Novel allosteric PARP1 inhibitors for the treatment of BRCA-deficient leukemia

Article abstract of DOI:10.1007/s00044-020-02537-0

The successful use of PARP1 inhibitors like olaparib (Loparza) in the treatment of BRCA1/2-deficient breast cancer has provided clinical proof-of-concept for applying personalized medicine based on synthetic lethality to the treatment of cancer. Unfortunately, all marketed PARP1 inhibitors act by competing with the cofactor NAD⁺ and resistance is already developing to this anticancer mechanism. Allosteric PARP1 inhibitors could provide a means of overcoming this resistance. A high throughput screen performed by Tulin et al. identified 5F02 as an allosteric PARP inhibitor that acts by preventing the enzymatic activation of PARP1 by histone H4. 5F02 demonstrated anticancer activity in several cancer cell lines and was more potent than olaparib and synergistic with olaparib in these assays. In the present study, we explored the structure–activity relationship of 5F02 by preparing analogs that possessed structural variation in four regions of the chemical scaffold. Our efforts led to lead molecule 7, which demonstrated potent anti-clonogenic activity against BRCA-deficient NALM6 leukemia cells in culture and a therapeutic index for the BRCA-deficient cells over their BRCA-proficient isogenic counterparts.

Full text of DOI:10.1007/s00044-020-02537-0

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02537-0
ORIGINAL RESEARCH
Novel allosteric PARP1 inhibitors for the treatment of BRCA-deficient
leukemia
Elizabeth Hewlett1 Monika Toma2 Katherine Sullivan-Reed2 John Gordon1 Tomasz Sliwinski3 Alexei Tulin4
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Wayne E. Childers1 Tomasz Skorski2
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Received: 19 November 2019 / Accepted: 25 March 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
The successful use of PARP1 inhibitors like olaparib (Loparza®) in the treatment of BRCA1/2-deficient breast cancer has
provided clinical proof-of-concept for applying personalized medicine based on synthetic lethality to the treatment of cancer.
Unfortunately, all marketed PARP1 inhibitors act by competing with the cofactor NAD+ and resistance is already
developing to this anticancer mechanism. Allosteric PARP1 inhibitors could provide a means of overcoming this resistance.
A high throughput screen performed by Tulin et al. identified 5F02 as an allosteric PARP inhibitor that acts by preventing
the enzymatic activation of PARP1 by histone H4. 5F02 demonstrated anticancer activity in several cancer cell lines and was
more potent than olaparib and synergistic with olaparib in these assays. In the present study, we explored the
structure–activity relationship of 5F02 by preparing analogs that possessed structural variation in four regions of the
chemical scaffold. Our efforts led to lead molecule 7, which demonstrated potent anti-clonogenic activity against BRCA-
deficient NALM6 leukemia cells in culture and a therapeutic index for the BRCA-deficient cells over their BRCA-proficient
isogenic counterparts.
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Keywords Allosteric BRCA Histone 4 PARP1 Synthetic lethality
Introduction
Tumor cells accumulate spontaneous and drug-induced
DNA damage, but they survive because of enhanced/altered
DNA repair activities (Bartkova et al. 2005). The hypothesis
that cancer cells are addicted to particular DNA repair
pathways is supported by selective elimination of tumor
cells by drugs/compounds targeting specific DNA repair
mechanisms (Nickoloff et al. 2017). DNA double-strand
breaks (DSBs), the most lethal DNA lesions, are usually
repaired by BRCA1/2 -mediated homologous recombina-
tion (BRCA-HR) in proliferating cells (Karanam et al.
2012). The enzyme poly (ADP-ribose) polymerase 1
(PARP1) may prevent accumulation of DNA DSBs by
playing a key role in base excision repair, single-strand
break repair, alternative nonhomologous end-joining (Alt-
NHEJ), and/or by facilitating MRE11-mediated recruitment
of RAD51 to promote stalled replication fork restart
(Metzger et al. 2013; Ying et al. 2012). The success of
PARP inhibitor (PARPi) olaparib (Lynparza®, Fig. 1) in
treating BRCA1/2-deficient breast tumors has established a
proof-of-concept of personalized cancer therapy utilizing
synthetic lethality (Lord et al. 2015). Moreover, we
These authors contributed equally: Elizabeth Hewlett, Monika Toma
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02537-0) contains supplementary
material, which is available to authorized users.
* Wayne E. Childers
wayne.childers@temple.edu
* Tomasz Skorski
tomasz.skorski@temple.edu
1
Moulder Center for Drug Discovery Research, Temple University
School of Pharmacy, 3307 N. Broad Street, Philadelphia, PA, USA
2
Sol Sherry Thrombosis Research Center, Lewis Katz School of
Medicine at Temple University, 3420 N. Broad Street,
Philadelphia, PA, USA
3
Laboratory of Medical Genetics, Faculty of Biology and
Environmental Protection, University of Lodz, Lodz, Poland
4
School of Medicine and Health Sciences, University of North
Dakota, 501 N. Columbia Rd., Grand Forks, ND, USA

Medicinal Chemistry Research
Fig. 1 Structures of marketed
PARP inhibitors and 5F02, an
allosteric PARP1 inhibitor that
acts by blocking histone
H4–stimulated activation of
the enzyme
demonstrated that PARPi’s trigger synthetic lethality also in
proliferating and quiescent tumor cells deficient in DNA-PK
mediated NHEJ (D-NHEJ) (Nieborowska-Skorska et al.
2017). Recently, we showed that quiescent and proliferating
cells from BRCA-HR and D-NHEJ-deficient hematopoietic
malignancies are sensitive to synthetic lethality triggered by
PARPi’s (Nieborowska-Skorska et al. 2017; Dasgupta et al.
2017; Maifrede et al. 2017).
Fig. 2 SAR strategy for the 5F02 chemical scaffold
Three drug design strategies for inhibiting PARP1 have
been pursued: competing with the cofactor NAD+,
obstructing PARP1 binding with DNA and preventing
enzymatic activation of PARP1 by histone H4 (Mal-
yuchenko et al. 2015; Kirsanov et al. 2014; Jain and Patel
2019). All of the marketed PARPi’s (Fig. 1) act by com-
peting with NAD+. Unfortunately, resistance to
NAD+-competitive PARP1 inhibitors is rapidly developing.
To overcome this problem, the Tulin group performed a
high throughput screen on a 50,000 compound library to
identify non-NAD-like PARP inhibitors (Thomas et al.
2016). In the course of that work, 5F02 (Fig. 1) and related
analogs were identified as a novel class of inhibitors of
histone H4-mediated PARP1 activation. To our knowledge,
this is the first report of compounds that inhibit PARP1
through this mechanism. In collaboration with the Tulin
group we demonstrated that 5F02 demonstrated antitumor
effects in a number of cell lines, exerted synergistic effect
with olaparib in those assays and was more potent than
olaparib in in vitro proliferation assays (Nieborowska-
Skorska et al. 2019).
relationship (SPR) around the chemical scaffold inherent in
5F02 we explored four regions of the molecule (Fig. 2),
namely the piperidine moiety, the requirement for a posi-
tively charged quaternary amine group, the requirement for
an ester functionality and the cyclododecyl substituent. To
assess the role of physicochemical properties we simulta-
neously tested all target molecules for their maximum
kinetic aqueous solubility in 2% DMSO/phosphate-buffered
saline and their stability in mouse and human liver micro-
somes in the presence and absence of NADPH. Our work
led to the identification of an analog within this novel series
that demonstrated selective anti-clonogenic activity against
a leukemia cell line that is BRCA-deficient compared with
BRCA-proficient isogenic counterparts.
Chemistry
Reagents were purchased from commercial suppliers
(Sigma-Aldrich, Combiblocks, Enamine, Fisher Scientific)
and used without further purification. Unless stated other-
wise, reactions were run in the presence of normal atmo-
spheric conditions. 1H NMR and 13C NMR data were
collected on a Bruker 400 MHz Avance III spectrometer at
We wished to expand on these innovative findings by
examining the structure–activity relationship (SAR) of the
interesting chemical scaffold presented by 5F02. To achieve
a better understanding of the SAR and structure-property

Medicinal Chemistry Research
ambient temperature in the identified solvent. 13C NMR
were run at 100 MHz. Peak positions are given in parts per
million downfield from tetramethylsilane as the internal
standard. LC–MS analysis was performed on an Agilent
Technologies 1200 series LC system coupled to a 6300
quadrapole MS. High resolution mass spectrometry analysis
was performed by the Mass Spectrometry Lab, School of
Chemical Sciences, University of Illinois at Urbana-
Champaign on a Waters QTOF Ultima ESI instrument.
Silica gel chromatography was performed using a Teledyne
ISCO Combiflash Rf system with UV detection at 220 and
254 nM. Reversed phase chromatography was performed on
a Gilson GX281 system using a C-18 column and a gradient
of acetonitrile in water with 0.1% trifluoroacetic acid
modifier. Detection was accomplished with UV at 220 and
254 nM. Purity (>99.5% unless otherwise noted) of all final
target compounds was confirmed by HPLC on two separate
systems: (1) normal phase on a silica gel column using a
gradient of ethyl acetate in hexane; (2) reversed phase on a
C-18 column using a gradient of acetonitrile in water.
Cyclododecyl 2-(piperidin-1-yl)acetate (1a) This com-
pound was obtained as a yellow oil; Yield = 38%. 1H NMR
(400 MHz, CDCl3) δ ppm: 5.05 (tt, J = 7.15, 4.89 Hz, 1H,
O–CH), 3.16 (m, 2H, N–CH2), 2.53 (m, 4H. C2, C6 of
piperidine –CH2), 1.73–1.58 (m, 4H, C3, C5 of piperidine
–CH2), 1.51–1.32 (m, 24H C4 of piperidine-CH2, cyclo-
dodecyl –CH2); ESIMS m/z (pos) 310.2 (100%) [M + H]+.
Cyclododecyl-2-(pyrrolidin-1-yl)acetate (2a) This com-
1
pound was obtained as a orange solid; Yield = 51%. H
NMR (400 MHz, CDCl3) δ ppm: 5.13–5.04 (m, 1H,
O–CH), 3.34 (s, 2H, N–CH2), 2.72–2.64 (m, 4H, C2, C5 of
pyrrolidine –CH2), 1.89–1.78 (m, 4H, C3, C4 of pyrrolidine
–CH2), 1.78–1.67 (m, 2H, C2, C12 of cyclododecyl –CH2),
1.58–1.48 (m, 2H, C2, C12 of cyclododecyl –CH2),
1.48–1.24 (m, 18H, remaining cyclododecyl –CH2); ESIMS
m/z (pos) 296.2 (100%) [M + H]+.
Cyclododecyl-2-(azepan-1-yl)acetate (3a) This compound
1
was obtained as a orange solid; Yield = 44%. H NMR
(400 MHz, CDCl3) δ ppm: 5.06–5.01 (m, 1H, O–CH), 3.36
(s, 2H, N–CH2), 2.68–2.63 (m, 4H, C2, C7 of azacyclo-
heptyl ring –CH2), 1.85–1.76 (m, 4H, C3, C6 of azacy-
cloheptyl ring –CH2), 1.74–1.64 (m, 2H, C3, C5 of
azacycloheptyl ring –CH2), 1.55–1.43 (m, 2H, C3, C5 of
azacycloheptyl ring –CH2), 1.42–1.19 (m, 22H, remaining
cyclododecyl –CH2); ESIMS m/z (pos) 324.3 (100%) (M +
H)+.
Cyclododecyl 2-chloroacetate
In
a round bottom flask, cyclododecanol (20.8 gm,
113 mmol) was dissolved in benzene (225 mL), and then
pyridine (11.4 mL, 141 mmol) was added. This mixture was
cooled to 0 °C and chloroacetyl chloride (12.7 gm,
113 mmol) was added dropwise. The stirred mixture was
then heated at 78 °C and allowed to stir for 3 h. Upon
completion, the reaction was allowed to cool to room
temperature. The mixture was diluted with diethyl ether and
washed with water, 10% aqueous HCl, and finally saturated
aqueous sodium bicarbonate. The organic layer was dried
over anhydrous magnesium sulfate and concentrated on a
rotary evaporator. The residuals were purified by chroma-
tography on silica gel (gradient of 0–10% hexanes in ethyl
acetate). The desired product (19.1 gm, 65%) was obtained
as a white solid. 1H NMR (400 MHz, CDCl3) δ ppm:
5.11–5.04 (m, 1H, O–CH), 4.00 (s, 2H, CH2–Cl), 1.78–1.67
(m, 2H, C2, C12 cyclododecyl –CH2), 1.57–1.46 (m, 2H,
C2, C12 cyclododecyl –CH2), 1.44–1.26 (m, 18H,
remaining cyclododecyl –CH2); ESIMS m/z (pos): 282.3
(100%)/284.3 (33%) [M + Na]+.
Cyclododecyl 2-(4,4-dimethylpiperidin-1-yl)acetate (4a)
This compound was obtained as a orange solid; Yield =
1
58%. H NMR (400 MHz, CDCl3) δ ppm: 5.00–4.96 (m,
1H, O–CH), 3.33 (s, 2H, N–CH2), 2.61–2.55 (m, 4H, C2,
C6 of piperidine –CH2), 1.83–1.72 (m, 4H, C3, C5 of
piperidine –CH2), 1.70–1.61 (m, 2H, C2, C12 of cyclodo-
decyl –CH2), 1.51–1.41 (m, 2H, C2, C12 of cyclododecyl
–CH2), 1.42–1.19 (m, 18H, remaining cyclododecyl –CH2),
0.85 (s, 6H, piperidine –CH3); ESIMS m/z (pos) 338.3
(100%) [M + H]+.
Cyclododecyl 2-morpholinoacetate (5a) This compound
1
was obtained as a yellow solid; Yield = 22%. H NMR
(400 MHz, CDCl3) δ ppm: 5.04–4.96 (m, 1H, O–CH),
3.76–3.74 (m 4H, morpholino O–CH2), 3.13 (s, 2H,
N–CH2), 2.62–2.60 (m, 4H, morpholino N–CH2),
1.68–1.61 (m, 2H, C2, C12 cyclododecyl –CH2), 1.48–1.41
(m, 2H, C2, C12 cyclododecyl –CH2), 1.36–1.23 (m, 18H,
remaining cyclododecyl –CH2); ESIMS m/z (pos) 312.2
(100%) [M + H]+.
General Procedure for the synthesis of compounds 1a–9a
In a reaction vial, cyclododecyl 2-chloroacetate (0.50 gm,
1.9 mmol) was combined with the appropriate cyclic sec-
ondary amine (2.5 mmol) and stirred at 100 °C for 48 h. The
reaction was cooled to room temperature and the mixture
were purified by silica gel chromatography (gradient of
0–40% hexanes in ethyl acetate) to provide the desired
products 1a–9a.
Cyclododecyl 2-thiomorphinoacetate (6a) This compound
1
was obtained as a yellow solid; Yield = 37%. H NMR
(400 MHz, CDCl3) δ ppm: 5.02–4.97 (m, 1H, O–CH), 3.34

Medicinal Chemistry Research
(s, 2H, N–CH2), 2.68–2.61 (m, 4H, thiomorpholinyl
N–CH2), 2.60–2.52 (m, 4H, thiomorpholinyl S–CH2),
1.73–1.64 (m, 2H, C2, C12 cyclododecyl –CH2), 1.55–1.47
(m, 2H C2, C12 cyclododecyl –CH2), 1.42–1.19 (m, 18H,
remaining cyclododecyl –CH2); ESIMS m/z (pos) 328.2
(100%) [M + H]+.
1-(2-(Cyclododecyloxy)-2-oxoethyl)-1-methylpiperidin-1-
ium iodide (1, 5F02) This compound was obtained as a
1
yellow solid; Yield = 75%. H NMR (400 MHz, CDCl3) δ
ppm: 5.14–5.07 (m, 1H, O–CH), 4.88 (s, 2H, N–CH2),
3.95–3.88 (m, 4H, C2, C6 of piperidinel –CH2), 3.41 (s, 3H,
N-CH3), 2.41–2.29 (m, 4H, C3, C5 of piperidine –CH2),
2.29–2.16 (m, 2H, C4 of piperidine –CH2), 1.77–1.69 (m,
2H, C2, C12 cyclododecyl –CH2), 1.55–1.43 (m, 2H, C2,
C12 cyclododecyl –CH2), 1.39–1.25 (m, 18H, remaining
cyclododecyl –CH2); MS (ESI) (m/z) 324.3 (M)+; 13C NMR
(100 MHz, CDCl3) δ ppm 162.5 (C=O), 60.4 (cyclodode-
cyl C1), 59.6 (N+–CH2), 51.9 (piperidinyl C2, 6), 47.0
(N+–CH3), 27.6 (cyclododecyl C2, 12), 22.5 (cyclododecyl
C4,10), 22.3 (cyclododecyl C5, 9), 21.9 (cyclododecyl
C6,8), 21.7 (cyclododecyl C7), 19.5 (cyclododecyl C3, 11),
19.1 (piperidinyl C4), 18.8 (piperidinyl C3, 5); ESIMS m/z
(pos) 324.3 [M]+; HRESIMS m/z (pos): 324.2896
C20H38NO2 (calcd. 324.2903).
Cyclododecyl 2-(piperazin-1-yl)acetate (7a) This com-
1
pound was obtained as a yellow solid; Yield = 8.5%. H
NMR (400 MHz, CDCl3) δ ppm: 5.04–4.96 (m, 1H,
O–CH), 3.13 (s, 2H, N–CH2), 2.62 (m, 4H, piperazine
N–CH2), 2.54–2.52 (m, 4H, piperazine N–CH2) 2.40–2.38
(m, 1H, piperazine NH), 1.68–1.61 (m, 2H, C2, C12
cyclododecyl –CH2), 1.48–1.41 (m, 2H, C2, C12 cyclodo-
decyl –CH2), 1.36–1.23 (m, 18H, remaining cyclododecyl
–CH2); ESIMS m/z (pos) 312.2 (100%) [M + H]+.
tert-Butyl 4-(2-(cyclododecyloxy)-2-oxoethyl)piperazine-1-
carboxylate (8a) This compound was obtained as a white
1
solid; Yield = 26%. H NMR (400 MHz, CDCl3) δ ppm:
1-(2-(Cyclododecyloxy)-2-oxoethyl)-1-methylpyrrolidin-1-
5.06–4.97 (m, 1H, O–CH), 3.48–3.32 (m, 4H, piperazine
N–CH2), 3.13 (s, 2H, N–CH2), 2.48–2.46 (m, 4H, piper-
azine N–CH2), 167–1.62 (m, 2H, C2, C12 cyclododecyl
–CH2), 1.47–1.42 (m, 2H, C2, C12 cyclododecyl –CH2),
1.43 (s, 9H, BOC–CH3) 1.38–1.24 (m, 18H, remaining
cyclododecyl –CH2); ESIMS m/z (pos) 411.3 (100%) [M +
H]+.
ium iodide (2) This compound was obtained as an orange
solid; Yield = 85%. H NMR (400 MHz, CDCl3) δ ppm:
1
5.10–5.03 (m, 1H, O–CH), 4.85 (s, 2H, N–CH2), 4.08–4.01
(m, 4H, C2, C5 of pyrrolidine –CH2), 3,41 (s, 3H, N–CH3),
2.39–2.26 (m, 2H, C3, C4 of pyrrolidine, –CH2), 2.26–2.12
(m, 2H, C3, C4 of pyrrolidine –CH2), 1.76–1.65 (m, 2H,
C2, C12 cyclododecyl –CH2), 1.54–1.44 (s, 2H, C2, C12
cyclododecyl –CH2), 1.38–1.21 (m, 18H, remaining
cyclododecyl –CH2); 13C NMR (100 MHz, CDCl3) δ ppm
164.4 (C=O), 76.3 (cyclododecyl C1), 65.8 (pyrrolidinyl
C2, 5), 62.7 (N+–CH2), 49.7 (N+–CH3), 29.1 (cyclododecyl
C3, 11), 23.9 (cyclododecyl C4, 10), 23.7 (cyclododecyl
C5, 9), 23.3 (cyclododecyl C6, 8), 23.1 (cyclododecyl C7),
21.5 (cyclododecyl C3, 11), 20.9 (pyrrolidinyl C3, 4);
ESIMS m/z (pos) 310.3 (100%) [M]+; HRESIMS m/z (pos):
310.2745 C19H36NO2 (calcd. 310.2746).
Cyclododecyl-2-(4-benzylpiperidin-1-yl)acetate (9a) This
1
compound was obtained as a white solid; Yield = 26%. H
NMR (400 MHz, CDCl3) δ ppm: 7.30–7.26 (m, 2H, Ar–H),
7.21–9.19 (m, 1H, Ar–H), 7.16–7.154 (m, 2H, Ar–H),
5.06–4.97 (m, 1H, O–CH), 3.13 (s, 2H, N–CH2), 2.85–2.83
(m, 2H, benzyl –CH2), 2.48–2.46 (m, 4H, C2, C6 of
piperiinel –CH2), 1.67–1.62 (m, 2H, C2, C12 cyclododecyl
–CH2), 1.47–1.43 (m, 1H, C4 of piperidine –CH),
1.39–1.34 (m, 2H, C2, C12 cyclododecyl –CH2), 1.36–1.22
(m, 22H, C3, C5 of piperidine –CH2, remaining cyclodo-
decyl –CH2); ESIMS m/z (pos) 411.3 (100%) (M + H)+.
1-(2-(Cyclododecyloxy)-2-oxoethyl)-1-methylazepan-1-ium
iodide (3) This compound was obtained as a yellow solid;
Yield = 68%. 1H NMR (400 MHz, CDCl3)
δ ppm:
General procedure for the synthesis of compounds 1–6, 8–9
5.10–5.03 (m, 1H, O–CH), 4.71 (s, 2H, N–CH2), 4.07–3.98
(m, 2H, C2, C7 of azacycloheptyl ring, –CH2), 3.92–3.83
(m, 2H, C2, C7 of azacycloheptyl ring, –CH2), 3.59 (s, 3H,
N–CH3), 2.06–1.86 (m, 4H, C3, C6 of azacycloheptyl ring
–CH2), 1.83–1.62 (m, 6H, C3, C4 of azacycloheptyl ring
and C2, C12 cyclododecyl –CH2), 1.55–1.43 (m, 2H, C2,
C12 cyclodedecyl –CH2), 1.41–1.18 (m, 18H, remaining
cyclododecyl –CH2); 13C NMR (100 MHz, CDCl3) δ ppm
163.1 (C=O), 76.06 (cyclododecyl C1), 65.9 (azacyclo-
heptyl C2, 7), 62.1 (N+–CH2), 51.4 (N+–CH3), 29.1
(cyclododecyl C2, 12), 26.7 (azacycloheptyl C3, 6), 23.9
(cyclododecyl C4, 10), 23.7 (cyclododecyl C5, 9), 23.3
(cyclododecyl C6, 8), 23.1 (cyclododecyl C7), 22.2
In a reaction vial, the appropriate amino-cyclododecyl
acetate 1a–9a (0.65 mmol) was dissolved in methanol
(2 mL), and then methyl iodide (0.72 mmol) was added.
The vial was sealed and the resulting mixture was heat to
65 °C and stirred at that temperature overnight. The
reaction mixture was then cooled to room temperature and
concentrated on a rotary evaporator and the resulting
residue triturated with diethyl ether. The resulting pre-
cipitate was collected by vacuum filtration, washed with
diethyl ether and dried in vacuo to provide the desired
products 1–6, 8–9.

Medicinal Chemistry Research
(cyclododecyl C3, 11), 20.9 (azacycloheptyl C4, 5); ESIMS
m/z (pos) 338.3 (100%) [M]+; HRESIMS m/z (pos):
338.3056 C21H40NO2 (calcd. 338.3059).
(cyclododecyl C6, 8), 23.1 (cyclododecyl C7), 20.7
(cyclododecyl C3, 11); ESIMS m/z (pos) 342.2 (100%)
[M]+; HRESIMS m/z (pos) (M+) 342.2462 C19H36NO2S
(calcd. 342.2467).
1-(2-(Cyclododecyloxy)-2-oxoethyl)-1,4,4-trimethylpiperi-
din-1-ium iodide (4) This compound was obtained as a
white solid; Yield = 71%. H NMR (400 MHz, DMSO) δ
4-(tert-Butoxycarbonyl)-1-(2-(cyclododecyloxy)-2-oxoethyl)-
1-methylpiperazin-1-ium iodide (8) This compound was
obtained as a yellow solid; Yield = 30%. 1H NMR
(400 MHz, CDCl3) δ ppm: 5.13–5.09 (m, 1H, O–CH),
4.04–3.90 (m, 4H, piperazinyl –CH2), 3.75 (s, 3H, N–CH3),
3.72–3.65 (m, 4H, piperazinyl –CH2), 1.78–1.73 (m, 2H,
C2, C12 cyclododecyl –CH2), 1.95–1.55 (m, 2H, C2, C12
cyclododecyl –CH2), 1.45 (s, 9H, BOC –CH3), 1.38–1.21
(m, 18H, remaining cyclododecyl –CH2); 13C NMR
(100 MHz, CDCl3) δ ppm 163.7 (ester C=O), 153.5 (BOC
C=O), 82.1 (BOC t-butyl quaternary C), 76.6 (cyclodode-
cyl C1), 60.9 (piperizinyl N–CH2). 60.3 (piperizinyl
N+–CH2), 47.9 (N+–CH3), 29.1 (cyclododecyl C1), 28.3
(BOC –CH3), 23.9 (cyclododecyl C4, 10), 23.7 (cyclodo-
decyl C5, 9), 23.3 (cyclododecyl C6,8), 23,2 (cyclododecyl
C7), 21.0 (cyclododecyl C3, 11); ESIMS m/z (pos) 425.3
(100%) [M]+; HRESIMS m/z (pos): 425.3375 C24H45N2O4
(calcd. 425.3379).
1
ppm: 5.11–5.02 (m, 1H, O–CH), 4.51 (s, 2H, N–CH2),
3.63–3.51 (m, 2H, C2, C6 of piperidine –CH2), 3.51–3.40
(m, 2H, C2, C6 of piperidine –CH2), 3.21 (s, 3H, N–CH3),
1.80–1.46 (m, 8H, C3, C5 of piperidine –CH2 and C2, C12
cyclododecyl –CH2), 1.42–1.22 (m, 18H, remaining
cyclododecyl –CH2), 1.02 (s, 3H, piperidine –CH3), 1.01 (s,
3H, piperidine –CH3); 13C NMR (100 MHz, DMSO-d6) δ
ppm 169.8 (C=O), 79.2 (cyclododecyl C1), 62.7
(N+–CH2), 60.1 (piperidinyl C2,6), 48.4 (N+–CH3), 36.7
(piperidinyl C3, 5), 34.2 (piperidinyl 4-CH3), 33.8 (cyclo-
dodecyl C2, 12), 32.3 (piperidinyl C4), 28.7 (cyclododecyl
C4, 10), 28.5 (cyclododecyl C5, 9), 28.1 (cyclododecyl C6,
8), 27.9 (cyclododecyl C7), 25.6 (cyclododecyl C3, 11);
ESIMS m/z (pos) 352.3 (100%) [M]+; HRESIMS m/z (pos):
352.3209 C22H42NO2 (calcd. 352.3216).
4-(2-(Cyclododecyloxy)-2-oxoethyl)-4-methylmorpholin-4-
ium iodide (5) This compound was obtained as a yellow
solid; Yield = 36%. H NMR (400 MHz, CDCl3) δ ppm:
4-(Benzyl)-1-(2-(cyclododecyloxy)-2-oxoethyl)-1-methylpi-
1
peridin-1ium iodide (9) This compound was obtained as a
1
5.15–5.08 (m, 1H, O–CH), 4.47 (s, 2H, N–CH2), 4.02–3.88
(m, 4H, morpholinyl O–CH2), 3.74–3.57 (m, 4H, morpho-
linyl N–CH2), 3.38 (s, 3H, N–CH3), 1.79–1.68 (m, 2H, C2,
C12 cyclododecyl –CH2), 1.57–1.46 (m, 2H, C2, C12
cyclododecyl –CH2), 1.41–1.19 (m, 18H, remaining
cyclododecyl –CH2); 13C NMR (100 MHz, CD3OD) δ ppm
165.4 (C=O), 76.7 (cyclododecyl C1), 63.2 (morpholinyl
O–CH2), 62.1 (morpholinyl N–CH2), 61.5 (N+–CH2), 49.0
(N+–CH3), 30.1 (cyclododecyl C2, 12), 25.0 (cyclododecyl
C4,10), 24.8, 24.6 (cyclododecyl C5, 9), 24.5 (cyclododecyl
C6, 8), 24.3 (cyclododecyl C7), 22.1 (cyclododecyl C3, 11);
ESIMS m/z (pos) 326.2 (100%) [M]+; HRESIMS m/z (pos):
326.2696 C19H36NO3 (calcd. C).
yellow solid; Yield = 44%. H NMR (400 MHz, DMSO) δ
ppm: 7.24–7.17 (m, 2H, Ar–H), 7.16–7.06 (m, 3H, Ar–H),
5.09–4.99 (m, 1H, O–CH), 4.97 (s, 2H, N–CH2), 4.03–3.93
(m, 4H, C2, C6 of piperidine –CH2), 3.44 (s, 3H, N–CH3),
2.68–2.55 (m, 2H, benzyl –CH2), 2.20–2.03 (m, 1H, C4 of
piperidine –CH), 1.87–1.75 (m, 2H, C3, C5 of piperidine
–CH2), 1.75–1.55 (m, 4H, C3, C5 of piperidine –CH2, C2,
C12 cyclododecyl –CH2), 1.52–1.37 (m, 2H, C2, C12
cyclododecyl –CH2), 1.37–1.16 (m, 18H, remaining
cyclododecyl –CH2); 13C NMR (100 MHz, CDCl3) δ ppm
164.0 (C=O), 138.2 (phenyl C1), 129.1 (phenyl C3, 5),
128.6 (phenyl C2, 6), 126.5 (phenyl C4), 75.8 (cyclodo-
decyl C1), 64.0 (N+–CH2), 61.6 (piperidinyl C2, 6), 45.3
(N+–CH3), 41.6 (benzyl –CH2), 34.2 (piperidinyl C4), 29.1
(cyclododecyl C2, 12), 26.0 (piperidinyl C3, 5), 23.8
(cyclododecyl C4, 10), 23.6 (cyclododecyl C5, 9), 23.3
(cyclododecyl C6, 8), 23.2 (cyclododecyl C7), 21.0
(cyclododecyl C3, 11); ESIMS m/z (pos) 414.4 (100%)
[M]+; HRESIMS m/z (pos): 414.3369 C27H44NO2 (calcd.
414.3372),
4-(2-(Cyclododecyloxy)-2-oxoethyl)-4-methylthiomorphilin-
4-ium iodide (6) This compound was obtained as a yellow
1
solid; Yield = 66%. H NMR (400 MHz, CDCl3) δ ppm:
5.17–5.09 (m, 1H, O–CH), 4.51 (s, 2H, N–CH2), 4.35–4.14
(m, 4H, thiomorpholinyl N–CH2), 3.99–3.79 (m, 4H, thio-
morpholinyl S–CH2), 3.56 (s, 3H, N–CH3), 1.81–1.67 (m,
2H, C2, C12 cyclododecyl –CH2), 1.59–1.47 (m, 2H, C2,
C12 cyclododecyl –CH2), 1.43–1.23 (m, 18H, remaining
cyclododecyl –CH2); 13C NMR (100 MHz, CDCl3) δ ppm
163.2 (C=O), 77.0 (cyclododecyl C1), 62.0 (thiomorpho-
linyl N–CH2), 57.7 (N+–CH2), 48.6 (N+–CH3), 37.9
(cyclododecyl C1), 28.7 (thiomorpholinyl S–CH2), 23.9
(cyclododecyl C4, 10), 23.8 (cyclododecyl C5, 9), 23.2
Synthesis of 1-(2-(cyclododecyloxy)-2-oxoethyl)-1-methylpi-
perazinium iodide (7) 4-(tert-Butoxycarbonyl)-1-(2-
(cyclododecyloxy)-2-oxoethyl)-1-methylpiperazin-1-ium
iodide (8, 0.04 gm, 0.07 mmol) was dissolved in a 1:1
mixture of dichloromethane and trifluoroacetic acid
(2 mL). The mixture was stirred at room temperature for

Medicinal Chemistry Research
15 min and then concentrated on a rotary evaporator. The
desires product (0.032 gm, 100%) was obtained as a
yellow solid by trituration with diethyl ether. H NMR
1-(2-(Cyclododecyloxy)-2-oxoethyl)-1-(1-propyl)piperidin-1-
ium iodide (11) This compound was obtained as a yellow
1
1
solid; Yield = 11%. H NMR (400 MHz, CDCl3) δ ppm:
(400 MHz, CDCl3) δ ppm: 5.11–5.04 (m, 1H, O–CH),
4.98 (s, 2H, N–CH2), 4.36–4.27 (m, 2H, C2, C6 of
piperazine –CH2), 4.14–4.07 (m, 2H, C2, C6 of piper-
azine –CH2), 4.00–3.92 (m, 1H, piperazine NH), 3.66 (s,
3H, N–CH3), 3.15–3.01 (m, 2H, C3, C5 of piperazine
–CH2), 2.98–2.88 (m 2H, C3, C5 of piperazine –CH2),
1.77–1.60 (m, 2H, C2, C12 cyclododecyl –CH2),
1.56–1.38 (m, 2H, C2, C12 cyclododecyl –CH2),
1.3.0–1.20 (m, 18H, remaining cyclododecyl –CH2); 13C
NMR (100 MHz, CDCl3) δ ppm 163.7 (C=O), 73.0
(cyclododecyl C1), 62.0 (N+–CH2), 61.0 (piperazinyl
N+–CH2), 46.1 (N+–CH3), 37.5 (piperazinyl NH–CH2),
29.2 (cyclododecyl C2, 12), 24.0 (cyclododecyl C4, 10),
23.4 (cycyldodecyl C5, 9), 23.3 (cyclododecyl C6, 8),
21.5 (cyclododecyl C7), 21.0 (cyclododecyl C3, 11);
ESIMS m/z (pos) 325.3 (100%) [M]+; HRESIMS m/z
(pos); 325.2850 C19H37N2O2 (calcd. 325.2855).
5.09–5.03 (m, 1H, O–CH), 4.47 (s, 2H, N–CH2), 4.28–4.15
(q, J = 7.4 Hz, 2H, propyl N–CH2), 3.85–3.74 (m, 2H, C2,
C6 of piperidine –CH2), 3.73–3.58 (m, 2H, C2, C6 of
piperidine –CH2), 1.93–1.66 (m, 10H, C3, C4, C5 piper-
idine –CH2, propyl –CH2, C2, C12 cyclodecyl –CH2),
1.53–1.38 (m, 2H, C2, C12 cyclododecyl –CH2), 1.32–1.27
(m, 18H, remaining cyclododecyl –CH2), 0.98 (t, J =
7.3 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ ppm 164.0
(C=O), 76.1 (cyclododecyl C1), 60.2 (N+–CH2), 60.0
(propyl N+–CH2), 57.4 (piperidinyl C2, 6), 29.1 (cyclodo-
decyl C2, 12), 23.8 (cyclododecyl C4, 10), 23.6 (cyclodo-
decyl C5, 8), 23.3 (cyclododecyl C6, 8), 23.1 (cyclododecyl
C7), 21.0 (cyclododecyl C3, 11), 20.3 (piperidinyl C4),
20.1 (piperidinyl C3, 5), 15.9 (propyl –CH2), 10.8 (propyl
–CH3); ESIMS m/z (pos) 352.3 (100%) [M]+. HRESIMS
m/z (pos): 352.3212 C22H42NO2 (calcd. 352.3216).
1-(2-(Cyclododecyloxy)-2-oxoethyl)-1-(1-butyl)piperidin-1-
General procedure for the synthesis of compounds 10–13
ium iodide (12) This compound was obtained as a yellow
solid: Yield = 19%. H NMR (400 MHz, CDCl3) δ ppm:
1
In a reaction vial 1a (0.20 gm, 0.65 mmol) was dissolved in
methanol (2 mL), and then the appropriate alkyl iodide
(0.72 mmol) was added. The vial was sealed and the
resulting mixture was heat to 65 °C and stirred at that
temperature overnight. The reaction was then cooled to
room temperature, concentrated on a rotary evaporator and
the resulting residue triturated with diethyl ether. The
resulting precipitate was collected by vacuum filtration,
washed with diethyl ether and dried in vacuo to obtain the
desired products 10–13.
5.13–5.04 (m, 1H, O–CH), 4.47 (s, 2H, N–CH2), 4.21–4.16
(m, 2H, propyl N–CH2), 3.85–3.82 (m, 2H, C2, C6 of
piperidine –CH2), 3.74–3.70 (m, 2H, C2, C6 of piperidine
–CH2), 1.96–1.80 (m, 6H, C3, C5 of piperidine –CH2, butyl
–CH2), 1.73–1.62 (m, 4H, C4 of piperidine, C2, C12
cyclododecyl –CH2), 1.54–1.45 (m, 2H, C2, C12 cyclodo-
decyl –CH2), 1.43–1.16 (m, 20H, butyl –CH2, remaining
cyclododecyl –CH2), 0.96 (t, J = 7.28 Hz, 3H, butyl –CH3);
13C NMR (100 MHz, CDCl3) δ ppm 163.8 (C=O), 75.9
(cyclododecyl C1), 57.4 (butyl N+–CH2), 55.7 (N+–CH2),
52.8 (piperidinyl C2, 6), 29.0 (cyclododecyl C2, 12), 24.0
(cyclododecyl C4, 10), 23.8 (cyclododecyl C5, 9), 23.6
(cyclododecyl C6, 8), 23.3 (cyclododecyl C7), 23.1
(cyclododecyl C3, 11), 22.9 (butyl C2), 21.2 (piperidinyl
C4), 21.0 (piperidinyl C3, 5), 20.0 (butyl C3), 13.6 (butyl
–CH3); MS (ESI) (m/z) 366.3 (M)+. ESIMS m/z (pos) 366.3
(100%) [M]+; HRESIMS m/z (pos): 366.2270 C23H44NO2
(calcd. 366.3372).
1-(2-(Cyclododecyloxy)-2-oxoethyl)-1-ethylpiperidin-1-ium
iodide (10) This compound was obtained as an orange
1
solid; Yield = 35%. H NMR (400 MHz, CDCl3) δ ppm:
5.14–5.07 (m, 1H, O–CH), 4.48 (s, 2H, N–CH2 (to car-
bonyl)), 4.26–4.20 (q, J = 8.0 Hz, 2H, ethyl –CH2),
3.92–3.81 (m, 4H, C2, C6 of piperidine –CH2), 1.93–1.81
(m, 6H, C3,C4, C5 of piperidine –CH2), 1.79–1.72 (m, 2H,
C2, C12 cyclododecyl –CH2), 1.56–1.49 (m, 2H, C2, C12
cyclododecyl –CH2), 1.40–1.28 (m, 18H, remaining
cyclododecyl –CH2), 1.35 (t, J = 8.0 Hz, 3H, ethyl –CH3);
13C NMR (100 MHz, CDCl3) δ ppm 164.0 (C=O), 76.4
(cyclododecyl C1), 59.7 (N+–CH2), 57.0 (ethyl –CH2),
54.0 (piperidinyl C2, 6), 29.2 (cyclododecyl C2, 12), 24.0
(cyclododecyl C4, 10), 23.8 (cyclododecyl C5, 9), 23.5
(cyclododecyl C6, 8), 23.3 (cyclododecyl C7), 21.1
(cyclododecyl C3, 11), 20.5 (piperidinyl C4), 20.2
(piperidinyl C3, 5), 8.3 (ethyl –CH3); ESIMS m/z (pos)
338.3 (100%) [M]+; HRESIMS m/z (pos): (M+): 338.3052
C21H40NO2 (calcd. 338.3059).
1-(2-(Cyclododecyloxy)-2-oxoethyl)-1-(1-pentyl)piperidin-1-
ium iodide (13) This compound was obtained as a yellow
1
solid; Yield = 5%. H NMR (400 MHz, CDCl3) δ ppm:
5.14–5.07 (m, 1H, O–CH), 4.51 (s, 2H, N–CH2), 4.36–4.26
(m, 2H, pentyl N–CH2), 3.86–3.77 (m, 2H, C2, C6 of
piperidine –CH2), 3.77–3.68 (m, 2H, C2, C6 of piperidine
–CH2), 1.86–1.83 (m, 4H, C3, C5 of piperidine –CH2),
1.77–1.71 (m, 4H, C4 of piperidine –CH2, pentyl –CH2),
1.69–1.62 (m, 2H, C2, C12 cyclododecyl –CH2), 1.57–1.49
(m, 4H, pentyl –CH2, C2, C12 cyclododecyl –CH2),
1.40–1.29 (m, 20H, pentyl –CH2, remaining cyclododecyl

Medicinal Chemistry Research
–CH2), 0.92–0.89 (m, 3H, pentyl –CH3); 13C NMR
(100 MHz, CDCl3) δ ppm 164.0 (C=O), 76.2 (cyclododecyl
C1), 60.0 (butyl N+–CH2), 57.8 (N+–CH2), 59.1 (piper-
idinyl C2, 6), 29.0 (cyclododecyl C2, 12), 28.3 (pentyl C3),
23.9 (pentyl C2), 23.7 (cyclododecyl C4, 10), 23.3 (cyclo-
dodecyl C5, 9), 23.1 (cyclododecyl C6, 8), 22.2 (cyclodo-
decyl C7), 21.8 (cyclododecyl C3, 11), 21.0 (piperidinyl
C3, 5), 20.3 (piperidinyl C4), 20.0 (butyl C3), 13.8 (butyl
–CH3); ESIMS m/z (pos) 380.3 (100%) [M]+; HRESIMS
m/z (pos): 380.3518 C24H46NO2 (calcd. 380.3529).
(2 mL), and then methyl iodide (97 μL, 0.77 mmol) was
added. The vial was sealed and the resulting mixture was
heat to 65 °C and stirred at that temperature overnight.
Upon return, the reaction mixture was concentrated on a
rotary evaporator and the resulting residue triturated with
diethyl ether. The resulting yellow solid was collected by
vacuum filtration, washed with diethyl ether and dried in
vacuo to provide the desired product (0.24 gm, 81%) as an
orange solid. 1H NMR (400 MHz, CDCl3) δ ppm:
5.21–5.15 (m, 1H, O–CH), 3.19 (s, 2H, N–CH2), 2.58 (s,
3H, N–CH3), 2.55–2.44 (m, 4H, C2, C6 of piperidine
–CH2), 1.85–1.74 (m, 2H, C3, C5 of piperidine –CH2),
1.69–1.40 (m, 22H, C3, C4, C5 of piperidine –CH2,
cyclodecyl –CH2); 13C NMR (100 MHz, CDCl3) δ ppm
163.8 (C=O), 77.6 (cyclodecyl C1), 61.8 (N+–CH2), 53.3
(piperidinyl C2,6), 48.5 (N+–CH3), 30.0 (cyclodecyl
C2,10), 24.9 (cyclodecyl C4,8), 24.8 (cyclodecyl C5, 7),
24.0 (cyclodecyl C6), 22.1 (cyclodecyl C3, 9), 20.5
(piperidinyl C2), 20.2 (piperidinyl C3, 5); ESIMS m/z (pos)
296.2 (100%) [M]+. HRESIMS m/z (pos): 296.2587
C18H34NO2 (calcd. 296.2590).
Synthesis of 1-(2-(cyclodecyloxy)-2-oxoethyl)-1-
methylpiperidin-1-ium iodide (14)
Cyclodecyl 2-chloroacetate In a round bottom flask,
cyclodecanol (Schenker and Gunthard 1952) (3.0 gm,
19.2 mmol) was dissolved in benzene (30 mL), and then
pyridine (1.9 mL, 24.0 mmol) was added. This mixture was
cooled to 0 °C and chloroacetyl chloride (1.53 mL,
19.2 mmol) was added dropwise. The stirred mixture was
then heated at 78 °C and allowed to stir for 3 h. Upon
completion, the reaction was allowed to cool to room
temperature. The mixture was diluted with diethyl ether and
washed with water, 10% aqueous HCl, and finally saturated
aqueous sodium bicarbonate. The organic layer was dried
over anhydrous magnesium sulfate and concentrated on a
rotary evaporator. The residuals were purified by chroma-
tography on silica gel (gradient of 0–10% ethyl acetate in
hexane). The desired product (3.70 gm, 83%) was obtained
as a white solid. 1H NMR (400 MHz, CDCl3) δ ppm:
5.15–5.09 (m, 1H, O–CH), 3.32 (s, 2H, CH2Cl), 2.12–2.04
(m, 2H, C2, C10 cyclodecyl –CH2), 1.75–1.70 (m, 2H, C2,
C10 cyclosdecyl –CH2), 1.58–1.52 (m, 14H, remaining
cyclododecyl –CH2); ESIMS m/z (pos) 255.1 (100%)/257.1
(33%) [M + Na]+.
Synthesis of 1-(2-(Cyclooctyloxy)-2-oxoethyl)-1-
methylpiperidin-1-ium iodide (15)
Cyclooctyl 2-chloroacetate In a round bottom flask,
cyclooctanol (2.46 gm, 19.2 mmol) was dissolved in ben-
zene (30 mL), and then pyridine (1.9 mL, 24.0 mmol) was
added. This mixture was cooled to 0 °C and chloroacetyl
chloride (1.53 mL, 19.2 mmol) was added dropwise. The
stirred mixture was then heated at 78 °C and allowed to stir
for 3 h. Upon completion, the reaction was allowed to cool
to room temperature. The mixture was diluted with diethyl
ether and washed with water, 10% aqueous HCl, and finally
saturated aqueous sodium bicarbonate. The organic layer
was dried over anhydrous magnesium sulfate and con-
centrated on a rotary evaporator. The residuals were purified
by chromatography on silica gel (gradient of 0–10% ethyl
acetate in hexane). The desired product (2.46 gm, 63%) was
isolated as a yellow oil. 1H NMR (400 MHz, CDCl3) δ ppm:
5.11–5.06 (m, 1H, O–CH), 3.35 (s, 2H, CH2Cl), 2.02–1.96
(M, 2H, C2, C10 cyclooctyl –CH2), 1.71–1.65 (m, 2H, C2,
C10 cyclooctyl –CH2), 1.49–1.35 (m, 10H, remaining
cyclooctyl –CH2); ESIMS m/z (pos) 227.1 (100%)/229.1
(33%) [M + Na]+.
Cyclodecyl 2-(piperidin-1-yl)acetate (14a) In a reaction
vial, cyclodecyl 2-chloroacetate (0.50 gm, 1.9 mmol) was
combined with piperidine (0.25 mL, 2.5 mmol) and stirred
at 100 °C for 48 h. The reaction was cooled to room tem-
perature and the mixture were purified by silica gel chro-
matography (0–40% ethyl acetate in hexane) to provide the
1
desired product 14a (0.24 gm, 38%) as a yellow oil. H
NMR (400 MHz, CDCl3) δ ppm: 5.18–5.14 (m, 1H,
O–CH), 3.22 (s, 2H, N–CH2), 2.53–2.47 (m, 4H, C2, C6 of
piperidine –CH2), 1.88–1.79 (m, 2H, C3, C5 of piperidine-
CH2), 1.69–1.40 (m, 22H, C3, C4, C5 of piperidine –CH2,
remaining cyclodecyl –CH2); ESIMS m/z (pos) 282.2
(100%) [M + H]+.
Cyclooctyl 2-(piperidin-1-yl)acetate (15a) In a reaction
vial, cyclooctyl 2-chloroacetate (0.5 gm, 2.15 mmol) was
combined with piperidine (0.28 mL, 2.79 mmol) and stirred
at 100 °C for 48 h. The reaction was cooled to room tem-
perature and the mixture were purified by silica gel chro-
matography (0–40% ethyl acetate in hexane) to provide the
1-(2-(Cyclodecyloxy)-2-oxoethyl)-1-methylpiperidin-1-ium
iodide (14) In a reaction vial, cyclodecyl 2-(piperidin-1-yl)
acetate (0.20 gm, 0.7 mmol) was dissolved in methanol
1
desired product 15a (0.28 gm, 46%) as a yellow oil. H

Medicinal Chemistry Research
NMR (400 MHz, CDCl3) δ ppm: 4.98–4.94 (m, 1H, O–CH),
3.19 (s, 2H, N–CH2), 2.50–2.44 (m, 4H, C2, C6 of piperidine
–CH2), 1.75–1.58 (m, 2H, C3, C5 of piperidine –CH2),
1.50–1.40 (m, 18H, C3, C4, C5 of piperidine –CH2,
cyclooctyl –CH2); ESIMS m/z (pos) 254.2 (100%) [M + H]+.
Upon return, the reaction mixture was concentrated on a
rotary evaporator and the resulting residue triturated with
diethyl ether. The resulting yellow solid was collected by
vacuum filtration, washed with diethyl ether and dried in
vacuo to provide the desired product (0.19 gm, 58%) as an
orange solid. 1H NMR (400 MHz, CDCl3) δ ppm:
4.82–4.75 (m, 1H, O–CH), 3.19 (s, 2H, N–CH2), 2.58 (s,
3H, N–CH3), 2.60–2.51 (m, 4H, C2, C6 of piperidine
–CH2), 1.86–1.77 (m, 2H, C3, C5 of piperidine –CH2),
1.72–1.60 (m, 6H, C2, C6 cyclohexyl –CH2, C3, C4, C5 of
piperidine –CH2), 1.56–1.20 (m, 8H, C2, C6 cyclohexyl
–CH2, remaining cyclohexyl –CH2); 13C-NMR (100 MHz,
CDCl3) δ ppm 163.6 (C=O), 76.1 (cyclohexyl C1), 61.7
(N+–CH2), 61.0 (piperidinyl C2, 6), 48.5 (N+–CH3), 31.3
(cyclohexyl C2, 6), 25.0 (cyclohexyl C5), 23.6 (cyclohexyl
C3, 5), 20.4 (piperidinyl C4), 20.2 (piperidinyl C3, 5);
ESIMS m/z (pos) 240.2 (100%) [M]+; HRESIMS m/z (pos):
240.1960 C14H26NO2 (calcd. 240.1964).
1-(2-(Cyclooctyloxy)-2-oxoethyl)-1-methylpiperidin-1-ium
iodide (15) In a reaction vial, cyclooctyl 2-(piperidin-1-yl)
acetate (0.2 gm, 0.71 mmol) was dissolved in methanol
(2 mL), and then methyl iodide (99 μL, 0.78 mmol) was
added. The vial was sealed and the resulting mixture was
heat to 65 °C and stirred at that temperature overnight.
Upon return, the reaction mixture was concentrated on a
rotary evaporator and the resulting residue triturated with
diethyl ether. The resulting yellow solid was collected by
vacuum filtration, washed with diethyl ether and dried in
vacuo to provide the desired product (0.22 gm, 74%) as a
yellow solid. 1H NMR (400 MHz, CDCl3) δ ppm:
4.95–4.90 (m, 1H, O–CH), 3.16 (s, 2H, N–CH2), 2.6–2.60
(s, 3H, N–CH3), 2.52 (m, 4H, C2, C6 of piperidine –CH2),
1.77–1.57 (m, 2H, C3, C5 of piperidine –CH2), 1.51–1.39
(m, 18H, C3, C4, C5 of piperidine –CH2, cyclooctyl –CH2);
13C NMR (100 MHz, CDCl3) δ ppm 163.5 (C=O), 78.8
(cyclooctyl C1), 61.7 (N+–CH2), 61.1 (piperidinyl C2, 6),
48.5 (N+–CH3), 31.5 (cyclooctyl C2, 8), 26.8 (cyclooctyl
C4, 6), 25.3 (cyclooctyl C5), 22.8 (cyclooctyl C3, 7), 22.7
(piperidinyl C4), 20.4 (piperidinyl C3, 5); ESIMS m/z (pos)
268.2 (100%) [M]+; HRESIMS m/z (pos): 268.2271
C16H30NO2 (calcd. 268.2277).
Synthesis of 1-(2-(Cyclopentyloxy)-2-oxoethyl)-1-
methylpiperidin-1-ium iodide (17)
Cyclopentyl 2-chloroacetate In a round bottom flask,
cyclopentanol (1.65 gm, 19.2 mmol) was dissolved in ben-
zene (30 mL), and then pyridine (1.9 mL, 24.0 mmol) was
added. This mixture was cooled to 0 °C and chloroacetyl
chloride (1.53 mL, 19.2 mmol) was added dropwise. The
stirred mixture was then heated at 78 °C and allowed to stir
for 3 h. Upon completion, the reaction was allowed to cool
to room temperature. The mixture was diluted with diethyl
ether and washed with water, 10% aqueous HCl, and finally
saturated aqueous sodium bicarbonate. The organic layer
was dried over anhydrous magnesium sulfate and con-
centrated on a rotary evaporator. The residuals were purified
by chromatography on silica gel (gradient of 0–10% ethyl
acetate in hexane). The desired product (1.72 gm, 55%) was
Synthesis of 1-(2-(Cyclohexyloxy)-2-oxoethyl)-1-
methylpiperidin-1-ium iodide (16)
Cyclohexyl 2-(piperidin-1-yl)acetate (16a) In a reaction
vial, cyclohexyl 2-chloroacetate (Toronto Research Chemi-
cals, Cat. # C992108) (0.5 gm, 2.83 mmol) was combined
with piperidine (0.37 mL, 3.68 mmol) and stirred at 100 °C
for 48 h. The reaction was cooled to room temperature and the
mixture were purified by silica gel chromatography (0–40%
ethyl acetate in hexane) to provide the desired product 16a
(0.31 gm, 49%) as a yellow oil. 1H NMR (400 MHz, CDCl3)
δ ppm 4.95–4.92 (m, 1H, O–CH), 3.17 (s, 2H, N–CH2),
2.51–2.46 (m, 4H, C2, C6 of piperidine –CH2), 1.81–1.70 (m,
2H, C3, C5 of piperidine –CH2), 1.51–1.42 (m, 14H, C3, C4,
C5 of piperidine –CH2, cyclohexyl –CH2); ESIMS m/z (pos)
226.2 (100%) [M + H]+.
1
isolated as a yellow liquid. H NMR (400 MHz, CDCl3) δ
ppm: 5.11–5.04 (m, 1H, O–CH), 3.30 (s, 2H, CH2Cl),
1.99–1.93 (m, 2H, C2, C5 cyclopentyl –CH2), 1.77–1.70
(m, 2H, C2, C5 cyclopentyl –CH2), 1.49–1.38 (m, 6H,
remaining cyclopentyl –CH2); ESIMS m/z (pos) 185.0
(100%)/187.0 (33%) [M + Na]+.
Cyclopentyl 2-(piperidin-1-yl)acetate (17a) In a reaction
vial, cyclopentyl 2-chloroacetate (0.5 gm, 3.07 mmol) was
combined with piperidine (0.40 mL, 4.0 mmol) and stirred
at 100 °C for 48 h. The reaction was cooled to room tem-
perature and the mixture were purified by silica gel chro-
matography (0–40% ethyl acetate in hexane) to provide the
1-(2-(Cyclohexyloxy)-2-oxoethyl)-1-methylpiperidin-1-ium
iodide (16) In a reaction vial, cyclohexyl 2-(piperidin-1-yl)
acetate (0.2 gm, 0.91 mmol) was dissolved in methanol
(3 mL), and then methyl iodide (136 μL, 1.00 mmol) was
added. The vial was sealed and the resulting mixture was
heat to 65 °C and stirred at that temperature overnight.
1
desired product 17a (0.33 gm, 51%) as a yellow oil. H
NMR (400 MHz, CDCl3) δ ppm 5.10–5.04 (m, 1H, O–CH),
3.20 (s, 2H, N–CH2), 2.52–2.44 (m, 4H, C2, C6 of piper-
idine –CH2), 1.80–1.75 (m, 2H, C3, C5 of piperidine

Medicinal Chemistry Research
–CH2), 1.50–1.38 (m, 12H, C3, C4, C5 of piperidine –CH2,
cyclopentyl –CH2); ESIMS m/z (pos) 212.2 (100%)
(M + H)+.
for 3 h. The reaction was then cooled to room temperature,
diluted with diethyl ether and the organic layer washed with
water, 10% aqueous HCl and saturated aqueous sodium
bicarbonate. The organic layer was dried over anhydrous
magnesium sulfate and concentrated on a rotary evaporator.
The residue was purified by silica gel chromatography
(gradient of 0–10% ethyl acetate in hexane) to provide the
desired product (0.66 gm, 32% over two steps) as a yellow
1-(2-(Cyclopentyloxy)-2-oxoethyl)-1-methylpiperidin-1-ium
iodide (17) In a reaction vial, cyclopentyl 2-(piperidin-1-
yl)acetate (0.2 gm, 0.94 mmol) was dissolved in methanol
(3 mL), and then methyl iodide (140 uL, 1.03 mmol) was
added. The vial was sealed and the resulting mixture was
heat to 65 °C and stirred at that temperature overnight.
Upon return, the reaction mixture was concentrated on a
rotary evaporator and the resulting residue triturated with
diethyl ether. The resulting yellow solid was collected by
vacuum filtration, washed with diethyl ether and dried in
vacuo to provide the desired product (0.16 gm, 49%) as an
yellow solid. 1H NMR (400 MHz, CDCl3) δ ppm:
5.20–5.15 (m, 1H, O–CH), 3.11 (s, 2H, N–CH2), 2.50–2.46
(m, 4H, C2, C6 of piperidine –CH2), 2.44 (s, 3H, N–CH3),
1.85–1.80 (m, 2H, C3, C5 of piperidine –CH2), 1.77–1.37
(m, 12H, C3, C4, C5 of piperidine –CH2, cyclopentyl
–CH2); 13C NMR (100 MHz, CDCl3) δ ppm 164.7 (C=O),
80.3 (cyclopentyl C1), 61.8 (N+–CH2), 53.3 (piperidinyl
C2, 6), 47.9 (N+–CH3), 32.6 (cyclopentyl C2, 5), 23.6
(cyclopentyl C3, 4), 20.4 (piperidinyl C4), 20.2 (piperidinyl
C3, 5); ESIMS m/z (pos) 226.1 (100%) [M]+ HRESIMS
m/z (pos): 226.1805 C13H24NO2 (calcd. 226.1807).
1
oil. H NMR (400 MHz, CDCl3) δ ppm: 4.80–4.75 (m 1H,
N–CH), 2.88 (s, 3H, N–CH3), 2.80 (s, 2H, CH2Cl),
1.72–1.61 (m, 2H, C2, C12 cyclododecyl –CH2), 1.58–1.20
(m, 20H, C2, C12 cyclododecyl –CH2, remaining cyclo-
dodecyl –CH2); ESIMS m/z (pos) 274.2 (100%)/276.2
(33%) [M + H]+.
N-Cyclododecyl-N-methyl-2-(piperidin-1-yl)acetamide In a
reaction vial, 2-chloro-N-cyclododecyl-N-methylacetamide
(0.60 gm, 2.2 mmol) was combined with piperidine
(0.28 mL, 2.8 mmol) and the sealed vial was stirred at
100 °C for 48 h. The reaction was cooled to room tem-
perature and mixture was purified by silica gel chromato-
graphy (gradient of 0–40% ethyl acetate in hexane) to
provide the desired product (0.085 gm, 12%) as a yellow
1
solid. The H NMR indicated the presence of rotational
isomers. ESIMS m/z (pos) 323.3 (100%) [M + H]+.
trans: 1H NMR (400 MHz, CDCl3) δ ppm: 4.81–4.74 (m,
1H, O–CH), 3.48 (br s, 2H, N–CH2), 3.17 (m, 2H, C2, C6
of piperidine –CH2), 2.80 (s, 3H, N–CH3), 1.68–1.48 (m,
10H, C2, C3, C4, C5, C6 of piperidine –CH2, C2, C12
cyclododecyl –CH2), 1.38–1.21 (m, 20H, C2, C12 cyclo-
dodecyl –CH2, remaining cyclododecyl –CH2);
Synthesis of 1-(2-(cyclododecyl(methyl)amino)-2-
oxoethoxy)-1-methylpiperidin-1ium iodide (18)
N-Methylcyclodedecanamine Cyclododecanone (3.0 gm,
16.5 mmol) and 2 M methylamine in tetrahydrofuran
(9.9 mL, 19.8 mmol) were dissolved in anhydrous tetra-
hydrofuran (40 mL). The mixture was cooled to 0 °C and
then treated with lithium aluminum hydride (1 M in tetra-
hydrofuran, 8.2 mL, 8.2 mmol). The reaction mixture was
allowed to come up to room temperature and then stirred
overnight. The reaction was quenched by dropwise addition
of saturated ammonium chloride and then extracted with
dichloromethane. The organic layer was washed with water
and brine, dried over anhydrous magnesium sulfate and
concentrated on a rotary evaporator. The crude product
(2.60 gm, 80%) was obtained as a white solid and was used
in the subsequent reaction without further purification.
ESIMS m/z (pos) 198.3 (100%) [M + H]+.
1
cis: H NMR (400 MHz, CDCl3) δ ppm: 4.28 (br s, 1H,
O–CH), 3.95 (br s, 2H, N–CH2), 2.73 (s, 3H, N–CH3),
2.48–2.40 (m, 4H, C2, C6 of piperidine –CH2), 1.82–1.73
(m, 4H, C3, C5 of piperidine –CH2), 1.57–1.45 (m, 4H, C4
of piperidine –CH2, C12 of cyclododecyl –CH2), 1.38–1.21
(m, 20H, C2, C12 of cyclododecyl –CH2, remaining
cyclododecyl –CH2).
1-(2-(Cyclododecyl(methyl)amino)-2-oxoethoxy)-1-methyl-
piperidin-1-ium iodide (18) In a reaction vial, N-cyclodo-
decyl-N-methyl-2-(piperidin-1-yl)acetamide
(0.20 gm,
0.62 mmol) was dissolved in methanol (2 mL), and then
methyl iodide (93 μL, 0.68 mmol) was added. The vial was
sealed and the resulting mixture was heat to 65 °C and
stirred at that temperature overnight. Upon return, the
reaction mixture was concentrated on a rotary evaporator
and the resulting residue triturated with diethyl ether. The
resulting yellow solid was collected by vacuum filtration,
washed with diethyl ether and dried in vacuo to provide the
desired product (0.24 gm, 84%) as an orange solid. 1H
NMR (400 MHz, CDCl3) δ ppm; 1H NMR (400 MHz,
CDCl3) δ ppm: 4.76–4.74 (m, 1H, O–CH), 4.68 (s, 2H,
2-Chloro-N-cyclododecyl-N-methylacetamide Crude
N-
methylcyclododecanamine (1.5 gm, 7.6 mmol) was dis-
solved in benzene (15 mL) and then pyridine (0.75 mL,
9.5 mmol) was added. The resulting stirred mixture was
cooled to 0 °C and treated dropwise with chloroacetyl
chloride (0.61 mL, 7.6 mmol). Upon complete addition the
reaction was heated to 78 °C and stirred at that temperature

Medicinal Chemistry Research
N–CH2), 4.09–4.03 (m, 2H, C2, C6 of piperidine –CH2),
3.79–3.74 (m, 2H, C2, C6 of piperidine), 3.44 (s, 3H,
N–CH3), 2.97 (s, 3H, N–CH3), 1.97–1.85 (m, 4H, C3, C5 of
piperidine –CH2), 1.82–1.74 (m, 2H, C2, C12 cyclododecyl
–CH2), 1.69–1.60 (C4 of piperidine –CH2), 1.53–1.44 (m,
2H, C2, C12 cyclododecyl –CH2), 1.39–1.25 (m, 18H,
remaining cyclododecyl –CH2); 13C NMR (100 MHz,
CD3OD) δ ppm 167.3 (C=O), 66.1 (N+–CH2), 51.8
(piperidinyl C2,6), 51.5 (cyclododecyl C1), 48.3 (N+–CH3),
31.4 (cyclododecyl C2, 12), 31.3 (amide N–CH3), 27.8
(cyclododecyl C4, 10), 27.4 (cyclododecyl C5, 9), 26.0
(cyclododecyl C6, 8), 26.3 (cyclododecyl C7), 25.7
(piperidinyl C4), 24.8 (cyclododecyl C3,11), 23.6 (piper-
idinyl C3,5); ESIMS m/z (pos) 337.3 (100%) [M+];
HRESIMS m/z (pos): 337.3210 C21H41N2O (calcd.
338.3219).
generated by adding 4 μL of 50× of five concentrations of
test compounds in DMSO to 40 μL of acetonitrile in UV
Star plates followed by 156 μL of the appropriate solu-
bility medium. Analysis and statistics were performed
using GraphPad® Prism v. 5.04. Data are reported as the
maximum concentration observed in the filtrate. The fol-
lowing solubility medium was used in the assays.
Phosphate buffer (pH 7.4): Millipore-defined universal
buffer—45 mM potassium phosphate, 45 mM sodium
acetate, 45 mM ethanolamine, pH = 7.4.
Experimental procedure for determining stability in
mouse and human liver microsomes
The clearance of test compounds in mouse or human liver
microsomes was determined at 37 °C. Assays were con-
ducted in 96-deep well polypropylene plates. Test com-
pounds (1 μM) were incubated in 0.5 mL of 100 mM
potassium phosphate buffer (pH 7.4) with 0.5 mg/mL
pooled liver microsomes from male CD-1 mice (Life
Technologies, Grand Island, NY) or pooled liver micro-
somes from humans (ThermoFisher, Waltham, MA), 2 mM
tetra-sodium NADPH and 3 mM magnesium chloride for
60 min at 37 °C with gentle shaking. At five time points,
75 μL of reaction mixture was transferred to 96-shallow
well stop plates on ice containing 225 mL of acetonitrile
with 0.1 μM propafenone. Control reactions (lacking
NADPH) were performed in a similar manner to demon-
strate NADPH dependency of compound loss. Standard
curves for test compounds were generated using five con-
centrations in duplicate that were processed as above but
with zero incubation time. Stop plates were centrifuged at
2000g for 10 min and then 170 μL of the supernatants were
transferred to a Waters Aquity® UPLC 700 μL 96-well
sample plate with cap mat (Waters, Milford, MA). The
amount of compound remaining in the supernatant was
quantified by LC/MS/MS using a Waters TQ MS (electro-
spray positive mode) coupled to a Waters Aquity® UPLC
(BEH column, C18 1.7 μm, 2.1 × 50 cm, gradient of acet-
onitrile/water/0.1% formic acid). Propafenone was used as
the internal standard. GraphPad® Prism v 5.04 was used for
nonlinear fitting of time course data to generate Clint
values.
In vitro physicochemical/ADME assays
In order to assess the effects of aqueous solubility on the
in vitro pharmacology of our compounds we assessed all
final target molecules for their maximum kinetic aqueous
solubility in 2% DMSO/phosphate-buffered saline. As a
further differentiator we also examined the stability of our
final target molecules in mouse and human liver micro-
somes in the presence and absence of NADPH as an indi-
cator of potential metabolic liability.
Experimental procedure for determining maximum
kinetic aqueous solubility
Solubility assays were performed using Millipore Multi-
Screen®HTS-PFC Filter Plates designed for solubility
assays (EMD Millipore, Billerica, MA). The 96-well
plates consist of two chambers separated by a filter.
Liquid handling was performed using JANUS® Verispan
and MTD workstations (Perkin Elmer, Waltham, MA).
Four microliter of drug solutions (10 mM in DMSO) are
added to 196 μL of the appropriate medium in the top
chamber to give a final DMSO concentration of 2% and a
theoretical drug concentration of 200 μM. Plates are
gently shaken for 90 min and then subjected to vacuum.
Insoluble drug is captured on the filter. One hundred sixty
microliters of the filtrate is transferred to 96-well Griener
UV Star® analysis plates (Sigma-Aldrich, St. Louis, MO)
containing 40 μL of acetonitrile. The drug concentration
in the filtrate is measured by UV absorbance on a Spec-
tromax® Plus microplate reader (Molecular Devices,
Sunnyvale, CA) using Softmax Pro software v. 5.4.5.
Absorbances at 5 wavelengths (280, 300, 320, 340, and
360 nM) were summed to generate the UV signal. Assays
were performed in triplicate. Standard curves were
General biology
In order to assess the ability of compounds to inhibit H4-
stimulated PARP1 activity, we developed a benchtop var-
iation of the high throughput screening assay used by the
Tulin group to identify 5F02 (Thomas et al. 2016). In this
assay, H4 stimulates PARP1 to poly-ADP-ribosylate the
histone. Compounds are assessed for their ability to inhibit

Medicinal Chemistry Research
this activity. Simultaneous with this assay, compounds were
assessed for two drug-like properties using plate-based high
throughput assays, namely maximum solubility in 2%
DMSO/phosphate-buffered saline and potential liability to
oxidative and hydrolytic metabolism as assessed by stability
in mouse and human liver microsomes. Compounds that
demonstrated reasonable potency and drug-like properties
in these assays were then assessed for antitumor effects and
BRCA selectivity. To test the effect of our various 5F02
analogs against BRCA-deficient and BRCA-proficient
tumors, BRCA-pathway proficient NALM6 acute lympho-
blastic leukemia cell line cells and BRCA-pathway deficient
RAD54−/− isogenic counterparts were employed as
described before.
et al. 2018). Briefly, test compounds were added to Nalm-6
isogenic RAD54−/− human leukemia cells or Nalm-6
RAD54+/+ parental cells (Horizon Discovery Group,
Cambridge, UK) for 3–5 days followed by plating in
methylcellulose. Clonogenic activity was assessed 7 days
after plating. Data were plotted and IC50 values determined
using the GraphPad® Prism v 5.04 nonlinear curve fitting
program.
Results and discussion
Compounds 1–17 were prepared based on the method of
Koslov et al. (1989) and using the general method depicted
in Scheme 1 and experimental details are provided in the
Supplementary information section. The required cycloalkyl
alcohols were acylated with chloroacetyl chloride. The
resulting intermediates was treated with the desired cyclic
secondary amine and those intermediates (1a–6a, 8a–17a)
were finally treated with the required alkyl iodide in
refluxing methanol to provide final target compounds 1–6
and 8–17. Methylation of intermediate 7a proved proble-
matic so the desired product 7 was ultimately obtained by
de- protection of the BOC-derivative 8 with trifluoroacetic
acid. To prepare compound 18 the method depicted in
Scheme 2 was employed. Cyclododecylmethylamine was
prepared from cyclododecanone via reductive amination
using a two-step sequence that involved imine formation
followed by reduction with lithium aluminum hydride. The
secondary amine was then converted to 18 using the
methodology depicted in Scheme 1.
The results of the SAR study on the piperidine moiety of
5F02 are shown in Table 1. 5F01 (1) displayed an IC50
value for inhibiting H4-stimulated PARP1 activity of
around 100 nM. Changes in the size of the piperidinyl ring
(compounds 2 and 4) induced an increase in H4/PARP1
inhibitory potency. Likewise, addition of additional lipo-
philic bulk in the 4-position of the piperidine ring (e.g., the
gem-di-methyl of compound 4, the tert-butyl group of
compound 8, and the benzyl moiety of compound 9)
enhanced potency somewhat, suggesting that the lipophilic
binding pocket that accommodates the 6-membered ring of
5F02 may be somewhat longer and wider than the volume
occupied by the chair comformation of that 6-membered
ring. The greatest enhancement in potency seen with
structural changes in this region of the molecule came with
the introduction of a hydrogen bond accepting oxygen atom
in the morpholine derivative (compound 5). This effect was
not mimicked with the thiomorpholine derivative (com-
pound 6), although without a structural model it is not
possible to ascertain if this is caused by the electronic nature
of the sulfur atom or the change in the ring shape and size
induced by the sulfur atom. All of the analogs within this
Experimental procedure for determining inhibition
of H4-stimulated PARP1 activation
Nunc® MaxiSorp™ 96-well plates were coated with
10 ng/well (50 μL) of recombinant human histone H4 in
phosphate-buffered saline (PBS) overnight at room tem-
perature. All subsequent procedures at room temperature.
Plates were washed (200 μL; 5 min/wash) three times and
then blocked for 1 h with PBS-T (PBS + 0.05% Tween
20) with 5% nonfat milk. Plates were then washed twice
with PBS-T and once with assay buffer (AB: 50 mM Tris/
HCl, pH 8.0; 25 mM MgCl2; 0.1% Triton® X-100). 20 μL/
well of multiple concentrations of test compounds in
assay buffer were then preincubated with 20 μL/well of
0.625 U/mL human Parp-1 (Trevigen #4668-500-01) for
15 min. The twenty minute reactions were initiated by the
addition of 10 μL/well of 50 μM NAD+ and stopped by
washing three times with PBS-T.
Plates were then incubated with 50 μL/well (1:4000 in
PBS-T/5% milk) of primary antibody (Tulip #1020; anti-
poly (ADP-Ribose) polymer; clone 10H) for 1 h. Plates
were washed three times and then incubated with 50 μL/
well (1:1500 in PBS-T/milk) of secondary HRP-conjugated
goat anti-mouse antibody for 1 h. Plates were washed three
times with PBS-T and once with PBS and then 50 μL/well
of SureBlue reagent was added. After 10 min, 50 μl/well of
1N hydrochloric acid stop solution was added. Absorbance
(A450-A650) was measured with a Molecular Devices
SpectraMax Plus® plate reader. IC50’s were determined
using the GraphPad® Prism v 5.04 nonlinear curve fitting
program.
Experimental procedure for measuring the effect of
drugs on RAD54−/− Nalm-6 human leukemia cells
and RAD54+/+ Nalm-6 parental cells
The protocol for the Nalm-6 cellular assays have been
described elsewhere (Karanam et al. 2012; Sullivan-Reed

Medicinal Chemistry Research
Scheme 1 Synthesis of compounds 2–17. Reagents and conditions: a chloroacetyl chloride, pyridine, benzene, reflux 3 h; b cyclic secondary
amine, 100 °C; c alkyl iodide, methanol, reflux; d 18 h; trifluoroacetic acid, r.t., 15 min
group demonstrated similar maximum aqueous solubility
(100–200 μM range in 2% DMSO/PBS) that were well
within the range tested in our assays. All of the analogs
tested, including 5F02, were found to be labile to metabo-
lism in both mouse and human liver microsomes in the
presence of NADPH, something that is not totally unex-
pected given the presence of the cycloalkyl group. How-
ever, all of the analogs within this group were stable in
microsomes in the absence of NADPH, suggesting that the
ester group is surprisingly stable to enzymatic hydrolysis.
To address the question of the necessity of the positive
charge on the quaternary nitrogen we tested the penultimate
intermediates (lacking the quaternary methyl group) of each
final target molecule. We had previously shown that the
des-methyl, non-quaternary intermediate leading to 5F02
was essentially inactive in the H4/PARP1 assay established
in the Tulin lab (Karpova et al. 2019).
A representative group of non-charged derivatives
Scheme 2 Synthesis of compound 18. Reagents and conditions: a 66%
(Intermediates 1a–9a) showed no inhibition of H4-
aq, methylamine, methanol, reflux, 2 h; b lithijm aluminum hydride,
stimulated PARP1 activity in our hands at concentrations
up to 10 μM (Table S1, Supplementary information sec-
tion). Thus, either the charge or the presence of the
tetrahydrofuran; c chloroacetyl chloride, pyridine, benzene, reflux 3 h;
d cyclic amine, 100 °C; e iodomethane, methanol, reflux, 18 h

Medicinal Chemistry Research
Table 1 In vitro H4/PARP1 inhibitory activity, maximum kinetic aqueous solubility and mouse and human liver microsomal stability of
compounds 1–9
Liver Microsomal Stability (t1/2, min)
H4/PARP
Inhibition
IC50, nM
Max. Aq.
Solubility
(uM)
Cd
#
Mouse
Human
R
logP
+ NADPH
- NADPH*
92%
+ NADPH*
- NADPH*
88%
1
2
3
5.11
4.66
5.56
104
52.4
54
4.9
3.8
1.8
3.4
< 2
< 2
177
125
140
98%
87%
92%
95%
4
5.52
45.6
2.6
84%
< 2
92%
95
5
6
7
3.65
4.32
3.34
34.5
156
466
5.1
4
100%
88%
100
< 2
< 2
4
87%
85%
100
200
110
185
13.3
8
9
5.08
6.79
55.8
61.8
4.1
5
100
100
< 2
< 2
83
87
200
113
aPercent compound remaining after 1 h @ 37 °C
quaternary alkyl group is necessary for potent inhibitory
activity.
(compound 12) derivatives, while not quite as potent as
5F02, were more potent than the ethyl analog. A size lim-
itation was reached with the n-pentyl group (compound 13),
which demonstrated a significant loss in potency compared
with the other derivatives in this group. Maximum aqueous
solubility for these compounds was similar to that seen with
5F02. Compounds 12 and 13, with the longer alkyl chains,
demonstrated increased stability in mouse liver microsomes
Since the initial SAR results indicated that the quaternary
nitrogen played some role in binding, we next explored the
size of the alkyl group on that quaternary nitrogen. The
results are shown in Table 2. The ethyl analog (compound
10) was less potent than 5F02 (compound 1). Interestingly,
the more flexible n-propyl (compound 11) and n-butyl

Medicinal Chemistry Research
in the presence of NADPH compared with compounds 1–
11. However, both compounds showed some loss of
material in liver microsomes in the absence of NADPH,
suggesting a potential liability to hydrolytic metabolism.
The next region of the 5F02 scaffold that we explored
was the cyclododecyl group. To accomplish this task we
systematically reduced the ring size in the order: 12 (1):10
(14):8 (15):6 (16):5 (17). The results are presented in
Table 3.
Reducing the ring size, on the whole, resulted in deri-
vatives with increased H4/PARP1 inhibitory potency,
although as with the other structural changes the increase in
potency was not dramatic. This limited sensitivity to
structural changes is not uncommon for small molecule
inhibitors of protein–protein interactions (Jin et al. 2014).
The greatest effect was seen with the cyclodecyl derivative
14 and the cyclopentyl analog 17, which both demonstrated
approximately twofold greater inhibitory potency than
5F02. Reducing the size of the cycloalkyl ring, however,
Table 2 In vitro H4/PARP1 inhibitory activity, maximum kinetic aqueous solubility and mouse and human liver microsomal stability of
compounds 10–13
Liver Microsomal Stability (t1/2, min)
H4/PARP
Inhibition
IC50, nM
Max. Aq.
Solubility
(uM)
Cd
#
Mouse
Human
R
logP
+ NADPH
- NADPH*
+ NADPH*
- NADPH*
1
Me
Et
5.11
5.52
5.97
6.42
6.87
104
174
142
136
258
4.9
4.7
92%
96%
100%
89%
70%
3.4
< 2
< 2
< 2
< 2
88%
87%
100%
100%
100%
177
200
194
186
196
10
11
12
13
n-Pr
8.9
n-Bu
n-Pentyl
16.2
16.0
aPercent compound remaining after 1 h @ 37 °C
Table 3 In vitro H4/PARP1 inhibitory activity, maximum kinetic aqueous solubility and mouse and human liver microsomal stability of
compounds 14–17
Liver Microsomal Stability (t1/2, min)
H4/PARP
Inhibition
IC50, nM
Max. Aq.
Solubility
(uM)
Cd
#
Mouse
Human
R
logP
+ NADPH
- NADPH*
92%
+ NADPH*
- NADPH*
1
5.11
104
4.9
3.4
88%
177
14
15
4.21
3.31
53.7
80.0
6.2
2.2
99%
97%
7.7
100%
95%
181
182
> 60
16
17
2.41
1.96
77.1
57.1
2.9
100%
97%
> 60
> 60
100%
90%
187
200
21.2
aPercent compound remaining after 1 h @ 37 °C

Medicinal Chemistry Research
had a beneficial effect on stability to oxidative metabolism.
While the cyclodecyl analog was still very labile to oxida-
tive metabolism in mouse and human liver microsomes,
compounds 15–17 were found to be stable in human liver
microsomes for up to 60 min (the longest time tested) and
the cyclopentyl derivative 17 also demonstrated increased
stability in mouse liver microsomes (t1.2 = 21.2 min). All of
these derivatives were stable in mouse and human liver
microsomes in the absence of NADPH, and these results
were not influenced by solubility limitations. Thus, the
Fig. 3 Structure and screening data for compound 18
Fig. 4 The effect of 5F02 allosteric analogs on the clonogenic activity
of RAD54 deficient and proficient leukemia cells. NALM6 parental
cells and NALM6 RAD54−/− isogenic counterparts (103/100 μL)
were treated for 72 h with the indicated compounds followed by
plating in methylcellulose as described before (Nieborowska-Skorska
et al. 2017). Results represent mean SD percentage of colonies in
comparison to untreated cells from triplicate experiments

Medicinal Chemistry Research
cyclocodecyl group of 5F02 appears to be a weak spot for
oxidative metabolism.
plasma protein binding and/or saturation of metabolism at
higher doses (Karpova et al. 2019). Surprisingly, the ester
group appears relatively stable to hydrolytic metabolism,
which could indicate that it is shielded from enzymatic
hydrolysis by the cycloalkyl group and/or the quaternary
amine moiety.
The last region of the molecule we explored was the ester
group. We had previously reported that substitution of the
ester with a secondary amide significantly reduced the H4/
PARP1 inhibitory activity of the 5F02 scaffold significantly
(Karpova et al. 2019). Hypothesizing that either a hydrogen
bond donor in this position might be detrimental to binding
or the presence of the secondary amide might be stabilizing
an unfavorable conformation compared with the ester group
(Lewin and Frucht 1975), we prepared the tertiary amide
derivative 18. Screening results are summarized in Fig. 3.
While not as potent as the homologous ester analogs 1–9,
compound 18 did inhibit H4/PARP1 activity to a greater
extent than the analogous secondary amide (Karpova et al.
2019), giving an IC50 value in our hands of 191 nM.
Unfortunately, the compound was still labile to oxidative
metabolism in liver microsomes.
Because no dramatic SAR trends were realized in our
SAR studies we elected to test most of the analogs in our
in vitro clonogenic cancer cell model. The model employs
the human isogenic leukemia cell line NALM6 wherein the
cells have been made BRCA-deficient by eliminating
RAD54 activity (RAD54−/−) (Fujita et al. 2013). Results
in this cell line are compared with results obtained with
RAD54+/+ parental cells which possess RAD54 activity
and are BRCA-proficient to ascertain the selectivity of
compounds for BRCA-deficient cancers. Results are pre-
sented in Fig. 4.
5F02 (1) demonstrated a therapeutic window for killing
BRCA-deficient cells (open circles) compared with BRCA-
proficient cells (closed circles). In general, compounds that
demonstrated a lower IC50 value in the in vitro H4-
stimulated PARP1 assay tended to be more potent in the
NALM6 model, although most of the compounds tested
showed little or no selectivity for BRCA-deficient cells over
the BRCA-proficient parental cells. The two exceptions to
this trend toward lack of selectivity were compounds 5 and
7, which were both more potent at killing BRCA-deficient
NALM6 RAD54−/− leukemia cells than the RAD54+/+
BRCA-proficient parental counterparts. The reasons for this
are not clear at present, but the results emphasize the lim-
itations of relying only on biochemical assays for profiling
anticancer compounds and the need to support biochemical
results with data generated in live tissue.
Conclusions
In conclusion, we have expanded our previous findings on
non-NAD like inhibitors of H4-stimulated PARP1 activity
to obtain a better understanding of the SAR and SPR within
this chemical scaffold. Compound 7 displayed potent,
BRCA-selective anti-cancer activity in the NALM6 human
leukemia cell line and a greater stability in mouse liver
microsomes compared with 5F02 (1) while compound 17
gave indication that manipulating the cycloalkyl group may
lead to compounds with improved metabolic stability.
Synergy studies with compound 7 and olaparib are currently
in progress, as are mechanistic studies to better understand
the mechanism for the selectivity seen with 5F02 and our
lead molecule. These results will be reported in the future.
Acknowledgements The authors would like to thank Dr Furong Sun
and the staff at the Mass Spectrometry Lab, School of Chemical
Sciences, University of Illinois at Urbana-Champaign for providing
the high resolution mass spectrometry data.
Funding This work was funded by the National Institutes of Health/
National Cancer Institute under R01 CA186238 to TS. MT was sup-
ported by an Etiuda6 scholarship awarded to her by the Polish National
Science Centre.
Author contributions EH and MT contributed equally to the paper.
EH designed and synthesized the molecules described in the paper and
contributed to the preparation of the paper. MT performed pharma-
cological experiments, prepared figures and contributed to the pre-
paration of the paper. KS-R performed pharmacological experiments.
JG performed the in vitro ADME studies. TS oversaw the efforts of
MT. WEC supervised the medicinal chemistry efforts, helped design
the molecules described in the paper and revised the paper. TS
supervised the pharmacology efforts, designed the pharmacology
protocols used in the paper and revised the paper. AT contributed to
the preparation and revision of the paper.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
The structural changes made to date resulted in only
limited improvements in metabolic stability and the trend
was seen more in mouse liver microsomes than in human
liver microsomes. Those changes that imparted the greatest
improvement in liver microsomal stability resulted in
compounds that lacked BRCA-deficient selectivity. That
being said, 5F02 possessed a longer than expected in vivo
half-life, which might be the result of its relatively high
interest.
Ethical approval All experiments carried out in the course of this work
were performed in compliance with OSHA regulations and under
protocols that were approved by the Temple University Institutional
Biosafety Committee.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.

Medicinal Chemistry Research
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