Analogs of penfluridol as chemotherapeutic agents with reduced central nervous system activity

Article abstract of DOI:10.1016/j.bmcl.2018.10.036

Several recent reports have highlighted the feasibility of the use of penfluridol, a well-known antipsychotic agent, as a chemotherapeutic agent. In vivo experiments have confirmed the cytotoxic activity of penfluridol in triple-negative breast cancer model, lung cancer model, and further studies have been proposed to assess its anticancer activity and viability for the treatment of glioblastomas. However, penfluridol anticancer activity was observed at a dosage significantly higher than that administered in antipsychotic therapy, thus raising the concern for the potential onset of CNS side effects in patients undergoing intensive pharmacological treatment. In this study, we evaluate the potential CNS toxicity of penfluridol side by side with a set of analogs.

Full text of DOI:10.1016/j.bmcl.2018.10.036

Accepted Manuscript
Analogs of penfluridol as chemotherapeutic agents with reduced central nervous
system activity
Md Ashraf-Uz-Zaman, Md Sanaullah Sajib, Luca Cucullo, Constantinos M.
Mikelis, Nadezhda A German
PII:
S0960-894X(18)30838-2
https://doi.org/10.1016/j.bmcl.2018.10.036
BMCL 26095
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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21 October 2018
23 October 2018
Please cite this article as: Ashraf-Uz-Zaman, M., Sanaullah Sajib, M., Cucullo, L., Mikelis, C.M., German, N.A.,
Analogs of penfluridol as chemotherapeutic agents with reduced central nervous system activity, Bioorganic &
Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.10.036
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Analogs of penfluridol as chemotherapeutic agents with reduced central nervous system activity.
Md Ashraf-Uz-Zaman1, Md Sanaullah Sajib1, Luca Cucullo1, Constantinos M. Mikelis1, Nadezhda A
German1.
1 Department of Pharmaceutical Sciences, Texas Tech University Health Sciences Center, School of
Pharmacy, Amarillo, TX, 79106, United States
Keywords: penfluridol; anticancer agent; central nervous system; repurposing; toxicity; optimization.
Abstract:
Several recent reports have highlighted the feasibility of the use of penfluridol, a well-known antipsychotic
agent, as a chemotherapeutic agent. In vivo experiments have confirmed the cytotoxic activity of
penfluridol in triple-negative breast cancer model, lung cancer model, and further studies have been
proposed to assess its anticancer activity and viability for the treatment of glioblastomas. However,
penfluridol anticancer activity was observed at a dosage significantly higher than that administered in
antipsychotic therapy, thus raising the concern for the potential onset of CNS side effects in patients
undergoing intensive pharmacological treatment. In this study, we evaluate the potential CNS toxicity of
penfluridol side by side with a set of analogs.
Graphical Abstract TOC:
Authors on this paper have filed a provisional patent application (INV 1365, TTU, USA) detailing this
work.

Traditionally, de novo drug discovery for cancer treatment is invariably associated with high cost for
preclinical and clinical studies which is estimated at approximately 1.042 billion dollars per drug1.
Therefore, repurposing approved drugs to extend their therapeutic viability to other diseases (such as
cancer treatment) is likely to afford the quickest and most cost-effective transition from bench to bedside2,
3. At the same time, to avoid potential side toxicity issues, it is crucial to gather all available toxicological
information associated with the use of a given drug4 and prevent scenarios similar to bevacizumab,
cetuximab and others5, the USA Food and Drug Administration (FDA) developed guidance for a systemic
approach to collect and report compound-associated toxicities6. This is especially important when the
proposed dosage and/or frequency of administration of a repurposed drug are substantially different
(higher) than those previously associated for its original therapeutic scope.
Among the many drugs being tested for their off-label activities, penfluridol, an oral antipsychotic agent7,
was investigated for its anticancer properties8. Several groups have reported that this compound reduces
cancer growth in vitro and in vivo9-15. Encouraging tumor-killing data and ability of this drug to cross the
blood-brain barrier (BBB) prompt the idea of using this agent in the treatment of glioblastoma12, 13 and
cancers that have high rates of metastasis in the brain, including metastatic triple-negative breast
cancer10 and lung cancers14. However, in vivo data also indicated that anticancer dosage should be
considerably higher (estimated at 50 mg of daily dosing in human)10-12, 15, 16 than those used for the
treatment of chronic schizophrenia and similar psychotic disorders (weekly oral dosing of 20 mg to 100
mg, 160 mg for resistant cases)17. Earlier, clinical data report neurological side effects attributed to the
antipsychotic use of penfluridol, including epilepsy, fatigue, dyskinesias, Parkinsonism, akathisia, dystonia
and depression, although the numbers are at the lower range18-20. Because penfluridol is capable of
penetrating the BBB we estimated that the dosage required to produce chemotherapeutic activities10-12
would substantially increase the incidence of neurological side effects already associated with its use,
thus impacting the sustainability and overall benefits associated with the treatment.
To assess these potential side-effects, we have evaluated the ability of penfluridol to inhibit major groups
of G-coupled protein receptors (GPCRs) that are expressed in the brain21. Our data have confirmed that
this compound has a multitarget CNS profile with the majority of Ki values being in the nanomolar range.

Table 1. Inhibitory activity of penfluridol at selected CNS receptors.
5HT1A
5HT1D
5HT2A
5HT2B
5HT2C
5HT5a
5HT6
5HT7
D1
D2
D3
D4
D5
Inhibition,
%
100
91.3
95.2
97.3
95.3
95.0
96.0
98.4
96.5
71.5
98.2
77.6
82.9
103
103
280
147
159
136
103
125
Ki, nM
356
3560
361
184
881
KOR
MOR
DOR
H1
H2
NET
SERT
DAT
Alpha 1D
Alpha 2B
Alpha 2C
Beta 3
Inhibition, %
Ki, nM
94.6
103
87.0
867
89.1
84.0
103
98.4
103
86.2
588
92.7
103
89.1
95.7
602
95.6
401
100
445
94.0
515
1714
1714
An initial literature analysis has shown that the available pharmacophore for CNS activity of penfluridol22,
23 somewhat diverges from the one reported for its anticancer property24, 25(Figure 1). Therefore, we have
hypothesized that by leveraging the pharmacophores activities to promote its anticancer properties while
reducing its GPCRs activities we can develop a more effective and safer chemotherapeutic penfluridol
analog. Multiple modifications of the right - side motif containing the 4-substituted piperidine ring
highlighted the importance of spatial orientation of the piperidine residue and hydroxy group for
anticancer and antipsychotic activity22-25. It appears that this part of the molecule interacts with both
targets via hydroxyl bonding and stabilizes the active conformation required for both types of biological
activity. Hence, we have prepared analogs with the modifications in the linker motif and the left part of the
original penfluridol structure. During the initial stage of our project, we have screened the obtained
derivatives for their ability to inhibit selected GPCRs and to inhibit the growth of cancer cells in vitro,
followed by evaluation of the top compound for its ability to cross the BBB in vivo. In addition, we have
identified the dosing regimen that is required to achieve a therapeutic dose of selected compounds in
mice.

Figure 1. Structural requirements for antipsychotic and anticancer activities of penfluridol and its analogs23, 24
.
The proposed derivatives were prepared in good yields using a previously published procedure24, 26-28 as
depicted in Schemes 1 through 3 and in schemes S1-S2 (supplemental material). In particular, the right
moiety of all analogs was prepared by reacting a Grignard reagent with the N-BOC piperidone, followed
by deprotection step in the presence of hydrochloric acid in ethanol24 (Scheme S1). As shown in Scheme
S2 (supplemental material), the left part of compounds was constructed using selected benzene
derivatives and the corresponding lactones to produce the intermediate 5 that was further utilized in
preparing a key intermediate 7 (Scheme 1). This compound was coupled with a moiety 2 (scheme S1,
supplemental material) to obtain the final diphenyl butyl- or diphenyl pentylpipreridines 8 and 6. In
scheme 2, synthesis of analogs 11a and 11b started with the amination of aryl chloride using 1,1’-
bis(diphenylphosphino)ferrocene (DPPH) as a catalyst.27 Next, the coupling of 9 with dibromo- or bromo-
chloroalkane afforded intermediate 10 which was further reacted with 2 to afford compounds 11a and 11b
in 10% and 24% overall yield respectively. Finally, in Scheme 3 monophenyl analog 13 was prepared in
three steps following the published procedure26 to afford the desired product in 8% overall yield. All
products were purified by flash chromatography and characterized using 1H and 13C NMR. The purity of
all compounds was at or above 95% (supplemental material).

Scheme 1: Reagents and conditions: (a) Pd/C, H2, EtOH, r.t. (b) 2, Na2CO3, Kl, CH3CN, reflux.
Scheme 2: Reagents and conditions: (a) 1,1′-Ferrocenediyl-bis(diphenylphosphine), Pd(dppf)Cl2.CH2Cl2, KtBuO,
anhydrous THF, 100 ºC; (b) NaH, THF, 80 ºC; (c) 2, Na2CO3, Kl, CH3CN, reflux.
Scheme 3: Reagents and conditions: (a) LiAlH4, THF, 0 °C to r.t.; (b) CBr4, Ph3P, DCM, 0 °C to r.t.; (c) 2, Na2CO3, KI,
CH3CN, reflux.
To confirm that the
proposed changes retain
similar cytotoxic as
penfluridol, analogs were
evaluated in vitro using
two cancer cell lines of
both mouse and human
origin: mouse-derived
luciferin-expressing Lewis
Lung Carcinoma (LLC luc)
cell line and human triple-
negative breast cancer cell
Figure 2. Synthesized analogs of penfluridol with the corresponding activity against
MDA MB231 and LLC cell lines.
line (MDA MB-231) using
the 3-[4, 5-dimethylthiazol-

2-yl]-2, 5-dimethyltetrazolium bromide (MTT) colorimetric assay29. All experiments were performed in
triplicates to ensure good reproducibility and penfluridol was used as a positive control. Data were
obtained at 24h, 48h, and 72 hours and IC50 values (µM) were calculated (supplementary materials).
Among all derivatives tested, compounds 8a, 8c and 11a showed the best activity (IC50 3.5 - 7.1 µM), with
little to no reduction when compared to penfluridol (IC50 4.3 - 5.1 µM). All three compounds have
modifications in the spacer linker including elongation of the chain by one carbon (8a and 8c) or
introduction of nitrogen atom (11a). An unsaturated linker with the elongated chain 6 (IC50 9.6 - 11.2 µM)
caused a two-fold reduced cytotoxicity when compared to penfluridol. Interestingly, when two favorable
features, such as elongated linker and N-containing linker, were combined, the activity has decreased
(11b, IC50 8.2 - 9.2µM). Next, modifications of the left motif, including unsubstituted phenyl moieties (8b)
and monophenyl analog (13) were marked by the reduced activity, whereas introduction of methoxy
group was tolerated (8c,
IC50 6-7 μM). Overall, in vitro cytotoxicity data have shown that all designed analogs were active against
selected cancer cell lines, supporting our hypothesis that these modifications will not alter significantly the
anticancer activity of the original penfluridol molecule.
Next, we have investigated the effect of the proposed changes on the inhibition of selected G-protein
coupled receptors (GPCRs). Receptor binding profile and Ki determinations were provided by the
National Institute of Mental Health’s Psychoactive Drug Screening Program, where primary binding assay
has identified compounds with significant inhibition effect of 50% and more21. These derivatives were
further
evaluated using a radioligand binding assay, and Ki values were calculated accordingly.
As shown in Tables 2–4, the greatest reduction in the inhibitory activity for spacer modified analogs was
observed for compound 11b. For some serotonin receptors, this N-butyl analog has shown decreased
inhibition (50% - 80%) when compared to penfluridol (95% - 100%) (Figure 2), and for other serotonin
receptor subtypes it retained inhibition levels at >90% but decreased the Ki valued by 3-16-fold (Table 3).
Comparable results were obtained for dopamine receptors, opioid receptors, NET, SERT and DAT
(Figure 3, Tables 4). Moreover, 11b has shown abolished activity at H1 and H2 receptors, significantly
improving CNS-toxicity profile of the original penfluridol molecule. Similar effect was observed for

compound 8a, a homologated analog of penfluridol. However, it appears that elongation of the chain
doesn’t reduce the ability of this compound to inhibit dopamine receptors (Table 4). On a contrary, 8a
produced significantly lower Ki values for D4 receptor subtype (Ki 25 nM) when compared to the original
penfluridol molecule (Ki 10000nM). An introduction of N-propyl linker, 11a, resulted in higher affinity of a
molecule to histamine receptors and in substantially increased binding to the D4 receptor subtype,
whereas the rest of the receptor’s groups were not affected. Available results for unsaturated analog 6
suggest that this compound has slight improvement in the CNS binding profile, although increased affinity
to D4 receptor is observed here as well.
In the second group of analogs with the modifications on the left side of the diphenylbutylpiperidine
structure, the most favorable profile is associated with the compound 8c. The data suggest a significant
reduction in the Ki values for the majority of the serotonin and dopamine receptor subtypes, opioid
receptors, and histamine receptors (Figure 3, Tables 2-4). The monophenyl analog 13 has shown no
substantial changes in the binding profile, whereas compound 8b lacking the 4-F substituent displays
increased ability to inhibit all dopamine receptor subtypes, MOR, H2, and DAT. These data were not in
line with the previously reported structure-activity relationship trends22, 23, where the importance of the
electron withdrawing groups at para-position was highlighted for the antipsychotic activity.
Inhibition of 5HTRs
inhibition of Dopamine Receptors
Inhibition of selected CNS receptors
600
400
200
0
800
1000
800
600
400
200
0
D5
D4
D3
D2
D1
DAT
SERT
NET
H2
5HT7
5HT6
600
400
200
0
5HT5a
5HT2C
5HT2B
5HT2A
5HT1D
5HT1a
H1
DOR
MOR
KOR
PFL 11a 8a
6
11b 8b 8c 13
a
a
6
c
3
1
L
b
b
8
a
a
6
c
3
1
L
b
b
8
1
8
8
F
1
1
8
8
F
1
1
1
1
1
P
P
Figure 3. Inhibition of selected CNS receptors by penfluridol and its analogs. 5HT– serotonin receptors, D –
dopamine receptors, KOR – kappa opioid receptor, MOR – mu opioid receptor, DOR – delta opioid receptor, H1-
histamine receptor 1, H2 – histamine receptor 2, NET – norepinephrine transporter, SERT – serotonin transporter,
DAT – dopamine transporter.
Overall, through the analysis of the receptor binding profiles, we were able to identify three compounds,
8a, 11b and 8c, with the reduced affinity to selected CNS receptors. Compounds 11b and 8c showed a
significant reduction in the inhibition of dopamine receptors, a group of receptors strongly associated with
the neurological side-effects produced by penfluridol. Similarly, 8a had pronounced effect on the inhibition
of serotonin receptors, while moderate changes in the affinity to dopamine receptors. Therefore, we

expect 8a, 11b and 8c to have diminished CNS toxicity if used as chemotherapeutic agents. Due to the
minimal changes in the penfluridol structure, we assumed that our analogs yielded minimal alterations in
the BBB permeability and no additional organ toxicity. To confirm this hypothesis, we have selected
compounds 8a (IC50 4.8 μM, MDA MB231; IC50 4.2μM, LLC) and 8c (IC50 6.1 μM, MDA MB231; IC50 7.1
μM, LLC) for
Table 2. Binding activity (Ki, nm) of penfluridol and its analogs at serotonin receptor subtypes. Data represent mean
inhibition (n=4). NA – not active; ND – not determined.
5HT1A
5HT1D
5HT2A
5HT2B
5HT2C
5HT5a
5HT6
5HT7
PFL
11a
8a
356
3560
752
361
184
177
423
2199
509
374
729
NA
881
10000
ND
10000
1680
1671
ND
280
1262
1395
1363
1094
481
164
402
1574
969
844
1000
1105
6059
1000
900
1712
1603
1126
2131
740
894
ND
NA
10000
812
6
11b
8b
1074
628
4152
NA
1670
1695
1400
ND
326
8c
10000
901
10000
4431
4973
ND
949
1120
13
889
2548
Table 3. Binding activity (Ki, nm) of penfluridol and its analogs at dopamine receptor subtypes. Data represent mean
inhibition (n=4). NA – not active; ND – not determined.
D1
D2
D3
D4
D5
PFL
11a
8a
147
300
798
159
148
194
136
474
209
10000
25
125
437
529
385
ND
ND
744
121
623
ND
6
317
550
38
552
782
62
1643
10000
58
11b
8b
8c
13
10000
135
10000
ND
417
460
437
ND
459
132

Table 4. Binding activity (Ki, nm) of penfluridol and its analogs at selected CNS receptors. Data represent mean
inhibition (n=4). NA – not active; ND – not determined.
KOR
MOR
DOR
H1
H2
NET
SERT
DAT
PFL
11a
8a
10000
667
867
902
356
328
10000
70
1714
1558
1413
ND
10000
814
ND
10000
252
546
526
NA
588
902
77
10000
903
1714
1191
529
2669
1021
10000
1328
10000
1705
428
ND
ND
ND
6
718
NA
11b
8b
10000
1526
10000
NA
458
518
1438
ND
10000
1180
1314
ND
10000
121
ND
278
NA
8c
8253
536
7031
718
622
ND
13
401
further evaluation in vivo. The LLC cell line is a syngeneic cell line for mice of the C57BL6 background
that have functional immune system30. Therefore, we have selected the C57BL6 mice (females) for our in
vivo experiments. In the initial study, three groups of animals were treated with penfluridol, 8a or 8c
compounds (5 mg/ kg) by intraperitoneal (i.p.) injection for seven consecutive days. This dosing regimen
was chosen to match the published in vivo data, where seven days were reported as the minimum length
of treatment for therapeutic effect in an in vivo lung cancer model31. Twenty-four hours after the last
injection, all mice were sacrificed, and organs were collected and stored at -80 ºC. LC-MS/MS analysis of
plasma, brain, lungs and adipose tissue was performed to identify concentration levels of the compounds
(Figure 4). The observed distribution pattern for analog 8a was very similar to the one displayed by
Figure 4. Concentration (nM) of tested drugs in selected organs after 7-day treatment of daily i.p administration
(5 mg/kg), n=5, *= P<0.05, ** = p< 0.01, *** = p<0.001, **** = p 0.0001.

penfluridol, where an accumulation of the drug occurred predominantly in the adipose tissue and lungs. At
the same time, compound 8a has higher level of the drug in the brain relative to penfluridol. Analog 8c, on
the other hand, displayed lower concentration of the drug in all tested samples, although the overall
distribution pattern was similar to penfluridol and 8a. Our preliminary analysis of the pharmacokinetic
profile of 8c has shown that the maximum concentration of drug in the brain (Figure S5, supplemental
material) is achieved at a 6-hour time point following the i.p. injection of 10 mg/kg dose. Currently, we are
investigating if lower levels of 8c in animal samples are associated with the faster metabolic degradation
of this compound when compared to penfluridol. In particular, we are looking at the stability of the 4-
methoxyphenyl moiety under experimental conditions.
None of the tested compounds had a significant effect on the organ weight (Figure S6, supplemental
material) supporting previously published data for penfluridol10. In addition, we performed clinical
chemistry analysis of blood samples of the animals treated with penfluridol, compound 8a, and compound
8c (Figure 5, Figure S7). Specifically we measured the levels of electrolytes, minerals, protein metabolism
(total protein, albumin, globulin, A/G ratio), kidney function (blood urea nitrogen - BUN), liver injury
Figure 5 Effect of drug treatment (5 mg/kg, daily i.p. dosing for 7 days) on A. Kidney function: blood urea nitrogen
(BUN); B. Liver injury: glutamate dehydrogenase (GLDH) and C. Liver injury: alkaline phosphatase (ALP), n=5, *=
p<0.05, ** = p< 0.01.
(including alanine aminotransferase - ALT, aspartate aminotransferase - AST, and glutamate
dehydrogenase - GLDH), cholestasis, alkaline phosphatase (ALP), bilirubin, pancreatic function
(amylase), and muscle injury (creatine kinase (CK), AST, ALT) 32. In penfluridol-treated animals our
results were consistent with the onset of hepatic stress or injury and inflammation of the GI tract. In fact,
only penfluridol - treated animals have shown decreased BUN levels and increased GLDH corresponding
to hepatic stress or injury, whereas decreased ALP levels may result from low zinc level or can be caused

by inflammation of GI tract. None of these changes was observed in mice treated with compound 8c, thus
suggesting that this analog have a better toxicity profile when compared to penfluridol.
To summarize, our study started with the evaluation of the potential CNS-related toxicity of penfluridol at
the doses proposed for the anticancer therapy. As we have shown, this compound inhibits a majority of
CNS-related GPCRs at the nanomolar level, raising concerns about a potential burden on a patient under
treatment. In our study we have identified 2 compounds (8a and 8c) with anticancer activity but lesser
CNS affinity (hence reduced CNS-related side effects) t when compared to penfluridol. In addition, these
compounds have shown no toxicity in mice. The metabolic stability of these compounds and their
utilization in metastatic triple-negative breast cancer model (8a) and lung cancer model (8c) is currently
under investigation and data will be presented in due course. Furthermore, comparative side by side
studies will be performed in the near future to evaluate the anticancer activity of these penfluridol analogs
against commercially available treatments.
Abbreviations:
ALP; alkaline phosphatase, creatine kinase; ALT; alanine aminotransferase, AST; aspartate
aminotransferase, BBB; blood brain barrier, BOC; tert-butoxycarbonyl, BUN; blood urea nitrogen test, CK;
creatine kinase, CNS; central nervous system, D1-5; dopamine receptor subtypes, DAT; dopamine
transporter, DCM; dichloromethane, DOR; delta opioid receptor, DPPH; 1,1’-
bis(diphenylphosphino)ferrocene, ESI electrospray ionization, EtOH; ethanol, FDA; Food and Drug
Administration, GI; gastrointestinal, GLDH; glutamine dehydrogenase, H1-2; histamine receptor subtypes,
5HT; serotonin receptors, IC50; half-maximum inhibitory concentration, i.p.; intraperitoneal injection, Ki;
inhibition constant, KOR; kappa opioid receptor; LC; liquid chromatography, LC-MS; liquid
chromatography-mass spectrometry, LLC luc; mouse-derived luciferin expressing Lewis lung carcinoma
cell line, MDA MB231; human triple-negative breast cancer cell line, MOR; mu opioid receptor, MTT; 3-
[4,5-dimethylthiazol-2-yl]-2,5-dimethyltetrazolium bromide, NET; norepinephrine transporter, NMR;
nuclear magnetic resonance, r.t.; room temperature, SERT; serotonin transporter, THF; tetrahydrofuran,
TLC; thin-layer chromatography.

Acknowledgments:
Ki determinations and receptor binding profiles were generously provided by the National Institute of
Mental Health's Psychoactive Drug Screening Program, Contract # HHSN-271-2013-00017-C (NIMH
PDSP). The NIMH PDSP is Directed by Bryan L. Roth MD, PhD at the University of North Carolina at
Chapel Hill and Project Officer Jamie Driscoll at NIMH, Bethesda MD, USA.
This work was supported in part by the Laura W. Bush Institute for Women’s Health Seed Grant,
TTUHSC, 2017.

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Highlights



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Anticancer doses of penfluridol inhibit a majority of G-protein coupled receptors.
Analysis of antipsychotic pharmacophore allowed to design less toxic analogs.
Confirmed in vitro cytotoxicity of analogs, in vivo permeability of the BBB.
Clinical chemistry analysis confirmed optimized toxicity profile.