Antimalarial activity of natural and synthetic prodiginines

Article abstract of DOI:10.1021/jm200543y

Prodiginines are a family of linear and cyclic oligopyrrole red-pigmented compounds. Herein we describe the in vitro antimalarial activity of four natural (IC₅₀ = 1.7-8.0 nM) and three sets of synthetic prodiginines against Plasmodium falciparum. Set 1 compounds replaced the terminal nonalkylated pyrrole ring of natural prodiginines and had diminished activity (IC ₅₀ > 2920 nM). Set 2 and set 3 prodiginines were monosubstituted or disubstituted at either the 3 or 5 position of the right-hand terminal pyrrole, respectively. Potent in vitro activity (IC₅₀ = 0.9-16.0 nM) was observed using alkyl or aryl substituents. Metacycloprodiginine and more potent synthetic analogues were evaluated in a P. yoelii murine patent infection using oral administration. Each analogue reduced parasitemia by more than 90% after 25 (mg/kg)/day dosing and in some cases provided a cure. The most favorable profile was 92% parasite reduction at 5 (mg/kg)/day, and 100% reduction at 25 (mg/kg)/day without any evident weight loses or clinical overt toxicity.

Full text of DOI:10.1021/jm200543y

ARTICLE
pubs.acs.org/jmc
Antimalarial Activity of Natural and Synthetic Prodiginines
Kancharla Papireddy,† Martin Smilkstein,†,‡ Jane Xu Kelly,†,‡ Shweta,† Shaimaa M. Salem,†
Mamoun Alhamadsheh,† Stuart W. Haynes,§ Gregory L. Challis,§ and Kevin A. Reynolds*,†
†Department of Chemistry, Portland State University, Portland, Oregon, United States
‡Medical Research Service, Department of Veterans Affairs Medical Center and Oregon Translational Research and
Drug Development Institute, Portland, Oregon, United States
§Department of Chemistry, University of Warwick, Coventry, U.K.
S Supporting Information
b
ABSTRACT:
Prodiginines are a family of linear and cyclic oligopyrrole red-pigmented compounds. Herein we describe the in vitro antimalarial
activity of four natural (IC50 = 1.7ꢀ8.0 nM) and three sets of synthetic prodiginines against Plasmodium falciparum. Set 1
compounds replaced the terminal nonalkylated pyrrole ring of natural prodiginines and had diminished activity (IC50 > 2920 nM).
Set 2 and set 3 prodiginines were monosubstituted or disubstituted at either the 3 or 5 position of the right-hand terminal pyrrole,
respectively. Potent in vitro activity (IC50 = 0.9ꢀ16.0 nM) was observed using alkyl or aryl substituents. Metacycloprodiginine and
more potent synthetic analogues were evaluated in a P. yoelii murine patent infection using oral administration. Each analogue
reduced parasitemia by more than 90% after 25 (mg/kg)/day dosing and in some cases provided a cure. The most favorable profile
was 92% parasite reduction at 5 (mg/kg)/day, and 100% reduction at 25 (mg/kg)/day without any evident weight loses or clinical
overt toxicity.
’ INTRODUCTION
prodiginines are also shown to have potent in vitro activity
against Plasmodium species, at much lower concentrations than
seen with mammalian cells.7aꢀe Despite potent and selective in
vitro activity against P. falciparum, the reported efficacy of
prodiginines in vivo has been incomplete, associated with
toxicity, or lacking altogether, discouraging their further con-
sideration as antimalarial candidates.
On careful review, however, the previous studies are each
limited in the identification or variety of compounds tested, by
the lack of in vivo correlation studies, or by the route of drug
administration in the animals studied. These limitations leave
open the question of whether or not there is suitable antimalarial
activity among these compounds. Furthermore, previous studies
have been limited to naturally occurring prodiginines, leaving
open the possibility that some synthetic analogues may have
improved in vitro activity or in vivo efficacy or reduced toxicity.
We therefore undertook a more comprehensive assessment of
Malaria remains a major global health threat and an enormous
economic burden to disease-endemic nations. Despite increased
attention and new commitment to malaria eradication, the
disease causes more than a million deaths each year, and drug
resistance to each new chemotherapeutic approach has devel-
oped, most notably in Plasmodium falciparum, the parasite
causing nearly all malaria-related deaths.1 On the heels of the
global spread of chloroquine-resistant (CQR) P. falciparum,
resistance has also quickly developed to a variety quinoline analo-
gues, to antifolates, to inhibitors of electron transport, and
perhaps most ominously, now to artemisinin.2a,b It is evident
that the search for effective novel antimalarial compounds must
be comprehensive and must include explorations of chemotypes
distinct from the prototypes in clinical use.
Prodiginines are a family of linear and cyclic oligopyrrole
red-pigmented compounds with antibacterial,3 anticancer,4 and
immunosuppressive activity,5 produced by actinomycetes and
other eubacteria. Some prodiginines induce apoptotic effects,
breaking genomic deoxyribonucleic acid (DNA) strands.6 The
Received: November 17, 2010
Published: July 08, 2011
r
2011 American Chemical Society
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Scheme 2. Synthesis of 3-Alkylated Pyrroles (10aꢀk)
Figure 1. Naturally occurring alkyl prodiginines (1ꢀ4).
Scheme 1. Synthesis of 2-Alkylated Pyrroles (6aꢀh)
gave the desired prodiginine analogues 18ꢀ25 (Table 1) and
26ꢀ43 (Tables 2 and 3).
A new synthetic strategy, outlined in Scheme 5, was used
to synthesize 3,5-disubstituted prodiginine analogues 46ꢀ55
(Table 2) and 56ꢀ75 (Table 3). The 2,4-disubstituted pyrroles
44 were synthesized from the corresponding 2- or 3-acylpyrroles
(5 or 9) with their corresponding acyl chlorides and AlCl3.14
Subsequent reduction with NaBH4 gave the desired 2,4-disub-
stituted pyrroles 45.9 Acid-catalyzed condensation of these
45 with 13 provided the desired 3,5-disubstituted prodiginine
analogues 46ꢀ75 (Tables 2 and 3).13
Biological Activity. The natural and synthetic prodiginines
were assayed for their in vitro antimalarial activity against
P. falciparum pansensitive D6 with chloroquine (CQ) as a ref-
erence drug. A direct comparative assessment of the antimalarial
activity of natural products prodigiosin (1), undecylprodiginine
(2), metacycloprodiginine (3), and streptorubin B (4) (Figure 1)
revealed they all had remarkably potent activity with very
low IC50 values (8, 7.7, 1.7, and 7.8 nM, respectively) against
P. falciparum strain D6 and were slightly more active than CQ
(11 nM). The natural prodiginine 3 was consistently observed to
be the most potent of the four natural products and demon-
strated that changes in the alkyl component of prodiginines
affected the in vitro antimalarial activity. On the basis of these
observations, a series of new analogues of prodiginines with
similar core structure were generated.
In the early stages, a set of analogues 18ꢀ25 (Table 1), in
which an indole, phenyl, thiophen, and furan replaced the
terminal nonalkylated pyrrole ring (left-hand side) of the core
moiety, were generated. In vitro analysis of the activity of these
compounds against P. falciparum pansensitive D6 demonstrated
activity (IC50 > 2920 nM) significantly diminished relative to that
of the natural prodiginines 1ꢀ4 (IC50 = 1.7ꢀ8.0 nM). This work
suggested that the left-hand side pyrrole ring of the prodiginines
is important for the potent antimalarial activity. We subsequently
tested a hypothesis that the alkyl chain on right-hand side pyrrole
of the prodiginines might represent an opportunity to make
potent and selective antimalarials with the desired “druglike”
properties. Subsequent analogues were synthesized (Schemes 4
and 5) to obtain a SAR for the alkyl groups at the terminal
alkylated pyrrole of the prodiginines.
the antiplasmodial activity of prodiginines, initially reassessing the
activity of four naturally occurring prodiginines 1ꢀ4 (Figure 1) and
subsequently assessing a series of synthetic analogues. Here we
report these results, including impressive in vitro potency, structureꢀ
activity relationship (SAR), and evidence of in vivo efficacy, including
curative efficacy in mice after oral administration.
’ RESULTS AND DISCUSSION
Synthesis. The synthesis of various 2-alkylpyrrole segments
6aꢀh (Scheme 1) was initiated by converting the readily
accessible pyrrole to the corresponding 2-acylpyrroles 5aꢀh.8
These acylpyrroles were then smoothly converted to 6aꢀh using
an excess of NaBH4 in 2-propanol under reflux.9
Synthesis of 3-alkylpyrrole segments 10aꢀk (Scheme 2) used
the same pyrrole starting material, which treated with phenyl-
sulfonyl chloride in the presence of NaOH provided a N-
phenylsulfonylpyrrole (7).10 Use of AlCl3 permitted the regio-
selective acylation of 7 at the 3 position with the acyl chlorides to
obtain the N-phenylsulfonyl-3-acylpyrroles 8aꢀk, which were
subsequently hydrolyzed under basic (5 N NaOH) conditions to
give 3-acylpyrroles 9aꢀk.11 In the final step 9aꢀk were reduced
to 10aꢀk using NaBH4 under reflux.9
By use of literature methodologies, the key intermediates
involved in the synthesis of prodiginines, 4-methoxy-2,20-bipyr-
role-5-carboxaldehyde (13) and its analogues 14ꢀ17, were
prepared via intermediate 12 from the commercially available
4-methoxy-3-pyrrolin-2-one (11) (Scheme 3).12 Acid-catalyzed
condensation of appropriate alkylpyrrole segments (2,4-di-
methylpyrrole, 6aꢀh, and 10aꢀk) with 13ꢀ17 (Scheme 4)13
One set of these analogues 26ꢀ38 (Table 2) and 39ꢀ43
(Table 3) were monosubstituted and contained different alkyl
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Scheme 3. Synthesis of 4-Methoxy-2,20-bipyrrole-5-carbaldehyde (13) and Its Analogues (14ꢀ17)
Scheme 4. Synthesis of Various Prodiginine Analogues (18ꢀ43)
chains or aryl groups at either the 3 or 5 position of the terminal
pyrrole ring (right-hand side) (Scheme 4). These compounds
were screened for their in vitro antimalarial activity against
P. falciparum strain D6, and the results are shown in Tables 2 and
3. Of the monoalkylated analogues, compounds 26, 27, 31, 32,
and 38 showed the poorest activity with IC50 values above 1700
nM against D6. Compounds 28, 30, and 33 all exhibited slightly
better activity with IC50 values 375, 300, and 460 nM, respec-
tively, against D6. Analogues 29, 34, 35, 36, and 37 were the most
potent of the monoalkylated prodiginine analogues with IC50
values of 80, 80, 28, 4.6, and 8.0 nM, respectively, against D6.
These observations and the activity of the natural product 2
demonstrate that the C6ꢀC11 alkyl chain length at either the 3 or
5 position of the right-hand side pyrrole ring is required for
optimal activity of monoalkylated prodiginines. The presence of
either an amine group (see 31 IC50 = 1700 nM (D6) versus 2
40 IC50 = 170 nM (D6)), the activity was poorer than that seen for
the more potent monoalkylated prodiginines (such as 2and 35ꢀ37).
The final set of prodiginine analogues generated had substit-
uents at both positions 3 and 5 of the terminal pyrrole ring (right-
hand side). The substituents were two alkyl chains (46ꢀ55), an
alkyl chain and an aryl group (56ꢀ65), or two aryl groups
(66ꢀ75). These compounds 46ꢀ75 (Tables 2 and 3) were
screened for in vitro animalarial activity against P. falciparum
strain D6. Among the dialkylated analogues, 49, 50, and 51
showed potent in vitro antimalarial activity with IC50 values of
1.7, 1.7, and 2.1 nM, respectively (Table 2), against D6. Other
dialkylated prodiginines 47, 48, 52, 53, and 55 also exhibited
excellent activity with IC50 values of 4.5, 2.9, 4.9, 6.2, and 5.3 nM,
respectively, against D6 and demonstrated that they are more
active than the monoalkylated prodiginines (2 and 26ꢀ38). The
two exceptions were analogues 46 (IC50 = 8900 nM) and 54
(IC50 = 92 nM), which contained a total alkyl chain length of 2
and 16 carbons, respectively, on the right-hand side pyrrole ring
of the core moiety. From both the mono- and dialkylated series
the data clearly show that prodiginines containing a combined
C8ꢀC15 alkyl chain length had the most potent in vitro
antimalarial activity (Figure 2).
IC50 = 7.7 nM (D6)) or methyl carboxylate (see 32 IC50
=
4500 nM (D6) versus 34 IC50 = 80 nM (D6)) at the end of the
alkyl chain leads to a decrease in activity. The 3-aryl monosub-
stituted prodiginine analogues 39ꢀ43 (Table 3) exhibited mod-
erate inhibitory activity with IC50 values of 83, 170, 65, 90,
and 56 nM, respectively, against D6. While the presence of a
halogen substituent appeared to lead to an increase in activity
(see 41 IC50 = 65 nM (D6) versus 39 IC50 = 83 nM (D6) and
The analogues 56ꢀ65, which contain one alkyl and one aryl
substituents on the right-hand pyrrole ring were also tested for in
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Table 1. In Vitro Antimalarial Activity of Prodiginine Analogues Where the Nonalkylated Terminal Pyrrole (Left-Hand Side) Has
Been Substituted
vitro antimalarial activity against P. falciparum strain D6. Among
these analogues, 62 (IC50 = 0.9 nM) and 63 (IC50 = 1.3 nM)
(Table 3) were the most potent and comparable to the most
potent dialkylated prodiginines 49ꢀ51 and the natural prodigi-
nine 3 (IC50 = 1.7 nM). In this series (56ꢀ65), all compounds
with the exception of 60 (IC50 = 16.0 nM) were extremely potent
(IC50 < 6.3 nM), significantly more than the corresponding
analogues 39ꢀ43 which have an aryl substituent but lack the
alkyl substituent. The in vitro activity results of chloro (56ꢀ61),
fluoro (62 and 63), and bromo (64 and 65) substituted
analogues of prodiginine indicated that the bromo substituted
analogues 64 and 65 were slightly less potent than chloro and
fluoro substituted analogues (Table 3).
The majority of the new synthetic prodiginines were also
tested alongside CQ against the multidrug-resistant (MDR)
Dd2. In all cases there was no significant change in the activity
of each analogue between the D6 and Dd2 (Tables 1ꢀ3),
indicating no cross-resistance with CQ. The most potent syn-
thetic prodiginines were active in the 1 nM range against Dd2,
compared to 135 nM for CQ.
A number of the synthetic prodiginines were tested against a
number of mammalin cell lines, and IC50 in the 500ꢀ2000 nM
range was not observed (data not shown). A general assay with
mitogen stimulated murine splenic lymphocytes was used to
evaluate all the synthetic prodiginines (Tables 1ꢀ3) and to
determine if synthetic changes led to reduced toxicity.15 The
majority of the synthetic prodiginines exhibited much greater
toxicity (IC50 < 125 nM) in this particular assay. There were
prodiginines that had slightly less toxicity (1000ꢀ4000 nM), but
these were in all cases also associated with markedly reduced
activitiy against P. falciparum and were still more toxic than CQ
(16 000 nM). Thus, by use of these particular in vitro assays, the
antimalarial activity and toxicity for the current set of synthetic
prodiginines exhibit comparable SAR.
The final set of analogues 66ꢀ75 contains two aryl substituent
groups on the right-hand side pyrrole ring. In this set, most of
the compounds (66, 67, 69ꢀ71, 74, and 75) exhibited
potent activity (IC50 < 8.3 nM). The exceptions were 68 (IC50
=
14.0 nM), which contains a bromine atom on both aryl substit-
uents, and 72 (IC50 = 12.6 nM) and 73 (IC50 = 14.7 nM) in
which each aryl group is dihalogenated (Table 3). A comparison
of the most potent compounds in this series with analogues
47ꢀ65 revealed that the in vitro activity for prodiginines bearing
two aryl substituents is slightly less than for disubsititued
prodiginines in which either one or both of the substituents are
alkyl chains. That said, a general pattern of more potent in vitro
activity for disubstutued prodignines compared to monosubsti-
tuted prodiginines was observed.
The in vivo antimalarial efficacy of the natural prodiginine 3
and a selection of the more potent synthetic analogues were
evaluated in a P. yoelii murine patent infection. In initial proof-
of-principle testing, administration of 25 (mg/kg)/day 3 by
gavage using a caprylic/capric triglyceride vehicle resulted in
greater than 99% reduction in parasitemia (0.28% vs 54.1% in
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Table 2. In Vitro Antimalarial Activity of Prodiginines Containing Alkyl Substituents at the 3 and 5 Positions of the Terminal
Pyrrole Ringa
a N.D.: not determined.
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Table 3. In Vitro Antimalarial Activity of Prodiginines Containing Aryl Substituents at Either the 3 Position or the 3 and 5
Positions of the Terminal Pyrrole Ringa
a N.D.: not determined.
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Scheme 5. Synthesis of 2,4-Disubstitued Pyrroles (45) and the Corresponding Prodiginine Analogues (46ꢀ75)
Table 4. In Vivo Antimalarial Efficacy of Prodiginine
Analogues in P. yoelii Murine Infection
% group mean % parasitemia
dose,
no. of treated
mice
weight,
reduction vs
compd (mg/kg)/day
day 3 vs day 0 controls, day3
controls
5
5
5
5
4
5
4
5
5
5
4
5
3
5
5
100.8
101.2
105.0
100.1
92.4
0
38
41
41
48
48
49
49
57
57
58
58
60
60
66
66
5
25
5
90
90
25
5
100
97
101.5
84.5
25
5
100
94
102.9
87.6
Figure 2. SAR of combined alkyl chain length for mono- and dialky-
lated prodiginines and in vitro antimalarial activity against D6 strain of
P. falciparum.
25
5
100
99
100.3
96.0
25
5
100
72
controls). At 100 (mg/kg)/day parasite clearance was complete
and was accompanied by temporary weight loss, and three of
four mice were cured. These data provided the first demonstra-
tion of oral effectiveness of prodiginines. Equally important,
unlike previous reports of parenteral dosing, efficacy after
gavage dosing was observed to be without evident toxicity.
These findings indicated that antiplasmodial oral efficacy might
be achievable and led to similar in vivo testing of some of the
more potent synthetic prodiginines. Examples of impressive
potency against murine malaria were observed and are briefly
summarized here (Table 4). Analogues 48, 49, 57, 58, and 66
each reduced parasitemia by more than 90%; the exceptions
were 41 (38%) and 60 (72%) after 5 (mg/kg)/day dosing,
without any evidence of weight loss or toxicity. 41, 48, 49, 57,
58, 60, and 66 each reduced parasitemia by g90% after 25
(mg/kg)/day dosing; however, weight loss was observed during
the dosing course with a few analogues (Table 4). However,
the weight reduction was temporary and for unknown rea-
sons (including dehydration, fasting of tested mice, or toxicity
100.2
93.4
25
5
100
92
107.5
102.5
25
100
at the higher dose). While an exhaustive in vivo analysis of
prodiginine analogues was not conducted, the temporary
weight loss was primarily associated with analogues with alkyl
substituents. The prodiginines 41 and 66 by contrast have no
alkyl substituents and have one or two aryl substituents. Of
these compounds tested, 66 had the most favorable profile: 92%
parasite reduction at 5 (mg/kg)/day, 100% reduction at 25
(mg/kg)/day without any evident weight loss or clinical overt
toxicity. Although testing consisted only of equidose compar-
isons between compounds, without full dose ranging or
efforts to define curative dose levels, it is worth mentioning
that there was one cure each in the 60 and 58 treated groups
(25 (mg/kg)/day).
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’ CONCLUSIONS
29.8 mmol), octanoyl chloride (7.8 g, 44.7 mmol), and zinc powder
(3.88 g, 59.7 mmol) in toluene (50 mL) was stirred at room
temperature for 4 h. The reaction mixture was quenched with
saturated sodium bicarbonate solution (50 mL) and extracted with
ethyl acetate (3 ꢁ 30 mL). The combined organic layer was washed
with water, dried over anhydrous Na2SO4, and evaporated under
reduced pressure. The crude product was chromatographed on
Although natural prodiginines 37d and heptylprodigiosin
(76)7e,16 have demonstrated remarkable in vitro potency against
growth of P. falciparum, the mode of action is unknown, and a
SAR has not been reported. Herein, we have prepared a set of
prodiginine analogues via simple and efficient pathways from
commercially available inexpensive starting materials and re-
agents. These standardized protocols can be used to generate a
large number of prodiginines in a gram scale from a single batch.
This current work has suggested that the nonalkylated terminal
pyrrole (left-hand side) of the natural prodiginines is required for
in vitro activity but that the substituents at the alkylated terminal
pyrole (right-hand side) can be varied. Specifically in vitro
activity comparable or better (IC50 e 1 nM) than most of the
natural prodiginines was observed for analogues with either alkyl
or aryl substituents at both the 3 and 5 positions of the right-hand
side pyrrole, In addition these synthetic prodiginines are equally
effective against P. falciparum pansensitive D6 and MDR Dd2.
Previous results of in vivo testing, have also cast doubt on
the suitability of prodiginines as antimalarial candidates. Against
P. berghei in mice, subcutaneous injection of natural prodiginines
1,7a 76,7e 2, 3, butylcycloheptylprodiginine (77), and cyclono-
nylprodiginine (78)7b,16 have each been shown to extend
survival, however, either without cure or at doses resulting in
more toxic deaths than cures. Natural prodiginine 1 was shown to
be completely ineffective for antimalarial activity, even at toxic
doses, for oral treatment of Rhesus monkeys infected with
P. cynomolgi.7c In the current study it has been demonstrated
for the first time that prodiginines can be administered orally and
produce marked parasite clearance, including cures in some
cases, without evident weight lose and toxicity. Nonetheless,
preliminary in vitro assays indicate concerns associated with
the toxicity of prodiginines, and the quest for analogues with
improved antimalarial activity but reduced toxicity continues.
1
silica gel to afford the title compound 5e (3.16 g, 55%). H NMR
(CDCl3, 400 MHz) δ 9.71 (br s, 1H), 7.02 (m, 1H), 6.91 (m, 1H),
6.27 (m, 1H), 2.75 (t, J = 7.5 Hz, 2H), 1.70 (m, 2H), 1.33 (m, 8H),
0.92 (t, J = 7.1 Hz, 3H).
Representative Procedure for the Synthesis of 2-Octyl-
1H-pyrrole (6e). To a stirred solution of 5e (1.0 g, 5.18 mmol) in
150 mL of 2-propanol (IPA) at 25 °C was added slowly sodium
borohydride (NaBH4) (1.34 g, 36.26 mmol), and the reaction mixture
was heated at reflux for 10 h. The hot reaction mixture was poured into
150 mL of iceꢀwater, and the solution was acidified with 10% aqueous
HCl. The suspension was extracted with dichloromethane (3 ꢁ 50 mL).
The combined organic extracts were washed with water, brine and dried
over anhydrous Na2SO4. The solvent was evaporated under reduced
pressure, and the crude product was chromatographed on silica gel to
afford the title compound 6e (714 mg, 77%). 1H NMR (CDCl3,
400 MHz) δ 7.79 (br s, 1H), 6.56 (m, 1H), 6.04 (m, 1H), 5.83 (m,
1H), 2.50 (t, J = 7.6 Hz, 2H), 1.53 (m, 2H), 1.28 (m, 10H), 0.79 (t, J =
7.0 Hz, 3H); 13C NMR (CDCl3, 100 MHz) δ 132.9, 116.0, 108.2, 104.8,
31.9, 29.7, 29.5, 29.4, 29.3, 27.8, 22.7, 14.1.
Synthesis of Compound 7. Pyrrole (10 g, 149 mmol) was added
to a well-agitated suspension of NaOH (17.9 g, 447 mmol) in 100 mL of
1,2-dichloroethane. This mixture was then cooled to 0 °C and stirred for
10 min. Then a solution of phenylsulfonyl chloride (31.5 g, 173 mmol)
in 20 mL of 1,2-dichloroethane was added dropwise over a period of
20 min. Thirty minutes after the completion of addition, the mixture was
allowed to come to room temperature and left stirring overnight. The
reaction was quenched by pouring the mixture into 300 mL of distilled
water. The organic layer was separated, and the aqueous layer was
extracted with dichloromethane (3 ꢁ 100 mL). The combined organic
extract was washed with distilled water to neutrality and dried over
anhydrous Na2SO4. Removal of the solvent in vacuo gave 7 as a white
crystalline solid (21.6 g, 70%). This solid was subsequently washed with
hot hexane to give greater than 65% overall yield.
Representative Procedure for the Synthesis of (1-Benze-
nesulfonyl-1H-pyrrol-3-yl)-(4-chlorophenyl)methanone (8i).
To a stirred suspension of anhydrous AlC13 (3.85 g, 29 mmol) in 50 mL
of 1,2-dichloroethane at 25 °C was added slowly octanoyl chloride (5.0 g,
27 mmol). The resulting solution was stirred at 25 °C for 10 min. A
solution of 7 (5.0 g, 24 mmol) in 10 mL of 1,2-dichloroethane was added
slowly, and the mixture was stirred at 25 °C for 5 h. The reaction was
quenched with ice and water, and the product was extracted into
dichloromethane (3 ꢁ 100 mL). The combined organic layer was washed
with brine, dried over anhydrous Na2SO4, and evaporated under
reduced pressure to afford crude product 8i (8.0 g, 100%) as a white
solid. The crude product was used for the next step without further
purification.
Representative Procedure for the Hydrolysis of 8i. A
solution of 8i (8.0 g, 24 mmol) in 100 mL of 1,4-dioxane was stirred
with 100 mL of 5 N NaOH at 25 °C for 20 h. The organic layer was
collected, and the aqueous layer was thoroughly extracted with ethyl
acetate. The combined extracts were washed with brine, dried over
anhydrous Na2SO4, and evaporated under reduced pressure to give
(4-chlorophenyl)-(1H-pyrrol-3-yl)methanone (9i) as a solid material
(4.6 g, 100%).
’ EXPERIMENTAL SECTION
Purification of Natural Prodiginines (1ꢀ4). Natural product
1 was purchased from the developmental therapeutics program NCI/
NIH. By use of minor modifications to literature procedures, 2 and
4 were isolated from S. coelicolor M51117 and 3 was isolated from
S. longisporus ruber.13a
General. 1H NMR spectra were recorded with a Bruker AMX-400
MHz spectrometer using CDCl3. 13C NMR spectra were recorded at
100 MHz with CDCl3. Chemical shifts are reported as values in
ppm relative to CHCl3 (7.26) in CDCl3, and TMS was used as internal
standard. HRMS (ESI) data were recorded on a high-resolution
(30 000) thermo LTQ-Orbitrap Discovery hybrid mass spectrometer
(San Jose, CA). Reagents were from Aldrich and used as supplied.
Unless otherwise noted, reactions that required the use of anhydrous,
inert atmosphere techniques were carried out under an atmosphere of
argon/nitrogen. Chromatography was executed with silica gel (230ꢀ
400 mesh) and neutral alumina using mixtures of ethyl acetate and
hexane as eluents. Analytical HPLC analyses were performed on a
Supelco Discovery HS C18 column (4.6 mm ꢁ 250 mm) with a linear
elution gradient ranging from CH3OH/CH3CN/H2O (40%/10%/
50%) to CH3OH (100%) in 0.15% trifluoroacetic acid at a flow rate
of 1 mL/min. A purity of >95% has been established for all tested
compounds with the exception of 25, 26, and 33, which achieved 93%,
81%, and 86%, respectively.
Synthesis of (5-Bromo-3-methoxypyrrol-2-ylidene-
methyl)diethylamine (12). To a mixture of diethylformamide
(2.68 g, 26.5 mmol) and chloroform (10 mL) at 0 °C was added
Representative Procedure for the Synthesis of 1-(1H-
Pyrrol-2-yl)octan-1-one (5e). A mixture of pyrrole (2.0 g,
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dropwise a solution of phosphorus oxybromide (6.32 g, 22.1 mmol) in
chloroform (10 mL). The resulting suspension was stirred at 0 °C for
30 min, and the solvent was removed by rotary evaporation to obtain the
Vilsmeier complex as a white solid. After the sample was dried in vacuo
for 20 min, chloroform (10 mL) was added to the solid and the mixture
was cooled to 0 °C. A solution of 4-methoxy-3-pyrrolin-2-one (11)
(1.0 g, 8.8 mmol) in chloroform (20 mL) was added dropwise, and the
mixture was warmed to room temperature and then heated at 60 °C for
5 h. The mixture was poured onto iceꢀwater (75 mL), and the pH of the
aqueous solution was adjusted to pH 7ꢀ8 by treatment with 2 N NaOH.
Ethyl acetate (40 mL) was added to the resulting precipitate, and the
mixture was filtered over Celite to remove the black solid containing
phosphorus salts. The two layers were separated, and the aqueous layer
was extracted with EtOAc (3 ꢁ 100 mL). The organic layers were
combined, washed with brine (3 ꢁ 200 mL), dried over anhydrous
Na2SO4, filtered, and the solvent was removed by rotary evaporation to
furnish the crude bromoenamine 12. The residue was filtered over a pad
of silica gel (50 mL) using a 10% EtOAC/hexanes as eluent to obtain the
enamine as an oil, which upon drying in vacuo led to a solid (1.64 g,
72%). 1H NMR (CDCl3, 100 MHz) δ 6.90 (s, 1H), 5.50 (s, 1H), 4.05 (q,
J = 7.1 Hz, 2H), 3.65 (s, 2H), 3.30 (q, J = 7.1 Hz, 2H), 1.25 (t, J = 7.1 Hz,
3H), 1.20 (t, J = 7.1 Hz, 3H); 13C NMR (CDCl3, 100 MHz) δ165.3,
138.7, 133.5, 120.8, 96.4, 58.0, 51.2, 44.5, 18.3, 21.2.
Representative Procedure for the Preparation of Com-
pound 13. To a degassed solution of toluene (5 mL) were added
Pd(OAc)2 (303 mg, 1.3 mmol) and PPh3 (1.6 g, 6.1 mmol). The mixture
immediately turned bright yellow and was stirred at 70 °C for 30 min
under N2. A solution of 12 (3.5 g, 13.5 mmol) and N-Boc-2-pyrrole-
boronic acid (3.72 g, 17.6 mmol) in 10% water/dioxane (50 mL) was
degassed and purged with N2. The solution was transferred to a sus-
pension of Pd(PPh3)4 in toluene followed by the addition of Na2CO3
(4.31 g, 40.6 mmol). The mixture was stirred for 3 h at 100 °C and then
poured into water (100 mL). The pH of the solution was lowered to pH
7 with 2 N HCl. The brown precipitate was recovered by filtration over a
fritted disk funnel and washed with water and then acetone. The yellow
solid was washed with 10 mL of CHCl3 and then Et2O (2 ꢁ 10 mL). The
desiredproduct 13 was obtained asa yellow solid and usedwithout further
purification (2.32 g, 90%). 1H NMR (DMSO-d6, 400 MHz) δ 11.40 (br s,
1H), 11.20 (br s, 1H), 9.30 (s, 1H), 6.90 (t, J = 4.4 Hz, 1H), 6.75 (s, 1H),
6.25 (d, J = 4.4 Hz, 1H), 6.10 (d, J = 4.4 Hz, 1H), 3.80 (s, 3H).
anhydrous AlC13 (1.34 g, 10.1 mmol) in 30 mL of 1,2-dichloroethane at
25 °C was added slowly octanoyl chloride (1.34 g, 8.1 mmol). The
resulting solution was stirred at 25 °C for 10 min. A solution of 9i (1.3 g,
6.7 mmol) in 10 mL of 1,2-dichloroethane was added slowly, and the
mixture was stirred at 25 °C for 2 h. The reaction was quenched with ice
and water, and the product was extracted into dichloromethane (3 ꢁ
50 mL). The combined organic layer was washed with brine, dried over
anhydrous Na2SO4, and evaporated under reduced pressure to afford 44
(2.14 g, 100%) as a white solid. The crude product was washed with hot
1
hexane to give greater than 96% of overall yield. H NMR (CDCl3,
400 MHz) δ 10.26 (br s, 1H), 7.60 (dd, J = 1.5, 1.9 Hz, 1H), 7.32 (dd, J =
0.9, 1.5 Hz, 1H), 2.78 (m, 4H), 1.72 (m, 4H), 1.38ꢀ1.25 (m, 16H), 0.89
(m, 6H); 13C NMR (CDCl3, 100 MHz) δ 196.1, 192.2, 132.6, 127.5,
127.2, 115.2, 39.8, 38.1, 31.7 (2C), 29.4, 29.3, 29.1, 29.0, 25.1, 24.7 (2C),
22.6, 14.1 (2C).
Representative Procedure for the Synthesis of 2,4-Dioc-
tyl-1H-pyrrole (45). To a stirred solution of 44 (1.3 g, 4.0 mmol) in
150 mL of 2-propanol at 25 °C was added slowly sodium borohydride
(2.11 g, 57.0 mmol), and the reaction mixture was heated at reflux for 10
h. The hot reaction mixture was poured into 150 mL of iceꢀwater, and
the solution was acidified with 10% aqueous HCl. The suspension was
extracted with dichloromethane (3 ꢁ 50 mL). The combined organic
extracts were washed with water, brine, and dried over anhydrous
Na2SO4. The solvent was evaporated under reduced pressure to give
45, and the crude product was chromatographed on neutral alumina
1
to afford the title compound 45 (865 mg, 73%). H NMR (CDCl3,
400 MHz) δ 7.60 (br s, 1H), 7.40 (s, 1H), 5.76 (s, 1H), 2.53 (t, J =
7.6 Hz, 2H), 2.41 (t, J = 7.6 Hz, 2H), 1.57 (m, 4H), 1.33 (m, 18H), 0.87
(m, 6H); 13C NMR (CDCl3, 100 MHz) δ 132.8, 124.9, 112.8, 105.4,
31.9 (2C), 31.2, 29.7, 29.6, 29.5, 29.4, 29.3, 29.2, 27.9, 27.4, 27.2, 22.7
(2C), 14.1 (2C).
Representative Procedure for the Synthesis of 40-Meth-
oxy-50-(5-octyl-3-propyl-1H-pyrrol-2-ylmethylene)-1H,50H-
[2,20]bipyrrolyl, Hydrochloride (48). To a stirred suspension of 13
(100 mg, 0.52 mmol) and compound 45 (151 mg, 0.68 mmol) in
anhydrous methanol (10 mL) was added methanolic 2 N HCl (Catalytic
amount). The resulting bright colored solution was stirred for 5 h at
room temperature. The methanol was removed under reduced pressure.
The crude solid was dissolved in ethyl acetate (50 mL) and washed with
saturated NaHCO3 solution (2 ꢁ 25 mL). The organic layer was dried
over anhydrous Na2SO4. The solvent was removed under reduced
pressure, and the crude product was chromatographed on neutral
alumina as stationary phase and hexaneꢀethyl acetate as mobile phase
to afford the final prodiginine analogue 48 (143 mg, 69%). Most of these
prodiginine analogues were in the form of HCl salt (addition of ethereal
HCl to the pure prodiginine analogue). 1H NMR (CDCl3, 400 MHz) δ
12.79-12.60 (3 br s, 3H), 7.20 (m, 1H), 7.04 (s, 1H), 6.90 (m, 1H), 6.33
(m, 1H), 6.08 (br s, 2H), 4.01 (s, 3H), 2.92 (t, J = 7.7 Hz, 2H), 2.62 (t, J =
7.5 Hz, 2H), 1.75 (m, 2H), 1.64 (m, 2H), 1.41ꢀ1.31 (m, 10H), 0.97 (t,
J = 7.3 Hz, 3H), 0.87 (t, J = 7.1 Hz, 3H); 13C NMR (CDCl3, 100 MHz) δ
165.6, 153.4, 147.4, 145.5, 126.7, 124.2, 122.3, 120.1, 116.7, 113.2, 112.7,
111.6, 92.7, 58.7, 31.9, 29.4, 29.3, 29.2, 29.1, 28.4, 28.3, 24.2, 22.7, 14.1,
13.9. HRMS (ESI) calcd for C25H36N3O (M + H)+, 394.2853; found,
394.2853.
In Vitro Antimalarial Activity: P. falciparum Growth Inhibi-
tion. P. falciparum pansensitive D6 and MDR Dd2 (parasites MRA-285,
MRA-156, ATCC, Manassas, VA) were cultivated under standard condi-
tions, as previously described,18 and in vitro susceptibility to test
compounds was determined by measuring growth after 72 h in the
presence of varying concentrations of test compound or CQ (positive
control) or no drug; each condition was done in quadruplicate. Growth
inhibition was determined using the SYBR Green I based fluorescence
assay19 with minor modifications as previously described20 and expressed
as the compound concentration inhibiting growth by 50% (IC50).
Representative Procedure for the Synthesis of 40-Methoxy-
50-(3-octyl-1H-pyrrol-2-ylmethylene)-1H,50H-[2,20]bipyrrolyl(36).
To a stirred suspension of 13 (220 mg, 1.15 mmol) and compound 10d
(269 mg, 1.5 mmol) in anhydrous methanol (10 mL) was added
methanolic 2 N HCl (catalytic amount). The resulting bright colored
solution was stirred for 5 h at room temperature. The methanol was
removed under reduced pressure. The crude solid was dissolved in ethyl
acetate (50 mL) and washed with saturated NaHCO3 solution (2 ꢁ
25 mL). The organic layer was dried over anhydrous Na2SO4. The
solvent was removed under reduced pressure, and the crude product was
chromatographed on neutral alumina as stationary phase and hexa-
neꢀethyl acetate as mobile phase to afford the final prodiginine
1
analogue 36 (256 mg, 63%). H NMR (CDCl3, 400 MHz) δ 6.92
(s, 1H), 6.77 (m, 1H), 6.72 (dd, J = 1.2, 2.4 Hz, 1H), 6.60 (d, J = 2.4 Hz,
1H), 6.21 (dd, J = 0.9, 2.7 Hz, 1H), 6.06 (s, 1H), 5.97 (d, J = 2.5 Hz, 1H),
4.00 (s, 3H), 2.58 (t, J = 7.6 Hz, 2H), 1.57 (m, 2H), 1.28 (m, 10H), 0.90
(t, J = 6.6 Hz, 3H); 13C NMR (CDCl3, 100 MHz) δ 168.5, 159.8, 139.8,
134.6, 128.5, 126.8, 125.3, 122.5, 113.4, 112.6, 110.4, 110.2, 95.1, 58.4,
31.9, 31.4, 29.5 (2C), 29.3, 26.1, 22.7, 14.1. HRMS (ESI) calcd for
C22H29N3O (M + H)+, 352.2383; found, 352.2387. Most of these
prodiginine analogues were in the form of HCl salt (addition of ethereal
HCl to the pure prodiginine analogue).
Representative Procedure for the Synthesis of 1-(5-Octa-
noyl-1H-pyrrol-3-yl)octan-1-one (44). To a stirred suspension of
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In Vivo Antimalarial Efficacy: P. yoelii Murine Patent
Infection. Female outbred CF-1 mice (Charles River, Wilmington,
MA) were injected intravenously via tail vein with 5 ꢁ 105 erythrocytes
infected with P. yoelii Kenya (parasite MRA-428, ATCC, Manassas, VA),
and groups of five mice were then treated with test compound or vehicle
only, once each day for 3 days beginning 24 h after infection. All
compounds were administered by gavage in caprylic/capric triglyceride
vehicle (Miglyol 812, Sasol, Hamburg, Germany). One day after the final
dose, thin blood smears were made from all mice, stained using a
modified Giemsa method, and parasitemia ((infected erythrocytes/total
erythrocytes) ꢁ 100) was determined using microscopy and compared
to untreated controls. Mice without detectable parasitemia were ob-
served and monitored by serial examination of blood smears. Infected
mice were euthanized; those remaining parasite-free for 28 days were
considered cured.
Alamar Blue Assay for Mammalian Cell Viability. The
general cytotoxic effects of prodiginine analogues on host cells were
assessed by a functional assay as described previously15 using murine
splenic lymphocytes induced to proliferate and differentiate by con-
canavalin A. Splenic lymphocytes isolated from C57BL/6J mice were
washed twice in RPMI 1640 medium and resuspended in complete
RPMI containing 10% fetal bovine serum (FBS), 50 μg/mL penicillin/
streptomycin, 50 μM β-mercaptoethanol, and 1 μg/mL concanavalin A.
Cells (100 μL/well) were then seeded into 96-well flat-bottom tissue
culture plates containing drug solutions (100 μL) serially diluted with
complete culture medium to a final cell density of 2 ꢁ 105 per well. The
plates were then incubated for 72 h in a humidified atmosphere at 37 °C
and 5% CO2. An aliquot of a stock solution of resazurin (Alamar Blue,
prepared in 1ꢁ PBS) was then added at 20 μL per well (final
concentration, 10 μM), and the plates were returned to the incubator
for another 24 h. After this period, the fluorescence in each well was
measured in a Gemini EM plate reader with an excitation wavelength of
560 nm and an emission wavelength of 590 nm. IC50 values were
determined by nonlinear regression analysis of logistic concentrationꢀ
fluorescence intensity curves (GraphPad Prism software).
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Corresponding Author
*Mailing address: Office of Academic Affairs, Portland State
University, P.O. Box 751, Portland, Oregon, 97207-0751. Phone:
503 296 4829, Fax: 503 725 5262. E-mail: reynoldk@pdx.edu.
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’ ACKNOWLEDGMENT
This work was supported by a grant from the National
Institutes of Health (Grant GM077147).
’ ABBREVIATIONS USED
CQR, chloroquine-resistant; DNA, deoxyribonucleic acid; SAR,
structureꢀactivity relationship; CQ, chloroquine; IC50, half max-
imal inhibitory concentration; nM, nanomolar; MDR, multi-
drug-resistant
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