Discovery of selective SIRT2 inhibitors as therapeutic agents in B-cell lymphoma and other malignancies

Article abstract of DOI:10.3390/molecules25030455

Genetic ablation as well as pharmacological inhibition of sirtuin 2 (SIRT2), an NAD⁺dependent protein deacylase, have therapeutic effects in various cancers and neurodegenerative diseases. Previously, we described the discovery of a dual SIRT1/SIRT2 inhibitor called cambinol (IC50 56 and 59 μM, respectively), which showed cytotoxic activity against cancer cells in vitro and a marked anti-proliferative effect in a Burkitt lymphoma mouse xenograft model. A number of recent studies have shown a protective effect of SIRT1 and SIRT3 in neurodegenerative and metabolic diseases as well as in certain cancers prompting us to initiate a medicinal chemistry effort to develop cambinol-based SIRT2-specific inhibitors devoid of SIRT1 or SIRT3 modulating activity. Here we describe potent cambinol-based SIRT2 inhibitors, several of which show potency of ~600 nM with >300 to >800-fold selectivity over SIRT1 and 3, respectively. In vitro, these inhibitors are found to be toxic to lymphoma and epithelial cancer cell lines. In particular, compounds 55 (IC50 SIRT2 0.25 μM and <25% inhibition at 50 μM against SIRT1 and SIRT3) and 56 (IC50 SIRT2 0.78 μM and <25% inhibition at 50 μM against SIRT1 and SIRT3) showed apoptotic as well as strong anti-proliferative properties against B-cell lymphoma cells.

Full text of DOI:10.3390/molecules25030455

Article
Discovery of Selective SIRT2 Inhibitors as
Therapeutic Agents in B-Cell Lymphoma and
Other Malignancies
Sarwat Chowdhury 1, Smitha Sripathy 1, Alyssa Webster 1, Angela Park 1, Uyen Lao 1,
Joanne H. Hsu 1, Taylor Loe 1, Antonio Bedalov 1 and Julian A. Simon 1,2,
*
1
Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA;
csarwat@yahoo.com (S.C.); ssripath@fredhutch.org (S.S.); awebster@fredhutch.org (A.W.);
aypark12@gmail.com (A.P.); ulao@fredhutch.org (U.L.); jhhsu11@gmail.com (J.H.H.);
taylor.k.loe@gmail.com (T.L.); abedalov@fredhutch.org (A.B.)
2
Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
* Correspondence: jsimon@fredhutch.org; Tel.: +1-(206)-667-6241
Received: 26 December 2019; Accepted: 19 January 2020; Published: 21 January 2020
Abstract: Genetic ablation as well as pharmacological inhibition of sirtuin 2 (SIRT2), an NAD+-
dependent protein deacylase, have therapeutic effects in various cancers and neurodegenerative
diseases. Previously, we described the discovery of a dual SIRT1/SIRT2 inhibitor called cambinol
(IC50 56 and 59 µM, respectively), which showed cytotoxic activity against cancer cells in vitro and
a marked anti-proliferative effect in a Burkitt lymphoma mouse xenograft model. A number of
recent studies have shown a protective effect of SIRT1 and SIRT3 in neurodegenerative and
metabolic diseases as well as in certain cancers prompting us to initiate a medicinal chemistry effort
to develop cambinol-based SIRT2-specific inhibitors devoid of SIRT1 or SIRT3 modulating activity.
Here we describe potent cambinol-based SIRT2 inhibitors, several of which show potency of ~600
nM with >300 to >800-fold selectivity over SIRT1 and 3, respectively. In vitro, these inhibitors are
found to be toxic to lymphoma and epithelial cancer cell lines. In particular, compounds 55 (IC50
SIRT2 0.25 µM and <25% inhibition at 50 µM against SIRT1 and SIRT3) and 56 (IC50 SIRT2 0.78 µM
and <25% inhibition at 50 µM against SIRT1 and SIRT3) showed apoptotic as well as strong anti-
proliferative properties against B-cell lymphoma cells.
Keywords: sirtuin; SIRT2; acetylation; cancer; lymphoma
1. Introduction
Sirtuins are NAD+-dependent protein deacylases (this term is more accurate than the previously
widely used “deacetylases” since these enzymes remove other acyl modifications) that have emerged
as key regulators of diverse cellular processes such as chromatin modification, gene expression, DNA
repair, cell cycle control and cell survival [1,2]. The human genome encodes seven isoforms, sirtuins
1–7 of which sirtuin 2 (SIRT2) is primarily a cytoplasmic protein that is known to localize to the
nucleus especially during mitosis [3]. SIRT2 exhibits robust deacetylase and demyristoylase activities
against histone and non-histone substrates. Expressed ubiquitously, SIRT2 is prominently expressed
in brain and muscle tissues [4]. Genetic ablation of SIRT2 has been shown to have protective effects
in models of neurodegenerative disorders such as Huntington’s [5] and Parkinson’s diseases [6]. A
growing body of literature also suggests that SIRT2 may be a valid drug target in certain cancers.
SIRT2-mediated deacetylation has been shown to promote mutant K-ras oncogenic activity [7].
Similarly, SIRT2 enhances the stability of n-myc and c-myc oncogenes leading to cancer cell
Molecules 2020, 25, 455; doi:10.3390/molecules25030455
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proliferation [8]. Further, siRNA-mediated downregulation of SIRT2 was found to induce apoptosis
in gliomas [9], and hepatocellular and pancreatic carcinomas [10].
SIRT2 is known to process diverse substrates such as histone H4 lysine-16 (H4K16) [11], alpha-
tubulin [12], p53 [13], EP300 [14]. Foxo-family of proteins [14,15] and glucose-6-phosphate dehydrogenase
(G6PD) [16] among others. Owing to its regulatory roles in various cellular processes, perhaps it is not
surprising that the dual roles of SIRT2 as a cancer suppressor as well as an oncogene [17] have been
reported [18]. Recent consensus seems to reconcile these contradictory findings by taking into account
disease-specific contexts such as cancer sub-type, expression pattern of SIRT2 and its substrate proteins as
well as their roles in oncogenic signaling [19,20]. As with SIRT2, there are conflicting reports of cancer
suppressor and oncogenicactivities of SIRT1 and3[21] in various tumor types[22,23]. Additionally, SIRT1
activity has been demonstrated to confer neuroprotection in several age-related neurodegenerative
disorders including Parkinson’s and Alzheimer’s diseases [24,25] and metabolism related disorders [26].
The mitochondrial isoform SIRT3 deacylase activity is important for mitochondrial metabolism, and cell
survival [27]. Additionally, loss of function studies indicated SIRT3 has a tumor suppressor function
through modulation of reactive oxygen species (ROS) production [28]. Because of these potentially
confounding activities, identification of isoform-selective sirtuin inhibitors is the most practical approach
to validating SIRT2 as a drug target.
Historically, it has been difficult to synthesize isoform-selective sirtuin inhibitors primarily
because all sirtuin isoforms possess a core 260-amino acid catalytic domain which is highly conserved
from bacteria to humans [29,30]. The SIRT1 catalytic domain is 45% identical to SIRT2 and bears 69%
similarity [31]. Previous reported sirtuin inhibitors showed little or no isoform selectivity and had
modest potency. Nonetheless, dual SIRT1/2 inhibitors showed anti-cancer activity. For example,
EX527, sirtinol, and saleramide induced cell death in breast cancer cell line in vitro in a p53 dependent
manner [32]. Splitomicin [33] and its analogs showed anti-proliferative properties in MCF-7 breast
cancer cell line [34]. A thio-myristoylated dipeptide inhibitor of SIRT2 with nanomolar potency was
found to promote degradation of c-myc and have anti-cancer activity in multiple cell lines [35]. In
neurodegeneration models, AGK2, a sirtuin inhibitor with higher selectivity for SIRT2 (IC50 for SIRT1
>50 µM and SIRT2 23.5 µM, respectively) showed protection from alpha-synuclein toxicity in
Parkinson’s disease model [6]. Encouragingly, recent reports of sub-micromolar SIRT2-selective
inhibitors also show neuroprotection in a Parkinson’s disease model [36–38].
We previously reported a dual SIRT1/2 inhibitor called cambinol 1 (in vitro IC50 54 and 46 µM
respectively) [39] that sensitized cells to paclitaxel and etoposide and, was cytotoxic as a single agent
against B-cell lymphoma cell lines in vitro and xenograft models in vivo. As expected from the distinct
mechanism and no sequence homology between class I and II deacetylases, cambinol had no activity
against class I (HDAC1) or II (HDAC6) enzymes. Intriguingly, a follow-up study with cambinol-based
dual SIRT1/2 inhibitors demonstrated a strong correlation between SIRT2 inhibitory activity and
cytotoxicity in Namalwa Burkitt lymphoma cell line [40]. Following our long-standing interest in anti-
lymphoma agents, coupled with the need for isoform-selective sirtuin modulators, we launched a
medicinal chemistry effort to identify SIRT2 selective cambinol analogs and evaluate cytotoxicity against
B-cell lymphomas. Herein we report the discovery of open ring cambinol analogs, devoid of thio-
pyrimidinone moiety as SIRT2-specific inhibitors (Figure 1). Based on structure-activity relationship
(SAR) evaluation, potency was improved >200 fold; displaying IC50 at the sub-micromolar level as
racemates. Concurrently, high selectivity against SIRT1 and 3 isoforms was also achieved. These
compounds were found to be cytotoxic against B-cell lymphoma and epithelial cancer cell lines in vitro.
2. Results
Our previous efforts to synthesize and evaluate tetracyclic cambinol analogs as sirtuin inhibitors
showed that replacement of thio-pyrimidinone ring with other heterocycles (Figure 1) can impart
improved potency and isoform-selectivity, as exemplified by pyrazolone compound 2.

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Me
H
N
S
NH
NH
NH
O
OH
O
OH
OMe
1
2
SIRT1 = 56 mM
SIRT2 = 59 mM
SIRT3 = >200 mM
SIRT1 = 37 mM
SIRT2 = >200 mM
SIRT3 = >200 mM
Figure 1. Cambinol (1) and its pyrazolone analog 2 showing improved SIRT1 selectivity.
While exploring variants of the heterocyclic ring of cambinol keeping the hydroxynaphthalene and
phenyl ring moieties intact, we discovered that simple -ketoamide 4, generated by aminolysis of lactone
3 showed improved inhibitory potency (IC50 SIRT2 13 µM) as well as high selectivity (IC50 SIRT1, >200
µM) (Scheme 1). We hypothesized that by the virtue of open chain scaffold, these chiral cambinol analogs
might display enhanced receptor/small molecule interaction as compared to 1. Realizing the potential for
rapid diversification of amide sidechains as a means to improve potency, selectivity and to modulate
physicochemical properties of these compounds, we embarked on an SAR study to identify potent and
isoform-selective SIRT2 inhibitors. On account of keto-enol tautomerization leading to racemization, we
commenced an investigation with racemates of the desired cambinol analogs.
Me
Me
Me
O
O
O
OH
NH2
NH2
a
O
O
OH
O
OH
Br
Br
Br
3
4
5
Scheme 1. Synthesis of acyclic analogs. Reagents: (a) THF, NH4OH, rt, 14 h 80%.
2.1. Chemistry
Our general synthetic scheme calls for various substituted hydroxynaphthaldehydes 7 and -keto
esters (Scheme 3) as the starting materials. Scheme 2 shows a general synthesis route to 2-hydroxy-1-
naphthaldehydes 7.
H
O
a
OH
OH
R1
R1
6
7
H
O
b,c
OH
OH
HO
N
Cl
N
8
9
Scheme 2. Synthetic scheme of hydroxynaphthaldehyde and hydroxyquinoline carbaldehyde. Reagents
and Conditions: (a): Cl2CHOMe, TiCl4, CH2Cl2, 0–20 °C, 24 h; (b): (i) TFA, hexamethylenetetramine, 100
°C, 2 h; (ii) MeOH; (c) DMF, POCl3, 0 °C to rt, 10 h, H2O.

Molecules 2020, 25, 455
O2N
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H
a,b
Me
N
O
Me
O
O
Me
O
O
O
O
10
11
Scheme 3. Synthesis of ethyl 3-(4-acetamidophenyl)-3-oxopropanoate. Reagents and Conditions: (a)
EtOH, AcOH, Fe powder, HCl, reflux, 2 h; (b) CH2Cl2, AcCl, TEA, rt, 3 h.
Titanium-catalyzed Rieche formylation of commercially available substituted naphthols was used to
generate the corresponding naphthaldehydes 7 in good yields. Hydroxyquinoline carbaldehyde 8 was
obtained in two steps first by Reimer-Tiemann reaction 2,6-dihyoxyquinoline followed by chlorination
with POCl3 to give the desired product in quantitative yield over two steps.
While most -keto esters were obtained from commercial sources, compound 11 was prepared
in two steps from nitrobenzoyl propionate ester 10. Iron-mediated reduction of the nitro group first
gave the corresponding aniline in good yield. In the subsequent step, acetylation of the aniline
furnished the desired compound 11.
The core scaffolds were synthesized by one-pot morpholine-catalyzed Knoevenagel
condensation/lactonization of hydroxynaphthaldehydes 12 with -keto esters. The resultant
coumarin lactones 13, which precipitated from the reaction mixture, were obtained in good yields.
Selective 1,4-reduction by treatment with NaBH4 in anhydrous pyridine furnished the desired
saturated lactones, which were subjected to aminolysis to yield the desired products 15 (Scheme 4).
R2
R2
R2
O
O
O
O
O
NHR3
H
O
O
b
O
a
c
O
OH
OH
R1
R1
R1
R1
12
13
14
15
Scheme 4. Synthesis of SIRT2 inhibitors. Reagents and Conditions: (a) substituted benzoyl propionate
ester, piperidine, EtOH, reflux, 2 h; (b) NaBH4, pyridine, 20 °C, 3 h; HCl; (c) R3NH2, THF, TEA, rt, 8 h.
2.2. Structure Activity Relationship Study (SAR)
The newly synthesized open chain cambinol analogs were evaluated for in vitro SIRT1, 2 and 3
inhibitory potency using commercially available purified sirtuin enzymes (Cayman Chemical, Ann
Arbor, MI, USA) and luciferin-based SIRT-Glo assay (Promega Corp., Madison, WI, USA) employing a
p53 acetylated peptide as substrate. In the initial screening, single point inhibition was determined
using 50 µM test compound concentration to assess the potency and selectivity. We used cambinol as a
positive control showing 54% SIRT1 inhibition, 46% SIRT2, and 16% SIRT3 inhibition at 50 µM (Table
1) was used. Promising compounds were evaluated by a full dose-response curve to ascertain IC50
against each of the sirtuin isoforms.
Our exploration began with variation of phenyl ring substituents while keeping the 6-bromo-2-
hydroxy naphthyl group unchanged due to its favorable effect on isoform selectivity; a rationale we
derived from our previous study. SAR of the phenyl group (Table 1) showed a clear trend where 4-
substitution was favored over 3-position, and 2-substitution was found to be detrimental for inhibitory
activity (e.g., 4, 16 and 17). Inhibitory potency against SIRT2 increased with 4-substitution of progressively
larger/more hydrophobic groups (19, 20 and 22) while showing no measurable activity against SIRT1 at
50 µM. For selectivity against phylogenetically closest ortholog mitochondrial SIRT3 [41] 4-CF3 group
(e.g., 19), imparted the highest selectivity for SIRT2 versus SIRT3 (100 fold). Only 25-fold selectivity for
SIRT2 was observed both for Cl and Br-groups (20 and 22).

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Table 1. Optimization of the phenyl group (R1).
R1
O
NHR2
R1
R2 SIRT1 * SIRT2 * SIRT3 * SIRT2 **
O
OH
Br
1
Cambinol
4-Me-phenyl
54
9.0
38
46
89
70
3.0
57
95
100
89
93
91
72
96
40
52
58
16
n.d.
n.d.
n.d.
n.d.
30
59.0
13.0
n.d.
n.d.
n.d.
2.79
1.90
12.6
2.50
8.95
n.d.
12.4
n.d.
n.d.
n.d.
4
H
H
16
17
18
19
20
21
22
23
24
25
26
27
28
3-Me-phenyl
2-Me-phenyl
H
0.0
0.0
0.0
0.0
0.0
7.0
53
Phenyl
H
4-Chlorophenyl
4-Trifluoromethylphenyl
3-Trifluoromethylphenyl
4-Bromophenyl
3-Bromophenyl
4-Methoxyphenyl
4-Fluorophenyl
4-Pyridyl
H
H
5.0
H
15
H
42
H
n.d.
n.d.
39
H
34
H
35
Me
Me
Me
13
n.d.
n.d.
n.d.
3-Pyridyl
30
4 Acetamidophenyl
0.0
* Percentage inhibition at 50 µM compound. ** IC50 concentration for SIRT2 (µM).
SAR of the naphthyl substituents of the open ring analogs showed that substitution at 6-position by
hydrophobic group was crucial for activity (Table 2; compounds 23, 33, 34 vs. 29). Interestingly, cambinol
1, which lacks a substituent on the naphthyl ring inhibits SIRT2 moderately (IC50 59 µM).
Table 2. Optimization of the naphthyl group (R2).
R1
R2
R3
SIRT1 * SIRT2 * SIRT3 * SIRT2 **
23
29
30
31
32
33
34
35
3-Br
3-Br
H
H
53
12
91
14
32
37
16
97
97
84
n.d.
n.d.
n.d.
n.d.
n.d.
6.0
8.95
n.d.
n.d
3-Br
H
0.0
0.0
0.0
12
3-Br
H
n.d.
n.d.
0.59
0.77
12.1
3-Br
H
4-CF3
4-CF3
4-CF3
Me
Me
Me
0.0
0.0
>200
>200
* Percentage inhibition at 50 µM compound. ** IC50 concentration for SIRT2 (µM).

Molecules 2020, 25, 455
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This discrepancy could arise from a different binding mode of the acyclic compounds relative to
cambinol. The hydroxyquinoline group in 35 seems to be tolerated by SIRT2. However, this analog
showed lower potency than corresponding hydroxy-naphthalene compounds. Several other substitution
patterns were also evaluated (29–32) and did not yield potent inhibitors of either SIRT1 or 2.
Having found that substituents 4-Br, -Cl and -CF3 in the phenyl ring in this open-ring scaffold
give 20 to 30-fold higher potency over cambinol 1, we sought to identify the side chains that can
confer favorable SIRT2 selectivity and improve potency (Table 3). Various amines such as acyclic,
carbocyclic (data not shown) and heterocyclic amines were evaluated. It was observed that bulky
sidechains were detrimental for activity and conversely and sidechains derived from small acyclic
amines (33) and those terminated in polar head groups augmented potency (e.g., 36, 43 and 45). For
the 3-bromophenyl series, aliphatic acyclic substituents improved not only potency, but also
selectivity against SIRT1; keeping selectivity over SIRT3 unchanged (23 vs. 36, 43, and 44).
Table 3. Optimization of the sidechain (R2).
R1
R2
SIRT1 *
SIRT2 *
SIRT3 *
SIRT2 **
23
36
37
3-Br
3-Br
3-Br
NH2
53
0.0
28
91
94
86
41
43
46
8.95
4.75
7.62
38
39
3-Br
4-Br
1.0
13
22
30
n.d.
n.d.
n.d.
n.d.
40
41
42
4-Br
3-Br
3-Br
23
0.0
3.0
40
53
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
6.0
43
44
3-Br
3-Br
0.0
0.0
89
98
20
0.96
6.50
n.d.
20
45
4-CF3
4-CF3
NH2
0.0
10
100
88
5.0
1.90
n.d.
n.d.
46
33
4-CF3
4-CF3
28
35
88
98
n.d.
n.d.
n.d.
0.49
NHMe
* Percentage inhibition at 50 µM compound. ** IC50 concentration for SIRT2 (µM).
We next incorporated optimal side chain (R2 substituents) into the 6-bromonaphthyl and 4-
substituted phenyl core scaffold (Table 3). Gratifyingly, we obtained compounds with sub-
micromolar potencies against SIRT 2 while displaying high selectivity against SIRT1 and SIRT3. As
seen earlier (Table 1) 4-CF3 substitution gave highly potent compounds 49, 53 and 56 that showed
inhibitory potency below 300 nM as a racemic mixture with >400 over SIRT1 and >800-fold selectivity
over SIRT3. In general, with any given side chain, 4-CF3 and 4-Br groups yielded the most potent
SIRT2 inhibitors as shown in Table 4.

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Table 4. Optimization of the SIRT2 inhibitors (R1 and R2).
R1
R2
SIRT1 *
SIRT2 *
SIRT3 *
SIRT2 **
47
48
49
4-Cl
4-Br
0.0
18
15
98
96
99
5
0.96
0.52
0.27
29
27
4-CF3
50
51
52
53
4-Cl
4-Br
4.0
20
99
98
93
91
17
14
21
16
0.65
0.58
0.65
1.57
4-CF3
3-CF3
0.0
8.0
54
4-Cl
12
99
6
0.59
55
56
4-CF3
4-Br
0.0
12
100
95
20
22
0.25
0.78
NHMe
* Percentage inhibition at 50 µM compound. ** IC50 concentration for SIRT2 (µM).
2.3. Biological Evaluation of Sirtuin 2 Inhibitors
To ascertain SIRT2 inhibitory activity of our newly synthesized compounds in a cellular context, we
examined acetylation level of α-tubulin, a known SIRT2 substrate [12]. Non-small cell lung cancer cell line
NCI-H460 was treated with compound 55 at 0, 5, and 10 µM for 18 h. Cells were harvested, lysed and α-
tubulin acetylation was analyzed by western blot (Figure 2). As predicted, dose-dependent increase in
tubulin acetylation was observed upon exposure to 55 as compared to the vehicle-treated negative control
confirming inhibitory activity of the newly developed SIRT2 inhibitors in cells.
Figure 2. Determination of α-tubulin acetylation in NCI-H460 cells by western blot following 18 h
treatment with 55. GAPDH used as loading control.
With selective SIRT2 inhibitors in hand, we proceeded to evaluate cytotoxicity of our compounds
in Daudi, Raji (Burkitt lymphoma) and OCI-Ly8-LAM-53 (OCI, a representative of several Diffuse
Large B-Cell Lymphoma lines; DLBCL) cell lines. Briefly, cells were plated in 96-well plates and treated
with inhibitors at desired concentrations for 72 h. Cellular viability was determined by measuring the
amount of cellular ATP using CellTiter-Glo assay kit (Promega) and normalized against DMSO-treated
controls. While all SIRT2 inhibitors were found to be toxic to B-cell lymphoma cell lines (Table 5),
interestingly, primary carboxamide side chain containing inhibitors (e.g., 24, 22), which showed lower
potencies in in vitro enzyme assays, displayed higher cytotoxicity as compared to other more potent
inhibitors with polar side chains. Conversely, 4-CF3 phenyl substituted compounds displaying high in
vitro SIRT2 inhibitory activity (e.g., 49 and 55) were found to be less potent in the cell-based assay. N-

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methyl side chain containing compounds (33 and 56) appear to have more specific activity against
SIRT2 and exhibit potent cellular cytotoxicity. Selected inhibitors were further evaluated in an
expanded panel of cancer cell lines including breast cancer (MCF-7 and MDA-MB231), prostate cancer
(PC-3) and glioblastoma multiforme (U-251). SIRT2-specific inhibitors were found to be less toxic
against these cell lines compared to lymphoma cell lines. However, the trend in potency as a function
of the structure is consistent with the results obtained against lymphoma cell lines. These results
demonstrate toxicity of the new SIRT2 inhibitors in various cancers cell lines, B-cell lymphomas in
particular, as observed with the hit compound cambinol (1). Interestingly, the published SIRT2 inhibitor
AGK2 was only weakly active against the OCI DLBCL cell line (LC50 73.2 µM).
Table 5. In vitro SIRT2 inhibitory activity (IC50) and cytotoxicity (LC50) of SIRT2 inhibitors in B-cell
lymphoma and other cancer cell lines.
SIRT2
IC50
Daudi
LC50
Compound
Raji
OCI
MCF7
MDA-MB231
PC3
U251
19
22
33
43
47
48
49
50
51
52
54
55
56
2.8
5.0
7.3
8.2
1.6
n.d.
n.d.
n.d.
n.d.
n.d.
11.8
20.2
n.d.
n.d.
n.d.
18.0
21.6
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
14.7
23.5
n.d.
n.d.
n.d.
23.8
22.8
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
11.8
18.4
n.d.
n.d.
n.d.
16.5
17.2
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
15.8
15.7
n.d.
25.9
n.d.
17.3
21.1
n.d.
2.5
7.0
7.8
0.49
0.96
0.96
0.52
0.27
0.65
0.58
0.65
0.59
0.25
0.78
14.5
15.8
n.d.
10.5
12.0
n.d.
19.2
28.2
15.8
11.9
7.1
18.5
19.4
20.2
10.0
16.5
19.9
16.7
19.4
18.5
11.9
9.1
16.4
n.d.
9.7
5.0
13.1
11.7
10.2
25.5
11.2
24.9
5.7
* SIRT2 IC50 and toxicity LC50 (µM).
2.4. SIRT 2 Inhibitor as Anti-Proliferation and Apoptosis Inducing Agent in OCI-Ly8-LAM53 (OCI) Cells
Measurement of ATP, while an excellent surrogate marker for cell viability, does not shed light
on the mechanism of drug toxicity such as drug-induced cell death. To gain a better insight pertaining
to the cytotoxic effects of SIRT2 inhibition, we probed for induction of apoptosis by western blot
analysis as well as by annexin V staining assay upon exposure to the SIRT2 inhibitor [39]. OCI DLBCL
cells were treated with 56 for 24 h. Cells were lysed and analyzed by western blot assay for cleaved
poly-ADP-ribose polymerase (PARP), a well-accepted apoptosis marker [42] (Abcam, Cambridge,
UK). The result (Figure 4e) showed dose-dependent PARP cleavage with progressively higher
concentrations of 56 indicating SIRT2 inhibition promotes caspase-mediated apoptotic cell death. A
similar finding, induction of apoptosis (Q2) was observed by annexin V staining upon exposure to
56 (Figure 4a–d) while showing negligible cell population undergoing necrotic cell death (Q1). Taken
together, these results show that SIRT2 inhibition ensues dose-dependent induction of apoptosis.
3. Discussion
Our initial effort to identify SIRT2-selective cambinol analogs has led to the discovery of open
chain inhibitors derived from cambinol displaying submicromolar potency against SIRT2 and high
selectivity against SIRT1/3 in vitro. In this initial SAR, we explored substitution of three functional
groups: the phenyl group (box A), naphthyl group (box B) and the side chain (box C) (as shown in
Figure 4) while beta-keto and the hydroxyl group were kept constant. The beta-keto amide functionality
makes these inhibitors susceptible to racemization at the chiral center under physiological conditions.
Because of this, all of the compounds described herein were tested as racemates.

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Figure 3. Induction of apoptosis upon treatment of OCI cells with 56. (A–D) Annexin V staining, (E)
PARP cleavage. (A). Control cells treated with DMSO; (B). Cells treated with etoposide (5 µM); (C).
Cells treated with 10 mM 56; d). (D). Cells treated with 25 µM 56; Apoptotic cells appear in the upper
right-hand quadrant (A–D). (E). Induction of PARP cleavage (normalized ratio = cleaved PARP/actin).
SAR study revealed that 4-substituted phenyl group (box A) and 6-substituted 2-hydroxynaphthyl
(box B) connected by a three-carbon linker containing a ketone, (Figure 4) provides a hydrophobic core of
the most potent SIRT2 inhibitors. Docking studies with cambinol and its analogs published earlier
concluded [43] that cambinol and analogs bind in the substrate binding narrow cleft where the hydroxyl
group of the naphthyl ring system (box B) forms a key interaction with Glu116 in SIRT2.
Figure 4. General structure of the SIRT2 inhibitors.
A corollary drawn from the binding orientation[43] predicts that interaction with Gln167 and His187
in SIRT2 would impart both potency and selectivity since these residues are unavailable in the more open
binding pocket of SIRT1. Our observation from the SAR with the beta-keto amide side chains (box C)
agrees well the docking study showing that small polar sidechains enhance potency and impart high
selectivity against highly homologous SIRT1 and SIRT3. As a result, we were able to improve selectivity
for STIR2 by more than 200-fold over cambinol (1). Interestingly, lack of symmetry in the three
dimensional SIRT2 binding pocket [43] suggests that the potency of the two enantiomers of SIRT2
inhibitors would be different. This remains to be verified with non-enolizable cambinol analogs.

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Our previous reports [39,40] and studies by other groups have shown that pharmacological
inhibition or genetic downregulation of SIRT2 has an anti-cancer effect in various malignancies which
corroborates our observation in the present study [44–46]. Newly synthesized cambinol analogs having
high specificity for SIRT2 were found to be toxic to in a panel of cancer cell lines. Further, as with
cambinol, higher toxicity against B-cell lymphoma cell lines such as in Raji and Daudi (Burkitt
lymphoma) and OCI (DLBCL) were observed. Curiously, we found that -NH2 or -N(Me)H sidechain-
containing SIRT2 inhibitors while somewhat less- potent on the enzyme in-vitro assay, are more potent
in cellular context as compared their polar side chain containing analogs e.g., 55 vs. 46. While off-target
activities cannot be ruled out without further screening, this discrepancy could also arise from
differences in cell-permeability or efflux.
We observed that SIRT2 inhibition leads to dose-dependent apoptotic cell death as seen by PARP-
cleavage in western blot analysis and in annexin V assay (Figure 4). While uncovering the exact
mechanism is beyond the scope of the present study, several hypotheses found in the literature can be
invoked. SIRT2 inhibition may correct BCL6 and p53 acetylation imbalance, a known driver of
lymphomagenesis [39,47]. Additionally, SIRT2 inhibition has been shown to exert an anti-proliferative
effect by facilitating degradation of c-myc; [35] the oncogene which is constitutively active in several
Burkitt lymphomas. Blocking of cell proliferation through deactivation of G6PD by SIRT2-mediated
deacetylation is another potential mechanism [16]. Pleiotropic effects on other client proteins cannot be
ruled out as suggested by several groups in recent studies [45,48]. Finally, recent reports have
demonstrated that acyl modifications of lysine residues (e.g., myristoylation) may play a role in the
biological functions of the sirtuins. Only a detailed comprehensive evaluation of the myriad acylation/de-
acylation activities can shed light on the relevance of the various acyl lysine modifications.
In conclusion, we have developed cambinol analogs, a class of sirtuin inhibitors distinct from
other published compounds targeting class III deacylases. Our lead compounds display high SIRT2
inhibitory potency (as low as 250 nM) with excellent selectivity profiles against other sirtuin isoforms.
The compounds are cytotoxic to various cancer cell lines, B-cell derived lymphomas (Burkitt
lymphoma and DLBCL) in particular. From a medicinal chemistry standpoint, further optimization
of the cambinol scaffold with the goal of higher potency and improved physicochemical properties
is currently underway and will be reported in due course.
4. Materials and Methods
4.1 Enzyme Inhibition Assays
SIRT1, SIRT2, and SIRT3 were purchased from Cayman Chemical. Enzyme inhibition assays
were performed in 96-well black plates using the SIRT-Glo Assay (Promega Corp.) according to the
manufacturer’s instructions. Compounds were dissolved in 100% DMSO and to make 5- or 10-mM
stock solutions. For initial enzyme inhibition tests, 50 µM of inhibitory concentration was used. The
results are average of triplicate experiments (final DMSO concentration ~0.25%). IC50 values were
determined in triplicate and reported values are averages of two independent experiments.
4.2. Cell Viability Assays
Cell lines were obtained from ATCC (Manassas, VA, USA) and were grown under standard
conditions as recommended. For viability assays, 3000 cells per well were dispensed into 96-well
plates and treated with test compounds (final DMSO concentration 0.25%). Cells were incubated with
test compounds (or DMSO controls) for 72 h or 5 days as noted at 37 °C in 5% CO2 and viability was
determined using the CellTiter-Glo Assay (Promega Corp.) according to manufacturer’s instructions.
Assays were carried out in triplicate and reported values are averages of at least two independent
experiments.
4.3. General Chemistry Information
Thin layer chromatography was carried out using 60 F254 silica gel plates (Merck, Darmstadt,
Germany) using appropriate solvent mixtures. Solvents were ACS reagent grade and anhydrous
solvents (Sigma-Aldrich, St. Louis, Missouri, USA, and Acros Organics/Fisher Scientific, Pittsburgh,

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PA USA,) were used as received. Medium pressure chromatography for compound purification was
carried out using Isolera with Silicycle HP cartridges (Biotage, Charlotte, NC, USA,). LCMS was
performed using an 1100 HPLC system (Agilent, Santa Clara, CA, USA) equipped with a XTerra MS
C18 5 µm, 4.6 Å~ 50 mm column (Waters, Milford, MA, USA ) or a Poroshell 120 EC-C18 4.6 × 100
mm column (Agilent, Santa Clara, CA, USA) using an Agilent photodiode array detector and an in-
line Agilent 6130 single quadrupole mass spectrometer. Analytical HPLC method involved gradient
elution from 0 to 95% acetonitrile in water (0.1% formic acid) over 6 min. Agilent ChemStation
software (Agilent, Santa Clara, CA, USA) was used to develop methods. Final purity of the
1
compounds was determined by H-NMR (AV-300, Brucker, Billerica, MA, USA) or by analyzing
chromatogram of the products at 210, 254 and 280 nm.
4.4. Representative Procedure (I) for Preparation of Substituted 2-Hydroxy-1-Naphthalene Carbaldehydes (7)
6-Bromo-2-hydroxynaphthalene-1-carbaldehyde (AstaTech, Bristol, PA, USA). A solution of TiCl4 (1.0 M
in CH2Cl2, 27 mmol, 27 mL) in dichloromethane was treated with a solution of dichloromethyl methyl
ether (13.5 mmol) in 1,2-dichloroethane or dichloromethane (3 mL) at 0 °C for 15 min. A solution of
6-bromo-2-hydroxy naphthalene (13.5 mmol) in CH2Cl2 (30 mL) was added dropwise, and the
reaction was allowed to stir overnight with gradual warm up to the room temperature. The reaction
was quenched by adding 1 M HCl (10 mL). The aqueous layer was extracted with CH2Cl2 (×3) and
the organic layers were then combined, dried over Na2SO4, and reduced to dryness to afford a
reddish-brown residue which was further purified using medium pressure chromatography using a
gradient EtOAc/hexane solvent system (1−10% EtOAc) to yield 1.4 g (1.20 g, 5.8 mmol, 43%) of the
desired product as a white powder. 1H-NMR (300 MHz, DMSO-d6) δ 12.01–11.79 (bs, 1H), 10.76 (s, 1H),
8.91 (d, J = 9.0 Hz, 1H), 8.17 (d, J = 1.5 Hz, 1H), 8.11 (d, J = 9.0 Hz, 1H), 7.73 (dd, J = 9.0, 1.8 Hz, 1H), 7.30
(d, J = 9.0 Hz, 1H). LRMS: m/z = 248.9 (M − H)−.
4.5. Representative Procedure (II) for Preparation of Substituted 2-Benzoyl-3H-benzo-[f]chromen-3-ones (13)
8-Bromo-2-(3-bromobenzoyl)-3H-benzo[f]chromen-3-one (12). To a solution of substituted 6-bromo-2-
hydroxyl naphthaldehyde (124 mg, 0.5 mmol) in ethanol (5 mL) were added the corresponding ethyl-
(3-bromophenyl)-3-oxopropanoate 95.5 µL (0.5 mmol). Piperidine (5 drops) was added, and the
reaction was heated under reflux for 2 h. The reaction was allowed to cool, and the yellowish
precipitate obtained was collected by filtration and washed with ethanol several times to get the
condensation product 8-bromo-2-(3-bromobenzoyl)-3H-benzo[f]chromen-3-one, 200 mg (0.20 g, 0.44
1
mmol, 88%). H-NMR (300 MHz, DMSO-d6) δ 9.24 (s, 1H), 8.59 (d, J = 9.0 Hz, 1H), 8.43 (s, 1H), 8.17
(d, J = 9.0 Hz, 1H), 8.18 (s, 1H), 7.99 (d, J = 7.8 Hz, 1H), 7.90 (t, J = 7.5 Hz, 2H), 7.74 (d, J = 9.3 Hz, 1H),
7.53 (t, J = 7.8 Hz, 1H). LRMS m/z = 457.0 (M + H)+.
4.6. Representative Procedure (III) for Preparation of Substituted 2-(Benzoyl)-1H,2H-naphtho [2,1-b]pyran-3-
ones (14)
8-Bromo-2-(3-bromobenzoyl)-1H,2H-naphtho [2,1-b]pyran-3-one (13). The corresponding benzoyl
coumarin (0.20 g, 0.44 mmol) was dissolved in dry pyridine (2 mL). To this solution was added NaBH4
(1.25 equiv., 0.55 mmol, 20.9 mg), and the reaction was stirred at room temp for 3 h. The mixture was
then poured in cold 2 M hydrochloric acid (5 mL), which resulted in a white precipitate. The
precipitate was washed several times with water, dried under a vacuum to yield the corresponding
1,2-dihydrocoumarin, 8-bromo-2-(3-bromobenzoyl)-1H,2H-naphtho[2,1-b]pyran-3-one as a white
powder (0.17 g, 0.36 mmol, 82%) which was taken to the next step without purification. 1H-NMR (300
MHz, DMSO-d6) δ 8.29 (d, J = 7.8 Hz, 2H), 8.09 (d, J = 7.8 Hz, 1H), 8.03–7.87 (m, 3H), 7.71 (d, J = 9.9
Hz, 1H), 7.53 (t, J = 7.8 Hz, 1H), 7.42 (d, J = 9.0 Hz, 1H), 5.38 (dd, J = 11.1, 6.6 Hz, 1H), 3.81-3.54 (ddd,
J = 16.8, 11.7, 6.6 Hz, 2H). LRMS m/z 458.9 (M + H)+.
4.4. Representative Procedure (IV) for Preparation of Substituted 2-[(2-Hydroxynaphthalen-1-yl)methyl]-3-
oxo-3-phenylpropanamides
To a solution of 1,2-dihydrocoumarin, (0.29 mmol) in anhydrous THF (3 mL) was added
ammonium hydroxide solution (12 M) 223 µL or the desired amine (1.0 equiv., TEA 2.0 equiv., Sigma-

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Aldrich, St. Louis, MO, USA) and the reaction was stirred at rt for 8–14 h. Upon completion of the
reaction (as judged by TLC/LC-MS), the solution was concentrated in vacuo. The reaction mixture
was then purified using HP Silicycle columns using Biotage purification system using Hx/EtOAc
gradient elution.
2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-3-(4-methylphenyl)-3-oxopropanamide (5). 60.0 mg (0.15
mmol, 50%). 1H-NMR (300 MHz, DMSO-d6) δ 9.84 (s, 1H), 7.99 (d, J = 2.1 Hz, 1H), 7.96–7.85 (m, 3H),
7.64 (d, J = 9.0 Hz, 1H), 7.48 (dd, J = 9.0, 1.8 Hz, 2H), 7.27 (d, J = 8.1 Hz, 2H), 7.20 (d, J = 8.7 Hz, 1H),
6.92 (s, 1H), 4.69 (dd, J = 8.4, 5.1 Hz, 1H), 3.46 (ddd, J = 13.8, 8.4, 5.1 Hz, 2H), 2.35 (s, 3H). LRMS [ES]+:
m/z = 412.1 (M + H)+.
2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-3-(3-methylphenyl)-3-oxopropanamide (16). 6.8 mg (0.02
1
mmol, 92%). H-NMR (300 MHz, DMSO-d6) δ 9.99-9.75 (bs, 1H), 7.99 (d, J = 2.1 Hz, 1H), 7.93 (d, J =
9.3 Hz, 1H), 7.79–7.73 (m, 2H), 7.64 (d, J = 9.0 Hz, 1H), 7.52–7.46 (m, 2H), 7.44–7.31 (m, 2H), 7.20 (d, J
= 8.7 Hz, 1H), 6.98–6.88 (bs, 1H), 4.67 (dd, J = 8.1, 5.4 Hz, 1H), 3.46 (ddd, J = 13.8, 8.7, 5.4 Hz, 2H), 2.32
(s, 3H).LRMS [ES]+: m/z = 412.1 (M + H)+.
2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-3-(2-methylphenyl)-3-oxopropanamide (17). 58.2 mg (0.14
mmol, 93%). 1H-NMR (300 MHz, DMSO-d6) δ 9.85 (s, 1H), 8.00 (d, J = 1.8 Hz, 1H), 7.91 (d, J = 9.3 Hz,
1H), 7.65 (d, J = 8.7 Hz, 2H), 7.48 (dd, J = 9.3, 2.1 Hz, 1H), 7.41–7.32 (m, 2H), 7.27–7.15 (m, 3H), 6.87 (s,
1H), 4.59–4.49 (m, 1H), 3.51–3.41 (m, 2H), 2.29 (s, 3H). LRMS [ES]+: m/z = 412.1 (M + H)+.
2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-3-oxo-3-phenylpropanamide (18). 13.8 mg (0.03 mmol,
28%). 1H-NMR data for the major tautomer is provided. 1H-NMR (300 MHz, DMSO-d6) δ 9.86 (s, 1H),
8.01–7.95 (m, 2H), 7.92 (d, J = 9.0 Hz, 1H), 7.80 (d, J = 7.2 Hz, 1H), 7.67–7.61 (m, 3H), 7.52–7.43 (m, 3H),
7.20 (d, J = 8.7 Hz, 1H), 6.95 (s, 1H), 4.69 (dd, J = 8.4, 5.1 Hz, 1H), 3.60–3.38 (m, 2H). LRMS [ES]+: m/z =
398.0 (M + H)+.
2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-3-(4-chlorophenyl)-3-oxopropanamide (19). 21.8 mg (0.05 mmol,
42%). 1H-NMR data for the major isomer is provided. 1H-NMR (300 MHz, DMSO-d6) δ 9.95- 9.76 (bs, 1H),
8.05-7.86 (m, 4H), 7.64 (d, J = 9.0 Hz, 1H), 7.57–7.44 (m, 4H), 7.18 (d, J = 8.7 Hz, 1H), 6.97 (s, 1H), 4.66 (dd, J
= 8.4, 5.1 Hz, 1H), 3.47 (ddd, J = 13.8, 8.4, 5.4 Hz, 2H). LRMS [ES]+: m/z = 432.0 (M + H)+.
2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-3-(4-trifluoromethylphenyl)-3-oxopropanamide (20). 30.8
mg (0.07 mmol, 59%). 1H-NMR data for the major isomer is provided. 1H-NMR (300 MHz, DMSO-d6)
δ 9.89 (s, 1H), 8.12 (d, J = 8.4 Hz, 1H), 7.99 (d, J = 2.1 Hz, 1H), 7.95–7.80 (m, 4H), 7.64 (d, J = 8.7 Hz, 1H),
7.56–7.45 (m, 2H), 7.18 (d, J = 8.7 Hz, 1H), 7.12–6.98 (bs, 1H), 4.72 (dd, J = 8.4, 5.4 Hz, 1H), 3.49 (ddd, J
= 14.4, 8.4, 5.1 Hz, 2H). LRMS [ES]+: m/z = 466.0 (M + H)+.
2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-3-(3-trifluoromethylphenyl)-3-oxopropanamide (21). 35.5
mg (0.08 mmol, 68%). 1H-NMR data for the major isomer is provided. 1H-NMR (300 MHz, DMSO-d6)
δ 9.89 (bs, 1H), 8.22–8.12 m, 1H), 7.98 (d, J = 2.1 Hz, 1H), 7.94-7.85 (m, 2H), 7.69 (t, J = 7.8 Hz, 1H), 7.62
(d, J = 8.7 Hz, 1H), 7.58–7.53 (bs, 1H), 7.51–7.44 (m, 1H), 7.17 (d, J = 8.7 Hz, 1H), 6.99 (bs, 1H), 4.75 (dd,
J = 8.1, 5.7 Hz, 1H), 3.58–3.44 (m, 2H). LRMS [ES]+: m/z = 466.1 (M + H)+.
2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-3-(4-bromo)-3-oxopropanamide (22). 21.1 mg (0.02 mmol,
48%). LRMS [ES]+: m/z = 478.0 (M + H)+.
2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-3-(3-bromo)-3-oxopropanamide (23). 1H-NMR data for the
major isomer is provided. 1H-NMR (300 MHz, DMSO-d6) δ 9.98–9.87 (bs, 1H), 8.07 (s, 1H), 7.99 (d, J =
1.8 Hz, 1H), 7.95–7.86 (m, 2H), 7.82–7.74 (m, 1H), 7.64 (d, J = 8.7 Hz, 1H), 7.54–7.39 (m, 3H), 7.19 (d, J
= 9.0 Hz, 1H), 6.97 (bs, 1H), 4.66 (dd, J = 8.1, 5.1 Hz, 1H), 3.57–3.37 (m, 2H). LRMS [ES]+: m/z = 477.0
(M + H)+.
2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-3-(4-methoxy)-3-oxopropanamide (24). 44.0 mg (0.11
mmol, 85%). 1H-NMR (300 MHz, DMSO-d6) δ 10.02–9.57 (bs, 1H), 8.02–7.95 (m, 3H), 7.92 (d, J = 9 Hz,
1H), 7.64 (d, J = 8.7 Hz, 1H), 7.52-7.44 (m, 2H), 7.20 (d, J = 9.0 Hz, 1H), 6.98 (d, J = 8.7 Hz, 2H), 6.92 (bs,

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1H), 4.62 (dd, J = 8.4, 5.1 Hz, 1H), 3.82 (s, 3H), 3.38 (ddd, J = 13.5, 8.4, 5.1 Hz, 2H). LRMS [ES]+: m/z =
428.1 (M + H)+.
2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-3-(4-fluoro)-3-oxopropanamide (25). 19.5 mg (0.05 mmol,
1
37%). H-NMR (300 MHz, DMSO-d6) δ 9.87(s, 1H), 8.04 (dd, J = 9.0, 5.4 Hz, 2H), 7.99 (d, J = 2.1 Hz,
1H), 7.90(d, J = 2.3 Hz, 1H), 7.64 (d, J = 9.0 Hz, 1H), 7.53–7.44 (m, 1H), 7.30 (t, J = 9.0 Hz, 2H), 7.19 (d, J
= 9.0 Hz, 1H), 7.00–6.93 (bs, 1H), 4.66 (dd, J = 8.1, 5.4 Hz, 1H), 3.56–3.41 (m, 2H). LRMS [ES]+: m/z =
416.0 (M + H)+.
2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-3-oxo-3-(pyridin-4-yl)propenamide (26). 37.8 mg (0.09
mmol, 46%). LRMS [ES]+: m/z = 413.1 (M + H)+.
2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-3-oxo-3-(pyridin-3-yl)propenamide (27). 12.3 mg (0.03
mmol, 19%). 1H-NMR (300 MHz, DMSO-d6) δ 9.90 (s, 1H), 9.09–8.98 (bs, 1H), 8.82–8.68 (bs, 1H), 8.19
(td, J = 7.8, 1.8 Hz, 1H), 7.99 (d, J = 2.1 Hz, 1H), 7.82 (d, J = 9.3 Hz, 1H), 7.63 (d, J = 8.7 Hz, 1H), 7.54–
7.46 (m, 3H), 7.18 (d, J = 9.0 Hz, 1H), 4.69–4.56 (m, 1H), 3.54–3.42 (m, 2H), 2.33 (d, J = 4.5 Hz, 3H).
LRMS [ES]+: m/z = 413.0 (M + H)+.
2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-3-(4-acetamidophenyl)-3-oxopropanamide (28). 14.0 mg
(0.03 mmol, 26%). 1H-NMR (300 MHz, DMSO-d6) δ 10.09 (s, 1H), 9.86 (s, 1H), 8.11–8.05 (bs, 1H), 7.99
(d, J = 2.1 Hz, 1H), 7.91–7.78 (m, 3H), 7.64 (d, J = 8.7 Hz, 2H), 7.49 (dd, J = 9.0, 2.1 Hz, 1H), 7.37 (d, J =
8.1 Hz, 1H), 7.19 (d, J = 8.7 Hz, 1H), 4.63–4.50 (m, 1H), 3.54–3.43 (m, 2H), 2.31 (d, J = 4.5 Hz, 3H), 2.05
(s, 3H). LRMS [ES]+: m/z = 469.0 (M + H)+.
3-(3-Bromophenyl)-2-[(2-hydroxynaphthalen-1-yl)methyl]-3-oxopropanamide (29). 48.5 mg (0.12 mmol,
93%). 1H-NMR data for the major isomer is provided. 1H-NMR (300 MHz, DMSO-d6) δ 9.65 (s, 1H), 8.06
(t, J = 1.5 Hz, 1H), 7.92 (d, J = 8.4 Hz, 1H), 7.83–7.72 (m, 2H), 7.63 (d, J = 9.0 Hz, 1H), 7.57–7.52 (bs, 1H), 7.46–
7.34 (m, 3H), 7.28–7.22 (m, 1H), 7.14 (d, J = 8.7 Hz, 1H), 7.04–6.97 (bs, 1H), 4.70 (dd, J = 8.1, 5.4 Hz, 1H), 3.49
(ddd, J = 13.8, 8.1, 5.4 Hz, 2H). LRMS [ES]+: m/z = 398.0 (M + H)+.
3-(3-Bromophenyl)-2-[(2-hydroxy-6-methoxynaphthalen-1-yl)methyl]-3-oxopropanamide (30). 15.3 mg (0.36
mmol, 92%). LRMS [ES]+: m/z = 410.0 (M + H)+.
2-[(3-Bromo-2-hydroxynaphthalen-1-yl)methyl]-3-(3-bromophenyl)-3-oxopropanamide (31). 27.2 mg (0.06 mmol,
44%). 1H-NMR data for the major isomer is provided. 1H-NMR (300 MHz, DMSO-d6) δ 9.98 (s, 1H), 8.14-
8.08 (m, 2H), 7.99–7.92 (m, 1H), 7.83–7.66 (m, 3H), 7.63–7.53 (m, 1H), 7.47–7.32 (m, 2H), 7.25–7.13 (m, 1H),
7.12–6.97 (m, 1H), 4.78–4.63 (m, 1H), 3.56–3.45 (m, 2H). LRMS [ES]+: m/z = 476.00 (M + H)+.
2-[(7-Bromo-2-hydroxynaphthalen-1-yl)methyl]-3-(3-bromophenyl)-3-oxopropanamide (32). 61.7 mg (0.13
mmol, 100%). LRMS [ES]+: m/z = 476.00 (M + H)+.
2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-N-methyl-3-oxo-3-[4-(trifluoromethyl)phenyl]propenamide
(33). 33.2 mg (0.07 mmol, 69%). 1H-NMR data for the major isomer is provided. 1H-NMR (300 MHz,
DMSO-d6) δ 9.94–9.80 (bs, 1H), 8.08 (d, J = 8.1 Hz, 1H), 7.99 (d, J = 1.8 Hz, 1H), 7.98–7.90 (m, 1H), 7.87–
7.75 (m, 4H), 7.67–7.61 (m, 1H), 7.52–7.46 (m, 1H), 7.18 (d, J = 9.0 Hz, 1H), 4.64 (t, J = 7.2 Hz, 1H), 3.48
(d, J = 6.3 Hz, 2H), 2.31 (d, J = 4.5 Hz, 3H).
2-[(2-Hydroxy-6-methylnaphthalen-1-yl)methyl]-3-oxo-3-[4-(trifluoromethyl)phenyl]propenamide (34). 48.0
mg (0.11 mmol, 70%). 1H-NMR data for the major isomer is provided. 1H-NMR (300 MHz, DMSO-d6)
δ 9.59–9.40 (bs, 1H), 8.07 (d, J = 8.4 Hz, 2H), 7.99–7.89 (m, 1H), 7.86–7.72 (m, 2H), 7.67–7.61 (m, 1H),
7.56–7.47 (m, 2H), 7.44–7.38 (m, 1H), 7.23 (bd, J = 8.7 Hz, 1H), 7.08 (d, J = 8.7 Hz, 1H), 4.72–4.59 (m,
1H), 3.53–3.45 (m, 2H), 2.39 (s, 3H), 2.33 (d, J = 4.5 Hz, 3H). LRMS [ES]+: m/z = 416.1 (M + H)+.
2-[(2-Chloro-6-hydroxyquinolin-5-yl)methyl]-3-oxo-3-[4-(trifluoromethyl)phenyl]propenamide (35). 41.2 mg
1
(0.09 mmol, 75%). 1H-NMR data for the major isomer is provided. H-NMR (300 MHz, DMSO-d6) δ
10.28–10.10 (bs, 1H), 8.35 (d, J = 9.0 Hz, 1H), 8.10 (d, J = 8.4 Hz, 2H), 8.02–7.92 (m, 1H), 7.85 (d, J = 8.4
Hz, 2H), 7.71 (d, J = 9.0 Hz, 1H), 7.44 (dd, J = 9.6, 7.2 Hz, 2H), 4.74–4.57 (m, 1H), 3.53–3.42 (m, 2H),
2.28 (d, J = 4.5 Hz, 3H). LRMS [ES]+: m/z = 437.1 (M + H)+.

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2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-3-(3-bromophenyl)-N-(2-hydroxyethyl)-3-oxopropanamide
1
1
(36). 4.7 mg (0.01 mmol, 16%). H-NMR data for the major isomer is provided. H-NMR (300 MHz,
DMSO-d6) δ 10.03-9.49 (bs, 1H), 8.04 (s, 1H), 7.99 (s, 1H), 7.89 (d, J = 7.5 Hz, 1H), 7.85–7.74 (m, 2H),
7.63 (d, J = 9.0 Hz, 1H), 7.44 (d, J = 9.3 Hz, 1H), 7.40 (t, J = 7.8 Hz, 1H), 7.18 (d, J = 9.0 Hz, 2H), 4.71–
4.57 (m, 1H), 3.50–3.41 (m, 2H), 3.13–2.79 (m, 4H). LRMS [ES]+: m/z = 520.0 (M + H)+.
2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-3-(3-bromophenyl)-N-(3-hydroxypropyl)-3-oxopropanamide
(37). 10.4 mg (0.02 mmol, 21%). 1H-NMR data for the major isomer is provided. 1H-NMR (300 MHz,
DMSO-d6) δ 8.04 (s, 1H), 7.99 (bs, 1H), 8.10 (d, J = 7.8 Hz, 1H), 7.80 (d, J = 9.0 Hz, 2H), 7.68–7.57 (m,
2H), 7.52–7.38 (m, 3H), 7.20 (d, J = 9.0 Hz, 1H), 4.66–4.53 (m, 1H), 3.56–3.39 (m, 2H), 3.11–2.69 (m, 4H),
1.20–1.03 (m, 2H). LRMS [ES]+: m/z = 434.0 (M + H)+.
2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-1-(3-bromophenyl)-3-(morpholin-4-yl)propane-1,3-dione
(38). 25.9 mg (0.05 mmol, 52%). 1H-NMR data for the major isomer is provided. 1H-NMR (300 MHz,
DMSO-d6) δ 10.04 (s, 1H), 8.05 (d, J = 1.8 Hz, 1H), 7.90 (t, J = 1.8 Hz, 1H), 7.83–7.68 (m, 4H), 7.51 (dd, J
= 9.3, 2.1 Hz, 1H), 7.45 (t, J = 7.8 Hz, 1H), 7.22 (d, J = 8.7 Hz, 1H), 5.00 (dd, J = 9.3, 5.4 Hz, 1H), 3.60–
3.45 (m, 2H), 3.31–2.91 (m, 6H), 2.87–2.76 (m, 1H), 2.45–2.35 (m,1H). LRMS [ES]+: m/z = 546.0 (M + H)+.
2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-3-(3-bromophenyl)-N-(furan-3-ylmethyl)-3-oxopropanamide
(39). 34.4 mg (0.08 mmol, 17%). 1H-NMR data for the major isomer is provided. 1H-NMR (300 MHz,
DMSO-d6) δ 9.89 (s, 1H), 8.51 (t, J = 5.4 Hz, 1H), 8.04 (t, J = 1.5 Hz, 1H), 7.99 (d, J = 1.8 Hz, 1H), 7.88 (d,
J = 7.8 Hz, 1H), 7.84–7.74 (m, 2H), 7.63 (d, J = 8.7 Hz, 1H), 7.49–7.34 (m, 3H), 7.18 (d, J = 9.0 Hz, 1H),
6.23 (dd, J = 3.0, 1.8 Hz, 1H), 5.76 (d, J = 2.7 Hz, 1H), 4.70 (t, J = 6.0 Hz, 1H), 4.00 (dd, J = 15.6, 5.4, 5.1
Hz, 2H), 3.55–3.44 (m, 2H). LRMS [ES]+: m/z = 556 (M + H)+.
2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-3-(3-bromophenyl)-3-oxo-N-(pyridin-4-ylmethyl)-propenamide
1
1
(40). 30.7 mg (0.05 mmol, 31%). H-NMR data for the major isomer is provided. H-NMR (300 MHz,
DMSO-d6) δ 9.94 (bs, 1H), 8.73-8.60 (m, 1H), 8.24 (d, J = 5.1 Hz, 2H), 8.13 (s, 1H), 8.05 (s, 1H), 7.94 (d, J = 7.8
Hz, 1H), 7.84 (t, J = 9.3 Hz, 2H), 7.70 (d, J = 8.7 Hz, 1H), 7.51−7.41 (m, 2H), 7.23 (d, J = 9.0 Hz, 1H), 6.66 (d, J
= 4.8 Hz, 2H), 4.87−4.72 (m, 1H), 4.06−3.98 (m, 2H), 3.69−3.38 (m, 2H). LRMS [ES]+: m/z = 567.0 (M + H)+.
2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-3-(3-bromophenyl)-N-(2-methoxyethyl)-3-oxopropanamide
(41). 25.0 mg (0.47 mmol, 31%). 1H-NMR data for the major isomer is provided. 1H-NMR (300 MHz,
DMSO-d6) δ 9.91–9.75 (bs, 1H), 8.19–8.06 (m, 2H), 8.03–7.98 (m, 1H), 7.93 (d, J = 7.8 Hz, 1H), 7.83–7.74
(m, 2H), 7.64 (d, J = 8.7 Hz, 1H), 7.48 (bd, J = 9.3 Hz, 1H), 7.41 (t, J = 7.8 Hz, 1H), 7.20 (d, J = 9.0 Hz, 1H),
4.64 (dd, J = 8.4, 4.5 Hz, 1H), 3.44 (ddd, J = 13.8, 8.4, 4.5 Hz, 2H), 3.03–2.82 (m, 6H), 2.77–2.64 (m, 1H).
LRMS [ES]+: m/z = 534.1 (M + H)+.
2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-3-(3-bromophenyl)-3-oxo-N-(pyridin-3-ylmethyl)-
propenamide (42). 35.2 mg (0.06 mmol, 41%). 1H-NMR data for the major isomer is provided. 1H-NMR
(300 MHz, DMSO-d6) δ 9.90 (bs, 1H), 8.59 (t, J = 5.1 Hz, 1H), 8.38–8.32 (m, 1H), 8.20-8.13 (m, 1H), 8.07
(t, J = 1.5 Hz, 1H), 8.01 (d, J = 2.1 Hz, 1H), 7.93-7.77 (m, 2H), 7.66 (d, J = 8.7 Hz, 1H), 7.46 (dd, J = 9.0,
2.1 Hz, 1H), 7.40 (t, J = 7.8 Hz, 1H), 7.20 (d, J = 8.7 Hz, 1H), 7.10−7.04 (m, 1H), 7.02−6.95 (m, 1H), 4.70
(dd, J = 9.0, 4.5 Hz, 1H), 4.05−3.98 (m, 2H), 3.62−3.38 (m, 2H). LRMS [ES]+: m/z = 567.0 (M + H)+.
2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-3-(3-bromophenyl)-N-(2-carbamoylethyl)-3-oxopropanamide
1
1
(43). 35.4 mg (0.06 mmol, 31%). H-NMR data for the major isomer is provided. H-NMR (300 MHz,
DMSO-d6) δ 9.84 (s, 1H), 8.12–8.04 (m, 1H), 8.03–7.98 (m, 2H), 7.87 (d, J = 8.1 Hz, 1H), 7.783–7.74 (m, 2H),
7.63 (d, J = 9.0 Hz, 1H), 7.53–7.47 (m, 1H), 7.39 (t, J = 8.1 Hz, 1H), 7.17 (d, J = 8.7 Hz, 1H), 7.15−7.09 (bs, 1H),
6.77−6.67 (bs, 1H), 4.66–4.55 (m, 1H), 3.50−3.41 (m, 2H), 3.08−2.84 (m, 2H), 1.92 (dt, J = 7.2, 2.1 Hz, 2H).
2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-3-(3-bromophenyl)-N-[2-(methylcarbamoyl)ethyl]-3-oxo-
propanamide (44). 22.1 mg (0.04 mmol, 30%). 1H-NMR data for major isomers are provided. 1H-NMR
(300 MHz, DMSO-d6) δ 9.84 (d, J = 5.7 Hz, 1H), 8.12–7.96 (m, 3H), 7.94–7.69 (m, 3H), 7.68–7.45 (m, 3H),
7.44–7.33 (m, 1H), 7.22–7.13 (m, 1H), 4.68–4.54 (m, 1H), 3.52–3.41 (m, 2H), 3.08–2.84 (m, 2H), 2.59–2.45
(m, 3H), 1.98–1.83 (m, 2H). LRMS [ES]+: m/z = 561.0 (M + H)+.

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2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-1-(4-methylpiperazin-1-yl)-3-[4-(trifluoromethyl)phenyl]-
propane-1,3-dione (45). 30.1 mg (0.06 mmol, 49%). 1H-NMR (300 MHz, DMSO-d6) δ 10.13-1.04 (bs,1H), 8.05
(d, J = 2.1 Hz, 1H), 7.99–7.93 (m, 2H), 7.90–7.80 (m, 3H), 7.78–7.68 (m, 2H), 7.50 (dd, J = 9.0, 2.1 Hz, 1H), 7.23
(d, J = 9.0 Hz, 1H)), 5.12–4.97 (m, 1H), 3.64–3.46 (m, 2H), 3.23–2.93 (m, 3H), 2.72–2.58 (m, 1H), 2.03–1.88 (m,
1H), 1.86 (s, 3H), 1.40–1.23 (m, 1H), 0.92–0.78 (m, 1H). LRMS [ES]+: m/z = 549.1 (M + H)+.
2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-1-[4-(trifluoromethyl)phenyl]hex-5-yne-1,3-dione (46). 42.2
mg (0.08 mmol, 42%). LRMS [ES]+: m/z = 504.1 (M+H)+.
2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-3-(4-chlorophenyl)-N-(2-hydroxyethyl)-3-oxopropanamide
(47)/ 13.1 mg (0.03 mmol, 14%). 1H-NMR (300 MHz, DMSO-d6) δ 9.95–9.75 (bs,1H), 7.99 (d, J = 2.1 Hz,
2H), 7.96 (d, J = 8.4 Hz, 2H), 7.82 (d, J = 9.3 Hz, 1H), 7.64 (d, J = 9.0 Hz, 1H), 7.52 (d, J = 8.7 Hz, 2H),
7.48 (dd, J = 9.0, 2.1 Hz, 1H), 7.18 (d, J = 8.7 Hz, 1H), 4.67–4.58 (m, 1H), 4.47–4.38 (m, 1H), 3.50–3.42
(m, 2H), 3.10-2.80 (m, 3H). LRMS [ES]+: m/z = 476.0 (M + H)+.
2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-3-(4-bromophenyl)-N-(2-hydroxyethyl)-3-oxopropanamide
1
(48). 23.9 mg (0.05 mmol, 25%). H-NMR (300 MHz, DMSO-d6) δ 9.90–9.80 (s,1H), 7.99 (d, J = 2.1 Hz,
2H), 7.87 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 9.0 Hz, 1H), 7.70–7.60 (m, 3H), 7.48 (dd, J = 9.3, 2.1 Hz, 1H), 7.18
(d, J = 8.7 Hz, 1H), 4.68–4.58 (m, 1H), 4.48–4.36 (m, 1H), 3.50–3.42 (m, 2H), 3.11–2.78 (m, 3H). LRMS
[ES]+: m/z = 520.0 (M + H)+.
2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-N-(2-hydroxyethyl)-3-oxo-3-[4-(trifluoromethyl)phenyl]-
propenamide (49). 37.9 mg (0.07mmol, 41%). LRMS [ES]+: m/z = 510.0 (M + H)+.
2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-N-(2-carbamoylethyl)-3-(4-chlorophenyl)-3-oxopropanamide
1
1
(50). 36.0 mg (0.07 mmol, 42%). H-NMR data for major isomers are provided. H-NMR (300 MHz,
DMSO-d6) δ 9.81 (s, 1H), 8.04 (t, J = 5.7 Hz, 1H), 7.99 (d, J = 1.8 Hz, 1H), 7.92 (d, J = 8.7 Hz, 2H), 7.80
(d, J = 9.3 Hz, 1H), 7.59 (d, J = 9.0 Hz, 1H), 7.55–7.49 (m, 3H), 7.17 (d, J = 8.7 Hz, 1H), 7.14–7.06 (bs, 1H),
6.78–6.68 (bs, 1H), 4.63–4.55 (m, 1H), 3.51–3.40 (m, 2H), 3.10–2.81 (m, 2H), 1.96–1.86 (m, 2H). LRMS
[ES]+: m/z = 503.1 (M + H)+.
2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-3-(4-bromophenyl)-N-(2-carbamoylethyl)-3-oxopropanamide
1
1
(51). 24.1 mg (0.04 mmol, 26%). H-NMR data for major isomers are provided. H-NMR (300 MHz,
DMSO-d6) δ 9.82 (s, 1H), 8.08–8.01 (m, 1H), 7.99 (d, J = 1.8 Hz, 1H), 7.84 (d, J = 8.7 Hz, 2H), 7.79 (d, J =
9.3 Hz, 1H), 7.69–7.60 (m, 3H), 7.49 (dd, J = 9.0, 1.8 Hz, 1H), 7.17 (d, J = 9.0 Hz, 1H), 7.13–7.08 (bs, 1H),
6.77–6.68 (bs, 1H), 4.64–4.50 (m, 1H), 3.50–3.42 (m, 2H), 3.08–2.81 (m, 2H), 1.95–1.85 (m, 2H). LRMS
[ES]+: m/z = 547.0 (M + H)+.
2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-N-(2-carbamoylethyl)-3-oxo-3-[4-(trifluoromethyl)phenyl]-
propenamide (52). 46.3 mg (0.09 mmol, 48%). 1H-NMR (300 MHz, DMSO-d6) δ 9.89-9.74 (bs,1H), 8.07
(d, J = 8.4 Hz, 2H), 7.99 (d, J = 1.8 Hz, 1H), 7.84–7.74 (m, 3H), 7.68–7.58 (m, 2H), 7.49 (dd, J = 9.0, 2.1
Hz, 1H), 7.18 (dd, J = 9.0, 2.1 Hz, 1H),7.13–7.05 (bs, 1H), 6.77–6.67 (bs, 1H), 4.74–4.57 (m, 1H), 3.52–
3.41 (m, 2H), 3.08–2.83 (m, 2H), 1.95–1.86 (m, 2H). LRMS [ES]+: m/z = 537.1 (M + H)+.
2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-N-(2-carbamoylethyl)-3-oxo-3-[3-(trifluoromethyl)phenyl]-
propenamide (53). 25.7 mg (0.03 mmol, 16%). 1H-NMR data for major isomers are provided. 1H-NMR
(300 MHz, DMSO-d6) δ 9.83 (s,1H), 8.19–8.07 (m, 2H), 7.98 (d, J = 1.8 Hz, 1H), 7.95–7.55 (m, 5H), 7.50
(dd, J = 9.0, 2.1 Hz, 1H), 7.18 (d, J = 9.0 Hz, 1H), 7.13–7.05 (bs, 1H), 6.76–6.67 (m, 1H), 4.74–4.62 (m,
1H), 3.52–3.42 (m, 2H), 3.12–2.85 (m, 2H), 1.97–1.87 (m, 2H).
2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-3-(4-chlorophenyl)-N-[2-(methylcarbamoyl)ethyl]-3-oxo-
propanamide (54). 35.8 mg (0.07 mmol, 64%). 1H-NMR (300 MHz, DMSO-d6) δ 9.92–9.72 (bs,1H), 8.10–
7.87 (m, 4H), 7.87–7.76 (m, 1H), 7.70–7.60 (m, 1H), 7.56–7.43 (m, 4H), 7.26–7.11 (m, 1H), 4.66–4.52 (m,
1H), 3.53–3.40 (m, 2H), 3.10–2.82 (m, 2H), 2.55–2.45 (m, 3H), 1.98–1.84 (m, 2H). LRMS [ES]+: m/z = 517.1
(M + H)+.
2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-N-[2-(methylcarbamoyl)ethyl]-3-oxo-3-[4-(trifluoromethyl)-
phenyl]propenamide (55). 60.4 mg (0.12 mmol, 68%). 1H-NMR data for major isomers are provided. 1H-

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NMR (300 MHz, DMSO-d6) δ 9.80–9.74 (bs,1H), 8.08 (d, J = 7.8 Hz, 2H), 7.99 (d, J = 1.8 Hz, 1H), 7.80–
7.77 (m, 3H), 7.64 (d, J = 9.0 Hz, 1H), 7.53–7.45 (m, 2H), 7.17 (d, J = 9.0 Hz, 1H), 4.65 (t, J = 6.6 Hz, 1H),
3.47 (d, J = 6.6 Hz, 2H), 3.18–2.84 (m, 2H), 2.46 (d, J = 4.8 Hz, 3H), 1.95–1.84 (m, 2H). LRMS [ES]+: m/z
= 551.1 (M + H)+.
2-[(6-Bromo-2-hydroxynaphthalen-1-yl)methyl]-3-(4-bromophenyl)-N-methyl-3-oxopropanamide (56). 51.5
mg (0.11 mmol, 96%). 1H-NMR data for major isomers are provided. 1H-NMR (300 MHz, DMSO-d6) δ
9.98–9.38 (bs,1H), 7.99 (d, J = 1.5 Hz, 1H), 7.94–7.79 (m, 3H), 7.72–7.61 (m, 3H), 7.49 (dd, J = 9.3, 1.8 Hz, 1H),
7.19 (d, J = 8.7 Hz, 1H), 4.64-4.50 (t, J = 6.6 Hz, 1H), 3.52–3.41 (m, 2H), 2.31 (d, J = 4.2 Hz, 3H).
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, General Chemistry.
Author Contributions: Conceptualization, A.B. and J.A.S.; investigation, S.S., A.P., U.L., J.H.H., and T.L.; data
interpretation, S.S., A.P., U.L., J.H.H., T.L., A.B. and J.A.S., draft preparation, S.S., A.B. and J.A.S.; review and
editing, A.B. and J.A.S. All authors have read and agreed to the published version of the manuscript.
Funding: This research was supported by RO1-CA206462 and P30-CA015704 (National Institutes of
Health/National Cancer Institute).
Acknowledgments: We thank colleagues in the Fred Hutchinson Cancer Research Center Proteomics shared
resource for assistance with mass spectrometry as well as members of the Simon and Bedalov labs for helpful
comments and assistance.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 55 and 56 are available from the authors.
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