Discovery of sultam-containing small-molecule disruptors of the huntingtin–calmodulin protein–protein interaction

Article abstract of DOI:10.1007/s00044-020-02583-8

The aberrant protein–protein interaction between calmodulin and mutant huntingtin protein in Huntington’s disease patients has been found to contribute to Huntington’s disease progression. A high-throughput screen for small molecules capable of disrupting this interaction revealed a sultam series as potent small-molecule disruptors. Diversification of the sultam scaffold afforded a set of 24 analogs or further evaluation. Several structure–activity trends within the analog set were found, most notably a negligible effect of absolute stereochemistry and a strong beneficial correlation with electron-withdrawing aromatic substituents. The most promising analogs were profiled for off-target effects at relevant kinases and, ultimately, one candidate molecule was evaluated for neuroprotection in a neuronal cell model of Huntington’s disease.

Full text of DOI:10.1007/s00044-020-02583-8

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02583-8
ORIGINAL RESEARCH
Discovery of sultam-containing small-molecule disruptors of the
huntingtin–calmodulin protein–protein interaction
Nicholas J. Klus1 Khushboo Kapadia2 Peter McDonald3 Anuradha Roy3 Kevin J. Frankowski1 Nancy A. Muma2
Jeffrey Aubé
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1
Received: 15 May 2020 / Accepted: 30 May 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
The aberrant protein–protein interaction between calmodulin and mutant huntingtin protein in Huntington’s disease patients
has been found to contribute to Huntington’s disease progression. A high-throughput screen for small molecules capable of
disrupting this interaction revealed a sultam series as potent small-molecule disruptors. Diversification of the sultam scaffold
afforded a set of 24 analogs or further evaluation. Several structure–activity trends within the analog set were found, most
notably a negligible effect of absolute stereochemistry and a strong beneficial correlation with electron-withdrawing
aromatic substituents. The most promising analogs were profiled for off-target effects at relevant kinases and, ultimately, one
candidate molecule was evaluated for neuroprotection in a neuronal cell model of Huntington’s disease.
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Keywords Huntington’s disease High-throughput screening Neurodegeneration Structure–activity relationship studies
Introduction
the molecular basis for disease progression (Hague et al.
2005; Marsh 2019). The underlying pathology of HD arises
from an underlying gene mutation that encodes for a mutant
Huntingtin protein (mHtt) whose subsequent aggregation
leads to neuronal death. Currently available therapeutic
agents for the treatment of HD and other neurodegenerative
diseases treat only the symptoms associated with the dis-
ease, leaving the underlying pathology and progression
unaffected. The treatment disparity is especially apparent
for HD where both the underlying cause and the molecular
basis of the disease progression has been established, yet the
FDA-approved drugs available, such as tetrabenazine,
merely dampen the involuntary movements (chorea) asso-
ciated with HD (Paleacu 2007). Currently, the majority of
developmental HD drugs that aim to treat disease progres-
sion are macromolecular biologics that will require invasive
routes of administration (i.e., intrathecal or intracer-
ebroventricular) for these agents to reach the CNS (Berg
2017). This would be particularly problematic for the aging
population that exhibits the highest penetrance of the dis-
ease. Accordingly, small-molecule therapeutics that directly
address the primary HD lesion are highly sought. To this
end, herein we report preliminary efforts toward validating
drug-like agents that focus on a target protein–protein
interaction (PPI) that has previously been validated using a
polypeptide inhibitor.
Progressive neurodegenerative disorders such as Parkin-
son’s disease, Alzheimer’s disease, and Huntington’s dis-
ease (HD) have remained challenging therapeutic areas for
drug discovery in large part due to poor understanding of
We dedicate this paper to Professor Robert Hanzlik in recognition of
his many scientific achievements.
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02583-8) contains supplementary
material, which is available to authorized users.
* Nancy A. Muma
nmuma@ku.edu
* Jeffrey Aubé
jaube@unc.edu
1
Division of Chemical Biology and Medicinal Chemistry,
Center for Integrative Chemical Biology and Drug Discovery,
UNC Eshelman School of Pharmacy, 125 Mason Farm Road,
Chapel Hill, NC 27599, USA
2
Department of Pharmacology and Toxicology, University of
Kansas, 1251 Wescoe Hall Drive, Lawrence, KS 66045, USA
3
University of Kansas High-Throughput Screening Laboratory,
University of Kansas, 2034 Becker Drive, Lawrence, KS 66047,
USA

Medicinal Chemistry Research
A current hypothesis for the cause of mHtt aggregation is
transglutaminase (TG) upregulation that catalyzes the
calcium-dependent cross-linking (transamidation) of peptide-
bound glutamine residues on substrate proteins to the ε-
amino-group of lysine residues on either the same or different
proteins (Folk and Finlayson 1977; Karpuj et al. 1999).
These cross-linkages are implicated in tau protein oligo-
merization in Alzheimer’s disease patients and there is also
evidence to suggest that aberrant TG activity contributes to
the HD phenotype (Tucholski 1999). TG mRNA, protein
levels and activity have all been shown to be upregulated in
HD cell models (Karpuj et al. 1999; Lesort 1999, Zainelli
et al. 2005), and TG proteins co-localize with mHtt in
inclusion bodies found in HD patients (Zainelli et al. 2003).
Furthermore, inhibiting or knocking out TG in mouse models
increases their survival (Dedeoglu et al. 2002; Mastrober-
ardino et al. 2002) while inhibiting TG in a cellular model
also decreases cross-linking of mHtt and the cytotoxicity
associated with mHtt expression (Zainelli et al. 2005).
development, such as antisense mRNA or zinc-finger pro-
tease inhibitors, that attempt to interrupt the production of
mHtt at the transcriptional or translational level. Accord-
ingly, we sought to develop small-molecule probes capable
of disrupting this PPI to validate this interaction as a viable
small-molecule target.
Materials and methods
Chemistry
See SI for the synthesis, characterization, 1H and 13C NMR
spectra of all intermediates and 1H and 13C NMR spectra of
final compounds.
General Procedure A: HATU-promoted amide synthesis
A 1-dram vial equipped with a stir bar was charged with
40 mg of carboxylic acid 6–8, HATU (1.2 equiv), and
MeCN (10 mL per mmol acid) and the reaction stirred at rt
for 20 min. The requisite amine component (2.0 equiv) was
added and the reaction stirred for an additional 20 min.
DIPEA (3.0 equiv) was then added and the reaction stirred
overnight for 12–19 h. The reaction mixture was diluted
with EtOAc, washed with saturated, aqueous sodium
bicarbonate solution, and then brine. The organic layer was
dried over anhydrous Na2SO4, filtered, concentrated, and
purified via flash chromatography (30% EtOAc in hexanes)
to afford the amide product as a colorless oil.
Calmodulin (CaM) is a calcium (Ca2+)-binding protein
that, amongst its other roles in the cell, regulates TG
activity (Cheung 1982; Puszkin and Raghuraman 1985).
Studies have shown that CaM and TG proteins co-
immunoprecipitate with mHtt in transfected cell culture
models as well as intranuclear inclusions found in HD brain
samples (Zainelli et al. 2004). Furthermore, inhibition of
CaM in cells expressing mHtt and TG resulted in a
decrease of TG-catalyzed mHtt aggregation (Zainelli et al.
2004). Interestingly, although wild-type huntingtin does
not interact with CaM, mHtt has a much higher affinity for
CaM (Bao et al. 1996). The increased affinity for CaM by
mHtt alters CaM function and leads to aberrant biochem-
ical processes within the cell, including upregulation of TG
activity. Based on the above data, we believe that an
underlying driver of HD progression is the sequestration
and upregulation of TG by the mHtt–CaM heterodimer
leading to an increase of TG-catalyzed mHtt cross-linking.
Previous studies using short peptide sequences borrowed
from CaM have provided substantial evidence that the
disruption of the mHtt–CaM PPI is an attractive target for
the treatment of HD in vitro, in vivo, and in mouse models
(Dudek et al. 2008, 2010; Dai et al. 2009). In biochemical
and cell-based assays, disruption of this system leads to
both decreased TG-catalyzed modifications of mHtt and
mHtt-associated cytotoxicity (Dudek et al. 2008, 2010); in
mHtt mouse models, administration of the CaM fragment
significantly reduced body weight loss and improved motor
function as indicated by improved rotarod performance,
longer stride length, lower stride frequency, fewer low
mobility bouts, and longer travel distance than HD controls
(Dai et al. 2009). The inhibition of the mHtt–CaM PPI
could be a viable target for small molecules (Berg 2017),
and complementary to macromolecular therapies in
(S)-N-Benzyl-4-methyl-2-(5-methylene-1,1-dioxido-4,5-
dihydrobenzo[f][1,2]thiazepin-2(3H)-yl)pentanamide (9b)
Carboxylic acid 6 and benzylamine were reacted according to
General Procedure A to afford the amide product 9b as a
colorless oil (45.1 mg, 0.109 mmol, 85% yield). Rf = 0.4 (20%
1
EtOAc in hexanes). H NMR (400 MHz, CDCl3) δ 7.82 (dd,
J = 8.0, 1.4 Hz, 1H), 7.42 (td, J = 7.5, 1.4 Hz, 1H), 7.30 (ddd,
J = 7.4, 6.5, 1.5 Hz, 2H), 7.26–7.16 (m, 3H), 7.09–7.04 (m,
2H), 6.13 (s, 1H), 5.21 (dd, J = 24.0, 1.2 Hz, 2H), 4.43 (dd,
J = 8.5, 6.4 Hz, 1H), 4.28 (dd, J = 14.8, 5.9 Hz, 1H), 4.16 (dd,
J = 14.8, 5.7 Hz, 1H), 3.71 (ddd, J = 13.4, 9.2, 3.8 Hz, 1H),
3.51 (ddd, J = 14.1, 6.3, 4.2 Hz, 1H), 2.59 (ddd, J = 13.9, 6.3,
3.8 Hz, 1H), 2.45 (ddd, J = 13.7, 9.2, 4.2 Hz, 1H), 1.71 (ddd,
J = 14.4, 8.3, 6.4 Hz, 1H), 1.37 (ddd, J = 14.0, 8.5, 5.6 Hz,
1H), 1.30–1.15 (m, 1H), 0.74 (d, J = 4.4 Hz, 3H), and 0.72 (d,
J = 4.6 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 169.9, 146.9,
140.0, 139.7, 137.7, 132.8, 130.6, 128.8, 127.7, 127.6, 127.6,
126.0, 119.8, 59.2, 45.5, 43.8, 37.8, 35.7, 24.7, 22.8, and 22.0.
FTIR (thin film) 2955, 2359, 2341, 1661, and 1332 cm–1.
½αŠ22 − 77.7 (c 1.0, CHCl3). HRMS (ESI, m/z): [M + H]+
D
calcd for C23H29N2O3S+ 413.1893, found 413.1868.

Medicinal Chemistry Research
(S)-N-(4-Chlorobenzyl)-4-methyl-2-(5-methylene-1,1-
dioxido-4,5-dihydrobenzo[f][1,2]thiazepin-2(3H)-yl)
pentanamide (9c)
(S)-4-Methyl-N-(4-methylbenzyl)-2-(5-methylene-1,1-
dioxido-4,5-dihydrobenzo[f][1,2]thiazepin-2(3H)-yl)
pentanamide (9e)
Carboxylic acid 6 and 4-chlorobenzylamine were reacted
according to General Procedure A to afford the amide
product 9c as a colorless oil (51.3 mg, 0.115 mmol, 93%
yield). Rf = 0.3 (20% EtOAc in hexanes). 1H NMR
(400 MHz, CDCl3) δ 7.81 (dd, J = 8.2, 1.4 Hz, 1H),
7.47–7.39 (m, 1H), 7.30 (ddd, J = 7.8, 6.3, 1.5 Hz, 2H),
7.22–7.15 (m, 2H), 7.01 (d, J = 8.4 Hz, 2H), 6.24 (s, 1H),
5.25 (d, J = 1.1 Hz, 1H), 5.19 (d, J = 1.2 Hz, 1H), 4.43
(dd, J = 8.5, 6.4 Hz, 1H), 4.27–4.10 (m, 2H), 3.71 (ddd,
J = 13.5, 9.4, 3.7 Hz, 1H), 3.48 (ddd, J = 14.1, 6.2,
4.2 Hz, 1H), 2.58 (ddd, J = 13.9, 6.2, 3.7 Hz, 1H), 2.44
(ddd, J = 13.7, 9.5, 4.2 Hz, 2H), 1.70 (ddd, J = 14.4, 8.3,
6.4 Hz, 1H), 1.36 (ddd, J = 14.0, 8.5, 5.6 Hz, 1H),
1.27–1.14 (m, 1H), 0.72 (d, J = 3.3 Hz, 3H), and 0.71 (d,
J = 3.4 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 170.1,
146.8, 140.0, 139.6, 136.4, 133.4, 132.8, 130.6, 129.0,
128.9, 127.6, 126.0, 119.9, 59.2, 45.5, 43.0, 37.7, 35.7,
24.7, 22.8, and 21.9. FTIR (thin film) 2955, 2359, 2341,
Carboxylic acid 6 and 4-methylbenzylamine were reacted
according to General Procedure A to afford the amide
product 9e as a colorless oil (45.8 mg, 0.107 mmol, 87%
yield). Rf = 0.3 (20% EtOAc in hexanes). 1H NMR
(400 MHz, CDCl3) δ 7.82 (dd, J = 8.2, 1.4 Hz, 1H), 7.42
(td, J = 7.4, 1.4 Hz, 1H), 7.29 (ddd, J = 7.8, 6.4, 1.5 Hz,
2H), 7.03 (d, J = 7.7 Hz, 2H), 6.95 (d, J = 8.0 Hz, 2H),
6.07 (s, 1H), 5.24 (d, J = 1.1 Hz, 1H), 5.18 (d, J = 1.2 Hz,
1H), 4.41 (dd, J = 8.4, 6.4 Hz, 1H), 4.23 (dd, J = 14.6,
5.9 Hz, 1H), 4.10 (dd, J = 14.6, 5.5 Hz, 1H), 3.71 (ddd,
J = 13.4, 9.2, 3.8 Hz, 1H), 3.51 (ddd, J = 14.1, 6.4,
4.2 Hz, 1H), 2.64–2.52 (m, 1H), 2.51–2.39 (m, 1H), 2.26
(s, 3H), 1.70 (ddd, J = 14.4, 8.3, 6.4 Hz, 1H), 1.37 (ddd,
J = 14.0, 8.5, 5.6 Hz, 1H), 1.23 (dq, J = 13.1, 6.5 Hz,
1H), 0.73 (d, J = 4.4 Hz, 3H), and 0.71 (d, J = 4.5 Hz,
3H). 13C NMR (101 MHz, CDCl3) δ 169.8, 147.0, 140.1,
139.7, 137.3, 134.7, 132.8, 130.5, 129.5, 127.7, 127.6,
126.0, 119.8, 59.2, 45.5, 43.5, 37.8, 35.7, 24.7, 22.8,
22.0, and 21.2. FTIR (thin film) 2955, 2359, 2341, 1661,
1663, and 1331 cm–1. ½αŠ22:2 − 67.1 (c 1.0, CHCl3).
D
HRMS (ESI, m/z): m/z: [M + H]+ calcd for
and 1332 cm–1. ½αŠ22:4− 71.2 (c 1.0, CHCl3). HRMS (ESI,
D
C23H28ClN2O3S+ 447.1504, found 447.1489.
m/z): [M + H]+ calcd for C24H31N2O3S+ 427.2050, found
427.2042.
(S)-N-(4-Chloro-3-(trifluoromethyl)benzyl)-4-methyl-2-(5-
methylene-1,1-dioxido-4,5-dihydrobenzo[f][1,2]thiazepin-2
(3H)-yl)pentanamide (9d)
(S)-N-(4-Methoxybenzyl)-4-methyl-2-(5-methylene-1,1-
dioxido-4,5-dihydrobenzo[f][1,2]thiazepin-2(3H)-yl)
pentanamide (9f)
Carboxylic acid 6 and 4-chloro-3-(trifluoromethyl)benzy-
lamine were reacted according to General Procedure A to
afford the amide product 9d as a colorless oil (61.7 mg,
0.120 mmol, 97% yield). Rf = 0.3 (20% EtOAc in hexanes).
1H NMR (400 MHz, CDCl3) δ 7.80 (dd, J = 8.1, 1.3 Hz,
1H), 7.46–7.40 (m, 2H), 7.35 (d, J = 8.2 Hz, 1H), 7.32–7.26
(m, 2H), 7.25–7.20 (m, 1H), 6.41 (s, 1H), 5.26 (d, J =
1.1 Hz, 1H), 5.20 (d, J = 1.2 Hz, 1H), 4.44 (dd, J = 8.3,
6.6 Hz, 1H), 4.26 (dd, J = 6.2, 1.7 Hz, 2H), 3.72 (ddd, J =
13.6, 9.5, 3.6 Hz, 1H), 3.48 (ddd, J = 14.1, 6.1, 4.2 Hz, 1H),
2.59 (ddd, J = 13.9, 6.1, 3.8 Hz, 1H), 2.44 (ddd, J = 14.1,
9.6, 3.9 Hz, 1H), 1.70 (ddd, J = 14.0, 8.1, 6.6 Hz, 1H), 1.34
(ddd, J = 14.0, 8.3, 5.7 Hz, 1H), 1.25–1.12 (m, 1H), 0.72 (d,
J = 2.2 Hz, 3H), and 0.70 (d, J = 2.4 Hz, 3H). 13C NMR
(176 MHz, CDCl3) δ 170.3, 146.8, 140.0, 139.4, 137.3,
132.9, 132.0, 131.8, 131.3, 130.6, 128.5 (q, J = 31.44 Hz),
127.6, 126.7 (q, J = 5.19 Hz), 125.9, 122.8 (q, J = 273.30),
119.9, 59.1, 45.5, 42.6, 37.7, 35.7, 24.7, 22.6, and 22.0.
FTIR (thin film) 2957, 2359, 2341, 1668, 1318, 1168, and
Carboxylic acid 6 and 4-methoxylbenzylamine were reacted
according to General Procedure A to afford the amide
product 9f as a colorless oil (46.6 mg, 0.105 mmol, 85%
yield). Rf = 0.2 (20% EtOAc in hexanes). 1H NMR
(400 MHz, CDCl3) δ 7.82 (dd, J = 8.2, 1.4 Hz, 1H), 7.42
(td, J = 7.4, 1.4 Hz, 1H), 7.30 (ddd, J = 6.9, 6.3, 1.4 Hz,
2H), 7.02–6.97 (m, 2H), 6.78–6.72 (m, 2H), 6.06 (s, 1H),
5.24 (d, J = 1.1 Hz, 1H), 5.18 (d, J = 1.2 Hz, 1H), 4.41 (dd,
J = 8.5, 6.4 Hz, 1H), 4.20 (dd, J = 14.5, 5.8 Hz, 1H), 4.09
(dd, J = 14.6, 5.6 Hz, 1H), 3.73 (s, 4H), 3.51 (ddd, J = 14.1,
6.3, 4.1 Hz, 1H), 2.58 (ddd, J = 13.9, 6.4, 3.8 Hz, 1H), 2.45
(ddd, J = 13.6, 9.1, 4.0 Hz, 1H), 1.70 (ddd, J = 14.4, 8.3,
6.4 Hz, 1H), 1.37 (ddd, J = 14.0, 8.4, 5.5 Hz, 1H), 1.23 (td,
J = 12.9, 12.4, 6.1 Hz, 1H), 0.73 (d, J = 4.5 Hz, 3H), and
0.71 (d, J = 4.5 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ
169.8, 159.1, 146.9, 140.0, 139.7, 132.8, 130.5, 129.8,
129.0, 127.6, 126.0, 119.8, 114.2, 59.1, 55.4, 45.4, 43.2,
37.8, 35.7, 24.7, 22.8, and 21.9. FTIR (thin film) 2955,
1130 cm–1. ½αŠ22:4 − 51.3 (c 1.0, CHCl3). HRMS (ESI, m/z):
2359, 2341, 1662, and 1512 cm–1. ½αŠ22:1− 66.3 (c 1.0,
D
D
m/z: [M + H]+ calcd for C24H27ClF3N2O3S+ 515.1378,
CHCl3). HRMS (ESI, m/z): m/z: [M + H]+ Calcd for
found 515.1406.
C24H31N2O3S+ 433.1999, found 433.1992.

Medicinal Chemistry Research
(S)-N-(Cyclohexylmethyl)-4-methyl-2-(5-methylene-1,1-
dioxido-4,5-dihydrobenzo[f][1,2]thiazepin-2(3H)-yl)
pentanamide (9g)
product 10a as a colorless oil (54.2 mg, 0.113 mmol, 91%
yield). Rf = 0.4 (25% EtOAc in hexanes). 1H NMR
(400 MHz, CDCl3) δ 7.80 (dd, J = 8.1, 1.3 Hz, 1H),
7.48–7.38 (m, 1H), 7.32–7.26 (m, 4H), 6.93 (dd, J = 8.2,
2.1 Hz, 1H), 6.32 (d, J = 6.4 Hz, 1H), 5.26 (q, J = 1.0 Hz,
1H), 5.20 (d, J = 1.2 Hz, 1H), 4.43 (dd, J = 8.4, 6.5 Hz,
1H), 4.18 (dd, J = 6.0, 1.5 Hz, 2H), 3.78–3.66 (m, 1H), 3.48
(ddd, J = 14.1, 6.2, 4.2 Hz, 1H), 2.63–2.54 (m, 1H),
2.51–2.38 (m, 1H), 1.69 (ddd, J = 14.1, 8.2, 6.5 Hz, 1H),
1.36 (ddd, J = 14.0, 8.4, 5.6 Hz, 1H), 1.28–1.13 (m, 1H),
0.72 (d, J = 2.4 Hz, 3H), and 0.71 (d, J = 2.5 Hz, 3H). 13C
NMR (101 MHz, CDCl3) δ 170.2, 146.8, 140.0, 139.5,
138.2, 132.9, 132.7, 131.6, 130.7, 130.6, 129.7, 127.6,
127.0, 125.9, 119.9, 59.1, 45.5, 42.6, 37.6, 35.7, 24.7, 22.7,
and 21.9. FTIR (thin film) 2956, 2359, 2341, 1668, 1333,
Carboxylic acid 6 and 4-cyclohexylmethylamine were
reacted according to General Procedure A to afford the
amide product 9g as a colorless oil (40 mg, 0.096 mmol,
1
77% yield). Rf = 0.5 (20% EtOAc in hexanes). H NMR
(400 MHz, CDCl3) δ 7.85 (dd, J = 8.2, 1.4 Hz, 1H),
7.48–7.40 (m, 1H), 7.36–7.28 (m, 2H), 5.85 (s, 1H), 5.27 (d,
J = 1.1 Hz, 1H), 5.22 (d, J = 1.3 Hz, 1H), 4.37 (dd, J = 8.5,
6.3 Hz, 1H), 3.73 (ddd, J = 14.1, 9.2, 3.7 Hz, 1H), 3.51
(ddd, J = 14.1, 6.4, 4.1 Hz, 1H), 2.99–2.79 (m, 2H),
2.68–2.54 (m, 1H), 2.52–2.39 (m, 1H), 1.76–1.42 (m, 7H),
1.41–0.96 (m, 5H), and 0.84–0.66 (m, 8H). 13C NMR
(101 MHz, CDCl3) δ 170.0, 147.9, 140.1, 139.7, 132.8,
130.6, 127.6, 126.0, 119.8, 59.2, 45.9, 45.5, 37.8, 35.8,
30.8, 26.4, 25.9, 25.9, 24.7, 22.8, and 22.0. FTIR (thin film)
and 1169 cm–1. ½αŠ21:9 + 58.8 (c 1.0, CHCl3). HRMS (ESI,
D
m/z): m/z: [M + H]+ calcd for C23H27Cl2N2O3S+ 481.1114,
found 481.1114.
2922, 2539, 2341, 1661, and 1332 cm–1. ½αŠ22:4 − 68.5 (c
D
1.0, CHCl3). HRMS (ESI, m/z): [M + H]+ calcd for
(R)-N-Benzyl-4-methyl-2-(5-methylene-1,1-dioxido-4,5-
dihydrobenzo[f][1,2]thiazepin-2(3H)-yl)pentanamide (10b)
C23H35N2O3S+ 419.2363, found 419.2361.
(S)-N-(3,4-Dichlorophenethyl)-4-methyl-2-(5-methylene-1,1-
dioxido-4,5-dihydrobenzo[f][1,2]thiazepin-2(3H)-yl)
pentanamide (9h)
Carboxylic acid 7 and benzylamine were reacted accord-
ing to General Procedure A to afford the amide product
10b as a colorless oil (42.1 mg, 0.102 mmol, 83% yield).
1
Rf = 0.4 (25% EtOAc in hexanes). H NMR (400 MHz,
Carboxylic acid 6 and 3,4-dichlorophenethylamine were
reacted according to General Procedure A to afford the
amide product 9h as a colorless oil (55 mg, 0.11 mmol, 90%
yield). Rf = 0.4 (30% EtOAc in hexanes). 1H NMR
(400 MHz, CDCl3) δ 7.88 (ddd, J = 7.7, 1.3, 0.6 Hz, 1H),
7.51 (td, J = 7.5, 1.4 Hz, 1H), 7.41–7.32 (m, 3H), 7.24 (d,
J = 2.1 Hz, 1H), 6.99 (dd, J = 8.2, 2.1 Hz, 1H), 6.06–5.99
(m, 1H), 5.32 (d, J = 1.1 Hz, 1H), 5.27 (d, J = 1.2 Hz, 1H),
4.39 (dd, J = 8.2, 6.6 Hz, 1H), 3.70 (ddd, J = 13.5, 9.6,
3.6 Hz, 1H), 3.47 (ddd, J = 14.1, 6.0, 4.2 Hz, 1H),
3.42–3.30 (m, 2H), 2.65 (ddt, J = 13.3, 6.1, 3.3 Hz, 3H),
2.55–2.43 (m, 1H), 1.73 (ddd, J = 13.9, 8.1, 6.6 Hz, 1H),
1.33 (ddd, J = 14.0, 8.3, 5.7 Hz, 1H), 1.23–1.14 (m, 1H),
and 0.75 (t, J = 6.8 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ
170.1, 146.9, 140.0, 139.6, 138.9, 132.9, 132.5, 130.8,
130.7, 130.6, 130.6, 128.3, 127.6, 126.0, 119.8, 59.2, 45.5,
CDCl3) δ 7.82 (dd, J = 8.1, 1.4 Hz, 1H), 7.46–7.38 (m,
1H), 7.32–7.26 (m, 2H), 7.26–7.16 (m, 3H), 7.09–7.03 (m,
2H), 6.14 (s, 1H), 5.24 (d, J = 1.2 Hz, 1H), 5.18 (d, J =
1.2 Hz, 1H), 4.43 (dd, J = 8.5, 6.4 Hz, 1H), 4.28 (dd, J =
14.8, 5.9 Hz, 1H), 4.16 (dd, J = 14.8, 5.7 Hz, 1H), 3.71
(ddd, J = 13.4, 9.2, 3.8 Hz, 1H), 3.51 (ddd, J = 14.1, 6.4,
4.2 Hz, 1H), 2.58 (ddd, J = 13.9, 6.4, 3.7 Hz, 1H),
2.52–2.39 (m, 1H), 1.71 (ddd, J = 14.4, 8.3, 6.4 Hz, 1H),
1.37 (ddd, J = 14.0, 8.5, 5.6 Hz, 1H), 1.31–1.16 (m, 1H),
0.73 (d, J = 4.4 Hz, 3H), and 0.72 (d, J = 4.6 Hz, 3H). 13
C
NMR (101 MHz, CDCl3) δ 169.9, 146.9, 140.0, 139.7,
137.7, 132.8, 130.6, 128.8, 127.7, 127.6, 127.6, 126.0,
119.8, 59.2, 45.5, 43.8, 37.8, 35.7, 24.7, 22.8, and 22.0.
FTIR (thin film) 2955, 2359, 2341, 1662, 1335, and
1169 cm–1. ½αŠ21:6 + 67.5 (c 1.0, CHCl3). HRMS (ESI, m/
D
z): m/z: [M + H]+ calcd for C23H29N2O3S+ 413.1893,
40.5, 37.7, 35.8, 34.8, 24.7, 22.7, and 22.0. FTIR (thin film)
found 413.1870.
22:5
2955, 2868, 1666, 1469, 1331, and 1167 cm–1. ½αŠ
−
D
66.7 (c 1.0, CHCl3). HRMS (ESI, m/z): m/z: [M + H]+
(R)-N-(4-Chlorobenzyl)-4-methyl-2-(5-methylene-1,1-
dioxido-4,5-dihydrobenzo[f][1,2]thiazepin-2(3H)-yl)
pentanamide (10c)
calcd for C24H29Cl2N2O3S+ 495.1270, found 495.1262.
(R)-N-(3,4-Dichlorobenzyl)-4-methyl-2-(5-methylene-1,1-
dioxido-4,5-dihydrobenzo[f][1,2]thiazepin-2(3H)-yl)
pentanamide (10a)
Carboxylic acid 7 and 4-chlorobenzylamine were reacted
according to General Procedure A to afford the amide
product 10c as a colorless oil (48.4 mg, 0.124 mmol, 88%).
1
Carboxylic acid 7 and 3,4-dichlorobenzylamine were reac-
Rf = 0.4 (25% EtOAc in hexanes). H NMR (400 MHz,
ted according to General Procedure A to afford the amide
CDCl3) δ 7.81 (dd, J = 8.1, 1.4 Hz, 1H), 7.43 (td, J = 7.5,

Medicinal Chemistry Research
1.3 Hz, 1H), 7.30 (ddd, J = 7.9, 6.2, 1.5 Hz, 2H), 7.19
(s, 1H), 7.17 (d, J = 2.0 Hz, 1H), 7.01 (d, J = 8.4 Hz, 2H),
6.25 (d, J = 6.9 Hz, 1H), 5.25 (d, J = 1.2 Hz, 1H), 5.19 (d,
J = 1.2 Hz, 1H), 4.43 (dd, J = 8.5, 6.4 Hz, 1H), 4.26–4.13
(m, 2H), 3.71 (ddd, J = 13.5, 9.4, 3.7 Hz, 1H), 3.48 (ddd,
J = 14.0, 6.2, 4.2 Hz, 1H), 2.58 (ddd, J = 13.9, 6.2, 3.7 Hz,
1H), 2.51–2.38 (m, 1H), 1.70 (ddd, J = 14.4, 8.3, 6.4 Hz,
1H), 1.36 (ddd, J = 14.1, 8.5, 5.5 Hz, 1H), 1.21 (dt, J =
14.0, 6.1 Hz, 2H), 0.72 (d, J = 3.3 Hz, 3H), and 0.71 (d,
J = 3.4 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 170.1,
146.8, 140.0, 139.6, 136.4, 133.4, 132.8, 130.6, 129.0,
128.9, 127.6, 126.0, 119.9, 59.2, 45.5, 43.0, 37.7, 35.7,
24.7, 22.8, and 21.9. FTIR (thin film) 2956, 1663, 1333,
(ddt, J = 7.8, 7.2, 0.9 Hz, 1H), 7.32–7.26 (m, 2H),
7.05–7.00 (m, 2H), 6.95 (d, J = 7.9 Hz, 2H), 6.08 (s, 1H),
5.24 (d, J = 1.2 Hz, 1H), 5.18 (d, J = 1.3 Hz, 1H), 4.41 (dd,
J = 8.5, 6.4 Hz, 1H), 4.23 (dd, J = 14.7, 5.9 Hz, 1H), 4.10
(dd, J = 14.6, 5.5 Hz, 1H), 3.71 (ddd, J = 13.4, 9.2, 3.8 Hz,
1H), 3.51 (ddd, J = 14.1, 6.4, 4.2 Hz, 1H), 2.59 (ddd, J =
13.9, 6.4, 3.8 Hz, 1H), 2.45 (ddd, J = 13.8, 9.3, 4.2 Hz, 1H),
2.26 (s, 3H), 1.70 (ddd, J = 14.3, 8.3, 6.4 Hz, 1H), 1.37
(ddd, J = 14.0, 8.5, 5.6 Hz, 1H), 1.30–1.15 (m, 1H), 0.73 (d,
J = 4.3 Hz, 3H), and 0.71 (d, J = 4.5 Hz, 3H). 13C NMR
(101 MHz, CDCl3) δ 169.8, 147.0, 140.0, 139.7, 137.3,
134.7, 132.8, 130.5, 129.5, 127.7, 127.6, 126.0, 119.8,
59.2, 45.5, 43.5, 37.8, 35.7, 24.7, 22.8, 22.0, and 21.2.
FTIR (thin film) 2923, 2359, 23412, 1668, 1334, and
and 1168 cm–1. ½αŠ20:9 + 69.2 (c 1.0, CHCl3). HRMS (ESI,
D
m/z): m/z: [M + H]+ calcd for C23H28ClN2O3S+ 447.1504,
1168 cm–1. ½αŠ21:1 + 68.7 (c 1.0, CHCl3). HRMS (ESI, m/z):
D
found 447.1476.
m/z: [M + H]+ calcd for C24H31N2O3S+ 427.2050, found
427.2048.
(R)-N-(4-Chloro-3-(trifluoromethyl)benzyl)-4-methyl-2-(5-
methylene-1,1-dioxido-4,5-dihydrobenzo[f][1,2]thiazepin-2
(3H)-yl)pentanamide (10d)
(R)-N-(4-Methoxybenzyl)-4-methyl-2-(5-methylene-1,1-
dioxido-4,5-dihydrobenzo[f][1,2]thiazepin-2(3H)-yl)
pentanamide (10f)
Carboxylic acid 7 and 4-chloro-3-(trifluoromethyl)ben-
zylamine were reacted according to General Procedure A
to afford the amide product 10d as a colorless oil
(54.9 mg, 0.124 mmol, 86%). Rf = 0.4 (25% EtOAc in
hexanes). 1H NMR (400 MHz, CDCl3) δ 7.80 (dd, J = 8.1,
1.3 Hz, 1H), 7.46–7.40 (m, 2H), 7.35 (d, J = 8.2 Hz, 1H),
7.32–7.26 (m, 2H), 7.22 (dd, J = 8.2, 2.2 Hz, 1H), 6.41 (s,
1H), 5.26 (d, J = 1.1 Hz, 1H), 5.20 (d, J = 1.2 Hz, 1H),
4.44 (dd, J = 8.3, 6.6 Hz, 1H), 4.32–4.17 (m, 2H), 3.72
(ddd, J = 13.5, 9.5, 3.6 Hz, 1H), 3.48 (ddd, J = 14.1, 6.1,
4.2 Hz, 1H), 2.59 (ddd, J = 13.9, 6.1, 3.6 Hz, 1H),
2.52–2.39 (m, 1H), 1.70 (ddd, J = 14.4, 8.1, 6.6 Hz, 1H),
1.34 (ddd, J = 14.0, 8.3, 5.7 Hz, 1H), 1.27–1.11 (m, 1H),
Carboxylic acid 7 and 4-methoxylbenzylamine were reacted
according to General Procedure A to afford the amide
product 10f as a colorless oil (52.3 mg, 0.124 mmol, 96%).
Rf = 0.3 (25% EtOAc in hexanes). ½αŠ21:1 + 64.0 (c 1.0,
D
1
CHCl3). H NMR (400 MHz, CDCl3) δ 7.81 (dd, J = 8.1,
1.4 Hz, 1H), 7.45–7.39 (m, 1H), 7.33–7.26 (m, 2H),
7.01–6.97 (m, 2H), 6.77–6.73 (m, 2H), 6.07 (s, 1H), 5.24 (d,
J = 1.1 Hz, 1H), 5.18 (d, J = 1.3 Hz, 1H), 4.40 (dd, J = 8.5,
6.4 Hz, 1H), 4.20 (dd, J = 14.6, 5.8 Hz, 1H), 4.09 (dd, J =
14.6, 5.6 Hz, 1H), 3.74-3.66 (complex, 4H), 3.51 (ddd, J =
14.1, 6.4, 4.2 Hz, 1H), 2.58 (ddd, J = 13.5, 6.3, 3.7 Hz, 1H),
2.50–2.39 (m, 1H), 1.69 (ddd, J = 14.3, 8.3, 6.4 Hz, 1H),
1.37 (ddd, J = 14.0, 8.5, 5.6 Hz, 1H), 1.22 (td, J = 12.5,
11.5, 5.8 Hz, 1H), 0.73 (d, J = 4.2 Hz, 3H), and 0.71 (d,
J = 4.4 Hz, 4H). 13C NMR (101 MHz, CDCl3) δ 169.8,
159.1, 146.9, 140.0, 139.7, 132.8, 130.5, 129.8, 129.0,
127.6, 126.0, 119.8, 114.2, 59.2, 55.4, 45.4, 43.2, 37.8,
35.7, 24.7, 22.8, and 22.0. FTIR (thin film) 2966, 1662,
0.72 (d, J = 2.2 Hz, 3H), and 0.70 (d, J = 2.3 Hz, 3H). 13
C
NMR (176 MHz, CDCl3) δ 170.4, 146.8, 140.0, 139.4,
137.3, 132.9, 132.0, 131.8, 131.3, 130.6, 128.6 (q, J =
32.08 Hz), 127.6, 126.8 (q, J = 5.25 Hz), 125.9, 122.5 (q,
J = 273.35 Hz), 119.9, 59.1, 45.5, 42.6, 37.6, 35.8, 24.7,
22.6, and 22.0. FTIR (thin film) 2957, 1667, 1318, 1169,
and 1130 cm–1. ½αŠ20:9 + 52.4 (c 1.0, CHCl3). HRMS (ESI,
1512, and 1333 cm–1. ½αŠ21:1 + 64.0 (c 1.0, CHCl3). HRMS
D
D
m/z): m/z: [M + H]+ calcd for C24H27ClF3N2O3S+
(ESI, m/z): m/z: [M + H]+ calcd for C24H31N2O4S+
515.1378, found 515.1413.
443.1999, found 443.1973.
(R)-4-Methyl-N-(4-methylbenzyl)-2-(5-methylene-1,1-
dioxido-4,5-dihydrobenzo[f][1,2]thiazepin-2(3H)-yl)
pentanamide (10e)
(R)-N-(Cyclohexylmethyl)-4-methyl-2-(5-methylene-1,1-
dioxido-4,5-dihydrobenzo[f][1,2]thiazepin-2(3H)-yl)
pentanamide (10g)
Carboxylic acid 7 and 4-methylbenzylamine were reacted
according to General Procedure A to afford the amide
product 10e as a colorless oil (50.8 mg, 0.119 mmol, 96%
yield). Rf = 0.4 (25% EtOAc in hexanes). 1H NMR
(400 MHz, CDCl3) δ 7.81 (dd, J = 8.1, 1.6 Hz, 1H), 7.42
Carboxylic acid 7 and 4-cyclohexylmethylamine were
reacted according to General Procedure A to afford the
amide product 10g as a colorless oil (43 mg, 0.103 mmol,
83% yield). Rf = 0.4 (25% EtOAc in hexanes). H NMR
(400 MHz, CDCl3) δ 7.86–7.82 (m, 1H), 7.47–7.41 (m, 1H),
1

Medicinal Chemistry Research
7.35–7.29 (complex, 2H), 5.85 (m, 1H), 5.27 (d, J = 1.1 Hz,
1H), 5.22 (d, J = 1.3 Hz, 1H), 4.37 (dd, J = 8.5, 6.4 Hz,
1H), 3.78–3.68 (m, 1H), 3.55–3.45 (m, 1H), 2.99–2.78 (m,
2H), 2.67–2.54 (m, 1H), 2.53–2.38 (m, 1H), 1.76–1.45
(complex, 7H), 1.40–0.98 (complex, 5H), and 0.84–0.66
(complex, 8H). 13C NMR (101 MHz, CDCl3) δ 170.0,
147.0, 140.1, 139.7, 132.8, 130.6, 127.6, 126.0, 119.8,
59.2, 45.9, 45.5, 37.8, 35.8, 30.8, 26.4, 25.9, 25.9, 24.7,
22.8, and 22.0. FTIR (thin film) 2922, 2851, 2359, 2341,
0.86 (d, J = 5.1 Hz, 3H), and 0.84 (d, J = 5.0 Hz, 3H). 13C
NMR (101 MHz, CDCl3) δ 199.4, 169.3, 139.2, 137.8,
136.1, 132.9, 132.6, 131.8, 131.6, 130.6, 129.6, 129.6,
126.9, 125.5, 58.9, 42.4, 42.4, 39.7, 37.2, 24.6, 22.6, and
21.7. FTIR (thin film) 3365, 2957, 1663, 1638, and
1338 cm–1. ½αŠ21:8 − 41.4 (c 1.0, CHCl3). HRMS (ESI, m/z):
D
m/z: [M + H]+ calcd for C22H25Cl2N2O4S+ 483.0907;
found 483.0907.
1661, and 1334 cm–1. ½αŠ22:6 + 67.5 (c 1.0, CHCl3). HRMS
(S)-N-Benzyl-2-(1,1-dioxido-5-oxo-4,5-dihydrobenzo[f][1,2]
thiazepin-2(3H)-yl)-4-methylpentanamide (11b)
D
(ESI, m/z): m/z: [M + H]+ calcd for C23H35N2O3S+
419.2363, found 419.2344.
Carboxylic acid 8 and benzylamine were reacted accord-
ing to General Procedure A to afford the amide product
11b as a colorless oil (35.0 mg, 0.123 mmol, 68.7%). Rf =
0.3 (25% EtOAc in hexanes). 1H NMR (400 MHz,
CDCl3) δ 7.82 (dd, J = 7.5, 1.6 Hz, 1H), 7.70 (dd, J = 7.5,
1.5 Hz, 1H), 7.54 (dtd, J = 19.5, 7.5, 1.5 Hz, 2H),
7.26–7.17 (complex, 4H), 7.08–7.05 (m, 2H), 6.07–5.99
(m, 2H), 4.45 (dd, J = 8.6, 6.6 Hz, 1H), 4.26 (dd, J =
14.7, 5.9 Hz, 1H), 4.14 (dd, J = 14.7, 5.6 Hz, 1H),
3.60–4.52 (m, 1H), 3.51–3.42 (m, 1H), 3.29–3.20 (m,
1H), 3.18-3.08 (m, 1H), 1.69–1.59 (m, 1H), 1.52–1.42 (m,
1H) 1.41–1.27 (m, 1H), 0.80 (d, J = 2.5 Hz, 3H), and 0.78
(d, J = 2.4 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 199.8,
169.2, 139.3, 137.5, 136.4, 133.0, 131.9, 129.7, 128.9,
127.8, 125.8, 59.1, 43.7, 42.6, 39.8, 37.6, 24.8, 22.8, and
21.8. FTIR (thin film): 3320, 2956, 2926, 1359, and
(R)-N-(3,4-Dichlorophenethyl)-4-methyl-2-(5-methylene-1,1-
dioxido-4,5-dihydrobenzo[f][1,2]thiazepin-2(3H)-yl)
pentanamide (10h)
Carboxylic acid 7 and 3,4-dichlorophenethylamine were
reacted according to General Procedure A to afford the
amide product 10h as a colorless oil (58.2 mg, 0.117 mmol,
95%). Rf = 0.4 (25% EtOAc in hexanes). 1H NMR
(400 MHz, CDCl3) δ 7.84–7.80 (m, 1H), 7.44 (td, J = 7.6,
1.4 Hz, 1H), 7.35–7.26 (m, 3H), 7.17 (d, J = 2.1 Hz, 1H),
6.93 (dd, J = 8.2, 2.1 Hz, 1H), 5.97 (s, 1H), 5.25 (t, J =
1.0 Hz, 1H), 5.21 (d, J = 1.2 Hz, 1H), 4.33 (dd, J = 8.3,
6.6 Hz, 1H), 3.64 (ddd, J = 13.5, 9.5, 3.6 Hz, 1H),
3.45–3.21 (m, 3H), 2.59 (ddt, J = 13.3, 6.0, 3.3 Hz, 3H),
2.41 (dddd, J = 13.8, 9.6, 4.2, 1.0 Hz, 1H), 1.66 (ddd, J =
14.0, 8.1, 6.6 Hz, 1H), 1.27 (ddd, J = 14.0, 8.3, 5.7 Hz, 1H),
1.20–1.07 (m, 1H), and 0.68 (t, J = 6.8 Hz, 6H). 13C NMR
(101 MHz, CDCl3) δ 170.0, 146.7, 139.9, 139.5, 138.7,
132.7, 132.4, 130.6, 130.5, 130.5, 130.4, 128.2, 127.4,
125.8, 119.6, 59.1, 45.3, 40.3, 37.5, 35.6, 34.6, 24.5, 22.5,
and 21.8. FTIR (thin film) 2956, 1667, 1332, and
1168 cm–1. ½αŠ20:8 + 63.9 (c 1.0, CHCl3). HRMS (ESI, m/z):
m/z: [M + H]+Dcalcd for C24H29Cl2N2O3S+ 495.1270, found
495.1250.
1682 cm−1. ½αŠ21:4 − 43.6 (c 1.0, CHCl3). HRMS (ESI, m/
D
z): m/z: [M + H]+ calcd for C22H27N2O4S+ 415.1686,
found 415.1686.
(S)-N-(4-Chlorobenzyl)-2-(1,1-dioxido-5-oxo-4,5-
dihydrobenzo[f][1,2]thiazepin-2(3H)-yl)-4-
methylpentanamide (11c)
Carboxylic acid 8 and 4-chlorobenzylamine were reacted
according to General Procedure A to afford the amide product
11c as a colorless oil (33.0 mg, 0.068 mmol, 56% yield). Rf =
0.3 (25% EtOAc in hexanes). 1H NMR (400 MHz, CDCl3) δ
7.88 (dd, J = 7.8, 1.3 Hz, 1H), 7.77 (dd, J = 7.6, 1.4 Hz, 1H),
7.61 (dtd, J = 22.5, 7.5, 1.4 Hz, 2H), 7.28–7.21 (m, 2H),
7.11–7.03 (m, 2H), 6.18 (t, J = 5.8 Hz, 1H), 4.51 (dd, J = 8.5,
6.6 Hz, 1H), 4.32–4.16 (m, 2H), 3.67–3.56 (m, 1H),
3.57–3.46 (m, 1H), 3.37–3.28 (m, 1H), 3.27–3.15 (m, 1H),
1.77–1.65 (m, 1H), 1.58–1.46 (m, 1H), 1.46–1.31 (m, 1H),
0.85 (d, J = 4.0 Hz, 3H), and 0.84 (d, J = 3.9 Hz, 3H). 13C
NMR (101 MHz, CDCl3) δ 199.6, 169.4, 139.5, 136.3, 136.2,
133.6, 133.0, 131.9, 129.8, 129.1, 129.0, 125.7, 59.1, 43.0,
42.6, 39.8, 37.5, 24.8, 22.8, and 21.8. FTIR (thin film) 3366,
(S)-N-(3,4-Dichlorobenzyl)-2-(1,1-dioxido-5-oxo-4,5-
dihydrobenzo[f][1,2]thiazepin-2(3H)-yl)-4-
methylpentanamide (11a)
Carboxylic acid 8 and 3,4-dichlorobenzylamine were reac-
ted according to General Procedure A to afford the amide
product 11a as a colorless oil (38.5 mg, 0.080 mmol, 65%
yield). Rf = 0.3 (25% EtOAc in hexanes). 1H NMR
(400 MHz, CDCl3) δ 7.87 (dd, J = 7.7, 1.2 Hz, 1H), 7.76
(dd, J = 7.6, 1.4 Hz, 1H), 7.61 (dtd, J = 22.0, 7.5, 1.4 Hz,
2H), 7.34 (d, J = 8.2 Hz, 1H), 7.24 (d, J = 2.1 Hz, 1H), 6.99
(dd, J = 8.2, 2.1 Hz, 1H), 6.27 (t, J = 6.0 Hz, 1H), 4.52 (dd,
J = 8.5, 6.6 Hz, 1H), 4.29–4.16 (m, 2H), 3.67–3.55 (m, 1H),
3.57–3.46 (m, 1H), 3.39–3.27 (m, 1H), 3.28–3.16 (m, 1H),
1.77–1.65 (m, 1H), 1.58–1.46 (m, 1H), 1.44–1.32 (m, 1H),
2957, 1663, 1533, and 1336 cm–1. ½αŠ21:8 − 45.9 (c 1.0,
D
CHCl3). HRMS (ESI, m/z): m/z: [M + H]+ calcd for
C24H25ClN2O4SH 449.1224; found 449.1296.

Medicinal Chemistry Research
(S)-N-(4-Chloro-3-(trifluoromethyl)benzyl)-2-(1,1-dioxido-5-
oxo-4,5-dihydrobenzo[f][1,2]thiazepin-2(3H)-yl)-4-
methylpentanamide (11d)
yield). Rf = 0.2 (25% EtOAc in hexanes). 1H NMR
(400 MHz, CDCl3) δ 7.88 (dd, J = 7.6, 1.4 Hz, 1H), 7.77
(dd, J = 7.6, 1.5 Hz, 1H), 7.61 (dtd, J = 19.1, 7.5, 1.5 Hz,
2H), 7.10–7.02 (m, 2H), 6.85–6.78 (m, 2H), 6.03 (t, J =
5.7 Hz, 1H), 4.49 (dd, J = 8.6, 6.6 Hz, 1H), 4.25 (dd, J =
14.5, 5.8 Hz, 1H), 4.13 (dd, J = 14.5, 5.5 Hz, 1H), 3.79 (s,
3H), 3.68–3.57 (m, 1H), 3.59–3.47 (m, 1H), 3.37–3.25 (m,
1H), 3.26–3.14 (m, 1H), 1.77–1.64 (m, 1H), 1.59–1.47 (m,
1H), 1.46–1.34 (m, 1H), 0.86 (d, J = 2.4 Hz, 3H), and 0.84
(d, J = 2.3 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 199.8,
169.1, 159.2, 139.3, 136.4, 132.9, 131.9, 129.7, 129.6,
129.2, 125.8, 114.2, 59.1, 55.4, 43.2, 42.6, 39.8, 37.6, 24.8,
22.8, and 21.8. FTIR (thin film) 3318, 2956, 1660, and
1512 cm–1. ½αŠ21:8 − 40.1 (c 1.0, CHCl3). HRMS (ESI, m/z):
m/z [M + H]+D calcd for C23H29N2O5S+ 445.1792; found
445.1792.
Carboxylic acid 8 and 4-chloro-3-(trifluoromethyl)benzy-
lamine were reacted according to General Procedure A to
afford the amide product 11d as a colorless oil (44.0 mg,
0.085 mmol, 69% yield). Rf = 0.3 (25% EtOAc in hexanes).
1H NMR (400 MHz, CDCl3) δ 7.92–7.87 (m, 1H),
7.80–7.75 (m, 1H), 7.62 (dtd, J = 19.3, 7.5, 1.4 Hz, 2H),
7.49 (d, J = 2.1 Hz, 1H), 7.42 (d, J = 8.2 Hz, 1H), 7.29 (dd,
J = 8.4, 2.2 Hz, 1H), 6.32 (t, J = 6.0 Hz, 1H), 4.53 (dd, J =
8.4, 6.8 Hz, 1H), 4.40–4.23 (m, 2H), 3.66–3.55 (m, 1H),
3.57–3.45 (m, 1H), 3.41–3.29 (m, 1H), 3.28–3.16 (m, 1H),
1.79–1.67 (m, 1H), 1.56–1.44 (m, 1H), 1.43–1.31 (m, 1H),
and 0.88–0.80 (m, 6H). 13C NMR (176 MHz, CDCl3) δ
199.6, 169.6, 139.4, 137.1, 136.3, 133.0, 132.1, 132.0,
131.9, 131.5, 129.7, 128.7 (q, J = 31.43 Hz), 126.8 (q, J =
5.19 Hz), 125.5, 122.7 (q, J = 273.33 Hz), 59.0, 42.6, 42.5,
39.9, 37.4, 24.8, 22.7, and 21.8. FTIR (thin film) 3398,
(S)-N-(Cyclohexylmethyl)-2-(1,1-dioxido-5-oxo-4,5-
dihydrobenzo[f][1,2]thiazepin-2(3H)-yl)-4-
methylpentanamide (11g)
2959, 1664, 1532, and 1319 cm–1. ½αŠ21:9 − 46.2 (c 1.0,
D
CHCl3). HRMS (ESI, m/z): m/z: [M + H]+ calcd for
Carboxylic acid 8 and 4-cyclohexylmethylamine were
reacted according to General Procedure A to afford the
amide product 11g as a colorless oil (28.0 mg, 0.123 mmol,
C23H25ClF3N2O4S+ 517.1170; found 517.1171.
1
(S)-2-(1,1-Dioxido-5-oxo-4,5-dihydrobenzo[f][1,2]thiazepin-
2(3H)-yl)-4-methyl-N-(4-methylbenzyl)pentanamide (11e)
54% yield). Rf = 0.3 (25% EtOAc in hexanes). H NMR
(400 MHz, CDCl3) δ 7.90–7.86 (m, 1H), 7.74–7.70 (m, 1H),
7.62–7.53 (m, 2H), 5.76 (s, 1H), 4.40 (dd, J = 8.5, 6.6 Hz,
1H), 3.58 (ddd, J = 12.9, 7.9, 4.8 Hz, 1H), 3.47 (ddd, J =
13.1, 8.1, 4.8 Hz, 1H), 3.28 (ddd, J = 14.8, 8.1, 4.8 Hz, 1H),
3.14 (ddd, J = 14.8, 7.9, 4.8 Hz, 1H), 2.96–2.78 (m, 2H),
1.69–1.39 (complex, 8H), 1.38–0.97 (complex, 6H), 0.79
(d, J = 1.4 Hz, 3H), and 0.78 (d, J = 1.3 Hz, 3H). 13C NMR
(101 MHz, CDCl3) δ 199.8, 169.3, 139.5, 136.5, 132.9,
131.9, 129.8, 125.7, 59.2, 45.8, 42.7, 39.8, 37.7, 37.6, 30.8,
26.4, 25.8, 24.8, 22.8, and 21.9. FTIR (thin film): 3382,
Carboxylic acid 8 and 4-methylbenzylamine were reacted
according to General Procedure A to afford the amide
product 11e as a colorless oil (37.9 mg, 0.088 mmol, 72%
yield). Rf = 0.3 (25% EtOAc in hexanes). 1H NMR
(400 MHz, CDCl3) δ 7.88 (dd, J = 7.6, 1.4 Hz, 1H), 7.77
(dd, J = 7.4, 1.6 Hz, 1H), 7.61 (dtd, J = 19.7, 7.5, 1.4 Hz,
2H), 7.10 (d, J = 8.3 Hz, 2H), 7.02 (d, J = 8.0 Hz, 2H), 6.04
(s, 1H), 4.49 (dd, J = 8.6, 6.6 Hz, 1H), 4.29 (dd, J = 14.6,
5.8 Hz, 1H), 4.15 (dd, J = 14.6, 5.5 Hz, 1H), 3.69–3.57 (m,
1H), 3.59–3.48 (m, 1H), 3.37–3.26 (m, 1H), 3.26–3.14 (m,
1H), 2.33 (s, 3H), 1.77–1.65 (m, 1H), 1.59–1.47 (m, 1H),
1.47–1.35 (m, 1H), 0.86 (d, J = 2.3 Hz, 3H), and 0.84 (d,
J = 2.2 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 199.7,
169.0, 139.2, 137.3, 136.3, 134.3, 132.8, 131.7, 129.6,
129.4, 127.6, 125.6, 59.0, 43.4, 42.5, 39.7, 37.4, 24.6, 22.7,
21.7, and 21.0. FTIR (thin film) 3314, 2956, 2924, 2869,
2923, 2851, 1660, and 1431. ½αŠ21:7 − 42.2 (c 0.50, CHCl3).
D
HRMS (ESI, m/z): m/z: [M + H]+ calcd for C22H33N2O4S+
421.2156; found 421.2154.
(S)-N-(3,4-Dichlorophenethyl)-2-(1,1-dioxido-5-oxo-4,5-
dihydrobenzo[f][1,2]thiazepin-2(3H)-yl)-4-
methylpentanamide (11h)
1662, and 1339. ½αŠ21:7 − 46.8 (c 1.0, CHCl3). HRMS (ESI,
Carboxylic acid 8 and 3,4-dichlorophenethylamine were
reacted according to General Procedure A to afford the
D
m/z): m/z [M + H]+ calcd for C23H29N2O4S+ 429.1843;
found 429.1843.
amide product 11h as a colorless oil (53.3 mg,
0.107 mmol, 87% yield). Rf = 0.2 (25% EtOAc in hex-
1
(S)-2-(1,1-Dioxido-5-oxo-4,5-dihydrobenzo[f][1,2]thiazepin-
2(3H)-yl)-N-(4-methoxybenzyl)-4-methylpentanamide (11f)
anes). H NMR (400 MHz, CDCl3) δ 7.94–7.89 (m, 1H),
7.81–7.77 (m, 1H), 7.68–7.60 (m, 2H), 7.35 (d, J = 8.2 Hz,
1H), 7.22 (d, J = 2.1 Hz, 1H), 6.98 (dd, J = 8.2, 2.1 Hz,
1H), 5.93–5.80 (m, 1H), 4.41 (dd, J = 8.3, 6.8 Hz, 1H),
3.59–3.26 (complex, 5H), 3.18 (ddd, J = 14.6, 7.8, 4.8 Hz,
1H), 2.76–2.59 (m, 2H), 1.68 (ddd, J = 14.0, 8.1, 6.8 Hz,
Carboxylic acid 8 and 4-methoxylbenzylamine were reacted
according to General Procedure A to afford the amide
product 11f as a colorless oil (40.7 mg, 0.092 mmol, 75%

Medicinal Chemistry Research
1H), 1.42 (ddd, J = 14.1, 8.3, 5.8 Hz, 1H), 1.34–1.22 (m,
1H), and 0.81 (d, J = 6.6 Hz, 6H). 13C NMR (101 MHz,
CDCl3) δ 199.6, 169.4, 139.6, 138.7, 136.3, 133.0, 132.6,
132.0, 130.8, 130.8, 130.7, 129.8, 128.2, 125.8, 59.2,
42.6, 40.4, 39.8, 37.4, 34.7, 24.8, 22.7, and 21.9. FTIR
(thin film) 3381, 2957, 1682, 1532, and 1336 cm–1.
Counterscreen assay 2: AlphaScreen TruHits™ assay
The AlphaScreen TruHits kit (PerkinElmer) was used to
identify compound interference using the vendor protocol.
The Streptavidin donor beads (10 μg/mL) and the biotin
acceptor beads (5 μg/mL) were diluted in the IB containing
10 mM Tris-HCl, pH 8.0, 1 mM CaCl2, 150 mM NaCl,
0.1% BSA, and 20% glycerol. The TruHits bead mix
(30 μL) was incubated with the compounds for 2 h at room
temperature. The assay plates were read using the Enspire
Plate reader (PerkinElmer) using AlphaScreen default label.
½αŠ21:9− 35.7 (c 1.0, CHCl3). HRMS (ESI, m/z): m/z: [M +
D
H]+ calcd for C23H27Cl2N2O4S+ 497.0990; found
497.1063.
Biology
Compounds
Cytotoxicity assay
All synthesized compounds were solvated in 100% DMSO.
For the AlphaScreen and cytotoxicity assays, compounds
(final concentrations, 300, 100, 33.3, 11.1, 3.7, 1.2, 0.4, 0.1,
0.046, 0.015, 0.005, 0.002, and 0 μM) were transferred
acoustically using ECHO 555 (Labcyte Inc.) to 384-well
white Alpha assay plates (PerkinElmer) or to white poly-
styrene plates for cytotoxicity screens (Griener). All assays
were performed in triplicate.
The SH-SY5Y cells (ATCC® CRL-2266) were plated in
384-well microplates at 6000 cells/well in 1:1 mixture of
ATCC-formulated Eagle’s Minimum Essential Medium,
Catalog No. 302003, and F12 Medium in 10% FBS. The
PC12 73Q cell line was grown in Kaighn’s modification of
Ham’s F12 media (ATCC#30-2004) supplemented with
15% horse serum and 2.5% FBS in the presence of G418
and Zeocin. The PC12 73Q cells were seeded at 8000 cells/
well in poly-D-lysine coated plates (Corning). The PC12
73Q cell line can be induced to express full-length human
huntingtin protein with 73 glutamine repeats under the
control of a RheoSwitch (HD Community Biorepository,
CHDI Foundation, and the Coriell Institute for Medical
Research). In this assay, cells were not induced to express
huntingtin protein. Media and vehicle control wells were
included in each assay plate. After 24 h of incubation with
compounds at 37 °C, cytotoxicity was measured on Enspire
plate reader (PerkinElmer) using the luminescence-based
CellTiter-Glo reagent (Promega Inc.). Percent cytotoxicity
was normalized to DMSO controls.
CaM/mHtt Alpha-Screen binding assay
The compound activity was tested in triplicate in the His-
mHtt and GST-CaM AlphaScreen assay that was opti-
mized in the KU-HTS lab. All assays were performed in
384-well formats in a final volume of 30 μL/well and
0.6% DMSO. All proteins and beads were diluted in the
interaction buffer (IB) containing 10 mM Tris-HCl,
pH 8.0, 1 mM CaCl2, 150 mM NaCl, 0.1% BSA, and
20% glycerol. Briefly, 10 nM His-mu-mHtt was pre-
incubated with library compounds (15 μM) for 30 min
followed by the addition of GST–CAM (10 nM). After
60 min at room temperature, a mixture of the acceptor
Histidine (Nickel Chelate, 10 μg/mL and donor Glu-
tathione beads (15 μg/mL)) was added together. After 2 h
at room temperature, Alpha counts were measured using
Enspire Multilabel Plate Reader (PerkinElmer Inc.) using
AlphaScreen default label. Percent inhibition of the
AlphaScreen assay was normalized to DMSO positive
and negative controls.
CaM-dependent kinase assay
To determine if the compounds inhibit the functioning of
CaM, the activity of two CaM-dependent enzymes, cal-
cium/CaM-dependent kinase 2γ (CaMK2γ, Human,
recombinant; C-terminal truncation), and death-associate
protein kinase 1 (DAPK1, Human, recombinant; amino
acids 1–363) were assessed. The assay was performed in
white opaque 384-well plates (Corning, NY) using the
ADP-Glo kinase activity assay kit (Promega, Madison, WI)
and DAPK1 and CAMK2γ kinase enzyme system kit
(Promega, Madison, WI). The assay was carried out
according to the manufacturer’s protocol with slight mod-
ifications. The DAPK1 reaction was initiated with 25 ng
DAPK1, 5 µM ATP, and 0.1 μg/μL myelin basic protein
(native Swine) substrate in the presence of test compounds
9c, 9d, 9h, 10c, and 10h and incubated at room temperature
for 60 min. The CaMK2γ reaction was initiated with 12 ng
Counterscreen assay 1: His–GST protein interaction assay
The purified His-tagged GST protein (15 nM) was incu-
bated with the compounds for 30 min at room temperature
in IB containing 10 mM Tris-HCl, pH 8.0, 1 mM CaCl2,
150 mM NaCl, 0.1% BSA, and 20% glycerol. A mixture of
the acceptor Histidine (Nickel Chelate, 5 μg/mL and donor
Glutathione beads (5 μg/mL)) was added together. The
plates were read using Enspire Plate reader.

Medicinal Chemistry Research
CaMK2γ, 25 µM ATP, and 0.2 μg/μL. Autocamtide-2 sub-
strate in the presence of test compounds 9c, 9d, 9h, 10c, and
10h and incubated at room temperature for 60 min. Each
test compound was tested at a single concentration of
100 µM (dissolved in DMSO). Kinase enzyme activity was
measured by detecting newly synthesized ATP via a lumi-
nescent readout.
Results and discussion
Fig. 1 Structure and high-throughput screening activity of the sultam
hit, KUC102204N, 9a
High-throughput screening
To identify small-molecule disruptors of the mHtt–CaM
PPI, the University of Kansas High-Throughput Screening
Laboratory interrogated their in-house compound collection
for hit molecules that inhibited the binding of mHtt to CaM
using an AlphaScreen platform (Ullman et al. 1994). The
compound collection (ca. 225,000 compounds) comprised
small molecules sourced from numerous vendors and col-
lections, including the University of Kansas Chemical
Methodology and Library Development (KU CMLD)
Center. The KU CMLD center was a synthetic chemistry
initiative whose raison d’être was to pioneer new synthetic
methods for parallel synthesis and demonstrate their utility
toward the construction of novel compound sets. The utility
of such compound sets to identify tractable hits for various
therapeutic targets has been demonstrated by us (Fran-
kowski et al. 2011) and others (Basu et al. 2011; Brown
et al. 2011; Huryn et al. 2011).
Among the most potent inhibitors of the CaM–mHtt
interaction was a compound synthesized in the KU CMLD,
KUC102204N (Fig. 1). KUC102204N is an amino acid-
derived sultam core synthesized via a “Click, Click,
Cyclize” reaction sequence developed in the laboratory of
KU CMLD investigator Paul Hanson (Rayabarapu et al.
2009; Zhou et al. 2009). We used the reported route
(Rayabarapu et al. 2009) to resynthesize the hit compound
for activity and selectivity confirmation, as well as for the
synthesis of new analogs to explore the structure–activity
relationship (SAR) trends of the series.
Scheme 1 General synthesis of sultam analogs, reagents, and condi-
tions: (a) L- or D-methyl leucinate, Et3N, DCM, 2 h, 59–61% yield (b)
PPh3, DIAD, but-3-en-1-ol, DCM, 3 h, 80–95% yield (c) Pd(OAc)2,
PPh3, Et3N, MeCN, µW, 1 h, 74–84% yield (d) LiOH, 3:1:1 THF:
MeOH:H2O, 2 h, 70–85% yield (e) O3, Me2S, DCM, 2 min, 94% yield
(f) HATU, DIPEA, NH2R, MeCN, 18 h, 54–97% yield
cyclization to produce the bicyclic scaffold that was sapo-
nified with LiOH to provide carboxylic acids 6 and 7. The
olefin-containing acid 6 was converted to the ketone 8 via
ozonolysis and HATU-promoted amide coupling afforded
the target analogs.
SAR studies
Using the Topliss decision tree (Topliss 1972), a sys-
tematic approach was taken to investigate the effects of ring
substitution on activity. We constructed a comprehensive
matrix by coupling eight different amines with three car-
boxylic acid scaffolds (the natural and unnatural amino
acid-derived exo-methylenes 6 and 7, and ketone 8). All
analogs were screened for inhibition of CaM–mHtt binding
and counterscreened for assay interference and cytotoxicity
(Table 1).
The hit compound, KUC102204N, was synthesized using
the protocols developed by Hanson and coworkers with
only minor modifications (Scheme 1) (see the Supporting
Information for synthetic details of all intermediates). Bro-
mobenzylsulfonyl chloride was coupled to the methyl ester
of either (R)- or (S)-leucine to afford a sulfonamide, which
was subsequently alkylated under Mitsunobu conditions to
produce olefins 2 and 3. Microwave irradiation in the pre-
sence of Pd(OAc)2 effected an intramolecular Heck
The resynthesized sample of the screening hit,
KUC102204N (9a) retained activity in disrupting the

Medicinal Chemistry Research
Table 1 Structure–activity relationship studies for
analog series
a
sultam
activity and the 4-methyl analogs 9e and 10e were slightly
less active than the unsubstituted benzyl. Replacing the
unsubstituted benzyl group with a methylenecyclohexyl
group led to analogs of comparable potency (cf. 9b and 10b
vs. 9g and 10g). Extending the linker length by replacing
3,4-dichlorobenzyl with 3,4-dichlorophenylethyl led to the
analogs 9h and 10h with approximately twofold greater
potency than the screening hit.
We next synthesized a complementary series where the
methylene on the sultam ring was replaced with a ketone.
Having established negligible effect from configuration of
the amino acid side chain, we only synthesized the L-
leucine derivatives. The aryl-based SAR trends identified
above were again observed, though the keto-analogs were
generally ~0.5-fold as potent as the methylene-containing
analogs, with two exceptions. The 4-methoxylbenzyl keto-
analog 11f retained marginal activity in disrupting the
mHtt–CaM PPI and the methylenecyclohexyl keto-analog
11g was only 0.5-fold as potent as the benzyl keto-analog
11b. The keto-analogs 11a to 11h were all found to possess
greater cytotoxicity than any of the olefin analogs. Lastly,
the compounds were tested in assay interference counter-
screens and were found to be free of any liabilities (see the
Supporting Information, Fig. S-1).
Taken together, the SAR results demonstrate that this
series of small molecules sultams is indeed capable of dis-
rupting the mHtt–CaM interaction; the most potent analog
10h possessed potency of 0.50 µM. Furthermore, several
preliminary SAR trends emerged. Electron-withdrawing
groups on the aryl group were found to be beneficial for
activity, while converting the olefin to a ketone slightly
diminished the activity. Interestingly, the configuration of
the leucine side chain did not significantly affect activity
and we found that the two-carbon linked phenethyl was
more favorable than the one-carbon linked benzyl. The SAR
trends identified here would be useful in guiding the design
of future analogs in this series for disrupting the
mHtt–CaM PPI.
A subset of potent analogs that also possessed minimal
cytotoxicity (9c, 9d, 9 h, 10c and 10 h) was also tested for
off-target activity against other CaM-dependent kinases to
ensure that the innate functions of CaM were not disrupted
upon compound treatment. To determine if the test com-
pounds affect Ca+2/CaM-dependent DAPK1 and CaMK2γ
activity, we performed an in vitro kinase enzyme activity
assay for each kinase in the presence of CaM. Percent
enzyme activity was calculated by normalizing the enzyme
activity obtained for each compound to the enzyme
activity obtained in the absence of any compound. Grati-
fyingly, the compounds did not inhibit DAPK1 at com-
pound concentrations up to 100 μM and only one analog
(9h) had a statistically significant impact on CaM2Kγ
activity (Fig. 2).
Compound
AlphaScreen CaM–mHtt, IC50 Cytotoxicity
(µM)
(SH-SY5Y cells,
24 h) (µM)
9a
1.03
>300
(KUC102204N)
10a
9b
0.98
12.4
12.7
2.8
>300
>300
10b
9c
87
94
86
2
3
9
10c
9d
3
1.38
1.8
>300
>300
102
10d
9e
15
7
10e
9f
15.1
>300
>300
16.6
12.2
0.55
0.5
64 2.5
>300
10f
9g
>300
101
99
5
10g
9h
4
>300
>300
39.8
43.8
41.4
44.2
43.4
44.6
40.1
32.3
10h
11a
11b
11c
11d
11e
11f
11g
11h
2.14
14.59
11.19
2.03
24.5
52.56
31.03
0.97
mHtt–CaM PPI and was found to possess no measurable
toxicity to SHY5Y cells. We also synthesized and tested the
enantiomer of KUC102204N (10a), which was found to
have comparable activity and toxicity.
We next investigated the effect of modifying the amine
side chain. Replacement of the 3,4-dichlorobenzyl group
with a benzyl group afforded analogs 9b and 10b, which
were significantly less potent in disrupting the mHtt–CaM
PPI; again, the enantiomeric sets were approximately
equipotent. The 4-chlorobenzyl analogs 9c and 10c
regained much of the lost potency relative to series b,
though both enantiomers were slightly cytotoxic (ca.
90 μM). The 4-chloro-3-(trifluoromethyl)benzyl analogs 9d
and 10d further regained potency, though both enantiomers
were still less potent than the hit and its enantiomer (9a and
10a, respectively). In contrast to the above-noted electron-
withdrawing group-containing congeners, analogs contain-
ing either 4-methyl or 4-methoxy were significantly less
potent. Thus, the strongly electron-donating 4-methox-
ybenzyl analogs 9f and 10f lost all mHtt–CaM disruption

Medicinal Chemistry Research
Fig. 2 Compounds were tested in vitro to determine if a DAPK1 and b
CaMK2g enzyme activity was impacted. Each compound was tested at
a single concentration of 100 µM. Data are presented as mean stan-
dard error of the mean. Each experiment was run three times with at
least two replicates per experiment. Compounds 9c, 9d, 10c, and 10h
did not significantly inhibit DAPK1 or CaMK2g enzyme activity.
Compound 9h showed a statistically significant effect on inhibition of
CaMK2g enzyme activity but not DAPK1 activity as compared with
control. *p = 0.0138
Fig. 3 Compounds 9d and 9h do
not show protection against
mHtt toxicity
Analogs 9d and 9h were further evaluated in a neuronal
cell model for HD neurodegeneration (Fig. 3). Unfortu-
nately, the activity in the AlphaScreen assay did not translate
to therapeutically relevant activity in this cellular model,
with barely measurable activity even at 10 μM. Analog 9h
was evaluated for preliminary pharmacokinetic properties
and found to possess a kinetic solubility of 0.6 μM and
permeability (PAMPA assay) of 7.6 × 10−7 cm/s. Based on
these data, the poor solubility (extracellular solubility barely
above the IC50) and mediocre PAMPA permeability could
be sufficient to prevent effective intracellular concentrations
and may likely be responsible for the poor correlation
between screening assay and cellular model.
indicated a significant difference in toxicity due to the
induction of mHtt but no significant rescue of the toxicity
with compound 9d. Similarly, one-way ANOVA analysis of
compound 9h [([(F (6,99) = 12.46, p < 0.0001))] showed a
significant difference among the treatment groups. How-
ever, the Dunnett’s multiple comparison test did not show
any significant effect of 9h in rescuing the toxicity asso-
ciated with mHtt expression although there was a significant
toxicity in the presence of mHtt as compared with control.
Conclusion
Compounds 9d and 9h were tested for protection against
mHtt toxicity at three different concentrations in PC12 cells.
Data are presented as mean standard error of the mean.
Each experiment was run at least three times. For compound
9d, Shapiro–Wilk test showed that data were not normal
and log transformation of the data set did not achieve nor-
mality. Hence, a Kruskal–Wallis nonparametric test was
performed on the data set. Kruskal–Wallis analysis of
compound 9d [(Kruskal–Wallis statistic (7,140) = 81.48,
p < 0.0001)] showed significant difference among the
treatment groups. Dunn’s multiple comparison post hoc test
These results demonstrate that small molecules are capable
of disrupting the mHtt–CaM interaction in vitro. Although
this particular series did not translate into useful cellular
activity, it represents a start toward validating aberrant PPIs
as a viable target for small-molecule therapy and paves the
way for future investigations into the utilization of small
molecules as a treatment for HD.
Acknowledgements The authors thank Paul R. Hanson and colleagues
for populating the KU screening collection with the initial hit and other
unique molecules. The authors are grateful to Michael D. Cameron at

Medicinal Chemistry Research
the Scripps Research Institute DMPK Core for pharmacokinetic
property evaluation and the University of North Carolina’s Department
of Chemistry Mass Spectrometry Core Laboratory for their assistance
with mass spectrometry analysis. Q Exactive HF-X system HRMS
determinations were supported by the National Science Foundation
under Grant No. (CHE-1726291). This work was supported by the
National Institute of Neurological Disorders and Stroke (Award
NS088059).
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Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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