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D. J. Mack et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5177–5181
F
F
O
(CO2H)2
CH3CN
reflux
Golgicide A (GCA)
N
N
H
N
10:1 syn:trans
(racemic)
+
H2N
F
F
Scheme 1. Synthesis of golgicide A (GCA).
not known how significant each of its five functional groups (two
fluorines, piperidine and pyridine nitrogens, as well as the cyclo-
pentene olefin) are in contributing to its biological activity. More-
over, no studies have been reported regarding the potential for
GCA to inhibit mosquito metabolism. Furthermore, since golgicide
A is now sold commercially,13 it is important to know which of the
four compounds in the commercial preparation are the most active
(Scheme 2). This racemic diastereomeric mixture, which results
from the acid catalyzed Povarov reaction, is primarily composed
of the cis-diastereomer (1 and 2) in favor of the trans-diastereomer
(3 and 4). The first question we set out to answer was, are all four
components of Golgicide A (GCA 1–4) equally potent with respect
to their COPI vesicle inhibitory activity? It seemed unlikely to us
that this would be the case as the fused rigid tricyclic architecture
of GCA would present the pyridine substituent in a dramatically
different non-flexible fashion to the target. Furthermore, if we
were to assume that the major cis-components (1 and 2) of GCA
were responsible for its biological response, it was still quite likely
that the two enantiomeric rigid semi-bowl shaped structures
would interact with different efficiency to their chiral target. We
addressed this challenge by synthesizing GCA, separating the dia-
stereomers (1 + 2 from 3 + 4) using a combination of chromatogra-
phy and crystallization. The enantiomers were separated from each
other using chiral HPLC-chromatography.14 These pure compounds
(GCA-1, GCA-2 and GCA-3) were tested against GCA (mixture)
using DMSO as a control in a mosquito ovarian development assay
(Table 1). This mosquito biological assay was performed by micro-
injecting each of the compounds into blood fed Ae. aegypti mosqui-
toes and measuring follicle lengths in the developing ovaries 48 h
post blood meal. As we predicted, the individual components of
the GCA mixture responded in a dramatically different fashion.
The minor racemic trans-diastereomer (GCA-3) was shown to be
close to inactive compared to GCA. When the two enantiomers
(GCA-1 vs GCA-2) of the major cis-diastereomer were compared
to each other the significance of chirality was shown to be strongly
on display, with one enantiomer (GCA-1) performing poorly, while
the other enantiomer (GCA-2) was superior to all four components
as well as the commercial GCA mixture. With this new insight into
golgicide-A (GCA), we set out to unambiguously determine the
absolute configuration of the active component (GCA-2). We were
fortunate to be able to grow X-ray quality crystals of GCA-2, which
in turn allowed us to determine its absolute configuration shown
in Scheme 2.15
GCA, which is not a rationally designed inhibitor, surfaced as a
candidate from an activity screen of a known compound library.11
Having demonstrated the significance of substitution (trans vs cis)
and chirality on inhibitory activity, we next set out to decipher the
importance of each one of its substituents. The carbon skeleton of
most drug architectures sets the conformational and three dimen-
sional boundaries by which its substituents can interact with the
biological target. With the functional groups being the most likely
candidates responsible for target binding, we decided to evaluate
how important the contribution of each one of GCA’s five func-
tional groups (two aryl fluorides, piperidine and pyridine nitrogen
atoms and the cyclopentene olefin) was to the overall biological
activity using the same in vivo biological assay (Table 1). The ana-
logs we first synthesized are shown in Scheme 3. Each analog has
one of the key GCA functional groups either replaced or substituted
(piperidine nitrogen). These analogs were readily accessed by one
of two approaches. GCA analogs 4–6 were made by reducing or
substituting the major GCA-syn isomer (1 + 2), while analogs 7–9
took advantage of the flexibility of the Povarov reaction.16 When
these new analogs were subjected to the same ovarian develop-
ment assay as GCA’s 1–3, it was revealed that all but one functional
group contribute substantially to inhibitory activity. Removing the
pyridine nitrogen (GCA-8) proved to be beneficial. This most active
GCA derivative provided us with an opportunity for pursuing fur-
ther analogs inspired by this new hit structure.
Inspired by GCA-8 we synthesized GCA analogs 10–17 (GCA-13,
not shown, has the 4-fluoro group replaced with a trifluoromethyl
group), wherein the 3-pyridine group has been replaced with
heterocycles (10 and 12), alkyl- (11) or aryl (14–17) groups. Two
analogs from this new set surfaced as noteworthy based on the
in vivo mosquito reproduction assay. GCA-12, wherein the pyridine
nitrogen is in the 2-position compared to the 3-position for GCA
proved to be equipotent to GCA, but inferior to lead candidate
GCA-8. Substituting the phenyl group of GCA-8 with a 4-trifluoro-
methyl group (GCA-17) proved to be a favorable substitution that
resulted in a slight improvement in inhibitory activity.
Our analysis of this new data suggested that less polar aromatic
replacements for the 3-pyridine group of GCA were performing
better than other substitutions. With these hit structures, we
decided to explore the potential of increasing the size of this sub-
stituent further in a flexible chainlike fashion with the hope of
gaining useful secondary target interactions. Towards that end
we decided to functionalize the amine group of GCA-16 with a di-
GCA = 1+2 and 3+4 in a 10:1 ratio
Minor GCA (trans)
Major GCA (cis)
F
F
F
F
N
N
H
N
N
H
N
N
H
N
N
H
F
F
F
F
3
4
1 (GCA-1)
2
(GCA-2)
3+4 (GCA-3)
F
GCA-2 is the most
active component
of the GCA mixture
N
N
H
F
2 (GCA-2)
Scheme 2. Four components of golgicide A – Crystal structure and absolute configuration of most active component (GCA-2).
MacK, Daniel J.
