Optimization of non-ATP competitive CDK/cyclin groove inhibitors through REPLACE-mediated fragment assembly

Article abstract of DOI:10.1021/jm3013882

A major challenge in drug discovery is to develop and improve methods for targeting protein-protein interactions. Further exemplification of the REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment) strategy for generatin

Full text of DOI:10.1021/jm3013882

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Article
Optimization of non-ATP competitive CDK/cyclin groove
Inhibitors through REPLACE mediated Fragment Assembly
Campbell McInnes, Shu Liu, Padmavathy Nandha Premnath, Tracy Perkins,
Joshua K Bolger, Lindsay Odom Kirkland, and George A Kontopidis
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm3013882 • Publication Date (Web): 16 Jan 2013
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Optimization of non-ATP competitive CDK/cyclin groove Inhibitors through
REPLACE mediated Fragment Assembly.
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Shu Liu#, Padmavathy Nandha Premnath#, Joshua K. Bolger,
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Tracy Perkins, Lindsay O. Kirkland, George Kontopidis1 and Campbell McInnes*
Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy,
University of South Carolina, Columbia, SC, 29208
1Department of Biochemistry, Veterinary School, University of Thessaly,
Trikalon 224, Karditsa 43100, Greece
# These authors made equal contributions to this manuscript.
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ABSTRACT
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A major challenge in drug discovery is to develop and improve methods for targeting proteinꢀ
protein interactions. Further exemplification of the REPLACE strategy for generating inhibitors of
proteinꢀprotein interactions demonstrated that it can be used to optimize fragment alternatives of
key determinants, to combine these in an effective way and was achieved for compounds targeting
the CDK2 substrate recruitment site on the cyclin regulatory subunit. Phenylheterocyclic isosteres
replacing a critical chargeꢀcharge interaction provided new structural insights for binding to the
cyclin groove. In particular, these results shed light onto the key contributions of a Hꢀbond observed
in crystal structures of Nꢀterminally capped peptides. Furthermore the structureꢀactivity relationship
of a bisarylether Cꢀterminal capping group mimicking dipeptide interactions, was probed through
ring substitutions, allowing increased complementarity with the primary hydrophobic pocket. This
study further validates REPLACE as an effective strategy for converting peptidic compounds to
more pharmaceutically relevant compounds.
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INTRODUCTION
CDKs, the cyclin regulatory subunits and their natural inhibitors, the CDK tumor suppressor
proteins (CDKIs), are central to cell cycle regulation and their functions are commonly altered in
tumor cells. Deregulation of CDK2 and CDK4 through inactivation of CDKIs such as p16INK4a
,
p21WAF1, p27KIP1, and p57KIP2 can override the G1 checkpoint 1, 2 and lead to transformation. CDKs
interact with certain cell cycle substrates through the cyclin binding motif (CBM) and form a
complex with the cyclin groove of the G1 and S phase cyclins, a surface binding site involving a
proteinꢀprotein interaction. It has been shown that CDK isoform and substrate selective inhibition
can be achieved through the use of peptides that block recruitment of both pRb and E2F and
potently inhibit CDK2/cyclin A2 kinase activity3. Inhibition of CDKs through the cyclin provides
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an approach to obtain selectivity against other protein kinases and to specifically block the activity
of the G1 and S phase CDKs (only these contain a functional cyclin binding groove). In particular,
CDKs including CDK7 and 9, that regulate the RNA polymeraseꢀII (RNAPII) transcription cycle
should be unaffected by Cyclin Groove Inhibitory (CGI) compounds. Although it has been shown
that cancer cells depend on the RNAPII cycle to express antiꢀapoptotic genes and that inhibition of
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transcriptional CDKs leads to potent antiꢀtumor agents , it is at the same time likely that this will
lead to effects in normal cells and may be responsible for toxicities observed with CDK inhibitors
that have been clinically evaluated.
The cyclin binding motif represents a consensus of the cyclin groove binding sequences found
in many cell cycle and tumor suppressor proteins 3, 5. CGI peptides in transducible form have been
shown to induce cell cycle arrest and selective apoptosis in tumor cells in vitro6ꢀ8. These
permeabilized peptides also act as antiꢀtumor agents in that when administered directly to a SVT2
mouse tumor model, significant tumor growth inhibition was obtained and histological analysis
showed that tumors underwent apoptosis9. The significant antiꢀtumor effects obtained through these
peptide studies, provides strong impetus for further drug development with the aim of obtaining
more drugꢀlike small molecule CGI inhibitors. To this end, we developed a unique strategy for
iteratively modifying and replacing peptide determinants in a stepꢀwise fashion 10.
In the search for more drugꢀlike compounds and in order to iteratively convert effective
peptidic cyclin groove inhibitors into more pharmacologically appropriate compounds, the
REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment)
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strategy was applied to discover mimetics . The peptide sequence, HAKRRLIF was previously
identified as an optimized variant of the native p21WAF1 cyclin binding motif sequence 5, 11. It
contains three major binding determinants including Ala2 interacting with a minor hydrophobic
pocket, Arg4 which forms a chargeꢀcharge interaction with acidic cyclin residues, and the LeuꢀIleꢀ
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Phe motifette contacting the primary hydrophobic pocket (Figure 1A). As shown by peptide SAR
and truncation of the Nꢀterminal residues, loss of interaction with secondary hydrophobic pocket
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can result in significant decreases in potency (RRLIF is 10 fold less potent that HAKRRLIF)11, 12
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Using the REPLACE methodology, low molecular weight fragments are docked into the
vacated space of a ligandꢀreceptor complex structure which has been created by truncation of
peptide determinant. After fragments with requisite chemical functionality are selected through
prioritization through scoring of the nonꢀbonded interactions of the docked complexes, they are then
chemically ligated onto the truncated peptide through solid phase parallel synthesis. This approach
has been validated through in vitro screening of FLIP fragment ligated inhibitory peptides (FLIPs)
where a hit rate of approximately 20% was obtained (the actual number of active FLIPS compared
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to the total number synthesized and tested) . A residue critical for binding and highly sensitive to
substitution (Arg4) was successfully replaced with a 5ꢀmethylꢀ1ꢀphenylꢀ1Hꢀ1,2,4ꢀtriazoleꢀ3ꢀ
carboxylic acid Nꢀterminal capping group (Ncap). Recently REPLACE has been applied and
validated against another antiꢀcancer kinase drug target and more drugꢀlike non ATP competitive
PLK1 inhibitors obtained through fragment replacement 13.
In addition to the Nꢀterminal REPLACE efforts, a 3ꢀphenoxybenzylamine mimic for the Cꢀ
terminal phenylalanine was identified, thereby providing a fragment based starting point for further
lead optimization. In this present study, the optimization and structureꢀactivity relationship
determination for the aforementioned N and Cꢀterminal capping groups was undertaken. Further
validation for the REPLACE strategy was obtained not only through identification of partial ligand
alternatives but also in combination of these optimized fragments through appropriate ligation
methods. Efforts to maximize binding of the identified fragments in the context of the peptide were
established before incorporating N and Cꢀterminal groups into a single molecule that is considerably
more drugꢀlike and significantly less peptidic in nature. Exemplification of the REPLACE approach
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provides a significant advance in targeting proteinꢀprotein interactions, a major challenge in drug
discovery.
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RESULTS
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N-terminal Partial Ligand Alternatives: Synthesis of derivatives and isosteres of 1-phenyl-1H-1,2,4-
triazole-3 carboxamide.
Various heterocyclic isosteres were designed based on the success applying REPLACE to the
cyclin groove and in the identification of the 5ꢀmethylꢀ1ꢀphenylꢀ1Hꢀ1,2,4ꢀtriazoleꢀ3ꢀcarboxylic acid
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capping group to replace Arg4 of the peptide . These were pursued further to introduce structural
diversity and to improve drugꢀlike properties. In order to more extensively validate the REPLACE
strategy and to investigate the optimization of Nꢀterminal capping groups in the Fragment Ligated
Inhibitory Peptide (FLIP) context, structural modification of the phenyltriazole scaffold was
undertaken. Furthermore in order to determine the sensitivity and importance of the heterocyclic
ring for activity and to establish the optimal requirements of the phenyl ring substitution for binding
to the cyclin groove, replacement core structures were synthesized and included 5ꢀmethyl ꢀ1ꢀ
phenylꢀ1Hꢀ1,2,4ꢀtriazoleꢀ3ꢀcarboxylic acid (phenyltriazole), 5ꢀmethylꢀ1ꢀphenylꢀ 1Hꢀpyrazole ꢀ3ꢀ
carboxylic acid (phenylpyrazole), 1ꢀphenylꢀ1Hꢀpyrroleꢀ3ꢀcarboxylic acid (phenylpyrrole), 2ꢀ
phenylꢀ2Hꢀimidazoleꢀ4ꢀcarboxylic acid (phenylimidazole), 5ꢀphenylfuranꢀ2ꢀcarboxylic acid
(phenylfuran), and 2ꢀphenylthiazoleꢀ4ꢀcarboxylic acid (phenylthiazole) as shown in table 1. The
phenylpyrazole and phenylfuran isosteres in particular were selected as versatile core structure that
could be exploited in order to generate diverse substitutions of the phenyl ring contacting the
secondary hydrophobic pocket.
The phenyltriazole derived capping groups were generated in a diazonium reaction starting
with the appropriate aniline and ethyl 2ꢀchloroacetoacetate, followed by cyclization in the presense
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of acetaldehyde oxime (Scheme 1)14, 15. Synthesis of the phenylpyrazole scaffold was achieved
through (Scheme 2) reaction of ethylacetopyruvate with the corresponding substituted phenyl
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hydrazine . Initial attempts involved base catalysis of the reaction upon which two isomers were
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obtained. The desired isomer was identified and confirmed through 1ꢀD NOE analysis where
irradiation of the R4 methyl group led to an enhancement of the two ortho aromatic hydrogens. This
reaction was further optimized by performing the cyclization in acidic conditions thereby
protonating the hydrazine and suppressing formation of the nonꢀdesired regioisomer. The versatile
pyrazole synthesis allowed generation of a variety of analogs including the unsubstituted phenyl,
the 3ꢀmethoxy and 4ꢀmethoxy phenyl as well as the 3,5ꢀdichloro, 3 chloro and the 4 chlorophenyl
compounds. The phenylfuran Ncaps were also synthesized through a diazotization of the requisite
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aniline followed by reaction with furoic acid (scheme 3) , the phenylpyrrole generated through
cyclization of appropriate aniline with 2,5ꢀdimethoxytetrahydrofuranꢀ3ꢀcarbaldehyde in acetic acid
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(Scheme 4) , and the phenylimidazole through cyclization of 3ꢀfluoroꢀN'ꢀhydroxybenzimidamide
and ethylpropiolate (scheme 5) 19.
Structure-activity relationship of substituted phenylheterocyclic isosteres as N-terminal capping
groups.
Prior to testing of the library of phenylheterocyclic Ncaps, studies on the binding affinity of
the native p21WAF1 pentapeptide RRLIF were undertaken. This peptide is an important reference
point since modified peptides and FLIPs are compared directly to the native ligand. The results
showed IC50 values of 1.4 and 16.1 µM for CDK2/cyclin A2 and CDK4/cyclin D1 in the
fluorescence polarization assay respectively demonstrating that RRLIF binds about 10 fold tighter
to cyclin A. With this established, direct comparison of the efficacy of the various heterocycles was
determined through the competitive binding of FLIP variants with identical phenyl substitutions. To
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this end, variants of each heterocyle were generated with 3,5ꢀdichloro and 4ꢀchloro phenyl
modifications and incorporated to make the FLIPs. The resulting SAR around the heterocyclic
scaffolds determined that while isosteric ring systems are presented, significant differences are
observed in the in vitro potencies as measured in the fluorescence polarization (FP) binding assay
(Table 1). After testing of each of these, the phenyltriazole series was revealed to be the most
successful among all six isosteric capping groups (5773, 5774) where these were found to have IC50
values for CDK2/cyclin A2 of 4 ꢁM and 11.5 ꢁM, indicating slight loss of potency compared to
RRLIF. In contrast, a potency increase for 5774 was observed for the binding to CDK4/cyclin D
relative to RRLIF (12 ꢁM vs. 16.1 ꢁM) whereas 5773 has a decreased inhibitory activity with an
IC50 of 27.3 ꢁM. Direct comparison of the corresponding phenylpyrazole FLIPs 5763 (3,5ꢀ
dichloro, 21.8 ꢁM) and 5765 (4 chloro, 33.7 ꢁM) against CDK2/cyclin A2 indicates that a 3ꢀ5 fold
potency loss is obtained for the FLIP bioisostere incorporating one less nitrogen. With the exception
of the phenylimidazole scaffold, all other heterocyclic derivatives were found to be marginally or
completely inactive at the concentrations tested. The 3ꢀfluorophenylimidazole containing FLIP
analog (5852) was found to be of comparable potency to the corresponding phenylpyrazole (5771)
for both CDK2/cyclin A2 and CDK4/cyclin D1. These results as a whole suggest that appropriate
positioning of a viable Hꢀbond donor atom in the heterocyclic ring is a key determinant of activity.
Further to the direct comparison of identically substituted heterocyclic variants, the SAR of
the phenyl ring and its interaction with the secondary hydrophobic pocket were examined. Firstly in
the phenyltriazole series, substitutions included the 3ꢀchloro, 3ꢀmethyl and the unsubstituted phenyl
ring. Of these analogs, the 3ꢀchloro was determined to be the most active (5906, 5.7 µM) and of
comparable potency to the 35DCPT. Despite the similar size of the 3ꢀmethyl derivative (5907), it
was found to be of considerably lower activity and was less active than the unsubstituted version.
This was found to be true both in the CDK2/cyclin A2 and the CDK4/cyclin D1 contexts. Further
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testing of the derivatives synthesized in the phenylpyrazole series provided additional insights into
the structureꢀactivity relationship. As indicated from the data and consistent with the SAR for the
triazole, the 3,5ꢀdichloro analog (5763) is a stronger binder to CDK2/cyclin A2 than the 4ꢀchloro
(5765) however the opposite is true for activity against CDK4/cyclin D1. In the latter case, the
relative potencies are reversed compared to cyclin A in that 5765 is approximately twoꢀfold more
potent than 5763. In the pyrazole context, the unsubstituted analog (5762, IC50 = 40.3 ꢁM for
CDK2/cyclin A2 and 54.2 ꢁM for CDK4/cyclin D1) was of similar potency to the 4ꢀchloro analogs.
The 3ꢀchloro derivative (5764) was found to be the most potent in this series being 2ꢀfold more
active for CDK2/cyclin A2 relative to the 3,5ꢀdichloro (5763). In addition the 3ꢀfluoro substituted
phenylpyrazole FLIP (5771) was also made and found to be a weaker binder for CDK2/cyclin A2
(IC50 = 29.6 ꢁM) and comparable inhibitor for CDK4/cyclin D1 (IC50 = 65.9 ꢁM) relative to 5764.
The 3ꢀmethoxy analog (5766) is about 6 fold less potent than the 3ꢀchloro (5764) and is also a
marginal inhibitor of CDK4/cyclin D1. The 4ꢀmethoxy substitution (5766) was observed to be
detrimental since no appreciable inhibition of either CDK complex was evident.
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C-terminal Partial Ligand Alternatives (Ccaps): derivatives of 1-phenyl-1H-1,2,4-triazole-3
carboxamide.
Previously the computational enrichment strategy was applied to identify potential nonꢀpeptidic
replacements for the Cꢀterminal residues (Ile7 and Phe8) previously verified as critical determinants
for binding to the cyclin groove. A SAR study of various bis aryl compounds revealed that 3ꢀ
phenoxybenzylamine was the most effective Cꢀcapping group (Table 2). Further to this 3ꢀ
phenoxybenzylamine and its isostere (4ꢀphenoxypyridinꢀ2ꢀyl)methanamine were introduced as Cꢀ
cap core structures to examine the binding requirements with the primary hydrophobic pocket.
Structural modifications were undertaken to determine if potency enhancements observed after
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addition of halogens to the aromatic sidechain of phenylalanine in the peptide context were relevant
to this Ccap 11, 12. In this effort, phenyltriazole Ncaps were utilized since these were shown to be the
most effective as described (Table 1). Combination of the Nꢀcap and Cꢀcaps was undertaken to
corroborate that the activities identified in the peptide context were retained in the less peptidic and
more drugꢀlike compound. The Cꢀcap core structures synthesized and tested were made in two very
similar contexts presenting no significant structural differences: phenoxybenzylamine (PBA) (Table
2, 5807 and 5849) and 3ꢀphenoxypyridylmethylamine (PPMA) (Table 2, 5824, 5823, 5822, 5825,
and 5848). Substitutions incorporated primarily halogens at the 3 and 4 positions of the phenoxy
group interacting with the lipophilic pocket but also included a 2ꢀmethyl and 3,5ꢀdichloro
substituted versions.
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Overall, the binding affinities of the seven FLIPs for CDK2/cyclin A2 (table 2) were in general
shown to be greater than for CDK4/cyclin D1. IC50 values for the active compounds range from
18.1 ꢁM to 106.1 ꢁM. A compound with 1ꢀ(4ꢀchlorophenyl)ꢀ5ꢀmethylꢀ1Hꢀ1,2,4ꢀtriazoleꢀ3ꢀ
carboxylic acid (4CPT) as the Nꢀcap structure and 3PBA as the Cꢀcap, (Table 2, 5807) was found to
be the weakest inhibitor of CDK2/cyclin A2. Testing of the FLIP compound bearing a fluoro
substitution at 3ꢀposition (PPMA, 5824) resulted in an IC50 of 53.2 ꢁM and the 3ꢀchloro substituted
analog (5848) had comparable potency. Movement of the fluoro substitution to the 4ꢀposition
(5823) provided a 4ꢀfold increase in binding activity (18.1 ꢁM) and resulted in the most potent
compound in this series however the 4ꢀchloro did not result in a similar enhancement (5822, IC50 =
54.4 ꢁM). Incorporation of a methyl group at the 2ꢀposition (5825) led to an IC50 value of 60 ꢁM
and therefore was not as successful as other analogs. Testing of the 3,5ꢀdichloro PBA derivative
(5849) in the 3,5ꢀDCPT context suggested a different activity profile from the other compounds and
indicating that this FLIP is not a potent inhibitor either for CDK4/cyclin D1 or CDK2/cyclin A2. In
order to assess drugꢀlikeness of the most active N and C cap combination, ClogP and polar surface
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area were calculated (ChemBioOffice 12, CambridgeSoft) for RRLIF and SCCP5823 and found to
be ꢀ2.75 , 5.3 and 303, 199 Å2 respectively.
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DISCUSSION
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The cyclin binding groove is composed of a primary hydrophobic pocket (interacting with Leu6
and Phe8 of HAKRRLIF), a secondary hydrophobic pocket (binding to Ala2) as well as an acidic
region (contacting Lys3 and Arg4). The REPLACE strategy has been applied in order to identify Nꢀ
and Cꢀcaps replacements for peptide determinants and generate nonꢀpeptidic inhibitors with greater
drugꢀlikeness. The SAR of phenylheterocyclic Nꢀcaps and bisarylether Cꢀcaps was therefore studied
to optimize binding affinity for cyclin A and D and further explore the structural basis for binding
and protein and ligand determinants in the regions described above.
Previously it has been shown in SAR studies that peptide affinity can be significantly impacted
through substitution of the Nꢀterminal residues contacting the secondary hydrophobic pocket and
acidic region in both cyclin A2 and D1 contexts and also that this can lead to preferential binding to
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one cyclin over the other . In order to explore this further and to optimize inhibitor binding, an
extensive SAR was generated for the phenylheterocyclic capping groups described. In prior studies
crystallography revealed that the phenyltriazole Ncap makes significant complementarity with the
CBG through interactions with the secondary hydrophobic pocket and through observed Hꢀbonds to
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the side chains of Trp217 and Gln254 . The 1ꢀphenylꢀ1Hꢀ1,2,4ꢀtriazoleꢀ3 carboxylic acid Ncap
represents an excellent template to generate isosteres with very similar steric complementarity while
investigating the requirements for substitution of the aryl ring and the Hꢀbond interactions observed
in the crystal structure. The isosteric series provides a rare opportunity to directly compare similarly
substituted heterocyclic isosteres and investigate direct contributions of key Hꢀbond interactions. In
the first instance, this analysis provides considerable insights into need for the heteroatoms in the
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1,2,4ꢀtriazole ring and in particular the Hꢀbond observed between N2 and the indole NH of Trp217.
The inactivity of the phenylfuran series and the general decreased potency of the phenylpyrazole (2ꢀ
5 fold relative to the phenyltriazoles) suggest not only a role for N2 as a binding determinant but
also for the N4 heteroatom. As the sp2 hybridized oxygen atom of a furan in general is not a good
Hꢀbond acceptor due to participation of the lone pairs in the aromatic character of the ring and
increased electronegativity compared to nitrogen, the decreased activity of this series is in line with
expectations. While the triazole ring also is aromatic, the total of 3 nitrogen lone pairs are available
and therefore any individual one is more available to participate in Hꢀbonding. The lower relative
potency of the phenylpyrazole series, which possess the N2 acceptor atom, but lack the N4
heteroatom was however an unexpected result. Crystal structures of the phenyltriazoles bound to
cyclin A did not suggest a role for the N4 atom in binding and the potency loss is not easily
explained since the pyrazole and triazole rings would be expected to have similar physicochemical
properties. A probable contribution of N4 to binding is suggested by modeling studies in which a
water molecule was placed between this atom and the carboxylate sidechain of Asp283 or Asp129
in two cyclins (Figure 2). This putative water forms appropriate interactions in forming a bridging
Hꢀbond between the ligand and the cyclin in the correct geometry and distance, suggests a role for
N4 and therefore explains the decreased potency observed in the phenylpyrazole series. The
phenylpyrrole containing FLIPs place an Hꢀbond donor adjacent to the indole NH and
unsurprisingly do not retain detectable levels of activity. While the phenylimidazole capped peptide
generated did not have an identically substituted ring (3ꢀfluoro) to the other analogs in this
evaluation, it can nonetheless be compared to the counterpart synthesized in the phenylpyrazole
series. Both of these FLIP isosteres possess similar activity suggesting that their Hꢀbonding
properties to the indole NH are equivalent. Overall, comparison of the phenylheterocyclic Nꢀ
capping groups provides key insights into the contributions of Hꢀbonds observed in crystal
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structures with cyclin A while suggesting a structural rationale for molecular differences between
similar bioisosteres.
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Further insights into the SAR of the aryl ring of the phenylheterocycles and also the molecular
determinants of binding to the minor hydrophobic pocket of the cyclin groove is provided through
the data obtained for the triazole and pyrazole series. Overall the activity of phenyltriazole
containing FLIPs and phenylpyrazole compounds were well aligned in terms of their relative
potency in binding to both CDK2/cyclin A2 and CDK2/cyclin D1 with the phenyltriazoles generally
being more potent. Selectivity for cyclin A or cyclin D was observed depending on the aryl
substitution. The 3,5ꢀdichloro analogs (phenyltriazole, 5773; phenylpyrazole, 5763) are more potent
than their 4ꢀchloro counterparts in the binding to CDK2/cyclin A2 whereas 4ꢀchloro substituted
FLIPS (phenyltriazole, 5774; phenylpyrazoles, 5765) showed greater affinity to CDK4/cyclin D1
thus reversing the order of selectivity. Preferential binding to cyclin A by 3ꢀsubstituted Nꢀcaps and
for cyclin D by 4ꢀchloro derivatives was therefore observed in both series of phenylheterocyclic
isosteres. In order to determine the structural basis for cyclin selectivity, modeled complexes of the
3,5 and 4ꢀchloro subsituted phenyltriazole FLIPs bound to cyclin D1 were generated based on
crystal structures of these analogs in complex with cyclin A and apo structures of cyclin D1.
Previously the cyclin grooves of cyclins A2 and D1 have been systematically compared and shown
to have significant differences in the volume of the secondary hydrophobic pocket and leading to
selectivity in the peptide context depending on the size of the contacting sidechain20. The smaller
pocket results in displacement of the 3ꢀsubstituted analogs from the subsite at the expense of
hydrophobic complementarity and thus interact with weaker affinity for cyclin D1 in contrast to that
for cyclin A (Figure 2). The improved binding of the 4ꢀsubstitutions (relative to the 3 and 3,5ꢀ
subsituted derivatives) to cyclin D1 can also be explained by a binding mode more conducive to
close contact of the phenyl ring with the minor pocket and interactions of the 4ꢀchloro atom with
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Glutamates 66 and 70. The decreased potency of the 3ꢀfluoro derivatives relative to the 3ꢀchloro in
both cyclin contexts is in line with the expectation based on forming the best complementarity with
the volume of the minor hydrophobic pocket. The activity decrease observed with the 3ꢀmethyl
analog in the triazole context is harder to explain although the slightly different volume of the
methyl may be less optimal for interaction. Furthermore the increase in activity of the 3ꢀchloro
relative to the dichloro only in the pyrazole context can likely be explained by a slightly different
binding mode as a result of the additional Hꢀbond in the phenyltriazole FLIPs. In this alternate
conformation, the 5 substitution leads to a steric clash and less favorable interactions result from the
necessity of avoiding these nonꢀoptimal contacts. Marginal binding of the methoxy substituted
pyrazole compounds is consistent with results obtained for the other 3 and 4 modified FLIPs and the
expected interactions of the larger substituents with the available space for interaction.
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After the optimization of the phenylheterocyclic Ncaps, the study of bisarylether as Cꢀcaps was
carried out to gain insights into the binding of fragments to the primary hydrophobic pocket. Nꢀcaps
and Cꢀcaps were incorporated into a single molecule in order to further investigate and validate the
key aspect of REPLACE that individual PLAs found in the peptide context can then be combined to
generate less peptidic and more drugꢀlike compounds. In the first instance, combination of the 4ꢀ
CPT and 3ꢀPBA as N and Ccaps for an ArgꢀLeu dipeptide (Table 2, 5807) suggested that potency
loss is occurring through a variant binding mode. Addition of halogen atoms to the phenoxy group
of the Ccap however results in recovery of lost binding and in the particular case of the 4ꢀfluoro
derivative, the in vitro activity is less than 2ꢀfold of the expected potency for combination of the
two capping groups while substantially improving the drugꢀlike character of the cyclin groove
inhibitor. Drugꢀlikeness is improved by incorporation of the bisarylether system which replaces two
residues with a small molecule capping group. Similar to the spacing function of Ile in the peptide
context (RRLIF increased activity relative to RRLF), the Cꢀcaps have an aromatic ring as a spacer
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group.
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Further modeling studies of the PBA and PBMA Ccap core structures indicates the structural
basis for increased binding affinity of the halogen substituted FLIPs (Figure 1D). The bisaryl ether
Ccap replaces the interaction of the Phe sidechain and its contacts with the primary hydrophobic
pocket adjacent to those of the Leu residue. Previous peptide SAR studies have demonstrated that 3
and 4 halogen substituted Phe residues have increased potencies through more optimal contacts with
this subꢀsite 11, 12. As can be seen (Table 2), all 3 and 4 modified Ccaps resulted in better binding to
the cyclin groove compared to 3PBA (5807) and are therefore consistent with this result. The 5 fold
increase observed for the 4ꢀfluoro (5823) indicates that the phenoxy aryl ring adopts a very similar
conformation to that in the peptide context (i.e. RRLIX where X is 4ꢀfluorophenylalanine) and
therefore leads to comparable increase in complementarity with this pocket. It is perhaps an even
more optimal geometry since the only a 2ꢀfold increase is observed in the peptide context. The
larger 4ꢀchloro substitution (5822) decreases the FLIP potency therefore suggesting the fluorine is
the optimal size. The lack of activity of the Cꢀcapped cyclin groove inhibitors for CDK4/cyclin D1
is largely consistent with the previous observation that the phenylalanine subsite (in addition to the
differences in the secondary hydrophobic pocket) has a decreased volume relative to cyclin A. As
none of the bisꢀaryl ethers have appreciable activity, it is apparent that interactions of the Ccap with
the smaller lipophilic site are suboptimal and therefore lead to significantly reduced activity (Figure
1D).
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In this study, further validation for the REPLACE strategy has been obtained from the
standpoint that partial ligand alternatives can be optimized when linked to and in the context of the
peptide. In addition, both N and C PLAs have been combined to create a more drug like and less
peptidic compound. With respect to the pentapeptide, three residues of the cyclin groove inhibitor
have been removed in introducing small molecule functionality into the molecule. Specifically, the
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most potent analog (5823) has physicochemical properties that are more consistent with Lipinski’s
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21
and other rules for drugꢀlikeness. It has a ClogP of 5.3 (between ꢀ0.4 and +5.6 for Ghose
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modified rules 22) in contrast to the RRLIF value of ꢀ2.75, a polar surface area of 100 Å2 less than
the parent peptide and 6 and 10 Hꢀbond donors and acceptors respectively. Further REPLACE
efforts centered on switching out the Arg and Leu residues for a nonꢀpeptidic core will lead to
additional optimization in terms of potency, stability and cellular permeability through bringing the
above parameters further in line. In particular, the inclusion of citrullene, the non charged isostere
of arginine and betahomoleucine will be undertaken. These have been shown in the peptide context
to retain potency 23and will further improve stability and drugꢀlikeness. These results also
demonstrate that in the iterative conversion of peptides into small molecules, potential changes in
binding mode, while detrimental, can be circumvented by reoptimization of proteinꢀligand affinity
in the molecule combining individual partial ligand alternatives. As a whole, this validation of
REPLACE further serves to exempify this strategy in drug discovery efforts to generate potent and
selective proteinꢀprotein interaction inhibitors of kinaseꢀsubstrate interactions.
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EXPERIMENTAL SECTION
All solvents and reagents were used as obtained. 1H NMR and 13C NMR spectra were recorded with
a Varian Mercury 300 and 400 Spectrometer, respectively. Mass spectra were measured with
Micromass QTOF: Tandem quadrupleꢀtime of flight mass spectrometer electrospray ionization
(ESI) and VG 70S: Doubleꢀfocusing magnetic sector mass spectrometer (EI). Analytical purities of
evaluated compounds were >95% unless stated otherwise. The following analytic method (unless
stated otherwise) was used on a Waters Alliance 2695 HPLC with a 2996 diodeꢀarray detector and
equipped a C18 (2) 100 A, 250 x 4.6mm, 5ꢁ column (Phenomenox Luna). A gradient from 100%
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water (0.1% trifluoroacetic acid) to 60% acetonitrile (0.1% trifluoroacetic acid) was run over 30
mins and held for 4 mins.The chromatograms were extracted at 226 and 254 nm.
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Preparation of phenyltriazole N-capping groups (Scheme 1)14, 15 (5843, 5773, 5774, 5906, 5907)
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Ethyl 2ꢀchloroꢀ2ꢀ(2ꢀ(3,5ꢀdichlorophenyl)hydrazono)acetate (1)
To a solution of 3,5ꢀDichloroaniline (1.62g, 10mmol) in 10 ml methanol, 10 ml of 6 N hydrochloric
acid was added while maintaining the temperature at 0°C. Sodium nitrate (1.38g, 20mmol) was then
slowly added as a solid. The reaction mixture was stirred for 15 mins at 0°C after which sodium
acetate was added as a solid to adjust the reaction to pH 5. Subsequent to this, a solution of ethyl 2ꢀ
chloroacetoacetate (1.64, 10mmol) in methanol was slowly added while maintaining the
temperature at 0°C, after which the reaction mixture was allowed to warm to ambient temperature
and stirred for 12 h. The reaction was monitored by TLC using 25% ethyl acetate in hexanes, for
completion. Methanol was then removed under reduced pressure, diethyl ether was added and the
organic layer was separated and washed with saturated sodium bicarbonate and water prior to
drying over sodium sulfate. The organic layer was concentrated and the product crystalized from
ethanol to give an orange solid 2.29g (77.7%).
1H NMR (methanolꢀd4, 300 MHz) δ (ppm) 7.2 (s, 3H), 7.0 (s, 1H), 4.32ꢀ4.39 (q, J=3.52, 3H) 1.35ꢀ
1.4 (t, J=3.41Hz, 3H).
MS (EI+) 294
Ethyl 1ꢀ(3,5ꢀdichlorophenyl)ꢀ5ꢀmethylꢀ1Hꢀ1,2,4ꢀtriazoleꢀ3ꢀcarboxylate (2)
The step 1 product, Ethyl 2ꢀchloroꢀ2ꢀ(2ꢀ(3,5ꢀdichlorophenyl)hydrazono)acetate (1) (2.29g,
7.7mmol), and acetaldehyde oxime (0.473g, 7.7mmol) were dissolved in toluene and heated to
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reflux for 2h after which triethylamine (0.81g, 7.7mmol) was added. The reaction was monitored by
TLC using 25% ethyl acetate in hexanes. After completion, the reaction mixture was then
concentrated and partitioned between ethyl acetate and water. The layers were separated and the
aqueous layer was washed with ethyl acetate. The combined organic layers were washed with water
and brine, dried with sodium sulfate, filtered, and then concentrated. The residue was crystallized
from diethylether/hexanes to give the product as fine, tan needles weighing 1.97g (84.5%).
1H NMR (chloroformꢀD, 300 MHz) δ (ppm) 7.45 (s, 2H), 7.49 (s, 2H) 4.46ꢀ4.53 (q, J=3.36Hz, 2H)
2.62 (s, 3H), 1.41ꢀ1.46 (t, J=3.35Hz, 3H)
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MS (EI+) 300
1ꢀ(3,5ꢀdichlorophenyl)ꢀ5ꢀmethylꢀ1Hꢀ1,2,4ꢀtriazoleꢀ3ꢀcarboxylic acid (3)
The step 2 product, Ethyl 1ꢀ(3,5ꢀdichlorophenyl)ꢀ5ꢀmethylꢀ1Hꢀ1,2,4ꢀtriazoleꢀ3ꢀcarboxylate (2)
(1.9g, 6.33mmol) was refluxed in sodium hydroxide (2.02g, 50.64mmol), 19ml of ethanol and
19ml of water for 2h and the reaction was monitored by TLC (35% ethyl acetate in hexanes). After
the completion of reaction, the reaction mixture was cooled, the alcohol was evaporated and diluted
with water. The reaction mixture was acidified with 1N hydrochloric acid and stirred to precipitate
the product.
1H NMR (chloroformꢀD, 400 MHz) δ (ppm) 7.86 (t, J=1.04Hz) 7.8 (d, J= 1.12Hz3H) 2.54 (s, 3H)
13C NMR (Dimethyl sulfoxideꢀD6, 400 MHz) δ (ppm) 160.6, 154.5, 154.11, 138.5, 135.1, 134.7,
129.0, 123.8
HRMS EI+ observed 270.9914, calculated 270.9915, 0.4ppm
Preparation of phenylpyrazole N-capping groups (Scheme 2)16 (5762-5767, 5771)
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Ethyl 1ꢀ(3,5ꢀdichlorophenyl)ꢀ5ꢀmethylꢀ1Hꢀpyrazoleꢀ3ꢀcarboxylate (4)
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1 g (4.68mmol) of 3,5ꢀdichlorophenyl hydrazine was dissolved in 25 ml of acetonitrile in a 100 ml
round bottom flask, triethylamine (0.47g, 6.9mmol) and ethyl aceto pyruvate (0.74g, 4.68mmol)
were added to the reaction mixture under stirring and left to continue overnight. The reaction was
monitored by TLC (25% ethyl acetate in hexanes) for completion. After the reaction was complete,
the reaction mixture was diluted with dichloromethane and washed with water. The organic layer
was separated, dried over sodium sulfate and concentrated. The crude mixture was then purified by
flash chromatography (Biotage SP4) using a SNAP 100g column with a gradient run starting from
6% ethylacetate: 94% hexanes to 50% ethyl acetate and 50% hexanes over 15 column volumes to
yield a white solid 0.39g (21% yield).
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1H NMR (chloroformꢀD3, 300MHz) δ (ppm) 7.431 (s, 3H), 6.744 (s, 1H), 4.49ꢀ4.44 (t, J=7.0Hz,
2H), 2.39 (s, 3H), 1.39ꢀ1.42 (t, J=2.54Hz, 3H),
MS (EI+) 298
1ꢀ(3,5ꢀdichlorophenyl)ꢀ5ꢀmethylꢀ1Hꢀpyrazoleꢀ3ꢀcarboxylic acid (5)
The ester obtained from step1, ethyl 1ꢀ(3,5ꢀdichlorophenyl)ꢀ5ꢀmethylꢀ1Hꢀpyrazoleꢀ3ꢀcarboxylate
(4) (0.39g, 1.3mmol) was dissolved in 10 ml methanol. The ester solution was treated with a 10 ml
solvent mixture of tetrahydrofuran, 20% potassium hydroxide and methanol (1:1:1). The solution
was stirred at room temperature overnight and after completion, as evidenced by TLC (25%
ethylacetate in hexanes), methanol was evaporated and the reaction solution was partitioned
between ethylacetate and water. After washing with ethyl acetate, the aqueous layer was collected
and acidified to pH 2 using 1N hydrochloric acid. The acidified aqueous layer was then partitioned
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between dichloromethane and additional water. The organic layer was dried over sodium sulfate
and concentrated to get solid product 0.23g (100%).
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1H NMR (dimethyl sulfoxideꢀD6, 400MHz) δ (ppm) 7.78ꢀ7.79 (t, J=0.937Hz,) 7.8 (d, J=1.14, 1H),
6.74 (s, 1H), 2.50 (s, 3H)
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13C NMR (dimethyl sulfoxideꢀD6, 400MHz) δ (ppm) 162.9, 144.5, 141.4, 140.8, 134.5, 128.0,
123.5, 11.9
HRMS EI+ Observed 269.9959, calculated 269.9963, 1.5ppm
Preparation of phenylfuran N-capping groups (Scheme 3)17 (5768, 5769)
Synthesis of 5ꢀ(3,5ꢀdichlorophenyl)furanꢀ2ꢀcarboxylic acid (6)
A mixture of 3,5ꢀdichloro aniline (0.84g, 5mmol), hydrochloric acid (15%, 3ml ) and water (4.5ml),
was heated until a clear solution was obtained. This solution was then cooled to 0oC, diazotized
with aqueous sodium nitrite (30%, 1.2ml) and filtered. To the filtered solution, water (2.5ml) and
furoic acid (0.56g, 5mmol) were added. An aqueous solution of cupric chloride (0.125g in 10 ml
water) was added dropwise and stirred for 4h at room temperature and kept aside for 16h. The
resulting solid was filtered off, suspended in water and purified by flash chromatography (Biotage
SP4) using a SNAP 100g column with a gradient 0% to 40% ethylacetate for 5 column volumes, the
concentration of ethyl acetate at 40% was kept constant for 2 column volumes and then increased
to 100% in 5 column volume. The purified product obtained weighed 0.36g (28%)
1H NMR (dimethyl sulfoxideꢀD6, 400MHz) δ (ppm) 7.85 (d, 2H), 7.65 (t, 2H), 7.36 (d, J=3.88Hz,
1H), 7.40 (d, 3.96, 1H)
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13C NMR (dimethyl sulfoxideꢀD6, 400MHz) δ (ppm) 159.0, 152.9, 145.0, 134.9, 132.2, 128.0,
122.7, 119.7, 110.5
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HRMS EI+ Observed 255.9698, calculated 255.9694, 1.6ppm
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Preparation of phenylpyrrole N-capping groups (Scheme 4) 18 (5775, 5776)
1ꢀ(4ꢀchlorophenyl)ꢀ1Hꢀpyrroleꢀ3ꢀcarbaldehyde (7)
4ꢀchloroaniline (0.51g, 4 mmol), 2,5ꢀdimethoxytetrahydrofuranꢀ3ꢀcarbaldehyde (0.76g, 4.8 mmol),
acetic acid (16ml) were stirred in a round bottom flask until a red colored solution was observed.
The reaction mixture was then refluxed at 120oC for 15 mins and a dark reddish brown solution was
obtained. The reaction was monitored by TLC (25% ethyl acetate: 75% hexanes) and worked up
when complete. The reaction mixture was cooled, extracted with ethyl acetate and the organic layer
was washed with saturated sodium bicarbonate, dried over sodium sulfate and concentrated to get
0.77g (94%)
1H NMR (chloroformꢀD3, 300MHz) δ (ppm) 9.85 (s, 1H), 7.62ꢀ7.63 (t, J=1.8Hz, 1H), 7.44ꢀ7.47 (d,
J=9Hz, 2H), 7.34ꢀ7.37 (d,J=9.3Hz, 2H) 7.05 (t, J=0.9Hz, 1H), 6.57ꢀ6.58 (q, J=1.5Hz, 1H)
MS (EI+) 204
1ꢀ(4ꢀchlorophenyl)ꢀ1Hꢀpyrroleꢀ3ꢀcarboxylic acid (8)
1ꢀ(4ꢀchlorophenyl)ꢀ1Hꢀpyrroleꢀ3ꢀcarbaldehyde(7) (0.76g, 3.58mmol) obtained from step 1, silver
nitrate (1216.26mg, 7.16mmol), 6 N sodiumhydroxide (15ml) and methanol (5.8ml) were refluxed
for 5h and the reaction was monitored by TLC for completion. The reaction mixture was filtered
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through celite and the aqueous layer was acidified to get a white precipitate. The precipitate was
filtered off, dissolved in ethyl acetate, and dried over sodium sulfate to get the product 0.6g (65%)
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1H NMR (dimethyl sulfoxideꢀd6, 400 MHz) δ (ppm) 7.95 (t, J=0.93, 1H), 7.71ꢀ7.73 (d, J=4.6Hz,
2H), 7.53ꢀ7.56 (d, J= 4.7Hz, 2H), 7.43ꢀ7.44 (t, J= 1.33Hz1H), 6.61ꢀ6.62 (q, J=0.88Hz, 1H)
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13C NMR (dimethyl sulfoxideꢀd6, 400 MHz) δ (ppm) 165.0, 137.9, 130.5, 129.5, 123.9, 121.7,
120.7, 118.6, 111.5 HRMS
HRMS (EI+) observed 221.0242, calculated 221.0244, 0.9ppm
Preparation of phenylimidazole N-capping group (Scheme 5)19 (5852)
Ethyl 2ꢀ(3ꢀfluorophenyl)ꢀ1Hꢀimidazoleꢀ4ꢀcarboxylate (9)
The reactants 3ꢀfluoroꢀN'ꢀhydroxybenzimidamide (1g, 6.49mmol) and ethylpropiolate (0.636g,
6.49mmol) were dissolved in methanol and heated at reflux for 3 days. Toluene was then added and
the methanol was distilled off. The reaction was heated at reflux for 6h after which the toluene was
removed by rotary evaporation and the residue was chromatographed on silica gel twice (2%
methanol/dichloromethane and 20% ethyl acetate/ Hexanes). The residue was dissolved in phenyl
ether and heated to 200°C for 25 mins and then poured into cyclohexane and extracted with 1N
hydrochloric acid. The acid layer was basified with sodium bicarbonate and extracted with ethyl
acetate. After drying with sodium sulfate, it was filtered, concentrated and purified by flash
chromatography on silica gel (Ethylacetate/Hexanes gradient) to give the desired product as an offꢀ
white solid.
2ꢀ(3ꢀfluorophenyl)ꢀ1Hꢀimidazoleꢀ4ꢀcarboxylic acid (10)
The step 2 product, ethyl 2ꢀ(3ꢀfluorophenyl)ꢀ1Hꢀimidazoleꢀ4ꢀcarboxylate (9) (0.3g, 1.281mmol was
refluxed in sodium hydroxide (0.45g, 11.25 mmol), ethanol (5ml) and water (5ml) for 2h and the
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reaction was monitored by TLC. After the completion of reaction, the reaction mixture was cooled,
the alcohol was evaporated and diluted with water. The reaction mixture was acidified with 1N
hydrochloric acid and stirred to precipitate the product.
9
1H NMR dimethyl sulfoxideꢀD6, 400MHz) δ (ppm) 13.28 (d, 1H), 7.52 (q, 2H) 7.24(t, 2H)
13C NMR (DMSO, 400MHz) δ (ppm) 163.57, 161.16, 133.87, 132.02, 131.00, 130.92. 124.90,
121.42, 115.68, 111.98
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HRMS ES+ Observed 207.0576, calculated 207.0570, 2.9ppm
(4-(3-chlorophenoxy)pyridine-2-yl)methanamine (5848).
This material was sourced commerically from UORSYS Ltd and fully characterized in house as
follows:
1H NMR (chloroformꢀD3, 400 MHz) δ (ppm) 3.95, s 2H
1H 7.25, m, 1H 7.37, m, 1H 8.45, d, 1H
, 6.75, m, 1H, 6.87, d, 1H, 7.01, m, 1H, 7.13, m,
,
,
,
13C NMR (chloroformꢀD3, 400 MHz) δ (ppm) 164.8, 164.4, 154.9, 151.0, 135.5, 130.9, 125.6,
121.2, 118.9, 110.7, 109.7, 47.7
HRMS EI+ observed 234.0561, calculated 234.0560, 0.4 ppm
Peptide & FLIP Synthesis
All peptides and Nꢀterminally capped FLIPs were synthesized and purified using standard Fmoc
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chemistry by GenScript (Piscataway, NJ). HPLC and MS were used to confirm the purity and
structure of each peptide (see Supplementary Information Table 1). Compounds in Table 2 were
synthesized by the following procedure used for compound 5926: The Nꢀterminally capped and
protected ArgꢀLeu peptide (16.1 mg, 0.02 mmol) was dissolved in DCM and (4ꢀ(3ꢀ
chlorophenoxy)pyridineꢀ2ꢀyl)methanamine was added along with HBTU (16.7 mg, 0.02 mmol)
and DIPEA (6.5 mg, 0.02 mmol). The reaction was stirred and monitored until TLC/HPLC
indicated the complete consumption of starting material. Once complete, the reaction mixture was
concentrated and then partitioned between EtOAc and H2O. The organic layer was washed with 1N
NaOH, 1N HCl, and brine and dried with Na2SO4. The resulting product was deprotected with
95:2.5:2.5 TFA:H2O:TIPS over night, then concentrated and triturated with cold diethyl ether. The
final N and C capped peptide was purified using semiꢀpreparative HPLC (0ꢀ80 CH3CN over 20
mins).
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Fluorescent Polarization Binding Assay
CDK4/cyclin D1 FP assay This assay was performed using black 384ꢀwell plates using a
previously described procedure24 with the following modifications. To each well were added: 5 ꢁl
CDK4/cyclin D1 (0.3 ꢁg/well purified recombinant human kinase complex from Invitrogen), 5 ꢁl
compound solution, 5 ꢁl 30 nM fluoresceinylꢀAhxꢀProꢀValꢀLysꢀArgꢀArgꢀLeuꢀ(3ClPhe)ꢀGly tracer
peptide. Compounds and kinase complexes were diluted using assay buffer (25 nM HEPES pH 7,
10 nM NaCl, 0.01% Nonidet Pꢀ40, 1mM dithiothretiol (DTT). Plate was centrifuged for 1 min at
500 rpm and then incubated with shaking for 45 mins at room temperature. Fluorescence
polarization was read on DTX880 multimode detector (Beckman Coulter, Brea, CA) fitted with 485
nm/535 nm excitation/emission filters and a dichroic mirror suitable for fluorescein. Relative mp
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was calculated for each concentration tested using the equation showing below. IC50 values were
determined by logarithmic regression by correlating relative mps and testing concentrations.
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ʚ
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MP compound ꢀMP(DMSO, protein, tracer)
Relative mp = MP DMSO, protein ꢀMP(DMSO, protein, tracer)
ʚ
ʛ
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CDK2/cyclin A2 FP assay This assay was performed using black 384ꢀwell plates. To each well
were added: 5 ꢁl CDK4/cyclin D1 (0.3 ꢁg/well purified recombinant human kinase complex), 5 ꢁl
compound solution, 5 ꢁl 30 nM fluoresceinylꢀAhxꢀProꢀValꢀLysꢀArgꢀArgꢀLeuꢀPheꢀGly tracer
peptide. Compounds and kinase complexes were diluted using assay buffer (25 nM HEPES pH 7,
10 nM NaCl, 0.01% Nonidet Pꢀ40, 1mM dithiothretiol (DTT). Plate was centrifuged for 1 min at
500 rpm and then incubated with shaking for 45 mins at room temperature. Fluorescence
polarization was read on DTX880 multimode detector (Beckman Coulter, Brea, CA) fitted with 485
nm/535 nm excitation/emission filters and a dichroic mirror suitable for fluorescein. Relative mp
was calculated for each concentration tested using the equation showing below. IC50 values were
determined by logarithmic regression by correlating relative mps and testing concentrations.
ACKNOWLEDGMENTS. We thank Dr’s. Michael Walla and William Cotham in the
Department of Chemistry and Biochemistry at the University of South Carolina for assistance with
Mass Spectrometry, Helga Cohen and Dr. Perry Pellechia for NMR spectrometry. Dr Michael
Wyatt and Ms. Kara Estes provided assistance with the FP assay. This work was partly funded by
the National Institutes of Health through the research project grant, 5R01CA131368.
AUTHOR INFORMATION
Corresponding Author: Campbell McInnes: Eꢀmail: mcinnes@sccp.sc.edu; Phone: (803) 576ꢀ5684
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Notes: The authors declare no competing financial interest.
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FIGURE LEGEND
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Figure 1. REPLACE mediated conversion of the octapeptide cyclin groove inhibitor HAKRRLIF
into an N and Cꢀterminally capped dipeptide. (Structures were modeled from PDB ID 2UUE).
Figure 2. Modeled structure of SCCP5773 in complex with CDK4/cyclin D1 (brown carbon atoms)
overlayed with the crystal structure of the same Ncap bound to CDK2/cyclin A2 (yellow carbon
atoms). A Connolly surface representation of cyclin A is shown along with the putative water
molecule contributing bridging hydrogen bonds.
Scheme 1. Synthesis of phenyltriazole Nꢀcapping groups
Scheme 2. Synthesis of phenylpyrazole Nꢀcapping groups
Scheme 3. Synthesis of phenylfuran Nꢀcapping groups
Scheme 4. Synthesis of phenylpyrrole Nꢀcapping groups
Scheme 5. Synthesis of phenylimidazole Nꢀcapping groups
SUPPORTING INFORMATION
Supplementary Table 1 provides analytical characterization data for FLIPs shown in Table 2. This
material is available free of charge via the Internet at http://pubs.acs.org.
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TABLE OF CONTENT GRAPHIC
Fragment based Design of NonꢀATP competitive CDK Inhibitors using REPLACE
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