Discovery of potent 2,4-difluoro-linker poly(ADPribose) polymerase 1 inhibitors with enhanced water solubility and in vivo anticancer efficacy

Article abstract of DOI:10.1038/aps.2017.104

Poly (ADP-ribose) polymerase 1 (PARP1) is overexpressed in a variety of cancers, especially in breast and ovarian cancers; tumor cells that are deficient in breast cancer gene 1/2 (BRCA1/2) are highly sensitive to PARP1 inhibition. In this study, we identified a series of 2,4-difluorophenyl-linker analogs (15-55) derived from olaparib as novel PARP1 inhibitors. Four potent analogs 17, 43, 47, and 50 (IC50=2.2-4.4 nmol/L) effectively inhibited the proliferation of Chinese hamster lung fibroblast V-C8 cells (IC50=3.2-37.6 nmol/L) in vitro, and showed specificity toward BRCA-deficient cells (SI=40-510). The corresponding hydrochloride salts 56 and 57 (based on 43 and 47) were highly water soluble in pH=1.0 buffered salt solutions (1628.2 μg/mL, 2652.5 μg/mL). In a BRCA1-mutated xenograft model, oral administration of compound 56 (30 m?kg-1d-1, for 21 d) exhibited more prominent tumor growth inhibition (96.6%) compared with the same dose of olaparib (56.3%); in a BRCA2-mutated xenograft model, oral administration of analog 43 (10 m?kg-1d-1, for 28 d) significantly inhibited tumor growth (69.0%) and had no negative effects on the body weights. Additionally, compound 56 exhibited good oral bioavailability (F=32.2%), similar to that of olaparib (F=45.4%). Furthermore, the free base 43 of the hydrochloride salt 56 exhibited minimal hERG inhibition activity (IC50=6.64 μmol/L). Collectively, these data demonstrate that compound 56 may be an excellent drug candidate for the treatment of cancer, particularly BRCA-deficient tumors.

Full text of DOI:10.1038/aps.2017.104

Acta Pharmacologica Sinica 2017: 1–12
© 2017 CPS and SIMM All rights reserved 1671-4083/17
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Original Article
Discovery of potent 2,4-difluoro-linker poly(ADP-
ribose) polymerase 1 inhibitors with enhanced water
solubility and in vivo anticancer efficacy
Wen-hua CHEN1, #, Shan-shan SONG2, #, Ming-hui QI3, #, Xia-juan HUAN2, Ying-qing WANG2, Hualiang JIANG1, 4, Jian DING2,
2,
1,
Guo-bin REN3, , Ze-hong MIAO , Jian LI
*
*
*
1Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai
200237, China; 2Division of Antitumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, Shanghai 201203, China; 3Laboratory of Pharmaceutical Crystal Engineering & Technology, School
of Pharmacy, East China University of Science and Technology, Shanghai 200237, China; 4State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
Abstract
Poly (ADP-ribose) polymerase 1 (PARP1) is overexpressed in a variety of cancers, especially in breast and ovarian cancers; tumor cells
that are deficient in breast cancer gene 1/2 (BRCA1/2) are highly sensitive to PARP1 inhibition. In this study, we identified a series
of 2,4-difluorophenyl-linker analogs (15−55) derived from olaparib as novel PARP1 inhibitors. Four potent analogs 17, 43, 47, and
50 (IC50=2.2−4.4 nmol/L) effectively inhibited the proliferation of Chinese hamster lung fibroblast V-C8 cells (IC50=3.2−37.6 nmol/L)
in vitro, and showed specificity toward BRCA-deficient cells (SI=40−510). The corresponding hydrochloride salts 56 and 57 (based
on 43 and 47) were highly water soluble in pH=1.0 buffered salt solutions (1628.2 μg/mL, 2652.5 μg/mL). In a BRCA1-mutated
xenograft model, oral administration of compound 56 (30 mg·kg-1·d-1, for 21 d) exhibited more prominent tumor growth inhibition
(96.6%) compared with the same dose of olaparib (56.3%); in a BRCA2-mutated xenograft model, oral administration of analog 43
(10 mg·kg-1·d-1, for 28 d) significantly inhibited tumor growth (69.0%) and had no negative effects on the body weights. Additionally,
compound 56 exhibited good oral bioavailability (F=32.2%), similar to that of olaparib (F=45.4%). Furthermore, the free base 43 of
the hydrochloride salt 56 exhibited minimal hERG inhibition activity (IC50=6.64 μmol/L). Collectively, these data demonstrate that
compound 56 may be an excellent drug candidate for the treatment of cancer, particularly BRCA-deficient tumors.
Keywords: PARP-1 inhibitor; olaparib; breast cancer; BRCA-deficient tumors
Acta Pharmacologica Sinica advance online publication, 3 Aug 2017; doi: 10.1038/aps.2017.104
lance, cell cycle progression, DNA damage response initia-
Introduction
Poly (ADP-ribose) polymerases (PARPs) are ADP-ribosyltrans- tion, apoptosis, and transcription regulation[5]. PARP1 is the
ferases that cleave nicotinamide adenine dinucleotide (NAD+) most abundant among the PARP family members and plays
into ADP-ribose and nicotinamide and then transfer the ADP- an essential role in the repair of DNA single-strand breaks
ribose units to a variety of target proteins (such as histones, (SSB)[6, 7]. PARP1 is responsible for more than 90% of ADP-
topoisomerases, DNA polymerases, and DNA ligases) or to ribosylation occurring in cells and is evolutionarily conserved
themselves[1-4]. PARPs are involved in a number of impor- in all advanced eukaryotes[8, 9]. PARP1 is a 113 kDa enzyme
tant biological processes, such as genome integrity surveil- that contains three major structural domains, including a
42 kDa DNA-binding domain in the N-terminal region with
two zinc fingers, a 55 kDa catalytic domain in the C-termi-
# These authors contributed equally to this work.
To whom correspondence should be addressed.
E-mail jianli@ecust.edu.cn (Jian LI);
zhmiao@simm.ac.cn (Ze-hong MIAO);
rgb@ecust.edu.cn (Guo-bin REN)
nal region and an auto-modification domain in the central
*
region[10-12]. The DNA-binding domain can recognize, and
bind damaged DNA single-strand breaks and consequently
stimulate the polymerization of ADP-ribose, thus leading to
unwinding of DNA from histones and exposing the damaged
Received 2016-12-09 Accepted 2017-04-04

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Chen WH et al
DNA for repair. The catalytic domain utilizes NAD+ as a bond interactions between the fluorine atom and drug tar-
substrate to construct linear and branched polymers of ADP- gets. In addition, fluorine is a powerful tool for modulating
ribose on its targets, and thus performs a pivotal role in DNA the pKa of proximal functional groups and the electron densi-
damage repair.
ties of (hetero)aromatic rings[36-38]. The pKa of an ionizable
PARP1 is overexpressed in a variety of cancers, and the center in a drug molecule determines the lipophilic profile,
expression of PARP1 has been associated with overall prog- which in turn influences solubility, permeability, and protein
nosis in cancers, especially breast and ovarian cancers[11, 12]
;
binding. Changes in pKa can manifest as changes in potency,
therefore, PARP1 has emerged as an attractive anticancer selectivity, toxicity, and pharmacokinetic (PK) properties,
drug target for anchoring DNA damage. Cell lines deficient including absorption, distribution, metabolism, and excretion
in the breast cancer genes 1/2 (BRCA1/2, two known tumor (ADME)[39-43]. Evidently, the incorporation of fluorine into
suppressor genes whose mutations are associated with breast, drug molecules is a commonly used strategy to increase phar-
ovarian, prostate, and pancreatic cancers) were 1000-fold more macological activity and optimize the ADME profile[44]
.
sensitive to PARP inhibition than either wild-type or heterozy-
The discovery and investigation of olaparib have indi-
gous mutant cells. Therefore, the inhibition of PARP1 induces cated that the phthalazine fragment is essential and that the
highly selective killing of BRCA1/2-deficient tumor cells. In piperazine segment is flexible to support PARP1 inhibition
this regard, many small molecules targeting PARP1 have been potency[24]. Given the therapeutic potential of olaparib as a
developed as chemosensitizers used in combination with ion- PARP1 inhibitor, we designed a novel series of 2,4-difluoro-
izing radiation, as DNA-damaging chemotherapeutic agents linkers for analog 15 derived from olaparib (Figure 2) to obtain
or as stand-alone therapies (synthetic lethal) to kill cancer cells novel olaparib analogs. Under the premise of minimizing
defective in DNA repair mechanisms (especially BRCA1/2 structural changes, we introduced the 4-F substituent on the
mutant breast or ovarian tumors)[13-21]
.
phenyl linker. The molecular docking studies (described
Many PARP inhibitors have previously been described, below) showed that not only the fluorine atom at position 4
most of which are analogs of nicotinamide[7, 22, 23]. Several but also the fluorine atom at position 2 of analog 15 formed
PARP inhibitors (Figure 1) have been used in late-phase a hydrogen bond with Glu763 and Ser864, respectively, thus
(phases 2 and 3) clinical trials and approved for some ovar- indicating that the 2,4-difluorophenyl-linker analogs of 1
ian cancers[2]; these inhibitors include olaparib (1, AZD-2281; might improve biological activity. To explore the SAR (struc-
AstraZeneca/Kudos)[24], rucaparib (2, AG-014699; Clovis/ ture-activity relationship) of the 2,4-difluorophenyl-linker
Pfizer)[25, 26], niraparib (3, MK-4827; Tesaro/Merck)[27, 28], veli- analogs of 1, we designed a novel series of 2,4-difluorophenyl-
parib (4, ABT-888; AbbVie)[29-31], and talazoparib (5, BMN 673; linker analogs (15−55) derived from 1 and comprehensively
BioMarin/LEAD/Medivation)[32]
.
evaluated whether a specific type of substituent (R, Figure 2)
The fluorine atom plays an important and unique role in on the terminal nitrogen of piperazine could improve phar-
influencing molecular conformation. From the perspective of macological activity and modulate water solubility. Finally,
steric effects, the influence of fluorine is anticipated to be mar- a highly potent PARP1 enzymatic inhibitor, 56, which has
ginal, and fluorine is a small atom with a van der Waals radius excellent in vivo anti-tumor efficacy, good oral bioavailability,
of 1.47 Å, which is close to the 1.20 Å radius of hydrogen[33-35]
.
enhanced water solubility and a good safety profile was iden-
Moreover, the high electronegativity of fluorine results in tified as a unique candidate compound for the development of
highly polarized C−F bonds and in new potential hydrogen antitumor drugs.
Figure 1. PARP inhibitors currently in late-phase (phases 2 and 3) clinical trials or already approved for clinical use.
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Chen WH et al
3
Figure 2. Chemical modification strategies (15–55) for lead compound olaparib.
Figure 3. Molecular docking studies on 1 and 15. Docked poses of 1 (green sticks) (A) and 15 (green sticks) (B) in the NAD+ binding site (cyan and pink
cartoon). Hydrophilic interacting residues are represented with yellow carbons. Hydrogen bonding distances (Å) are represented with black numbers.
Hydrogen bonds are shown with yellow dash lines.
1-methylbenzene (6) in the presence of N-iodosuccinimide
Materials and methods
Reagents
in trifluoroacetic acid at 0 °C for 24 h, and a 73% yield was
Reagents and solvents were purchased from Adamas-Beta, achieved. Further treatment of compound 7 with cuprous
J&K, Energy Chemical, Target Molecule, TCI, Alfa Aesar, and cyanide from N,N-dimethylformamide under reflux for 20 h
Acros, and were used without further purification. Analytical gave rise to 2,4-difluoro-5-methylbenzonitrile (8), with a 62%
thin-layer chromatography (TLC) was performed with HSGF yield. The oxidation of compound 8 with chromic anhydride
254 (150–200 μm thickness; Yantai Huiyou Co, China), and from the mixture of acetic anhydride and glacial acetic acid
the spots were visualized with UV light. The yields were not at 0 °C for 4 h yielded compound 9, with a 30% yield. The
optimized. Melting points were measured in a capillary tube hydrolysis of compound 9 provided 2,4-difluoro-5-formyl-
with an SGW X-4 melting point apparatus without correc- benzonitrile (10). Dimethyl (3-oxo-1,3-dihydroisobenzofuran-
tion. Nuclear magnetic resonance (NMR) spectroscopy was 1-yl)phosphonate (12) was prepared from the reaction of
performed on a Bruker AMX-400 NMR (IS as TMS). Chemical 2-formyl-benzoic acid with dimethyl phosphonate and meth-
shifts were determined in parts per million (ppm, δ) down- anesulfonic acid in sodium methoxide at 0 °C for 6 h, with
field from tetramethylsilane. Proton coupling patterns were a 52% yield. The coupling of compound 10 and compound
described as follows: singlet (s), doublet (d), triplet (t), quartet 12 afforded compound 13, through use of triethylamine and
(q), multiplet (m), or doublet of doublets (dd). High-resolution tetrahydrofuran at room temperature for 5 h, with an 86%
mass spectra (HRMS) were determined with electric ioniza- yield. The 2,4-difluoro-5-((4-oxo-3,4-dihydroph-thalazin-1-yl)
tion (EI) and electrospray ionization (ESI) produced by Waters methyl)benzoic acid (14) was prepared from compound 13
GCT Premier and Waters LCT mass spectrometers. HPLC with hydrazine hydrate and sodium hydroxide at 70−90 °C for
analysis of analogs 15–55 was performed on an Agilent 1100 21 h, with a 75% yield. The subsequent condensation of acid
with a quaternary pump and a diode array detector (DAD). 14 with an appropriate piperazine substrate in the presence
The peak purity was verified on the basis of UV spectra. All of HOBt, EDCI and dichloro-methane produced the target
analogs were confirmed to be ≥95% pure.
analogs 15–55, with yields of 10%−60%[25]. All analogs were
confirmed to be ≥95% pure (Supplementary Table S1).
Chemistry
As outlined in Scheme 1, 1,5-difluoro-2-iodo-4-methylbenzene PARP1 enzyme inhibition assays
(7) was prepared from commercially available 2,4-difluoro- The inhibition of the tested compounds on PARP1 enzymatic
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Chen WH et al
Scheme 1. Synthesis of analogues 15−55. aReagents and conditions: (a) NIS, TFA, 0 °C, 24 h; (b) DMF, CuCN, reflux, 20 h; (c) CrO3, (CH3CO)2O,
CH3COOH, 0 °C, 4 h; (d) EtOH, H2O, reflux, 2 h; (e) MeONa, (CH3O)2PHO, CH3SO3H, 0 °C, 2 h; (f) THF, Et3N, 15 °C, 1 h; (g) (i) NaOH, 90 °C, 1 h; (ii) NH2-
NH2·H2O, 70 °C, 18 h; (h) HOBt, EDCI, DIPEA, piperazine, DMA, overnight.
activity in a cell-free system was measured with enzyme- for 168 h. The IC50 values were measured with a sulforhoda-
linked immunosorbent assays (ELISA)[45-47]. Briefly, NAD+ (6 mine B colorimetric assay.
μmol/L) and the activator deoxyoligonucleotide (100 μg/mL)
were diluted in 70 μL of reaction buffer and added to each Determination of water solubility
well of a histone-pre-coated 96-well plate, and this was fol- The water solubility of 1, 17, 43, 47, 50, and 56−58 was deter-
lowed by the addition of 10 μL of the compound or a solvent mined through an HPLC method. Stock solutions (800
control. The reaction was started by addition of 20 μL of μg/mL) of the samples were prepared in methanol. Then, 10
recombinant human PARP1 (10 ng/well) for 1.5 h. After cells μL of the diluted solutions (50, 100, 200, 400, and 800 μg/mL)
were washed with PBST, 100 μL of anti-PARP polyclonal anti- were injected into the HPLC system to assess linearity. Cali-
body (Trevigen) was added, and the mixture was incubated bration curves were plotted as peak area versus the concentra-
for another 1.5 h. After washing the cells, the second anti- tion of the sample. Next, 10 mg of the sample was added to
body, goat anti-rabbit IgG horseradish peroxidase, was added, a 5-mL centrifuge tube, and 1 mL of pure water or a buffered
and the mixture incubated for an additional 30 min. Finally, a salt solution (pH=7.4 or 1.0) was pipetted into the tube. If the
solution of 0.03% H2O2 and 2 mg/mL OPD in 0.1 mol/L citrate solution was unsaturated and remained clear, more testing
buffer (pH 5.4) was added, and the mixture was incubated for was needed. After the solution was stirred for 24 h, it was fil-
10 min. The reaction was stopped by the addition of 2 mol/ tered with a syringe, and the HPLC system was injected with
L H2SO4. A490 was measured with a multi-well spectropho- the same 10 μL. Water solubility was calculated by compari-
tometer (SPECTRA MAX190). The inhibition rate of PARP1 son of the peak area of the tested compound to the calibration
enzymatic activity was calculated as (A490control-A490treated
A490control)×100%. The concentration required to inhibit 50%
of PARP1 enzymatic activity (IC50) was determined with the Pharmacokinetic analysis
/
curves.
Logit method.
Compounds 1 and 56 were tested in pharmacokinetic stud-
ies using Sprague-Dawley (male) rats obtained from Shang-
Cytotoxicity assays
hai Sippr-BK Laboratory Animal Co Ltd. The animals were
The V-C8 and V79 cells obtained from Leiden University were housed in a room with controlled temperature and humidity
seeded on 96-well plates, cultured overnight and treated with and were allowed free access to food and water. For studying
gradient concentrations of the tested compounds for 72 h. The compounds 1 and 56, the rats were divided into 2 groups (3
IC50 values were measured with a Cell Counting Kit-8 (CCK-8) rats/group), and each group received the compounds intrave-
assay (Dojindo Laboratories, Japan)[47, 48]
.
nously at a dose of 10 mg/kg or orally at a dose of 20 mg/kg,
The UWB1.289 and UWB1.289+BRCA1 cells obtained from respectively. Compounds 1 and 56 were dissolved in DMSO
ATCC were seeded on 96-well plates, cultured overnight and (5%) and 15% HP-β-CD water (95%) to make a 5 mg/mL stock
treated with gradient concentrations of the tested compounds concentration. Blood samples from the orally treated rats and
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Chen WH et al
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the intravenously injected rats were collected at 0.083, 0.25, 0.5, analog 15 not only displayed good enzymatic potency com-
1, 2, 4, 6, 8, and 24 h post-dose. All blood samples were cen- pared with that of olaparib (IC50=16.1 nmol/L vs 7.3 nmol/L)
trifuged at 8000 revolutions per minute at 4 °C for 6 min. The but also showed better cell inhibition activity (IC50=167.1
plasma was collected and stored at -80 °C. All plasma sam- nmol/L vs 263.6 nmol/L) in the V-C8 cytotoxicity assay (Table
ples were analyzed within one week after collection. Sample 1). Encouraged by this result, we turned our attention to
analysis was performed via LC-MS/MS using an Agilent 1100 explore SAR by incorporating various substituents (R) on the
chromatography system with a Phenomenex Gemini-NX C18 N-atom of the piperazine ring of analog 15.
column (5 μm, 50 mm×2.1 mm). The mobile phase consisted
First, a small set of analogs with various acyl substituents
of mixtures of 0.1% formic acid in acetonitrile and 0.1% formic (16−20, Table 1) on the terminal nitrogen of the piperazine
acid in water and was run in gradient-mode at a flow rate of ring were examined. The enzymatic inhibitory activity of the
0.6 mL/min. Mass spectra were detected with an API4000 acetyl analog 16 (IC50=30.0 nmol/L) was 2-fold less than that
triple quadruple equipped with an ESI source. Internal stan- of analog 15. Replacing the cyclopropane with tetrahydrofu-
dards were used to track the responses of analytes in plasma ran or morpholine resulted in analogs 18 and 19, respectively,
samples. Plasma concentration-time data were analyzed, and which had similar enzymatic inhibitory activities (IC50=12.5
the pharmacokinetic parameters were calculated.
nmol/L and 14.0 nmol/L, respectively). Interestingly, the
benzoyl (17) and 4-methylbenzoyl (20) analogs showed greater
enzymatic potencies than that of olaparib (IC50=3.5 nmol/L
Xenograft experiments
Human breast cancer MDA-MB-436 xenografts in nude mice and 4.6 nmol/L vs 7.3 nmol/L).
were used to evaluate the anticancer activities of 1, 56 and 57,
To further develop this 2,4-difluorophenyl linker, we next
and human pancreatic cancer Capan-1 xenografts were used explored analogs 21−25, in which the acyl fragment was
to evaluate the anticancer activities of 5 and 43. The model replaced by alkyl groups (Table 1). Analogs 24 and 25 showed
was established by the transplantation of 5×106 MDA-MB-436 similar enzymatic activities to that of 15 (IC50=15.3 nmol/L
cells or Capan-1 cells that were subcutaneously injected into and 10.4 nmol/L vs 16.1 nmol/L). However, analogs 21−23
the right armpit of each of the 4-5-week-old female BALB/cA replaced the acyl fragment with smaller methyl, ethyl, and
nude mice. When the average tumor volume reached approxi- propyl groups through enzymatic potency reductions that
mately 150 mm3, the mice were selected on the basis of tumor were approximately 2−10-fold. These data indicated a favor-
volume and randomly assigned to vehicle, 1, 5, 56, 57 and 43 able role for bulky substituents on the terminal nitrogen of the
groups. For 21 consecutive days, the animals in the 56 and 57 piperazine ring. Thus, we next introduced either arylmethyl
groups were given daily oral doses of 56 and 57 (1 mg·kg-1·d-1, (26−29) or aryl (30) groups on the terminal nitrogen of the
10 mg·kg-1·d-1 and 30 mg·kg-1·d-1, respectively). The animals in piperazine ring (Table 1). Analog 26, which had a benzyl
the positive control (1) group were given an oral dose of olapa- substitution, demonstrated an enzymatic inhibitory activity
rib (30 mg/kg). For 28 consecutive days, the animals in the (IC50=21.1 nmol/L) similar to that of 15. Analogs 27−29, which
43 group were given an oral dose of 43 (10 mg·kg-1·d-1) daily. had heteroarylmethyl substitutions, showed approximately
For 10 consecutive days, the animals in the positive control 2−3-fold less enzymatic inhibitory activity than that of 15.
(5) group were given an oral dose of 5 (0.5 mg/kg) daily. The Significantly, analog 30, which had a phenyl ring on the ter-
animals in the vehicle group received a daily oral treatment of minal nitrogen of the piperazine ring, displayed an enzymatic
0.5% carboxymethylcellulose sodium. During the treatment potency (IC50=8.9 nmol/L) similar to that of olaparib.
period, the implanted tumors were measured with a caliper
Considering the phenyl substituent advantage at the termi-
twice weekly. The maximum width (X) and length (Y) of the nal nitrogen of the piperazine ring, we next tested the efficacy
tumors were measured, and the volume (V) was calculated of different substituted phenyl analogs (31−42, Table 2). Unex-
using the formula: V=(X2Y)/2. Then, relative tumor volume pectedly, electron-withdrawing or electron-donating substitu-
(RTV) was calculated as follows: RTV=Vt/V0, where V0 rep- ents on the phenyl ring did not differentially affect activity (36
resents the tumor volume at the beginning of the treatments, vs 38 vs 41). However, substituted positions within the phenyl
and Vt represents the tumor volume after treatment. The ani- ring substantially improved the activity, and substitution at
mal body weights were also measured at the same time. The the para-position of the phenyl ring showed the best enzy-
experiments followed the Institutional Ethical Guidelines of matic inhibitory activity, as compared with the substitutions
the Animal Care and Use Committee (Shanghai Institute of at the ortho-position (31 vs 33, 39 vs 40 and 41 vs 42) and meta-
Materia Medica, Chinese Academy of Sciences, China).
position (35 vs 37).
To extend the substitution at the terminal nitrogen of the
piperazine, we replaced the phenyl ring with other heteroaryl
rings (43−55, Table 3). Adding a pyrid-2-yl group (43) sig-
Results and discussion
Lead optimization and SAR
All synthesized analogs were first evaluated for PARP1 enzy- nificantly enhanced the enzymatic inhibitory activity (43 vs 1
matic activity inhibition by using olaparib as the reference and 30). Thus, we explored the inhibitory effects of different
standard in the assay. Given the importance of the fluorine substitutions on the pyridine ring (44−49, Table 3). The incor-
atom, analog 15 was designed by replacing the middle 2-fluo- poration of a substituent, either electron-donating (47−49) or
rophenyl linker with a 2,4-difluorophenyl linker. As a result, electron-withdrawing (44−46), at diverse positions in the pyri-
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Chen WH et al
Table 1. In vitro activity of analogues 15−30.
PARP1 enzyme
IC50 (nmol/L)
Cell activity IC50 (nmol/L)a
PARP1 enzyme
IC50 (nmol/L)
Cell activity IC50 (nmol/L)a
V-C8 (BRCA2-/-) V79 (BRCA2+/+
)
No
1
R
V-C8 (BRCA2-/-)
V79 (BRCA2+/+
)
No
R
7.3 0.5
263.6
167.1
>10000
15
16.1 2.4
>10000
23
24
25
26
24.1 15.1
15.3 1.4
10.4 0.9
21.1 2.7
16
17
18
30.0 5.1
3.5 0.3
13.9
5106.9
12.5 3.3
19
20
14.0 3.2
27
28
34.6 4.8
4.6 1.4
99.2
2693.5
35.2*
21
22
157.4 29.4
61.3 20.9
29
30
49.4*
8.9 5.7
1135.8
5734.0
*Single test results.
dine ring was not beneficial and resulted in various losses of 54) substituents improved the inhibitory activity; (3) among
enzymatic inhibitory activity. Intriguingly, we observed that the studied sets of the R substituents (Tables 1−3), the potency
the double heteroatom-containing heteroaryl rings (50, pyrim- increased in the order of heteroaryl>aryl>acyl>(aryl)alkyl.
idinyl and 54, thiazolyl) exhibited more promising potencies
than that of analog 43, and analog 54 had the strongest effect. Cell proliferation inhibition of potent analogs
Not surprisingly, the size of the heteroaryl ring was very From the results described above, seventeen analogs (17, 20,
important for the enzymatic inhibitory activity, and substitu- 30, 35−39, 41, 43−44, 46−48, 50, and 54−55) were identified
tions (52−53) as well as fused aromatic rings (55) were not tol- as potent PARP1 inhibitors with IC50 values lower than 10
erable.
nmol/L. Therefore, these compounds were further evalu-
Analysis of the data shown in Tables 1−3 revealed some ated for their inhibition of cell proliferation. As shown in
noteworthy observations of the SAR for analogs 1 and 15−55: Tables 1−3, the selected analogs together with the reference
(1) replacement of the middle 2-fluorophenyl linker with the compound (1, olaparib) were tested in a matched pair of well-
2,4-difluorophenyl linker was tolerated (1 vs 15); (2) an R sub- characterized Chinese hamster lung fibroblast V79 (wild type)
stituent at the terminal nitrogen of the piperazine ring was and V-C8 (BRCA2 deficient) cell lines.
critical for high potency, and (aryl)alkyl substituents (21−29)
Interestingly, the benzoyl and 4-methylbenzoyl analogs
were not beneficial, but acyl (especially aroyl for 17 and 20), (17 and 20) showed similar enzymatic potency, but analog 17
aryl (especially para-substituted phenyl for 36, 38, and 41) and had a better cell inhibitory activity (IC50=13.9 nmol/L vs 99.2
heteroaryl (especially unsubstituted heteroaryl for 43, 50, and nmol/L) and a better selectivity against BRCA2-deficient cells
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Chen WH et al
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Table 2. In vitro activity of analogues 31−46.
PARP1 enzyme
IC50 (nmol/L)
Cell activity IC50 (nmol/L)a
V-C8 (BRCA2-/-) V79 (BRCA2+/+
PARP1 enzyme
IC50 (nmol/L)
Cell activity IC50 (nmol/L)a
V-C8 (BRCA2-/-) V79 (s)
No
1
R
)
No
R
7.3 0.5
263.6
>10000
31
12.0 4.6
37
38
39
7.0 1.1
2.1 1.7
5.8 2.2
8579.9
1585.1
1108.4
2745.8
1593.4
852.5
32
33
27..9 11.8
64.1 25.5
34
33.9 4.1
40
24.3 7.7
35
36
6.8 4.8
3.1 2.3
2916.4
72.9
1549.4
568.4
41
42
3.1 1.6
3254.2
>10000
10.6 2.7
(SI=367 vs 27). Although analogs 30, 35, 37, and 46 demon- To further investigate the selectivity of six analogs (1, 30, 35,
strated enzymatic inhibitory activity (IC50=6.8−9.1 nmol/L) 43−44, 47, and 50), we tested their inhibitory activity against
similar to that of 1, they had much less potent cellular activity both the BRCA1-deficient human ovarian carcinoma cells
in the V-C8 assays (IC50>700 nmol/L). Moreover, a similar UWB1.289 and BRCA1-complemented human ovarian car-
trend was also observed for analogs 38−39, 41, and 54, which cinoma UWB1.289+BRCA1 cells. As shown in Table 4, these
displayed good enzymatic potency (IC50=1.6−5.8 nmol/L), analogs selectively killed the BRCA1-deficient human ovar-
although the cellular activity determined in the V-C8 assays ian carcinoma cells, especially analog 47, which had excel-
was decreased 3−12-fold compared with that of olaparib. lent selectivity compared with that of 1 [IC50(BRCA1+/+)/
Additionally, analogs 36, 44, 48 and 55 presented both good IC50(BRCA1-/-)=5.6 vs 10.2].
enzymatic and cellular inhibitory activities. However, they
also showed modest proliferation inhibition in wild-type V79 Water solubility evaluation
cells (IC50<3500 nmol/L), thus leading to worse selectivity Because compound water solubility is an important property
against BRCA2-deficient cells compared with that of olaparib of drug potency, we next tested the water solubility of the four
(SI=~38 vs 8−30). Four of the most potent inhibitors (17, 43, potent PARP1 inhibitors in pure water (17, 43, 47, and 50). As
47, and 50) not only displayed better enzymatic (IC50=2.2−4.4 shown in Table 5, all four analogs showed poor solubility com-
nmol/L) and cellular (IC50=3.2−37.6 nmol/L) inhibitory activi- pared with that of olaparib (1.9−2.3 μg/mL vs 103.9 μg/mL),
ties but also were more selective (SI=40−510), thus confirming and the lower solubility may have been due to the higher
that the tested PARP1 inhibitors selectively killed BRCA2- number of lipophilic terminal (hetero)aryl groups. Consid-
deficient cells. Therefore, we focused on these four analogs for ering the basic groups (tertiary amine) in the structures of
further development.
analogs 43, 47, and 50, we explored the solubility of the hydro-
chloride salts to improve their water solubility. As expected,
the corresponding hydrochloride salts of analogs 43 and 47
Selectivity of analogs on BRCA1-/- and BRCA1+/+ cells
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Chen WH et al
Table 3. In vitro activity of analogues 47−55.
PARP1 enzyme
IC50 (nmol/L)
Cell activity IC50 (nmol/L)a
PARP1 enzyme
IC50 (nmol/L)
Cell activity IC50 (nmol/L)a
V-C8 (BRCA2-/-) V79 (BRCA2+/+
)
No
1
R
V-C8 (BRCA2-/-)
V79 (BRCA2+/+
)
No
R
7.3 0.5
263.6
>10000
43
2.7 0.6
37.6
2737.8
50
51
2.2 1.3
3.2
1632.9
44
45
4.6 2.3
27.9 7.8
41.9
978.6
14.8 4.8
52
23.1 7.1
46
47
9.1 4.5
4.4 2.1
774.8
9.6
>10000
385.1
53
54
33.7 14.2
1.6 0.7
615.6
267.0
>10000
2662.1
48
49
3.9 2.1
115.1
3481.6
55
4.3 1.8
33.9 15.7
Table 4. Selectivity of analogues on BRCA1-/- and BRCA1+/+ cells.
Cell activity IC50 (μmol/L) (mean SD)
tested the solubility of 43, 47, 50, 56, 57 and 58 in pH=7.4 and
pH=1.0 buffered salt solutions (BSS). As shown in Table 6, the
solubility of each pair of compounds in pH=7.4 was identical;
however, the corresponding salts (56 and 57) displayed better
solubility than their bases (43 and 47) in pH=1.0 buffer (414.4
μg/mL vs 1628.2 μg/mL, 1526.2 μg/mL vs 2652.5 μg/mL,
respectively). Hence, we presumed that 56 and 57 would have
accelerated absorption in the stomach (pH=1−3). Considering
their excellent in vitro biological and water solubility profiles,
56 and 57 were further evaluated in an in vivo xenograft tumor
model.
No
UWB1.289
(BRCA1-/-)
UWB1.289+BRCA1
IC50 (BRCA1+/+)/
(BRCA2+/+
)
IC50 (BRCA1-/-)
1
4.3 0.05
23.1 11.6
2.5 0.3
2.7 0.04
0.4 0.1
44.2 5.8
25.3 1.8
14.0 4.3
11.3 1.1
0.5 0.1
10.2
1.1
5.6
4.1
1.3
30
35
43
44
47
50
0.008 0.002
0.4 0.05
0.5 0.2
2.3 0.07
65.6
6.1
In vivo antitumor studies
To assess the in vivo antitumor effects of 56 and 57 as single
agents, nude mice with subcutaneous BRCA1-mutated human
breast cancer MDA-MB-436 xenografts were treated with 56 or
57 once daily for 21 consecutive days (Figure 4). Oral admin-
istration of a 30 mg/kg dose of 56 significantly inhibited the
growth of MDA-MB-436 xenografts, with an excellent growth
inhibition rate of 96.6% at the end of treatment. Remark-
(56 and 57) presented significantly increased solubility values,
which were 2193.0 µg/mL and 3601.0 µg/mL, respectively.
The solubility was increased almost 21- and 35-fold compared
with that of olaparib, yet the water solubility of the hydro-
chloride salt 58 was still less than 10 µg/mL. Next, we further
Acta Pharmacologica Sinica

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Chen WH et al
9
Table 5. The water solubility of selected analogues.
No
Analog
S (µg/mL)
No
Analog
S (µg/mL)
1
103.9
50
0.4
17
1.9
56
2193.0
43
47
2.3
2.0
57
58
3601.0
3.0
The mice treated with oral 57 at a 30 mg/kg dose showed
a potent effect against tumor growth, with a growth inhibi-
tion rate of 77.6% at the 21-day time-point, an effect relatively
weaker than that of 56 overall. Furthermore, 56 and 57 at the
1 mg/kg, 10 mg/kg and 30 mg/kg doses were well tolerated,
and no lethality or significant weight loss was observed dur-
ing the 21 consecutive treatment days (Figure 4).
Table 6. The water solubility of selected analogues in pH=7.4 and pH=1.0
buffer.
No
S (µg/mL)
No
S (µg/mL)
pH=7.4
43
47
50
1.9
4.7
<1.0
56
57
58
2.4
4.7
<1.0
Because 56 displayed excellent antitumor efficacy in sub-
cutaneous BRCA1-mutated human breast cancer MDA-
MB-436 xenografts, we next evaluated the inhibitory activities
of 43 (the corresponding base of 56) and 56 against Capan-1
(BRCA2-/-) cells. The results showed that 43 presented had
inhibitory activity than 56 (IC50=5.6 vs 34.4 nmol/L); there-
fore, we further assessed the in vivo antitumor effects of 43 in
the subcutaneous BRCA2-mutated human pancreatic cancer
Capan-1 xenografts. Nude mice with subcutaneous BRCA2-
mutated human pancreatic cancer Capan-1 xenografts were
treated with 43 once daily for 28 consecutive days (Figure 4C).
Oral administration of a 10 mg/kg dose of 43 significantly
inhibited the growth of Capan-1 xenografts, with a partial
growth inhibition rate of 69.0% at the end of treatment, simi-
larly to the results for Talazoparib (5) at the 0.5 mg/kg dose
(which resulted in a growth inhibition rate of 70.4%). How-
ever, the mice in the 0.5 mg/kg dose group that were treated
with Talazoparib (5) were in poor condition and experienced
pH=1.0
43
47
50
414.4
1526.2
132.3
56
57
58
1628.2
2652.5
145.3
ably, a 10 mg/kg dose of 56 still showed a growth inhibition
rate of 79.5% at the end of treatment, and this treatment was
more effective than olaparib (1) at the 30 mg/kg dose (which
resulted in a growth inhibition rate of 56.3%). When the dose
of 56 was decreased to 1 mg/kg, the treatment resulted in a
modest growth inhibition rate (43.2%), which was only slightly
lower than that with the 30 mg/kg dose of olaparib. Those
data were consistent with the in vitro cytotoxicity results of
compounds 1 and 56 (IC50: 90.5 nmol/L for 1 and 15.7 nmol/L
for 56) in MDA-MB-436 cells.
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Chen WH et al
Figure 4. Analogues 56 and 57 exhibited antitumor activity against BRCA1-mutated MDA-MB-436 xenografts in nude mice. And 43 also showed anti-
tumor effect against BRCA2-mutated Capan-1 xenografts. Nude mice with MDA-MB-436 and Capan-1 xenografts received and oral dose of the vehicle
(0.2 mL/20 g body weight), the positive control (1), 56 or 57 once each day for 21 consecutive days and positive control (5), 43 once each day for 28
consecutive days. Tumor volume and the body weight of the nude mice were measured at the indicated time points. A (56), B (57), and C (43) represent
the changes in relative tumor volume (RTV; calculated from the corresponding tumor volume) over the treatment time and the changes in body weight
over the treatment time. Note: The same controls (vehicle and olaparib) were used for 56 and 57, but for clarity and readability, we plotted the data
from the 56 groups and the 57 groups separately.
weight loss at the 10-day time-point; therefore, the adminis- poorer oral half-life (0.6 h vs 1.0 h), displayed faster plasma
tration was discontinued. The mice were allowed to recover clearance (3.0 L·h-1·kg-1 vs 1.8 L·h-1·kg-1) and had a larger vol-
until day 21, at which point treatment was administered until ume distribution (3.5 L/kg vs 3.0 L/kg). Moreover, the safety
the end of the experiment.
evaluation experiment (see the detailed assay in the Supple-
mentary Information) showed that the corresponding base 43
possessed low hERG inhibition activity (IC50=6.64 μmol/L).
Pharmacokinetic and in vitro safety properties
The excellent in vitro and in vivo biological properties of the Altogether, these data demonstrate that 56 may be a viable
hydrochloride salt 56 demonstrated that 56 was a good drug candidate for discovering potential therapeutic drugs with
candidate that targeted PARP1. Therefore, we next assessed specificity toward BRCA-deficient tumors.
the pharmacokinetic properties of 56 in an in vivo rat model.
As shown in Table 7, 56 and olaparib (1) demonstrated similar
Conclusions
oral bioavailability (F) after oral exposure in Sprague-Dawley In summary, we discovered a novel series of 2,4-difluorophe-
rats, with oral bioavailability values of 32.2% and 45.4%, nyl-linker analogs (15−55) derived from olaparib for use as
respectively. Compared with olaparib, 56 had a slightly PARP1 inhibitors. On the basis of the structure of the lead
Acta Pharmacologica Sinica

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Chen WH et al
11
compound, olaparib, 41 completely novel 2,4-difluoro-based logs 15–55, the hERG inhibition assay methods and the molec-
analogs were synthesized and tested in a PARP1 enzymatic ular docking details are available on the Acta Pharmacologica
inhibitory assay. Seventeen analogs, 17, 20, 30, 35−39, 41, 43, Sinica website.
44, 46−48, 50, 54 and 55, showed potent PARP1 inhibitory
activities (IC50<10 nmol/L). The most potent analog (54) dem-
onstrated a PARP1 inhibitory capability approximately five
times higher than that of olaparib. Preliminary SARs showed
that the 2,4-difluorophenyl linker of the new analogs inher-
ited the PARP1 inhibitory activity, which was contributed by
a 2-fluorophenyl linker. Additionally, terminal small steric
aroyl, para-substituted phenyl, and unsubstituted heteroaryl
substituents substantially improved the potency.
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