8,8-Dialkyldihydroberberines with potent antiprotozoal activity

Article abstract of DOI:10.1021/np300638f

Semisynthetic 8,8-dialkyldihydroberberines (8,8-DDBs) were found to possess mid- to low-nanomolar potency against Plasmodium falciparum blood-stage parasites, Leishmania donovani intracellular amastigotes, and Trypanosoma brucei brucei bloodstream forms. For example, 8,8-diethyldihydroberberine chloride (5b) exhibited in vitro IC₅₀ values of 77, 100, and 5.3 nM against these three parasites, respectively. In turn, two 8,8-dialkylcanadines, obtained by reduction of the corresponding 8,8-DDBs, were much less potent against these parasites in vitro. While the natural product berberine is a weak DNA binder, the 8,8-DDBs displayed no affinity for DNA, as assessed by changes in the melting temperature of poly(dA·dT) DNA. Selected 8,8-DDBs showed efficacy in mouse models of visceral leishmaniasis and African trypanosomiasis, with 8,8-dimethyldihydroberberine chloride (5a) reducing liver parasitemia by 46% in L. donovani-infected BALB/c mice when given at an intraperitoneal dose of 10 mg/kg/day for five days. The 8,8-DDBs may thus serve as leads for discovering new antimalarial, antileishmanial, and antitrypanosomal drug candidates.

Full text of DOI:10.1021/np300638f

Article
pubs.acs.org/jnp
8,8-Dialkyldihydroberberines with Potent Antiprotozoal Activity
Molla Endeshaw,† Xiaohua Zhu,† Shanshan He,† Trupti Pandharkar,† Emily Cason,‡
Kiran V. Mahasenan,† Hitesh Agarwal,† Chenglong Li,† Manoj Munde,§ W. David Wilson,§ Mark Bahar,†
Raymond W. Doskotch,† A. Douglas Kinghorn,† Marcel Kaiser,⊥,∥ Reto Brun,⊥,∥ Mark E. Drew,†,‡
and Karl A. Werbovetz*,†
†Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210,
United States
‡Department of Microbial Infection and Immunity, College of Medicine, The Ohio State University, Columbus, Ohio 43210, United
States
§Department of Chemistry, Georgia State University, Atlanta, Georgia 30302, United States
⊥Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, CH-4002 Basel, Switzerland
∥University of Basel, CH-4002 Basel, Switzerland
S
* Supporting Information
ABSTRACT: Semisynthetic 8,8-dialkyldihydroberberines (8,8-DDBs) were
found to possess mid- to low-nanomolar potency against Plasmodium
falciparum blood-stage parasites, Leishmania donovani intracellular amastigotes,
and Trypanosoma brucei brucei bloodstream forms. For example, 8,8-
diethyldihydroberberine chloride (5b) exhibited in vitro IC50 values of 77,
100, and 5.3 nM against these three parasites, respectively. In turn, two 8,8-
dialkylcanadines, obtained by reduction of the corresponding 8,8-DDBs, were
much less potent against these parasites in vitro. While the natural product
berberine is a weak DNA binder, the 8,8-DDBs displayed no affinity for DNA,
as assessed by changes in the melting temperature of poly(dA·dT) DNA.
Selected 8,8-DDBs showed efficacy in mouse models of visceral leishmaniasis
and African trypanosomiasis, with 8,8-dimethyldihydroberberine chloride (5a)
reducing liver parasitemia by 46% in L. donovani-infected BALB/c mice when
given at an intraperitoneal dose of 10 mg/kg/day for five days. The 8,8-DDBs
may thus serve as leads for discovering new antimalarial, antileishmanial, and antitrypanosomal drug candidates.
(5b), revealed it to be a mixture of compounds. Resynthesis
based on the original procedure11 resulted in the isolation and
structural characterization of a novel compound, 5,6-didehydro-
8,8-diethyl-13-oxodihydroberberine (4), where oxidation had
taken place at C-13 and between C-5 and C-6 of the
protoberberine core.12 Compound 4 retained the potent
antiparasitic activity of the initial sample and displayed efficacy
in a murine visceral leishmaniasis model.12 However, 4 could be
given to mice only at 1 mg/kg/day when administered ip. Thus,
a critical step in the further progression of molecules related to
4 as antiparasitic candidates is to decrease toxicity while
retaining antiparasitic efficacy.
lthough berberine (1) was first described in the 19th
Acentury, this compound continues to be investigated for
its biological properties, as accounts of the anticancer,1
antidiabetic,2 and antiviral3 properties of this plant quaternary
alkaloid were recently published. Berberine also possesses
activity against protozoans that cause malaria,4,5 leishmania-
sis,6,7 and African trypanosomiasis.8 The clinically used drugs
available to treat these parasitic infections display one or more
limitations, including toxicity, expense, the need for parenteral
administration, and decreased efficacy due to resistance.
Berberine has poor bioavailability,9 requiring large oral doses
to produce a pharmacological effect,6,10 and the in vitro potency
of berberine is lower than that of standard antiprotozoal
drugs.5,8 Thus, improvements in the potency and physicochem-
ical properties of berberine are required before related
compounds can be considered as candidates against malaria,
leishmaniasis, and/or African trypanosomiasis.
Cheng et al. reported the 8,8-dialkyldihydroberberines (8,8-
DDBs), 5a−5c, and investigated their antidiabetic activity.13
Compound 5a promoted glucose uptake and AMPK
phosphorylation in L6 myoblasts and reduced glucose levels
Our previous evaluation of berberine analogues synthesized
many years earlier11 resulted in the identification of a sample
with exceptional in vitro antiprotozoal potency. Analysis of this
derivative, initially thought to be 8,8-diethyldihydroberberine
Special Issue: Special Issue in Honor of Lester A. Mitscher
Received: September 17, 2012
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Article
a
Scheme 1
a
Reagents and conditions: (a) aq NaOH or KOH, reflux; (b) POCl3, reflux; (c) 1-EtMgBr, Et2O, reflux; 2-H2SO4, ice, then HCl (g); (d) 1-MeMgBr
(3 M in Et2O), EtMgBr (3 M in Et2O), or n-PrMgBr (2 M in THF), Et2O, reflux; 2-NH4OH, then HCl(g) in EtOAc, rt; (e) 1-MeMgBr or EtMgBr
(3 M in Et2O), Et2O, reflux; 2-NH4OH; 3-NaBH4, MeOH.
a
Table 1. In Vitro Activity (nM) of Berberine and 8,8-DDBs
b
c
d
compound
IC50 vs Pf
IC50 vs Ld
IC50 vs Tbb
IC50 vs Vero cells
e
e
1
800 310
20
17 000 3000
290 40
1100 100
2.0 0.0
>200 000
4
4
39 000 4000
8800 1800
18 000 1000
19 000 1000
5a
5b
5c
6a
6b
63 10
77 34
200 50
4600 400
5.3 0.9
100 10
30
7300 600
1700
1
550 60
53
9
f
g
7300 3100
52 000 4000
230 10
ND
ND
f
0
8900 300
ND
ND
ND
ND
h
CQ
8.3 1.7
ND
ND
i
AmB
46 10
ND
ND
j
Sur
ND
130 10
k
Podo
ND
ND
ND
17
1
a
b
c
d
e
Mean standard error (n ≥ 3 unless noted otherwise). P. falciparum. L. donovani. T. brucei brucei. Mean range (n = 2), results taken from ref
f
g
h
i
j
k
12. Mean range (n = 2). Not determined. Chloroquine. Amphotericin B. Suramin. Podophyllotoxin.
in diabetic mice. Cheng et al. also reported that 5a has an oral
bioavailability of 10%, much higher than that of berberine or
dihydroberberine. In the present study, the 8,8-DDBs 5a−5c
and the canadine derivatives 6a and 6b were prepared
semisynthetically and evaluated for their in vitro antiprotozoal
activity. The in vivo antileishmanial and antitrypanosomal
efficacy of the compounds with the highest in vitro potency
were also evaluated.
synthesized by borohydride-mediated reduction of the 8,8-
DDB-free bases obtained after dialkylation of 3 in good overall
yield (69−72% from 3). 1H and 13C NMR spectra were
consistent with those previously reported for 5a−5c,13 and
HPLC analysis indicated that target compounds 5a−5c, 6a, and
6b were obtained in ≥95% purity. Detailed methods and
characterizations for the target compounds are provided in the
Supporting Information.
In vitro biological activities of the 8,8-DDBs 4 and 5a−5c
and the 8,8-dialkylcanadines 6a and 6b together with those of 1
are given in Table 1. Against erythrocyte-stage P. falciparum
3D7 parasites, compounds 4 and 5c displayed the best efficacy,
with the range of potency being less than 4-fold among the 8,8-
DDBs. Compounds 4 and 5c exhibited in vitro antimalarial
potency within 4-fold of chloroquine against this susceptible
strain. The diethyl compound 5b was the most potent 8,8-DDB
against intracellular L. donovani, exhibiting approximately 2-fold
lower potency than the antileishmanial drug amphotericin B
and 2-fold greater potency than 5a. In turn, the diethyl
derivatives 4 and 5b were the most potent molecules in this
series against bloodstream-form Trypanosoma brucei brucei,
displaying low nanomolar IC50 values and superior in vitro
RESULTS AND DISCUSSION
■
Compounds 5a−5c were synthesized from 1 through the
intermediates 8-oxoberberine (2) and 8-chloroberberine (3) in
a similar manner to 4, using slight modifications of literature
procedures (Scheme 1).11−13 A suspension of 3 in diethyl ether
was treated with methyl, ethyl, and n-propyl Grignard reagents
followed by ammonium hydroxide workup13 to provide the
corresponding 8,8-DDB-free bases. The acidic workup used
previously likely leads to oxidation of the 8,8-DDB product, as
observed in the synthesis of 4.12 The 8,8-DDB-free bases were
unstable and were thus converted into their stable salts in 48−
66% yield by bubbling HCl gas through a solution of the free
bases in EtOAc. Canadine derivatives 6a and 6b were
B
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antitrypanosomal efficacy compared to suramin. The carbonyl-
containing compound 4 exhibited 2.5-fold greater potency than
5b, while the dipropyl and dimethyl derivatives 5c and 5a were
27-fold and 2300-fold less potent than 4, respectively. The 8,8-
dialkylcanadines 6a and 6b were much less active than the
corresponding 8,8-DDBs against all three parasites. As with 8,8-
DDBs 5a and 5b, dimethyl canadine (6a) is far less potent
against African trypanosomes than diethyl congener 6b (230-
fold). In general, the in vitro selectivity indexes for the 8,8-
DDBs (IC50 value vs Vero cells/IC50 value vs parasites) were
excellent. The selectivity indexes for 4 and 5b were greater than
100 against L. donovani and over 3000 against T. b. brucei, while
these indexes were greater than 600 for 4 and 5c against P.
falciparum.
Given their outstanding in vitro potency, compounds 5a−5c
were evaluated for their toxicity to female BALB/c mice to
determine an appropriate dosing regimen for in vivo efficacy
studies. Compounds 5a−5c displayed no overt toxicity when
given ip to mice at 10 mg/kg/day × 5. Administration of 5a at
10 mg/kg/day × 5 starting at 7 days postinfection by the ip
route to female BALB/c mice infected with L. donovani
parasites resulted in 46% inhibition of liver parasitemia when
assessed at 14 days postinfection. Compounds 5b and 5c were
less effective in this model when given at the same dose and
assessed under the same conditions (Table 2).
known. The antitrypanosomal drug pentamidine cures a subset
of infected animals in this model when given ip at 20 mg/kg/
day × 4.14
Since previous studies demonstrated the binding of 1 to
DNA,15,16 we examined the DNA binding properties of the 8,8-
DDBs (Table 4). The change in melting temperature (ΔTm)
Table 4. Increase in Poly(dA·dT) Melting by 1 and 8,8-
DDBs
compound
ΔTm (°C)
1
3.5
0.5
0.3
0.5
0.0
4
5a
5b
5c
caused by 1 was modest, consistent with earlier work.15 Little to
no increase in the ΔTm of poly(dA)·poly(dT) DNA was
observed in the presence of 4 and 5a−5c, suggesting that these
compounds are not DNA binders.
Considering the lack of DNA binding of the 8,8-DDBs and
their superior antiparasitic activity compared to 1, we examined
the conformations of 1 and 8,8-diethyl derivatives 4 and 5b by
molecular modeling. For the energy-minimized conformations
of 1, 4, and 5b, the dimethoxyaryl (“D”) ring present in all
analogues was superimposed (see Scheme 1 for the labeling and
numbering of the protoberberine ring system). A larger
dihedral angle between the boxed atoms of the “C” ring was
observed for 4 (15.8°) and 5b (8.5°) compared to 1 (0.1°).
Thus, both 4 and 5b prefer a more bent geometry than 1 due to
the loss in aromaticity of ring C.
While 4 displayed exceptional in vitro potency against
malaria parasites, Leishmania, and African trypanosomes and
exhibited efficacy in a mouse model of visceral leishmaniasis,
this compound was toxic to mice at relatively low doses.12
However, removing the C-13 carbonyl and the C-5−C-6
double bond resulted in the 8,8-DDBs 5a−5c, which displayed
in vitro antiprotozoal potency comparable to that of 4. The C-
13 carbonyl and the C-5−C-6 double bond present in 4 are
thus not absolute requirements for antiparasitic activity. Despite
their in vitro potency, moderate to weak in vivo efficacy was
observed with 8,8-DDBs in murine models of visceral
leishmaniasis and African trypanosomiasis. As mentioned
earlier, Cheng et al. reported that the oral bioavailability of
5a was 10%, superior to that of 1 or dihydroberberine,13 but
there is no further information regarding the pharmacokinetic
properties of 5a or the other 8,8-DDBs examined here. Since a
compound’s pharmacokinetic profile has a profound impact on
its in vivo efficacy, a detailed investigation of such properties for
8,8-DDBs is required to rationalize the apparent disconnect
between in vitro and in vivo antiparasitic efficacy.
Table 2. In Vivo Efficacy of 8,8-DDBs in a Murine Model of
Visceral Leishmaniasis
dose
(mg/kg)
% inhibition of liver parasitemia (mean SD,
compound
n = 4)
5a
10 × 5 ip
10 × 5 ip
10 × 5 ip
10 × 5 ip
46
3
5b
29 11
32 10
5c
miltefosine
97
2
On the basis of their potency against African trypanosomes
in vitro, 4 and 5b were evaluated for efficacy in female NMRI
mice infected with T. b. rhodesiense STIB900 parasites. Infected
animals were treated with compounds or vehicle daily from day
3 to 6 postinfection; then parasitemia was assessed on day 7
postinfection and twice weekly thereafter. When administered
ip at 1 mg/kg/day × 4, compound 4 reduced parasitemia in all
infected mice on day 7, but each animal in this group displayed
heavy parasitemia on day 10 (Table 3). Treatment with
compound 5b at 10 mg/kg/day × 4 ip produced a more
variable response in infected mice, with two animals having
cleared parasitemia by day 7 and relapsing as late as day 10 and
18 postinfection. One animal in the group treated with 5b was
found dead on day 7 postinfection; the cause of death is not
Table 3. In Vivo Efficacy of 4 and 5b against T. b. rhodesiense
Compounds 5a−5c were approximately 2- to 4-fold more
toxic to Vero cells in vitro compared to 4 (Table 1), but could
be administered to BALB/c mice at 10-fold higher doses than 4
by the ip route. The 8,8-DDBs evaluated here may elicit cell
type- or organ system-specific toxicity that is not reproduced by
assays performed on Vero cells or the peritoneal macrophage
host cells we employed. The problem of correlating in vitro and
in vivo toxicity data has been noted previously.17 Identification
of the biological targets of the 8,8-DDBs is needed to better
characterize the effects of these compounds on both parasites
and host cells. Berberine is active against numerous cellular
STIB900
dose
cured/
compound
(mg/kg)
infected
day of relapse
a
b
a
a
control
untreated
1 × 4 ip
10 × 4 ip
0/3
0/4
0/4
7
7
7
7
7
7
b
b
b
b
4
7
7
5b
18
10
dead on
d7
a
b
Control mice displayed heavy parasitemia on day 7. Mice were
weakly positive for trypanosomes on day 7 and heavily positive on day
10.
C
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targets and also binds to DNA.15,16 Since the 8,8-DDBs did not
affect the melting temperature of poly(dA·dT) DNA (Table 4),
it is unlikely that these compounds either act on parasites by
binding directly to DNA or possess mutagenic effects in cells.
The lack of planarity of compounds 4 and 5b (Figure 1) may
point apparatus and are uncorrected. Thin-layer chromatography was
conducted on precoated TLC plates from E. Merck, and compounds
were visualized with UV light. Nuclear magnetic resonance spectra
were obtained at 300 or 400 MHz for 1H and 75 or 100 MHz for 13
C
using instruments from Bruker. Purity of the target compounds was
assessed using either a Phenomenex Gemini 5 μm C18 Axis packing
reversed-phase column (250 × 4.1 mm) or a Merck LichroCART
Lichrospher 100 RP-18 10 μm reversed-phase column employing a
Hitachi HPLC system (L-2130) and a diode array detector (L-2455)
at a constant flow rate of 1 mL/min. Mobile phase A consisted of
water containing 0.1% trifluoroacetic acid, while mobile phase B
consisted of acetonitrile containing 0.1% trifluoroacetic acid. The
gradient employed ran from 0% to 100% mobile phase B over 22 min,
then from 100% to 0% mobile phase B over 8 min (total run time of
30 min). Details of the synthetic procedures and compound
characterizations are available as Supporting Information.
In Vitro Assays. In vitro antimalarial,21 antileishmanial,22 and
antitrypanosomal23 efficacy testing and cytotoxicity evaluation24 of the
compounds was carried out as described previously. The binding of
test compounds to poly(dA)·poly(dT) DNA was assessed by a
method reported earlier.25
Evaluation of in Vivo Efficacy. The efficacy of test and control
compounds against L. donovani LV82 parasites in female BALB/c mice
was examined by methods described previously.26 Evaluation of
compounds for efficacy in female NMRI mice infected with T. b.
rhodesiense STIB900 parasites was performed according to the
procedure outlined by Wenzler et al.14 in which infected animals
were treated with test compounds or vehicle daily from day 3 to 6
postinfection, with the minor modification that parasitemia was
recorded by tail blood examination on day 7 postinfection and twice
weekly thereafter. The day of relapse was recorded after detection of
parasitemia; mice with a heavy parasite load were euthanized.
Protocols for in vivo experiments were approved by either the
Institutional Animal Care and Use Committee at The Ohio State
University (L. donovani model) or the veterinary authorities of the
Canton Basel-Stadt, Switzerland (T. b. rhodesiense model).
Figure 1. Energy-minimized conformations of 1 (cyan), 4 (coral), and
5b (gray) superimposed by ring D. Dihedral angles were calculated for
the indicated atoms of the “C” ring (yellow boxes). The largest
dihedral angle of these three compounds, occurring in 4, is shown in
red.
prohibit the binding of these semisynthetic berberine
derivatives to DNA. Berberine13,18 and the 8,8-DDBs 5a−
5c13 promote the phosphorylation of the metabolic regulatory
protein AMP-dependent protein kinase (AMPK). The presence
of AMPK subfamily genes was proposed in kinetoplastid
parasites,19 and homologues of AMPK subunits β and γ were
partially characterized in T. brucei.20 Further studies are
required to formulate a hypothesis concerning the antiparasitic
mechanism(s) of action of the 8,8-DDBs. If the antiparasitic
target(s) of the 8,8-DDBs is(are) distinct from the mammalian
target(s) resulting in toxicity, it may be possible to increase the
in vivo selectivity of these compounds while optimizing their
efficacy.
Molecular Modeling. Compound coordinates were built using the
Maestro v9.1 interface of Schrodinger Suite 2010. After initial
̈
geometry cleanup, compounds were subjected to Macromodel energy
minimization (Batchmin v9.8). Potentials from OPLS force field 2005
and a constant dielectric of 1.0 were used with water as solvent. A
maximum of 500 steps with a convergent threshold of 0.05 rmsd was
applied for the PRCG minimization method.
Given the shortcomings of the current drugs used against
malaria, leishmaniasis, and African trypanosomiasis, a series of
compounds capable of providing leads against one or more of
these diseases should be of great interest. The 8,8-DDBs
possess several favorable attributes as an antiparasitic class.
First, they display outstanding in vitro potency and selectivity
against the relevant parasites. Second, the 8,8-DDBs reported
here can be prepared in three steps from the relatively
inexpensive plant alkaloid berberine. Third, they possess
activity in murine models of visceral leishmaniasis and African
trypanosomiasis, providing proof of concept that 8,8-DDBs
display in vivo antiparasitic efficacy. Finally, distinctions in the
emerging antiparasitic structure−activity relationship for 8,8-
DDBs and 8,8-dialkylcanadines indicate that it should be
possible to design related molecules with selectivity for one
protozoan compared to another, boding well for the
identification of highly specific antiparasitic agents. A broader
investigation of the 8,8-DDBs and their analogues is thus
warranted in the search for new drug candidates against
protozoan parasites.
ASSOCIATED CONTENT
* Supporting Information
Details of the synthetic procedures and compound character-
izations are available free of charge via the Internet at http://
pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Tel: +1 614 292 5499. Fax: +1 614 292 2435. E-mail:
werbovetz.1@osu.edu.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported financially by the Ohio State
University Public Health Preparedness for Infectious Diseases
program.
DEDICATION
EXPERIMENTAL SECTION
General Experimental Procedures. All reagents and solvents
were from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated.
Melting points were recorded on a Thomas-Hoover capillary melting
■
■
Dedicated to Dr. Lester A. Mitscher, of the University of
Kansas, for his pioneering work on the discovery of bioactive
natural products and their derivatives.
D
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