Synthesis and Structure-Activity Relationships of Tambjamines and B-Ring Functionalized Prodiginines as Potent Antimalarials

Article abstract of DOI:10.1021/acs.jmedchem.5b00560

Synthesis and antimalarial activity of 94 novel bipyrrole tambjamines (TAs) and a library of B-ring functionalized tripyrrole prodiginines (PGs) against a panel of Plasmodium falciparum strains are described. The activity and structure-activity relationsh

Full text of DOI:10.1021/acs.jmedchem.5b00560

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Article
Synthesis and Structure–Activity Relationships of Tambjamines
and B-Ring Functionalized Prodiginines as Potent Antimalarials
Papireddy Kancharla, Jane X. Kelly, and Kevin A. Reynolds
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00560 • Publication Date (Web): 25 Aug 2015
Downloaded from http://pubs.acs.org on August 29, 2015
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Synthesis and Structure–Activity Relationships of
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Tambjamines and BꢀRing Functionalized Prodiginines as
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Potent Antimalarials
Papireddy Kancharla,† Jane Xu Kelly,†,‡ and Kevin A. Reynolds†*
†Department of Chemistry, Portland State University, Portland, Oregon 97201, United States
‡Department of Veterans Affairs Medical Center, Portland, Oregon 97239, United States
*K.A.R.: mailing address, Finance & Administration, Portland State University, P.O. Box 751,
Portland, Oregon, 97207ꢀ0751; tel, 503 725 3886; fax, 503 725 5800; eꢀmail, reynoldk@pdx.edu
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ABSTRACT
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Synthesis and antimalarial activity of 94 novel bipyrrole tambjamines (TAs) and a library of Bꢀ
ring functionalized tripyrrole prodiginines (PGs) against a panel of Plasmodium falciparum
strains are described. The activity and structureꢀactivity relationships demonstrate that the ringꢀC
of PGs can be replaced by an alkylamine, providing for TAs with retained/enhanced potency.
Furthermore, ringꢀB of PGs/TAs can be substituted with short alkyl substitutions either at 4ꢀ
position (replacement of OMe) or 3ꢀ and 4ꢀpositions without impacting potency. Eight
representative TAs and two PGs have been evaluated for antimalarial activity against multidrugꢀ
resistant P. yoelii in mice in the dose range of 5–100 mg/kg × 4 days by oral administration. The
KAR425 TA offered greater efficacy that previously observed for any PG, providing 100%
protection to malariaꢀinfected mice until day 28 at doses of 25 and 50 mg/kg × 4 days, and was
also curative in this model in a single oral dose (80 mg/kg). This study presents the first account
of antimalarial activity in tambjamines.
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INTRODUCTION
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Malaria is a global parasitic infectious disease caused by Plasmodium parasites, among which
Plasmodium falciparum (Pf) is the most dangerous one, with the highest rates of complications
and mortality. It has been estimated that there are 584,000 people died from this disease in 2013
and the burden is heaviest in the African Region, where an estimated 90% of all malaria deaths
occur, and in children aged under 5 years, who account for 78% of all deaths.1 On the heels of
the global spread of chloroquineꢀresistant P. falciparum (CQRPf), resistance has also quickly
developed to a variety of quinoline analogues, to antifolates, to inhibitors of electron transport,
and perhaps most ominously, now to artemisinin.2,3 Therefore, novel medicinal agents are
urgently needed to overcome the emergence and spread of resistance.
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Prodiginines (PGs, 1a–c), tambjamines (TAs, 2a–b), and modified prodiginines (streptorubin
B (3a), metacycloprodiginine (3b) and marineosins (4 and 5)) belong to a family of
pyrrolylpyrromethene (PPM) alkaloids (Figure 1) derived from bacterial and marine sources.4ꢀ7
These structurally distinctive natural products can be envisioned to arise via a bifurcated process
from a common precursor, 4ꢀmethoxyꢀ2,2ʹꢀbipyrroleꢀ5ꢀcarboxaldehyde (MBC; 6, Figure 1) and
the corresponding alkylpyrrole and/or alkylamine.7ꢀ10 The natural and synthetic PPM products
are undergoing intense scrutiny in the medicinal chemistry because of both their wide range of
biological activities and modes of action (antimicrobial,11ꢀ14 immunosuppressive,15ꢀ22
antitumor,11,12,23 anticancer,6,24ꢀ30 antimalarial7,31ꢀ38 transmembrane anion transport,28ꢀ30,39ꢀ45 and
DNA intercalation46,47). Certain PGs and TAs have also been observed to bind duplex DNA and
can cleave this biomolecule in the presence of Cu(II).4,48 Some of these compounds have shown
clinical potential, and in particular, PG analogue, GX15ꢀ070 has completed phase II clinical
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trials for the treatment of small cell lung cancer and is engaged in multiple clinical trials for the
treatment of other cancer conditions.49,50
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Figure 1. Structures of PPM natural products (1−5) and their common biosynthetic precursor (6)
As a part of an ongoing interest in developing new antiparasitic agents, we reisolated the
natural PGs 1a, and 3a from Streptomyces coelicolor M511, and 3b from S. longisporusruber
(Figure 1).51,52 These natural PGs exhibited great potency with very low IC50 values against P.
falciparum strains, a potency only slightly more than chloroquine (CQ).37 The natural PG 3b
provided an excellent in vivo efficacy against multidrugꢀresistant P. yoelii in mice by oral route,
and it was curative in this model at 100 mg/kg/day, and three of four mice were cured. This data
provided the first demonstration of oral effectiveness of PGs.37 Recently we also have isolated
the modified prodiginines, marineosins (5) and their pathway intermediates 23ꢀ
hydroxyundecylprodiginine (1b), 23ꢀketoundecylprodiginine (1c) and premarineosin (4) through
heterologous expression of the entire mar gene cluster and/or gene replacement mutants in a
heterologous host, S. venezuelae.7 Of these, the compound 4 antimalarial activity compares
favorably with the most potent naturally occurring PGs and CQ.
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The structural and functional diversity and promising antimalarial activity of these natural PGs
and marineosins spurred us to synthesize various analogues of these lead molecules to obtain
more active compounds. We recently reported the antimalarial activity of a large library of
synthetic PGs.37,38 This work has shown that a terminal nonalkylated pyrrole (ringꢀA), and 3,5ꢀ
dialkyl substitutions on the other terminal alkylated pyrrole (ringꢀC) of a natural tripyrrole PGs
core structure are crucial for the potent antimalarial activity. A number of the synthetic PGs were
effective at lower concentrations (IC50 = 0.9–16.0 nM) against P. falciparum strains and their
potency was more than the natural PGs and CQ. However, preliminary in vitro assays indicate
concerns associated with the toxicity of PGs.
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Our work on the potent antimalarial activity of PGs,37,38 to date have been limited to SAR
studies of Aꢀ and Cꢀring functionalized PGs. With a few exceptions,20,24,25,30,38 there have been
no reports of a comprehensive series of TAs and Bꢀring functionalized PGs being prepared and
evaluated for biological activities. In particular, the antimalarial activities of the TAs have not
been reported to the best of our knowledge. These toxicity concerns for PGs and the intriguing
biological activities of these PPM scaffolds have spurred us to expand the structural and
functional diversity. Therefore, we have undertaken syntheses of novel TAs and Bꢀring
functionalized PGs for enhanced antimalarial activity and reduced toxicity. To that end, we have
developed new methods for the synthesis of various 2,2ʹꢀbipyrroleꢀ5ꢀcarboxaldehydes,53 and
utilized in the generation of the novel TAs and Bꢀring functionalized PGs. Here we report the
synthesis, and structure–activity relationships (SARs) of TAs and Bꢀring functionalized PGs.
The results show TAs with impressive in vitro potency and low toxicity, and demonstrate that a
tripyrrole structure is not required for activity. Furthermore evidence of in vivo efficacy with
TAs, including curative efficacy in mice after oral administration is reported.
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RESULTS AND DISCUSSION
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Chemistry. The key precursors 6–43, which are involved in the synthesis of prodiginines
(PGs) and tambjamines (TAs) (Scheme 10), are depicted in Figures 2 and 3. By use of literature
methodologies, MBC (6) and analogue 21 were prepared from readily available 4ꢀmethoxyꢀ3ꢀ
pyrrolinꢀ2ꢀone in two steps54 and 2,2ʹꢀbipyrroleꢀ5ꢀcarboxaldehydes 7, 8, and 10−18 were
synthesized by our recent methods.53 The syntheses of various new pyrrole carboxaldehydes 9,
19, 20, and 22–39 are outlined in Schemes 1−9.
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Figure 3. Key precursors (21–43) for the synthesis of Aꢀ and Bꢀring functionalized PGs and TAs
Synthesis of 4ꢀ(4ꢀchlorophenyl)ꢀ[2,2ʹꢀbipyrrole]ꢀ5ꢀcarboxaldehyde (9). Synthesis of the
aryl substituted 3ꢀpyrrolinꢀ2ꢀone 48, a key synthon in the synthesis of bipyrroleꢀcarboxaldehyde
9, was began with the coupling of Bocꢀglycine (44) with 2,2ꢀdimethylꢀ1,3ꢀdioxaneꢀ4,6ꢀdione
(meldrumʹs acid) to afford the acylated meldrumʹs acid, which was further converted into the
desired intermediate 45, by an intramolecular cyclization and a subsequent decarboxylation
(Scheme 1).55 The compound 45 was treated with pꢀtoluenesulfonyl chloride in the presence of
N,Nꢀdiisopropylethylamine (DIPEA) to give the tosylated product 46, in 89% yield, which was
further subjected to Suzukiꢀcoupling reaction with 4ꢀchlorophenylboronic acid to give the Nꢀ
Bocꢀ4ꢀarylꢀ3ꢀpyrrolinꢀ2ꢀone 47. The desired 4ꢀarylꢀ3ꢀpyrrolinꢀ2ꢀone 48 was obtained in
excellent yield by deprotection of the NꢀBoc group of 47 with trifluoroacetic acid.56 Using the
reported Vilsmeier formylation method,54 48 was then smoothly transformed to 5ꢀbromoꢀ3ꢀ(4ꢀ
chlorophenyl)ꢀpyrroleꢀ2ꢀcarboxaldehyde 49, which when further subjected to Suzuki coupling
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with NꢀBocꢀ2ꢀpyrroleboronic acid followed by deprotection of the NꢀBoc group gave the desired
2,2ʹꢀbipyrroleꢀ5ꢀcarboxaldehyde 9, in 59% yield (Scheme 1).53
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Scheme 1. Synthesis of 4ꢀ(4ꢀchlorophenyl)ꢀ[2,2ʹꢀbipyrrole]ꢀ5ꢀcarboxaldehyde (9)
Synthesis of 2,2ʹꢀbipyrroleꢀ5ꢀcarboxaldehyde (19). In 1988, Borger and Patel synthesized
the 2,2ʹꢀbipyrroleꢀ5ꢀcarboxaldehyde (19) in seven steps.12 In this work, we successfully
accomplished 19 in two oneꢀpot sequences from easily available pyrrole (50), as shown in
Scheme 2. To that end, compound 50 was consecutively treated with Nꢀchlorosuccinimide (NCS)
and Vilsmeier reagent (POCl3/DMF, in situ generation) under controlled temperatures to obtain
the 5ꢀchloroꢀpyrroleꢀ2ꢀcarboxaldehyde (51) in good yield.57 The Suzuki crossꢀcoupling of 51
with NꢀBocꢀ2ꢀpyrroleboronic acid followed by deprotection of the NꢀBoc group, provided the
desired bipyrroleꢀcarboxaldehyde 19 in 45% isolated yield (Scheme 2).
Scheme 2. Synthesis of 2,2ʹꢀbipyrroleꢀ5ꢀcarboxaldehyde (19)
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Synthesis of 3ꢀ(pyrrolꢀ2ꢀyl)ꢀ4,5,6,7ꢀtetrahydroꢀisoindoleꢀ1ꢀcarboxaldehyde (20). The key
intermediate 53 was prepared via BartoneZard’s method, using 1ꢀnitroꢀ1ꢀcyclohexene (52) as a
starting material (Scheme 3).53,58,59 Upon treating with NaOH in ethylene glycol under reflux, 53
was smoothly converted to 4,5,6,7ꢀtetrahydroꢀisoindole (54) in 90% yield by successive
hydrolysis and decarboxylation of the ester group.53 Using the standard Vilsmeier formylation
method, 54 was then transformed to 4,5,6,7ꢀtetrahydroꢀisoindoleꢀ1ꢀcarboxaldehyde (55), which
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when further treated with 1,3ꢀdibromoꢀ5,5ꢀdimethylhydantoin (DBDMH)53 in THF at ꢀ78 C to
room temperature provided the 3ꢀbromoꢀ4,5,6,7ꢀtetrahydroꢀisoindoleꢀ1ꢀcarboxaldehyde (56).
Subsequently, Suzuki crossꢀcoupling reaction between 56 and NꢀBocꢀ2ꢀpyrroleboronic acid and
further deprotection of the NꢀBoc group led to the desired bipyrroleꢀcarboxaldehyde 20 in good
yield (Scheme 3).
Scheme 3. Synthesis of 3ꢀ(pyrrolꢀ2ꢀyl)ꢀ4,5,6,7ꢀtetrahydroꢀisoindoleꢀ1ꢀcarboxaldehyde (20)
Synthesis of isomeric [2,3ʹꢀbipyrrole]ꢀ5ʹꢀcarboxaldehydes (22–25). To investigate the ringꢀ
A positional effect on antimalarial activity, the isomeric bipyrroleꢀcarboxaldehydes 22–25 were
prepared, as shown in Scheme 4. Pyrroleꢀ2ꢀcarboxaldehyde (40) and 3,5ꢀdimethylꢀpyrroleꢀ2ꢀ
carboxaldehyde (42) were obtained from commercial sources, and the 3ꢀmethylꢀpyrroleꢀ2ꢀ
carboxaldehyde (57) and 3ꢀethylꢀpyrroleꢀ2ꢀcarboxaldehyde (58) were prepared according to our
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reported procedures.53 These pyrroleꢀ2ꢀcarboxaldehydes were then converted into the
corresponding 4ꢀbromoꢀpyrroleꢀ2ꢀcarboxaldehydes 59–62, via a regioselective bromination at 4ꢀ
position using DBDMH in THF in good yields (Scheme 4).53 These 4ꢀbromoꢀpyrroleꢀ2ꢀ
carboxaldehydes 59–62, were further subjected to Suzukiꢀcoupling reaction with NꢀBocꢀ2ꢀ
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pyrroleboronic acid, and a subsequent treatment with LiOH in THF/MeOH (1:1) at 60 C,
resulted in the desired isomeric bipyrroleꢀcaraboxaldehydes 22–25 (Scheme 4).
Schemeꢀ4. Synthesis of isomeric [2,3ʹꢀbipyrrole]ꢀ5ʹꢀcarboxaldehydes (22–25)
Synthesis of MBCʹs analogues (26–31) containing herteroaryl/aryl groups in the place of
ringꢀA. To probe the exact role of the 2ꢀpyrrolyl moiety (ringꢀA) on activity, we have prepared
various key carboxaldehyde precursors 26–31, in which the ringꢀA is completely replaced by
various heterocycles and/or aryl moieties and the ringꢀB is substituted with short alkyl groups
(Scheme 5). The 5ꢀbromoꢀ3,4ꢀdimethylꢀpyrroleꢀ2ꢀcarboxaldehyde (65) was prepared in 6 steps
according to the literature methods from acetaldehyde (63) and nitroethane (64),53 and it was
subsequently subjected to Suzukiꢀcoupling reaction with various boronic acids and further
deprotection of the Boc/TIPS group led to the corresponding carboxaldehydes 26–31 (Scheme
5).
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Schemeꢀ5. Synthesis of MBCʹs analogues containing heteroaryl/aryl groups in the place of ringꢀA (26–
31)
Synthesis of 3,4ꢀdimethylꢀ[2,2ʹꢀbipyrrole]ꢀ5ꢀcarboxaldehydes where the ringꢀA contains
Cꢀalkyl groups (32–36). To investigate the effect of the ringꢀA alkyl substituents pattern on
potency, we have prepared various alkylated bipyrroleꢀcarboxaldehyde precursors 32–36, as
shown in Schemes 6 and 7. The 2ꢀacetylꢀpyrrole (66a), 2,4ꢀdimethylpyrrole (67c), and 3ꢀethylꢀ
2,4ꢀdimethylpyrrole (67d) were obtained from commercial sources, and the 2ꢀisobutyrylꢀpyrrole
(66b) was prepared according to the literature methods.60 The compounds 66a and 66b were then
converted into the corresponding 2ꢀalkylꢀpyrroles 67a and 67b, respectively, using LiAlH4 in
THF under reflux (Scheme 6).61 By using standard procedures, the NꢀBocꢀprotected pyrroles
68a–68d were prepared in excellent yields from 67a–67d using diꢀtertꢀbutyl dicarbonate
(Boc2O) in the presence of 4ꢀ(dimethyl amino)pyridine (DMAP), and subsequently these were
converted into the corresponding 5ꢀalkylꢀ(1ꢀtertꢀbutoxycarbonylpyrrolꢀ2ꢀyl)boronic acids 69a–
69d.62 The resultant boronic acids 69a–69d were carried forward into the Suzukiꢀcoupling
reaction with 65 without further purification to afford their corresponding [2,2ʹꢀbipyrrole]ꢀ5ꢀ
carboxaldehydes 32–35 in good yields (Scheme 6).
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59
60
Schemeꢀ6. Synthesis of 3,4ꢀdimethylꢀ[2,2ʹꢀbipyrrole]ꢀ5ꢀcarboxaldehydes where the ringꢀA contains Cꢀ
alkyl groups (32–35)
We have also developed a simple and convenient method for the synthesis of NꢀBocꢀ4ꢀethylꢀ2ꢀ
pyrrolboronic acid (71) via a regioselective boronylation of NꢀBocꢀ3ꢀethylꢀpyrrole (70),53 using
nꢀBuLi/2,2,6,6ꢀtetramethylpiperidine, and trimethyl borate (Scheme 7, Experimental Section).
Further investigations to expand the substrate scope of the regioselective boronylation as well as
mechanistic studies are underway in our laboratory. Finally the 4ʹꢀethylꢀ3,4ꢀdimethylꢀ[2,2ʹꢀ
bipyrrole]ꢀ5ꢀcarboxaldehyde (36) was prepared in good yield via Suzukiꢀcoupling of 65 with
boronic acid 71, followed by the deprotection of NꢀBoc group with LiOH (Scheme 7). The final
compound 36 was fully characterized by extensive 2D NMR analysis (see Supplementary
Information).
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Scheme 7. Synthesis of 4ʹꢀethylꢀ3,4ꢀdimethylꢀ[2,2ʹꢀbipyrrole]ꢀ5ꢀcarboxaldehyde (36)
Synthesis of 3ꢀ(imidazolꢀ2ꢀyl)ꢀ4,5,6,7ꢀtetrahydroꢀisoindoleꢀ1ꢀcarboxaldehyde (37). To
investigate the role of ringꢀA with an extra nitrogen atom on potency, we have replaced the ringꢀ
A by imidazole moiety, as in 37 (Scheme 8). The NꢀBocꢀpyrrole 72 was prepared in 95% yield
from compound 55 using Boc2O/DMAP, and subsequently the aldehyde group was protected by
trimethyl orthoformate under acidic conditions to obtain the desired intermediate 73. The
compound 73 was further reacted with triisopropyl borate/LDA in THF, and followed by
aqueous solution of KHSO4/NH4Cl at room temperature to provide the desired boronic acid 74 in
excellent yield.53 Finally, the Suzuki crossꢀcoupling reaction between 74 and 2ꢀbromoꢀimidazole
(75), and subsequent deprotection of the NꢀBoc group led to the desired carboxaldehyde 37 in
65% isolated yield (Scheme 8).
Scheme 8. Synthesis of 3ꢀ(imidazolꢀ2ꢀyl)ꢀ4,5,6,7ꢀtetrahydroꢀisoindoleꢀ1ꢀcarboxaldehyde (37)
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Synthesis of 3ꢀmethylꢀ4,5,6,7ꢀtetrahydroꢀisoindoleꢀ1ꢀcarboxaldehyde (38) and 5,5ʹꢀ
methylenebis(4ꢀethylꢀ3ꢀmethylꢀpyrroleꢀ2ꢀcarboxaldehyde) (39). We wanted to study the
analogues of TAs without ringꢀA, therefore, two representative pyrrole aldehydes 38 and 39
(Scheme 9) were synthesized. Initially, 1ꢀmethylꢀ4,5,6,7ꢀtetrahydroꢀisoindole (77) was
synthesized from ethylꢀ4,5,6,7ꢀtetrahydroꢀisoindoleꢀ1ꢀcarboxylate (53) via an unstable
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o
intermediate 76, using LiAlH4 in THF at 0 C to room temperature in 85% isolated yield. The
resultant alkylꢀpyrrole 77 was further converted to 3ꢀmethylꢀ4,5,6,7ꢀtetrahydroꢀisoindoleꢀ1ꢀ
carboxaldehyde (38) by Vilsmeier reagent (POCl3/DMF) (Scheme 9). Conversely, the bis(3ꢀ
ethylꢀ4ꢀmethylꢀpyrrolꢀ2ꢀyl)methane (79) was prepared from diethylꢀ5,5′ꢀmethylenebis(4ꢀethylꢀ3ꢀ
methylꢀ2ꢀpyrrolecarboxylate) (78) in excellent yields via a successive hydrolysis and a
decarboxylation of the ester groups. Further Vilsmeier formylation of 79 provided the desired
dicarboxaldehyde 39 in 73% isolated yield (Scheme 9).
Scheme 9. Synthesis of 3ꢀmethylꢀ4,5,6,7ꢀtetrahydroꢀisoindoleꢀ1ꢀcarboxaldehyde (38) and 5,5’ꢀ
methylenebis(4ꢀethylꢀ3ꢀmethylꢀpyrroleꢀ2ꢀcarboxaldehyde) (39)
Synthesis of novel PGs (85–98) and TAs (99–187). By using our standardized procedures,
the monoꢀ and dialkyl/alkylaryl pyrroles 80–84 were synthesized (Figure 4).37 The acidꢀ
catalyzed condensation of either the alkyl pyrroles 80–84 or the commercially available
alkyl/arylamines with various bipyrroleꢀcarboxaldehydes and analogues 6–43, provided the
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desired PGs 85, 86, 88–98, and TAs 99–187, respectively, in good to excellent isolated yields
(Scheme 10). The PG 85 was further treated with MeI/NaH in DMF to provide the N,Nꢀdimethyl
PG 87 in 85% isolated yield (Scheme 10).
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50
51
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53
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60
Figure 4. Potential substrates (80–84) for the synthesis of PGs
Schemeꢀ10. Synthesis of novel PGs (85–98) and TAs (99–187)
Synthesis of TA like Analogues (190, 191 and 194−196). Distinct syntheses were designed
and executed to obtain a different class of TA like analogues 190, 191 and 194−196, in which the
crucial ringꢀB of TAs is completely replaced by an alkylamide/amine linkage (Scheme 11). To
that end, compound 188 was synthesized via a standard condensation method (EDCl/DMAP)
from 44 and 1ꢀadamantylamine in 85% yield. Removal of the Boc group of 188 by trifluoroacetic
acid:water (1:1) provided the intermediate 189 in good yield,63 which was further utilized in a
condensation reaction with pyrroleꢀ2ꢀcarboxylic acid to furnish the desired product 190.
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Treatment of 190 with LiAlH4 in THF at 0 oC to reflux conditions gave the 191 in 82% isolated
yield (Scheme 11). Conversely, analogues 194–196, were also synthesized, as shown in Scheme
11. The pyrroleꢀ2ꢀcarboxaldehyde (40) was subjected to HornerꢀWadsworthꢀEmmons (HWE)
reaction with methyl diethylphosphonoacetate in the presence of NaH to obtain the methylꢀ3ꢀ
(pyrrolꢀ2ꢀyl)acrylate (192),64,65 which when hydrolyzed under basic (LiOH.H2O) conditions,
furnished the 2ꢀpyrrolyl acrylic acid 193. Condensation of 193 with 189 in the presence of
EDCl/DMAP led to the corresponding condensed product 194, which was further treated with
NaBH4/NiCl2.6H2O to give the saturated product 195. Treatment of 195 with LiAlH4 in THF at 0
oC to reflux conditions provided the desired product 196 in 78% yields (Scheme 11).
3
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Scheme 11. Synthesis of novel analogues (190, 191 and 194–196)
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Biological Activity. In this work, the structureꢀactivity relationships (SARs) focused on
various substitutions and positions of the ringꢀA, and ꢀB and the nature of the alkylamines of
TAs, and ringꢀB of PGs. Specifically, the modifications to the ringꢀB of TAs and PGs were
designed in order to understand the structural requirements, as well as the necessity of the ringꢀB
for the potent antimalarial activity. We have synthesized various series of novel TAs and Bꢀring
functionalized PGs, and evaluated for antimalarial activity against the chloroquineꢀsensitive
(CQS) D6, and the chloroquineꢀresistant (CQR) Dd2 and 7G8 strains of Pf with chloroquine (CQ)
as a reference drug.66,67 In parallel, the cytotoxicity of the most potent antimalarial PGs and TAs
(IC50 < 250 nM) was tested against hepatocellular HepG2 cancer cell line using mefloquine
(MQ) as a control drug (see Tables 1–6).68,69
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60
In Vitro Antimalarial Activity of PGs (85–98). In our previous work, synthetic PG 85 had
shown an excellent potency against Pf strains D6 (CQS) and Dd2 (CQR) with great IC50 values
(Table 1), and had the most favorable profile: 92% parasite reduction at 5 mg/kg/day, 100%
reduction at 25 mg/kg/day in a P. yoelii murine patent infection without any evident weight loss
or clinical overt toxicity.37 To explore the Nꢀalkyl effect on potency, initially we synthesized two
Nꢀmethylated analogues 86 and 87 of the 85 (Table 1). These compounds 86 and 87 led to a
large decrease in the antimalarial activity (IC50 > 2250 nM) against three Pf strains D6, Dd2 and
7G8, demonstrating that both pyrrole NH groups (ringꢀA and ꢀC) of the PGs are required for
potent antimalarial activity and that support our previous findings.38 To investigate the
importance of the methoxy group (OMe) on ringꢀB, two analogues 88 and 89, in which the OMe
group is replaced by 4ꢀchlorophenyl moiety and hydrogen (complete removal of OMe),
respectively, were prepared and examined for in vitro antimalarial activity. A dramatic loss of
potency was observed for both compounds 88 and 89, which have an IC50 of > 2500 nM against
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all tested Pf strains (Table 1). Interestingly, while replacing the OMe group by ethyl unit as in 90
also led to the reduced potency (90: IC50 =101 nM versus 1a: IC50 = 7.2 nM against D6), the
reduction was modest (14ꢀfold). This result demonstrated that a short aliphatic substitution at 4ꢀ
position on the ringꢀB could replace the OMe group and retain activity. Together, these results
highlighted the importance of the OMe or short alkyl group on the ringꢀB of PGs for potent
antimalarial activity, support our previous findings.38
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We next investigated whether substitutions at 2 and 3 positions of the ring B are tolerated. A
series of novel Bꢀring functionalized PGs 91−98, in which the ringꢀB is substituted with either
monoꢀ and/or diꢀsubstituents at 3ꢀ and 4ꢀpositions, were generated and examined for their in
vitro antimalarial activity (Table 1). A significant loss of potency (IC50 >1500 nM) was observed
for 91 and 92, containing an isopropyl, and tertꢀbutyl groups, respectively, at 3ꢀposition on the
ringꢀB. The adverse effect of the substitutions at 3ꢀposition on the ringꢀB was further confirmed
by the introduction of the chloro (Cl) substitution at 3ꢀposition of 90, as with the analogue 93,
which had an IC50 of > 2500 nM against all strains (90: IC50 =101 nM versus 93: IC50 > 2500
nM), suggesting that the rigid bulky substitutions or chlorine moiety (EWG) at 3ꢀposition are not
preferred (Table 1). To further investigate the impact of the short alkyl substituents at both the 3ꢀ
and 4ꢀpositions on ringꢀB, a set of mixed analogues 94−98, which contain the 3ꢀethyl/4ꢀmethyl
groups on the ringꢀB, was examined. Analogues 94 and 95, which have monoꢀalkyl groups at 5ꢀ
position of the ringꢀC, showed a roughly 20ꢀfold drop in activity as compared to
undecylprodiginine (1a) (Table 1). Conversely, the analogue 96 containing a monoalkyl group at
3ꢀposition on the ringꢀC, showed higher potency (3ꢀfold) than 95 against all tested Pf strains,
while it had 9ꢀfold lesser potency than the corresponding OMe group containing analogue (IC50
= 4.6 nM against D637). Interestingly, the analogue 97, which has 3ꢀalkyl and 5ꢀalkylaryl
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substituents on the ringꢀC, showed equipotent to the 85. While the analogue 98, which has 3,5ꢀ
dialkylaryl substituents on ringꢀC, showed ~5ꢀfold lower potency when compared to the
corresponding OMe group containing analogue 85 (Table 1), again these results are consistent
and support the findings that the 3,5ꢀdisubstitutions on ringꢀC are very important for potent
activity.37 In summary, these SAR analyses of the ringꢀB functionalized PGs demonstrate that
the short alkyl substitutions are well tolerated at 3/4ꢀpositions on the ringꢀB.
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11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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29
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31
32
33
34
35
36
37
38
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60
Table 1. In Vitro Antimalarial Activity and Cytotoxicity of PGs (85–98)
antimalarial activity
(IC50 in nM)a
compd
R1
R2
R3
R4
D6
Dd2
7G8
cytotoxicity
(IC50 in nM)a
HepG2
SIb
(D6)
cLogPc
H
OMe
H
H
ꢀ
ꢀ
6.1
4.8
5.5
> 2500
> 2500
> 2500
> 2500
51
> 250000
> 40983
4.8
5.1
5.3
7.7
5.2
5.7
6.3
6.7
5.8
6.1
4.8
4.9
6.7
85
86
87
88
89
90
91
92
93
94
95
96
97
Me
OMe
2250 > 2500
> 2500 > 2500
> 2500 > 2500
> 2500 > 2500
ntd
nt
ꢀ
Me
OMe
Me
H
ꢀ
ꢀ
H
4ꢀClC6H4
ꢀ
nt
ꢀ
ꢀ
nꢀC11H23
nꢀC11H23
nꢀC11H23
nꢀC11H23
nꢀC11H23
nꢀC11H23
nꢀC8H17
H
H
H
H
H
iꢀPr
nt
H
Et
H
101
66
18939
nt
187
ꢀ
H
1586
1500
> 2500
> 2500
> 2500
145
H
H
tꢀBu > 2500 > 2500
nt
ꢀ
H
H
Et
Me
Me
Me
Cl
Et
Et
Et
Et
> 2500 > 2500
nt
ꢀ
162
127
41
190
216
53
62000
71000
57200
82024
383
559
1395
12619
H
132
nꢀC8H17
61
nꢀC7H15
4ꢀFC6H4CH2 Me
6.5
7.0
5.9
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4ꢀClC6H4CH2 4ꢀClC6H4CH2 Me
Et
28
7.2
13
nt
42
7.5
115
nt
42
7.0
130
nt
30600
nt
1093
6.7
4.2
3.7
5.3
98
ꢀ
ꢀ
ꢀ
1a
nt
CQ
21800
MQ
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aIC50 values are the average of at least three determinations, each carried out in triplicate (± 10%). In order to
compare results run on different days, and with different batches of each stain; CQ was run as a positive control. All
results obtained were ‘normalized’ to the CQ values of 13 nM for D6, 115 nM for Dd2 and 130 nM for 7G8.
bSI (selectivity index) = IC50 (cytotoxicity)/IC50 (D6)
ccLogP values were calculated using ChemBioDraw Ultra software (version 14), dnt = not tested
In Vitro Antimalarial Activity of 4ꢀSubstituted BꢀRing Functionalized TAs (99–129).
Having determined the substituents impact on the antimalarial activity of the PGs (through this
and previous work37,38), we subsequently tested a hypothesis that the complete replacement of
the rightꢀhand side alkylated pyrrole (ringꢀC) of PGs by alkylamines, providing the TAs, might
represent an opportunity to make potent and selective antimalarials with the desired “druglike”
properties. Specifically, lower molecular weight (MWT) and lipophilic properties (LogP) are the
two key characteristics that determine adsorption, distribution, metabolism, excretion and
toxicity (ADMET) liabilities, with some ADMET parameters depending more on MWT and
some on LogP.70 Subsequent TA analogues 99−129 (Table 2), which have lower MWT (< 400)
and cLogP (< 4.2, except 114), were generated to obtain a SAR for the alkylamines in the place
of ringꢀC and substituents at 4ꢀposition on the ringꢀB.
Initially, a series of new TAs 99–113, which have various alkyl/arylamines in the place of
ringꢀC and the OMe group at the 4ꢀposition on the ringꢀB (as in natural products), were
synthesized and evaluated for their in vitro antimalarial activity against Pf strains and the results
are shown in Table 2. TAs 99−102 containing the nꢀalkylamines in the place of ringꢀC, exhibited
good activity against all Pf strains, specifically, analogues 100 and 102 showed the highest
potencies (IC50 < 50 nM) (Table 2). To probe the effect of cycloalkylamines in the place of ringꢀ
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C/nꢀalkylamines on activity, we synthesized another set of TAs 103–109 (Table 2). Of these
cycloalkylated TAs, analogues 108 and 109, which have the cyclooctylamine and 1ꢀ
adamantylamine moieties, respectively, were the most potent antimalarial candidates (108: IC50
< 7.1 nM, and 109: IC50 < 3.8 nM against all tested Pf strains, see Table 2) with good selectivity
and these results are more comparable to the potent PG 85 (IC50 < 4.5 nM), and the natural PG
1a (IC50 < 7.0 nM). These results, clearly demonstrated that the elongation of the cycloalkyl ring
size (from cyclopropyl, 103: IC50 = 2500 nM to 1ꢀadamantyl, 109: IC50 < 3.1 nM) lead to an
increase in activity (Table 2 and Figure 5). The greatest loss of potency (IC50 > 2500 nM) was
observed in 110, in which ringꢀC is replaced by piperidine moiety, suggesting that the free NH is
required for the potent antimalarial activity. Replacement of cyclohexyl moiety with
benzylpiperidine as with 111 led to slightly reduced potency (106: IC50 = 49 nM versus 111:
IC50 = 127 nM against D6). The analogue 112, which contain a 4ꢀchloroaniline in the place of
ringꢀC showed the moderate activity (Table 2). These results unequivocal demonstrate that the
ringꢀC of PGs can be replaced by alkylamines, providing the novel TAs with retained and/or
enhanced antimalarial and cytotoxic properties.
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To investigate the importance of the OMe group on ringꢀB of TAs, another set of TAs 113–
119, in which the OMe group is replaced by 4ꢀchlorophenyl moiety, was generated and
examined for their in vitro antimalarial activity (Table 2). In vitro analysis of the activity of these
compounds 113–119 against Pf, demonstrated activity (IC50 > 250 nM) significantly diminished
when compared to the corresponding OMe group containing TAs (100, 102, and 105–109). This
work suggested that the bulky aromatic substitution at 4ꢀposition on the ringꢀB had an adverse
effect on antimalarial activity. Interestingly the replacement of the OMe group with short alkyl
substituents (methyl/ethyl) also reduced the potency of the compounds 120–122, 124 and 125
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(IC50 > 250 nM) (Table 2). Conversely, the adamantly analogues 123 and 126, in which the OMe
group is replaced by methyl and ethyl groups on the ringꢀB, respectively, showed a substantially
higher potency against D6 strain (109: IC50 = 3.1 nM, versus 123: IC50 = 1.3 nM, 126: IC50 = 2.5
nM) with great selectivity. Complete removal of the OMe group on ringꢀB as with the analogues
127–129, resulted in the total loss of activity (127, 128: IC50 > 2500 nM vs 107: IC50 = 23 nM,
108: IC50 = 4.8 nM, and 129: IC50 = 341 nM vs 109: IC50 = 3.1 nM, 123: IC50 = 1.3 nM, 126:
IC50 = 2.5 nM against D6). Together, these results again demonstrate that the substituents at 4ꢀ
position on the ringꢀB have an important role in potent antimalarial activity, and the OMe group
can be replaced by short alkyl substituents (methyl/ethyl), when 1ꢀadamantylamine exists in the
place of ringꢀC.
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12
13
14
15
16
17
18
19
20
21
22
23
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26
27
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31
32
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34
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36
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43
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46
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49
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51
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57
58
59
60
Table 2. In Vitro Antimalarial Activity and Cytotoxicity of 4ꢀSubstituted BꢀRing
Functionalized TAs (99–129)
antimalarial activity
(IC50 in nM)a
SIb
(D6)
cLogPc
0.08
0.9
compd
99
R1
R2
D6
210
34
Dd2
159
37
7G8
74.6
25
cytotoxicity
(IC50 in nM)a HepG2
OMe
OMe
OMe
OMe
OMe
nꢀC4H9
nꢀC6H13
nꢀC8H17
nꢀC11H23
23000
26700
ntd
109
785
ꢀ
100
101
102
103
345
55
177
53
69
1.7
23
9800
nt
178
ꢀ
3.0
2400
2500
946
− 0.9
OMe
591
497
156
nt
ꢀ
− 0.4
104
OMe
OMe
68
49
84
71
45
30
30500
15000
448
306
− 0.03
0.4
105
106
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OMe
OMe
OMe
23
4.8
3.1
34
7.1
2.6
15
7.5
3.8
10100
9700
3300
439
2021
1064
0.8
1.2
0.7
107
108
109
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19
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31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
OMe
OMe
> 2500
127
> 2500
244
> 2500
207
nt
ꢀ
− 0.05
110
111
> 250000
> 1968 0.5
OMe
4ꢀClC6H4
nꢀC6H13
255
1129
664
368
314
564
663
510
nt
nt
nt
nt
ꢀ
ꢀ
ꢀ
ꢀ
1.1
3.8
5.9
2.9
112
113
114
115
4ꢀClC6H4
4ꢀClC6H4
4ꢀClC6H4
> 2500
> 2500
> 2500
nꢀC11H23
1218
4ꢀClC6H4
4ꢀClC6H4
1025
963
> 2500
1250
415
348
nt
nt
ꢀ
ꢀ
3.3
3.7
116
117
4ꢀClC6H4
4ꢀClC6H4
832
1135
316
126
nt
nt
ꢀ
ꢀ
4.1
3.6
118
119
> 250
> 250
Me
Me
nꢀC11H23
1167
1469
515
nt
nt
ꢀ
ꢀ
4.2
2.0
120
121
> 250
> 250
> 250
Me
Me
> 250
1.3
> 250
15
> 250
4.3
nt
ꢀ
2.4
1.8
122
123
6900
5308
Et
Et
Et
> 250
> 250
2.5
> 250
> 250
16
> 250
> 250
7.7
nt
nt
ꢀ
ꢀ
2.4
2.8
2.2
124
125
126
6100
2440
H
H
> 2500
> 2500
> 2500
> 2500
> 2500
> 2500
nt
nt
ꢀ
ꢀ
1.8
2.2
127
128
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Journal of Medicinal Chemistry
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2
3
4
H
341
295
235
70000
205
1.6
129
5
6
7
8
9
13
nt
115
nt
130
nt
nt
ꢀ
ꢀ
3.7
5.3
CQ
21800
MQ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
aIC50 values are the average of at least three determinations, each carried out in triplicate (± 10%). In order to
compare results run on different days, and with different batches of each stain; CQ was run as a positive control. All
results obtained were ‘normalized’ to the CQ values of 13 nM for D6, 115 nM for Dd2 and 130 nM for 7G8.
bSI (selectivity index) = IC50 (cytotoxicity)/IC50 (D6)
ccLogP values were calculated using ChemBioDraw Ultra software (version 14), dnt = not tested
1000
900
800
700
600
D6
500
Dd2
7G8
400
300
200
100
0
3
4
5
6
7
8
10
Carbons involved in cycloalkyl moiety (3–10)
Figure 5. SAR of TAs (103–109) containing various cycloalkyl groups and in vitro antimalarial
activity against Pf strains D6, Dd2, and 7G8
In Vitro Antimalarial Activity of 3ꢀSubstituted BꢀRing Functionalized TAs (130–141).
Having established the substitution pattern at 4ꢀposition on the ringꢀB and the terminal
alkylamines (cycloheptylꢀ, cyclooctylꢀ, and 1ꢀadamantylamines) as optimal, we then examined
the effects of substitution pattern at 3ꢀposition, where the 4ꢀposition is vacant on the ringꢀB of
the TAs (Table 3). To that end, we generated a series of novel TAs 130–141, in which the 3ꢀ
position on the ringꢀB is occupied with alkyl groups and screened for their antimalarial activity
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against Pf strains (Table 3). The greatest loss of potency was observed when the short alkyl
(methyl/ethyl) groups moving from 4ꢀposition (121–126, Table 2) to the 3ꢀposition (130–141,
Table 3). Moreover, the adamantly analogues 132 and 135, showed a significant decline in
activity (132: IC50 = 106 nM vs 123: IC50 = 1.3 nM; and 135: IC50 = 117 nM, vs 126: IC50 = 2.5
nM against D6), and the analogue 141, had an almost total loss of activity (IC50 > 2500 nM). The
one exception is the adamantly analogue 138, containing an isopropyl group at 3ꢀposition on the
ringꢀB, which showed the better potency (IC50 < 30 nM) against all tested Pf strains with good
selectivity. These results show that generally alkyl substitutions at 3ꢀposition versus the 4ꢀ
position, adversely affects the potency irrespective of the terminal alkylamines.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Tableꢀ3. In Vitro Antimalarial Activity and Cytotoxicity of 3ꢀSubstituted BꢀRing Functionalized
TAs (130–141)
antimalarial activity
(IC50 in nM)a
SIb
(D6)
cLogPc
2.1
compd
R1
R2
D6
Dd2
7G8
cytotoxicity
(IC50 in nM)a HepG2
Me
2107
> 2500
2147
ntd
ꢀ
ꢀ
130
Me
Me
1376
106
> 250
170
1778
95
nt
2.5
2.0
131
132
30000
283
Et
Et
Et
1305
1276
117
523
> 250
45
1456
1326
90
nt
nt
ꢀ
ꢀ
2.5
3.0
2.4
133
134
135
15200
130
iꢀPr
> 2500
1968
1980
nt
ꢀ
2.9
136
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5
6
iꢀPr
iꢀPr
1079
26
665
20
1480
31
nt
ꢀ
3.3
2.7
137
138
18500
711
7
8
9
tꢀBu
tꢀBu
tꢀBu
> 2500
> 2500
> 2500
> 2500
> 2500
> 2500
> 2500
> 2500
> 2500
nt
nt
nt
ꢀ
ꢀ
ꢀ
3.3
3.8
3.2
139
140
141
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13
nt
115
nt
130
nt
nt
ꢀ
ꢀ
3.7
5.3
CQ
21800
MQ
aIC50 values are the average of at least three determinations, each carried out in triplicate (± 10%). In order to
compare results run on different days, and with different batches of each stain; CQ was run as a positive control. All
results obtained were ‘normalized’ to the CQ values of 13 nM for D6, 115 nM for Dd2 and 130 nM for 7G8.
bSI (selectivity index) = IC50 (cytotoxicity)/IC50 (D6)
ccLogP values were calculated using ChemBioDraw Ultra software (version 14), dnt = not tested
In Vitro Antimalarial Activity of 3,4ꢀDisubstituted BꢀRing Functionalized TAs (142–165).
Exploration of the SARs around the ringꢀB of TAs indicated that the substitutions at 4ꢀposition
were greatly favored compared to the 3ꢀposition (Tables 2 and 3). This finding is exemplified by
the poor activity of the 3ꢀsubstituted analogues (130–141) with the exception of 138. We next
investigated whether substitutions at both the 3ꢀ and 4ꢀpositions are tolerated. We synthesized a
series of 3,4ꢀdisubstituted Bꢀring functionalized TAs 142–149, which have 3ꢀethyl, and 4ꢀmethyl
groups on the ringꢀB (Table 4). Of these 3,4ꢀdisubstituted TAs, analogues 142–144, 148, and
149 with an nꢀalkyl, cyclopropyl, benzylpiperidine and morpholine moieties, respectively,
showed the diminished activity (Table 4). Conversely, the analogues 145 and 146, which have
cycloheptyl and cyclooctyl moieties, respectively, showed the highest potencies (Table 4) than
those of the corresponding 3ꢀ and 4ꢀmonoalkyl substituted analogues (see Tables 2 and 3).
Significantly, the adamantly analogue 147, showed comparable potency to that of the
corresponding 4ꢀalkyl/methoxy substituted analogues (147: IC50 = 5.5 nM versus 109: IC50 = 3.1
nM, 123: IC50 = 1.3 nM, 126: IC50 = 2.5 nM against D6), and this potency is 5–20ꢀfold greater
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than the corresponding 3ꢀalkyl substituted analogues (147: IC50 = 5.5 nM versus 132: IC50 = 106
nM, 135: IC50 = 117 nM, 138: IC50 = 26 nM against D6). Interchange of the methyl and ethyl
groups between 3ꢀ and 4ꢀpositions on the ringꢀB as in 150–152 resulted in a ~2ꢀfold decrease in
potency (IC50 of 150–152 vs IC50 of 145–147). We were encouraged that the short alkyl
substitutions at both the 3ꢀ and 4ꢀpositions on the ringꢀB were well tolerated with comparable
and/or enhanced activities. This allowed for a variety of different analogues to be synthesized
with representative examples (153–165, Table 4). The analogues 153, 154, 156, 157, 159, and
160, which contain the same alkyl groups (methyl/ethyl/ꢀ(CH2ꢀCH2)2ꢀ) at both 3ꢀ and 4ꢀpositions
on the ringꢀB, and cycloheptyl/cyclooctylamines in the place of ringꢀC, were shown comparable
and/or greater potency to the dissimilar alkyl groups at both 3ꢀ and 4ꢀpositions containing TAs.
Significantly, the adamantly analogues 155 (IC50 < 2.4 nM), 158 (IC50 < 2.5 nM), and 161 (IC50
< 7.5 nM) showed enhanced (2–8ꢀfold) or comparable potency against all tested Pf strains when
compared to 147 (IC50 < 5.5 nM) and 152 (IC50 < 19 nM). The biggest potency loss occurred
(IC50 > 2250 nM) when we introduced a chlorine atom at 3ꢀposition on the ringꢀB as in 162–165
(IC50 of 156–158 vs 163–165, Table 4), and it is consistent with the observation that the chlorine
atom (EWG) has an adverse effect at 3ꢀposition on the ringꢀB of PGs. Collectively, from the
monoalkylated (Tables 2 and 3) and 3,4ꢀdialkylated TAs (Table 4) the data clearly showed that
the 3,4ꢀdisubstituted TAs containing cycloheptyl/cyclooctyl groups have significantly improved
potency than the corresponding monoalkylated TAs (Tables 2 and 3), and these potencies were
comparable to the corresponding OMe group containing analogues (Table 2). Notably, all the
adamantyl analogues, which have short (alkyl/methoxy) groups at 4ꢀposition (Table 2) and
dialkyl groups at 3/4ꢀpositions (Table 4) on ringꢀB, showed the greatest activity with good
selectivity.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
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Tableꢀ4. In Vitro Antimalarial Activity and Cytotoxicity of 3,4ꢀDisubstituted BꢀRing
Functionalized TAs (142–165)
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
antimalarial activity
(IC50 in nM)a
SIb
(D6)
ꢀ
ꢀ
ꢀ
cLogPc
2.0
compd
142
R1
Et
Et
Et
R2
R3
D6
Dd2
7G8
cytotoxicity
(IC50 in nM)a HepG2
ntd
Me
Me
Me
nꢀC4H9
nꢀC8H17
883
680
260
1166
> 2500
633
244
nt
nt
3.7
143
2047
2500
1.1
144
Et
Me
62
55
60
19200
310
2.7
145
Et
Et
Me
Me
56
60
75
18900
3300
337
600
3.1
2.6
146
147
5.5
4.3
3.6
Et
Et
Me
Me
Et
> 2500
1576
855
nt
nt
ꢀ
ꢀ
2.4
0.3
2.7
148
149
150
> 2500 > 2500 > 2500
Me
150
200
117
15800
105
Me
Me
Et
Et
111
19
201
14
128
14
23900
4500
215
237
3.1
2.6
151
152
Me
Me
60
38
47
21300
355
2.3
153
Me
Me
Me
Me
56
31
45
18100
6400
323
2.7
2.2
154
155
2.4
1.7
1.5
2667
Et
Et
54
30
88
16900
313
3.1
156
Et
Et
Et
Et
39
26
58
13000
3900
333
3.6
3.0
157
158
1.6
1.0
2.5
2437
–(CH2ꢀCH2)2–
35
39
23
6200
177
2.6
159
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