The structural requirements of histone deacetylase inhibitors: C4-modified SAHA analogs display dual HDAC6/HDAC8 selectivity

Article abstract of DOI:10.1016/j.ejmech.2017.10.076

Histone deacetylase (HDAC) enzymes govern the post-translational acetylation state of lysine residues on protein substrates, leading to regulatory changes in cell function. Due to their role in cancers, HDAC proteins have emerged as promising targets for

Full text of DOI:10.1016/j.ejmech.2017.10.076

Accepted Manuscript
The structural requirements of histone deacetylase inhibitors: C4-modified SAHA
analogs display dual HDAC6/HDAC8 selectivity
Ahmed T. Negmeldin, Joseph R. Knoff, Mary Kay H. Pflum
PII:
S0223-5234(17)30878-4
10.1016/j.ejmech.2017.10.076
EJMECH 9868
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 June 2017
Revised Date: 28 October 2017
Accepted Date: 28 October 2017
Please cite this article as: A.T. Negmeldin, J.R. Knoff, M.K.H. Pflum, The structural requirements of
histone deacetylase inhibitors: C4-modified SAHA analogs display dual HDAC6/HDAC8 selectivity,
European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.10.076.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
The structural requirements of histone deacetylase
inhibitors: C4-modified SAHA analogs display dual
HDAC6/HDAC8 selectivity
a,b
a
*a
Ahmed T. Negmeldin , Joseph R. Knoff , and Mary Kay H. Pflum
a
Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, Michigan 48202,
United States
b
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Cairo University, Cairo
11562, Egypt
∗
Corresponding author: Tel.: +1-313-577-1515; fax: +1-313-577-8822; e-mail: pflum@wayne.edu
Abbreviations used:
HDAC, histone deacetylase; SAHA, suberoylanilide hydroxamic acid; NAD, Nicotinamide adenine
dinucleotide; DNA, deoxyribonucleic acid; FDA, Food and Drug Administration; ELISA, enzyme-linked
immunosorbent assay; TMSCl, Trimethylsilyl chloride; DCM, Dichloromethane; THF, Tetrahydrofuran;
NaH, Sodium hydride; DCC; N,N'-Dicyclohexylcarbodiimide; DMAP, 4-Dimethylaminopyridine;
DIPEA, N,N-Diisopropylethylamine; TBTU, N,N,N′,N′-Tetramethyl-O-(benzotriazol-1-yl)uronium
tetrafluoroborate; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; GAPDH,
Glyceraldehyde
3-phosphate
dehydrogenase;
MTT,
3-(4,5-Dimethylthiazol-2-yl)-2,5-
Diphenyltetrazolium Bromide; EDCI, N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride;
NaHMDS, Sodium bis(trimethylsilyl)amide; LiAlH
chloride; LiCl, Lithium chloride.
4
, Lithium aluminum hydride; MSCl, Methanesulfonyl
1

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Highlights
-
Analogs of the non-selective FDA-approved drug SAHA (Vorinostat) demonstrated
selectivity for HDAC6 and HDAC8 over HDAC1, 2, and 3
-
The best compound, (R)-C4-benzyl SAHA, displayed IC values of 48 and 27 nM with
50
HDAC6 and HDAC8, respectively.
-
-
-
(R)-C4-benzyl SAHA maintained 520- to 1300-fold selectivity for HDAC6 and HDAC8.
The HDAC6 selectivity of C4-benzyl SAHA was reproduced in cancer cells
Docking studies provided a structural rationale for the HDAC6/HDAC8 selectivity of C4-
benzyl SAHA
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Abstract
Histone deacetylase (HDAC) enzymes govern the post-translational acetylation state of lysine
residues on protein substrates, leading to regulatory changes in cell function. Due to their role in
cancers, HDAC proteins have emerged as promising targets for cancer treatment. Four HDAC
inhibitors have been approved as anti-cancer therapeutics, including SAHA (Suberoylanilide
hydroxamic acid, Vorinostat, Zolinza). SAHA is a nonselective HDAC inhibitor that targets most of the
eleven HDAC isoforms. The nonselectivity of SAHA might account for its clinical side effects, but
certainly limits its use as a chemical tool to study cancer-related HDAC cell biology. Herein, the
nonselective HDAC inhibitor SAHA was modified at the C4 position of the linker to explore activity
and selectivity. Several C4-modified SAHA analogs exhibited dual HDAC6/8 selectivity. Interestingly,
(R)-C4-benzyl SAHA displayed 520- to 1300-fold selectivity for HDAC6 and HDAC8 over HDAC1, 2,
and 3, with IC50 values of 48 and 27 nM with HDAC6 and 8, respectively. In cellulo testing of the
inhibitors was consistent with the observed in vitro selectivity. Docking studies provided a structural
rationale for selectivity. The C4-SAHA analogs represent useful chemical tools to understand the role
of HDAC6 and HDAC8 in cancer biology and exciting lead compounds for targeting of both HDAC6
and HDAC8 in various cancers.
Keywords:
Histone Deacetylase, HDAC inhibitor, dual HDAC6/HDAC8 selective inhibitor, SAHA (Vorinostat),
HDAC isoform selectivity
3

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1. Introduction
Histone deacetylase (HDAC) proteins are key enzymes involved in epigenetic regulation of gene
expression. Specifically, HDAC-mediated deacetylation of acetyllysine residues on nucleosomal
histones leads to tight binding to genomic DNA, which affects accessibility and transcription [1-2]. In
addition, HDAC proteins influence protein-protein interaction, protein-DNA interaction, protein
localization, and protein stability through deacetylation of non-histone substrates [3-4]. The eighteen
human HDAC proteins are divided into four classes according to their homology with yeast proteins,
size, cellular localization, and number of catalytic active sites [5]. Class III (SIRT1-7) HDAC proteins
+
are NAD -dependent. Classes I (HDAC1, 2, 3 and 8), II (HDAC4, 5, 6, 7, 9, and 10), and IV
(HDAC11) HDAC proteins are metal-dependent, and are the focus of this work [5].
HDAC proteins regulate the expression of several cancer-related proteins involved in cell signaling,
transcription, and tumor suppression through the deacetylation of nucleosomal histone proteins [6-7].
Overexpression of HDAC proteins results in unregulated transcription and aberrant protein activity
and function, which is linked to several diseases, including cancer [7]. For example, HDAC1 was
overexpressed in lung [8], breast [9], and colon cancers [10]. HDAC2 was overexpressed in colorectal
cancer[11]. HDAC8 was highly expressed in neuroblastoma patients, leading to cancer progression
and poor survival rates [12]. In addition, selective inhibition of HDAC8 induced apoptosis in leukemia
and T-cell lymphoma cell lines [13]. Class II HDAC6 was overexpressed in oral squamous cell
carcinoma and ovarian cancer [14-15]. Overexpression of both HDAC6 and HDAC8 was linked to
breast cancer metastasis and invasion [16].
Due to their key role in cancer, several anti-cancer agents targeting HDAC proteins have been
developed [17]. HDAC inhibitors promoted apoptosis and reduced proliferation and migration through
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their effect on both histone and non-histone substrates.[17-19]. Several HDAC inhibitors have been
approved by the FDA for treatment of cancer, and several others are in clinical trials [20]. SAHA
(suberoylamide hydroxamic acid, Vorinostat, Zolinza™), and Belinostat (PXD101, Belodaq™, Figure
1) are FDA-approved for treatment of T-cell lymphoma [20-22], while Panobinostat (LBH-589,
Farydak™, Figure 1) was approved for treatment of multiple myeloma [23]. SAHA is a nonselective
inhibitor that targets most of the eleven metal-dependent HDAC isoforms [24]. The nonselectivity of
the FDA-approved drugs, including SAHA, might explain the side effects observed in the clinic, such
as cardiac arrhythmia and thrombocytopenia [25-26]. Moreover, the use of SAHA as a chemical tool
to study the role of specific HDAC isoforms in cancer cell biology is limited due to its nonselectivity.
To overcome the limitations of nonselective drugs, several isoform selective HDAC inhibitors have
been developed, with some in clinical trials. As illustrative examples, entinostat (MS-275, Figure 1) is
selective for HDAC1, 2, and 3 [24, 27], whereas tubastatin (Figure 1) is HDAC6-selective [28-29].
Recently, several dual HDAC6/8 selective inhibitors have been reported, including BRD-73954 and
valpropylhydroxamic acid (Figure 1) [30-31]. HDAC inhibitors that target one or two HDAC isoforms
will be valuable for development of new drugs with minimal side effects [32-35]. In addition, recent
reports suggested that inhibition of two HDAC isoforms is desirable by maintaining synergistic
therapeutic effects in various cancers [30, 36-37]. Related to this work, dual inhibition of HDAC6 and
HDAC8 might have potential application in breast cancer angiogenesis and metastasis [13, 30].
Moreover, selective HDAC inhibitors will be useful as chemical tools to study cancer-related HDAC
cell biology.
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Figure 1: Chemical structures of the FDA-approved drugs SAHA, Belinostat, and Panobinostat, the
C4-modified SAHA analogs reported here, and several isoform selective HDAC inhibitors discussed
in the text (MS-275, Tubastatin, PCI-34051, BRD-73954, Valpropylhydroxamic acid, Aminotetralin 32,
C2-hexyl SAHA, and C5-benzyl SAHA).
To understand the structural requirements of HDAC inhibitors, we previously synthesized SAHA
analogs substituted in the linker region at carbon 2 (C2), carbon 3 (C3), carbon 5 (C5), or carbon 6
(C6) (Figure 1) [38-41]. C2-hexyl SAHA (Figure 1) showed 49- to 300-fold HDAC6/8 dual selectivity
over HDAC1, 2, and 3, with 0.6 and 2.0 µM potency against HDAC6 and HDAC8, respectively [42].
Among the C5-SAHA analogs, C5-benzyl SAHA (Figure 1) displayed 8- to 21-fold HDAC6/8
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selectivity with IC50 values of 270 and 380 nM with HDAC6 and HDAC8, respectively [41]. Some of
the C3-modified SAHA analogs displayed preference for HDAC6 over HDAC1 and 3 [39], while some
of the C6-modified SAHA analogs inhibited HDAC1 and 6 over HDAC3 [40]. In addition, SAHA
analogs modified at the hydroxamic acid moiety had a preference for HDAC1 [43]. In this work, SAHA
analogs modified at the C4 position were synthesized and screened in vitro and in cellulo for their
activity and selectivity. The C4-modified SAHA analogs showed high selectivity towards HDAC6 and
8
over HDAC1, 2, and 3, with nanomolar potency against HDAC6 and HDAC8. Docking studies
provided a structural rationale for the observed selectivity. These studies emphasize that modification
of the SAHA linker can enhance isoform selectivity. In addition, the HDAC6/8 dual selective C4-SAHA
analogs reported here have the potential to be useful pharmacological tools for biomedical research
and lead compounds for anti-cancer drug development.
2. Results and discussion
2.1. Synthesis of C4-modified SAHA analogs
Synthesis of the C4-SAHA analogs started with a cross metathesis reaction of methyl-4-pentenoate
(2) with crotonaldehyde (3) using second generation Grubbs' catalyst to afford the α,β-unsaturated
aldehyde (4) (Scheme 1). Different substituents were appended to 4 via 1,4-addition using
organolithium cuprates, followed by Horner–Wadsworth–Emmons reaction with benzyl
phosphonoacetate (5) to give the unsaturated benzyl esters (6a-f). Reduction and hydrogenolysis of
6a-f gave free acids (7a-f), which were coupled with aniline to afford 8a-f. Finally, esters (8a-f) were
reacted with hydroxylamine to afford the C4-substituted SAHA derivatives (1a-f) as racemic mixtures.
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Scheme 1. Synthesis of C4-SAHA analogs (1a-f)
2.2. In vitro screening of C4-modified SAHA analogs.
SAHA analogs 1a-f were tested for global HDAC inhibition with HeLa cell lysates as the source of
all HDAC proteins (Table 1). SAHA also included as a broad spectrum inhibitor, while Tubastatin and
BRD-73954 were tested as isoform selective inhibitors. HDAC activity was measured using the
commercially available HDAC-Glo™ I/II substrate (Promega). The results of the screening showed
that all of the synthesized derivatives were less potent than SAHA (Tables 1 and S1, and Figure
S141). The most potent derivative was C4-methyl SAHA (1a), which showed an IC value of 3.3 µM.
50
Compared to the parent molecule SAHA, C4-methyl SAHA is 18-fold less potent, while the rest of the
analogs showed 78- to 344-fold reduction in potency. Both tubastatin and BRD-73954 also showed
36- to 60-fold less potency compared to SAHA (9.9 and 6.7 µM IC50 values). Because HeLa cell
lysates contain all HDAC isoforms, the poor potency of the C4-SAHA analogs suggests that they
might be selective for specific isoforms, similar to tubastatin and BRD-73954.
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Table 1: IC values for SAHA, Tubastatin, BRD-73954, and C4-SAHA analogs (1a-1f) with HeLa
5
0
a
cell lysates.
Compound
SAHA
Tubastatin
BRD-73954
IC50 (µM)
0.20 ± 0.02
9.9 ± 0.4
6.7 ± 0.2
3.3 ± 0.1
14 ± 1
1
a (methyl)
1b (ethyl)
1c (n-butyl)
53 ± 2
1
1
1
d (n-hexyl)
e (phenyl)
f (benzyl)
60 ± 1
65 ± 6
62 ± 1
a
Mean IC value and standard error of at least three independent trials are shown (Figure S141 and
50
Table S1).
To assess selectivity, an initial screen was performed with analogs 1a-f and the parent molecule
SAHA at a single concentration against HDAC1, 2, 3, and 6 using an ELISA-based HDAC activity
assay [28]. These select isoforms were chosen due to their high deacetylation activity among the
family members and their representation of both class I (HDAC1, 2, and 3) and class II (HDAC6).
SAHA, as expected, showed no selectivity among HDAC1, 2, 3, and 6, inhibiting their activity to a
similar extent (Figure 2). Interestingly, all C4-SAHA analogs (1a-f) displayed more potent inhibition
against HDAC6 compared to HDAC1, HDAC2, and HDAC3 (Figure 2 and Table S2). The analogs
that showed the greatest difference in potency with HDAC6 versus the other isoforms were C4-n-
butyl (1c) and C4-benzyl (1f). The C4-methyl SAHA analog (1a) showed the smallest difference in
potency comparing HDAC6 to HDAC1 and HDAC3 (Figure 2 and Table S2). This single concentration
screen suggested that C4-modification of the SAHA structure results in selectivity for HDAC6.
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HDAC1
HDAC2
HDAC3
HDAC6
1
00
8
6
4
2
0
0
0
0
0
SAHA 1a
1b
1c
1d
1e
1f
(
Me) (Et) (Bu) (Hex) (Ph) (Bn)
Figure 2: In vitro isoform selectivity screening of C4-modified SAHA analogs (1a-f) against HDAC1,
HDAC2, HDAC3, and HDAC6 using an ELISA-based HDAC activity assay [28]. Analogs 1a-f were
tested at 0.75, 0.75, 2.5, 1.25, 2.5, and 5 µM final concentration, respectively. SAHA was tested at 1
µM concentration [28]. Mean percent deacetylase activities from a minimum of two independent trials
with standard errors were plotted (Table S2).
To further assess selectivity, IC50 values for derivatives 1b-f were determined with HDAC1, HDAC2,
HDAC3, HDAC6, and HDAC8 isoforms (Table 2). HDAC8 was included due to its similar active site
structure compared to HDAC6 [31]. For comparison, the non-selective parent molecule SAHA and
the HDAC6-selective inhibitor tubastatin (Figure 1) were also tested as control compounds (Table 2)
[
28]. As expected, the non-selective inhibitor SAHA showed similar low nanomolar IC50 values with
HDAC1, 2, 3, 6, but a 6- to 27-fold reduction in potency against HDAC8 [28]. In contrast, the HDAC6-
selective inhibitor tubastatin displayed 87- to 130-fold selectivity for HDAC6 over HDAC1, 2, and 3,
and 11-fold selectivity for HDAC6 over HDAC8, which is consistent with prior studies [28, 42]. As
expected based on the single concentration screen, analogs 1b-f displayed preference for HDAC6
and HDAC8, with 28- to 740-fold selectivity compared to HDAC1, 2, and 3 (Tables 2 and S10).
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Importantly, analogs 1b-f maintained low nanomolar IC50 values in the 57 to 290 nM range with
HDAC6 and HDAC8 (Tables 2), similar to SAHA. Among the analogs, C4-benzyl SAHA (1f)
displayed the highest selectivity, with 210- to 740-fold selectivity for HDAC6 and 8 over HDAC1, 2,
and 3 (Tables 2 and S10), and potent inhibition with low nanomolar IC values (140 and 57 nM with
50
HDAC6 and HDAC8, respectively, Table 2). Similarly, C4-n-butyl (1c) and C4-n-hexyl (1d) SAHA
demonstrated 170- to 480-fold HDAC6/8-selectivity compared to HDAC1, 2, and 3 isoforms, with low
nanomolar IC50 values (Tables 2 and S10). The C4-ethyl (1b) SAHA analog displayed the weakest
selectivity among the analogs, with 28- and 46-fold selectivities for HDAC6 and 8 over HDAC1, 2, and
3
(Tables 2 and S10). Comparing the IC50 values of the analogs to the parent SAHA (Table 2), the
higher HDAC6 selectivities are due to comparable potency against HDAC6 (2.5 to 3-fold reduction),
but dramatic loss of potency against HDAC1, 2 and 3 (51- to 2,100- fold reduction, Table 2).
Remarkably, the analogs displayed enhanced potency with HDAC8 compared to the parent SAHA
compound (2- to 9-fold enhancement, Table 2), which lead to the observed HDAC8 selectivities,
along with HDAC6.
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Table 2: IC50 values for SAHA, Tubastatin, SAHA analogs 1b-1f, (R)-1f, and (S)-1f against
HDAC1, 2, 3, 6 and 8, and EC values with U937 cells.
50
a
b,c
IC50 values (nM)
EC50 (µM)
U937 cells
540 ± 10 0.88 ± 0.13
Compound
HDAC1
33 ± 1
2,700 ± 200
HDAC2
96 ± 10
3,900 ± 400
̶
HDAC3
20 ± 1
2,900 ± 500
̶
HDAC6 HDAC8
33 ± 3
31 ± 4
d
SAHA
d
Tubastatin
330 ± 10
83 ± 8
PCI-34051
̶
̶
̶
210 ± 30
1b (ethyl)
4,400 ± 300
4,900 ± 400
6,000 ± 1200 160 ± 10 130 ± 3
̶
1
c (n-butyl) 15,000 ± 1000 18,000 ± 2000 23,000 ± 3000
88 ± 7
74 ± 2
79 ± 3
34 ± 2
16 ± 3
1
d (n-hexyl) 35,000 ± 1000 38,000 ± 3000 30,000 ± 3000 140 ± 10
1e (phenyl) 11,000 ± 1000 12,000 ± 1000 23,000 ± 2000 110 ± 10 290 ± 20
̶
1
f (benzyl) 29,000 ± 1000 32,000 ± 2000 42,000 ± 4000 140 ± 10
57 ± 2
27 ± 2
150 ± 10
28 ± 7
(R)-1f
25,000 ± 2000 36,000 ± 3000 27,000 ± 2000
40,000 ± 1000 51,000 ± 1000 37,000 ± 2000
48 ± 8
95 ± 9
̶
̶
(
S)-1f
Mean IC50 value and standard error of at least three independent trials are shown (Figures S142-
b
S148 and Tables S3-S9). Mean EC value and standard error of at least three independent trials
50
c
are shown. Data in table is associated with Figures S150 and Table S11. "
determined. Previously reported IC values using the same assay procedure [28, 42].
̶
" represents not
d
50
In terms of a structure-activity relationship (SAR) analysis, modifying SAHA at the C4 position with
long alkyl substituents led to enhanced selectivity; the C4-hexyl 1d analog with the longest alkyl chain
demonstrated elevated selectivity (210- to 480-fold) compared to the C4-butyl 1c analog (170- to 310-
fold), which were both more selective than the C4-ethyl analog 1b with the smallest alkyl chain (28-
and 46-fold, Tables 2 and S10). For these alkyl analogs, selectivity was due to discrimination against
HDAC1, 2, and 3, with the least selective C4-ethyl analog 1b demonstrating greater potency to
HDAC1, 2, and 3 (4.4 - 6.0 µM, Table 2) compared to the most selective C4-hexyl analog 1d (30 – 38
µM, Table 2). The length of the substituent also influenced the selectivities of the analogs with aryl
groups at the C4 position; the lack of a methylene in C4-phenyl analog 1e led to decreased selectivity
(38- to 350-fold) compared to the C4-benzyl analog 1f (210- to 740-fold). Similar to the alkyl series,
the reduced selectivity of C4-phenyl analog 1e was due to greater potency with HDAC1 and 2 (11
and 12 µM, Table 2) compared to C4-benzyl analog 1f (29 and 32 µM, Table 2), in addition to
decreased potency with HDAC8 (290 nM, Table 2) compared to C4-benzyl analog 1f (57 nM, Table
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2). In total, the SAR analysis indicated that longer substituents at the C4 position led to greater
HDAC6/8 selectivity primarily due to discrimination against HDAC1, 2, and 3.
Compared to previously reported inhibitors, the observed HDAC6 selectivities of the analogs 1c,
1d, and 1f (at least 170-, and 210-fold) were higher than the selectivity observed with the HDAC6-
selective inhibitor tubastatin (at least 87-fold, Tables 2 and S10). Moreover, analogs 1c, 1d, and 1f
showed comparable HDAC8 selectivities (at least 200-, 380-, and 510-fold) relative to the HDAC8-
selective inhibitor PCI-34051 (Figure 1) (at least 400-fold) [44]. Finally, C4-benzyl analog 1f showed
higher dual HDAC6/8 selectivity (at least 210- and 510-fold) compared to the known HDAC6/8-
selective inhibitor BRD-73954 (75- and 250-fold selectivity to HDAC6, and 8 over HDAC1, 2, and 3)
[
30].
2.3. In cellulo selectivity testing
To further assess the observed HDAC6 selectivity in cellulo, the C4-benzyl (1f) SAHA analog was
tested for inhibition of HDAC activities in cells. The inhibition of HDAC6 was monitored by detecting
the levels of its known substrate acetyl-α-tubulin (AcTub), whereas Class I HDAC proteins (HDAC1,
2, and 3) inhibition was monitored through the levels of their known substrate acetyl-histone H3
(AcH3). SAHA or the analog 1f were incubated with U937 leukemia cells before lysis and western blot
analysis of protein acetylation. As expected, SAHA showed increased levels of both acetyl-α-tubulin
and acetyl-histone H3 (Figure 3A, lane 2), which is consistent with its non-selective inhibition of
HDAC1, 2, 3, and 6 isoforms. In contrast, C4-benzyl (1f) showed a dose-dependent increase in levels
of acetyl-α-tubulin (Figure 3A, lanes 3-5, AcTub) compared to the DMSO control (Figure 3A, lane 1),
which was elevated compared to the levels of acetyl histone H3 (Figure 3A, lanes 3-5, AcH3).
Greater acetylation of the HDAC6 substrate tubulin compared to the class I HDAC substrate histone 3
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in cells (Figure 3A) is consistent with the HDAC6 selectivity of 1f observed in the in vitro screening
(Table 2 and Figure 1).
Figure 3: A) Cell based selectivity testing of the C4-benzyl SAHA analog 1f. Western blot analysis of
acetyl-histone H3 (AcH3) and acetyl-α-tubulin (AcTub) is shown after treatment with SAHA or the C4-
benzyl SAHA analog 1f. U937 cells were treated with DMSO (1%), SAHA (5 µM), or increasing
concentrations of C4-benzyl SAHA (1f) analog (20-60 µM), before lysis, SDS-PAGE separation,
transfer to a PVDF membrane, and western analysis with AcH3 or AcTub antibodies. GAPDH levels
in the samples were also probed as a gel load control. A DMSO control sample was included for
comparison to inhibitor treated samples. Repetitive trials are shown in Figure S149. B) Cytotoxicity of
tubastatin (Tub) and PCI-34051 (PCI) alone or in combination using an MTT assay with the U937 cell
line. Concentrations close to the EC values of each inhibitor were used (80 µM for tubastatin and
50
200 µM for PCI-34051). Mean percentage cell viability from three independent trials with standard
errors was plotted (Table S12).
2.4. In vitro cancer cell growth inhibition
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To assess the cytotoxic effect of the HDAC6/8 selective inhibitors in cancer cells, select SAHA
derivatives were tested with the leukemia cell lines, U937 and Jurkat, due to the prominent role of
HDAC6 in leukemia [45]. Initially, as controls, the nonselective parent SAHA, along with the HDAC6-
selective inhibitor tubastatin and the HDAC8-selective inhibitor PCI-34051, were tested. SAHA
showed an EC50 value of 0.88 µM (Table 2), which is consistent with the cytotoxicity previously
reported [46]. The non-selective inhibition of all the HDAC proteins by SAHA likely contributes to its
high cytotoxicity. In contrast, the isoform selective inhibitors tubastatin and PCI-34051 displayed
lower cytotoxicity compared to SAHA, with 83 and 210 µM EC50 values, respectively (Table 2). To
provide evidence that dual inhibition of both HDAC6 and HDAC8 enhances anti-proliferative activity,
U937 cells were incubated alone or with both tubstatin and PCI-34051 at concentrations near their
EC50 values (Figure 3B). The combination of both tubastatin and PCI-34051 reduced cell viability
compared to each inhibitor alone, which suggests that dual HDAC6/8 inhibition provides beneficial
anti-viability effects.
Next, the EC50 values of analogs, C4-n-butyl (1c), C4-n-hexyl (1d), and C4-benzyl (1f), were
determined with the U937 cell line [45]. SAHA analogs 1c, 1d, and 1f displayed 34, 16, and 28 µM
EC50 values, respectively, with the U937 cell line (Table 2). The analogs were 18- to 39-fold less
cytotoxic compared to SAHA. To confirm the cytotoxicity study in U937 cells, SAHA and the analogs
were also tested for cytotoxicity with the Jurkat cell line. The analogs showed reduced cytotoxicity
compared to SAHA (Figure S151, Table S13), consistent with the study in U937 cells. The reduced
cytotoxicity of tubastatin, PCI-34051, and the C4-modified SAHA analogs in U937 and Jurkat cells
compared to SAHA might be due to their selectivity toward HDAC6 and/or 8. Consistent with this
hypothesis, similar micromolar cytotoxicities were also observed with the several HDAC6-selective
inhibitors in previous reports [37, 47-48].
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2.5. Enantioselective synthesis and screening of (R)- and (S)-C4-benzyl SAHA analog (1f)
Since all analogs were synthesized and screened as racemic mixtures, single enantiomers of the
most selective analog C4-benzyl SAHA 1f were synthesized to test selectivities. The asymmetric
syntheses of both enantiomers were carried out utilizing Evan's oxazolidinone chiral auxiliary (R)-8
(Scheme 2) [49]. To ultimately prepare (R)-1f, 4-pentenoyl chloride was reacted with chiral auxiliary
(R)-8 using n-butyllithium, which yielded pentenoyl oxazolidinone intermediate (R)-9 (Scheme 2A).
Enantioselective reaction of benzyl bromide with (R)-9 gave the diastereomeric intermediate (RS)-10.
To ultimately prepare (S)-1f, a similar procedure was employed (Scheme 2B), but reacting 3-
phenylpropanoyl chloride with chiral auxiliary (R)-8, followed by enantioselective reaction with allyl
bromide to give diastereomeric intermediate (RR)-10. Both diastereomers were obtained with high
1
diastereomeric ratios (dr), which were assessed by HNMR analysis (99:1 dr for (RS)-10, and 97:3 dr
for (RR)-10, Figures S79 and S86).
To remove the chiral auxiliary from (RS)-10 and (RR)-10, each diastereomer was reacted with
lithium aluminum hydride, which produced alcohols (S)-11 and (R)-11 (Scheme 2). To assess the
optical purity and enantiomeric excess (ee) of the alcohol intermediates (S)-11 and (R)-11, Mosher
esters
were
synthesized
by
coupling
each
alcohol
with
(R)-(+)-α-Methoxy-α-
trifluoromethylphenylacetic acid ((R)-MTPA) using EDCI and DMAP (Scheme S1) [50]. Analysis of the
1
HNMR spectra showed that both Mosher esters were observed in high diastereomeric ratios (99:1 dr
for (S)-11-(R)-MTPA, and 98:2 dr for (R)-11-(R)-MTPA, Figures S94, S95, S100, and S101), which
implies that the alcohol intermediates (S)-11 and (R)-11 were obtained with high enantiomeric ratios
(98% ee for (S)-11 and 96% ee (R)-11).
Scheme 2. Enantioselective synthesis of intermediate alcohols (S)-11 (a) and (R)-11 (b).
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To synthesize enantiopure C4-benzyl SAHA analogs (R)-1f and (S)-1f, both alcohols (S)-11 and
R)-11 were then converted to methanesulfonate esters, followed by substitution with dimethyl
(
malonate to give diesters (R)-13 and (S)-13, respectively (Schemes 3 and S2). Krapcho
decarboxylation afforded methyl ester intermediates (S)-14 and (R)-14. Cross metathesis of the
methyl esters with N-phenyl acrylamide (15) using Grubbs' second generation catalyst afforded ester
amides (S)-16 and (R)-16 [51]. Finally, reduction followed by substitution with hydroxyl amine gave
both enantiomers of C4-benzyl SAHA analog, (R)-1f and (S)-1f (Schemes 3 and S2).
Scheme 3. Synthesis of (R)-C4-benzyl SAHA analog
To assess the selectivities of enantiomers (R)-C4-benzyl SAHA (R)-1f and the (S)-C4-benzyl
SAHA (S)-1f, IC values of both were determined with HDAC1, 2, 3, 6, and 8 (Table 2). Similar to
50
the racemic mixture, both enantiomers showed dual HDAC6/HDAC8 selectivity. (R)-C4-benzy
SAHA (R)-1f displayed more potent inhibition against HDAC6 and HDAC8 compared to the (S)-C4-
17

ACCEPTED MANUSCRIPT
benzyl SAHA (S)-1f, with IC values of 48 and 27 nM for (R)-1f and 95 and 150 nM for (S)-1f
50
(Table 2). In addition, (R)-1f showed greater fold preference for HDAC6 and 8 over HDAC1, 2, and
3
(520- to 1300-fold) compared to the racemic mixture (210- to 740-fold). In contrast, (S)-1f showed
lower fold selectivities (240- to 540-fold) compared to both (R)-1f and the racemic mixture (Tables 2
and S10). In summary, in vitro screening revealed that both (R)-1f and (S)-1f are highly selective for
HDAC6 and 8 over HDAC1, 2, and 3 with nanomolar potency. However, (R)-1f was more potent
and selective than (S)-1f (Tables 2 and S10).
2.6. Docking studies
To rationalize the experimental results, docking studies with both enantiomers of C4-benzyl SAHA
(
1f) were performed using the AutoDock 4.2 and Autodock tools programs [52-53]. The recently
reported crystal structure of HDAC6 (PDB: 5G0H) [54] along with HDAC3 crystal structure (PDB:
A69) [55] were used in these studies. SAHA, the parent molecule, was also examined with both
4
crystal structures for comparison and to validate the docking procedure. With HDAC6, both
enantiomers of C4-benzyl SAHA (1f) were positioned similarly in the active site compared to SAHA,
with the hydroxamic acid moiety near the catalytic metal, the linker region in the 11 Å channel, and
the anilide group in the solvent exposed region. In addition, the hydroxamic acid groups bound within
1.9-2.7 Å of the active site residues (H573, H574, and Y745) and the active site catalytic zinc atom
(Figures 4A and S153A), similar to that of the parent molecule SAHA (1.7-2.7 Å) (Figure S152A).
Superimposition of each enantiomer with SAHA in the HDAC6 active site showed similar binding of
the hydroxamic acid moiety (Figures 4C and S153C). The docking studies with the HDAC6 crystal
structure are consistent with the high binding affinity of C4-benzyl SAHA and SAHA in vitro (Table 2).
18

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Figure 4: Docked poses of (R)-C4-benzyl SAHA (R)-1f in the crystal structures of HDAC6 (A,C) and
HDAC3 (B, D) using Autodock 4.2. A and B) Binding distances between the hydroxamic acid atoms
and active site residues (numbered in figure) or the metal are displayed in Angstroms. The atomic
2+
radius of the metal (Zn ) in was set at 0.5 Å for clarity. Atom color-coding: (R)-C4-benzyl SAHA
2
+
(
(
(
C=green/white; O=red; N=blue; H=White); amino acids (C=deep teal; O=red, N=blue); Zn metal ion
grey sphere). C and D) Shown is the superimposition of SAHA (yellow) and (R)-C4-benzyl SAHA
red) in the crystal structures of HDAC6 (C) or HDAC3 (D), with the metal ion (Zn ) represented as a
2+
grey sphere (1.39 Å radius).
To explain the observed selectivities, (R)-1f and (S)-1f were also docked into the HDAC3 crystal
structure. Longer and weaker binding interactions were observed with (R)-1f and (S)-1f in the HDAC3
structure (2.1-5.0 Å, Figures 4B and S153B) compared with the HDAC6 structure (1.9-2.7 Å).
Superimposition of each enantiomer with SAHA in the HDAC3 structure showed a shift in the position
of the hydroxamic acid moieties away from the catalytic metal compared to SAHA (Figure 4D and
19

ACCEPTED MANUSCRIPT
S153D). The docking analysis suggests that the presence of a bulky substituent on the C4 position
weakens binding to HDAC3 due to steric clash with the relatively narrow and long 11Å channel of
HDAC3 (Figures S154B, see arrows). In particular, the steric clash of the bulky substituent with
active site residues positions the hydroxamic acid moieties away from the metal binding region
(Figures 4B, S153B and S154B). In contrast, HDAC6 maintains a wider and shorter V-shaped 11Å
channel (Figure S154A, see arrow), which can accommodate relatively large C4 substituents without
affecting hydroxamic acid-metal binding (Figures 4A, S153A and S154A).
Structural analysis of prior HDAC6/8 selective inhibitors are consistent with our docking analysis
suggesting that HDAC6 maintains a wider and shorter 11Å channel compared to HDAC1, 2, and 3
[
29, 54]. Valpropylhydroxamic acid (Figure 1) with a propyl substituent in the linker region showed
selectivity for HDAC6 and 8, although with micromolar potency and low fold selectivity (9-17-fold
selectivity against with HDAC1, 2, and 3) [56]. Inhibitors bearing a bicyclic or phenyl ring in the linker
displayed dual HDAC6 and HDAC8 selectivity (BRD-73953 and Aminotetralin 32, Figure 1) [30, 36-
37]. Finally, SAHA analogs with a hexyl or benzyl substituent at the C2 or C5 positions in the linker
also demonstrated dual HDAC6/8 selectivity (Figure 1) [42]. Based on these prior reports and our
docking studies, we hypothesize that substitutuents at the C4 position of SAHA can be
accommodated in the relatively wider and shorter V-shaped 11Å cavity of HDAC6 active site (Figures
4A, S153A, 4C, S153C, and S154A), but not the narrower and relatively longer 11Å cavity of HDAC1,
HDAC2, and HDAC3 (Figures 4B, S153B, 4D, S153D, and S154B). In addition, the size of the
substituent plays a critical role in the selectivity. For example, analogs bearing smaller substituents
(as methyl or ethyl in 1a or 1b) demonstrated lower selectivity (Table S1) compared to analogs
comprising bulkier substituents (as butyl, hexyl, phenyl, and benzyl 1c-1f) (Figure 2 and Table 2). In
total, the docking results are consistent with previously reported structural studies suggesting that the
wider HDAC6 active site entrance is the source of selectivity.
20

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3. Conclusions
In conclusion, SAHA analogs modified at the C4 position were synthesized and screened for
potency and selectivity. C4-SAHA analogs showed up to 1300-fold dual selectivity for HDAC6 and
HDAC8 over HDAC1, HDAC2, and HDAC3. The best analogs were C4-n-butyl SAHA (1c) and C4-
benzyl SAHA (1f). C4-benzyl SAHA (1f) showed the highest fold selectivity with 210- to 740-fold
selectivity for HDAC6 and 8 compared to HDAC1, 2, and 3, and 140 and 57 nM IC50 with HDAC6 and
HDAC8. Interestingly, the fold selectivity of the C4-benzyl SAHA analog was higher than the recently
reported dual HDAC6/8 selective inhibitors (at least 23-, 75-, and 79-fold) [30, 36-37]. Furthermore, in
cellulo testing of the C4-benzyl analog showed consistency with the in vitro screening.
Enantioselective synthesis and screening of both enantiomers of the C4-benzyl SAHA revealed that
(R)-C4-benzyl SAHA is more potent and selective than the (S) enantiomer, with 48 and 27 nM IC50
with HDAC6 and HDAC8, and 520- to 1300-fold selectivity for HDAC6 and 8 over HDAC1, 2, and 3.
The dual HDAC6/HDAC8 selective C4-SAHA analogs reported in this work can be useful as
biological tools to understand the role of HDAC6 and HDAC8 in cancer, and as well as lead
compounds towards development of more effective anti-cancer drugs. More generally, these studies
with SAHA analogs suggest that modifying current drugs can significantly improve their properties.
4. Experimental
4.1. Materials and Instrumentation
Unless otherwise noted, chemicals were purchased from Sigma-Aldrich, Acros Organics, or Fisher
Scientific. “Iron-free” glassware was prepared by rinsing glass vessels with a 5M aqueous HCl
solution, followed by washing with distilled de-ionized water. “Iron-free” silica gel was prepared by
washing with 5M aqueous HCl, followed by washing with distilled de-ionized water until colorless, and
subsequently drying under air or in the oven at 80°C. NMR spectra were taken on a Varian or Agilent
1
4
00 or 600 MHz instruments. HNMR spectra showed NMR solvents peaks at 3.3 ppm (for CD OD)
3
13
and at 4.9 ppm (for trace amounts of water in NMR solvent), while CNMR spectra showed NMR
21

ACCEPTED MANUSCRIPT
solvent peaks at 77 (for CDCl ) or at 47 (for CD OD) [57]. Infrared (IR) spectra were taken on Perkin
3
3
Elmer Spectrum Two ATR-FTIR. Low resolution mass spectra (LRMS) were taken on Waters ZQ LC-
SQMS, while high resolution mass spectra (HRMS) spectra were taken on a Waters LCT-MS premier
TOF. HPLC analysis to assess the purity of final compounds was performed with a Waters 1525
binary HPLC pump and Waters 2998 Photodiode Array detector. The separation was performed on a
reverse phase HPLC analytical column (YMC America, 250 x 4.6 mmI.D, 4µm, 8 nm) using a gradient
of 90% to 10% of buffer A over 30 minutes (buffer A = 0.1% HPLC grade TFA in water; buffer B =
HPLC grade acetonitrile) at a flow rate of 1.0 mL/min at room temperature. The peaks were detected
at wavelength 254 nm. The synthesized final compounds were found to be ≥97% pure (Figures S48,
S53, S58, S63, S68, and S73). Flash chromatography was performed with 40-60 micron LC60A silica
gel (Davisil).
4.2. Synthesis procedures
4.2.1. Synthesis procedures for 1a-1f
4.2.1.1 Synthesis of methyl (E)-6-oxohex-4-enoate (4): The compound was synthesized according
to the reported procedure, with the use of a different catalystm [58]. Briefly, in a flame dried 2-neck
00 mL flask, purged with argon, Grubb's catalyst second generation (26.65 mg, 0.0314 mmol, 0.5
1
mol%) was dissolved in dry dichloromethane (20 mL). Crotonaldehyde 3 (2.6 mL, 31.38 mmol) and
methyl pent-4-enoate 2 (0.78 mL, 6.28 mmol) were added, and the reaction was stirred with reflux for
3.5 hours under argon. The reaction was then cooled to room temperature, concentrated, and
purified by silica gel flash chromatography (ethyl acetate:hexanes 1:4) to afford aldehyde 4 as an
orange oily product (97%). The spectral data for the synthesized compound was consistent with the
reported data in literature [58].
4.2.1.2 Synthesis of 1-benzyl 8-methyl (E)-5-ethyloct-2-enedioate (6b): In a 200 mL, 2-neck flame
dried flask, copper(I)bromide dimethyl sulfide (2.17 g, 10.56 mmol) was dissolved in dry THF (20
mL). Air was purged with argon, and then the temperature was reduced to -15°C. Ethyllithium (12.35
mL of a 1.7M solution in dibutyl ether, 756 mg, 21.0 mmol) was added drop wise with stirring and
the mixture was allowed to stir for additional 20 minutes at -15°C. The temperature was then
reduced to -78°, followed by drop wise addition of chlorotrimethylsilane (3.44 g, 31.66 mmol)
22

ACCEPTED MANUSCRIPT
and methyl pent-4-enoate 4 (500 mg, 3.52 mmol). The reaction was stirred for 5 hours at -78°C. The
reaction was then quenched by addition of a saturated ammonium chloride:ammonia solution
(
1:1) portion wise until the reaction color turned blue. The organic layer was collected, and the
aqueous layer was extracted with ethyl acetate (3 x 30 mL). The combined organic layers were dried over
anhydrous Na SO , filtered, and rotavaped to an oily crude product, which was used in the next
2
4
reaction without purification.
In a 200 mL 2-neck flame dried flask, air was purged with argon, and NaH (169 mg of 60% NaH in
mineral oil, 7.04 mmol) dissolved in dry THF (20 mL) was added. The reaction was then cooled to 0°C
and benzyl diethyl phosphonoacetate 5b (1.80 mL, 7.04 mmol) was added drop wise with stirring.
The reaction was allowed to stir for 15 minutes at 0°C, then the crude product from the previous
reaction was added. The reaction was stirred for another 30 minutes at 0°C, followed by stirring
for 90 minutes at room temperature. The reaction was quenched with a saturated ammonium
chloride solution (20 mL). The organic layer was collected, and the aqueous layer was extracted with
ethyl acetate (3 x 25 mL). The combined organic layers were dried over anhydrous MgSO , filtered, and
4
concentrated. The product was purified by flash silica gel chromatography (ethyl acetate:hexanes
1
1
=
7
:9) to afford 6b (375 mg, 35% over two steps). HNMR (400 MHz, CD OD)
δ
(ppm): 0.86-0.90 (t, J
3
7.2 Hz, 3H), 1.28-1.34 (m, 2H), 1.47 (m, 1H), 1.55-1.61 (m, 2H), 2.18-2.22 (m, 2H), 2.30-2.34 (t, J =
.2 Hz, 2H), 3.63 (s, 3H), 5.15 (s, 2H), 5.89-5.93 (d, J = 16 Hz, 1H), 6.92-6.98 (m, 1H), 7.29-7.35
13
(
m, 4H). CNMR (100 MHz, CD OD)
δ
(ppm): 9.69, 25.31, 27.86, 30.79, 35.24, 35.33, 38.07, 50.66,
3
6
1
5.69, 122.02, 127.80 (2C), 128.15, 136.28, 148.47, 166.31, 174.4. IR: 2957, 2931, 2875, 1723,
-
1
+
+
655, 1437 cm . LRMS (ESI, m/z): calculated for [M+H] C H O H , 305.2, found 305.2; calculated
18
24
4
+
+
24 4
for [M+Na] C18H O Na , 327.2, found 327.1.
4.2.1.3. Synthesis of 1-benzyl 8-methyl (E)-5-ethyloct-2-enedioate (6c): The procedure was
similar to that of 6b except the following reagents were used: copper(I)bromide dimethyl sulfide (4.34
g, 21.10 mmol), n-butyllithium (16.86 ml of a 2.5 M solution in hexanes, 2.7 g, 42.2 mmol),
chlorotrimethylsilane (6.87 g, 63.3 mmol), methyl pent-4-enoate 4 (1 g, 7.03 mmol), NaH (478 mg of
60% NaH in mineral oil, 11.95 mmol) and benzyl diethyl phosphonoacetate 5b (3.42 g, 11.95 mmol).
The reaction was left to stir at room temperature for 10 hours. The product was purified by flash
silica gel chromatography (5% ethyl acetate in hexanes) to afford 6c (602 mg, 26% over two steps).
1
HNMR (400 MHz, CD OD)
δ (ppm): 0.91 (t, J = 6.8 Hz, 3H), 1.29 (m 7H), 1.58 (m, 3H), 2.22 (t, J =
3
23

ACCEPTED MANUSCRIPT
6
1
3
3
.0 Hz, 2H), 2.33 (t, J = 7.2 Hz, 2H), 3.63 (s, 3H), 5.16 (s, 2H), 5.91 (d, J = 15.2 Hz, 1H), 6.97 (dt,
13
H), 7.34 (m, 4H). CNMR (100 MHz, CD
3
OD) δ (ppm): 12.96, 22.05, 28.35, 28.38, 30.78, 32.60,
5.77, 36.51, 50.64, 65.68, 122.01, 127.76 (2C), 128.12, 136.29, 148.47, 166.33, 174.44. IR: 3057,
-
1
+
033, 2954, 2928, 2860, 1720, 1654, 1456, 1436 cm . LRMS (ESI, m/z): calculated for [M+Na]
+
C H O Na , 355.19, found 355.16.
20
28
4
4.2.1.4. Synthesis of 1-benzyl 8-methyl (E)-5-phenyloct-2-enedioate (6e): The procedure was
similar to that of 6b except the following reagents were used: copper(I)bromide dimethyl sulfide (4.61
g, 22.40 mmol), phenyllithium (22.4 ml of a 2M in dibutyl ether, 44.8 mmol), chlorotrimethylsilane
(
8.53 mL, 76.24 mol), methyl pent-4-enoate 4 (1.06 g, 7.49 mmol), NaH (0.51 g of 60% NaH in
mineral oil, 12.70 mmol) and benzyl dimethyl phosphonoacetate 5a (2.67 mL, 12.70 mmol). The
reaction was heated to reflux for 1 hour 45 minutes. The product was purified by column
chromatography (diethyl ether:petroleum ether 1:6 to 1:4) to afford 6e as an orange yellow oil (1.64 g,
1
6
2% over two steps). HNMR (400 MHz, CDCl ): 1.87 (m, 1H), 2.05 (m, 1H), 2.15 (m, 2H), 2.53 (t, J =
3
7.6 Hz, 2H), 2.67 (m, 1H), 3.61 (s, 3H), 5.14 (s, 2H), 5.82 (d, J = 15.2 Hz, 1H), 6.88 (dt, J = 15.6 Hz,
13
and 7.2 Hz, 1H), 7.12 (d, J = 7.6 Hz, 2H), 7.22 (m, 1H), 7.37 (m. 7H). CNMR (100 MHz, CDCl3):
30.94, 31.96, 39.61, 44.50, 51.56, 66.05, 115.33, 120.33, 126.81, 127.53, 128.15, 128.53, 128.71,
-
1
1
36.04, 142.81, 147.45, 166.19, 173.82. IR: 3063, 3030, 2951, 1718, 1654, 1495, 1454, 1437 cm .
+
+
+
LRMS (ESI, m/z): calculated for [M+H] C H O H , 353.2; found 353.4; calculated for [M+Na]
22
24
4
+
C H O Na , 375.2; found 375.3.
22
24
4
4
1
0
.2.1.5. Synthesis of 8-methoxy-5-ethyl-8-oxooctanoic acid (7b): In a 50 mL flask, 6b (375 mg,
.2 mmol) was dissolved in MeOH (20 mL), then Pd(OH) (87 mg of 20 wt. % Pd(OH) on carbon,
2
2
.12 mmol) was added. The air inside the flask was purged with argon (three times), then with
hydrogen gas (three times). The reaction was stirred under hydrogen for 4 hours. The reaction was
filtered through a celite plug, and the solvent was evaporated. The product was purified by silica gel
1
flash chromatography (ethyl acetate:hexanes 1:1.5) to afford 7b (299 mg, 94%); HNMR (400 MHz,
CD OD)
δ (ppm): 0.84 (t, J = 7.2 Hz, 3H), 1.28 (m, 5H), 1.60 (m, 4H), 2.30 (m, 4H), 3.66 (s, 3H)
3
13
1
1.40 (bs, 1H). CNMR (100 MHz, CD OD) δ (ppm): 10.58, 21.69, 25.28, 28.00, 31.47, 32.11, 34.27,
3
-
1
3
8.12, 51.55, 174.52, 179.89. IR: 2956, 2930, 2873, 1738, 1709, 1614, 1459, 1439 cm . LRMS (ESI,
+
+
m/z): calculated for [M+Na] C H O Na , 239.1, found 239.2.
11
20
4
24

ACCEPTED MANUSCRIPT
4.2.1.6. Synthesis of 8-methoxy-8-oxo-5-phenyloctanoic acid (7e): The procedure was similar to
that of 7b except the following reagents were used: 6e (1.40 g, 3.98 mmol), Pd(OH) (1.12 g of 20 wt.
2
%
Pd(OH) on carbon, 1.59 mmol). The product was purified by column chromatography (ethyl
2
1
acetate:petroleum ether 1:6 then 1:3) to afford 7e (764 mg, 73%); HNMR (400 MHz, CD OD): 1.40
3
(m, 2H), 1.63 (m, 2H), 1.80 (m, 1H), 1.98 (m, 1H), 2.10 (m, 2H), 2.20 (m, 2H), 2.54 (m, 1H), 3.60 (s,
1
3
3
4
1
H), 7.17 (m, 3H), 7.28 (m, 2H); CNMR (100 MHz, CDCl
3
): 22.30, 31.80, 32.20, 33.95, 35.90,
5.15, 51.80, 126.40, 127.50, 128.30, 143.95, 174.30, 179.90. IR: 3028, 2948, 1734, 1705, 1603,
-
1
+
+
494, 1453, 1437 cm . LRMS (ESI, m/z): calculated for [M+H] C H O H , 265.1; found 265.4;
15
20
4
+
+
calculated for [M+Na] C H O Na , 287.1; found 287.4.
15
20
4
4.2.1.7. Synthesis of methyl 4-methyl-8-oxo-8-(phenylamino)octanoate (8a): The procedure was
similar to 6b except the following: copper(I)bromide dimethyl sulfide complex (2.26 g, 10.98 mmol),
dry THF (40 mL), methyllithium (13.7 ml of a 1.6M solution in diethyl ether, 482 mg, 21.9 mmol),
chlorotrimethylsilane (3.58 g, 32.94 mmol), methyl pent-4-enoate 4 (520 mg, 3.66 mmol), NaH (293
mg of 60% NaH in mineral oil, 7.32 mmol), dry THF (20 mL), and benzyl diethyl phosphonoacetate
5
b (2.10 g, 7.32 mmol). The reaction was stirred for 3.5 hours. The product was used in the next
step without purification.
The procedure was similar to 7b except the following: crude 6a from prior step, MeOH (20 mL)
and Pd(OH) (413 mg of 20 wt. % Pd(OH) on carbon, 0.59 mmol). The reaction was stirred under
2
2
hydrogen for 3.5 hours. The reaction was filtered through a celite plug, and the solvent was
evaporated. The crude product was used in the following reaction.
The crude product 7a was dissolved in acetonitrile (10 mL), followed by addition of DIPEA (946 mg,
7
.32 mmol) and TBTU (1.76 g, 5.49 mmol), and the reaction was left to stir for 20 minutes. Aniline
0.51 g, 5.49 mmol) was added, and the reaction was left to stir for 4.5 hours. The reaction was
quenched with 10% aqueous HCl (20 mL). The aqueous layer was extracted with ethyl acetate (4 x 20
mL). The combined organic extracts were washed with a saturated NaHCO (10 mL) and then dried
over anhydrous Na SO , filtered, and evaporated. The product was purified by silica flash
(
3
2
4
1
chromatography (ethyl acetate:hexanes 1:4) to afford 8a (434 mg, 43% over four steps). HNMR (400
MHz, CD OD) (ppm): 0.90 (d, J = 6.4 Hz, 3H), 1.21 (m, 1H), 1.43 (m, 3H), 1.74 (m, 3H), 2.67 (m,
δ
3
13
4
H), 3.63 (s, 3H), 7.06 (t, J = 7.6 Hz, 1H), 7.28 (t, J = 8.4 Hz, 2H), 7.53 (d, J = 8.8 Hz, 2H). CNMR
25

ACCEPTED MANUSCRIPT
(
100 MHz, CD OD)
δ
(ppm): 18.15, 22.90, 31.13, 31.50, 31.98, 35.79, 36.71, 50.61, 119.83, 123.69,
3
128.36, 138.50, 173.12, 174.72. IR: 3302, 3137, 3061, 2953, 2940, 2869, 1736, 1662, 1600, 1542,
-
1
+
+
1
499, 1442 cm . LRMS (ESI, m/z): calculated for [M+H] C H NO H , 278.18, found 278.17;
16
23
3
+
+
calculated for [M+Na] C H O Na , 300.16, found 300.10.
16
23
3
4.2.1.8. Synthesis of methyl 4-ethyl-8-oxo-8-(phenylamino)octanoate (8b): The procedure was
similar to the last step of 8a except the following reagents were used: DIPEA (319 mg, 2.47mmol),
TBTU (594 mg, 1.85 mmol) and aniline (172 mg, 185 mmol). The extraction was done with
dichloromethane (4 x 20 mL). The product was purified by silica gel flash chromatography (ethyl
1
acetate:hexanes 1:4) to afford 8b (250 mg, 75%). HNMR (400 MHz, CD OD)
δ (ppm): 0.87 (t, J = 7.2
3
Hz, 3H), 1.34 (m, 5H), 1.60 (m, 2H), 1.68 (m, 2H), 2.33 (m, 4H), 3.64 (s, 3H), 7.07 (t, 1H), 7.29 (t, J =
1
3
7
3
3
.2 Hz, 2H), 7.53 (d, J = 7.2 Hz, 2H). CNMR (100 MHz, CD
3
OD) δ (ppm): 9.35, 22.23, 24.83, 27.55,
0.56, 31.63, 36.48, 37.83, 50.32, 119.55, 123.40, 128.06, 138.19, 172.82, 174.49. IR: 3302, 3198, 3137,
-1
062, 2955, 2930, 2862, 1737, 1663, 1600, 1542, 1499, 1442 cm . LRMS (ESI, m/z): calculated for
+
+
+
+
[
M+H] C H O H , 292.19, found 292.19; calculated for [M+Na] C H O Na , 314.17, found 314.17.
17
25
3
I7 25
3
4.2.1.9. Synthesis of methyl 4-butyl-8-oxo-8-(phenylamino)octanoate (8c): The procedure was
similar to that of 7b except the following reagents were used: 6c (1.2 g, 3.6 mmol) Pd(OH) (504 mg
2
of 20 wt. % Pd(OH) on carbon, 0.72 mmol). The crude product was used in the following reaction.
2
The procedure was similar to the last step of 8a except the following reagents were used: Crude 7c
from prior reaction, DIPEA (464 mg, 3.59 mmol), TBTU (865 mg, 2.69 mmol) and aniline (250 mg, 2.69
mmol). The product was purified by silica gel flash chromatography (ethyl acetate:hexanes 1:6) to
1
afford 8c (250 mg, 44% over two steps). HNMR (400 MHz, CD OD)
δ
(ppm): 0.89 (t, J = 6.4 Hz,
3
3H), 1.31 (m, 9H), 1.60 (m, 2H), 1.68 (m, 2H), 2.32 (m, 4H), 2.82 (s, 3H), 7.06 (t, J = 7.2 Hz, 1H), 7.28
13
(
t, J = 8 Hz, 2H), 7.53 (d, J = 8 Hz, 2H). CNMR (100 MHz, CD OD)
δ
(ppm): 13.07, 22.48, 22.66,
3
28.29, 28.46, 30.84, 32.37, 32.55, 36.56, 36.77, 50.63, 119.84, 123.69, 128.36, 138.49, 173.10,
-
1
1
74.77. IR: 3302, 3198, 3137, 3041, 2953, 2928, 2859, 1737, 1661, 1600, 1541, 1499, 1441 cm .
+
+
+
LRMS (ESI, m/z): calculated for [M+H] C H NO H , 320.22, found 320.21; calculated for [M+Na]
19
29
3
+
C H NO Na , 342.20, found 342.17.
19
29
3
26

ACCEPTED MANUSCRIPT
4.2.1.10. Synthesis of methyl 4-hexyl-8-oxo-8-(phenylamino)octanoate (8d): The procedure was
similar to that of 6b except the following reagents were used: 4 (460 mg, 3.24 mmol) dry THF (20 mL),
copper(I)bromide dimethyl sulfide (2 g, 9.73 mmol), n-hexyllithium (8.46 ml of a 2.3 M solution in
hexanes, 1.792 g, 19.46 mmol), chlorotrimethylsilane (3.17 g, 29.19 mmol) and methyl pent-4-
enoate 4 (461 mg, 3.24 mmol), NaH (259 mg of 60% NaH in mineral oil, 6.48 mmol), and benzyl
diethyl phosphonoacetate 5b (1.85 g, 6.48 mmol). The reaction was heated to reflux for 4.5 hours.
The product was purified by silica gel flash chromatography (ethyl acetate:hexanes 1:10). The
purified product was used in the following reaction.
The procedure was similar to that of 7b except the following reagents were used: 6d from prior
reaction, Pd(OH) (228 mg of 20 wt. % Pd(OH) on carbon, 0.32 mmol). The crude product was used
2
2
in the following reaction.
The procedure was similar to that of 8a except the following: 7d from prior reaction, acetonitrile (15
mL), DIPEA (838 mg, 6.48 mmol), TBTU (1.56 g, 4.86 mmol) and aniline (452 mg, 4.86 mmol). The
reaction was quenched with 10% aqueous HCl (10 mL). The product was purified by silica gel flash
1
chromatography (ethyl acetate:hexanes 1:6) to afford 8d (214 mg, 19% over four steps). HNMR (400
MHz, CD OD)
δ
(ppm): 0.88 (t, J = 6.8 Hz, 3H), 1.31 (m, 13H), 1.60 and 1.68 (overlapped quartet and
3
quintet, J = 7.6 and 7.6 Hz, 4H), 2.32 (m, 4H), 3.64 (s, 3H), 7.07 (t, J = 7.2 Hz, 1H), 7.28 (t, J = 7.6
13
Hz, 2H), 7.53 (d, J = 7.6 Hz, 2H). CNMR (100 MHz, CD OD)
δ
(ppm): 13.02, 22.30, 22.47, 26.14,
3
2
1
8.32, 29.34, 30.85, 31.61, 32.35, 32.86, 36.57, 36.75, 50.61, 119.86, 123.70, 128.34, 138.47, 173.13,
-
1
74.81. IR: 3294, 3138, 3061, 2926, 2856, 1794, 1659, 1599, 1541, 1442 cm . LRMS (ESI, m/z):
+
+
+
+
calculated for [M+H] C H NO H , 348.25, found 348.26; calculated for [M+Na] C H NO Na ,
21
33
3
21 33
3
370.24, found 370.22.
4.2.1.11. Synthesis of methyl 8-oxo-4-phenyl-8-(phenylamino)octanoate (8e): 8-Methoxy-8-oxo-
5-phenyloctanoic acid 7e (0.763 g, 2.89 mmol) was dissolved in dichloromethane (25 mL), then
aniline (0.32 mL, 3.47 mmol) was added, followed by 4-(dimethylamino)pyridine (0.424 g, 3.47 mmol).
The mixture was stirred until 4-(dimethylamino)pyridine was completely dissolved, then dicyclohexyl
carbdiimide (0.716 g, 3.47 mmol) was added, and the reaction was stirred for 4 hours at room
temperature. The reaction was quenched with 10% aqueous HCl (40 mL), the organic layer was
washed with a saturated NaHCO3 solution, and then brine, dried over anhydrous Na SO ,
2
4
concentrated, and purified by column chromatography (acetone:petroleum ether 1:6) to give 8e (704
27

ACCEPTED MANUSCRIPT
1
mg, 72%). HNMR (400 MHz, CD OD): 1.46 (m, 2H), 1.65 (m, 2H), 1.80 (m, 1H), 1.98 (m, 1H), 2.11
3
(m, 2H), 2.28 (m, 2H), 2.53 (m, 1H), 3.55 (s, 3H), 7.05 (t, J = 7.6 Hz, 1H), 7.16 (m, 3H), 7.26 (m, 4H),
1
3
7
5
3
.52 (d, J = 8.4 Hz, 2H); CNMR (100 MHz, CD OD): 23.59, 31.55, 31.58, 35.92, 36.50, 45.05,
3
0.59, 119.83, 123.71, 126.10, 127.41, 128.21, 128.39, 138.48, 144.07, 172.96, 174.34. IR: 3301,
-
1
197, 3135, 3061, 3027, 2949, 2865, 1734, 1663, 1600, 1543, 1499, 1442 cm . LRMS (ESI, m/z):
+
+
+
+
calculated for [M+H] C H NO H , 340.2; found 340.2; calculated for [M+Na] C H O Na , 362.2;
21
25
3
21 25
3
found 362.2.
4.2.1.12. Synthesis of methyl 4-benzyl-8-oxo-8-(phenylamino)octanoate (8f):
The procedure was similar to that of 6b except the following reagents were used: 4 (500 mg, 3.52
mmol), dry THF (20 mL), copper(I)bromide dimethyl sulfide (2.17 g, 10.56 mmol), benzylmagnesium
chloride (21.11 ml of a 1.0 M solution in methyl THF, 3.189 g, 21.12 mmol), chlorotrimethylsilane
(
3.44 g, 31.68 mmol), NaH (282 mg of 60% NaH in mineral oil, 7.04 mmol) and benzyl diethyl
phosphonoacetate 5b (2.02 g, 7.04 mmol). The reaction was heated to reflux for 1 hour 45 minutes.
The crude product was purified by silica gel flash chromatography (ethyl acetate:hexanes 1:9). The
purified product was used in the following reaction.
2
The procedure was similar to that of 7b except the following: 6f from prior reaction, Pd(OH) (247
mg of 20 wt. % Pd(OH) on carbon, 0.35 mmol). The reaction was stirred for 4.5 hours under
2
hydrogen, then it was filtered, and the solvent was evaporated. The crude product was used in the
following reaction.
The procedure was similar to that of 8a except the following: crude 7f from prior reaction, DIPEA
(464 mg, 3.59 mmol), TBTU (865 mg, 3.59 mmol), and aniline (250 mg, 2.69 mmol). The reaction was
stirred for 4 hours 45 minutes. The combined organic extracts were dried over anhydrous Na CO .
2
3
The product was purified by silica gel flash chromatography (ethyl acetate:hexanes 1:3) to afford 8f
1
(
340 mg, 27% over four steps). HNMR (400 MHz, CD OD)
δ
(ppm): 1.35 (m, 2H), 1.60 and 1.72
3
(
overlapped m and m, 5H), 2.34 (m, 4H), 2.55 (d, J = 6.8 Hz, 2H), 3.61 (s, 3H), 7.10 (m, 4H), 7.21 (t,
13
J = 7.6 Hz, 2H), 7.28 (t, J = 7.2 Hz, 2H), 7.51 (d, J = 7.6 Hz, 2H). CNMR (100 MHz, CD
3
OD)
δ
(ppm): 22.35, 28.00, 30.84, 31.97, 36.66, 38.87, 39.61, 50.62, 119.90, 123.71, 125.47, 127.86,
128.34, 128.79, 138.44, 140.65, 173.04, 174.65. IR: 3302, 3026, 2929, 2863, 1734, 1661, 1599, 1542,
-
1
+
+
1
498, 1441 cm . LRMS (ESI, m/z): calculated for [M+H] C H NO H , 354.21, found 354.22;
22
27
3
+
+
calculated for [M+Na] C H NO Na , 376.19, found 376.21.
22
27
3
28