Aldol-Based Build/Couple/Pair Strategy for Ring Synthesis
A R T I C L E S
IV HDACs are the zinc-dependent hydrolases. Class I HDACs
include 1, 2, 3, and 8, all of which have been well documented
to exert deacetylase activity on histone substrates as well as a
more limited set of non-histone substrates. Class II HDACs can
be divided into class IIa members, which include HDACs 4, 5,
7 and 9, and class IIb members, which include HDACs 6 and
10. Class III HDACs (sirtuins; SIRTs 1-7) are NAD(+)-
dependent enzymes which exhibit a non-overlapping sensitivity
to most structural classes of inhibitors.55b
lysine and a C-terminal aminomethyl coumarin (AMC) deriva-
tive (Ac)Leu-Gly-Lys-acetyl-AMC).60 Upon incubation with
substrate and trypsin, HDAC 2 removes the acetyl group,
allowing for trypsin to cleave the tripeptide substrate, liberating
the AMC fluorophore, whose fluorescence increases signifi-
cantly. The assay was conducted in a kinetic mode using 4.6
µM substrate (the concentration of substrate that produces half-
maximal velocity (Km) for HDAC 2), 2.2 nM HDAC 2, 150
nM trypsin, and 17 µM compound.61 A total of 22 506 aldol-
derived library compounds were tested in the HDAC 2
biochemical screen, including 1604 RCM library compounds
derived from scaffolds (2R,5S,6R,12R)-30a and (2R,5S,6S,12R)-
30e, which vary only with respect to their stereochemistry at
C-6.62 Of all the library compounds tested, the RCM macro-
lactams produced the most interesting hits. The primary screen-
ing results for the RCM compounds in the HDAC 2 assay are
shown in Figure 4. Using a hit cutoff of 24% inhibition, 32
hits were identified (2.0% hit rate). These 32 compounds were
retested in 8-point 2-fold dose-response (32 µM-250 nM),
and 26 had confirmed activity (81% retest rate). Interestingly,
the most potent compounds with confirmed activity were the
products of reductive alkylation with AL11 (piperonaldehyde)
at R2. Meanwhile, the nature of the building block at R1 appeared
to be less critical for HDAC inhibition. All sulfonyl chlorides
(SC1-8) and formaldehyde (AL1) resulted in confirmed
activity, with IC50 values below 50 µM. (The one urea derivative
(R1 ) IS7) was inactive upon retest.) With respect to stereo-
chemistry, only one diastereomer was preferred in favor of the
syn-aldol-derived macrocycle (2R,5S,6R,12R)-30a.
In light of its predicted physicochemical properties (ALogP
4.4, cLogD 2.9, PSA 83) and high solubility in aqueous buffer
(>450 µM solubility in PBS pH 7), the product of reductive
alkylation with formaldehyde (AL1) at R1 and piperonaldehyde
(AL11) at R2 was selected for further SAR exploration (32,
Figure 5). Follow-up of the screening results was expanded to
include all stereoisomers of 32 tested at dose against HDACs
1, 2, and 3. As highlighted in Figure 5, SSAR for HDAC
inhibition emerged.63 First, a stereoisomer with improved
potency against HDAC 2 was identified, (2S,5R,6R,12R)-32
(BRD-4805), which had an IC50 of 6.6 µM. This is greater than
a 10-fold improvement as compared to the lead stereoisomer
identified in the pilot screen, (2R,5S,6R,12R)-32, which was only
weakly active against HDAC 2 upon retesting (IC50 ) 80 µM).
To further validate the activity of BRD-4805, the compound
was resynthesized in solution starting from macrolactam ent-
8g using the five-step sequence shown in Scheme 4. Preparation
of the analogue using solution-phase techniques obviates the
need for protecting group manipulations and loading onto solid
support. Follow-up testing of the resynthesized (and HPLC-
purified) compound in the HDAC 2 assay confirmed its activity.
To date, most published HDAC inhibitors belong to a limited
number of chemical classes: carboxylic acids (e.g., valproic
acid), hydroxamic acids (e.g., SAHA; Vorinostat), thiols (e.g.,
FK228; Istodax), and 2-aminobenzamides (e.g., MS-275; En-
tinostat). Additional classes include the keto epoxides (e.g.,
trapoxin), which were instrumental in the discovery of HDACs,
ethyl ketones (e.g., apicidine), o-hydroxybenzamides, trifluoro-
methyl ketones, and R-ketoamides. Based on the structures of
these inhibitors, a general model for HDAC inhibition has been
put forth consisting of “cap-linker-chelator” functionalities,
which is supported by structural models of HDACs and bound
inhibitors and numerous structure-activity relationship studies.57
While several class-specific HDAC inhibitors exist, such as the
thiophene-substituted benzamides that inhibit only class I
HDACs 1 and 2 or the recently described biphenyl hydroxam-
ates that are selective for class IIa HDACs,58 none of these
inhibitors are HDAC isoform specific, limiting the precision of
the functional conclusions that can be drawn about the cellular
effects and therapeutic potential of HDAC inhibition. Addition-
ally, all of the known classes of inhibitors are substrate
competitive inhibitors that are presumably chelating the active-
site zinc as a primary component to their binding activity. Given
the highly conserved nature of the HDAC active site between
different class members, the discovery of HDAC inhibitors that
are non-competitive in nature or that bind to HDACs with novel
binding modes may provide further insight into the regulation
and function of HDACs as well as attractive starting points for
obtaining isoform selectivity. Beyond applications in oncology,
where HDAC inhibitors are the subject of both clinical and basic
research, we have recently shown that HDAC 2, a class I
HDAC, is directly implicated in learning and memory.59
To test for HDAC 2 inhibitory activity, we used a high-
throughput coupled biochemical assay. The substrate for HDAC
2 was a tripeptide based on histone H4 Lys12 with an acetylated
(52) The use of phenylsilane as the π-allyl scavenger was also explored;
however, the formation of minor amounts of N-allyl side product was
observed.
(53) See Supporting Information for a detailed account of purity and yield
for all library members according to scaffold/building block.
(54) Badertscher, M.; Bischofberger, K.; Munk, M. E.; Pretsch, E. A.
J. Chem. Inf. Comput. Sci. 2001, 41, 889–893.
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(61) This concentration of compound was chosen for the primary screens
based upon the fact that robotic pin transfer used for compound
handling led to the transfer of 100 nL of compound at 5 mM into a
reaction volume of 30 µL. See details provided in Suppporting
Information describing compound handling.
(62) At the time the HDAC 2 assay was run, only a subset of the RCM
library was available for screening. Therefore, compounds derived from
only 2 of the 16 stereoisomeric RCM scaffolds were tested during
the primary screen. The remainder of the compounds screened were
derived from the SNAr and click libraries.
(63) All analogues were resynthesized and subjected to HPLC purification
prior to testing.
(58) Tessier, P.; Smil, D. V.; Wahhab, A.; Leit, S.; Rahil, J.; Li, Z.; De´ziel,
R.; Besterman, J. M. Bioorg. Med. Chem. Lett. 2009, 19, 5684–5688.
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