Synthesis and biological evaluation of santacruzamate A analogues for anti-proliferative and immunomodulatory activity

Article abstract of DOI:10.1016/j.bmc.2016.08.040

Santacruzamate A (SCA) is a natural product isolated from a Panamanian marine cyanobacterium, previously reported to have potent and selective histone deacetylase (HDAC) activity. To optimize the enzymatic and cellular activity, 40 SCA analogues were synthesized in a systematic exploration of the zinc-binding group (ZBG), cap terminus, and linker region. Two cap group analogues inhibited proliferation of MCF-7 breast cancer cells, with analogous increased degranulation of cytotoxic T cells (CTLs), while one cap group analogue reduced CTL degranulation, indicative of suppression of the immune response. Additional testing of these analogues resulted in reevaluation of the previously reported SCA mechanism of action. These analogues and the resulting structure–activity relationships will be of interest for future studies on cell proliferation and immune modulation.

Full text of DOI:10.1016/j.bmc.2016.08.040

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of santacruzamate A analogues
for anti-proliferative and immunomodulatory activity
Samantha M. Gromek a, James A. deMayo a, Andrew T. Maxwell a, Ashley M. West a, Christopher M. Pavlik a,
Ziyan Zhao b, Jin Li a, Andrew J. Wiemer a, Adam Zweifach b, Marcy J. Balunas a,
⇑
a Division of Medicinal Chemistry, Department of Pharmaceutical Sciences, University of Connecticut, 69 N. Eagleville Rd, Storrs, CT 06269, USA
b Department of Molecular and Cell Biology, University of Connecticut, 91 N. Eagleville Rd, Storrs, CT 06269, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Santacruzamate A (SCA) is a natural product isolated from a Panamanian marine cyanobacterium,
previously reported to have potent and selective histone deacetylase (HDAC) activity. To optimize the
enzymatic and cellular activity, 40 SCA analogues were synthesized in a systematic exploration of the
zinc-binding group (ZBG), cap terminus, and linker region. Two cap group analogues inhibited prolifera-
tion of MCF-7 breast cancer cells, with analogous increased degranulation of cytotoxic T cells (CTLs),
while one cap group analogue reduced CTL degranulation, indicative of suppression of the immune
response. Additional testing of these analogues resulted in reevaluation of the previously reported SCA
mechanism of action. These analogues and the resulting structure–activity relationships will be of inter-
est for future studies on cell proliferation and immune modulation.
Received 27 June 2016
Revised 17 August 2016
Accepted 22 August 2016
Available online xxxx
Keywords:
Santacruzamate A
Natural product analogues
Enzyme assays
Anti-proliferative activity
Immune modulation
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
affinity with DNA and leading to repression of gene transcription.
Regulation of acetylation and deacetylation patterns allows for
Development of new cancer chemotherapeutic compounds is a
fundamentally challenging endeavor requiring considerations at
the in vitro, in vivo, preclinical, and clinical stages. Ideal com-
pounds for drug discovery and development target cancerous cells
while maintaining low toxicity to normal cells, with an ongoing
shift away from discovery of new compounds with broad cytotox-
icity to those with selective activity, including compounds that
specifically regulate gene expression via epigenetic modulation,
that target signaling pathways, or that enhance immune system
function.1,2 Recently, epigenetic modulators such as inhibitors of
DNA methyltransferase (DNMT) and histone deacetylase (HDAC)
have received increased attention as anti-cancer agents, although
their clinical utility has thus far been limited to leukemias and
lymphomas.3,4 Likewise, there has been considerable attention
towards the development of small molecule cancer therapeutics
that affect immune system targets, although there are still very
few currently available agents.5–8
acute control of differentiation, growth arrest, and apoptosis.9–11
Moreover, hyper-deacetylation can result in silencing of tumor
suppression genes, loss of apoptosis, and initiation and propaga-
tion of tumorogenesis.12 Four HDAC inhibitors are currently
approved by the United States Food and Drug Administration (US
FDA) including suberoylanilide hydroxamic acid (SAHA, Vorinos-
tatÒ, Fig. 1), romidepsin (IstodaxÒ), panobinostat (FarydakÒ), and
belinostat (BeleodaqÒ), all of which are approved for patients with
refractory cancers (lymphoma or myeloma).13 HDACs exist in sev-
eral classes and with many isozymes, with current research
focused on the discovery and development of isozyme selective
epigenetic modulators to avoid the side effects inherent in non-
selective inhibitors.14–16
Targeted therapies that induce anti-cancer immunity have been
the focus of intensive research efforts, with recent efforts to dis-
cover new small molecule cancer immunotherapeutics.8 Compo-
nents of the adaptive and innate immune systems are capable of
recognizing and killing cancer cells, initiated by tumor antigens
that activate cytotoxic T lymphocytes (CTLs) through T cell recep-
tors or by cell-surface molecules that activate natural killer cells.17
Although there are several clinically available monoclonal antibod-
ies that modulate immune function, imiquimod (AldaraÒ) is one of
the only small molecule immunomodulators approved for cancer
patients in the US and is limited to cutaneous malignancies.7
HDACs and histone acetyltransferases (HATs) are key enzymes
regulating the packaging of DNA around histones, having extensive
impacts on gene transcription. HDAC enzymes remove acetyl
groups from histone lysine residues, resulting in enhanced binding
⇑
Corresponding author. Tel.: +1 860 486 3051; fax: +1 860 486 6857.
E-mail address: marcy.balunas@uconn.edu (M.J. Balunas).
http://dx.doi.org/10.1016/j.bmc.2016.08.040
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Gromek, S. M.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.040

2
S. M. Gromek et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Figure 1. Santacruzamate A (SCA, 1), SAHA, and the SCA-SAHA hybrid compound reported previously,18 and their overlaid 3D structures (SCA in yellow, SAHA in green, and
SCA-SAHA hybrid in blue).
We initially identified santacruzamate A (SCA, 1, Fig. 1) as a
bioactive natural product isolated from the marine cyanobac-
terium, cf. Symploca sp.18 SCA consists of three structural moieties
O
O
O
H
N
H
N
(iii)
(i)
H2N
N
H
R1
R1
OH
OH
except as
noted in 8
2a-11a
including an ethyl carbamate terminus, a modified c-aminobutyric
acid (GABA) linker, and a phenethylamine cap group and has sev-
eral structural features in common with SAHA (Fig. 1), which led
to an initial investigation of SCA for HDAC inhibition. SCA, SAHA,
and most HDAC inhibitors contain three basic structural motifs
including a zinc-binding group (ZBG), an aliphatic linker, and a sur-
face recognition cap group (Fig. 1). In contrast to SCA, SAHA ends in
a hydroxamate terminus and has a longer 6-carbon linker directly
attached to the phenylamine cap. SAHA binds to HDAC with the
phenyl cap group resting on top of the enzyme pocket, the aliphatic
linker traversing through the small channel, and the hydroxamate
binding to the zinc at the bottom.19 Because most hydroxamic
acids suffer from pan-isozyme activity and several clinical liabili-
ties (e.g., poor oral absorbance, rapid hydrolysis yielding poor
pharmacokinetics, and strong non-specific affinity for metallopro-
teins),20 we were enthusiastic about SCA given the carbamate ter-
minus. With its relatively small, linear modified peptide structure,
SCA is easily amenable to synthetic modifications and advanced
biological evaluation to fully explore structure–activity relation-
ships. In an effort to optimize the enzyme activity and to enhance
the cellular activity, we performed a systematic exploration of the
three chemical regions of the SCA structure including modification
of the zinc-binding group (ZBG), cap terminus, and linker region,
resulting in 40 derivatives reported herein that were evaluated
for anti-proliferative and immunomodulatory activity.
O
O
O
O
O
3
O
O
5
2
4
O
O
O
O
O
F
R 1
=
O
O
O
6
7
8 (ii)
O
S
10
S
O
9
O
11
Scheme 1. Synthesis of santacruzamate A N-carbamate analogues. Reagent and
conditions: (i) ClCO2-R1, K2CO3 or NaOH, THF, 0 °C to rt, 70–90%; (ii) Boc2O, NaOH,
THF, 97%; (iii) phenethylamine, EDC–HCl, TEA, cat. DMAP, CH2Cl2, 43–93%.
N-Carbamate modifications (Scheme 1) included changing the
ethyl carbamate to a methyl (5), a larger tert-butyl (8), elongating
to a propyl (2) and butyl (3), elongating and adding unsaturation (6
and 9), an even larger phenyl (4), fluorination (7), and incorporat-
ing
a thiocarbamate (10 and 11). Carbamate intermediates
(Scheme 1, 2a–5a, 7a–11a) were all prepared via analogous syn-
thetic methodology involving coupling of GABA with the respective
chloroformate.21–23 Compound 8a was formed using standard di-
tert-butyl dicarbonate (Boc2O) protection.24 To prepare compound
10a, prior to coupling with GABA, sodium ethoxide was added to
thiophosgene and reacted to obtain the ethyl chlorothioformate
10a. All resulting carbamates were coupled to phenethylamine
using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC),
trimethylamine (TEA), and catalytic 4-dimethylaminopyridine
(cat. DMAP) yielding analogues 2–11.
2. Results and discussion
2.1. Chemistry
Ten additional ZBG analogues were also prepared. To explore
the possibility of incorporation of bi-dentate binding, compounds
13 and 14 were synthesized (Scheme 2). A simpler urea analogue
was also prepared (12). In another series, the carbamate was
exchanged for amide bonds with various termini (Scheme 3, 15–
19). In addition, due to the potent zinc chelating affinity of the ter-
minal thioester of largazole,25,26 we replaced the carbamate with
an acetylated thiol (Scheme 4, 20). Lastly, in an effort to investigate
the spatial orientation of the ethyl carbamate in the binding pocket
an inverted ethyl carbamate, 21, was synthesized (Scheme 5).
The three urea analogues (Scheme 2, 12–14) were synthesized
via 12a starting from GABA which was dimerized using CS2 to form
a thiourea.27 This thiourea intermediate was either carried through
directly or oxidized to the urea intermediate upon exposure to 30%
H2O2 and KOH to form 12b.28 Intermediate 12b was then con-
verted through to the ethyl urea following literature procedure,27
2.1.1. Synthesis of santacruzamate A zinc-binding group
analogues
Given the known importance of ZBG binding for HDAC inhibi-
tion, the first structural modifications we made were to the carba-
mate terminus of SCA (Schemes 1–5). With SAHA, the hydroxamate
allows for bi-dentate binding of zinc between the deprotonated ter-
minal hydroxyl and the carbonyl oxygen with the catalytic pocket
(see Fig. 3 and Section 2.3 for more details). This bi-dentate binding
facilitates the strong metal binding characteristics of hydroxamate
bearing compounds. Herein, SCA is hypothesized to exhibit mono-
dentate coordination to the zinc and thus modification centered
on adjusting the electronics around the carbonyl oxygen of the
SCA carbamate. For this first phase we completed the synthesis of
20 compounds that differ in their ZBG terminus, leaving the three
carbon aliphatic linker and phenethylamine cap group unaltered.
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S. M. Gromek et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
O
O
R' = S = 12a
R' = O = 12b
O
H
N
H
N
(i)
(ii)
H2N
HO
NO
OH
OH
O
R'
O
O
H
N
H
N
(iii)
(v)
H
(iv)
N
N
12b
12d
OH
OH
12d
12c
O
O
O
H
H
N
N
N
H
12
O
O
O
H
H
(vi)
N
N
(vii)
N
N
12a
or 12b
OH
N
H
O
R'
O
R'
R' = S = 13a
R' = S = 13
R' = O = 14
R' = O = 14a
Scheme 2. Synthesis of santacruzamate A urea analogues. Reagents and conditions: (i) CS2, NaOH, H2O; (ii) 30% H2O2, KOH, H2O; (iii) KNO2, diluted H2SO4, 0 °C to rt; (iv) 68%
ethylamine/H2O, 100 °C; (v) phenethylamine, EDC–HCl, TEA, cat. DMAP, CH2Cl2, 0 °C to rt; (vi) 12a or 12b, cat. p-TsOH, NEAT 180 °C, 30 torr; (vii) 13a or 13b, phenethylamine,
EDC–HCl, TEA, cat. DMAP, CH2Cl2, 0 °C to rt.
O
O
O
(i, ii, or iii)
(iv)
H
N
H
N
H2N
R1
N
H
R1
O
OH
O
OH
15a -19a
R1
=
O
16 (ii)
17 (ii)
O
O
19 (iii)
18 (iii)
15 (i)
Scheme 3. Synthesis of santacruzamate A N-amide analogues. Reagents and conditions: (i) Ac2O, AcOH, Et2O; (ii) propionic anhydride or butyric anhydride, cat. H2SO4,
100 °C; (iii) R2-COCl, NaOH, THF/H2O, 0 °C to rt; (iv) phenethylamine, EDC–HCl, TEA, cat. DMAP, CH2Cl2.
O
O
bond in compounds 16a and 17a were generated via reaction with
butyric or propionic anhydride with cat. H2SO4, respectively. As
with the carbamates, the phenethylamine moiety was incorpo-
rated via EDC coupling to yield compounds 15–19.
To form compound 20 (Scheme 4), S-2-(chlorocarbonyl)ethyl
ethanethioate 20a was prepared by adding thioacetic acid to
acrylic acid, followed by addition of thionyl chloride following lit-
erature procedure.32 Intermediate 20a was then reacted with
GABA followed by subsequent coupling to phenethylamine as
described above. The inverted carbamate 21 (Scheme 5) was made
(i followed by ii)
OH
O
S
Cl
20a
O
H
O
(iii)
S
N
H2N
OH
OH
O
O
20b
O
H
(iv)
S
N
20b
N
H
O
O
20
Scheme 4. Synthesis of santacruzamate A thiol analogue. Reagents and conditions:
(i) thioacetic acid, 0 °C to rt; (ii) thionyl chloride, DCM, refluxed for 2 h at 40 °C; (iii)
20a, H2O, Na2CO3, 0 °C to rt; (iv) phenethylamine, EDC–HCl, TEA, cat. DMAP, CH2Cl2.
via opening c-butyrolactone with methanol and TEA to form a ter-
minal hydroxyl which was then converted to the active carbonate
via reaction with N,N0-disuccinimidyl carbonate and subsequently
reacted with aqueous ethylamine to form the inverted ZBG termi-
nus. The final step involved routine peptide coupling with
phenethylamine as described above.
which was subsequently coupled to phenethylamine to afford 12.
Compounds 13 and 14 were prepared by heating intermediates
12a or 12b with cat. p-TsOH at 30 mm Hg to form the cyclized
pyrrolidone compounds.27
For synthesis of N-amide compounds 15a–19a, each new func-
tionality was introduced as the acyl chloride via a modified Schot-
ten–Baumann reaction (Scheme 3).29–31 Formation of the N-amide
O
O
H
N
H
N
O
(i)
(ii)
O
O
R1
OH
H2N
except as
noted in 31
OH
22
O
O
___________________________________________________________________________
H
N
H
N
H
N
N
H
N
N
O
NHBoc
O
O
O
O
S
NO2
Cl
25
S
23
24
26
(i)
(ii)
O
O
O
O
HO
N
O
O
H
H
O
H
N
H
N
N
N
O
O
21a
21c
21b
R1
=
OTBS
30
29
OTBS
Cl
O
27
28
H
N
H
N
(iii)
(iv)
O
HN
H
N
21b
O
N
H
O
F
O
21
O
O
31 (iii)
33
32
Scheme 5. Synthesis of santacruzamate A inverted ethyl carbamate analogue.
Reagents and Conditions: (i) TEA, MeOH, reflux, 92%; (ii) methyl-4-hydroxybu-
tanoate, N,N0-disuccinimidyl carbonate, TEA, 79%; (iii) NHS-methyl-4-hydroxybu-
tanoate, 68% ethylamine, TEA, THF; (iv) phenethylamine, EDC–HCl, TEA, DMAP,
CH2Cl2, 56%.
Scheme 6. Synthesis of santacruzamate
A cap group analogues. Reagent and
conditions: (i) ethyl chloroformate, K2CO3, H2O, 0 °C to rt; (ii) cap-group R1,
EDC–HCl, TEA, cat. DMAP, CH2Cl2; (iii) EDC–HCl, HOBt, TEA, phenethyl-OH, DCM,
0 °C to rt.
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4
S. M. Gromek et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
2.1.2. Synthesis of santacruzamate A cap group analogues
The cap group has been theorized to be the initial sight of dock-
ing with the linker fitting into a hydrophobic groove that then ori-
ents the ZBG into its proper chelation position.4 On the cap end of
SCA several modifications were prepared (Scheme 6), while keep-
ing the GABA linker and ethyl carbamate of SCA unaltered. Two
of these modifications removed the phenyl ring altogether (23
and 24). Three compounds with chlorination or fluorination at var-
ious positions were expected to provide electron withdrawing
capacity (29, 30, 33). Additional modifications included replace-
ment of the amide bond with an ester (31), replacement of the phe-
Bioscience, Inc., San Diego, CA) with fluorogenic HDAC assay kits
from Active Motif (Carlsbad, CA). Samples of SCA (1) natural pro-
duct isolate and the synthetic compound from our previous
report18 were analyzed for compound stability prior to use for bio-
logical testing. Due to limited sample availability, SCA-SAHA
hybrid was re-synthesized to afford sufficient material for biologi-
cal evaluation. During our HDAC2 testing of these new analogues
we were unable to repeat the previous levels of activity (data not
shown) due to high variability in the assay results, possibly due
to variations in enzyme preparations.35 Therefore, we sent our
compounds to Reaction Biology Corp. (Malvern, PA) for testing in
their HDAC2 assay. Compounds were all screened in duplicate at
nethyl with
a thiazole ring (25), and replacement of the
phenethylamine with a methylated phenylalanine (32). Compound
26 was prepared to provide a larger terminus that was previously
shown to have antimalarial activity and HDAC inhibition.33 Com-
pounds 27 and 28 provided information regarding the effects of
an OTBS group either directly on the phenyl ring or one carbon
away.
1 lM and found to be inactive (Table 1), in contrast to our original
report of HDAC2 inhibition by SCA (1).18
These unexpected results led to further testing to attempt to
understand the discrepancies, including testing both synthetic
SCA (1) and the SCA-SAHA hybrid (Fig. 1) for activity on all of the
HDAC isozymes (Table 2). Both compounds were tested by BPS Bio-
Cap derivatives were all synthesized starting from GABA, with
conversion to the linked ethyl carbamate following standard acyla-
tion conditions via reaction with ethyl chloroformate using excess
potassium carbonate in water to afford intermediate 22. All cap
derivatives except 31 were coupled to their respective free acids
via previously described EDC coupling procedures. Preparation of
compound 31 utilized a PyBOP/HOBt mediated-coupling.34
sciences against HDACs 1–11 at two concentrations (10
M), with either SAHA or TSA (trichostatin A, a known HDAC
inhibitor) as positive controls. Santacruzamate A (1, synthetic)
inhibited all 11 HDACs at 10 M, with little to no inhibition at
M, indicating IC50 values for these isozymes all lie in the 5–
M range. The SCA-SAHA hybrid was also found to inhibit most
of HDAC isozymes at 10 M, with the exception of HDAC5 and
HDAC 7. At 5 M, the hybrid compound still had an inhibitory
lM and
5
l
l
5
10
l
l
l
l
2.1.3. Synthesis of santacruzamate A linker analogues
effect on HDAC6 (59% inhibition), but was inactive against the
Structure–activity investigation of the linker region involved
two specific types of modification (Scheme 7). The first of these
modified the length of the linker from 1 to 5 carbons (34–37),
while keeping the phenethylamine cap group and the ethyl carba-
mate linker unaltered. For the other type of linker modifications,
the overall chain length of the molecule was kept constant while
placement of the amide bond was varied from directly adjacent
to the phenyl (39) to one carbon from the carbamate (41) as well
as variations in between (38, 40). In addition, an analogue was pre-
pared without the GABA linker, shortening the compound to the
phenethylamine directly coupled to the ethyl carbamate (42). Syn-
thesis of intermediates 34a-41a was accomplished using commer-
cially available linker acids coupled with ethyl chloroformate
following standard acylation conditions using excess potassium
carbonate, followed by EDC coupling of the phenyl amines of var-
ious chain lengths to afford final compounds 34–41. For compound
42, the phenethylamine was coupled to ethyl chloroformate using
the standard acylation conditions described above.
other HDAC isozymes, indicative of IC50 values between 5 and
10
l
M for HDACs 1–4 and 8–11, IC50 values > 10
lM for HDACs 5
and 7, and an IC50 value < 5
lM for HDAC6. To explore other com-
mon epigenetic targets, SCA (1) was also tested for inhibition of sir-
tuins SIRT1-3 and SIRT5 (NAD+-dependent deacetylases) as well as
inhibition of DNA-methyltransferases DNMT1, DNMT3A/3L, and
DNMT3B/3L and found to be inactive at both 10 lM and 5 lM.
Due to the differences in enzymatic activity from our initial
reports to these current results, we elected to perform more bio-
logically relevant, Western blot analyses to determine the ability
of SCA (1) to cause changes in the histone acetylation status of
H3K9, H3K23, and H4K12 (Fig. 2), chosen as a representative small
panel of histone acetylation sites. Using the Molt-4 cell line, syn-
thetic SCA was not found to have an effect on acetylation of these
histones at 200
icant histone acetylation at 2.5
matic inhibition of HDACs by SCA at 10
l
M, as compared with SAHA, which induced signif-
M. Thus, although there was enzy-
M, it appears that this
l
l
does not translate to cellular inhibition of HDAC activity.
As a complement to HDAC screening, all analogues were also
screened for activity against several solid tumor and lymphoma
2.2. Biological evaluation
cell lines (Table 1). Initial screening was performed at 50 lM with
All analogues were tested in our laboratory for activity against
HDAC2 using newly purchased human recombinant enzyme (BPS
GI50 values obtained for active compounds. Compounds 27 and 28
were found to have activity against MCF-7 cells with GI50 values of
O
H
O
H
N
O
O
N
(
)m
(i)
(ii)
O
( )n
N
H
H2N
( )n
OH
( )n OH
O
O
(34a-41a)
(34-41)
34 n=1, m=2;
35 n=2, m=2;
36 n=4, m=2;
37 n=5, m=2;
38 n=4, m=1;
39 n=5, m=0;
40 n=2, m=4;
41 n=1, m=4;
H
N
NH2
O
(iii)
O
42
Scheme 7. Synthesis of santacruzamate A linker analogues. Reagents and conditions: (i) ethyl chloroformate, K2CO3, H2O, 0 °C to rt; (ii) phenethylamine, aniline,
benzenemethanamine, or benzenebutanamine, EDC–HCl, TEA, cat. DMAP, CH2Cl2, 0 °C to rt; (iii) ethyl chloroformate, TEA, CH2Cl2.
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S. M. Gromek et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
Table 1
Biological evaluation of santacruzamate A (1) analogues using enzymatic and cellular assays
Compd
Enyzmea
HDAC2
Solid Tumor Cellsb
MCF-7
Lymphoma Cellsb
HuT-78 Molt-4
PBMCb
CTLc % CD3
HCT116
MDA-MB-231
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
23
24
25
26
27
28
29
30
31
32
33
34
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
17.2
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
23.7
13.8
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
ntd
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
ntd
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
ntd
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
36.0
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
7.8
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
ntd
94
101
79
81
87
92
82
105
105
76
106
94
95
83
91
101
73
85
77
100
88
95
91
58
126
171
85
75
88
90
90
86
91
83
98
35
36
37
38
39
40
41
42
93
ntd
83
81
90
SCA (1)e
SCA (1)f
SCA-SAHAg
SAHAh
Doxorubicinh
ntd
77
>50
>50
29.1
>50
>50
>50
>50
>50
>50
113
21.4
10.7
a
b
c
IC50 values [
GI50 values [
l
l
M].
M].
% CD3-stimulated anti-LAMP fluorescence in cytotoxic T lymphocytes (CTLs), screened at 100
nt = not tested.
Santacruzamate A (1), natural product. Previously reported values: HDAC2 IC50 0.119 nM; HCT116 GI50 29.4
Santacruzamate A (1), synthetic. Previously reported values: HDAC2 IC50 0.112 nM; HCT116 GI50 28.3
SCA-SAHA hybrid. Previously reported values: HDAC2 IC50 3.5 nM; HCT116 GI50 2.3
SAHA and doxorubicin were utilized as positive controls for solid tumor cell assays.
lM.
d
e
f
l
M; HuT-78 GI50 1.4 l
M.18
l
M; HuT-78 GI50 1.3
l
M.18
g
h
l
M; HuT-78 GI50 0.7
l
M.18
Table 2
Inhibition of the Zn2+ dependent HDACs 1-11 by santacruzamate A (1) and the SCA-SAHA hybrid
Isozyme
SCA
SCA-SAHA Hybrid
SAHAa
10
TSAa
10
lM
5
lM
10
l
M
5
lM
l
M
100 lM
HDAC1
HDAC2
HDAC3
HDAC4
HDAC5
HDAC6
HDAC7
HDAC8
HDAC9
HDAC10
HDAC11
90
0
7
6
0.5
0.5
10 2.5
86 0.5
76 0.5
31 0.5
17 1.0
36 1.0
100 0.3
99 0.5
99 0.7
80 1.5
89 1.0
86 2.0
86
0
4
2.5
0
80 1.0
49 3.0
6
0
94 1.0
95 1.5
66
0
0
0
0.5
83 1.5
74 1.0
73 1.5
75
93
16 2.5
0
5
10
16
0
85
0
59 1.0
100
100
0
0
8.5
8.0
0
0
2.5
47 0.5
70 2.5
70 0.5
82 0.5
72 0.5
29
0
88 0.9
90 1.9
92 1.8
0
3.0
0
0
14 0.5
35
24 0.5
0
78 0.5
98 0.5
a
SAHA and TSA were utilized as positive controls. Values expressed as a % inhibition SEM.
Please cite this article in press as: Gromek, S. M.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.040

6
S. M. Gromek et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
sis that measures immunological activity of small molecules
[insufficient material of the SCA natural product isolate (1) was
available for this screen] (Table 1).39 Both anti-proliferative ana-
logues, 27 and 28, were found to increase lytic granule exocytosis
(26% and 71% augmentation of CD3-stimulated anti-LAMP fluores-
cence increases, respectively), corresponding to their levels of anti-
proliferative activity and indicative of enhancement of the immune
response. Compound 26, a cap group analogue with an extended
terminus and an electron-withdrawing nitro group, was found to
decrease granule exocytosis by 58%, indicative of suppression of
the immune response.
Figure 2. Effect of santacruzamate A and SAHA (VorinostatÒ, abbrevieated VOR
above) on the levels of acetylated histones H3K9, H3K23, and H4K12 in Molt-4 cells.
2.3. Molecular modeling
Due to the structural similarity of SCA and SAHA but the diver-
gence in enzymatic activity, we investigated the chemical differ-
ences in HDAC2 binding via molecular modeling. The primary
structural difference between SAHA and SCA involves the incorpo-
ration of a hydroxamate ZBG terminus for SAHA, which includes
carbonyl and hydroxyl oxygen moieties allowing for bidentate
chelation of the zinc ion via an octahedral metal geometry. Our
molecular model utilizes a previously published X-ray crystal
structure of HDAC2 with SAHA (PDB: 4LXZ)40 and overlays SCA
(1) within the existing binding pocket (Fig. 3). The model depicts
the optimal octahedral binding metal geometries of the zinc ion
in the HDAC2 binding pocket shown as yellow arrows (Fig. 3).
The zinc ion is displayed as a 6-coordinated ion with three binding
geometries filled by His183, Asp269 and Asp181, starting from the
left and proceeding counter clockwise. The remaining geometries
are labeled as potential ligand binding geometries, denoted as G0,
G00, and G000, starting from the left and proceeding clockwise.
In this model, SAHA is able to fill G0 and G00 but the zinc ion
would require an addition electron density donor. The binding
pocket is accessible to water molecules, which are believed to play
a role during HDAC2 catalytic deacetylation,41 suggesting that a
water molecule may occupy the remaining ligand binding location.
The ability of SAHA to fill G0 and G00 is facilitated in part by the pla-
nar geometry of the hydroxamate moiety and the rotatable bond
between C8 and C9. In addition to SAHA chelating the zinc ion,
potential hydrogen bonding further stabilizes the interaction with
HDAC2, including between Asp104 and the amide nitrogen nearest
to the phenyl cap, between His146 and the terminal hydroxamate
nitrogen, and between His145 and the terminal hydroxyl oxygen
(Fig. 3). The two terminal hydrogen bonds may form hydrogen
bonding and hydrogen shuttling networks by sharing or fully
23.7 and 13.8 lM, respectively (Table 1). Both compounds are tert-
butyldimethylsilyloxy (OTBS) cap group analogues with intact SCA
ethyl carbamate and GABA-derived linker groups. No activity was
found for synthetic compounds using HCT116 colon cancer cells
or MDA-MB-231 triple-negative breast cancer cells. Synthetic ana-
logues were also found to be inactive against HuT-78 cutaneous T
lymphoma cells, Molt-4 acute lymphoblastic leukemia cells, and
against non-cancerous peripheral blood mononuclear cells (PBMC).
Interestingly, the SCA natural product isolate (1) was found to have
activity against HCT116, HuT-78, and Molt-4 cells with GI50 values
of 17.2, 36.0, and 7.8 lM, respectively, indicative of a possible con-
taminant in the natural product sample, since the synthetic SCA
did not possess similar activity (this is in contrast to our previous
report in which the synthetic and natural product SCA were
equipotent, possibly due to experimental variations such as cell
density and/or media type). This contaminant would likely possess
potent anti-proliferative activity, given that the SCA isolate was
found to be 98.5% pure, as measured via LC–MS (Fig. S3A in Supple-
mentary material), and thus the contaminant would have to be
present in very low quantities, but with sufficient potency to be
able to cause biological activity. Given that the potent cytotoxic
dolastatins have previously been reported from several Symploca
species,36,37 we investigated the SCA natural product LC–MS and
found the main impurity to have an [M+H]+ of 774.5 (Fig. S3B in
Supplementary material), possibly consistent with dolastatin
17,38 although insufficient sample is available to confirm.
In addition to anti-proliferative activity, we were also interested
in the ability of these compounds to modulate the immune
response. Therefore, we tested all synthetic analogues in a pheno-
typic screen of cytotoxic T lymphocyte (CTL) lytic granule exocyto-
Figure 3. X-ray crystal structure of SAHA (green, left) bound to HDAC2 (gray, PDB: 4LXZ40), with santacruzamate A (1) (yellow, right) overlaid over the binding conformation
of SAHA with HDAC2. The zinc ion (dark gray) within the HDAC2 active site is shown with an idealized octahedral metal geometry (idealized metal geometries of the zinc ion
are shown as yellow arrows and geometries not filled by HDAC2 are labeled G0, G00, G000 and proceed clockwise starting from left) using the Chimera program.45 SAHA binds to
the zinc ion via chelation with G0 and G00, while santacruzamate A likely binds only to G0. Hydrogen bonds are depicted as orange lines. The surface of the HDAC2 active site,
within 4.1 Å of the ligands, was generated via Chimera and colored according to the corresponding heteroatom on enzyme surface.
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S. M. Gromek et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
transferring the hydrogen species between themselves and Asp186
and Asp179 adjacent to His146 and His145 respectively (see Fig. S2
in Supplementary material).42 The delocalization of electron den-
sity via hydrogen bonding and hydrogen shuttling within this net-
work may also further stabilize and enable the terminal oxygen
species to donate electron density to the zinc ion.
from a Baker cycle-tainer system. All glassware was flame-dried
under vacuum, and all reactions performed under an argon atmo-
sphere, unless otherwise noted. Flash chromatography performed
using a 60 Å porosity, 32–63 lM silica gel. NMR spectra were
obtained with a Bruker AVANCE 500 MHz spectrometer and analy-
sis was performed on ACD/Spectrus processor 2012. HRMS per-
formed at the University of Connecticut Mass Spectrometry
Facility on an AccuTOF (JEOL) and using DART (IonSense) ioniza-
tion source. Melting point was collected on a Mel-temp digital
melting point apparatus. LC–MS data collected on an Agilent ESI
single quadrupole mass spectrometer coupled to an Agilent 1260
HPLC system with a G1311 quaternary pump, G1322 degasser,
and a G1315 diode array detector using an Eclipse XDB-C18
Unlike SAHA, the terminal oxygen species of SCA (1) appears to
only allow for monodentate chelation (Fig. 3). For the metal geom-
etry to remain octahedral during SCA binding, an additional water
molecule would be required. This additional water molecule may
prevent SCA from obtaining a favorable binding orientation to
the zinc ion, although it may also be possible that without a biden-
tate chelator, the zinc ion may exist in a 5-coordination state.42 The
inability of SCA to form a favorable orientation to facilitate chela-
tion may be further inhibited by the extended planar geometry
of the terminal ethyl carbamate, potentially impeding the carbonyl
oxygen from obtaining optimal orientation. Another issue with SCA
binding involves the inability to stabilize the molecule in the bind-
ing pocket via hydrogen bonding with Asp104, His146, and His
145, since these residues are not situated properly when the carba-
mate terminus is coordinated with the zinc ion (Fig. 3). In addition,
since the terminal carbamate moiety and linker of SCA resembles
that of acetylated lysine, it may be possible that SCA acts as a sub-
strate and undergoes catalysis,41 and thus does not bind as a tradi-
tional HDAC inhibitor.
(4.6 ꢀ 150 mm, 5
lm) column using a gradient of 40–100% ace-
tonitrile with 0.1% formic acid over 30 min at a flow rate of
0.7 mL/min. Structural integrity of final target compounds were
determined by 1H and 13C NMR, melting point, and HRMS. Purity
of final target compounds were determined to be >95% by LC–MS.
4.2. Synthesis
4.2.1. General procedure for the preparation of final target
compounds
The intermediate free acid (1 equiv) was dissolved in CH2Cl2
(7 mL) followed by the addition of triethylamine (2.2 equiv). The
reaction was cooled to 0 °C and stirred for 30 min. Then phenethy-
lamine (1.1 equiv) and 4-dimethylaminopyridine (DMAP) (cat.,
0.1 equiv) were added to the solution followed by 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide hydrochloride (EDC–HCl)
(1.1 equiv) in five portions over 20 min. The solution stirred at
0 °C for 1 h then overnight at rt. The resulting solution was dis-
solved in an additional CH2Cl2 (25 mL), and washed with sat. NH4-
Cl (10 mL). The aqueous layer was extracted with CH2Cl2
(3 ꢀ 10 mL), organic layers combined, and sequentially washed
with 20 mL of each of the following: sat. NaHCO3, H2O, brine. The
organic layer was dried over Na2SO4 and concentrated to give a
residue which was further purified with Celite (100% ethyl acet-
ate). Then, the sample was dissolved in EtOAc, solution was heated,
and cooled to 0 °C. The solution was filtered to yield desired
product.
3. Conclusion
To optimize the enzymatic and cellular activity of santacruza-
mate A (1), we systematically altered the scaffold including chang-
ing the zinc-binding group, cap terminus, and linker regions. In the
process, we found enzymatic data that contrasted with our previ-
ously reported data, thus necessitating additional exploration of
biological activity and resulting in new data on this scaffold. Previ-
ous studies have shown that enzyme assays can be complicated by
numerous variables including source of enzyme, pH, temperature,
buffers, solvents, substrates, preparation of enzyme, concentra-
tions of assay components, time, among other variables.35 In addi-
tion, the need for fluorogenically labeled substrates can also affect
results, potentially leading to false-positive and/or false-negative
results.43 Also, HDAC assays rely on recombinant enzymes that
are isolated from their partner proteins and thus unlikely to exist
in their native conformations, resulting in an inability to translate
the results from these enzymatic assays to physiologically relevant
effects.44
4.2.2. Zinc-binding group analogues
4.2.2.1. Ethyl 3-(phenethylcarbamoyl)propylcarbamate (1)C. om-
pound
1
was synthesized following previously reported
procedure.18
We therefore followed up with substantial cellular testing in
solid tumor and lymphoma cell lines as well as molecular biologi-
cal tools to determine the effects of santacruzamate A and ana-
logues on anti-proliferative and immunomodulatory activity. In
these follow-up assays, several analogues were determined to be
biologically active including two silylated derivatives, compounds
27 and 28, which were found to exhibit both anti-proliferative
and immune stimulation activity, as well as compound 26 which
showed immune suppression activity. These compounds and the
resulting structure–activity relationships are of interest for future
studies and the molecular models that were reported herein will
be useful in guiding development of additional analogues.
4.2.2.2. Propyl (4-oxo-4-(phenethylamino)butyl)carbamate (2).
Converted to the N-phenylacetamide derivative using the general
procedure described and with the following specifications and alter-
ations: 2a (0.33 g, 1.71 mmol), TEA (0.58 mL, 3.78 mmol), phenethy-
lamine (0.24 mL, 1.89 mmol), DMAP (0.021 g, 0.17 mmol), EDC–HCl
(0.36 g, 1.89 mmol). Product was obtained as a white solid (yield:
0.42 g, 84%). Mp 98–99 °C. 1H NMR (500 MHz, CDCl3) d 7.12–7.25
(m, 5H), 6.00 (br s, 1H), 4.95 (br s, 1H), 3.92 (t, J = 6.7 Hz, 2H), 3.44
(q, 2H), 3.09 (q, J = 6.3 Hz, 2H), 2.75 (t, J = 7.0 Hz, 2H), 2.19 (t,
J = 7.0 Hz, 2H), 1.72 (pentet, 2H), 1.55 (sextet, J = 7.0 Hz, 2H), 0.85
(t, J = 7.4 Hz, 3H). 13C NMR (125 MHz, CDCl3) d 172.5, 157.2, 138.8,
128.7, 128.5, 126.4, 66.4, 40.6, 40.1, 35.6, 33.6, 26.1, 22.3, 10.3.
DART-HRMS (m/z) calculated for C16H25N2O3 [M+H]+ 293.1865,
found 293.1861.
4. Experimental section
4.1. General
4.2.2.3.
(3).
Butyl
(4-oxo-4-(phenethylamino)butyl)carbamate
Converted to the N-phenylacetamide derivative using the
All reagents and chemicals were purchased from Sigma–Aldrich
or Acros and used directly. Hexane, tetrahydrofuran (THF), diethyl
ether (Et2O), and dichloromethane (CH2Cl2) were used directly
general procedure described and with the following specifications
and alterations: 3a (0.35 g, 1.71 mmol), TEA (0.58 mL, 3.78 mmol),
phenethylamine (0.24 mL, 1.89 mmol), DMAP (0.02 g, 0.17 mmol),
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8
S. M. Gromek et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
EDC–HCl (0.36 g, 1.89 mmol). Product was obtained as a white solid
(yield: 0.41 g, 78%). Mp 88–89 °C. 1H NMR (500 MHz, CDCl3) d 7.12–
7.26 (m, 5H), 5.96 (br s, 1H), 4.91 (br s, 1H), 3.96 (t, J = 6.7 Hz, 2H),
3.44 (q, 2H), 3.10 (q, J = 6.2 Hz, 2H), 2.75 (t, J = 7.1 Hz, 2H), 2.10 (t,
2H), 1.72 (pentet, J = 6.8 Hz, 2H), 1.51 (pentet, 2H), 1.30 (sextet,
J = 7.5 Hz, 2H), 0.85 (t, J = 7.4 Hz, 3H). 13C NMR (125 MHz, CDCl3) d
172.7, 157.5, 139.1, 128.9, 128.8, 126.7, 64.9, 40.8, 40.3, 35.8, 33.9,
31.3, 26.4, 19.3, 13.9. DART-HRMS (m/z) calculated for C17H27N2O3
[M+H]+ 307.2022, found 307.2044.
2H), 3.22 (q, J = 6.4 Hz, 2H), 2.85 (t, J = 7.0 Hz, 2H), 2.20 (t,
J = 7.0 Hz, 2H), 1.84 (pentet, J = 6.8 Hz, 2H). 13C NMR (125 MHz,
CDCl3) d 172.6, 156.6, 139.0, 128.9, 128.8, 126.7, 82.7, 64.1, 40.8,
40.5, 35.8, 33.8, 26.0. DART-HRMS (m/z) calculated for C15H22FN2-
O3 [M+H]+ 297.1614, found 297.1638.
4.2.2.8. tert-Butyl (4-oxo-4-(phenethylamino)butyl)carbamate
(8).
Converted to the N-phenethylamine derivative using the
general procedure described and with the following specifications
and alterations: 8a (0.25 g, 1.23 mmol) dissolved in CH2Cl2 (3 mL),
TEA (0.34 mL, 2.46 mmol), phenethylamine (0.17 mL, 1.35 mmol),
DMAP (0.01 g, 0.08 mmol), EDC–HCl (0.26 g, 1.35 mmol). Product
was obtained as a white solid (yield: 0.31 g, 75%). Mp 94–95 °C.
1H NMR (500 MHz, CDCl3) d 7.13–7.25 (m, 5H), 5.97 (br s, 1H),
4.66 (br s, 1H), 3.45 (q, J = 6.9 Hz, 2H), 3.04 (d, J = 6.3 Hz, 2H),
2.76 (t, J = 6.9 Hz, 2H), 2.09 (t, J = 7.3 Hz, 2H), 1.70 (pentet, 2H),
1.36 (s, 9H). 13C NMR (125 MHz, CDCl3) d 172.5, 158.2, 138.9,
128.8, 128.6, 126.5, 79.3, 40.6, 39.7, 35.6, 33.7, 28.4, 26.4. DART-
HRMS (m/z) calculated for C17H27N2O3 [M+H]+ 307.2022, found
307.2012. Data matched that reported previously.46
4.2.2.4.
(4).
Benzyl
3-(phenethylcarbamoyl)propylcarbamate
Converted to the N-phenylacetamide derivative using the
general procedure described and with the following specifications
and alterations: 4a (0.1 g, 0.42 mmol) dissolved in CH2Cl2 (2 mL),
TEA (0.12 mL, 0.83 mmol), phenethylamine (0.06 mL, 0.48 mmol),
DMAP (0.005 g, 0.04 mmol), and EDC–HCl (0.09 g, 0.48 mmol). Pro-
duct was obtained as a white solid (yield: 0.14 g, 95%). Mp 115–
116 °C.1H NMR (500 MHz, CDCl3) d 7.20–7.38 (m, 10H), 5.91 (br s,
1H), 5.10 (s, 2H), 5.09 (br s, 1H), 3.52 (q, J = 6.7 Hz, 2H), 3.22 (q,
J = 6.3 Hz, 2H), 2.83 (t, J = 7.1 Hz, 2H), 2.18 (m, 2H), 1.82 (pentet,
J = 6.8 Hz, 2H). 13C NMR (125 MHz, CDCl3) d 172.6, 156.9, 138.9,
136.6, 128.8, 128.6, 128.5, 128.1, 128.1, 126. 5, 66.7, 40.7, 40.4,
35.7, 33.7, 26.1. DART-HRMS (m/z) calculated for C20H25N2O3 [M
+H]+ 341.1865, found 341.1832.
4.2.2.9. Prop-2-yn-1-yl (4-oxo-4-(phenethylamino)butyl)carba-
mate (9).
Converted to the N-phenylacetamide derivative
using the general procedure described and with the following
specifications and alterations: 9a (0.32 g, 1.71 mmol), TEA
(0.58 mL, 3.78 mmol), phenethylamine (0.24 mL, 1.89 mmol),
DMAP (0.021 g, 0.171 mmol), EDC–HCl (0.36 g, 1.89 mmol). Pro-
duct was obtained as a white solid (yield: 0.32 g, 65%). Mp 86 °C.
1H NMR (500 MHz, CDCl3) d 7.10–7.26 (m, 5H), 5.86 (br s, 1H),
5.21 (br s, 1H), 4.59 (d, 2H), 3.44 (q, 2H), 3.13 (q, 2H), 2.75 (t,
J = 7.0 Hz, 2H), 2.38 (t, 1H), 2.11 (t, 2H), 1.74 (pentet, 2H). 13C
NMR (125 MHz, CDCl3) d 172.7, 156.1, 139.0, 128.9, 128.8, 126.7,
78.5, 74.7, 52.6, 40.8, 40.6, 35.8, 33.8, 26.0. DART-HRMS (m/z) cal-
culated for C16H21N2O3 [M+H]+ 289.1552, found 289.1578.
4.2.2.5.
(5).
Methyl
3-(phenethylcarbamoyl)propylcarbamate
Converted to the N-phenylacetamide derivative using
the general procedure described and with the following specifica-
tions and alterations: 5a (0.23 g, 1.43 mmol) dissolved in CH2Cl2
(10 mL), phenethylamine (0.2 mL, 1.64 mmol), TEA (0.4 mL,
2.12 mmol), DMAP (0.01 g, 0.08 mmol), EDC–HCl (0.31 g,
1.64 mmol). Product was obtained as a white solid (yield: 0.35 g,
94%). Mp 109–110 °C.1H NMR (500 MHz, CDCl3) d 7.21–7.36 (m,
5H), 6.27 (br s, 1H), 5.34 (br s, 1H), 3.66 (s, 3H), 3.53 (q, 2H),
3.19 (q, J = 5.9 Hz, 2H), 2.84 (t, J = 7.2 Hz, 2H), 2.20 (t, J = 7.1 Hz,
2H), 1.82 (pentet, J = 6.8 Hz, 2H). 13C NMR (125 MHz, CDCl3) d
172.7, 157.6, 138.9, 128.7, 128.6, 126.5, 52.0, 40.7, 40.3, 35.7,
33.7, 26.1. DART-HRMS (m/z) calculated for C14H21N2O3 [M+H]+
265.1552, found 265.1543.
4.2.2.10. O-ethyl (4-oxo-4-(phenethylamino)butyl)carbamoth-
ioate (10).
Converted to the N-phenylacetamide derivative
using the general procedure described and with the following
specifications and alterations: 10a (0.5 g, 2.62 mmol) dissolved in
CH2Cl2 (10 mL), TEA (0.73 mL, 5.23 mmol), phenethylamine
(0.38 mL, 3.0 mmol), DMAP (0.03 g, 0.25 mmol), EDC–HCl (0.57 g,
3.0 mmol). Crude material was washed sequentially with 20 mL
of the following solutions: 1 M HCl, H2O, NaHCO3, and brine.
Resulting material was purified using SiO2 plug and titrated over-
night with hexanes. Product was obtained as a shimmering white
solid (yield: 0.7 g, 91%). 1H NMR (500 MHz, CDCl3) d 7.20–7.35
(m, 5H), 7.12 (br s, 1H), 5.79 (br s, 1H), 4.47 (q, 2H), 3.52–3.60
(m, 2H), 3.31 (q, J = 6.5 Hz, 2H), 2.84 (t, J = 7.0 Hz, 2H), 2.25 (t,
2H), 1.94 (pentet, 2H), 1.32 (t, 3H). 13C NMR (125 MHz, CDCl3) d
190.7, 172.8, 138.9, 128.9, 128.6, 126.8, 66.4, 45.11, 40.89, 35.77,
34.15, 24.2, 14.5. DART-HRMS (m/z) calculated for C15H23N2O2S
[M+H]+ 295.1480, found 295.1506.
4.2.2.6.
(6).
Allyl
(4-oxo-4-(phenethylamino)butyl)carbamate
Converted to the N-phenylacetamide derivative using
the general procedure described and with the following specifica-
tions and alterations: 6a (0.37 g, 2.0 mmol) dissolved in CH2Cl2
(5 mL), TEA (0.56 mL, 4.0 mmol), phenethylamine (0.28 mL,
2.2 mmol), DMAP (0.02 g, 0.2 mmol), EDC–HCl (0.42 g, 2.2 mmol),
concentrated in vacuo. The sample was recrystallized using diethyl
ether and hexane. Product was obtained as a white solid (yield:
0.47 g, 81%). Mp 90–91 °C. 1H NMR (500 MHz, CDCl3) d 7.19–7.34
(m, 5H), 6.04 (br s, 1H), 5.88–5.97 (m, 1H), 5.21–5.34 (m, 2H),
5.17 (br s, 1H), 4.56 (d, J = 5.4 Hz, 2H), 3.54 (q, 2H), 3.21 (q,
J = 6.5 Hz, 2H), 2.84 (t, J = 7.1 Hz, 2H), 2.19 (t, J = 6.9 Hz, 2H), 1.82
(pentet, J = 6.8 Hz, 2H). 13C NMR (125 MHz, CDCl3) d 172.5, 156.7,
138.9, 132.9, 128.7, 128.6, 126.5, 117.6, 65.5, 40.6, 40.3, 35.6,
33.7, 26.1. DART-HRMS (m/z) calculated for C16H23N2O3 [M+H]+
291.1709, found 291.1717.
4.2.2.11. S-Ethyl (4-oxo-4-(phenethylamino)butyl)carbamoth-
ioate (11).
Converted to the N-phenylacetamide derivative
using the general procedure described and with the following
specifications and alterations: 11a (0.4 g, 2.1 mmol) dissolved in
CH2Cl2 (6 mL), TEA (0.58 mL, 4.2 mmol), phenethylamine
(0.32 mL, 2.51 mmol), DMAP (0.026 g, 0.21 mmol), EDC–HCl
(0.48 g, 2.51 mmol). Product was obtained as a white solid (yield:
0.5 g, 83%). Mp 118–119 °C. 1H NMR (500 MHz, CDCl3) d 7.20–
7.35 (m, 5H), 5.99 (br s, 1H), 5.94 (br s, 1H), 3.55 (q, 2H), 3.31 (q,
J = 5.7 Hz, 2H), 2.92 (q, 2H), 2.85 (t, 2H), 2.20 (t, J = 6.8 Hz, 2H),
1.84 (pentet, J = 6.62 Hz, 2H), 1.30 (t, 3H). 13C NMR (125 MHz,
CDCl3) d 191.0, 172.5, 138.8, 128.7, 128.6, 126.5, 44.6, 40.7, 35.6,
4.2.2.7. 2-Fluoroethyl (4-oxo-4-(phenethylamino)butyl)carba-
mate (7).
Converted to the N-phenethylamine derivative
using the general procedure described and with the following
specifications and alterations: 7a (0.33 g, 1.71 mmol), TEA
(0.58 mL, 3.78 mmol), phenethylamine (0.24 mL, 1.89 mmol),
DMAP (0.02 g, 0.17 mmol), EDC–HCl (0.36 g, 1.89 mmol). Product
was obtained as a white solid (yield: 0.4 g, 79%). Mp 95 °C. 1H
NMR (500 MHz, CDCl3) d 7.20–7.35 (m, 5H), 5.86 (br s, 1H), 5.17
(br s, 1H), 4.54–4.65 (dt, 2H), 4.27–4.32 (dt, 2H), 3.52–3.58 (q,
Please cite this article in press as: Gromek, S. M.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.040

S. M. Gromek et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
9
33.8, 25.5, 24.3, 15.7 DART-HRMS (m/z) calculated for C15H23N2O2S
128.6, 126.5, 40.8, 39.0, 35.7, 33.9, 25.5, 23.2. DART-HRMS (m/z)
[M+H]+ 295.1480, found 295.1464.
calculated for C14H21N2O2 [M+H]+ 249.1603, found 249.1581.
4.2.2.12. 4-(3-Ethylureido)-N-phenethylbutanamide (12). Con-
verted to the N-phenylacetamide derivative using the general pro-
cedure described and with the following specifications and
alterations: 12d (0.012 g, 0.072 mmol) dissolved in CH2Cl2
(0.4 mL), TEA (0.022 mL, 0.16 mmol), phenethylamine (0.01 mL,
0.082 mmol), DMAP (0.8 mg, 0.007 mmol), EDC–HCl (0.016 g,
4.2.2.16. 4-Butyramido-N-phenethylbutanamide (16).
Con-
verted to the N-phenylacetamide derivative using the general pro-
cedure described and with the following specifications and
alterations: 16a (0.5 g, 2.89 mmol), TEA (0.81 mL, 5.78 mmol),
phenethylamine
(0.43 mL,
3.44 mmol),
DMAP
(0.035 g,
0.289 mmol), EDC–HCl (0.65 g, 3.44 mmol). Product was obtained
as a white solid (yield: 0.66 g, 83%). Mp 119–120 °C. 1H NMR
(500 MHz, CDCl3) d 7.20–7.34 (m, 5H), 6.35 (br s, 1H), 6.22 (br s,
1H), 3.54 (q, 2H), 3.27 (q, 2H), 2.85 (t, J = 7.3 Hz, 2H), 2.15–2.21
(m, 4H), 1.81 (pentet, 2H), 1.67 (sextet, 2H), 0.96 (t, 3H). 13C
NMR (125 MHz, CDCl3) d 173.7, 172.8, 151.7, 138.9, 128.7, 128.6,
126.5, 40.7, 38.9, 38.7, 35.7, 33.9, 25.6, 19.2, 13.8. DART-HRMS
(m/z) calculated for C16H25N2O2 [M+H]+ 277.1916, found 277.1948.
0.082 mmol). Product was obtained as
a white solid (yield:
0.015 g, 76%). 1H NMR (500 MHz, CDCl3) d 7.13–7.26 (m, 5H),
6.07 (br s, 2H), 4.78 (br s, 1H), 3.45 (q, 2H), 3.13 (m, 4H), 2.76 (t,
2H), 2.13 (t, 2H), 1.71 (pentet, 2H), 1.07 (t, 3H). 13C NMR
(125 MHz, CDCl3) d 173.3, 158.9, 139.0, 128.9, 128.8, 126.7, 40.9,
39.9, 35.8, 35.7, 33.8, 26.4, 15.6. DART-HRMS (m/z) calculated for
C
15H24N3O2 [M+H]+ 278.1869, found 278.1896. Data matched that
reported previously.46
4.2.2.17. N-Phenethyl-4-propionamidobutanamide (17). Con-.
verted to the N-phenylacetamide derivative using the general pro-
cedure described and with the following specifications and
alterations: 17a (0.50 g, 2.89 mmol), TEA (0.81 mL, 5.78 mmol),
phenethylamine (0.44 mL, 3.5 mmol), DMAP (0.03 g, 0.29 mmol),
EDC–HCl (0.66 g, 3.46 mmol). Product was obtained as a white
solid (yield: 0.52 g, 68%). Mp 117–118 °C. 1H NMR (500 MHz,
CDCl3) d 7.19–7.33 (m, 5H), 6.32 (br s, 1H), 6.23 (br s, 1H), 3.54
(q, 2H), 3.27 (q, J = 6.0 Hz, 2H), 2.85 (t, J = 7.1 Hz, 2H), 2.21 (m,
4H), 1.81 (pentet, J = 6.6 Hz, 2H), 1.16 (t, J = 7.6 Hz, 3H). 13C NMR
(125 MHz, CDCl3) d 174.4, 172.8, 138.9, 128.7, 128.6, 126.5, 40.7,
38.9, 35.7, 33.9, 29.8, 25.5, 9.9. DART-HRMS (m/z) calculated for
4.2.2.13.
lamino)butanamide (13).
etamide derivative using the general procedure described and
with the following specifications and alterations: 13a (0.1 g,
0.43 mmol) dissolved in CH2Cl2 (3 mL), TEA (0.13 mL 1.0 mmol),
N-Phenethyl-4-(2-oxo-pyrrolidine-1-thiocarbony-
Converted to the N-phenylac-
phenethylamine
(0.06 mL,
0.5 mmol),
DMAP
(0.005 g,
0.043 mmol), EDC–HCl (0.1 g, 0.5 mmol). The sample was dis-
solved in CH2Cl2, solution was heated, and product precipitated
from solution with the addition of hexane. Solution cooled to
ꢁ20 °C overnight and filtered. Product was obtained as a white
solid (yield: 0.075 g, 53%). Mp 98–99 °C. 1H NMR (500 MHz,
CDCl3) d 10.79 (br s, 1H), 7.21–7.35 (m, 5H), 5.71 (br s, 1H),
4.23 (t, J = 7.1 Hz, 2H), 3.69 (q, 2H), 3.55 (q, 2H), 2.85 (t, 2H),
2.72 (t, 2H), 2.23 (t, J = 7.1 Hz, 2H), 1.98–2.09 (m, 4H). 13C NMR
(125 MHz, CDCl3) d 180.8, 176.7, 171.8, 138.8, 128.8, 128.7,
126.5, 51.1, 44.6, 40.6, 35.7, 34.5, 33.7, 24.2, 16.9. DART-HRMS
C
15H23N2O2 [M+H]+ 263.1760, obs. 263.1784.
4.2.2.18. 3,3-Dimethyl-N-(4-oxo-4-(phenethylamino)butyl)bu-
tanamide (18). Converted to the N-phenylacetamide deriva-
tive using the general procedure described and with the
following specifications and alterations: 18a (1.04 g, 4.85 mmol)
dissolved in CH2Cl2 (30 mL), TEA (1.35 mL, 9.7 mmol), phenethy-
lamine (0.7 mL, 5.6 mmol), DMAP (0.06 g, 0.49 mmol), EDC–HCl
(1.06 g, 5.56 mmol). Product was obtained as an off white wet solid
(yield: 1.32 g, 89%). Mp 87–88 °C. 1H NMR (500 MHz, CDCl3) d
7.20–7.32 (m, 5H), 6.44 (br s, 1H), 6.17 (br s, 1H), 3.53 (q, 2H),
3.24 (q, J = 6.2 Hz, 2H), 2.85 (t, 2H), 2.20 (t, J = 6.8 Hz, 2H), 1.80
(pentet, J = 6.6 Hz, 2H), 1.04 (s, 9H). 13C NMR (125 MHz, CDCl3) d
172.8, 172.4, 138.9, 128.7, 128.6, 126.5, 50.6, 40.7, 38.8, 35.7,
34.0, 30.8, 29.8, 25.7. DART-HRMS (m/z) calculated for
(m/z) calculated for
334.1604.
C
17H24N3O2S [M+H]+ 334.1589, found
4.2.2.14. N-Phenethyl-4-(2-oxo-pyrrolidine-1-carbonylamino)
butanamide (14). Converted to the N-phenylacetamide
derivative using the general procedure described and with the fol-
lowing specifications and alterations: 14a (0.085 g, 0.4 mmol) dis-
solved in CH2Cl2 (1 mL), TEA (0.11 mL, 0.8 mmol), phenethylamine
(0.055 mL, 0.44 mmol), DMAP (0.005 g, 0.04 mmol), EDC–HCl
(0.08 g, 0.44 mmol). The sample was dissolved in CH2Cl2, solution
was heated, and product precipitated from solution with the addi-
tion of hexane. Solution cooled to ꢁ20 °C overnight and filtered.
Product was obtained as a white solid (yield: 0.05 g, 40%). Mp
68–69 °C. 1H NMR (500 MHz, CDCl3) d 8.48 (br s, 1H), 7.22–7.34
(m, 5H), 6.03 (br s, 1H), 3.86 (t, J = 7.1 Hz, 2H), 3.55 (q, J = 6.8 Hz,
2H), 3.32 (q, J = 6.6 Hz, 2H), 2.85 (t, J = 7.1 Hz, 2H), 2.62 (t, 2H),
2.19 (t, J = 7.1 Hz, 2H), 2.05 (pentet, 2H), 1.87 (pentet, 2H). 13C
NMR (125 MHz, CDCl3) d 177.1, 172.2, 153.4, 139.0, 128.8, 128.6,
126.4, 45.7, 40.6, 38.8, 35.7, 33.7, 33.4, 26.2, 17.0. DART-HRMS
(m/z) calculated for C17H24N3O3 [M+H]+ 318.1818, found 318.1823.
C
18H29N2O2 [M+H]+ 305.2229, found 305.2214.
4.2.2.19.
(19).
N-(4-Oxo-4-(phenethylamino)butyl)benzamide
Converted to the N-phenylacetamide derivative using
the general procedure described and with the following specifica-
tions and alterations: 19a (0.21 g, 1.0 mmol), TEA (0.3 mL,
2.12 mmol), phenethylamine (0.14 mL, 1.14 mmol), DMAP
(0.012 g, 0.1 mmol), EDC–HCl (0.22 g, 1.14 mmol). Product was
obtained as a white solid (yield: 0.26 g, 83%). Mp 106–107 °C. 1H
NMR (500 MHz, CDCl3) d 7.85 (d, 2H), 7.16–7.50 (m, 8H), 6.43 (br
s, 2H), 3.44–3.51 (m, 4H), 2.79 (t, J = 7.3 Hz, 2H), 2.27 (t, 2H),
1.93 (pentet, 2H). 13C NMR (125 MHz, CDCl3) d 173.2, 167.8,
138.8, 134.4, 131.4, 128.7, 128.6, 128.5, 127.0, 126.5, 40.75, 39.9,
35.6, 34.2, 25.0. DART-HRMS (m/z) calculated for C19H23N2O2 [M
+H]+ 311.1760, found 311.1740.
4.2.2.15. 4-Acetamido-N-phenethylbutanamide (15).
Con-
verted to the N-phenylacetamide derivative using the general pro-
cedure described and with the following specifications and
alterations: 15a (0.2 g, 1.38 mmol), TEA (0.38 mL, 2.74 mmol),
phenethylamine (0.2 mL, 1.58 mmol), DMAP (0.01 g, 0.082 mmol),
EDC–HCl (0.3 g, 1.58 mmol). Product was obtained as a white solid
(yield: 0.25 g, 73%). Mp 112–113 °C. 1H NMR (500 MHz, CDCl3) d
7.21–7.35 (m, 5H), 6.06 (br s, 2H), 3.56 (q, 2H), 3.28 (q, 2H), 2.86
(t, J = 7.3 Hz, 2H), 2.21 (t, 2H), 1.82 (pentet, J = 6.6 Hz, 2H), 1.65
(s, 3H). 13C NMR (125 MHz, CDCl3) d 172.9, 170.8, 138.9, 128.7,
4.2.2.20. S-(3-Oxo-3-((4-oxo-4-(phenethylamino)butyl)amino)
propyl)ethanethioate (20).
Converted to the N-phenylac-
etamide derivative using the general procedure described and with
the following specifications and alterations: 20b (0.45 g,
1.93 mmol) dissolved in CH2Cl2 (5 mL), TEA (0.54 mL, 3.86 mmol),
Please cite this article in press as: Gromek, S. M.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.040

10
S. M. Gromek et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
phenethylamine (0.29 mL, 2.3 mmol), DMAP (0.024 g, 0.19 mmol),
EDC–HCl (0.44 g, 2.3 mmol). Product was obtained as a white solid
(yield: 0.58 g, 89%). Mp 100–101 °C. 1H NMR (500 MHz, CDCl3) d
7.21–7.24 (m, 3H), 7.31–7.34 (m, 2H), 6.14 (br s, 1H), 5.95 (br s,
1H), 3.55 (m, 2H), 3.29 (m, 2H), 3.16 (t, 1H), 2.86 (t, 2H), 2.49 (t,
2H), 2.34 (s, 3H), 2.20 (m, 2H), 1.82 (pentet, 2H). 13C NMR
(125 MHz, CDCl3) d 195.7, 172.6, 170.9, 138.8, 128.7, 128.6,
126.5, 40.6, 39.1, 36.4, 35.6, 34.0, 30.5, 25.4, 25.0. DART-HRMS
and alterations: 22 (0.30 g, 1.71 mmol), TEA (0.57 mL, 4.11 mmol),
phenethylamine replaced with 2-aminothiazole (0.19 g,
1.89 mmol), DMAP (0.021 g, 0.171 mmol), EDC–HCl (0.36 g,
1.89 mmol). Product was obtained as a white powder (yield:
0.32 g, 73%). Mp 173–174 °C. 1H NMR (500 MHz, CD3OD) d 7.41
(d, J = 2.9 Hz, 1H), 7.08 (d, J = 2.9 Hz, 1H), 4.05 (q, J = 3.0 Hz, 2H),
3.17 (t, 2H), 2.51 (t, 2H), 1.89 (pentet, J = 6.7 Hz, 2H), 1.20 (t, 3H).
13C NMR (125 MHz, CD3OD) d 170.3, 157.4, 156.7, 135.6, 111.7,
(m/z) calculated for
C
17H25N2O3S [M+H]+ 337.1586, found
59.0, 38.3, 31.0, 23.9, 12.2. DART-HRMS (m/z) calculated for C10-
337.1583.
H
16N3O3S [M+H]+ 258.0912, found 258.0906.
4.2.2.21.
(21).
4-Oxo-4-(phenethylamino)butyl
21c (0.6 g, 3.15 mmol) was dissolved in THF (10 mL)
ethylcarbamate
4.2.3.4.
oxobutyl)carbamate (26).
Ethyl
(4-((4-(3-nitrophenyl)thiazol-2-yl)amino)-4-
Converted to the 3-nitrophenyl-
and to this was added aqueous LiOH (0.75 g, 3.15 mmol, 1 M) at
rt for 90 min. The reaction mixture was neutralized with 6 N HCl
and concentrated. The aqueous layer was extracted with EtOAc
(4 ꢀ 20 mL), washed with brine, dried over Na2SO4, and concen-
trated. Resulting solid was dissolved in EtOAc, solution was heated,
and cooled to 0 °C. The solution was filtered to yield desired pro-
duct and used in the next reaction without further purification.
Material was converted to the N-phenylacetamide derivative using
the general procedure described and was obtained as a white solid
(yield: 0.21 g, 56%). Mp 110–111 °C. 1H NMR (500 MHz, CDCl3) d
7.20–7.35 (m, 5H), 6.07 (br s, 1H), 5.04 (br s, 1H), 4.11 (q,
J = 7.0 Hz, 2H), 3.54 (q, 2H), 3.20 (q, J = 6.3 Hz, 2H), 2.84 (t,
J = 7.1 Hz, 2H), 2.19 (t, J = 7.0 Hz, 2H), 1.81 (pentet, J = 6.8 Hz, 2H),
1.25 (t, 3H). 13C NMR (125 MHz, CDCl3) d 172.7, 157.3, 139.1,
128.9, 128.8, 126.7, 61.0, 40.8, 40.3, 35.8, 33.9, 26.3, 14.8. DART-
HRMS (m/z) calculated for C15H23N2O3 [M+H]+ 279.1709, found
279.1736.
thiazol-2-amine derivative using the general procedure described
and with the following specifications and alterations: 22 (0.3 g,
1.71 mmol), TEA (0.57 mL, 4.11 mmol), phenethylamine replaced
with 3-nitrophenyl-thiazol-2-amine (0.42 g, 1.89 mmol), DMAP
(0.021 g, 0.171 mmol), EDC–HCl (0.36 g, 1.89 mmol). Product was
obtained as a white powder (yield: 0.42 g, 65%). Mp 167–168 °C.
1H NMR (500 MHz, CDCl3) d 10.38 (br s, 1H), 8.73 (s, 1H), 8.17 (d,
J = 7.9 Hz, 1H), 7.59 (t, 1H), 7.30 (t, 1H), 6.91 (s, 1H), 5.01 (br s,
1H), 4.34 (q, 2H), 3.85 (t, 2H), 2.60 (t, 2H), 2.07 (pentet,
J = 7.6 Hz, 2H), 1.37 (t, 3H). 13C NMR (125 MHz, CDCl3) d 170.6,
164.5, 158.3, 148.9, 147.6, 136.3, 131.8, 129.8, 122.6, 121.2,
109.8, 61.5, 40.0, 33.5, 26.7, 14.8. DART-HRMS (m/z) calculated
for C16H19N4O5S [M+H]+ 379.1076, found 379.1043.
4.2.3.5. Ethyl (4-((4-(((tert-butyldimethylsilyl)oxy)methyl)phe-
nyl)amino)-4-oxobutyl)carbamate (27).
Converted to the
toluene-OTBS derivative using the general procedure described
and with the following specifications and alterations: 22 (0.3 g,
1.71 mmol), TEA (0.57 mL, 4.11 mmol), phenethylamine replaced
4.2.3. Cap group analogues
4.2.3.1.
(23).
Ethyl
(4-(isopentylamino)-4-oxobutyl)carbamate
with
4-(((tert-butyldimethylsilyl)oxy)methyl)aniline
(0.45 g,
Converted to the N-isopentylacetamide derivative using
1.89 mmol), DMAP (0.021 g, 0.171 mmol), EDC–HCl (0.36 g,
1.89 mmol). Product was obtained as a white powder (yield:
0.089 g, 13%). Mp 102.7–103 °C. 1H NMR (500 MHz, CDCl3) d 8.38
(br s, 1H), 7.57 (d, J = 7.9 Hz, 2H), 7.29 (d, 2H), 4.99 (br s, 1H),
4.72 (s, 2H), 4.15 (q, J = 7.1 Hz, 2H), 3.33 (q, J = 5.8 Hz, 2H), 2.42
(t, J = 6.7 Hz, 2H), 1.93 (pentet, J = 6.5 Hz, 2H), 1.26 (t, J = 7.1 Hz,
3H), 0.96 (s, 9H), 0.11 (s, 6H). 13C NMR (125 MHz, CDCl3) d 171.2,
157.9, 137.3, 137.2, 126.9, 119.8, 64.9, 61.3, 40.0, 34.8, 29.8, 27.1,
26.1, 14.8, ꢁ5.0. DART-HRMS (m/z) calculated for C20H35N2O4Si
[M+H]+ 395.2366, found 395.2343.
the general procedure described and with the following specifica-
tions and alterations: 22 (0.3 g, 1.71 mmol), TEA (0.57 mL,
4.11 mmol), phenethylamine replaced with isopentylamine
(0.22 mL, 1.89 mmol), DMAP (0.021 g, 0.171 mmol), EDC–HCl
(0.36 g, 1.89 mmol). Product was obtained as a white powder
(yield: 0.19 g, 46%). Mp 99 °C. 1H NMR (500 MHz, CDCl3) d 6.12
(br s, 1H), 5.13 (br s, 1H), 4.11 (q, J = 7.0 Hz, 2H), 3.21–3.29 (m,
4H), 2.22 (t, J = 7.0 Hz, 2H), 1.83 (pentet, J = 6.8 Hz, 2H), 1.62 (sex-
tet, J = 6.7, 13.39 Hz, 1H), 1.38–1.41 (q, 2H), 1.24 (t, J = 7.1 Hz, 3H),
0.92 (d, J = 6.6 Hz, 6H). 13C NMR (125 MHz, CDCl3) d 172.7, 157.4,
60.9, 40.4, 38.6, 38.1, 33.9, 26.4, 26.1, 22.6, 14.8. DART-HRMS (m/
z) calculated for C12H25N2O3 [M+H]+ 245.1865, found 245.1870.
4.2.3.6.
Ethyl
(4-((4-((tert-butyldimethylsilyl)oxy)phenyl)
Converted to the ani-
amino)-4-oxobutyl)carbamate (28).
line-OTBS derivative using the general procedure described and
with the following specifications and alterations: 22 (0.3 g,
1.71 mmol), TEA (0.57 mL, 4.1 mmol), phenethylamine replaced
with 4-((tert-butyldimethylsilyl)oxy)aniline (0.42 g, 1.89 mmol),
DMAP (0.021 g, 0.171 mmol), EDC–HCl (0.36 mL, 1.89 mmol). Pro-
duct was obtained as a light brown solid (yield: 0.32 g, 49%). Mp
112 °C. 1H NMR (500 MHz, CDCl3) d 8.11 (br s, 1H), 7.36–7.38 (d,
J = 8.6 Hz, 2H), 6.72–6.74 (d, 2H), 4.86 (br s, 1H), 4.07 (q,
J = 7.4 Hz, 2H), 3.26 (q, J = 6.2 Hz, 2H), 2.32 (t, 2H), 1.85 (pentet,
J = 6.5 Hz, 2H), 1.18 (t, J = 7.1 Hz, 3H), 0.92 (s, 9H), 0.12 (s, 6H)
13C NMR (125 MHz, CDCl3) d 172.9, 157.1, 154.7, 135.5, 129.0,
122.5, 61.1, 40.7, 33.6, 28.6, 26.3, 21.0, 14.8. DART-HRMS (m/z) cal-
culated for C19H33N2O4Si [M+H]+ 381.2210, found 381.2197.
4.2.3.2. Ethyl (4-((2-((tert-butoxycarbonyl)amino)ethyl)amino)-
4-oxobutyl)carbamate (24).
Converted to the N-Boc-aceta-
mide derivative using the general procedure described and with
the following specifications and alterations: 22 (0.3 g, 1.71 mmol),
TEA (0.57 mL, 4.11 mmol), phenethylamine replaced with tert-
butyl (2-aminoethyl)carbamate (0.3 g, 1.89 mmol), DMAP
(0.021 g, 0.171 mmol), EDC–HCl (0.36 g, 1.89 mmol). Product was
obtained as a white solid (yield: 0.004 g, 8%). Mp 102–103 °C. 1H
NMR (500 MHz, CDCl3) d 6.54 (br s, 1H), 5.23 (br s, 1H), 5.03 (br
s, 1H), 4.14 (q, J = 7.0 Hz, 2H), 3.38 (q, J = 5.5 Hz, 2H), 3.30 (t, 2H),
3.24 (t, 2H), 2.25 (t, J = 6.9 Hz, 2H), 1.85 (pentet, J = 6.7 Hz, 2H),
1.46 (s, 9H), 1.26 (t, J = 7.1 Hz, 3H). 13C NMR (125 MHz, CDCl3) d
172.9, 157.1, 154.7, 79.6, 61.0, 40.7, 40.1, 37.7, 33.6, 28.6, 26.3,
14.8. DART-HRMS (m/z) calculated for
C
14H28N3O5 [M+H]+
4.2.3.7. Ethyl (4-((4-chlorophenethyl)amino)-4-oxobutyl)carba-
318.2029, found 318.2043.
mate (29).
Converted to the N-(para-chloro)-phenethylamine
derivative using the general procedure described and with the fol-
lowing specifications and alterations: 22 (0.30 g, 1.71 mmol), TEA
(0.57 mL, 4.11 mmol), phenethylamine replaced with para-
chloro-phenethylamine (0.26 mL, 1.89 mmol), DMAP (0.021 g,
4.2.3.3. Ethyl (4-oxo-4-(thiazol-2-ylamino)butyl)carbamate
(25). Converted to the N-aminothiazole derivative using the
general procedure described and with the following specifications
Please cite this article in press as: Gromek, S. M.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.040

S. M. Gromek et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
11
0.171 mmol), EDC–HCl (0.36 g, 1.89 mmol). Product was obtained
as a white powder (yield: 0.28 g, 51%). Mp 118–119 °C. 1H NMR
(500 MHz, CDCl3) d 7.27–7.29 (d, 2H), 7.14–7.15 (d, J = 8.3 Hz,
2H), 6.19 (br s, 1H), 5.02 (br s, 1H), 4.11 (q, J = 7.1 Hz, 2H), 3.51
(q, 2H), 3.19 (q, J = 6.1 Hz, 2H), 2.81 (t, J = 7.1 Hz, 2H), 2.19 (t,
2H), 1.80 (pentet, J = 6.7 Hz, 2H), 1.25 (t, J = 7.1 Hz, 3H). 13C NMR
(125 MHz, CDCl3) d 172.8, 157.4, 137.6, 132.4, 130.3, 128.9, 61.0,
40.7, 40.2, 35.2, 33.8, 26.5, 14.8. DART-HRMS (m/z) calculated for
as a white powder (yield: 0.18 g, 28%). Mp 117–118 °C. 1H NMR
(500 MHz, CDCl3) d 7.18 (t, 2H), 7.01 (t, 2H), 6.09 (br s, 1H), 4.97
(br s, 1H), 4.12 (q, J = 7.0 Hz, 2H), 3.52 (q, 2H), 3.20 (q, J = 6.2 Hz,
2H), 2.82 (t, J = 7.1 Hz, 2H), 2.20 (t, 2H), 1.81 (pentet, J = 6.7 Hz,
2H), 1.25 (t, J = 7.1 Hz, 3H) 13C NMR (125 MHz, CDCl3) d 172.8,
162.8, 160.9, 134.7, 130.4, 130.3, 115.6, 115.5, 61.0, 40.9, 40.3,
35.0, 33.8, 26.4, 14.8. DART-HRMS (m/z) calculated for C15H22FN2-
O3 [M+H]+ 297.1614, found 297.1638.
C
15H22N2ClO3 [M+H]+ 313.1319, found 313.1340.
4.2.4. Linker analogues
4.2.3.8. Ethyl (4-((3-chlorophenethyl)amino)-4-oxobutyl)carba-
mate (30). Converted to the N-(meta-chloro)-phenethylamine
4.2.4.1.
(34).
Ethyl
(2-oxo-2-(phenethylamino)ethyl)carbamate
Converted to the N-phenethylamine derivative using
derivative using the general procedure described and with the fol-
lowing specifications and alterations: 22 (0.3 g, 1.71 mmol), TEA
(0.57 mL, 4.11 mmol), phenethylamine replaced with meta-
chloro-phenethylamine (0.27 mL, 1.89 mmol), DMAP (0.021 g,
0.171 mmol), EDC–HCl (0.36 g, 1.89 mmol). Product was obtained
as a white powder (yield: 0.4 g, 74%). Mp 89–90 °C. 1H NMR
(500 MHz, CDCl3) d 7.29 (d, 2H), 7.15 (d, J = 8.3 Hz, 2H), 6.19 (br
s, 1H), 5.02 (br s, 1H), 4.11 (q, J = 7.1 Hz, 2H), 3.51 (q, 2H), 3.19
(q, J = 6.1 Hz, 2H), 2.81 (t, J = 7.1 Hz, 2H), 2.19 (t, 2H), 1.80 (pentet,
J = 6.7 Hz, 2H), 1.25 (t, J = 7.1 Hz, 3H). 13C NMR (125 MHz, CDCl3) d
172.8, 157.4, 137.6, 132.4, 130.3, 128.9, 61.0, 40.7, 40.2, 35.2, 33.8,
26.5, 14.8. DART-HRMS (m/z) calculated for C15H22N2ClO3 [M+H]+
313.1319, found 313.1341.
the general procedure described and with the following specifica-
tions and alterations: 34a (0.22 g, 1.5 mmol), TEA (0.42 mL,
3.1 mmol), phenethylamine (0.21 mL, 1.71 mmol), DMAP
(0.018 g, 0.15 mmol), EDC–HCl (0.32 g, 1.71 mmol). Product was
obtained as a white powder (yield: 0.21 g, 56%). Mp 100–101 °C.
1H NMR (500 MHz, CDCl3) d 7.20–7.34 (m, 5H), 6.20 (br s, 1H),
5.37 (br s, 1H), 4.15 (q, J = 7.1 Hz, 2H), 3.81 (d, J = 5.8 Hz, 2H),
3.56 (q, J = 6.9 Hz, 2H), 2.84 (t, J = 7.0 Hz, 2H), 1.27 (t, J = 7.1 Hz,
3H). 13C NMR (125 MHz, CDCl3) d 169.3, 157.0, 138.8, 128.9,
128.8, 126.8, 61.6, 44.8, 40.8, 35.8, 14.7. DART-HRMS (m/z) calcu-
lated for C13H19N2O3 [M+H]+ 251.1396, found 251.1383.
4.2.4.2. Ethyl (3-oxo-3-(phenethylamino)propyl)carbamate
(35).
Converted to the N-phenethylamine derivative using
4.2.3.9.
(31).
Phenethyl
4-((ethoxycarbonyl)amino)butanoate
the general procedure described and with the following specifica-
tions and alterations: 35a (0.3 g, 1.71 mmol), TEA (0.57 mL,
4.11 mmol), phenethylamine (0.24 mL, 1.89 mmol), DMAP
(0.021 g, 0.171 mmol), EDC–HCl (0.36 g, 1.89 mmol). Product was
obtained as a white powder (yield: 0.21 g, 47%). Mp 105–106 °C.
1H NMR (500 MHz, CDCl3) d 7.20–7.35 (m, 5H), 5.63 (br s, 1H),
5.29 (br s, 1H), 4.12 (q, J = 7.0 Hz, 2H), 3.56 (q, J = 6.9 Hz, 2H),
3.46 (q, J = 6.0 Hz, 2H), 2.84 (t, J = 7.0 Hz, 2H), 2.37 (t, J = 5.8 Hz,
2H), 1.26 (t, J = 7.1 Hz, 3H). 13C NMR (125 MHz, CDCl3) d 171.4,
157.0, 138.9, 128.89, 128.87, 126.8, 60.9, 40.8, 37.2, 36.3, 35.8,
Converted to the N-phenylacetamide derivative using
the general procedure described and with the following specifica-
tions and alterations: 22 (0.3 g, 1.71 mmol), HOBt (0.26 g,
1.89 mmol), TEA (0.57 mL, 4.11 mmol), phenethylamine replaced
with 2-phenylethan-1-ol (0.21 g, 1.71 mmol), EDC–HCl (0.36 g,
1.89 mmol). Product was obtained as a white powder (yield:
0.32 g, 67%). Mp 122–123 °C. 1H NMR (500 MHz, CDCl3) d 7.12–
7.27 (m, 6H), 4.62 (br s, 1H), 4.23 (t, J = 7.1 Hz, 2H), 4.03 (d,
J = 6.8 Hz, 2H), 3.09 (d, J = 6.2 Hz, 2H), 2.87 (t, J = 6.8 Hz, 2H), 2.26
(t, J = 7.1 Hz, 2H), 1.72 (t, J = 6.8 Hz, 2H), 1.16 (t, J = 6.8 Hz, 3H).
13C NMR (125 MHz, CDCl3) d 172.0, 162.6, 160.7, 157.3, 134.6,
130.2, 115.4, 115.3, 60.8, 40.8, 40.1, 34.9, 33.6, 26.3, 14.6. DART-
HRMS (m/z) calculated for C15H22NO4 [M+H]+ 280.1549, found
280.1553.
14.8. DART-HRMS (m/z) calculated for
265.1552, found 265.1538.
C
14H21N2O3 [M+H]+
4.2.4.3. Ethyl (5-oxo-5-(phenethylamino)pentyl)carbamate
(36). Converted to the N-phenethylamine derivative using
the general procedure described and with the following specifica-
tions and alterations: 36a (0.32 g, 1.71 mmol), TEA (0.57 mL,
4.11 mmol), DMAP (0.021 g, 0.171 mmol), EDC–HCl (0.36 g,
1.89 mmol). Product was obtained as a white powder (yield:
0.36 g, 72%). Mp 102–103 °C. 1H NMR (500 MHz, CDCl3) d 7.11–
7.25 (m, 5H), 5.53 (br s, 1H), 4.71 (br s, 1H), 4.02 (q, J = 6.7 Hz,
2H), 3.45 (q, J = 6.7 Hz, 2H), 3.09 (q, J = 6.2 Hz, 2H), 2.74 (t,
J = 7.0 Hz, 2H), 2.07 (t, J = 7.3 Hz, 2H), 1.56 (pentet, J = 7.5 Hz, 2H),
1.41 (pentet, J = 7.1 Hz, 2H), 1.16 (t, J = 7.0 Hz, 3H) 13C NMR
(125 MHz, CDCl3) d 172.9, 156.9, 139.0, 128.9, 128.8, 126.7, 60.9,
40.7, 40.4, 36.1, 35.9, 29.6, 22.7, 14.8. DART-HRMS (m/z) calculated
for C16H25N2O3 [M+H]+ 293.1865, found 293.1866.
4.2.3.10. Methyl (4-((ethoxycarbonyl)amino)butanoyl)pheny-
lalaninate (32).
Converted to the N-methyl-l-phenylalanine
derivative using the general procedure described and with the fol-
lowing specifications and alterations: 22 (0.3 g, 1.71 mmol), TEA
(0.57 mL, 4.11 mmol), methyl-l-phenylalanine (0.34 g, 1.89 mmol),
DMAP (0.021 g, 0.171 mmol), EDC–HCl (0.36 g, 1.89 mmol). Pro-
duct was obtained as a white powder (yield: 0.36 g, 63%). Mp
129–130 °C. 1H NMR (500 MHz, CDCl3) d 7.26–7.33 (m, 3H),
7.13–7.15 (d, J = 7.2 Hz, 2H), 6.35 (br s, 1H), 4.94 (br s, 1H), 4.88–
4.92 (q, 1H), 4.13 (q, J = 7.0 Hz, 2H), 3.75 (s, 3H), 3.17–3.21 (dd,
J = 5.7, 13.91 Hz, 2H), 3.07–3.11 (dd, 1H), 2.22–2.26 (dt, J = 2.7,
7.1 Hz, 2H), 1.80 (pentet, J = 6.8 Hz, 3H), 1.26 (t, J = 7.1 Hz, 3H)
13C NMR (125 MHz, CDCl3) d 172.4, 171.6, 157.2, 136.2, 129.4,
128.8, 127.3, 60.9, 53.4, 52.5, 40.1, 38.0, 33.5, 26.1, 14.8. DART-
HRMS (m/z) calculated for C17H25N2O5 [M+H]+ 337.1763, found
337.1732.
4.2.4.4.
(37).
Ethyl
(6-oxo-6-(phenethylamino)hexyl)carbamate
Converted to the N-phenethylamine derivative using
the general procedure described and with the following specifica-
tions and alterations: 37a (0.35 g, 1.71 mmol), TEA (0.57 mL,
4.11 mmol), phenethylamine (0.24 mL, 1.89 mmol), DMAP
(0.021 g, 0.171 mmol), EDC–HCl (0.36 g, 1.89 mmol). Product was
obtained as a white powder (yield: 0.35 g, 67%). Mp 99–100 °C.
1H NMR (500 MHz, CDCl3) d 7.18–7.32 (m, 5H), 5.64 (br s, 1H),
4.78 (br s, 1H), 4.09 (q, J = 6.8 Hz, 2H), 3.52 (q, J = 6.8 Hz, 2H),
3.15 (q, J = 6.2 Hz, 2H), 2.81 (t, J = 7.0 Hz, 2H), 2.12 (t, J = 7.5 Hz,
2H), 1.61 (pentet, J = 7.6 Hz, 2H), 1.48 (pentet, J = 7.3 Hz, 2H),
1.21–1.33 (m, 5H). 13C NMR (125 MHz, CDCl3) d 173.0, 156.9,
4.2.3.11. Ethyl (4-((3-fluorophenethyl)amino)-4-oxobutyl)car-
bamate (33).
Converted to the N-(m-fluoro)-phenethylamine
derivative using the general procedure described and with the fol-
lowing specifications and alterations: 22 (0.3 g, 1.71 mmol), TEA
(0.57 mL, 4.11 mmol), phenethylamine replaced with m-fluoro-
phenethylamine
(0.25 mL,
1.89 mmol),
DMAP
(0.021 g,
0.171 mmol), EDC–HCl (0.36 g, 1.89 mmol). Product was obtained
Please cite this article in press as: Gromek, S. M.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.040

12
S. M. Gromek et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
139.1, 128.9, 128.8, 126.7, 60.8, 40.8, 40.7, 36.6, 35.9, 29.9, 26.4,
25.4, 14.8. DART-HRMS (m/z) calculated for C17H27N2O3 [M+H]+
307.2022, found 307.2026.
29.2, 28.7, 14.7. DART-HRMS (m/z) calculated for C15H23N2O3 [M
+H]+ 279.1709, found 279.1713.
4.2.4.9. Ethyl phenethylcarbamate (42).
Converted to the N-
4.2.4.5.
(38).
Ethyl
(5-(benzylamino)-5-oxopentyl)carbamate
phenylacetamide derivative using the general procedure described
and with the following alterations: phenethylamine (0.31 mL,
2.5 mmol) dissolved in CH2Cl2 (10 mL) followed by the addition
of TEA (0.69 mL, 5.0 mmol) which was cooled to 0 °C. Ethyl chloro-
formate (0.21 mL, 2.20 mmol) added dropwise at 0 °C, stirred for
24 h at rt, and concentrated. Resulting material dissolved in CH2Cl2
and washed sequentially with 15 mL of the following solutions:
1 M HCl, H2O, sat. NaHCO3, H2O, and brine. Organic layer concen-
trated and dissolved in EtOAc. The solution was heated followed
by the addition of cold hexane to result in precipitation. Solution
cooled to 0 °C and precipitate collected through filtration. Product
was obtained as a white powder (yield: 0.12 g, 29%). Mp 35–36 °C.
1H NMR (500 MHz, CDCl3) d 7.20–7.36 (m, 5H), 4.71 (br s, 1H), 4.14
(q, J = 7.0 Hz, 2H), 3.46 (t, 2H), 2.84 (t, J = 6.9 Hz, 2H), 1.26 (t, 3H).
13C NMR (125 MHz, CDCl3) d 156.8, 139.0, 128.9, 128.7, 126.7,
60.9, 42.3, 36.4, 14.8. DART-HRMS (m/z) calculated for C11H16NO2
[M+H]+ 194.1181, found 194.1209.
Converted to the N-phenmethylamine derivative using
the general procedure described and with the following specifica-
tions and alterations: 36a (0.32 g, 1.71 mmol), TEA (0.57 mL,
4.11 mmol), phenylmethanamine (0.21 mL, 1.89 mmol), DMAP
(0.021 g, 0.171 mmol), EDC–HCl (0.36 g, 1.89 mmol). Product was
obtained as a white powder (yield: 0.35 g, 65%). Mp 102–103 °C.
1H NMR (500 MHz, CDCl3) d 7.18–7.27 (m, 5H), 5.99 (br s, 1H),
4.76 (br s, 1H), 4.35 (d, J = 5.7 Hz, 2H), 3.97 (q, J = 6.6 Hz, 2H),
3.10 (q, J = 6.1 Hz, 2H), 2.17 (t, J = 7.5 Hz, 2H), 1.62 (pentet,
J = 7.5 Hz, 2H), 1.45 (pentet, J = 7.1 Hz, 2H), 1.13 (t, J = 7.0 Hz, 3H).
13C NMR (125 MHz, CDCl3) d 172.9, 157.0, 138.6, 128.9, 127.9,
127.7, 60.9, 43.8, 40.3, 36.0, 29.7, 22.8, 14.8. DART-HRMS (m/z) cal-
culated for C15H23N2O3 [M+H]+ 279.1709, found 279.1696. Data
matched that reported previously.46
4.2.4.6.
(39).
Ethyl
(6-oxo-6-(phenylamino)hexyl)carbamate
Converted to the N-aniline derivative using the general
procedure described and with the following specifications and
alterations: 37a (0.35 g, 1.71 mmol), TEA (0.57 mL, 4.11 mmol),
aniline (0.17 mL, 1.89 mmol), DMAP (0.021 g, 0.171 mmol), EDC–
HCl (0.36 g, 1.89 mmol). Product was obtained as a white powder
(yield: 0.29 g, 60%). Mp 107–108 °C. 1H NMR (500 MHz, CDCl3) d
7.53 (br s, 1H), 7.45–7.46 (d, J = 8.0 Hz, 2H), 7.21–7.24 (t,
J = 7.8 Hz, 2H), 7.02 (t, J = 7.3 Hz, 1H), 4.70 (br s, 1H), 4.03 (q,
J = 6.8 Hz, 2H), 3.09 (q, J = 6.2 Hz, 2H), 2.28 (t, J = 7.4 Hz, 2H), 1.67
(pentet, J = 7.6 Hz, 2H), 1.46 (pentet, J = 7.2 Hz, 2H), 1.32 (pentet,
2H), 1.15 (t, J = 7.0 Hz, 3H). 13C NMR (125 MHz, CDCl3) d 171.5,
157.0, 138.3, 129.1, 124.3, 120.0, 60.9, 40.8, 37.6, 29.9, 26.4, 25.2,
4.3. Biological evaluation
4.3.1. HDAC enzyme assay
All compounds were screened at 1 lM for inhibition of HDAC2
at Reaction Biology (Malvern, PA) using proprietary protocols. The
substrate for HDAC2 was a fluorogenic peptide from p53 residues
379–382 (RHKKAC) at 50
(TSA) was tested in a 10-dose IC50 with 3-fold serial dilution start-
ing at 10 M. TSA exhibited an IC50 value of 25.8 nM.
Synthetic SCA and SCA/SAHA hybrid were also screened at
M and 5 M for inhibition against HDAC 1-11 at BPS Bio-
science (San Diego, CA) using proprietary protocols. HDAC 1-3, 6,
and 10 used 10 M HDAC substrate 3. HDAC 4-5, 7-9, and 11 used
M HDAC substrate Class 2a. Reference compound of either tri-
lM. Reference compound trichostatin A
l
10
l
l
14.8. DART-HRMS (m/z) calculated for
C
15H23N2O3 [M+H]+
279.1709, found 279.1721. Data matched that reported
l
previously.46
2
l
chostatin A (TSA) at 100
each HDAC isozyme.
lM or SAHA at 10 lM was tested against
4.2.4.7. Ethyl (3-oxo-3-((4-phenylbutyl)amino)propyl)carba-
mate (40).
Converted to the N-phenylbutylamine derivative
using the general procedure described and with the following
specifications and alterations: 35a (0.3 g, 1.71 mmol), TEA
4.3.2. Cell line maintenance
4.3.2.1. Solid tumor.
Human colon cancer cells (HCT116),
(0.57 mL,
4.11 mmol),
4-phenylbutan-1-amine
(0.3 mL,
breast cancer cells (MCF-7), and triple negative breast cancer cells
(MDA-MB-231) were obtained from the American Type Culture
Collection (ATCC, Manassas, VA). HCT116 cells were cultured in
McCoy’s 5A media (Gibco) supplemented with 10% FBS (Gibco)
and 1ꢀ penicillin/streptomycin (Cellgro). MCF-7 cells were cul-
tured in DMEM media (UConn Cell Culture Facility) supplemented
with 10% FBS and 1ꢀ penicillin/streptomycin. MDA-MB-231 cells
were cultured in the same supplemented media as MCF-7 with
addition of 1ꢀ glucose. All cells were grown in Corning Cell Cul-
ture, canted neck T75 (Fisher Scientific) in an Autoflow IR water-
jacketed CO2 incubator (37 °C, 5% CO2). Experiments performed
in Celltreat Tissue Culture Treated 96 well flat bottom plates.
DMSO was used to prepare all drug solutions and the final DMSO
concentration was not greater than 1%.
1.89 mmol), DMAP (0.021 g, 0.17 mmol), EDC–HCl (0.36 g,
1.90 mmol). Product was obtained as a white powder (yield:
0.25 g, 49%). Mp 105–106 °C. 1H NMR (500 MHz, CDCl3) d 7.07–
7.22 (m, 5H), 5.58 (br s, 1H), 5.23 (br s, 1H), 4.02 (q, J = 6.9 Hz,
2H), 3.38 (q, J = 6.2 Hz, 2H), 3.20 (q, 2H), 2.56 (t, J = 7.6 Hz, 2H),
2.30 (t, J = 5.8 Hz, 2H), 1.58 (pentet, 2H), 1.46 (pentet, 2H), 1.15
(t, J = 7.0 Hz, 3H). 13C NMR (125 MHz, CDCl3) d 171.3, 157.0,
142.2, 128.56, 128.54, 126.0, 60.9, 39.5, 37.2, 36.4, 35.6, 29.3,
28.8, 14.8. DART-HRMS (m/z) calculated for C16H25N2O3 [M+H]+
293.1865, found 293.1893.
4.2.4.8. Ethyl (2-oxo-2-((4-phenylbutyl)amino)ethyl)carbamate
(41).
Converted to the N-phenbutylamine derivative using the
general procedure described and with the following specifications
and alterations: 34a (0.22 g, 1.5 mmol), TEA (0.42 mL, 3.0 mmol),
4-phenylbutan-1-amine (0.26 mL, 1.65 mmol), DMAP (0.015 g,
0.15 mmol), EDC–HCl (0.32 g, 1.65 mmol). Product was obtained
as a white powder (yield: 0.039 g, 9%). Mp 101–102 °C. 1H NMR
(500 MHz, CDCl3) d 7.18–7.31 (m, 5H), 6.27 (br s, 1H), 5.47 (br s,
1H), 4.15 (q, J = 7.1 Hz, 2H), 3.83 (d, J = 5.4 Hz, 2H), 3.30 (q,
J = 6.6 Hz, 2H), 2.65 (t, J = 7.5 Hz, 2H), 1.67 (pentet, J = 7.6 Hz, 2H),
1.56 (pentet, 2H), 1.27 (t, J = 7.1 Hz, 3H). 13C NMR (125 MHz, CDCl3)
d 169.2, 157.1, 142.2, 128.56, 128.52, 126.0, 61.6, 44.8, 39.5, 35.6,
4.3.2.2. Lymphocytic lines.
Human cutaneous T lymphocyte
cells (HuT-78) and human T lymphoblast cells (Molt-4) were
obtained from ATCC (Manassas, VA). HuT-78 and Molt-4 were cul-
tured as described previously at 37 °C with 5% CO2.47 HuT-78 cells
were grown in Hyclone RPMI-1640 medium supplemented with
10% FBS and the addition of 1ꢀ penicillin/streptomycin. Molt-4
cells were grown in Hyclone RPMI-1640 medium supplemented
with 10% fetal clone III. Human peripheral blood mononuclear cells
(hPBMCs) were purified by density centrifugation from human
Please cite this article in press as: Gromek, S. M.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.040

S. M. Gromek et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
13
blood that was purchased from Research Blood Components
(Brighton, MA) as described previously.48 Briefly, hPBMCs were fro-
zen in freezing media (10% DMSO, 10% FBS, 80% media) in liquid
nitrogen until needed. The cells were re-suspended prior to use
at 1 million per mL in RPMI-1640 supplemented with 10% heat-
inactivated FBS, 1ꢀ HEPES, pyruvate, non-essential amino acids,
and 2-mercaptoethanol (b-mercaptoethanol, BME) and added to
96 well plates.
4.3.5. Cytotoxic T-lymphocyte assay
The cytotoxic T-lymphocyte (CTL) assay was performed as pre-
viously described.39 Briefly, TALL-104 CTL human leukemia cells
were prepared and maintained as previously described.49 Com-
pounds were prepared at a stock concentration of 10 mM in DMSO
for a final testing concentration of 100 lM. Due to the high-
throughput nature of the assay, screening was performed once
with dose response performed in duplicate for active compounds.
Compounds (1
of cell solution (100
bated at 37 °C for 30 min prior to addition of 10 lL stimulation
lL each) were added to wells followed by addition
l
L/well; 2.5 ꢀ 106 cells/mL). Plates were incu-
4.3.3. Cell viability assay
4.3.3.1. Solid tumor.
Cytotoxicity of santacruzamate A ana-
solution, which was comprised of experimental saline (ES) and
anti-LAMP antibody, supplemented with either anti-CD3 beads
logues in the HCT116, MCF-7, and MDA-MB-231 cell lines were
evaluated using a standard MTS-PMS assay. Briefly, a 96 well plate
was seeded at 3,000 cells per well. Before the addition of test com-
pounds, plates were incubated at 37 °C with 5% CO2 for 24 h. Then,
(experimental wells and positive control wells), 20
lM thapsi-
gargin [TG] and 1 M phorbol 12-myristate 13-acetate [PMA] to
l
generate maximal exocytosis, or non-stimulating anti-CD8 beads
to serve as negative controls). Plates were subject to a secondary
incubation at room temperature for 50 min in the dark with con-
stant rotation before being fixed with 1% paraformaldehyde (PFA)
and subject to flow cytometric analysis, conducted on a two-laser
BD FACSCalibur at the UConn Flow Cytometry Facility. To calculate
effects on exocytosis, anti-LAMP staining fluorescence intensity
was measured from cells determined based on forward and side
scatter to have a single bead bound. Responses were normalized
to control anti-CD3 levels using the equation:
1
l
L of test compound was added to the well and incubated for
another 72 h. All test compounds were screened at 50 M in
HCT116 and MCF-7 cell line and at 100 M in MDA-MB-231. Pos-
itive controls for experiments were doxorubicin at 10 M and
M for HCT116 and MDA-MB-231, respectively. SAHA at 20
l
l
l
5
l
lM
was used as a control for the MCF-7 cell line. MTS and PMS
reagents were prepared according to the protocol below and mixed
at a ratio of 20:1 MTS:PMS. 20 lL of the MTS/PMS solution was
added to each well. The plate was incubated for another 3 h and
then the absorbance of the wells were measured using a Synergy
H1 Hybrid Reader (Biotek) at 490 nm. A minimum of two indepen-
dent experiments were performed, each in triplicate.
% response ¼ 100 ꢂ ðCD3test ꢁ CD8ctrlÞ=ðCD3ctrl ꢁ CD8ctrl
Þ
where test indicates responses from a test sample, and ‘ctrl’ indi-
cates a control sample. Using this analysis, compounds that do
not affect granule exocytosis give responses of ꢃ100%, compounds
that inhibit exocytosis give responses <100% and compounds that
augment exocytosis give responses >100%.
4.3.3.2. Preparation of MTS and PMS reagents.
MTS reagent
(Promega Celltiter 96) was dissolved in sterile PBS (UConn Cell Cul-
ture Facility) to make a 2 mg/mL solution. Phenazine methosulfate
(PMS) reagent (Sigma) was dissolved in sterile PBS to make a
0.92 mg/mL solution. MTS and PMS reagents were stored at
ꢁ20 °C until needed.
Acknowledgments
Funds for this research were provided by a National Instituties
of Health (NIH) Fogarty International Center (FIC) K01 Interna-
tional Research Scientist Development Award (IRSDA) (K01
TW008002, M.J.B., P.I.) and by a NIH FIC supplement to the Interna-
tional Cooperative Biodiversity Group (ICBG) grant based in
Panama (U01 TW006634, W.H. Gerwick, P.I., M.J.B., P.I. for supple-
ment). Support was also received from an American Association of
Colleges of Pharmacy grant (AG140125, A.J.W., P.I.). We acknowl-
edge M. K. Hadden for access to cell culture facilities and Georgina
Appiah-Pippim, Brendan Stewart, and Anne Sung for assistance
with compound management and organization. Molecular graph-
ics and analyses were performed with the University of California,
San Francisco Chimera package, developed by the UCSF Resource
for Biocomputing, Visualization, and Informatics (supported by
NIGMS P41-GM103311).
4.3.3.3. Lymphocytic lines.
Cytotoxicity of the santacruza-
mate A analogues in the HuT-78, Molt-4, and hPBMCs was evalu-
ated by the Quanti-Blue (QB) Cell Viability Assay Kit from
BioAssay Systems (Hayward, CA). For the QB Cell Viability Assay
Kit, a 96 well plate was seeded with 100
plate was seeded with 50
L of cells at a concentration of 105
cells/mL for Molt-4 and HuT-78 cells and 106 cells/mL for hPBMCs.
All test compounds were screened at 50 M. After 70 h of incuba-
tion, 10 L of the QB detection reagent was added into each well
lL of cells or a 384 well
l
l
l
for the final 2 h of incubation. The fluorescence of the wells was
measured using a Perkin-Elmer VICTOR X plate reader at kex 530 -
nm and kem 590 nm. In dose response experiments, a linear regres-
sion was used to obtain IC50 values. All experiments were repeated
three independent times.
4.3.4. Western blot analysis
A. Supplementary data
Molt-4 cells were re-suspended at 6 ꢀ 106 cells/4 mL in fresh
media. Cells were treated for 24 h with the indicated concentra-
tions of compounds. Cells were lysed with ice cold lysing buffer
(25 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium
deoxycholate, 0.1% SDS) and total protein was quantified with a
BCA assay (ThermoFisher) according to manufacturer’s protocol.
Equivalent masses of proteins were resolved by SDS–PAGE on
16% gels, transferred to nitrocellulose, and blotted with antibod-
ies to acetylated histones (Acetyl-Histone H3 (K9) #9649,
Acetyl-Histone H3 (K23) #8848, or Acetyl-Histone H4 (K12)
#2591) from Cell Signaling Technologies (Danvers, MA). The
JLA20 actin antibody was obtained from the University of Iowa
Developmental Studies Hybridoma Bank and used as a loading
control.
Supplementary data (individual models of SAHA and san-
tacruzamate A binding without enzyme overlay, theoretical hydro-
gen bonding and shuttling of SAHA and HDAC2, analysis of SCA NP
purity, as well as intermediate synthesis and characterization and
spectra for final compounds) associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.bmc.
2016.08.040.
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