Endoxifen is a new potent inhibitor of PKC: A potential therapeutic agent for bipolar disorder

Article abstract of DOI:10.1016/j.bmcl.2010.02.024

Protein kinase C (PKC) plays a major role in regulation of both pre and postsynaptic neurotransmission. Excessive activation of PKC results in symptoms related to bipolar disorder. Tamoxifen, a widely used breast cancer drug is known to inhibit PKC and demonstrate antimanic properties in human. We describe herein the synthesis of endoxifen, a tamoxifen active metabolite and compared its PKC inhibitory activity with that of tamoxifen. Endoxifen exhibited fourfold higher potency compared to tamoxifen.

Full text of DOI:10.1016/j.bmcl.2010.02.024

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2665–2667
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Endoxifen is a new potent inhibitor of PKC: A potential therapeutic agent
for bipolar disorder
*
Shoukath M. Ali, Ateeq Ahmad, Syed Shahabuddin, Moghis U. Ahmad, Saifuddin Sheikh, Imran Ahmad
Jina Pharmaceuticals, Inc. 28100 N. Ashley Circle, Suite 103, Libertyville, IL 60048, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Protein kinase C (PKC) plays a major role in regulation of both pre and postsynaptic neurotransmission.
Excessive activation of PKC results in symptoms related to bipolar disorder. Tamoxifen, a widely used
breast cancer drug is known to inhibit PKC and demonstrate antimanic properties in human. We describe
herein the synthesis of endoxifen, a tamoxifen active metabolite and compared its PKC inhibitory activity
with that of tamoxifen. Endoxifen exhibited fourfold higher potency compared to tamoxifen.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 12 December 2009
Accepted 5 February 2010
Available online 23 February 2010
Keywords:
Endoxifen
PKC
Bipolar disorder
Chemical synthesis
Bipolar disorder (BPD) is a chronic debilitating illness character-
ized by drastic swings in mood, energy and functional ability, that
affects adult population.1 Until recently, not much was known
about the molecular and cellular mechanisms of BPD. Recent data
suggests that mania is associated with overactive protein kinase C
(PKC) intracellular signaling which plays a major role in regulation
of both pre and postsynaptic neurotransmission.2 Interestingly, the
current first line therapy of mania, lithium and valproate indirectly
inhibit PKC.3 In addition, recent preclinical and clinical studies
with the relatively selective PKC inhibitor Z-tamoxifen add support
to the relevance of the PKC target in BPD. Animal studies demon-
strate that tamoxifen significantly reduced manic behavior in ro-
dents.4 Preliminary investigations of PKC inhibitor tamoxifen in
the treatment of acute mania showed anti-manic effects compared
to placebo.5 Recently, two groups confirmed antimanic properties
of tamoxifen in double-blind, placebo controlled clinical studies.6
Thus, a growing body of work in both preclinical and clinical
indicates that PKC signalling may play an important factor in the
pathophysiology and treatment of BPD. The development of
CNS-penetrant PKC inhibitors such as tamoxifen may have consid-
erable benefit for this devastating illness.7 However, there is a large
inter-individual and ethnic variability in tamoxifen bioavailability
and function due to CYP2D6 genetic polymorphism which exten-
sively metabolizes tamoxifen into active metabolites, 4-hydroxy
tamoxifen and 4-hydroxy-N-desmethyl tamoxifen (endoxifen).8
Alternative PKC inhibitors that are independent of CYP2D6 action
such as endoxifen may provide better opportunity for the treat-
ment of BPD and other diseases. Here we have synthesized endox-
ifen (Fig. 1) and showed its PKC inhibitory activity in vitro for the
first time. We have also compared its PKC activity with known PKC
inhibitor tamoxifen.
Previously reported endoxifen synthesis involved demethyla-
tion of 4-hydroxytamoxifen.9 However, there are no reports for
its synthesis starting from commercially available starting
materials. Here, we report the synthesis of endoxifen starting from
commercially available 4-bromophenol, a-phenyl butyric acid and
2-chloroethoxybenzene. The synthetic scheme is depicted in
Scheme 1. 1-[4-(2-Chlororethoxy)phenyl]-2-phenyl-butan-1-one
3 was synthesized by refluxing solution of 2-phenylbutyric acid 2
and thionyl chloride in 1,2-dichloroethane (EDC) for an hour before
it was cooled to room temperature and added drop wise to a stir-
ring solution of 2-chloroethoxybenzene 1 and anhydrous alumi-
num chloride in EDC at 0 °C. After the completion of reaction the
workup was done by pouring the reaction solution in HCl/H2O
solution (1:1) and extracted with EDC. The crude product was puri-
fied by flash chromatography (hexane/ethyl acetate) yielding 3 in
88% yield. Alternatively, 3 was also synthesized in 96% yield by
* Corresponding author. Tel.: +1 847 573 0771.
E-mail address: imran@jinapharma.com (I. Ahmad).
Figure 1. Structure of endoxifen.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.024

2666
S. M. Ali et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2665–2667
isomer having the triplet at d 3.86 was assigned as Z-isomer and
the isomer with d 4.03 was identified as E-isomer.
Effect of synthesized endoxifen on PKC activity was evaluated
in vitro and compared with that of tamoxifen using a PKC kinase
activity ELISA assay kit (Assay Designs, Ann Arbor, MI) which con-
tained a PKC Substrate Microtiter Plate, Active PKC, ATP, Phospho-
specific Substrate Antibody, Anti-Rabbit IgG-HRP conjugate, TMB
substrate, Stop solution, Wash and Dilution Buffer. The assay was
carried out according to manufacturer’s instructions. Briefly,
50 ll of a reaction mix containing Kinase Assay Dilution Buffer,
0.025, 0.05, 0.1, or 0.2 mM endoxifen and 10 ng PKC was added
to each well of a presoaked PKC Substrate Microtiter Plate. To com-
pare PKC inhibition activity with tamoxifen, 50 ll of the Dilution
Buffer reaction mix containing 0.025, 0.05, 0.1, or 0.2 mM tamoxi-
fen (Toronto Research Chemicals, North York, ON, Canada) and
10 ng PKC was added to another set of wells. Fifty microliter of Ki-
nase Assay Dilution Buffer alone or with 10 ng PKC was used as
negative and positive controls, respectively. The reaction was initi-
ated by adding 10 ll of diluted ATP to each well. Following incuba-
tion at 30 °C for up to 90 min, the reaction was stopped by
emptying contents of the plate. Forty microliters of Phosphospeci-
fic Substrate Antibody was added to each well followed by 60 min
incubation at room temperature. All wells were washed four times
with Wash Buffer and 40
gate was added. After 30 min incubation at room temperature,
wells were again washed and 60 l of TMB Substrate was added.
Following further incubation at room temperature for 30 min,
20 l of Stop Solution was added and absorbance was read at
450 nm in a microplate reader (SpectraMax M2, Molecular Devices,
Sunnyvale, CA). The results were analyzed using SoftMaxÒ Pro 5
software.
ll of diluted Anti-Rabbit IgG: HRP Conju-
l
Scheme 1. Reagents and conditions: (a) method; (i) 2, SOCl2, EDC; 3, AlCl3, EDC; (b)
method; (ii) TFAA, rt; (c) concd H2SO4, rt; (d) (i) 6, Mg, THF; reflux?rt; (ii) add 3 in
THF, reflux; (e) concd HCl, MeOH, reflux; (f) methylamine, 2-propanol, reflux.
l
mixing 1, 2, and trifluoroacetic anhydride (TFAA) and stirring for
10 h. The reaction solution was neutralized with saturated sodium
bicarbonate solution before it was extracted with ethyl acetate and
concentrated. The crude product after crystallization in hexane at
0 °C provided 3 in 96% yield. The tetrahydopyran (THP) protecting
group was introduced into 4-bromophenol 4 by stirring with
3 M equiv of 3,4-dihydro-2H-pyran 5 and catalytic amount of con-
centrated sulfuric acid at 0 °C under nitrogen for 30 min. The reac-
tion solution was diluted with hexane–water. After workup, the
product was isolated in 91% yield by crystallization in small
amount of cold hexane. Compounds 3 and 6 were reacted under
Grignard’s reaction conditions (Mg/THF) to provide an intermedi-
ate 7 which was heated under acidic condition (HCl and methanol)
to undergo simultaneously dehydration and deprotection reaction.
After conventional workup, the crude product was purified on sil-
ica gel column and eluted with dichloromethane–hexane mixture
(1:1) to afford 1-[4-(2-chloroethoxy)phenyl-(4-hydroxyphenyl)-
2-phenyl but-1-ene] 8 as E- and Z-geometric isomers in 86.5%
yield. Compound 8 was then transformed into compound 9 by
treatment with 40% methylamine (aqueous) in isopropyl alcohol
and refluxed for 48 h. The solvent was removed and the residue
was extracted with ethyl acetate and washed with 5% aq sodium
bicarbonate solution followed by water and brine solution.
Endoxifen inhibited PKC activity in concentration dependent
manner. The percentage PKC inhibition ranged between 12% and
78% with endoxifen concentration between 0.025 and 0.2 mM,
respectively. In comparison, tamoxifen was found less potent PKC
inhibitor at 0.1 and 0.2 mM resulting 14% and 25% PKC inhibition,
respectively; lower concentrations of tamoxifen (0.025 and
0.05 mM) showed negligible PKC inhibition. Figure 2 shows endox-
ifen and tamoxifen induced PKC inhibition at 0.2 mM. The study
demonstrated that endoxifen is about fourfold more potent PKC
inhibitor than tamoxifen, and suggests its pivotal role in BPD.
In summary, we have synthesized endoxifen that inhibited PKC
significantly in comparison with tamoxifen. Preclinical and clinical
evidence demonstrate that PKC is an important target-perhaps the
first mechanistically distinct drug target for BPD and tamoxifen is a
relatively selective PKC inhibitor. This is the first report demon-
strating endoxifen to be potentially a superior candidate for BPD
treatment especially for those with defects in activating enzymes
After drying over anhydrous sodium sulfate, the organic extract
was concentrated in vacuo and subsequently triturated with ethyl
acetate/hexane (1:1) to provide a mixture (E:Z, 5.8:4.2, HPLC) of
geometric E- and Z-isomers of endoxifen in 52% yield. Z-Endoxifen
was isolated in pure form by fractional crystallization in benzene.
The product and the intermediates were characterized10 by 1H
NMR, 13C NMR, high resolution Mass spectrometry. The Z-configu-
ration was assigned based on the characteristic chemical shift9b,11
of OCH2 in 1H NMR. In all tamoxifen derivatives studies, the Z-iso-
mers (trans), the OCH2 protons of the side chain have a chemical
shift (d) less than 4.00 ppm owing to the through-space shielding
influence of the vicinal 2-phenyl substituents. Based on this, the
Figure 2. Percentage inhibition of PKC activity by 0.2 mM endoxifen and tamoxifen
in vitro.

S. M. Ali et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2665–2667
2667
7. DiazGranados, N.; Zarate, C. A., Jr. Curr. Psychiatry Rep. 2008, 10, 510.
for tamoxifen metabolism. Repeated daily oral administration of
endoxifen up to 4 mg/kg and 8 mg/kg body weight in rats and
mice, respectively, for 28 days showed no signs of toxicity (unpub-
lished observation). Efficacy studies with endoxifen will be the
subject of further investigation.
8. Information P. NOLVADEXÒ Product Monograph (Tamoxifen citrate). Copyright,
Astrazeneca Canada, Inc. 2000.
9. (a) Stearns, V.; Johnson, M. D.; Rae, J. M.; Morocho, A.; Novielli, A.; Bhargava,
P.; Hayes, D. F.; Desta, Z.; Flockhart, D. A. J. Natl. Cancer Inst. 2003, 95, 1758;
(b) Sun, D.; Sharma, A. K.; Dellinger, R. W.; Blevins-Primeau, A. S.; Balliet, R.
M.; Chen, G.; Boyiri, T.; Amin, S.; Lazarus, P. Drug Metab. Dispos. 2007, 35,
2006.
10. Compound 6: mp 56–57 °C; 1H NMR (400 MHz, CDCl3): d 1.68 (m, 3H), 1.83 (m,
2H), 1.98 (m, 1H), 3.57 (m, 1H), 3.85 (m, 1H), 5.36 (t, J = 3.2 Hz, 1H), 6.93 (AB,
J = 8.79, J = 2.14, 2H), 7.35 (AB, J = 8.79, J = 2.14 Hz, 2H). 13C NMR (100 MHz,
CDCl3): d 18.6, 25.1, 30.2, 61.9, 96.5, 113.8, 118.3, 132.2, 156.2; HRMS (ESI),
References and notes
1. (a) Goodwin, F. K.; Jamison, K. R. Manic-Depressive Illness; Oxford University
Press: New York, 1990; (b) Weissman, M. M.; Leaf, P. J.; Tishler, G. L.; Blazer, D.
G.; Karno, M.; Bruce, M. L.; Florio, L. P. Psychol. Med. 1988, 18, 141.
2. (a) Wang, H. Y.; Friedman, E. Biol. Psychiatry 1996, 40, 568; (b) Manji, H. K.;
Lenox, R. H. Biol. Psychiatry 1999, 46, 1328.
3. Friedman, E.; Hoau Yan, W.; Levinson, D.; Connell, T. A.; Singh, H. Biol.
Psychiatry 1993, 33, 520.
4. (a) Einat, H.; Manji, H. K. Biol. Psychiatry 2006, 59, 1160; (b) Narita, M.; Aoki, T.;
Ozaki, S.; Yajima, Y.; Suzuki, T. Neuroscience 2001, 103, 309.
5. (a) Kulkarni, J.; Garland, K. A.; Scaffidi, A.; Headey, B.; Anderson, R.; de Castella,
A.; Fitzgerald, P.; Davis, S. R. Psychoneuroendocrinology 2006, 31, 543; (b)
Bebchuk, J. M.; Arfken, C. L.; Dolan-Manji, S.; Murphy, J.; Hasanat, K.; Manji, H.
K. Arch. Gen. Psychiatry 2000, 57, 95.
6. (a) Yildiz, A.; Guleryuz, S.; Ankerst, D. P.; Ongur, D.; Renshaw, P. F. Arch. Gen.
Psychiatry 2008, 65, 255; (b) Zarate, C. A., Jr.; Singh, J. B.; Carlson, P. J.; Quiroz, J.;
Jolkovsky, L.; Luckenbaugh, D. A.; Manji, H. K. Bipolar Disord. 2007, 9, 561.
expected 278.991, observed 278.9984 (M+Na+). Compound 3: mp 68–70 °C; 1
H
NMR (400 MHz, CDCl3): d 0.89 (t, J = 7.5 Hz, 3H), 1.85 (m, 1H), 2.18 (m, 1H),
3.78 (t, J = 5.9 Hz, 2H), 4.22 (t, J = 5.9 Hz, 2H,), 4.38 (t, J = 7.26 Hz, 1H), 6.86 (AB,
J = 9 Hz, J = 2.14 Hz, 2H), 7.18 (m, 1H), 7.28 (m, 4H), 7.96 (AB, J = 8.78 Hz,
J = 1.93 Hz, 2H). 13C NMR (100 MHz, CDCl3): d 12.3, 27.1, 41.5, 55.1, 67.9, 114.2,
126.8, 128.2, 128.8, 130.6, 130.9, 139.9, 161.7, 198.5. HRMS (ESI), expected
325.0966, observed 325.0976 (M+Na+). Compound 9 (Z-endoxifen): mp 139–
143 °C; 1H NMR (400 MHz, DMSO-d6): d 0.85 (t, J = 7.24 Hz, 3H), 2.29 (s, 3H),
2.42 (q, J = 7.2 Hz, 2 H), 2.74 (t, J = 5.59 Hz, 3H), 3.86 (t, J = 5.56 Hz, 2H), 6.58 (d,
J = 8.78 Hz, 2H), 6.71 (d, J = 8.56 Hz, 2H), 6.75 (d, J = 8.68 Hz, 2H), 6.98 (d,
J = 8.2 Hz, 2H), 7.08–7.13 (m, 3H), 7.15–7.19 (m, 2H), 9.38 (br, 1H); HRMS (ESI),
expected 374.2115, observed 374.2116 (M+H+).
11. McCague, R.; Leclercq, G.; Legrosm, N.; Goodman, J.; Blackburn, G. M.; Jarman,
M.; Foster, A. B. J. Med. Chem. 1989, 32, 2527.