antitumor activities
when compared with SAHA.
’ RESULTS AND DISCUSSION
Chemistry. We designed a series of tetrahydroisoquinoline
derivatives with the general structure shown in Figure 2, based on
the structure of lead compound 7d. Compounds 22a, 22b, and
22c were synthesized using the procedures described in
Figure 2. Design idea and a general structure of the target compounds.
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Scheme 1. Synthesis of Compounds 22aꢀca
a Reagents and conditions: (a) (CH2O)n, 37% HC1, CH3OCH2CH2OCH3, 72ꢀ75 °C, 18 h, 58%; (b) (Boc)2O, 1 N NaOH, THF, 95%; (c) 10%
PdꢀC, H2, Et3N, MeOH, 88%; (d) R,NH2, DCC, HOBT, anhydrous THF, 84ꢀ91%; (e) BrCH2COOCH3, K2CO3, anhydrous DMF, 61ꢀ67%;
(f) TFA, Et3N, DCM, 94ꢀ96%; (g) 3-phenylpropionic acid, TBTU, Et3N, THF, 60ꢀ67%; (h) NH2OK, CH3OH, 56ꢀ59%.
Scheme 1.24 The tetrahydroisoquinoline ring of 16 was con-
structed from the starting material 3,5-diiodo-L-tyrosine (15) by
reaction with formaldehyde. Boc protection and hydrogenolytic
removal of the two iodo groups led to 18. Amidation of 18
followed by phenol hydroxyl group alkylation with bromoacetate
gave 20. Subsequent N-deprotection of 20aꢀc with trifluoroa-
cetic acid and condensation with 3-phenylpropionic acid af-
forded compounds 21aꢀc. The ester groups of 21aꢀc were
treated with NH2OK in methanol to get target compounds
22aꢀc.
22b, and 22c (Scheme 1). The HDAC8 inhibition assay of these
three compounds revealed that 22a with a 4-methoxyphenyl as
the R1 group was the most potent (Table 1). Besides, as a result
of the 3-phenylpropanoyl being the R2 group, all three com-
pounds possessed a lower IC50 than that of 7d. These results
encouraged us to keep the 4-methoxyphenyl group fixed and
replace the Boc group of 7d with other functional groups.
Compounds synthesized in Scheme 2 were specifically de-
signed to probe the effect of the N-substituent. HDAC8 inhibi-
tion assay showed that most of these derivatives were more
potent than the lead compound 7d, except 26a, 26e, 26g, 26h,
25l, 25m, and 25n (Table 2). It was remarkable that compounds
with a Boc group (compounds 25aꢀh) were more potent than
their corresponding deprotected analogues (compounds 26aꢀh)
without exception. For example, the IC50 of compound 25a was
0.514 μM, much lower than that of compound 26a (2.14 μM).
This indicates that the hydrophobicity of the HDACi cap group
is very beneficial to the HDAC inhibitory activity. Compared
with their corresponding tertiary amine analogues 25l, 25m, and
25n, compounds 22a, 25i, and 25j were almost 1 order of
magnitude more potent, which showed that reduction of the
amide group to a tertiary amine group was somewhat detrimental
to activity.
To further investigate the antiproliferative activities of these
derivatives, several potent compounds were selected to test their
effects on the viability of A549 (lung cancer) and MDA-MB-231
(breast cancer) cell lines with SAHA as the positive control. The
results presented in Table 3 showed that the antiproliferative
activities of these derivatives were similar to that of SAHA. It is
remarkable that compound 25e has an IC50 that is 40% lower
than that of SAHA against the MDA-MB-231 cell line. Encour-
aged by its promising in vitro activity, compound 25e was
progressed to an in vivo experiment. Considering that the
MDA-MB-231 cell line was to some extent more sensitive to
our compounds, especially to 25e, we established a subcutaneous
MDA-MB-231 xenograft model. Because compound 25e exhib-
ited similar cytotoxicity against normal human fibroblast cells
relative to SAHA (data not reported), we compared antitumor
N-Deprotection of 20a with trifluoroacetic acid afforded a key
intermediate 23 (Scheme 2). Subsequent derivatization on the
secondary amine of 23 under conditions b, c, or d gave
compounds 24aꢀn, which were treated with NH2OK in metha-
nol to give compounds 25aꢀn. Finally, compounds 26aꢀh were
achieved by removing the Boc groups of 25aꢀh in HCl-saturated
ethyl acetate.
Three kinds of dipeptide or dipeptidomimetic 28aꢀc were
prepared according to the classical peptide synthesis method
involving esterification, condensation, and saponification
(Scheme 3). Then 28aꢀc were installed onto the intermediate
23 to afford compounds 29aꢀc, which were finally transformed
into target hydroxamate compounds 30aꢀc and 31aꢀc.
The key steps from amino acid 27 to aldehydes 32a and 32b
followed reported procedures.25 Reductive amination of 23 with
32a, or 32b using sodium triacetoxyborohydride, followed by
ammonolysis of the ester group led to our target compounds 34a
and 34b, respectively (Scheme 4).
Biological Results. On the basis that all zinc ion-dependent
HDACs were highly conserved in their active sites and HDAC8 is
more available in our group, first, we still used HDAC8 as the
enzyme source to screen our target compounds efficiently.
Previously obtained SAR and molecular docking information
of compound 7d and its homologues indicated that the 4-meth-
oxyphenyl group was optimal for the R1 group, and the R2 group
(for example, tert-butoxycarbonyl group in 7d) was also a
significant contributor to HDAC inhibitory activity.24 In order
to further verify this conclusion, we evaluated compounds 22a,
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Scheme 2. Synthesis of Compounds 25aꢀn 26aꢀha
a Reagents and conditions: (a) TFA, Et3N, DCM, 94%; (b) carboxylic acids or Boc-protected amino acids, TBTU, Et3N, THF, 60ꢀ67%; (c) microwave,
R2Br, K2CO3, DMF, 75ꢀ81%; (d) benzyl bromide, K2CO3, DMF, 86%; (e) NH2OK, CH3OH, 52ꢀ58%; (f) HCl, anhydrous EtOAc, 88ꢀ93%.
effects of 25e and SAHA at the same dosage, 90 mg/kg, which is
close to the toxic dosage in mice of SAHA (100 mg/kg).26 In
detail, once tumors became about 100 mm3, mice were dosed
intraperitoneally with either 25e (90 mg/kg once a day) or
SAHA (90 mg/kg once a day) for 21 days. During the treatment,
no significant body weight difference among these groups of mice
and no signs of evident toxicity were detected. The relative
increment ratio (T/C) of 25e was 63%; meanwhile SAHA was
64%. Both the SAHA-treated group and the 25e-treated group
were significantly different from the control group (p < 0.05) by
Student’s two-tailed t test. As presented in Figure 3, compound
25e was demonstrated to have in vivo antitumor efficacy
comparable to that of SAHA.
Further optimization and modification focused on compound
25e were conducted. First, to check the role of the Boc group in
25e, three kinds of dipeptide or dipeptidomimetic residues
(28aꢀc) were used to substitute the Boc-isoleucine residue of
25e. These compounds were also tested against HDAC8 and
showed similar (30a, 30b, and 30c) or much higher (31a, 31b)
potency relative to 25e (Table 4). However, further cellular
activity evaluation revealed that their antiproliferative activities
were not as good as 25e (Table 5), probably because of less cell
membrane permeability compared to 25e. These results indi-
cated that the geometric confirmation and lipophilicity of the Boc
group were beneficial; only minor modification was tolerated in
the optimization of 25e.
Then, compounds 34a and 34b were designed and synthe-
sized (Scheme 4). Compared with 25e, 34a could have better
water solubility in the form of a salt. Considering that the tertiary
butyloxycarbonyl group (Boc group) of 34a was sensitive to
acidic conditions and thus might be destroyed by gastric acid, and
that the carbamate of 34a might be metabolized,27,28 we devel-
oped compound 34b. The enzyme inhibition results in Table 4
are in line with aforementioned SAR in which reduction of the
amide group to the tertiary amine group resulted in decreased
activity. General in vitro antiproliferative abilities of 34a and 34b
were also inferior to that of 25e but were superior to those of
30aꢀc, 31aꢀc (Table 5). Finally, another set of experiments in a
MDA-MB-231 xenograft model was conducted to compare the
in vivo antitumor efficacy of 25e, 34a, and 34b, with SAHA as the
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Scheme 3. Synthesis of Compounds 30aꢀc and 31aꢀca
a Reagents and conditions: (a) HCl, CH3OH, 96%; (b) Boc-protected amino acids, isobutyl chloroformate, NMM, THF, 74ꢀ84%; (c) 2 M NaOH,
C2H5OH, 86-91%; (d) TBTU, Et3N, THF, 61ꢀ67%; (e) NH2OK, CH3OH, 52ꢀ57%; (f) HCl, anhydrous EtOAc, 88ꢀ93%.
Scheme 4. Synthesis of Target Compounds 34a and 34ba
a Reagents and conditions: (a) (Boc)2O, 1 M NaOH, THF, 89%; (b) N,O-dimethylhydroxylamine hydrochloride, TBTU, Et3N, THF, 85%; (c) LiAlH4,
THF, 81%; (d) HCl, CH3OH, 96%; (e) 3,3-dimethylbutyric acid, isobutyl chloroformate, NMM, THF, 87%; (f) 2 M NaOH, C2H5OH, 80%;
(g) (CH3COO)3BHNa, 1,2-dichloroethane, 81ꢀ85%; (h) NH2OK, CH3OH, 55ꢀ58%.
positive control. Figure 4 shows the results in which significant
tumor growth delays were observed relative to the untreated
group for all of our compounds. With the exception of 34b, all
compound-treated groups were significantly different from the
control group as shown by Student’s two-tailed t test. Among
these analogues, 34a was the most effective, with a T/C value of
47% (p < 0.01). Meanwhile 25e (T/C = 67%, p < 0.05) exhibited
potency similar to that of SAHA (T/C = 70%, p < 0.05), which
agreed well with our preceding in vivo results. The poor in vivo
efficacy of 34b (T/C = 87%, p > 0.05) was unexpected. We
postulated that 34b dosed as the hydrochloride might have
decreased intraperitoneal absorption. However, detailed research
is warranted to confirm our interpretation.
The in vitro antiproliferative activity against several solid
tumor cell lines (A549, HeLa, ES-2, and MDA-MB-231) and
in vivo anticancer activity in a MDA-MB-231 xenograft model
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Table 1. HDAC Inhibition Activity of Compounds 7d,
22aꢀc, and SAHA
the tyrosine 100 residue in the rim of the HDAC8 active site
(Figure 5). For the R2 group, modification and optimization
could dramatically influence the hydrophobicity and steric
conformation and thus the inhibitory potency for HDAC8.
Generally, the more hydrophobic the R2 group, the more
potent the compound. This is supported by the fact that the
hydrochloride salts (compounds 26aꢀh, 31c) were all less
potent than their corresponding Boc-protected analogues
(compounds 25aꢀh, 30c), because removal of the Boc group
greatly decreased the hydropobicity. Besides, the result that
tertiary amine analogues (25l, 25m, and 25n) were not as
potent as their parent compounds (22a, 25i, and 25j) against
HDAC8 also supports the importance of hydrophobicity to
some extent. However, the hydrochloride salts 31a and 31b,
which were the most potent compounds against HDAC8 in
this series, seemed to be inconsistent with the aforementioned
analysis. To rationalize these exceptions, possible binding
modes of 31a, 31b, and 31c were compared with their Boc-
protected analogues 30a, 30b, and 30c by docking studies
(Figure 6). Compared with 30a (Figure 6a), compound 31a
(Figure 6d) could form two more hydrogen bonds with
carbonyl oxygen atom of proline 273 through amide NꢀH
and deprotected free NꢀH atoms. Further, relative to 30b
(Figure 6b), compound 31b (Figure 6e) could form three
additional strong hydrogen bonds with carbonyl oxygen
atoms of proline 273 and methionine 274 through amide
NꢀH and deprotected free NꢀH atoms. We postulated that
the additional hydrogen bonds might be the key factors that
made 31a and 31b more effective against HDAC8 compared
to their corresponding parent compounds 30a and 30b.
However, 31c (Figure 6f) could not form more hydrogen
bonds relative to 30c (Figure 6c). Moreover, different from
30a, 30b, 30c, 31a, and 31b, compound 31c could not form a
πꢀπ interaction with HDAC8 because the 4-methoxyphenyl
group of 31c was perpendicular to the tyrosine 100 residue.
Therefore, the IC50 value of 31c (1.44 ( 0.39 μM) was 10-fold
larger than that of 30c (0.147 ( 0.03 μM) and almost 2 orders
of magnitude larger than that of 31a (0.047 ( 0.01 μM) and
31b (0.068 ( 0.02 μM). Although the antiproliferative
activities of 31a and 31b were not as good as their enzyme
inhibitory activities, probably because of poor cellular uptake,
these two compounds could be used to probe the biological
function of HDACs.
Figure 7 reveals that the low energy conformations of
compounds 25e, 34a, and 34b, which were optimized by the
Sybyl/Sketch module, were almost the same because of their
structural similarity, but as shown in Figure 8, the binding
mode of compound 34b was quite different from its analogues
25e and 34a. In detail, the simulative pharmacophoric con-
formations of 25e and 34a were both similar to their calculated
low energy conformations, especially 34a, which was almost
accordant to its low energy conformation. However, the
simulative pharmacophoric conformation of 34b was quite
different from its calculated low energy conformation. This
might be the reason why compound 34a exhibited the most
potent in vivo anticancer activity but compound 34b was
essentially ineffective in the xenograft model. The real binding
modes of our compounds with HDAC8 should be confirmed
by further X-ray cocrystal studies. Moreover, the in vivo antic-
ancer activities of these analogues must be closely related to
their pharmacokinetic profiles. At present, related research is in
progress in our lab.
a Cited from ref 24. b Results expressed as the mean ( standard deviation
of at least three separate determinations.
indicate that this series of derivatives are not HDAC8-selective
inhibitors, because HDAC8 selective inhibitors, such as PCI-
34051, scarcely affect the growth and viability of solid tumor
cells.29 In order to explore the HDAC isoform selectivity profile
of the tetrahydroisoquinoline-based hydroxamic acid derivatives,
selected compounds 25e, 30aꢀc, 31aꢀc, 34a, and 34b were
progressed to enzyme inhibition assays against HeLa cell nuclear
extract (which contains primarily HDAC1 and HDAC2) and
HDAC6 (representative class II HDACs). The results presented
in Table 6 indicate that our tetrahydroisoquinoline-based hydro-
xamic acid scaffold exhibited little selectivity against individual
HDACs.
Preliminary investigation of the antiproliferative mechanism
was performed by treating MDA-MB-231 cells with 25e and 34a
at different concentrations for 48 h, using SAHA as the positive
control. Cell distribution by flow cytometric analysis revealed
that compounds 25e and 34a mainly induced G1 phase arrest at
low concentrations (0.5 μM); meanwhile, at increased concen-
trations (2 μM and 5 μM), more cells were accumulated at G2
phase (data not shown). Our results indicate that the action
model of 25e and 34a on MDA-MB-231 cell cycle arrest is similar
to that of SAHA.
Molecule Docking and Discussion. The SAR information
summarized from the present research was consistent with our
previously reported results. For the R1 group in the general
structure of these analogues (Figure 2), the 4-methoxyphenyl
group was optimum, which could form a πꢀπ interaction with
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Table 2. HDAC Inhibition Activity of Compounds 25aꢀ25n, 26aꢀ26h
a Results expressed as the mean ( standard deviation of at least three separate determinations.
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Table 3. Antiproliferative Activities of Representative Com-
pounds against A549 and MDA-MB-231 Cell Lines
Table 5. Antiproliferative Activities of Representative Com-
pounds against Several Tumor Cell Lines
IC50 (μM)a
IC50 (μM)a
compd
A549
MDA-MB-231
compd HeLa
K562
NB4
HL60
47.6
ES-2
5.0
MDA-MB-231
22a
25b
25e
25h
25i
4.80
12.7
0.94
0.84
117
1.52
1.29
0.79
1.58
4.45
3.05
2.77
1.31
25e
30a
30b
30c
31a
31b
31c
34a
34b
SAHA
37.1
184
1.47
2.32
2.78
12.3
6.59
20.4
51.7
7.0
1.50
5.15
63.3
68.2
9.28
69.9
>200
3.87
5.57
1.52 >200
0.79
NDb
ND
76.8 ND
167
>200
>200
118
ND
ND
ND
ND
ND
69.8 ND
ND
25j
5.95
3.58
0.74
ND
ND
42.7
25.7 ND
6.21
ND
ND
25k
SAHA
102
ND
ND
29.2
7.20
2.20
2.54
1.31
a Values are the mean of three experiments. The standard derivations are
<20% of the mean.
63.5 10.0
147 3.32
a Values are the mean of three experiments. The standard derivations are
<20% of the mean. b Not determined.
Figure 3. Antitumor activity of 25e and SAHA against MDA-MB-231
human tumor xenografts implanted in mice, expressed as mean tumor
volume.
Figure 4. Antitumor activity comparison of 25e, 34a, 34b, and SAHA
against MDA-MB-231 human tumor xenografts implanted in mice,
expressed as mean tumor volume.
Table 4. HDAC Inhibition Activity of 25e and Its Derivatives
drug SAHA. More detailed studies of the antitumor profiles of
these promising compounds are underway in our lab. Using 25e,
34a, and 34b as leads, continued efforts are underway to search
more potent HDACi.
30aꢀc, 31aꢀc, 34a, and 34b
compd
IC50 of HDAC8 (μM)a
compd
IC50 of HDAC8 (μM)a
30a
30b
30c
34a
25e
0.192 ( 0.04
0.201 ( 0.06
0.147 ( 0.03
0.263 ( 0.04
0.139 ( 0.011
31a
31b
31c
34b
0.047 ( 0.01
0.068 ( 0.02
1.44 ( 0.39
0.333 ( 0.04
’ EXPERIMENT SECTION
Materials and Methods. All commercially available starting
materials, reagents, and solvents were used without further purification
unless otherwise stated. All reactions were monitored by TLC with 0.25
mm silica gel plates (60GF-254). UV light, iodine stain, and ferric
chloride were used to visualize the spots. Silica gel or C18 silica gel was
used for column chromatography purification. Microwave-aided synth-
esis was performed using a CEM Discover-S microwave synthesis
system. 1H NMR spectra were recorded on a Bruker DRX spectrometer
at 600 MHz, δ in parts per million and J in hertz, using TMS as an
internal standard. High-resolution mass spectra were conducted by
Shandong Analysis and Test Center in Ji’nan, China. ESI-MS spectra
were recorded on an API 4000 spectrometer. Melting points were
determined uncorrected on an electrothermal melting point apparatus.
All tested compounds are >95% pure by HPLC analysis, performed on a
Agilent 1100 HPLC instrument using a Phenomenex Synergi 4 μ
Hydro-RP 80A column (250 mm ꢁ4.6 mm) according to one of the
a Results expressed as the mean ( standard deviation of at least three
separate determinations.
’ CONCLUSION
A series of tetrahydroisoquinoline-based hydroxamic acids
have been described as HDAC inhibitors in our previous work.
In our present research, further derivatization that focused on the
most potent compound 7d led to another novel series of
compounds exhibiting low micromolar to mid-nanomolar IC50
values in the in vitro HDAC inhibition assay and antiproliferative
assay. Several compounds, 25e, 34a, and 34b, were evaluated for
their in vivo antitumor study in a MDA-MB-231 xenograft mice
model and showed efficacy comparable to the that of approved
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Table 6. HDAC Inhibition Activity and Isoform Selectivity of 25e, 30aꢀc, 31aꢀ31c, 34a, and 34b
IC50 of HeLa nuclear
IC50 of HDAC6
IC50 of HDAC8
HDAC8/HeLa nuclear
HDAC8/HDAC6
isoform selectivity
compd
extract (μM)a
(μM)a
(μM)
extract isoform selectivity
25e
30a
30b
30c
31a
31b
31c
34a
34b
SAHA
0.058
0.065
0.128
0.214
0.108
0.073
0.071
0.447
0.297
0.107
0.047
0.141
0.116
0.164
0.191
0.163
0.169
0.094
0.158
0.195
0.139
0.192
0.201
0.147
0.047
0.068
1.44
2.40
2.95
1.57
0.69
0.44
0.93
2.03
0.59
1.12
13.8
2.96
1.36
1.73
0.90
0.25
0.42
0.85
2.80
2.11
7.60
0.263
0.333
1.48
a Values are the mean of at least two separate determinations.
Figure 5. Superposition of several tetrahydroisoquinoline-derived HDACi with potent HDAC8 inhibitory activity (compounds 22a, 25b, 25e, 25h, 25i,
25j, 25k, 30a, 30b, 30c, 31a, 31b, 31c, 34a, 34b) revealed their proposed binding modes at the HDAC8 active site. The zinc ion is shown as a red sphere.
following methods. Method A: compounds 22aꢀc, 25aꢀn, 30aꢀc, 34a,
and 34b were eluted with 50% acetonitrile/50% water (containing 0.1%
formic acid) over 20 min, with detection at 254 nm and a flow rate of
1.8 mL/min. Method B: compounds 26aꢀh and 31aꢀc were eluted
with 25% acetonitrile/75% water (containing 0.1% formic acid) over
20 min, with detection at 254 nm and a flow rate of 1.5 mL/min.
(S)-7-Hydroxy-6,8-diiodo-Tic hydrochloride (16), (S)-2-(tert-Butoxy-
carbonyl)-7- hydroxy-6,8-diiodo-Tic (17), (S)-2-(tert-Butoxycarbonyl)-7-
hydroxy-Tic (18), (S)-tert-Butyl 7-Hydroxy-3-((4-methoxyphenyl)carba-
moyl)-3,4-dihydroisoquinoline -2(1H)-carboxylate (19a) and its analo-
gues (19b, 19c), and (S)-tert-Butyl 7-(2-Methoxy-2-oxoethoxy)-3-((4-
methoxyphenyl)carbamoyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate
(20a) and its analogues (20b, 20c) were synthesized as described
previously.24 Dipeptides 28aꢀc were synthesized according to the
procedures described previously.31 Aldehydes 32a and 32b were obtained
from their corresponding carboxylic acid compounds according to a
similar literature method.25
General Procedure for the Preparation of 21a and Its
Analogues 21b, 21c, 24aꢀk, 29aꢀc. (S)-Methyl 2-(3-(4-
Methoxyphenylcarbamoyl)-2-(3-phenylpropanoyl)-1,2,3,4-
tetrahydroisoquinolin-7-yloxy)acetate (21a). To a solution of
20a (4.71 g, 10.0 mmol) in anhydrous dichloromethane (40 mL) was
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Figure 6. Proposed binding preferences comparison of compounds 30a (a), 30b (b), 30c (c), 31a (d), 31b (e), and 31c (f) at HDAC8. Protein is
represented by the molecular surface, and the amino acid residues are represented in stick mode. The dashed lines stand for the hydrogen bonds. The
pictures were produced using UCSF chimera software30 (atom types: H, white; N, blue; O, red; S, yellow).
Figure 7. Calculated low energy conformations of 25e (a), 34a (b), and 34b (c).
added trifluoroacetic acid (15 mL). When the reaction was finished,
saturated Na2CO3 was added to the solution until the pH became
weakly basic. The mixture was separated with a separatory funnel, and
the organic layer was washed with distilled water, dried over MgSO4, and
evaporated under vacuum to obtain 3.45 g of secondary amine 23
as a white solid. This product was used for the subsequent reaction
without further purification. 1H NMR (DMSO-d6) δ 2.72 (s, 1H), 2.74
(dd, J = 10.2 Hz, J = 15.6 Hz, 1H), 2.91 (dd, J = 4.8 Hz, J = 15.6 Hz, 1H),
3.54 (dd, J = 4.8 Hz, J = 10.2 Hz, 1H), 3.69 (s, 3H), 3.72 (s, 3H),
3.87ꢀ3.95 (m, 2H), 4.74 (s, 2H), 6.64 (d, J = 2.4 Hz, 1H), 6.72 (dd,
J =2.4 Hz, J = 8.4 Hz, 1H), 6.88 (d, J= 9.0 Hz, 2H), 7.05 (d, J= 8.4 Hz, 1H),
7.58 (d, J = 9.0 Hz, 2H), 9.75 (s, 1H). ESI-MS m/z: 371.3 [M þ H]þ.
At room temperature, to a solution of 3-phenylpropionic acid (0.75 g,
5.0 mmol) in anhydrous THF (20 mL) was added Et3N (0.56 g,
5.5 mmol) followed by 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluro-
nium tetrafluoroborate (TBTU, 1.78 g, 5.5 mmol). After 15 min, the amine
compound 23 (1.85 g, 5.0 mmol) was added. Stirring was continued for 8 h,
and then THF was evaporated with the residue being taken up in EtOAc
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Figure 8. Proposed binding mode comparison of compounds 25e (a), 34a (b), and 34b (c) at HDAC8. Protein is represented by the molecular surface,
and the zinc ion is shown in red (atom types: polar H, sky-blue; N, dark blue; O, red).
(40 mL). The EtOAc solution was washed with saturated Na2CO3 (3 ꢁ
10 mL) and brine (3 ꢁ 10 mL), dried over MgSO4, and evaporated
under vacuum. The crude product was purified by flash column
chromatography (petroleum ether/EtOAc 2:1) to give desired com-
pound 21a (1.53 g, 61% yield) as a colorless oil, which crystallized upon
standing. 1H NMR (DMSO-d6) δ 2.78ꢀ2.87 (m, 4H), 3.02ꢀ3.16 (m,
2H), 3.68 (s, 3H), 3.70 (s, 3H), 4.66 (d, J = 15.6 Hz, 1H), 4.72 (d,
J = 15.6 Hz, 1H), 4.74 (s, 2H), 5.01ꢀ5.04 (m, 1H), 6.77ꢀ6.88 (m, 2H),
7.09ꢀ7.12 (m, 1H), 7.15ꢀ7.29 (m, 7H), 7.35ꢀ7.39 (m, 2H), 9.74 (s,
1H). ESI-MS m/z: 503.2 [M þ H]þ.
Methyl 2-((S)-2-((S)-2-(tert-Butoxycarbonylamino)-4-methyl-
pentanoyl)-3-(4-methoxyphenylcarbamoyl)-1,2,3,4-tetra-
hydroisoquinolin-7-yloxy)acetate (24f). Colorless oil crystal-
lized upon standing, 63% yield.
Methyl 2-((S)-2-((S)-2-(tert-Butoxycarbonylamino)pro-
panoyl)-3-(4-methoxyphenylcarbamoyl)-1,2,3,4-tetrahydro-
isoquinolin-7-yloxy)acetate (24g). Colorless oil crystallized upon
standing, 67% yield.
Methyl 2-((S)-2-((S)-2-(tert-Butoxycarbonylamino)-3-methyl-
butanoyl)-3-(4-methoxyphenylcarbamoyl)-1,2,3,4-tetrahy-
droisoquinolin-7-yloxy)acetate (24h). Colorless oil crystallized
upon standing, 66% yield.
(S)-Methyl 2-(3-(4-Methoxyphenylcarbamoyl)-2-(2-phenyl-
acetyl)-1,2,3,4-tetrahydroisoquinolin-7-yloxy)acetate (24i).
Colorless oil crystallized upon standing, 61% yield.
(S)-Methyl 2-(3-(Phenethylcarbamoyl)-2-(3-phenylpro-
panoyl)-1,2,3,4-tetrahydroisoquinolin-7-yloxy)acetate (21b).
Colorless oil crystallized upon standing, 67% yield. 1H NMR (DMSO-d6) δ
2.48ꢀ2.85 (m, 8H), 2.95ꢀ3.08 (m, 2H), 3.68 (s, 3H), 4.44ꢀ4.65 (m, 2H),
4.72 (s, 2H), 4.98ꢀ5.00 (m, 1H), 6.75ꢀ6.80 (m, 3H), 7.04ꢀ7.08 (m, 2H),
7.16ꢀ7.31 (m, 8H), 7.98 (s, 1H). ESI-MS m/z: 501.3 [M þ H]þ.
(S)-Methyl 2-(3-(Phenylcarbamoyl)-2-(3-phenylpropanoyl)-
1,2,3,4-tetrahydroisoquinolin-7-yloxy)acetate (21c). Colorless
oil crystallized upon standing, 60% yield. 1H NMR (DMSO-d6) δ 2.81ꢀ
2.88 (m, 4H), 3.07ꢀ3.15 (m, 2H), 3.67 (s, 3H), 4.63 (d, J = 15.6 Hz,
1H), 4.69 (d, J = 15.6 Hz, 1H), 4.71 (s, 2H), 5.02ꢀ5.04 (m, 1H), 6.77ꢀ
6.86 (m, 2H), 7.01ꢀ7.03 (m, 1H), 7.11ꢀ7.31 (m, 8H), 7.45ꢀ7.52
(m, 2H), 9.99 (s, 1H). ESI-MS m/z: 473.2 [M þ H]þ.
(S)-Methyl 2-(2-Benzoyl-3-(4-methoxyphenylcarbamoyl)-
1,2,3,4-tetrahydroisoquinolin-7-yloxy)acetate (24j). Colorless
oil crystallized upon standing, 67% yield.
(S)-Methyl 2-(3-(4-Methoxyphenylcarbamoyl)-2-(4-phenyl-
butanoyl)-1,2,3,4-tetrahydroisoquinolin-7-yloxy)acetate (24k).
Colorless oil crystallized upon standing, 66% yield.
(S)-tert-Butyl 2-((2S,3S)-1-((S)-7-(2-Methoxy-2-oxoethoxy)-
3-(4-methoxyphenylcarbamoyl)-3,4-dihydroisoquinolin-
2(1H)-yl)-3-methyl-1-oxopentan-2-ylcarbamoyl)pyrrolidine-
1-carboxylate (29a). Colorless oil crystallized upon standing, 64%
(S)-Methyl 2-(2-(2-(tert-Butoxycarbonylamino)acetyl)-
3-(4-methoxyphenylcarbamoyl)-1,2,3,4-tetrahydroisoqui-
nolin-7-yloxy)acetate (24a). Colorless oil crystallized upon standing,
67% yield. 1H NMR (DMSO-d6) δ 1.30 þ 1.36 (s, 9H, cis/trans), 3.07ꢀ
3.19 (m, 2H), 3.68 (s, 3H), 3.70 (s, 3H), 3.93ꢀ4.04 (m, 2H), 4.62 (d, J =
15.6 Hz, 1H), 4.71 (d, J = 15.6 Hz, 1H), 4.72 (s, 2H), 4.93ꢀ4.95
(m, 1H), 6.77ꢀ6.86 (m, 5H), 7.12ꢀ7.14 (m, 1H), 7.36ꢀ7.40 (m, 2H),
9.88 (s, 1H). ESI-MS m/z: 528.2 [M þ H]þ.
1
yield. H NMR (DMSO-d6) δ 0.71ꢀ0.93 (m, 6H), 1.03ꢀ1.43 (m,
11H), 1.54ꢀ1.87 (m, 4H), 2.05ꢀ2.13 (m, 1H), 2.98ꢀ3.36 (m, 4H),
3.69 (s, 3H), 3.70 (s, 3H), 4.16ꢀ4.29 (m, 1H), 4.54ꢀ4.72 (m, 1H), 4.70
(s, 2H), 4.74ꢀ4.86 (m, 2H), 4.90ꢀ5.15 (m, 1H), 6.72ꢀ6.86 (m, 4H),
7.02ꢀ7.13 (m, 1H), 7.35ꢀ7.53 (m, 2H), 7.95ꢀ8.44 (m, 1H),
9.43ꢀ10.07 (m, 1H). ESI-MS m/z: 681.4 [M þ H]þ.
Methyl 2-((S)-2-((S)-2-(tert-Butoxycarbonylamino)-3-phenyl-
propanoyl)-3-(4-methoxyphenylcarbamoyl)-1,2,3,4-tetra-
hydroisoquinolin-7-yloxy)acetate (24b). Colorless oil crystal-
lized upon standing, 60% yield. 1HNMR(DMSO-d6) δ1.27 þ 1.20 (s, 9H,
cis/trans), 2.80ꢀ2.91 (m, 2H), 3.00ꢀ3.15 (m, 2H), 3.68 (s, 3H), 3.70
(s, 3H), 4.65 (d, J = 15.6 Hz, 1H), 4.76 (d, J = 15.6 Hz, 1H), 4.70 (s, 2H),
4.80ꢀ5.01 (m, 2H), 6.70ꢀ6.96 (m, 4H), 7.05ꢀ7.30 (m, 5H),
7.37ꢀ7.49 (m, 4H), 9.85 (s, 1H). ESI-MS m/z: 618.3 [M þ H]þ.
Methyl 2-((S)-2-((S)-2-(tert-Butoxycarbonylamino)-3-(4-hy-
droxyphenyl)propanoyl)-3-(4-methoxyphenylcarbamoyl)-1,2,
3,4-tetrahydroisoquinolin-7-yloxy)acetate (24c). Colorless oil
crystallized upon standing, 64% yield.
(S)-tert-Butyl 2-((S)-7-(2-Methoxy-2-oxoethoxy)-3-(4-meth-
oxyphenylcarbamoyl)-1,2,3,4-tetrahydroisoquinoline-2-
carbonyl)pyrrolidine-1-carboxylate (24d). Colorless oil crystal-
lized upon standing, 61% yield.
Methyl 2-((S)-2-((2S,3S)-2-(tert-Butoxycarbonylamino)-3-
methylpentanoyl)-3-(4-methoxyphenylcarbamoyl)-1,2,3,4-
tetrahydroisoquinolin-7-yloxy)acetate (24e). Colorless oil crys-
tallized upon standing, 59% yield.
Methyl 2-((S)-2-((2S,3S)-2-((S)-2-(tert-Butoxycarbonylamino)-
3-methylbutanamido)-3-methylpentanoyl)-3-(4-methoxy-
phenylcarbamoyl)-1,2,3,4-tetrahydroisoquinolin-7-yloxy)-
acetate (29b). Colorless oil crystallized upon standing, 61% yield. 1H
NMR (DMSO-d6) δ 0.71ꢀ0.96 (m, 12H), 1.10ꢀ1.19 (m, 1H), 1.20ꢀ
1.29 (m, 1H), 1.32 þ 1.37 (s, 9H, cis/trans), 1.68ꢀ1.78 (m, 1H), 1.81ꢀ
2.00 (m, 1H), 2.98ꢀ3.25 (m, 2H), 3.68 (s, 3H), 3.69 (s, 3H), 3.75ꢀ3.95
(m, 1H), 4.50ꢀ5.13 (m, 4H), 4.71 (s, 2H), 6.60ꢀ6.90 (m, 4H), 7.05ꢀ7.12
(m, 1H), 7.33ꢀ7.52 (m, 2H), 7.88 (s, 1H), 8.09 (s, 1H), 9.87 (s, 1H).
ESI-MS m/z: 683.4 [M þ H]þ.
tert-Butyl 4-((2S,3S)-1-((S)-7-(2-Methoxy-2-oxoethoxy)-
3-(4-methoxyphenylcarbamoyl)-3,4-dihydroisoquinolin-
2(1H)-yl)-3-methyl-1-oxopentan-2-ylcarbamoyl)piperidine-
1-carboxylate (29c). Colorless oil crystallized upon standing, 67% yield.
1H NMR (DMSO-d6) δ0.80ꢀ0.96 (m, 6H), 1.09ꢀ1.17 (m, 1H), 1.31ꢀ1.37
(m, 1H), 1.39 (s, 9H), 1.46ꢀ1.58 (m, 2H), 1.66ꢀ1.70 (m, 2H), 1.72ꢀ1.81
(m, 1H), 2.38ꢀ2.57 (m, 1H), 2.58ꢀ2.77 (m, 2H), 2.96ꢀ3.25 (m, 2H), 3.67
(s, 3H), 3.69 (s, 3H), 3.85ꢀ4.01 (m, 2H), 4.51ꢀ5.09 (m, 4H), 4.72 (s, 2H),
6.72ꢀ6.93 (m, 4H), 7.07ꢀ7.16 (m, 1H), 7.33ꢀ7.51 (m, 2H), 8.23 (s, 1H),
9.86 (s, 1H). ESI-MS m/z: 695.4 [M þ H]þ.
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General Procedure for the Preparation of 24l and Its Analo-
gue 24m. (S)-Methyl 2-(3-(4-Methoxyphenylcarbamoyl)-2-(3-
phenylpropyl)-1,2,3,4-tetrahydroisoquinolin-7-yloxy)acetate
(24l). A sealed tube containing compound 23 (0.15 g, 0.41 mmol),
potassium carbonate powder (0.084 g, 0.61 mmol), (3-bromopro-
pyl)benzene (0.12 g, 0.61 mmol), and DMF (4 mL) was subjected to
microwave irradiation at 70 °C for 20 min, at 100 W. After cooling, the
mixture was poured into 20 mL of water and extracted with EtOAc (3 ꢁ
10 mL). The organic layers were combined, washed with brine (3 ꢁ
10 mL), dried over MgSO4, and evaporated under vacuum. The residue
was purified by flash column chromatography (petroleum ether/EtOAc
3:1) to give desired compound 24l (0.16 g, 81% yield) as colorless oil.
1H NMR (DMSO-d6) δ 1.82ꢀ1.85 (m, 2H), 2.58ꢀ2.60 (m, 4H),
2.91ꢀ2.94 (m, 2H), 3.51ꢀ3.52 (m, 1H), 3.67 (s, 3H), 3.69 (d, J = 15.6
Hz, 1H), 3.71 (s, 3H), 4.03 (d, J = 15.6 Hz, 1H), 4.73 (s, 2H), 6.72ꢀ6.74
(m, 2H), 6.86 (d, J = 9.0 Hz, 2H), 7.05ꢀ7.06 (m, 1H), 7.12ꢀ7.24 (m,
5H), 7.51 (d, J = 9.0 Hz, 2H), 9.74 (s, 1H).
(H2O/MeOH 3:7) to give desired compound 22a (0.59 g, 59% yield)
as a white powder. Mp: 174ꢀ176 °C. 1H NMR (DMSO-d6) δ 2.79ꢀ
2.87 (m, 4H), 3.03ꢀ3.18 (m, 2H), 3.70 (s, 3H), 4.42 (s, 2H), 4.65 (d, J =
15.6 Hz, 1H), 4.72 (d, J = 15.6 Hz, 1H), 5.01ꢀ5.03 (m, 1H), 6.76ꢀ6.87
(m, 2H), 7.09ꢀ7.12 (m, 1H), 7.15ꢀ7.29 (m, 7H), 7.35ꢀ7.39 (m, 2H),
8.97 (s, 1H), 9.74 (s, 1H), 10.81 (s, 1H). HRMS (AP-ESI) m/z calcd for
C28H30N3O6 [M þ H]þ 504.2135, found 504.2141. Retention time:
2.6 min.
(S)-7-(2-(Hydroxyamino)-2-oxoethoxy)-N-phenethyl-2-
(3-phenylpropanoyl)-1,2,3,4-tetrahydroisoquinoline-3-car-
1
boxamide (22b). White powder, 56% yield. Mp: 146ꢀ148 °C. H
NMR (DMSO-d6) δ 2.52ꢀ2.86 (m, 8H), 3.10ꢀ3.20 (m, 2H), 4.40 (s,
2H), 4.53 (d, J = 15.6 Hz, 1H), 4.65 (d, J = 15.6 Hz, 1H), 4.98ꢀ5.00 (m,
1H), 6.76ꢀ6.80 (m, 3H), 7.03ꢀ7.06 (m, 2H), 7.14ꢀ7.30 (m, 8H), 7.90
(s, 1H), 8.96 (s, 1H), 10.81 (s, 1H). HRMS (AP-ESI) m/z calcd for
C29H31N3NaO5 [M þ Na]þ 524.2161, found 524.2198. Retention
time: 2.9 min.
(S)-Methyl 2-(3-(4-Methoxyphenylcarbamoyl)-2-phenethyl-
1,2,3,4-tetrahydroisoquinolin-7-yloxy)acetate (24m). Color-
less oil, 75% yield.
(S)-7-(2-(Hydroxyamino)-2-oxoethoxy)-N-phenyl-2-(3-phenyl-
propanoyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide
(22c). White powder, 56% yield. Mp: 167ꢀ169 °C. 1H NMR (DMSO-d6)
δ 2.81ꢀ2.88 (m, 4H), 3.08ꢀ3.17 (m, 2H), 4.42 (s, 2H), 4.63 (d, J =
15.6 Hz, 1H), 4.73 (d, J = 15.6 Hz, 1H), 5.02ꢀ5.04 (m, 1H), 6.76ꢀ6.88
(m, 2H), 7.01ꢀ7.03 (m, 1H), 7.10ꢀ7.30 (m, 8H), 7.46ꢀ7.50 (m, 2H), 8.96
(s, 1H), 9.99(s, 1H), 10.81(s, 1H). HRMS(AP-ESI) m/zcalcd for C27H28-
N3O5 [M þ H]þ 474.2029, found 474.2036. Retention time: 2.9 min.
(S)-tert-Butyl 2-(7-(2-(Hydroxyamino)-2-oxoethoxy)-3-(4-
methoxyphenylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-
2-oxoethylcarbamate (25a). White powder, 52% yield. Mp: 178ꢀ
180 °C. 1H NMR (DMSO-d6) δ 1.31 þ 1.37 (s, 9H, cis/trans), 3.08ꢀ3.20
(m, 2H), 3.70 (s, 3H), 3.94ꢀ4.04 (m, 2H), 4.42 (s, 2H), 4.62 (d, J = 15.6
Hz, 1H), 4.71 (d, J = 15.6 Hz, 1H), 4.93ꢀ4.95 (m, 1H), 6.76ꢀ6.87 (m,
5H), 7.11ꢀ7.14 (m, 1H), 7.37ꢀ7.41 (m, 2H), 8.97 (s, 1H), 9.87 (s, 1H),
10.83 (s, 1H). HRMS (AP-ESI) m/z calcd for C26H33N4O8 [M þ H]þ
529.2298, found 529.2300. Retention time: 2.1 min.
tert-Butyl (S)-1-((S)-7-(2-(Hydroxyamino)-2-oxoethoxy)-
3-(4-methoxyphenylcarbamoyl)-3,4-dihydroisoquinolin-
2(1H)-yl)-1-oxo-3-phenylpropan-2-ylcarbamate (25b). White
powder, 55% yield. Mp: 125ꢀ127 °C. 1H NMR (DMSO-d6) δ 1.26 þ
1.29 (s, 9H, cis/trans), 2.79ꢀ2.92 (m, 2H), 3.00ꢀ3.16 (m, 2H), 3.70 (s,
3H), 4.44 (s, 2H), 4.66 (d, J = 15.6 Hz, 1H), 4.78 (d, J = 15.6 Hz, 1H),
4.80ꢀ5.00 (m, 2H), 6.71ꢀ6.95 (m, 4H), 7.06ꢀ7.32 (m, 5H), 7.37ꢀ
7.49 (m, 4H), 8.96 (s, 1H), 9.84 (s, 1H), 10.81 (s, 1H). HRMS (AP-ESI)
m/z calcd for C33H38N4NaO8 [M þ Na]þ 641.2587, found 641.2603.
Retention time: 4.9 min.
tert-Butyl (S)-1-((S)-7-(2-(Hydroxyamino)-2-oxoethoxy)-
3-(4-methoxyphenylcarbamoyl)-3,4-dihydroisoquinolin-
2(1H)-yl)-3-(4-hydroxyphenyl)-1-oxopropan-2-ylcarbamate
(25c). White powder, 54% yield. Mp: 152ꢀ154 °C. 1H NMR (DMSO-
d6) δ 1.28 þ 1.32 (s, 9H, cis/trans), 2.67ꢀ2.89 (m, 2H), 3.02ꢀ3.14 (m,
2H), 3.71 (s, 3H), 4.48 (s, 2H), 4.62ꢀ4.97 (m, 4H), 6.58ꢀ6.72 (m, 3H),
6.79ꢀ6.87 (m, 3H), 6.95ꢀ7.23 (m, 4H), 7.41ꢀ7.44 (m, 2H), 8.95 (s,
1H), 9.18 (s, 1H), 9.83 (s, 1H), 10.81 (s, 1H). HRMS (AP-ESI) m/z calcd
for C33H38N4NaO9 [M þ Na]þ 657.2536, found 657.2541. Retention
time: 2.7 min.
(S)-tert-Butyl 2-((S)-7-(2-(Hydroxyamino)-2-oxoethoxy)-
3-(4-methoxyphenylcarbamoyl)-1,2,3,4-tetrahydroisoqui-
noline-2-carbonyl)pyrrolidine-1-carboxylate (25d). White
powder, 52% yield. Mp: 140ꢀ142 °C. 1H NMR (DMSO-d6) δ 1.31 þ
1.37 (s, 9H, cis/trans), 1.77ꢀ1.94 (m, 4H), 2.24ꢀ2.36 (m, 2H), 2.97ꢀ
3.19 (m, 2H), 3.70 (s, 3H), 4.40 (s, 2H), 4.55ꢀ4.92 (m, 4H), 6.78ꢀ6.86
(m, 3H), 6.93ꢀ6.97 (m, 1H), 7.11ꢀ7.27 (m, 1H), 7.38ꢀ7.49 (m, 2H),
8.96 (s, 1H), 9.88 (s, 1H), 10.82 (s, 1H). HRMS (AP-ESI) m/z calcd for
C29H36N4NaO8 [M þ Na]þ 591.2431, found 591.2448. Retention
time: 3.9 min.
(S)-Methyl 2-(2-Benzyl-3-(4-methoxyphenylcarbamoyl)-
1,2,3,4-tetrahydroisoquinolin-7-yloxy)acetate (24n). A mix-
ture of 23 (0.4 g, 1.08 mmol), potassium carbonate powder (0.22 g, 1.62
mmol), and benzyl bromide (0.28 g, 1.62 mmol) in anhydrous DMF
(15 mL) was stirred at room temperature for 4 h. The mixture was
poured into 60 mL of water and extracted with EtOAc (3 ꢁ 20 mL). The
organic layers were combined, washed with brine (3 ꢁ 15 mL), dried
over MgSO4, and evaporated under vacuum. The residue was purified by
flash column chromatography (petroleum ether/EtOAc 3:1) to give
1
desired compound 24n (0.43 g, 86% yield) as colorless oil. H NMR
(DMSO-d6) δ 2.97ꢀ3.06 (m, 2H), 3.60ꢀ3.65 (m, 3H), 3.67 (s, 3H),
3.71 (s, 3H), 3.76ꢀ3.89 (m, 2H), 4.75 (s, 2H), 6.61 (d, J = 2.4 Hz, 1H),
6.72 (dd, J = 2.4 Hz, J = 8.4 Hz, 1H), 6.87 (d, J = 7.2 Hz, 2H), 7.07 (d, J =
8.4 Hz, 1H), 7.26 (t, J = 7.2 Hz, 1H), 7.34 (d, J = 7.2 Hz, 2H), 7.41 (d, J =
7.2 Hz, 2H), 7.54 (d, J = 7.2 Hz, 2H), 9.86 (s, 1H).
General Procedure for the Preparation of 33a and Its
Analogues 33b. Methyl 2-((S)-2-((2S,3S)-2-(tert-Butoxycarbo-
nylamino)-3-methylpentyl)-3-(4-methoxyphenylcarbamoyl)-
1,2,3,4-tetrahydroisoquinolin-7-yloxy)acetate (33a). To a
solution of compound 23 (0.37 g, 1.0 mmol) and 32a (0.24 g, 1.1 mmol)
in 1,2-dichloroethane (20 mL) was added sodium triacetoxyborohy-
dride (0.32 g, 1.5 mmol). The mixture was stirred at room temperature
for 4 h, and the solvent was evaporated under vacuum. The residue was
taken up by EtOAc (30 mL), washed with saturated Na2CO3 (3 ꢁ
10 mL) and brine (3 ꢁ 10 mL), dried over MgSO4, and evaporated to
get the crude product, which was purified by flash column chromatog-
raphy (petroleum ether/EtOAc 3:1) to give desired compound 33a
(0.46 g, 81% yield) as colorless oil crystallized upon standing.
Methyl 2-((S)-2-((2S,3S)-2-(3,3-Dimethylbutanamido)-3-
methylpentyl)-3-(4-methoxyphenylcarbamoyl)-1,2,3,4-tet-
rahydroisoquinolin-7-yloxy)acetate (33b). Colorless oil crystal-
lized upon standing, 85% yield.
General Procedure for the Preparation of 22a and Its
Analogues 22b, 22c, 25aꢀ25n, 30aꢀ30c, 34a, 34b. (S)-7-
(2-(Hydroxyamino)-2-oxoethoxy)-N-(4-methoxyphenyl)-
2-(3-phenylpropanoyl)-1,2,3,4-tetrahydroisoquinoline-3-
carboxamide (22a). To a solution of compound 21a (1.0 g, 2.0 mmol)
in 10 mL of anhydrous methanol was added a solution of NH2OK (0.14 g,
6 mmol) in 3.5 mL of anhydrous methanol. The mixture was stirred for
0.5 h, and the solvent was evaporated under vacuum. The residue was
acidified with 2 N HCl until pH 5ꢀ6 and then extracted with EtOAc
(3 ꢁ 10 mL). The organic layers were combined, washed with brine
(3 ꢁ 10 mL), dried over MgSO4, and evaporated with the residue
being purified by C18 reversed-phase column chromatography
2834
dx.doi.org/10.1021/jm101605z |J. Med. Chem. 2011, 54, 2823–2838

Journal of Medicinal Chemistry
ARTICLE
tert-Butyl (2S,3S)-1-((S)-7-(2-(Hydroxyamino)-2-oxoethoxy)-
3-(4-methoxyphenylcarbamoyl)-3,4-dihydroisoquinolin-
2(1H)-yl)-3-methyl-1-oxopentan-2-ylcarbamate (25e). White
powder, 58% yield. Mp: 138ꢀ140 °C. 1H NMR (DMSO-d6) δ 0.85 (t,
J=7.2 Hz, 3H), 0.91 (d, J = 6.6 Hz, 3H), 1.13ꢀ1.17 (m, 1H), 1.24 þ 1.36
(s, 9H, cis/trans), 1.48ꢀ1.54 (m, 1H), 1.73ꢀ1.82 (m, 1H), 2.98ꢀ3.13
(m, 2H), 3.70 (s, 3H), 4.45 (s, 2H), 4.65 (d, J = 15.6 Hz, 1H), 4.83ꢀ4.86
(m, 1H), 5.01 (d, J = 15.6 Hz, 1H), 5.03ꢀ5.06 (m, 1H), 6.75ꢀ6.95 (m,
4H), 7.10ꢀ7.28 (m, 2H), 7.41ꢀ7.44 (m, 2H), 8.97 (s, 1H), 9.87 (s, 1H),
10.82 (s, 1H). HRMS (AP-ESI) m/z calcd for C30H40N4NaO8 [M þ
Na]þ 607.2744, found 607.2765. Retention time: 4.1 min.
tert-Butyl (S)-1-((S)-7-(2-(Hydroxyamino)-2-oxoethoxy)-
3-(4-methoxyphenylcarbamoyl)-3,4-dihydroisoquinolin-
2(1H)-yl)-4-methyl-1-oxopentan-2-ylcarbamate (25f). White
powder, 52% yield. Mp: 118ꢀ120 °C. 1H NMR (DMSO-d6) δ 0.93 (d,
J = 6.6 Hz, 3H), 0.99 (d, J = 6.6 Hz, 3H), 1.31 þ 1.36 (s, 9H, cis/trans),
1.45ꢀ1.53 (m, 2H), 1.59ꢀ1.70 (m, 1H), 3.02 (dd, J = 15.0 Hz, 6.6 Hz,
1H), 3.13 (dd, J = 15.6 Hz, 6.0 Hz, 1H), 3.70 (s, 3H), 4.41 (s, 2H),
4.56ꢀ4.97 (m, 4H), 6.75ꢀ6.91 (m, 4H), 7.02ꢀ7.25 (m, 2H), 7.40ꢀ
7.44 (m, 2H), 8.97 (s, 1H), 9.85 (s, 1H), 10.82 (s, 1H). HRMS (AP-ESI)
m/z calcd for C30H40N4NaO8 [M þ Na]þ 607.2744, found 607.2764.
Retention time: 4.7 min.
tert-Butyl (S)-1-((S)-7-(2-(Hydroxyamino)-2-oxoethoxy)-
3-(4-methoxyphenylcarbamoyl)-3,4-dihydroisoquinolin-
2(1H)-yl)-1-oxopropan-2-ylcarbamate (25g). White powder,
55% yield. Mp: 181ꢀ183 °C. 1H NMR (DMSO-d6) δ 1.19 (d, J = 6.6
Hz, 3H), 1.24 þ 1.35 (s, 9H, cis/trans), 3.01 (dd, J = 15.6 Hz, 6.6 Hz,
1H), 3.12 (dd, J = 15.6 Hz, 6.0 Hz, 1H), 3.70 (s, 3H), 4.41 (s, 2H),
4.54ꢀ5.00 (m, 4H), 6.75ꢀ6.85 (m, 3H), 6.91ꢀ6.93 (m, 1H), 7.03ꢀ
7.17 (m, 2H), 7.29ꢀ7.46 (m, 2H), 8.97 (s, 1H), 9.82 (s, 1H), 10.82 (s,
1H). HRMS (AP-ESI) m/z calcd for C27H34N4NaO8 [M þ Na]þ
565.2274, found 565.2297. Retention time: 2.4 min.
2.62ꢀ2.67 (m, 2H), 3.10ꢀ3.22 (m, 4H), 3.70 (s, 3H), 4.44 (s, 2H),
4.59ꢀ5.05 (m, 3H), 6.75ꢀ6.86 (m, 4H), 7.10ꢀ7.23 (m, 5H),
7.28ꢀ7.40 (m, 3H), 8.96 (s, 1H), 9.74 (s, 1H), 10.82 (s, 1H). HRMS
(AP-ESI) m/z calcd for C29H31N3NaO6 [M þ Na]þ 540.2111, found
540.2134. Retention time: 3.2 min.
(S)-7-(2-(Hydroxyamino)-2-oxoethoxy)-N-(4-methoxy-
phenyl)-2-(3-phenylpropyl)-1,2,3,4-tetrahydroisoquinoline-
3-carboxamide (25l). White powder, 57% yield. Mp: 110ꢀ112 °C.
1H NMR (DMSO-d6) δ 1.83ꢀ1.86 (m, 2H), 2.59ꢀ2.61 (m, 4H),
2.91ꢀ2.94 (m, 2H), 3.51ꢀ3.53 (m, 1H), 3.68 (d, J = 15.6 Hz, 1H), 3.71
(s, 3H), 4.03 (d, J = 15.6 Hz, 1H), 4.40 (s, 2H), 6.72ꢀ6.75 (m, 2H), 6.86
(d, J = 9.0 Hz, 2H), 7.05ꢀ7.06 (m, 1H), 7.13ꢀ7.25 (m, 5H), 7.50 (d, J =
9.0 Hz, 2H), 8.96 (s, 1H), 9.75 (s, 1H), 10.80 (s, 1H). HRMS (AP-ESI)
m/z calcd for C28H32N3O5 [M þ H]þ 490.2342, found 490.2506.
Retention time: 1.6 min.
(S)-7-(2-(Hydroxyamino)-2-oxoethoxy)-N-(4-methoxy-
phenyl)-2-phenethyl-1,2,3,4-tetrahydroisoquinoline-3-car-
1
boxamide (25m). White powder, 56% yield. Mp: 96ꢀ98 °C. H
NMR (DMSO-d6) δ 2.76ꢀ2.86 (m, 4H), 2.90ꢀ2.94 (m, 2H), 3.58ꢀ
3.60 (m, 1H), 3.70 (s, 3H), 3.79 (d, J = 15.6 Hz, 1H), 4.07 (d, J = 15.6 Hz,
1H), 4.41 (s, 2H), 6.74ꢀ6.76 (m, 2H), 6.84 (d, J = 9.0 Hz, 2H), 7.05ꢀ
7.07 (m, 1H), 7.16ꢀ7.27 (m, 5H), 7.38 (d, J = 9.0 Hz, 2H), 8.96 (s, 1H),
9.56 (s, 1H), 10.81 (s, 1H). HRMS (AP-ESI) m/z calcd for C27H30-
N3O5 [M þ H]þ 476.2185, found 476.2145. Retention time: 1.6 min.
(S)-2-Benzyl-7-(2-(hydroxyamino)-2-oxoethoxy)-N-(4-meth-
oxyphenyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide
(25n). White powder, 53% yield. Mp: 90ꢀ92 °C. 1H NMR (DMSO-d6)
δ 2.96ꢀ3.05 (m, 2H), 3.60ꢀ3.66 (m, 3H), 3.71 (s, 3H), 3.76ꢀ3.88 (m,
2H), 4.38 (s, 2H), 6.61 (d, J = 2.4 Hz, 1H), 6.75 (dd, J = 2.4 Hz, J = 8.4
Hz, 1H), 6.87 (d, J = 7.2 Hz, 2H), 7.08 (d, J = 8.4 Hz, 1H), 7.27 (t, J = 7.2
Hz, 1H), 7.34 (d, J = 7.2 Hz, 2H), 7.41 (d, J = 7.2 Hz, 2H), 7.53 (d, J = 7.2
Hz, 2H), 8.94 (s, 1H), 9.87 (s, 1H), 10.77 (s, 1H). HRMS (AP-ESI) m/z
calcd for C26H27N3NaO5 [M þ Na]þ 484.1848, found 484.1840.
Retention time: 1.5 min.
tert-Butyl (S)-1-((S)-7-(2-(Hydroxyamino)-2-oxoethoxy)-
3-(4-methoxyphenylcarbamoyl)-3,4-dihydroisoquinolin-
2(1H)-yl)-3-methyl-1-oxobutan-2-ylcarbamate (25h). White
(S)-tert-Butyl 2-((2S,3S)-1-((S)-7-(2-(Hydroxyamino)-2-oxo-
ethoxy)-3-(4-methoxyphenylcarbamoyl)-3,4-dihydroiso-
quinolin-2(1H)-yl)-3-methyl-1-oxopentan-2-ylcarbamoyl)-
pyrrolidine-1-carboxylate (30a). White powder, 52% yield. Mp:
129ꢀ131 °C. 1H NMR (DMSO-d6) δ 0.72ꢀ0.94 (m, 6H), 1.02ꢀ1.43
(m, 11H), 1.55ꢀ1.88 (m, 4H), 2.06ꢀ2.13 (m, 1H), 2.97ꢀ3.35 (m, 4H),
3.70 (s, 3H), 4.14ꢀ4.27 (m, 1H), 4.44 (s, 2H), 4.53ꢀ4.70 (m, 1H),
4.73ꢀ4.87 (m, 2H), 4.91ꢀ5.16 (m, 1H), 6.73ꢀ6.87 (m, 4H), 7.02ꢀ
7.12 (m, 1H), 7.34ꢀ7.52 (m, 2H), 7.94ꢀ8.43 (m, 1H), 8.96 (s, 1H),
9.43ꢀ10.07 (m, 1H), 10.81 (s, 1H). HRMS (AP-ESI) m/z calcd for
C35H47N5NaO9 [M þ Na]þ 704.3363, found 704.3271. Retention
time: 3.8 min.
1
powder, 58% yield. Mp: 143ꢀ145 °C. H NMR (DMSO-d6) δ 0.86
(d, J = 6.6 Hz, 3H), 0.86 (d, J = 6.6Hz, 3H), 1.26 þ 1.36 (s, 9H, cis/trans),
1.22ꢀ1.24 (m, 1H), 3.01 (dd, J = 14.4 Hz, 6.6 Hz, 1H), 3.11 (dd, J = 14.4
Hz, 6.0 Hz, 1H), 3.70(s, 3H), 4.36-4.41(m, 1H), 4.42 (s, 2H), 4.64 (d, J =
15.0 Hz, 1H), 4.80ꢀ4.82 (m, 1H), 4.96 (d, J = 15.0 Hz, 1H), 6.75ꢀ6.86
(m, 4H), 6.94ꢀ6.7.21 (m, 2H), 7.41ꢀ7.45 (m, 2H), 8.97 (s, 1H), 9.87 (s,
1H), 10.82 (s, 1H). HRMS (AP-ESI) m/z calcd for C29H38N4NaO8 [M
þ Na]þ 593.2587, found 593.2640. Retention time: 3.6 min.
(S)-7-(2-(Hydroxyamino)-2-oxoethoxy)-N-(4-methoxy-
phenyl)-2-(2-phenylacetyl)-1,2,3,4-tetrahydroisoquinoline-
3-carboxamide (25i). White powder, 59% yield. Mp: 141ꢀ143 °C.
1H NMR (DMSO-d6) δ 3.07ꢀ3.17 (m, 2H), 3.70 (s, 3H), 3.91 (s, 2H),
4.41 (s, 2H), 4.68 (d, J = 15.6 Hz, 1H), 4.81 (d, J = 15.6 Hz, 1H), 4.94ꢀ
4.98 (m, 1H), 6.74ꢀ6.85 (m, 2H), 7.07ꢀ7.14 (m, 1H), 7.20ꢀ7.32 (m,
7H), 7.36ꢀ7.40 (m, 2H), 8.96 (s, 1H), 9.80 (s, 1H), 10.82 (s, 1H). HRMS
(AP-ESI) m/z calcd for C27H28N3O6 [M þ H]þ 490.1978, found
490.1941. Retention time: 2.2 min.
tert-Butyl (S)-1-((2S,3S)-1-((S)-7-(2-(Hydroxyamino)-2-oxo-
ethoxy)-3-(4-methoxyphenylcarbamoyl)-3,4-dihydroiso-
quinolin-2(1H)-yl)-3-methyl-1-oxopentan-2-ylamino)-3-
methyl-1-oxobutan-2-ylcarbamate (30b, 57% yield). White
1
powder. Mp: 131ꢀ133 °C. H NMR (DMSO-d6) δ 0.72ꢀ0.98 (m,
12H), 1.11ꢀ1.19 (m, 1H), 1.21ꢀ1.32 (m, 1H), 1.33 þ 1.37 (s, 9H, cis/
trans), 1.69ꢀ1.79 (m, 1H), 1.81ꢀ1.92 (m, 1H), 2.98ꢀ3.25 (m, 2H),
3.69 (s, 3H), 3.77ꢀ3.97 (m, 1H), 4.40 (s, 2H), 4.51ꢀ5.10 (m, 4H),
6.61ꢀ6.92 (m, 4H), 7.06ꢀ7.15 (m, 1H), 7.33ꢀ7.53 (m, 2H), 7.89 (s,
1H), 8.06 (s, 1H), 8.95 (s, 1H), 9.87 (s, 1H), 10.81 (s, 1H). HRMS (AP-
ESI) m/z calcd for C35H50N5O9 [M þ H]þ 684.3609, found 684.3629.
Retention time: 5.0 min.
(S)-2-Benzoyl-7-(2-(hydroxyamino)-2-oxoethoxy)-N-(4-
methoxyphenyl)-1,2,3,4-tetrahydroisoquinoline-3-carbox-
amide (25j). White powder, 53% yield. Mp: 133ꢀ135 °C. 1H NMR
(DMSO-d6) δ 3.08ꢀ3.26 (m, 2H), 3.70 (s, 3H), 4.42 (s, 2H), 4.51 (d,
J = 15.6 Hz, 1H), 4.59 (d, J = 15.6 Hz, 1H), 4.95ꢀ5.00 (m, 1H), 6.75ꢀ
6.90 (m, 2H), 7.07ꢀ7.19 (m, 1H), 7.27ꢀ7.52 (m, 9H), 8.95 (s, 1H),
10.00 (s, 1H), 10.76 (s, 1H). HRMS (AP-ESI) m/z calcd for C26H26N3O6
[M þ H]þ 476.1822, found 476.1829. Retention time: 2.0 min.
tert-Butyl 4-((2S,3S)-1-((S)-7-(2-(Hydroxyamino)-2-oxo-
ethoxy)-3-(4-methoxyphenylcarbamoyl)-3,4-dihydroiso-
quinolin-2(1H)-yl)-3-methyl-1-oxopentan-2-ylcarbamoyl)-
piperidine-1-carboxylate (30c). White powder, 55% yield. Mp:
135ꢀ137 °C. 1H NMR (DMSO-d6) δ 0.80ꢀ0.96 (m, 6H), 1.09ꢀ1.16
(m, 1H), 1.30ꢀ1.38 (m, 1H), 1.39 (s, 9H), 1.49ꢀ1.59 (m, 2H),
(S)-7-(2-(Hydroxyamino)-2-oxoethoxy)-N-(4-methoxy-
phenyl)-2-(4-phenylbutanoyl)-1,2,3,4-tetrahydroisoquino-
line-3-carboxamide (25k). White powder, 52% yield. Mp: 140ꢀ
142 °C. 1H NMR (DMSO-d6) δ 1.80ꢀ1.87 (m, 2H), 2.54ꢀ2.58 (m, 2H),
2835
dx.doi.org/10.1021/jm101605z |J. Med. Chem. 2011, 54, 2823–2838

Journal of Medicinal Chemistry
ARTICLE
1.68ꢀ1.71 (m, 2H), 1.72ꢀ1.80 (m, 1H), 2.37ꢀ2.55 (m, 1H),
2.58ꢀ2.77 (m, 2H), 2.97ꢀ3.26 (m, 2H), 3.68 (s, 3H), 3.84ꢀ4.00 (m,
2H), 4.40 (s, 2H), 4.50ꢀ5.10 (m, 4H), 6.73ꢀ6.93 (m, 4H), 7.07ꢀ7.16
(m, 1H), 7.34ꢀ7.50 (m, 2H), 8.23 (s, 1H), 9.24 (s, 1H), 9.85 (s, 1H),
10.81 (s, 1H). HRMS (AP-ESI) m/z calcd for C36H50N5O9 [M þ H]þ
696.3609, found 696.3634. Retention time: 4.1 min.
88% yield. Mp: 190ꢀ192 °C. 1H NMR (DMSO-d6) δ1.89ꢀ2.00 (m, 4H),
3.02ꢀ3.26 (m, 4H), 3.70 (s, 3H), 4.42 (s, 2H), 4.50ꢀ5.06 (m, 4H),
6.83ꢀ6.87 (m, 3H), 6.99ꢀ7.00 (m, 1H), 7.20ꢀ7.21 (m, 1H), 7.40ꢀ7.46
(m, 2H), 8.65 (br s, 2H), 9.71 (br s, 1H), 10.22 (s, 1H), 10.87 (s, 1H).
HRMS (AP-ESI) m/z calcd for C24H29N4O6 [M þ H]þ 469.2087, found
469.2153. Retention time: 2.7 min.
tert-Butyl (2S,3S)-1-((S)-7-(2-(Hydroxyamino)-2-oxoethoxy)-
3-(4-methoxyphenylcarbamoyl)-3,4-dihydroisoquinolin-
2(1H)-yl)-3-methylpentan-2-ylcarbamate (34a). White powder,
(S)-2-((2S,3S)-2-Amino-3-methylpentanoyl)-7-(2-(hydroxy-
amino)-2-oxoethoxy)-N-(4-methoxyphenyl)-1,2,3,4-tetra-
hydroisoquinoline-3-carboxamide Hydrochloride (26e).
White powder, 91% yield. Mp: 176ꢀ178 °C. 1H NMR (DMSO-d6) δ
0.85 (t, J = 7.2 Hz, 3H), 1.08 (d, J = 6.6 Hz, 3H), 1.18ꢀ1.22 (m, 1H),
1.58ꢀ1.62 (m, 1H), 1.91ꢀ1.96 (m, 1H), 3.03 (dd, J = 15.6 Hz, 8.4 Hz,
1H), 3.18 (dd, J = 15.6 Hz, 6.0 Hz, 1H), 3.71 (s, 3H), 4.42 (s, 2H),
4.53ꢀ4.98 (m, 4H), 6.83ꢀ6.88 (m, 3H), 7.04ꢀ7.05 (m, 1H), 7.20ꢀ
7.21 (m, 1H), 7.44ꢀ7.46 (m, 2H), 8.08 (s, 3H), 8.98 (br s, 1H), 10.07 (s,
1H), 10.88 (s, 1H). HRMS (AP-ESI) m/z calcd for C25H33N4O6 [M þ
H]þ 485.2400, found 485.2594. Retention time: 4.8 min.
1
58% yield. Mp: 98ꢀ100 °C. H NMR (DMSO-d6) δ 0.80ꢀ0.89 (m,
6H), 1.03ꢀ1.07 (m, 1H), 1.34 þ 1.36 (s, 9H, cis/trans), 1.38ꢀ1.39 (m,
1H), 1.42ꢀ1.51 (m, 1H), 2.42ꢀ2.46 (m, 1H), 2.68ꢀ2.70 (m, 1H), 2.91ꢀ
2.99 (m, 2H), 3.59ꢀ3.61 (m, 2H), 3.66ꢀ3.69 (m, 1H), 3.70 (s, 3H),
3.98ꢀ4.00 (m, 1H), 4.40 (s, 2H), 6.59ꢀ6.79 (m, 3H), 6.84ꢀ6.86 (m,
2H), 7.03ꢀ7.06 (m, 1H), 7.49ꢀ7.53 (m, 2H), 8.95 (s, 1H), 9.67 (s, 1H),
10.79 (s, 1H). HRMS (AP-ESI) m/z calcd for C30H42N4NaO7 [M þ
Na]þ 593.2951, found 593.2978. Retention time: 2.6 min.
(S)-2-((2S,3S)-2-(3,3-Dimethylbutanamido)-3-methylpentyl)-
7-(2-(hydroxyamino)-2-oxoethoxy)-N-(4-methoxyphenyl)-
1,2,3,4-tetrahydroisoquinoline-3-carboxamide (34b). White
powder, 55% yield. Mp: 119ꢀ121 °C. 1H NMR (DMSO-d6) δ 0.75ꢀ
0.85 (m, 6H), 0.94ꢀ0.96 (m, 9H), 1.02ꢀ1.10 (m, 1H), 1.23ꢀ1.43 (m,
1H), 1.52ꢀ1.57 (m, 1H), 1.91ꢀ2.09 (m, 2H), 2.37ꢀ2.77 (m, 2H), 2.85ꢀ
3.01 (m, 2H), 3.58ꢀ3.95 (m, 2H), 3.70 (s, 3H), 3.98ꢀ4.00 (m, 1H),
4.10ꢀ4.12 (m, 1H), 4.40 (s, 2H), 6.64ꢀ6.87 (m, 4H), 7.04ꢀ7.05 (m,
1H), 7.39ꢀ7.58 (m, 3H), 8.96 (s, 1H), 9.74 (s, 1H), 10.80 (s, 1H).
HRMS (AP-ESI) m/z calcd for C31H45N4O6 [M þ H]þ 569.3339,
found 569.3681. Retention time: 2.5 min.
General Procedure for the Preparation of 26a and Its Analo-
gues 26bꢀ26h, 31aꢀ31c (S)-2-(2-Aminoacetyl)-7-(2-(hydroxy-
amino)-2-oxoethoxy)-N-(4-methoxyphenyl)-1,2,3,4-tetra-
hydroisoquinoline-3-carboxamide Hydrochloride (26a). To a
solution of compound 25a (0.26 g, 0.5 mmol) in dry EtOAc (8 mL) was
added a solution of EtOAc (8 mL) saturated by dry HCl gas. The reaction
solution was stirred at room temperature for 3 h at which time a precipitate
appeared. The suspension was filtered, with the filter being washed with
ether, to give desired compound 26a (0.21 g, 90% yield) as a white powder.
Mp: 193ꢀ195 °C. 1H NMR (DMSO-d6) δ 3.12ꢀ3.14 (m, 2H), 3.70 (s,
3H), 4.08ꢀ4.09 (m, 2H), 4.42 (s, 2H), 4.65ꢀ4.70 (m, 2H), 5.00ꢀ5.01 (m,
1H), 6.80ꢀ6.90 (m, 4H), 7.15ꢀ7.16 (m, 1H), 7.37ꢀ7.42 (m, 2H), 8.19
(br s, 3H), 8.98 (br s, 1H), 9.90 (s, 1H), 10.86 (s, 1H). HRMS (AP-ESI)
m/z calcd for C21H24N4NaO6 [M þ Na]þ 451.1594, found 451.1600.
Retention time: 1.9 min.
(S)-2-((S)-2-Amino-3-phenylpropanoyl)-7-(2-(hydroxy-
amino)-2-oxoethoxy)-N-(4-methoxyphenyl)-1,2,3,4-tetra-
hydroisoquinoline-3-carboxamide Hydrochloride (26b).
White powder, 91% yield. Mp: 188ꢀ190 °C. 1H NMR (DMSO-d6) δ
2.99ꢀ3.08 (m, 2H), 3.13ꢀ3.24 (m, 2H), 3.72 (s, 3H), 4.45 (s, 2H),
4.65ꢀ4.90 (m, 4H), 6.72ꢀ6.98 (m, 4H), 7.19ꢀ7.34 (m, 5H), 7.40ꢀ
7.51 (m, 3H), 8.45 (br s, 3H), 8.96 (br s, 1H), 10.09 (s, 1H), 10.83 (s,
1H). HRMS (AP-ESI) m/z calcd for C28H31N4O6 [M þ H]þ 519.2244,
found 519.2287. Retention time: 5.9 min.
(S)-2-((S)-2-Amino-3-(4-hydroxyphenyl)propanoyl)-7-(2-
(hydroxyamino)-2-oxoethoxy)-N-(4-methoxyphenyl)-1,2,3,4-
tetrahydroisoquinoline-3-carboxamide Hydrochloride (26c).
White powder, 92% yield. Mp: 180ꢀ182 °C. 1H NMR (DMSO-d6) δ
2.87ꢀ3.05 (m, 2H), 3.06ꢀ3.21 (m, 2H), 3.72 (s, 3H), 4.45 (s, 2H),
4.51ꢀ4.90 (m, 4H), 6.66ꢀ6.90 (m, 5H), 7.00ꢀ7.24 (m, 4H), 7.39ꢀ7.50
(m, 2H), 8.19 (br s, 3H), 8.97 (br s, 1H), 9.39 (s, 1H), 10.10 (s, 1H), 10.85
(s, 1H). HRMS (AP-ESI) m/z calcd for C28H31N4O7 [M þ H]þ
535.2193, found 535.2262. Retention time: 3.0 min.
(S)-2-((S)-2-Amino-4-methylpentanoyl)-7-(2-(hydroxy-
amino)-2-oxoethoxy)-N-(4-methoxyphenyl)-1,2,3,4-tetra-
hydroisoquinoline-3-carboxamide Hydrochloride (26f). White
powder, 93% yield. Mp: 168ꢀ170 °C. 1H NMR (DMSO-d6) δ 0.95 (d, J =
6.6 Hz, 3H), 1.08 (d, J = 6.6 Hz, 3H), 1.60ꢀ1.71 (m, 2H), 1.89ꢀ1.91 (m,
1H), 3.06 (dd, J = 15.0 Hz, 7.2 Hz, 1H), 3.18 (dd, J = 15.6 Hz, 6.0 Hz, 1H),
3.70 (s, 3H), 4.42 (s, 2H), 4.55ꢀ4.87 (m, 4H), 6.83ꢀ6.86 (m, 3H), 7.03ꢀ
7.05 (m, 1H), 7.19ꢀ7.20 (m, 1H), 7.44ꢀ7.47 (m, 2H), 8.22 (s, 3H), 8.96
(s, 1H), 10.11 (s, 1H), 10.88 (s, 1H). HRMS (AP-ESI) m/zcalcd for C25H33-
N4O6 [M þ H]þ 485.2400, found 485.2468. Retention time: 4.5 min.
(S)-2-((S)-2-Aminopropanoyl)-7-(2-(hydroxyamino)-2-oxo-
ethoxy)-N-(4-methoxyphenyl)-1,2,3,4-tetrahydroisoquino-
line-3-carboxamide Hydrochloride (26g). White powder, 90%
yield. Mp: 194ꢀ196 °C. 1H NMR (DMSO-d6) δ 1.44 (d, J = 6.6 Hz,
3H), 3.01 (dd, J = 14.4 Hz, 8.4 Hz, 1H), 3.20 (dd, J = 14.4 Hz, 6.0 Hz,
1H), 3.71 (s, 3H), 4.42 (s, 2H), 4.45ꢀ4.89 (m, 4H), 6.83ꢀ6.87 (m, 3H),
6.98ꢀ7.01 (m, 1H), 7.20ꢀ7.22 (m, 1H), 7.42ꢀ7.48 (m, 2H), 8.15 (s,
3H), 8.96 (br s, 1H), 10.06 (s, 1H), 10.83 (s, 1H). HRMS (AP-ESI) m/z
calcd for C22H27N4O6 [M þ H]þ 443.1931, found 443.1954. Retention
time: 2.3 min.
(S)-2-((S)-2-Amino-3-methylbutanoyl)-7-(2-(hydroxyamino)-
2-oxoethoxy)-N-(4-methoxyphenyl)-1,2,3,4-tetrahydroiso-
quinoline-3-carboxamide Hydrochloride (26h). White powder,
91% yield. Mp: 178ꢀ180 °C. 1H NMR (DMSO-d6) δ 0.93 (d, J = 6.6 Hz,
3H), 1.01 (d, J = 6.6 Hz, 3H), 2.23ꢀ2.26 (m, 1H), 3.02 (dd, J = 15.0 Hz,
9.0 Hz, 1H), 3.20 (dd, J = 15.0 Hz, 6.0 Hz, 1H), 3.71 (s, 3H), 4.41ꢀ4.52
(m, 1H), 4.42 (s, 2H), 4.54 (d, J = 15.0 Hz, 1H), 4.68ꢀ4.70 (m, 1H),
4.97 (d, J = 15.0 Hz, 1H), 6.84ꢀ6.88 (m, 3H), 7.04ꢀ7.05 (m, 1H),
7.20ꢀ7.22 (m, 1H), 7.45ꢀ7.47 (m, 2H), 8.09 (s, 3H), 8.99 (br s, 1H),
10.10 (s, 1H), 10.88 (s, 1H). HRMS (AP-ESI) m/z calcd for C24H31-
N4O6 [M þ H]þ 471.2244, found 471.2271. Retention time: 3.4 min.
(S)-7-(2-(Hydroxyamino)-2-oxoethoxy)-N-(4-methoxy-
phenyl)-2-((2S,3S)-3-methyl-2-((S)-pyrrolidine-2-carboxamido)-
pentanoyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide
Hydrochloride (31a). White powder, 93% yield. Mp: 173ꢀ175 °C.
1H NMR (DMSO-d6) δ 0.73ꢀ0.99 (m, 6H), 1.06ꢀ1.19 (m, 1H),
1.26ꢀ1.34 (m, 1H), 1.71ꢀ1.84 (m, 2H), 1.85ꢀ1.95 (m, 2H), 2.20ꢀ
2.39 (m, 1H), 3.02ꢀ3.29 (m, 4H), 3.70 (s, 3H), 4.20ꢀ4.31 (m, 1H),
4.44 (s, 2H), 4.54ꢀ4.69 (m, 1H), 4.72ꢀ4.88 (m, 2H), 4.90ꢀ5.10 (m,
1H), 6.74ꢀ6.97 (m, 4H), 7.09ꢀ7.20 (m, 1H), 7.35ꢀ7.47 (m, 2H), 8.49ꢀ
8.55 (m, 1H), 8.74ꢀ8.89 (m, 1H), 8.98 (br s, 1H), 9.95 (s, 1H), 10.02ꢀ
10.35 (m, 1H), 10.89 (s, 1H). HRMS (AP-ESI) m/z calcd for C30H40-
N5O7 [M þ H]þ 582.2928, found 582.2972. Retention time: 5.1 min.
(S)-2-((2S,3S)-2-((S)-2-Amino-3-methylbutanamido)-3-methyl-
pentanoyl)-7-(2-(hydroxyamino)-2-oxoethoxy)-N-(4-meth-
oxyphenyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide
Hydrochloride (31b). White powder, 88% yield. Mp: 176ꢀ178 °C.
(S)-7-(2-(Hydroxyamino)-2-oxoethoxy)-N-(4-methoxy-
phenyl)-2-((S)-pyrrolidine-2-carbonyl)-1,2,3,4-tetrahydroi-
soquinoline-3-carboxamide Hydrochloride (26d). White powder,
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1H NMR (DMSO-d6) δ 0.74ꢀ1.02 (m, 12H), 1.13ꢀ1.18 (m, 1H),
1.27ꢀ1.65 (m, 1H), 1.70ꢀ1.88 (m, 1H), 1.95ꢀ2.16 (m, 1H), 3.01ꢀ
3.29 (m, 2H), 3.64ꢀ3.87 (m, 1H), 3.70 (s, 3H), 4.44 (s, 2H), 4.54ꢀ5.30
(m, 4H), 6.75ꢀ6.96 (m, 4H), 7.10ꢀ7.19 (m, 1H), 7.35ꢀ7.48 (m, 2H),
8.18 (s, 3H), 8.63 (s, 1H), 8.98 (s, 1H), 9.96 (s, 1H), 10.87 (s, 1H).
HRMS (AP-ESI) m/z calcd for C30H42N5O7 [M þ H]þ 584.3084,
found 584.3158. Retention time: 7.5 min.
(S)-7-(2-(Hydroxyamino)-2-oxoethoxy)-N-(4-methoxy-
phenyl)-2-((2S,3S)-3-methyl-2-(piperidine-4-carboxamido)
pentanoyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide
Hydrochloride (31c). White powder, 92% yield. Mp: 180ꢀ182 °C.
1H NMR (DMSO-d6) δ 0.81ꢀ0.96 (m, 6H), 1.09ꢀ1.17 (m, 1H),
1.28ꢀ1.33 (m, 1H), 1.68ꢀ1.80 (m, 4H), 1.86ꢀ1.91 (m, 1H), 2.52ꢀ
2.65 (m, 1H), 2.58ꢀ2.78 (m, 2H), 3.00ꢀ3.18 (m, 2H), 3.21ꢀ3.29 (m,
2H), 3.70 (s, 3H), 4.28ꢀ5.05 (m, 4H), 4.43 (s, 2H), 6.75ꢀ7.01 (m, 4H),
7.11ꢀ7.21 (m, 1H), 7.43ꢀ7.57 (m, 2H), 8.26 (s, 1H), 8.56 (s, 2H), 9.38
(s, 1H), 9.84 (s, 1H), 10.89 (s, 1H). HRMS (AP-ESI) m/z calcd for
C31H42N5O7 [M þ H]þ 596.3084, found 596.3181. Retention time:
4.2 min.
In Vitro HDAC Inhibition Fluorescence Assay. In vitro activity
against HDAC8 was determined as previously described.24 Briefly,
HDAC8 solution (10 μL) was mixed with various concentrations of
compound sample (50 μL). Fluorogenic substrate (40 μL) was added,
and the mixture was incubated at 37 °C for 30 min and then stopped by
addition of 100 μL of developer containing trypsin and TSA. Fluores-
cence intensity was measured after 20 min using a microplate reader at
excitation and emission wavelengths of 390 and 460 nm, respectively.
The inhibition ratios were calculated from the fluorescence intensity
readings of tested wells relative to those of control wells, and the IC50
values were determined using a regression analysis of the concentration/
inhibition data. The procedures of HeLa nuclear extract assay and
HDAC6 (purchased from abcam Ltd.) assay were similar to that
of HDAC8.
In Vitro Antiproliferative Assay. All cell lines were maintained in
RPMI1640 medium containing 10% FBS at 37 °C in a 5% CO2
humidified incubator. Cell proliferation assay was determined by the
MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide)
method. Briefly, cells were passaged the day before dosing into a 96-well
cell plate, allowed to grow for a minimum of 4 h, and then treated with
different concentrations of compound sample for 48 h. A 0.5% MTT
solution was added to each well. After further incubation for another 4 h,
formazan formed from MTT was extracted by adding 200 μL of DMSO
for 15 min. Absorbance was then determined using an ELISA reader at
570 nm.
Molecular Docking Studies. Compounds were docked into the
active site of HDAC8 (PDB entry: 1T64) using Tripos SYBYL 8.0. The
residues in a radius of 12.0 Å around TSA in the cocrystal structure of
HDAC8 and TSA (PDB entry: 1T64) were selected as the active site.
Before the docking process, the protein structure was treated by deleting
water molecules, adding hydrogen atoms, modifying atom types, and
assigning AMBER7 FF99 charge. A 100-step minimization process was
performed to further optimize the protein structure. The molecular
structures were generated with the Sybyl/Sketch module and optimized
using Powell’s method with the Tripos force field with convergence
criterion set at 0.05 kcal/(Å mol) and assigned with the Gasteigerꢀ
H€uckel method. Molecular docking was carried out via the Sybyl/FlexX
module. Other docking parameters implied in the program were default
values.
’ ASSOCIATED CONTENT
Supporting Information. 1H NMR spectral information
S
b
of the key intermediate 23, representative target compounds 22a,
22b, 25c, 25e, 25f, 25h, 25l, 25m, 25n, 26a, 26e, 26f, 26h, 34a,
and 34b. HPLC analysis chromatograms of representative target
compounds 22a, 22b, 25c, 25e, 25f, 25h, 25l, 25m, 25n, 26a,
26e, 26f, 26h, 34a, and 34b. This material is available free of
charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: 86-531-88382264. Fax: 86-531-88382264. E-mail: wfxu@
yahoo.cn or haofangcn@sdu.edu.cn.
’ ACKNOWLEDGMENT
This work was supported by National Nature Science Foun-
dation of China (Grant No. 30772654 and No. 90713041) and
National High Technology Research and Development Program
of China (863 project; Grant No. 2007AA02Z314) and National
Science and Technology Major Project (Grant No. 2009-
ZX09103-118).
’ ABBREVIATIONS:
HDAC, histone deacetylase; HDACi, histone deacetylase inhi-
bitors; SAR, structureꢀactivity relationships; SAHA, suberoyla-
nilide hydroxamic acid; ZBG, zinc-binding group; Boc, tert-
butoxycarbonyl
In Vivo Antitumor Assay against MDA-MB-231 Xenograft.
For in vivo antitumor efficacy research, 5 ꢁ 106 human breast cancer
cells (MDA-MB-231) were inoculated subcutaneously in the right flank
of female athymic nude mice (5ꢀ6 weeks old, Slac Laboratory Animal,
Shanghai). Ten days after injection, tumors were palpable and mice were
randomized into treatment and control groups (six mice per group). The
treatment groups received 90 mg/kg/d compounds intraperitioneally
(i.p.), and the blank control group received an equal volume of PBS
solution containing 40% DMSO intraperitioneally. During treatment,
subcutaneous tumors were measured with a vernier caliper every three
days, and body weight was monitored regularly. Tumor volumes (V)
were estimated using the equation (V = ab2/2, where a and b stand for
the longest and shortest diameter, respectively). Relative increment ratio
(T/C) was calculated according to the following formula:
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