Thienopyrimidinone Based Sirtuin-2 (SIRT2)-Selective Inhibitors Bind in the Ligand Induced Selectivity Pocket

Article abstract of DOI:10.1021/acs.jmedchem.6b01690

Sirtuins (SIRTs) are NAD-dependent deacylases, known to be involved in a variety of pathophysiological processes and thus remain promising therapeutic targets for further validation. Previously, we reported a novel thienopyrimidinone SIRT2 inhibitor with good potency and excellent selectivity for SIRT2. Herein, we report an extensive SAR study of this chemical series and identify the key pharmacophoric elements and physiochemical properties that underpin the excellent activity observed. New analogues have been identified with submicromolar SIRT2 inhibtory activity and good to excellent SIRT2 subtype-selectivity. Importantly, we report a cocrystal structure of one of our compounds (29c) bound to SIRT2. This reveals our series to induce the formation of a previously reported selectivity pocket but to bind in an inverted fashion to what might be intuitively expected. We believe these findings will contribute significantly to an understanding of the mechanism of action of SIRT2 inhibitors and to the identification of refined, second generation inhibitors.

Full text of DOI:10.1021/acs.jmedchem.6b01690

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Article
Thienopyrimidinone based sirtuin-2 (SIRT2)–selective
inhibitors bind in the ligand induced 'selectivity pocket'
Sandeep Sundriyal, Sebastien Moniot, Zimam Mahmud, Shang Yao,
Paolo Di Fruscia, Christopher R. Reynolds, David T. Dexter, Michael J. E.
Sternberg, Eric W.-F. Lam, Clemens Steegborn, and Matthew J Fuchter
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01690 • Publication Date (Web): 30 Jan 2017
Downloaded from http://pubs.acs.org on January 30, 2017
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Thienopyrimidinone based sirtuin-2 (SIRT2)–selective inhibitors bind in the ligand induced
'
selectivity pocket'
ǂa
#a
†
†
ǂ
Sandeep Sundriyal , Sébastien Moniot , Zimam Mahmud , Shang Yao , Paolo Di Fruscia , Christopher R.
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Reynolds , David T. Dexter , Michael J. E. Sternberg , Eric W.ꢀF. Lam , Clemens Steegborn , Matthew J.
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Fuchter
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Department of Chemistry, Imperial College London, London SW7 2AZ, UK
†
Department of Surgery & Cancer, Imperial College London, Hammersmith Hospital Campus, Du Cane
Road, London W12 0NN, UK
#
Department of Biochemistry, University of Bayreuth, Universitaetsstrasse 30, 95447 Bayreuth, Germany
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Department of Life Sciences, Imperial College London, London SW7 2AZ, UK
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Centre for Neuroinflammation & Neurodegeneration, Division of Brain Sciences, Imperial College London,
London W12 0NN, UK
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ABSTRACT
Sirtuins (SIRTs) are NADꢀdependent deacylases, known to be involved in a variety of pathophysiological
processes and thus remain promising therapeutic targets for further validation. Previously, we reported a
novel thienopyrimidinone SIRT2 inhibitor with good potency and excellent selectivity for SIRT2. Herein,
we report an extensive SAR study of this chemical series and identify the key pharmacophoric elements and
physiochemical properties that underpin the excellent activity observed. New analogues have been identified
with submicromolar SIRT2 inhibtory activity and good to excellent SIRT2 subtypeꢀselectivity. Importantly,
we report a coꢀcrystal structure of one of our compounds (29c) bound to SIRT2. This reveals our series to
induce the formation of a previously reported ‘selectivity pocket’, but to bind in an inverted fashion to what
might be intuitively expected. We believe these findings will contribute significantly to understanding of the
mechanism of action of SIRT2 inhibitors and to the identification of refined, second generation inhibitors.
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INTRODUCTION
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The sirtuins (silent information regulator 2–related proteins) are enzymes that employ NAD to mediate their
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deacylase activity. Seven sirtuin isoforms (SIRT1ꢀ7) have been identified in humans, which differ in their
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cellular localization, function and specificity. Despite being labelled primarily as deacetylases, the sirtuins
appear to have a broader functional enzymatic role, with recent studies reporting their ability to remove
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succinyl, malonyl, myristoyl, palmitoyl and oxononanoyl groups ; with the substrate specificity
dependent on the enzyme in question. Sirtuin catalyzed deacylation occurs on histone substrates – with
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variable specificity
ꢀ and on a large number of non–histone proteins. Thus, as a consequence, the
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sirtuins play a significant role in various biological processes such as ageing,
inflammation,
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metabolism,
autophagy,
and DNA repair.
Since SIRT2 regulates the cell cycle during mitosis, it is unsurprising that its deregulation has been linked to
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a variety of cancers.
However, the role of this protein in cancer is complex and likely context specific.
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For example, while a selective SIRT2 suicide inhibitor was recently shown to result in proteolytic
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degradation of c–Myc, suggesting SIRT inhibition to be a strategy in cꢀMyc driven cancers, the loss of
SIRT2 function has conversely recently been reported to reprogramme cellular glycolytic metabolism (via
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PKM2 regulation), resulting in a tumour permissive phenotype. Aside from deregulation in cancer, SIRT2
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has been linked to type II diabetes , bacterial infections , cardiovascular diseases and neurological
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disorders
, thus underlining its potential therapeutic value in the context of drug discovery. Given the fact
that there is still much to be learnt about the precise role of SIRT2 in human biology and disease, the
availability of wellꢀcharacterised and selective inhibitors is of prime importance to assist with further
validation of this promising target.
A number of small molecule SIRT2 inhibitors have been reported (see Figure 1 for representative examples)
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including the physiological sirtuin inhibitor nicotinamide (1) and its derivatives,
sirtinol (2) and
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analogues, cambinol (3),
benzamide (4) and derivative (5) , AGK2 (6) , chromanꢀ4ꢀone
and
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bicyclic pyrazoles (7). Mechanismꢀbased ‘suicide’ SIRT2 inhibitors are also known.
For the majority of
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inhibitors, structural details of their binding site and interactions are still lacking. An exception to this are
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the aminothiazole analogues termed the ‘SirReals’ (such as 8), that were found by crystallography to
induce a new ‘selectivity pocket’ in SIRT2 to yield highly selective SIRT2 inhibitors. The SirReals were
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subsequently optimized using a structure–based approach.
Consistent with SIRT2 structural
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rearrangement upon ligand binding, propofol, an injectable hypnotic, was also found to bind to a new
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allosteric site in SIRT2 which was induced only in the presence of ADP–ribose.
We recently reported highly selective SIRT2 inhibitors based on the tetrahydrobenzo[4,5]thieno[2,3–
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d]pyrimidin–4(3H)–one scaffold, which were identified by in silico screening using a pharmacophore
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assembled from a previous study and literature compounds. The identified inhibitor, ICL–SIRT078 (19a,
Figure 1), was found to be highly selective for SIRT2 (SIRT2 data: IC (fluorogenic peptide) = 1.45 µM;
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IC (enzyme coupled SIRT–Glo) = 0.17 µM; K (tubulin–K40 peptide competitive) = 0.62 µM) and showed
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neuroprotection in a Parkinsonian neuronal cell death model. Only a limited number (9 in total) of analogues
were examined in the inital paper, particularly with no variation at the eastern N–3 region (see Figure 1) of
the inhibitor. A substrate competitive mechanism of action for 19a was inferred from biochemical
experiments, but the precise binding pose only predicted from computational docking.
Herein we report a much more extensive SAR study for this series, particularly with a variation at both the
N–3 and N–7’ positions. Through this, several potent analogues with sub–micromolar in vitro SIRT2
inhibitory activity have been identified, which retain excellent (>100 fold) selectivity for SIRT2 over SIRT1
and SIRT3. Importantly, we have solved a coꢀcrystal structure of one of our compounds bound to SIRT2.
This reveals our compounds to also induce formation of the ‘selectivity pocket’ in SIRT2, but to bind in an
inverted fashion to what might be intuitively expected from comparison of our series to the SirReal inhibitors
(
see Figure 1). Using the obtained structure, we have rationalized the SAR for our series, and determined an
optimum lipophilicity range for inhibitors binding in the induced pocket in SIRT2. As such, this data should
prove useful in the further development of refined inhibitors targeting the SIRT2 ‘selectivity pocket’.
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DISCUSSION
Structure activity relationship (SAR)
The newly synthesized analogues, based on our previously validated thienopyrimidinone scaffold, were
designed to contain a variety of N–3 (columns a – i, Table 1) and N–7’ substituents (rows 9 – 49, Table 1),
with the aim to conduct a relatively broad survey of the SAR features of this scaffold. These compounds
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were all synthesised according to the previously disclosed method (Scheme SS1 and SS2, Supporting
Information) and assessed as racemates. SIRT2 inhibitory activity was assessed using a validated SIRT2
assay, based on a fluorogenic peptide substrate derived from p53 (see Experimental Section). For this assay,
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suramin was used as a positive control and gave similar inhibitory activity to that observed previously.
Vehicle (DMSO) was used as the negative control. SIRT2 inhibitory activity was measured at two compound
concentrations, 1 µM (n=2) and 10 µM (n=2) for all compounds. Full (10–)dose response SIRT2 IC values
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(
n=2) were calculated only for selected derivatives that exhibited promising inhibitory activity at 1 µM.
A quick survey of the tested analogues (Table 1) immediately revealed the importance of a bulky aromatic
ring such as naphthalyl (columns a–d), quinolinyl (column e) or biphenyl (column i) at the N–3 position.
With limited exceptions, most of the compounds bearing a less bulky benzyl derivative – either an o– or m–
methoxybenzyl (columns f and h) or an unsubstituted benzyl (column g) – at N–3 displayed poor SIRT2
inhibition, in comparison to the analogues possessing a bicyclic ring or a biphenyl moiety at this position.
The most notable examples are 16a/d/e vs 16g/h; 18a/b/d/e vs 18g/h; 20a/b/d/e vs 20g/h; 23a/e vs 23f–h or
25a vs 25f–h. This SAR feature was observed in an early phase of our SAR study and hence we did not
pursue the further synthesis of benzyl based derivatives for other N–7’ R substituents, as evident from the
empty cells under columns f–h in Table 1.
The effect of substitution at the N–7’ position on SIRT2 inhibitory activity was found to be dependent on the
type of N–3 substituent. For example, the presence of a benzyl ring at N–7’ (row 9) yielded molecules with
only moderate activity in combination with almost all of the N–3 substituents except 8–quinolylmethylene
(9e, SIRT2 IC = 2.5 µM) and to a lesser extent 2–naphthyl (9d, SIRT2 IC = 5.45 µM). With more polar
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aromatic rings at N–7’, interesting trends were observed. Electron withdrawing halogenated rings at N–7’
rows 10–14) yielded poor to moderate inhibitors (SIRT2 % inhibition = 40–70% at 10 µM) with the
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naphthyl (columns a–d) or biphenyl–based (column i) N–3 substituents, whereas improved SIRT inhibitory
activity of these derivatives was observed when there was a 8–quinolylmethylene N–3 substituent (column
e). Examples include, 10e vs 10a–d; 12e vs 12a–d; 13e vs 13a–c; 14e vs 14a–d. Furthermore, 8–
quinolylmethylene N–3 substituted molecules exhibited very similar IC50 values between a substituted
benzyl group at N–7’ and halogentated rings at N–7’, for example 9e vs 10e, 12e–14e). A similar observation
was made with the 4–cyanobenzyl N–7’ substituent, which, in combination with 8–quinolylmethylene,
yielded 15e, a compound with submicromolar SIRT2 inhibitory activity (SIRT2 IC50 = 0.39µM).
Conversely, only moderate activity (SIRT2 % inhibition = 66–75% at 10 µM) was observed for compounds
bearing a 4–cyanobenzyl N–7’ substituent in the presence of other N–3 substituents (for example, 15a, 15b,
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15d and 15i).
The effect of an electron donating methoxy substitution on the N–7’ benzyl group seemed to improve the
activity of molecules possessing a 2–methoxy–naphthalen–1–yl N–3 substituent (column a) compared to the
other N–3 substituents. For example, 16a, 17a and 18a displayed a 9, 11 and 32 fold increase in potency,
over 9a, respectively. Interestingly, a similar improvement was not observed with the 8–quinolylmethyl N–3
substituent (column e). For instance, in comparison to the unsubstituted benzyl inhibitor (9e, SIRT2 IC =
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.50 µM), the o–methoxy benzyl analogue (16e) exhibited poorer potency (SIRT2 IC = 5.61 µM), while
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the p–methoxy benzyl analogue (18e) exhibited only a slight improvement in SIRT2 inhibition (SIRT2 IC50
1.83 µM). In general, analogues containing a p–methoxy benzyl N–7’ substituent consistently showed
better SIRT2 inhibition compared to the o–methoxy or m–methoxy analogues (18 vs 16/17). In particular,
8a exhibited potent SIRT2 inhibition compared to other methoxy substituted analogues with a sub–
micromolar SIRT2 IC value of 0.65 µM.
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The hit compound that initated this study, 19a , has a 3–pyridylmethylene substituent at the N–7’, and has
good inhibitory activity against SIRT2 (IC = 1.45 µM in the same assay format as this study). Changing the
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position of pyridyl nitrogen did not have any dramatic effect on the observed activity except that 2–pyridyl
(
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row 21) analogues exhibited slightly weaker SIRT2 inhibition compared to other two pyridyl substituents
apart from the derivative bearing a 7–quinolylmethylene substituent), for example 21a vs 19a/20a.
Compounds 19a and 20a with the 2–methoxy–naphthalen–1–yl substituent at N–3 were found to be most
potent in this category, with low micromolar activity (SIRT2 IC50 = 1.45 and 1.20 µM respectively).
Replacing the pyridine ring of N–7’with a pyrimidine ring (row 22) resulted in either poor (22a/f–h) or
moderate (22b/d/e) SIRT2 inhibition. Interestingly, analogues containing a 6–methoxypyridin–3–yl (row 23)
N–7' substituent exhibited activities similar to those with a 2ꢀpyridyl N–7' substituent (row 21).
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An aliphatic cyclic or saturated heterocyclic ring at N–3 (rows 24–26), led to a series of derivatives where
only a combination of these substituents with a 2–methoxy–naphthalen–1–yl N–7’ substituent (column a)
was found to give effective SIRT2 inhibitors. For example, analogues with pendent cyclohexyl (24a) or
tetrahydrofur–2–yl rings (25a) at N–7’ gave IC values lower than 2 µM in the presence of a 2–methoxy–
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naphthalen–1–yl N–3 substituent. A variety of derivatives bearing five membered heteroaromatic rings at N–
7’, such as pyrazole (row 27), furan (row 28), isoxazole (row 29) and thiophene (row 30), were also
evaluated. Most of the tested analogues in this series showed moderate to poor activity. An important outlier
however was isoxazole 29, with a 4–methoxy–naphthalen–1–yl group in the N–7’: 29c had a submicromolar
IC value 0.58 µM.
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Next, we evaluated the effect of number of linker carbons between core ring system and the N–7’
substituent. In general, increasing the linker chain from one (rows 9–30) to two carbons (rows 31–34) had a
slightly deleterious effect on the SIRT2 inhibition, for example 16a vs 33a; 19b vs 31b; 20a/b/d vs 32a/b/d.
Likewise, aromatic N–7' substituents without a linker carbon (rows 35–40) exhibited reduced potency,
especially in comparison to their counterparts attached with a one carbon linker. For example, analogues
35a/b/d vs 16a/b/d and 36b vs 18b. However, in combination with 8–quinolylmethyl N–3 substituent the
activity of analogues with these N–7' aromatic rings was somewhat rescued, as exemplified by 35e–40e;
most potent being 40e with a 4–fluorophenyl ring (SIRT2 IC 0.97 µM). This trend was not maintained
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however for analogues with aliphatic rings (rows 41–42) directly attached to the N–7’ position. Such
derivatives exhibited good activity in combination with 2–methoxy–naphthalen–1–yl (column a), 4–
methoxy–naphthalen–1–yl (column c) and biphenyl–3–yl (column i), at the N–3 position. Particularly,
analogues 41a, 41c, 41i, 42a and 42i exhibited SIRT2 IC values in the range of 0.73 – 1.74 µM. When the
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aliphatic ring at Nꢀ7’ was more polar, particularly containing a basic nitrogen atom directly attached to N–7'
via a two carbon linker, only moderately active (43a/c/i, 44i) or inactive analogues (43d/e and 44b/d/e) were
obtained.
Bulky N–7' substituents such as dihydro–1H–indenyl (45), tetrahydronaphthalen–1–yl (46) and
benzodioxolyl (47) were also found to be detrimental to the SIRT2 inhibition except when in combination
with the quinolin–8–yl substituent (column e) at the N–3 position. All three analogues, 45e–47e, exhibited
moderate SIRT2 inhibition, as compared to other analogues in these rows. Finally, two series of molecules
with less bulky and linear (rather than cyclic) N–7’ substituents were assessed (row 48 and 49) and were
found to possess poor SIRT2 inhibitory activity in most of the cases except 48c/i and 49i.
We also synthesized a few analogues where the fused cyclohexyl ring in the central thienopyrimidinone core
ring had been ring opened. These derivatives possessed a 2–methoxy–naphthalen–1–yl substituent at N–3
end and varying N–7' substituents (50–57, Table 2). These analogues were consistently found to be of
slightly lower potency in comparison to the corresponding analogues of the original scaffold, for example
50a vs 15a, 51a vs 18a, 52a vs 19a, 53a vs 20a, 54a vs 26a and 55a vs 42a. The only slightly reduced
activity of 51a–53a relative to the original scaffold suggests that the fused cyclohexyl ring and hence the
chiral centre at position 7 in the central core is not an essential feature of the SAR. Indeed, we previously
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found only a relatively small difference in the SIRT2 inhibitory values for enantiomers of 19a. Comparable
derivatives lacking a N–7' substituent (57a) or with a smaller dimethylamino (58a) substituent were found to
be inactive.
Overall, this SAR analysis suggests that a lipophilic and large bicyclic (column a–e) or biphenyl (column i)
N–3 substituent is a key requirement for SIRT2 inhibition for this series, while a wide variety of substituents
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with varying polarity and linker length are tolerated at N–7'. This suggests that the vector from Nꢀ7’ projects
into a relatively large pocket and that the N–7' substituent is not involved in any direction–dependent
interactions, such as hydrogen bonds or dipole–dipole contacts. The significant SIRT2 inhibition also
exhibited by analogues where the fused cyclohexyl ring of the core had been ring opened (50a–55a, Table 2)
also demonstrates the accommodating nature of the SIRT2 binding pocket in this region.
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Dependence of SIRT2 inhibitory activity on cLogP
One clear but confounding feature of the SAR above was that some N–7' substituents showed good activity
with only certain Nꢀ3 substituents, but not with others, and the ‘optimum’ Nꢀ3 substituent varied depending
on Nꢀ7’ (Table 1, vide supra). We reasoned that different combinations of N–7' and N–3 substituents might
be affecting the overall physicochemical parameters of a given molecule to making it suitable for binding to
the given target SIRT2 pocket. Since, the SAR analysis suggested potential SIRT2 pocket for these inhibitors
to be hydrophobic in nature we pursued a correlation between the calculated logP (cLogP) of the inhibitors
with their activity. Plotting the cLogP of analogues against their SIRT2 inhibitory activity (percentage
inhibition at 10 µM) suggested that the inactive analogues (<50 % inhibition) possess either high or low
cLogP values, when compared to the active inhibitors (>80% inhibition), which all have cLogP values
between 3.8 and 5.3 (Figure 2). We further categorized the analogues based on total aromatic ring count,
which clearly affects the cLogP of the analogues in this series. Interestingly, analogues with less than 4
aromatic rings showed a positive correlation (r = 0.701, Figure SF1, Supporting Information), while those
with 4 aromatic rings showed a negative correlation (r = –0.708, Figure SF2, Supporting Information) with
cLogP values. Further categorizing of the data based on the Nꢀ3 substituents revealed even higher correlation
between cLogP and activity of analogues with benzyl–based N–3 substituents (Figure SF3, Supporting
Information). Overall, with few exceptions most of the active analogues in this series lie in a narrow region
of cLogP and possess 3–4 aromatic rings. Analogues which do not fit this criteria are poor SIRT2 inhibitors.
This requirement is likely governed by the overall hydrophobic nature of the SIRT2 pocket to which these
inhibitors bind.
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This analysis explains several puzzling features of the SAR. For instance, most of the analogues based on
benzyl or methoxybenzyl (f–h) N–3 substituents possess lower cLogP, due to lower number of aromatic
rings (2–3) and thus are inactive. However, improved SIRT2 inhibition for these compounds can be observed
when N–7' is occupied by a lipophilic substituent, as in the case of 25f (27% inhibition@10 µM, cLogP =
2.78) vs 24f (79% inhibition@10 µM, cLogP = 4.23) and 45f (70% inhibition@10 µM, 4.58); 22g (18%
inhibition@10 µM, cLogP = 2.40) vs 14g (67% inhibition@10 µM , clogP = 4.57); 25h (3% inhibition@10
µM, clogP = 2.78) vs 18h (71% inhibition@10 µM , clogP = 3.82) and so on.
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Likewise, replacing the less lipophilic quinolin–8–yl (column e) with a biphenyl (column i) or
methoxynaphthyl (column a, c) at N–7' can improve the activity of analogues having a more polar N–3
substituent, for instance 42e (51% inhibition@10 µM, cLogP = 3.25) vs 42a (96% inhibition@10 µM,
cLogP = 4.06) and 42i (93% inhibition@10 µM, cLogP = 4.60); 43e (53% inhibition@10 µM, cLogP =
2.53) vs 43a (70% inhibition@10 µM, cLogP = 3.34), 43c (76% inhibition@10 µM, cLogP = 3.34) and 43i
(77% inhibition@10 µM, cLogP = 3.87); 44e (29% inhibition@10 µM, cLogP = 3.35) vs 44i (77%
inhibition@10 µM, cLogP = 4.70) . Conversely, replacement of quinolin–8–yl with more lipophilic N–3
substituents can also render active molecules inactive by raising the clogP values above the optimum level.
For example, 12e (88% inhibition@10 µM, cLogP = 4.89) vs 12a/c/d/i; 13e (94% inhibition@10 µM, cLogP
=
4.89) vs 13a/d/i; 14e (85% inhibition@10 µM, cLogP = 4.89) vs 14a/b/i; 15e (97% inhibition@10 µM,
cLogP = 4.12) vs 15a/b/d/i; 40e (90% inhibition@10 µM, cLogP = 4.67) vs 40a/c/i; 45e (91%
inhibition@10 µM, cLogP = 4.97) vs 45b/i and 46e (80% inhibition@10 µM, cLogP = 5.31) vs 46a/d/i.
X–ray structure and molecular docking study
During the course of this work, the aminothiazole based SIRT2 inhibitor 8 (Figure 1) was reported by Rumpf
77
et al. which shared some structural similarity with our original hit molecule 19a. Notably, both molecules
possess a naphthyl ring attached to one end of a heterocyclic central scaffold, with a nitrogen rich
heterocycle on the opposite end (see Figure 1). Corresponding to the SAR disclosed in this study, an
analogue of compound 8 possessing a benzyl ring (SirReal1) in place of the naphthyl ring was shown to be
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less active against SIRT2. Also, both series of molecules exhibited high SIRT2 selectivity over other
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isoforms that cannot be readily explained by ‘hotspot’ analysis of residues in the binding pocket. Based, on
these SAR similarities, we hypothesized that our thienopyrimidinone based inhibitors might also bind in the
SIRT2 active site by inducing the same 'selectivity pocket' as 8. Docking our highly active
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thienopyrimidinone analogues into the 8 bound SIRT2 structure (PDB 4RMH) (with the ligand removed)
indeed showed poses with overlap between the naphthyl groups of 8 and some of our active analogues.
However, the N–7' substituent was not found to occupy the 'selectivity pocket’ in many cases (data not
shown). Additionally, other SAR trends could not be readily explained by docking our analogues into this
structure (PDB 4RMH).
In parallel, we pursued characterization of SIRT2 inhibitor binding for our series by Xꢀray crystallography.
Pleasingly, we managed to obtain the SIRT2 structure co–crystallized with compound 29c. This new SIRT2
crystal form, obtained in the absence of substrate or coꢀsubstrate, belongs to a monoclinic space group and
contains two molecules of SIRT2 in the asymmetric unit (AU). Both copies share a very similar
conformation (rmsd 0.31 Å over 215 Cα atoms), which is also closely related to the structures of SIRT2 in
complex with 8 (rmsd ranging from 0.53 to 0.70 Å over 216 Cα atoms). Only one of the two SIRT2
molecules of the AU is bound to 29c (Figure 3a). The second SIRT2 molecule, although in a very similar
conformation, showed noisy electron density maps presumably arising from low occupancy and/or multiple
conformations of the inhibitor (racemate) and thus did not allow for confident modelling. For the wellꢀ
defined SIRT2 bound structure of 29c, the inhibitor was found to bind at the interface of the NAD–binding
domain and the zinc–binding domain, by inducing the 'selectivity pocket' in the extended C–site (Figure 3b
and Figure SF4, Supporting Information). This binding mode is consistent with the mechanism of inhibition
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observed for the hit compound that initated this study (19a) and closely related to the inhibition mode of 8.
However, the pose of 29c inside the pocket is inverted from what may be expected based on its structural
similarity to 8 (Figure 1) and our initial docking studies. Unlike 8, analogue 29c induces the 'selectivity
pocket' through its 2–methoxy–naphthalen–1–yl ring and positions itself deeper into the cavity created
between helices α6 (129ꢀ138) and α9 (206ꢀ215) of the zinc binding domain (Figure 3b and 3c), forming
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favourable hydrophobic and pi–pi contacts with the 'selectivity pocket' residues (Figure SF5, Supporting
Information). In order to accommodate the bulkier naphthyl ring of 29c, repositioning of α9 by 1.3 Å and
reorientation of the side chains of 'selectivity pocket' residues Tyr139, Pro140, Phe190, Leu206, Lys210 and
Phe214 are observed in comparison to the structure of SIRT2 with 8 bound (Figure SF6, Supporting
Information).
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The plane of the thienopyrimidinone ring of 29c is perpendicular to the 4–methoxy–naphthalen–1–yl ring,
+
and occupies a hydrophobic region adjacent to the NAD binding pocket, making contacts with Ile93, Pro94,
Phe96 and Ile169 (Figure SF5 and SF7, Supporting Information). The N–7’ dimethylisoxazole (DMI)
substituent of 29c extends towards the entry of the substrate binding pocket and interacts with Phe96 and
Phe119 through pi–pi as well as hydrophobic interactions (Figure SF5 and SF7 supporting information).
Like compound 8, overall binding of 29c in the SIRT2 active site is driven by pi–pi stacking, hydrophobic
interactions and van der Waals (vdW) contacts. Stacking on top of the thienopyrimidinone ring of 29c, the so
called “gate keeper” Phe96 side chain, which assists nicotinamide release and prevents backward reaction, is
+
blocked in a conformation compatible with NAD binding, consistent with the non competitive mode of
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inhibition for the co substrate observed for 19a. The DMI partially occupies the position of the acetyl group
of the substrate, also consistent with the competitive mode of inhibition of 19a with the substrate peptide
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(
figure SF8, Supporting Information).
The SIRT2 bound conformation of 29c and its interaction within the 'selectivity pocket' also explains the
observed SAR for this series. For instance, most of the analogues having a bicyclic ring (Table 1, columns
a–e) or a biphenyl (column i) at N–3 were found to have stronger SIRT2 inhibition compared to the
analogues with a benzyl or substituted benzyl group (column f–h) in this region. This might be due to the
stronger hydrophobic/vdW contacts exhibited by the bulky aromatic rings in the 'selectivity pocket' in case of
a–e/i compared to the less lipophilic benzyl group. This is further supported by the fact that analogues such
as 14g/h and 45f–h, are less active even though they lie in the optimum cLogP range (vide supra). Indeed,
docked poses of analogues with a benzyl N–3 substituent exhibit less contacts in the 'selectivity pocket'
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Figure SF5). However, docking scores of active and inactive analogues did not show a significant difference
data not shown) and could not be used to justify the different inhibitory activity of analogues with differing
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(
N–3 substituents. This is a well–recognized limitation of scoring functions used in molecular docking,
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especially due to the complexity derived from desolvation effects.
analogues (Table 1, column i) suggests that the biphenyl ring can also be accommodated inside the
selectivity pocket' in a manner similar to the naphthyl ring of 29c maintaining the pi–pi/hydrophobic
Interestingly, docking of biphenyl–3yl
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contacts, even though both groups differ in their overall shape (Fig. SF9, Supporting Information).
The groove occupied by the DMI moiety of 29c is wide and mostly lined by hydrophobic residues such as
Phe96, Phe119, Phe234, and Phe235. This explains why a wide variety of N–7’ substituents are wellꢀ
tolerated at the N–7' end of the thienopyrimidinone core (Table 1). SAR analysis for this series (ringꢀopened
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vs ring closed cyclohexyl fused core ring) and our previous SIRT2 inhibitor enantiomer data , suggests that
configuration of the chiral centre at position 7 does not have a substantial effect on SIRT2 inhibitory activity;
however electron density for only the R–isomer of 29c (R–29c) was observed in the SIRT2 binding pocket,
despite using the racemate for crystallization. Docking suggests that the S–isomer of 29c (S–29c) retains a
similar binding mode inside the SIRT2 pocket (Fig. SF10, Supporting Information) albeit with a slightly
lower docking score (–13.13 kcal/mol for R–29c vs –12.17 kcal/mol for S–29c). Similar docking results were
obtained for other potent analogues (Table ST1, Supporting Information) suggesting that the SIRT2 pocket
can accommodate either isomer with slight preference for the R configuration.
Overall, these studies suggest that the reported analogues induce 'selectivity pocket' in the active site of
SIRT2, in a similar fashion to the previously reported aminothiazole derivatives (such as 8), albeit in an
inverted fashion, and the overall binding is driven by the pi–pi and hydrophobic forces.
Since our thienopyrimidinone analogues share the same SIRT2 pocket as 8 and interact similarly and
principally via hydrophobic forces, we considered whether the activity of the aminothiazole series (cf 8)
might also have some dependence on the lipophilicity of this series. It is interesting that all highly active
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analogues (SIRT2 IC < 5 µM) of this previously reported series lie in a range of cLogP 4–6 and possess
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–4 aromatic rings, whereas inactive analogues (exhibiting SIRT2 inhibition only at 50 µM) are distributed
between a wider range of cLogP 3–7 (Fig. SF11, Supporting Information). It should be noted however, that
any observed lack of activity may also be due to the missing essential pharmacophore features, such as the
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DMP ring, as reported earlier for this series.
We observed high a correlation (r = 0.865, Fig. SF12,
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Supporting Information) between IC and cLogP values for the highly active analogues ( IC < 5 µM) in
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this series, which possess the key pharmacophore requirements. Together with the cLogP based analysis of
our series, these observations suggest an optimum lipophilicity is required by ligands to bind in this SIRT2
pocket and probably to induce the 'selectivity pocket'. This information can be used in future for the further
optimization of both SIRT2 inhibitor series.
Selectivity Study
81
In our previous study , we found 19a to be more than 100 times more selective for SIRT2 in comparison to
SIRT1, SIRT3 and SIRT5. To further evaluate the subtype selectivity of this series we evaluated the most
potent SIRT2 inhibitory compounds against SIRT1 and SIRT3 (Table 3). All assays used the same
fluorogenic peptide substrate derived from p53 (see Experimental Section) and full (10–)dose response
SIRT2 IC values (n=2) were calculated. Suramin was used as a positive control for SIRT1 and SIRT2 and
50
nictotinamide was used for SIRT3. All controls gave similar inhibitory activity to that observed previously.
Vehicle (DMSO) was used as the negative control. To our delight, 15e and 18a maintained excellent SIRT2
selectivity (>100 fold) over SIRT1 and SIRT3. In contrast, compound 29c maintained good selectivity for
SIRT2 over SIRT1 (>200 fold), however exhibits comparably poorer selectivity against SIRT3 (~30 fold).
Analogue 41c was found to be the least selective, with only 19 and 15 fold selectivity for SIRT2 vs SIRT1
and SIRT3, respectively. Therefore it would seem that this series generally maintains excellent selectivity for
SIRT2, except when a 4–methoxy–naphthalen–1–yl group is present in the N–3 position. Notably, N–3–
substituents are mainly responsible for inducing the 'selectivity pocket' that presumably imparts the isoform–
selectivity. Interestingly, we could not find any relevant differences between the sequences of SIRT1ꢀ3 in the
‘
selectivity pocket’ and the other enzyme parts contacting the ligand, which would rationalize the precise
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inhibitor selectivities observed. However, comparison of our SIRT2ꢀ29c complex to structures of SIRT1 and
SIRT3 reveal that the selectivity pocket of the latter two is different than that of SIRT2 due to notably
different conformations of residues 130ꢀ143 (SIRT2 numbering), which form helix α6 and the consecutive
loop (Figure SF13, Supporting Information). The isoform specificity thus appears to be caused indirectly, by
more distant isoenzyme features that influence the conformation of this sirtuin region.
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Tubulin hyperacetylation
To demonstrate the cellular activity of this series we studied the effect of active analogues to result in
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hyperacetylation of α–tubulin, a well–known SIRT2 substrate. The MCF–7 breast epithelial cancer cell line
was treated with different concentrations of 29c. Our previously published SIRT2 inhibitor from this series
8
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9a and 6, a different SIRT inhibitor chemotype, were used as positive controls. Additionally, analogue
25g lacking in vitro activity against SIRT2 (Table 1) was used as a negative control. To rule out the
possibility of increased acetylation of α–tubulin due to histone deacetylase activity by metalloꢀHDACs
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(
particularly HDAC6), Trichostatin, a pan HDAC inhibitor with no effect on SIRT2 activity, was employed
in the experiments. Treatment of cells with 29c resulted in increased levels of α–tubulin acetylation in a dose
dependent manner (Figure 4a) In contrast, no effect on tubulin acetylation levels was observed with 25g
(
Figure 4b) confirming that the enzymatic SIRT2 inhibition data obtained for our series correlates to α–
tubulin hyperacetylation. For prior inhibitors 19a and 6, tubulin hyperacetylation was only observed at a
treatment concentration of 20 µM or higher (Figure 4c–d), under the given conditions. While this might be
due to the slightly improved SIRT2 inhibitory potency of 29c (IC = 0.58 µM) over 19a (IC = 1.45 µM),
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clearly other physiochemical and stability differences between these two inhibitors will result in a difference
in the observed cell potency.
Conclusion
In conclusion, we have conducted an extensive SAR study of our previously reported thienopyrimidinone
SIRT2 inhibitor series and identified a number of inhibitors with submicromolar SIRT2 activity, while
retaining good to excellent selectivity over other the other SIRT isoforms tested. Our SAR analysis has
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revealed that a reasonably large variety of substituents can be tolerated at the N–7'–position, while a larger
lipophilic bicyclic/biphenyl ring is preferred at N–3 position. An X–ray crystal structure of SIRT2 in
complex with analogue 29c, revealed that our series of compounds also result in the induction of a
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previously described
'selectivity pocket' in the SIRT2 active site. This ligand induced 'selectivity pocket'
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likely explains the high selectivity of the tested analogues for SIRT2 over other subtypes of SIRT enzyme.
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Notably, our study adds to the growing examples of ligand induced SIRT2 conformational change
and
hence cautions against the reliability of molecular docking methods for binding site and orientation
prediction of SIRT2 inhibitors, using static structures. Consistent with the hydrophobic nature of 'selectivity
pocket' and the surrounding groove, we have found that active analogues in both thienopyrimidinone and
aminothiazole series (such as 8) possess cLogP within an optimum range. This finding should be useful for
further inhibitor optimization, since a simple to calculate descriptor like cLogP can be employed in
compound design and decision making.
EXPERIMENTAL SECTION
Chemistry. All chemicals and reagents were acquired from commercial sources and used as such unless
specified otherwise. All reactions were performed under an atmosphere of dry nitrogen unless otherwise
stated. Flash column chromatography was carried out using Merck Kiesegel 60 silica gel (230–400 mesh,
0.040 0.063 mm). Thin layer chromatography (TLC) was performed on aluminium plates using Merck
Kiesegel 60 F254 (230–400 mesh) fluorescent treated silica which were visualised under ultraviolet light
(
(
254 nm), or by staining with potassium permanganate or ninhydrin solution as appropriate. Chemical shifts
δ) are quoted in units of parts per million (ppm) referenced with the appropriate internal standard (TMS: δ =
0 ppm). Coupling constants (J) are given in Hertz (Hz). Coupling constants (J) are given in Hertz (Hz). The
1
H NMR spectra of representative compounds are reported as follows: ppm (multiplicity, coupling constants
J / Hz, number of protons). Synthetic schemes for the synthesis of analogues in Table 1 and Table 2 are
given in the supporting information. Purities of all the assayed compounds were determined by LCMS
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methods (described in Supporting Information) and were found to have ≥95% purity, except those specified
in Table ST2.
7ꢀ((3ꢀFluorobenzyl)amino)ꢀ3ꢀ(naphthalenꢀ1ꢀylmethyl)ꢀ5,6,7,8ꢀtetrahydrobenzo[4,5]thieno[2,3ꢀd]pyrimidinꢀ
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(3H)ꢀone (10b). H NMR (400 MHz, DMSOꢀd ) δ 8.43 (s, 1H), 8.19 (d, J = 8.2 Hz, 1H), 7.99 (d, J = 7.9
6
Hz, 1H), 7.89 (d, J = 8.3 Hz, 1H), 7.61 (dt, J = 17.8, 7.0 Hz, 2H), 7.45 (t, J = 7.7 Hz, 1H), 7.34 (q, J = 7.5
Hz, 1H), 7.25 – 7.16 (m, 2H), 7.10 – 6.98 (m, 2H), 5.68 (s, 2H), 3.82 (s, 2H), 3.13 – 3.00 (m, 2H), 2.94 (s,
1H), 2.85 – 2.72 (m, 1H), 2.58 (dd, J = 16.5, 7.8 Hz, 1H), 2.03 – 1.95 (m, 1H), 1.67 – 1.58 (m, 1H).
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1
ꢀ((3ꢀFluorobenzyl)amino)ꢀ3ꢀ(naphthalenꢀ2ꢀylmethyl)ꢀ5,6,7,8ꢀtetrahydrobenzo[4,5]thieno[2,3ꢀd]pyrimidinꢀ
1
(3H)ꢀone (10d). H NMR (400 MHz, DMSOꢀd6) δ 8.60 (s, 1H), 7.91 – 7.88 (m, 2H), 7.81 (d, J = 1.7 Hz,
H), 7.58 – 7.43 (m, 3H), 7.33 (td, J = 7.9, 6.1 Hz, 1H), 7.22 – 7.18 (m, 2H), 7.06 – 7.00 (m, 1H), 5.33 (s,
H), 3.81 (s, 2H), 3.10 – 2.98 (m, 2H), 2.95 – 2.88 (m, 1H), 2.79 – 2.70 (m, 1H), 2.59 – 2.53 (m, 1H), 2.00 –
.94 (m, 1H), 1.65 – 1.56 (m, 1H).
7ꢀ((2ꢀFluorobenzyl)amino)ꢀ3ꢀ(naphthalenꢀ1ꢀylmethyl)ꢀ5,6,7,8ꢀtetrahydrobenzo[4,5]thieno[2,3ꢀd]pyrimidinꢀ
1
4
(3H)ꢀone (11b). H NMR (400 MHz, DMSOꢀd6) δ 8.43 (s, 1H), 8.19 (d, J = 8.3 Hz, 1H), 7.99 (d, J = 7.8
Hz, 1H), 7.89 (d, J = 8.2 Hz, 1H), 7.66 – 7.57 (m, 2H), 7.52 – 7.43 (m, 2H), 7.31 – 7.25 (m, 1H), 7.18 – 7.12
m, 2H), 7.07 (d, J = 7.1 Hz, 1H), 5.68 (s, 2H), 3.84 (s, 2H), 3.07 (dd, J = 16.6, 5.4 Hz, 2H), 2.99 – 2.94 (m,
H), 2.83 – 2.75 (m, 1H), 2.59 (dd, J = 16.5, 7.7 Hz, 1H), 2.02 – 1.99 (m, 1H), 1.64 (dtd, J = 14.3, 9.1, 5.4
(
1
Hz, 1H).
7ꢀ((4ꢀChlorobenzyl)amino)ꢀ3ꢀ(naphthalenꢀ2ꢀylmethyl)ꢀ5,6,7,8ꢀtetrahydrobenzo[4,5]thieno[2,3ꢀd]pyrimidinꢀ
1
4
7
–
4
(3H)ꢀone (13b). H NMR (400 MHz, DMSOꢀd6) δ 8.60 (s, 1H), 7.91 – 7.88 (m, 3H), 7.81 (s, 1H), 7.53 –
.49 (m, 3H), 7.39 – 7.33 (m, 4H), 5.33 (s, 2H), 3.77 (s, 2H), 3.07 – 2.99 (m, 2H), 2.91 – 2.89 (m, 1H), 2.79
2.71 (m, 1H), 2.58 – 2.52 (m, 1H), 2.00 – 1.95 (m, 1H), 1.59 (dtd, J = 14.0, 8.9, 8.5, 5.2 Hz, 1H).
ꢀ(((3ꢀ(Naphthalenꢀ2ꢀylmethyl)ꢀ4ꢀoxoꢀ3,4,5,6,7,8ꢀhexahydrobenzo[4,5]thieno[2,3ꢀd]pyrimidinꢀ7ꢀ
1
yl)amino)methyl)benzonitrile (15d). H NMR (400 MHz, DMSOꢀd6) δ 8.60 (s, 1H), 7.96 – 7.85 (m, 3H),
7.81 (d, J = 1.6 Hz, 1H), 7.80 – 7.71 (m, 2H), 7.56 (d, J = 8.0 Hz, 2H), 7.55 – 7.43 (m, 3H), 5.33 (s, 2H),
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.87 (s, 2H), 3.10 – 2.96 (m, 2H), 2.93 – 2.87 (m, 1H), 2.79 – 2.71 (m, 1H), 2.581 – 2.51 (m, 1H), 2.00 –
.94 (m, 1H), 1.65 – 1.55 (m, 1H).
ꢀ(((4ꢀOxoꢀ3ꢀ(quinolinꢀ8ꢀylmethyl)ꢀ3,4,5,6,7,8ꢀhexahydrobenzo[4,5]thieno[2,3ꢀd]pyrimidinꢀ7ꢀ
1
yl)amino)methyl)benzonitrile (15e). H NMR (400 MHz, DMSOꢀd6) δ 9.00 (dd, J = 4.2, 1.8 Hz, 1H), 8.65
0
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2
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7
8
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(s, 1H), 8.43 (dd, J = 8.4, 1.8 Hz, 1H), 7.95 (d, J = 8.1 Hz, 1H), 7.76 (d, J = 7.9 Hz, 2H), 7.62 (dd, J = 8.3,
4
.2 Hz, 1H), 7.58 – 7.54 (m, 3H), 7.45 (d, J = 7.0 Hz, 1H), 5.76 (s, 2H), 3.87 (s, 2H), 3.07 – 3.00 (m, 2H),
.93 – 2.87 (m, 1H), 2.73 (dt, J = 16.8, 7.4 Hz, 1H), 2.58 – 2.54 (m, 1H), 2.00 – 1.95 (m, 1H), 1.65 – 1.55
2
(
m, 1H).
7
4
ꢀ((Pyridinꢀ3ꢀylmethyl)amino)ꢀ3ꢀ(quinolinꢀ8ꢀylmethyl)ꢀ5,6,7,8ꢀtetrahydrobenzo[4,5]thieno[2,3ꢀd]pyrimidinꢀ
1
(3H)ꢀone (19e). H NMR (400 MHz, DMSOꢀd6) δ 9.00 (dd, J = 4.4, 1.7 Hz, 1H), 8.65 (s, 1H), 8.54 (d, J =
2.2 Hz, 1H), 8.50 – 8.32 (m, 2H), 8.21 (s, 1H), 7.95 (d, J = 8.1 Hz, 1H), 7.76 (d, J = 7.7 Hz, 1H), 7.62 (dd, J
8.3, 4.2 Hz, 1H), 7.56 (t, J = 7.6 Hz, 1H), 7.45 (d, J = 7.2 Hz, 1H), 7.32 (dd, J = 7.8, 4.7 Hz, 1H), 5.76 (s,
=
2
2
7
H), 3.81 (s, 2H), 3.05 (dd, J = 16.5, 4.8 Hz, 2H), 2.95 – 2.90 (m, 1H), 2.74 (dt, J = 16.6, 7.9 Hz, 1H), 2.59 –
.53 (m, 1H), 2.02 – 1.95 (m, 1H), 1.65 – 1.56 (m, 1H).
ꢀ((Pyrimidinꢀ5ꢀylmethyl)amino)ꢀ3ꢀ(quinolinꢀ8ꢀylmethyl)ꢀ5,6,7,8ꢀtetrahydrobenzo[4,5]thieno[2,3ꢀ
1
d]pyrimidinꢀ4(3H)ꢀone (22e). H NMR (400 MHz, DMSOꢀd6) δ 9.05 (s, 1H), 9.00 (dd, J = 4.2, 1.8 Hz, 1H),
8.76 (s, 2H), 8.65 (s, 1H), 8.42 (dd, J = 8.2, 1.7 Hz, 1H), 8.24 (s, 1H), 7.95 (d, J = 8.1 Hz, 1H), 7.62 (dd, J =
8
2
1
3
.2, 4.2 Hz, 1H), 7.56 (t, J = 7.7 Hz, 1H), 7.45 (d, J = 7.2 Hz, 1H), 5.76 (s, 2H), 3.82 (s, 2H), 3.08 – 3.01 (m,
H), 2.96 – 2.92 (m, 1H), 2.75 (dt, J = 16.7, 6.9 Hz, 1H), 2.59 – 2.54 (m, 1H), 2.00 – 1.96 (m, 1H), 1.65 –
.56 (m, 1H).
ꢀ(Naphthalenꢀ2ꢀylmethyl)ꢀ7ꢀ(((tetrahydrofuranꢀ3ꢀyl)methyl)amino)ꢀ5,6,7,8ꢀ
1
tetrahydrobenzo[4,5]thieno[2,3ꢀd]pyrimidinꢀ4(3H)ꢀone (26d). H NMR (400 MHz, DMSOꢀd ) δ 8.59 (s,
6
1H), 7.98 – 7.85 (m, 2H), 7.81 (s, 1H), 7.50 (d, J = 7.9 Hz, 3H), 5.33 (s, 2H), 3.69 (dt, J = 13.7, 7.7 Hz, 2H),
3
.58 (q, J = 7.5 Hz, 1H), 3.39 – 3.31 (m, 2H), 3.11 – 2.95 (m, 2H), 2.90 (s, 1H), 2.82 – 2.68 (m, 1H), 2.62 –
2
.57 (m, 2H), 2.36 – 2.17 (m, 1H), 2.03 – 1.84 (m, 2H), 1.62 – 1.40 (m, 2H).
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7ꢀ(((3,5ꢀDimethylisoxazolꢀ4ꢀyl)methyl)amino)ꢀ3ꢀ((2ꢀmethoxynaphthalenꢀ1ꢀyl)methyl)ꢀ5,6,7,8ꢀ
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1
tetrahydrobenzo[4,5]thieno[2,3ꢀd]pyrimidinꢀ4(3H)ꢀone (29a). H NMR (400 MHz, DMSOꢀd6) δ 8.21 – 8.18
(
m, 1H), 8.05 – 8.02 (m, 2H), 7.91 (d, J = 8.1 Hz, 1H), 7.55 – 7.52 (m, 2H), 7.39 (t, J = 7.5 Hz, 1H), 5.55 (s,
H), 3.98 (s, 3H), 3.52 (s, 2H), 3.12 – 2.99 (m, 2H), 2.92 – 2.87 (m, 1H), 2.84 – 2.76 (m, 1H), 2.55 – 2.52
(m, 1H), 2.32 (s, 3H), 2.17 (s, 3H), 2.01 – 1.98 (m, 1H), 1.62 – 1.51 (m, 1H).
2
0
1
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8
9
0
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8
9
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0
3
ꢀ(Naphthalenꢀ1ꢀylmethyl)ꢀ7ꢀ((thiophenꢀ2ꢀylmethyl)amino)ꢀ5,6,7,8ꢀtetrahydrobenzo[4,5]thieno[2,3ꢀ
1
d]pyrimidinꢀ4(3H)ꢀone (30b). H NMR (400 MHz, DMSOꢀd6) δ 8.43 (s, 1H), 8.19 (d, J = 8.3 Hz, 1H), 7.99
(
d, J = 8.0 Hz, 1H), 7.89 (d, J = 8.3 Hz, 1H), 7.66 – 7.57 (m, 2H), 7.45 (t, J = 7.7 Hz, 1H), 7.36 (d, J = 5.1
Hz, 1H), 7.07 (d, J = 7.1 Hz, 1H), 6.98 – 6.94 (m, 2H), 5.68 (s, 2H), 4.00 (s, 2H), 3.11 – 2.96 (m, 3H), 2.78
dt, J = 16.9, 7.3 Hz, 1H), 2.58 (dd, J = 16.3, 7.6 Hz, 1H), 2.06 – 1.93 (m, 1H), 1.67 – 1.58 (m, 1H).
(
3ꢀ(Naphthalenꢀ2ꢀylmethyl)ꢀ7ꢀ((thiophenꢀ2ꢀylmethyl)amino)ꢀ5,6,7,8ꢀtetrahydrobenzo[4,5]thieno[2,3ꢀ
1
d]pyrimidinꢀ4(3H)ꢀone (30d). H NMR (400 MHz, DMSOꢀd6) δ 8.59 (s, 1H), 7.91 – 7.88 (m, 3H), 7.81 (s,
1
–
3
H), 7.58 – 7.44 (m, 3H), 7.35 (dd, J = 5.0, 1.3 Hz, 1H), 6.97 – 6.93 (m, 2H), 5.33 (s, 2H), 3.98 (s, 2H), 3.08
2.96 (m, 3H), 2.79 – 2.71, 2.58 – 2.54 (m, 1H), 2.00 – 1.96 (m, 1H), 1.63 – 1.54 (m, 1H).
ꢀ(Quinolinꢀ8ꢀylmethyl)ꢀ7ꢀ((thiophenꢀ2ꢀylmethyl)amino)ꢀ5,6,7,8ꢀtetrahydrobenzo[4,5]thieno[2,3ꢀ
1
d]pyrimidinꢀ4(3H)ꢀone (30e). H NMR (400 MHz, DMSOꢀd6) δ 9.01 (d, J = 3.8 Hz, 1H), 8.65 (s, 1H), 8.43
(d, J = 8.3 Hz, 1H), 8.16 (s, 1H), 7.95 (d, J = 8.1 Hz, 1H), 7.62 (dd, J = 8.3, 4.2 Hz, 1H), 7.56 (t, J = 7.7 Hz,
1
2
3
H), 7.44 (d, J = 7.2 Hz, 1H), 7.36 (d, J = 5.0 Hz, 1H), 6.98 – 6.93 (m, 2H), 5.76 (s, 2H), 3.98 (s, 2H), 3.06 –
.97 (m, 3H), 2.75 – 2.68 (m, 1H), 2.58 – 2.56 (m, 1H), 1.99 – 1.95 (m, 1H), 1.66 – 1.54 (m, 1H).
ꢀ([1,1'ꢀBiphenyl]ꢀ3ꢀylmethyl)ꢀ7ꢀ((thiophenꢀ2ꢀylmethyl)amino)ꢀ5,6,7,8ꢀtetrahydrobenzo[4,5]thieno[2,3ꢀ
1
d]pyrimidinꢀ4(3H)ꢀone (30i). H NMR (400 MHz, DMSOꢀd ) δ 8.60 (s, 1H), 7.68 (s, 1H), 7.63 (d, J = 7.6
6
Hz, 2H), 7.58 (d, J = 7.9 Hz, 1H), 7.51 – 7.35 (m, 4H), 7.39 – 7.28 (m, 2H), 7.00 – 6.90 (m, 2H), 5.22 (s,
2H), 3.97 (s, 2H), 3.11 – 2.94 (s, 3H), 2.80 – 2.71 (m, 1H), 2.59 – 2.50 (m, 1H), 2.02 – 1.93 (m, 1H), 1.66 –
1.51 (m, 1H).
3
ꢀ((4ꢀMethoxynaphthalenꢀ1ꢀyl)methyl)ꢀ7ꢀ((2ꢀmethoxyphenethyl)amino)ꢀ5,6,7,8ꢀ
1
tetrahydrobenzo[4,5]thieno[2,3ꢀd]pyrimidinꢀ4(3H)ꢀone (33c). H NMR (400 MHz, DMSOꢀd6) δ 8.36 (s,
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–
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H), 8.22 (d, J = 9.1 Hz, 2H), 8.12 (d, J = 8.4 Hz, 1H), 7.63 (t, J = 7.4 Hz, 1H), 7.56 (t, J = 7.6 Hz, 1H), 7.20
7.15 (m, 3H), 6.94 (dd, J = 8.1, 3.2 Hz, 2H), 6.86 (t, J = 7.4 Hz, 1H), 5.57 (s, 2H), 3.95 (s, 3H), 3.77 (s,
H), 3.11 – 3.03 (m, 3H), 2.85 – 2.77 (m, 3H), 2.73 (t, J = 7.7 Hz, 2H), 2.61 – 2.55 (m, 1H), 2.03 – 1.98 (m,
H), 1.65 – 1.56 (m, 1H).
0
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7ꢀ((2ꢀCyclohexylethyl)amino)ꢀ3ꢀ(naphthalenꢀ2ꢀylmethyl)ꢀ5,6,7,8ꢀtetrahydrobenzo[4,5]thieno[2,3ꢀ
1
d]pyrimidinꢀ4(3H)ꢀone (34d). H NMR (400 MHz, DMSOꢀd6) δ 8.62 (s, 1H), 7.91 – 7.85 (m, 3H), 7.81 (s,
1H), 7.52 – 7.49 (m, 3H), 5.33 (s, 2H), 3.12 – 3.07 (m, 3H), 2.80 – 2.74 (m, 3H), 2.69 – 2.55 (m, 1H), 2.04
(
t, J = 9.4 Hz, 1H), 1.68 – 1.59 (m, 5H), 1.38 – 1.28 (m, 3H), 1.23 – 1.10 (m, 3H), 0.92 – 0.84 (m, 2H).
7ꢀ((2ꢀMethoxyphenyl)amino)ꢀ3ꢀ(naphthalenꢀ2ꢀylmethyl)ꢀ5,6,7,8ꢀtetrahydrobenzo[4,5]thieno[2,3ꢀ
1
d]pyrimidinꢀ4(3H)ꢀone (35d). H NMR (400 MHz, DMSOꢀd6) δ 8.62 (s, 1H), 7.92 – 7.86 (m, 3H), 7.82 (d, J
= 1.7 Hz, 1H), 7.52 – 7.50 (m, 3H), 6.82 – 6.76 (m, 2H), 6.68 (d, J = 7.0 Hz, 1H), 6.60 – 6.54 (m, 1H), 5.35
(
s, 2H), 4.67 (d, J = 8.7 Hz, 1H), 3.87 – 3.78 (m, 1H), 3.75 (s, 2H), 3.17 – 3.06 (m, 2H), 2.96 – 2.88 (m, 1H),
2.76 (dd, J = 16.8, 8.4 Hz, 1H), 2.10 – 2.04 (m, 1H), 1.80 – 1.68 (m, 1H).
7ꢀ((3ꢀMethoxyphenyl)amino)ꢀ3ꢀ(naphthalenꢀ2ꢀylmethyl)ꢀ5,6,7,8ꢀtetrahydrobenzo[4,5]thieno[2,3ꢀ
1
d]pyrimidinꢀ4(3H)ꢀone (37d). H NMR (400 MHz, DMSOꢀd ) δ 8.62 (s, 1H), 7.90 (dd, J = 9.1, 4.8 Hz, 4H),
6
7.83 (s, 1H), 7.56 – 7.46 (m, 3H), 6.96 (t, J = 8.1 Hz, 1H), 6.27 – 6.21 (m, 1H), 6.19 (t, J = 2.2 Hz, 1H), 6.11
(dt, J = 7.9, 1.5 Hz, 1H), 5.63 (d, J = 8.2 Hz, 1H), 5.34 (s, 2H), 3.86 – 3.72 (m, 1H), 3.66 (s, 3H), 3.65 (s,
3
1
3
H), 3.17 – 3.07 (m, 4H), 2.93 (dd, J = 17.6, 8.8 Hz, 1H), 2.65 (dd, J = 16.8, 7.5 Hz, 1H), 2.10 – 2.00 (m,
H), 1.76 – 1.62 (m, 1H).
ꢀ(Quinolinꢀ8ꢀylmethyl)ꢀ7ꢀ(mꢀtolylamino)ꢀ5,6,7,8ꢀtetrahydrobenzo[4,5]thieno[2,3ꢀd]pyrimidinꢀ4(3H)ꢀone
1
(38e). H NMR (400 MHz, DMSOꢀd ) δ 9.01 (d, J = 3.8 Hz, 1H), 8.67 (s, 1H), 8.43 (d, J = 8.3 Hz, 1H), 7.96
6
(
d, J = 8.3 Hz, 1H), 7.67 – 7.53 (m, 2H), 7.46 (d, J = 6.9 Hz, 1H), 6.94 (t, J = 7.6 Hz, 1H), 6.45 – 6.42 (m,
2H), 6.34 (d, J = 7.4 Hz, 1H), 5.77 (s, 2H), 5.50 (d, J = 8.2 Hz, 1H), 3.76 (s, 1H), 3.16 – 3.07 (m, 2H), 2.95 –
2.86 (s, 2H), 2.17 (s, 3H), 2.08 – 2.00 (s, 1H), 1.71 – 1.63 (s, 1H).
3ꢀ(Naphthalenꢀ2ꢀylmethyl)ꢀ7ꢀ(pꢀtolylamino)ꢀ5,6,7,8ꢀtetrahydrobenzo[4,5]thieno[2,3ꢀd]pyrimidinꢀ4(3H)ꢀone
1
(39d). H NMR (400 MHz, DMSOꢀd ) δ 8.62 (s, 1H), 7.90 (dd, J = 9.0, 4.7 Hz, 3H), 7.83 (d, J = 1.7 Hz,
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H), 7.58 – 7.42 (m, 3H), 6.88 (d, J = 8.0 Hz, 2H), 6.55 (d, J = 8.4 Hz, 2H), 5.35 (m, 2H), 3.77 – 3.70 (s,
H), 3.17 – 3.02 (m, 2H), 2.97 – 2.82 (m, 1H), 2.63 (dd, J = 16.8, 8.0 Hz, 1H), 2.14 (s, 3H), 2.06 – 2.02 (m,
H), 1.68 (dt, J = 9.6, 7.1 Hz, 1H).
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ꢀ([1,1'ꢀBiphenyl]ꢀ3ꢀylmethyl)ꢀ7ꢀ((4ꢀfluorophenyl)amino)ꢀ5,6,7,8ꢀtetrahydrobenzo[4,5]thieno[2,3ꢀ
0
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1
d]pyrimidinꢀ4(3H)ꢀone (40i). H NMR (400 MHz, DMSOꢀd ) δ 8.63 (s, 1H), 7.69 (s, 1H), 7.64 (d, J = 7.6
6
Hz, 2H), 7.59 (d, J = 7.9 Hz, 1H), 7.52 – 7.40 (m, 3H), 7.42 – 7.29 (m, 2H), 6.90 (t, J = 8.7 Hz, 2H), 6.62
(
dd, J = 8.7, 4.5 Hz, 2H), 5.54 (d, J = 8.4 Hz, 1H), 5.24 (s, 1H), 3.73 (s, 1H), 3.14 – 3.09 (m, 2H), 2.91 (d, J
11.6 Hz, 2H), 2.05 (d, J = 11.5 Hz, 1H), 1.71 – 1.63 (m, 1H).
=
7
ꢀ(Cyclopentylamino)ꢀ3ꢀ((2ꢀmethoxynaphthalenꢀ1ꢀyl)methyl)ꢀ5,6,7,8ꢀtetrahydrobenzo[4,5]thieno[2,3ꢀ
1
d]pyrimidinꢀ4(3H)ꢀone (41a). H NMR (400 MHz, DMSOꢀd ) δ 8.24 (s, 1H), 8.18 (d, J = 8.6 Hz, 1H), 8.03
6
(d, J = 10.6 Hz, 2H), 7.91 (d, J = 8.2 Hz, 1H), 7.59 – 7.48 (m, 2H), 7.39 (t, J = 7.5 Hz, 1H), 5.55 (s, 2H),
3.98 (s, 3H), 3.28 – 3.16 (m, 2H), 3.12 – 2.93 (m, 4H), 2.84 – 2.76 (m, 1H), 2.01 – 1.96 (s, 1H), 1.81 – 1.75
(
m, 2H), 1.69 – 1.40 (m, 4H), 1.33 – 1.26 (m, 2H).
3ꢀ((2ꢀMethoxynaphthalenꢀ1ꢀyl)methyl)ꢀ7ꢀ((tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)amino)ꢀ5,6,7,8ꢀ
1
tetrahydrobenzo[4,5]thieno[2,3ꢀd]pyrimidinꢀ4(3H)ꢀone (42a). H NMR (400 MHz, DMSOꢀd ) δ 8.18 (d, J =
6
8.6 Hz, 1H), 8.03 (d, J = 9.7 Hz, 2H), 7.91 (d, J = 8.0 Hz, 1H), 7.58 – 7.49 (m, 2H), 7.39 (t, J = 7.4 Hz, 1H),
5.56 (s, 2H), 3.98 (s, 3H), 3.81 (dt, J = 11.6, 3.3 Hz, 2H), 3.13 – 2.96 (m, 3H), 2.82 – 2.77 (m, 2H), 2.68 –
2
.55 (m 1H), 1.99 – 1.91 (m, 1H), 1.78 – 1.73 (m, 2H), 1.61 – 1.50 (m, 1H), 1.25 – 1.17 (m. 2H).
3
ꢀ((4ꢀMethoxynaphthalenꢀ1ꢀyl)methyl)ꢀ7ꢀ((tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)amino)ꢀ5,6,7,8ꢀ
1
tetrahydrobenzo[4,5]thieno[2,3ꢀd]pyrimidinꢀ4(3H)ꢀone (42c). H NMR (400 MHz, DMSOꢀd ) δ 8.36 (s,
6
1H), 8.26 – 8.16 (m, 1H), 8.12 (d, J = 8.4 Hz, 1H), 7.63 (t, J = 7.6 Hz, 1H), 7.56 (t, J = 7.6 Hz, 1H), 7.16 (d,
J = 8.0 Hz, 1H), 6.93 (d, J = 8.0 Hz, 1H), 5.57 (s, 2H), 3.95 (s, 3H), 3.83 (d, J = 11.5 Hz, 2H), 3.17 (s, 1H),
3.14 – 3.00 (m, 2H), 2.88 – 2.79 (m, 2H), 2.68 – 2.63 (m, 1H), 1.98 (d, J = 13.3 Hz, 1H), 1.78 (t, J = 10.8
Hz, 2H), 1.64 – 1.49 (m, 1H), 1.31 – 1.17 (m, 2H).
3
ꢀ((2ꢀMethoxynaphthalenꢀ1ꢀyl)methyl)ꢀ7ꢀ((2ꢀmorpholinoethyl)amino)ꢀ5,6,7,8ꢀ
1
tetrahydrobenzo[4,5]thieno[2,3ꢀd]pyrimidinꢀ4(3H)ꢀone (43a). H NMR (400 MHz, DMSOꢀd ) δ 8.18 (d, J =
6
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.6 Hz, 1H), 8.08 – 7.99 (m, 2H), 7.91 (d, J = 7.2 Hz, 1H), 7.59 – 7.49 (m, 2H), 7.43 – 7.34 (m, 1H), 5.55 (d,
J = 2.4 Hz, 2H), 3.98 (s, 3H), 3.54 (t, J = 4.6 Hz, 4H), 3.12 – 2.67 (m, 7H), 2.42 – 2.25 (m, 6H), 2.02 – 1.92
m, 1H), 1.64 – 1.50 (m, 1H).
(
3
ꢀ(Naphthalenꢀ2ꢀylmethyl)ꢀ7ꢀ((1,2,3,4ꢀtetrahydronaphthalenꢀ1ꢀyl)amino)ꢀ5,6,7,8ꢀ
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tetrahydrobenzo[4,5]thieno[2,3ꢀd]pyrimidinꢀ4(3H)ꢀone (46d). H NMR (400 MHz, DMSOꢀd ) δ 8.61 (s,
6
1
2
6
7
H), 7.92 – 7.88 (m, 3H), 7.82 (s, 1H), 7.51 (d, J = 8.1 Hz, 3H), 7.34 (s, 1H), 7.15 – 6.96 (m, 3H), 5.34 (s,
H), 3.92 – 3.78 (m, 1H), 3.21 – 2.91 (m, 3H), 2.89 – 2.54 (m, 4H), 2.15 – 2.02 (m, 1H), 2.02 – 1.50 (m,
H).
ꢀ(Benzo[d][1,3]dioxolꢀ5ꢀylamino)ꢀ3ꢀ((4ꢀmethoxynaphthalenꢀ1ꢀyl)methyl)ꢀ5,6,7,8ꢀ
1
tetrahydrobenzo[4,5]thieno[2,3ꢀd]pyrimidinꢀ4(3H)ꢀone (47c). H NMR (400 MHz, DMSOꢀd ) δ 8.37 (s,
6
1H), 8.23 (d, J = 8.3 Hz, 1H), 8.13 (d, J = 8.5 Hz, 1H), 7.63 (t, J = 7.7 Hz, 1H), 7.56 (t, J = 7.5 Hz, 1H), 7.18
(
(
(
d, J = 8.0 Hz, 1H), 6.94 (d, J = 8.0 Hz, 1H), 6.66 (d, J = 8.3 Hz, 1H), 6.37 (d, J = 2.2 Hz, 1H), 6.14 – 6.04
m, 1H), 5.83 (s, 2H), 5.59 (s, 2H), 5.33 (d, J = 8.4 Hz, 1H), 3.96 (s, 3H), 3.79 – 3.64 (m, 1H), 3.21 – 3.04
m, 2H), 3.04 – 2.86 (m, 1H), 2.70 – 2.57 (m, 1H), 2.15 – 2.00 (m, 1H), 1.77 – 1.63 (m, 1H).
6
ꢀ(2ꢀAminoethyl)ꢀ3ꢀ((2ꢀmethoxynaphthalenꢀ1ꢀyl) methyl)ꢀ5ꢀmethylthieno [2, 3ꢀd] pyrimidinꢀ4(3H)ꢀone
(56a). [see SI, Scheme SS2] To a stirred solution of 63 (5.0 g, 9.82 mmol) in EtOH (50 mL), hydrazine
hydrate (15 mL) was added and the reaction mixture was heated at 90°C for 16 h. The progress of the
reaction was monitored by TLC. After completion the reaction was evaporated to dryness. The crude product
1
was purified by column chromatography using 7% MeOH/dichloromethane to afford 56a (2.5 g, 67.5%). H
NMR (400 MHz, DMSOꢀd ) δ 8.19 (d, J = 8.6 Hz, 1H), 8.07 – 7.99 (m, 2H), 7.95 – 7.87 (m, 1H), 7.60 –
6
7
.50 (m, 2H), 7.39 (ddd, J = 8.0, 6.8, 1.1 Hz, 1H), 5.55 (s, 2H), 3.98 (s, 3H), 2.86 – 2.70 (m, 4H), 2.43 (s,
3
H), 2.12 (br, s, 2H).
2ꢀ(4ꢀOxopentyl) isoindolineꢀ1,3ꢀdione (59). [see SI, Scheme SS2] To a stirred solution of 58 (20 g, 166
mmol) in DMF (150 mL), K CO (46 g, 333 mmol) and 5ꢀchloropentanꢀ2ꢀone (26.9 g, 183 mmol) was
2
3
added. The reaction mixture was heated at 80°C for 16 h. The progress of the reaction was monitored by
TLC. After completion of the reaction, the reaction mixture was quenched with ice water and filtered. The
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residue was washed with water, filtered and concentrated under reduced pressure. The crude material was
used for next reaction without purification to afford title compound 59 (20g, crude).
Ethyl 2ꢀaminoꢀ5ꢀ(2ꢀ(1,3ꢀdioxoisoindolinꢀ2ꢀyl)ethyl)ꢀ4ꢀmethylthiopheneꢀ3ꢀcarboxylate (60). [see SI, Scheme
SS2] To a stirred solution of compound 59 (15g, 64.9 mmol) and ethyl 2ꢀcyanoacetate (8.0g, 71.4mmol) in
EtOH (200 mL), morpholine (19.5 mL) and sulfur (2.3 g, 72 mmol) were added and stirred at 50°C for 16 h.
The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture
was quenched with water and extracted with EtOAc. The combined organic layer was washed with brine,
dried over anhydrous Na SO and concentrated under reduced pressure. The crude product was purified by
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29
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39
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
4
column chromatography using 40% EtOAc/hexane to afford title compound 60 (10.2g, 44%). LCMS: 358.99
M+H).
(
2ꢀ(2ꢀ(5ꢀMethylꢀ4ꢀoxoꢀ3,4ꢀdihydrothieno[2,3ꢀd]pyrimidinꢀ6ꢀyl)ethyl)isoindolineꢀ1,3ꢀdione (61). [see SI,
Scheme SS2] To a stirred solution of compound 60 (10.2g, 28.4 mmol) in formamide (120 mL), ammonium
formate (3.58g, 56.9 mmol) was added and stirred at 120°C for 16 h. The progress of the reaction was
monitored by TLC. After completion of the reaction, the reaction mixture was quenched with ice water and
precipitated solid was filtered. The crude product was purified by column chromatography using 5%
1
MeOH/Dichloromethane to afford title compound 61 (6.0g, 62.5%). H NMR (400 MHz, DMSOꢀd ) δ 12.3
6
(brs, 1H), 7.99(d, J = 8.4 Hz, 1H), 7.85(d, J = 7.8 Hz, 4H), 3.80 (t, 2H), 3.14 – 3.11 (m, 2H), 2.34 (s, 3H).
1ꢀ(Bromomethyl)ꢀ2ꢀmethoxynaphthalene (62). [see SI, Scheme SS2] To a stirred solution of 2ꢀmethoxyꢀ1ꢀ
naphthaldehyde (15g, 80.6 mmol) in EtOH (200 mL), NaBH (6.2g, 161 mmol) was added and stirred at
4
room temperature for 3 h. The progress of the reaction was monitored by TLC. After completion of the
reaction, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in water
and extracted with dichloromethane. The combined organic layer was washed with brine, dried over
anhydrous Na SO and concentrated under reduced pressure. The crude product was dissolved in
2
4
dichloromethane (200 mL), PBr (32.7g, 120 mmol) was added drop wise and the reaction mixture was
3
allowed to stir at room temperature for 1 h. After completion of the reaction, the reaction mixture was
quenched with water and extracted with dichloromethane. The combined organic layer was washed with
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brine, dried over anhydrous Na SO and concentrated under reduced pressure to afford title compound 62
2
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(
10.2g, crude).
2ꢀ(2ꢀ(3ꢀ((2ꢀMethoxynaphthalenꢀ1ꢀyl)methyl)ꢀ5ꢀmethylꢀ4ꢀoxoꢀ3,4ꢀdihydrothieno[2,3ꢀd]pyrimidinꢀ6ꢀ
yl)ethyl)isoindolineꢀ1,3ꢀdione (63). [see SI, Scheme SS2] To a stirred solution of 61 (6 g, 17.6 mmol) in
DMF (60 mL), Cs CO (11.6 g, 35.3 mmol) and 62 (6.6 g, 26.5 mmol) was added. The reaction mixture was
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allowed to stir at room temperature for 16 h. The progress of the reaction was monitored by TLC. After
completion of the reaction, the reaction mixture was quenched with ice water and filtered. Residue was
washed with water and filtered, concentrated under reduced pressure. The crude material was used for next
reaction without purification to afford title compound 63 (5.0g, crude). LCMS: 510.07(M+H).
SIRT enzymatic assays. Enzymatic assays were performed by Reaction Biology Corporation
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1
(
http://www.reactionbiology.com) as described before. In vitro SIRT assays were conducted by using the
fluorogenic peptide substrate from p53 residues 379–382 RHKK(Ac)ꢀAMC for SIRT1–3. The assay buffer
ꢀ1
contained 50 mM TrisꢀHCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl , 1 mg mL bovine serum
2
albumin and 1% DMSO. The protocol involved a twoꢀstep procedure. The fluorogenic substrate with the
acetylated lysine side chain was incubated with the SIRT enzyme to produce the deacetylated products,
which were then digested in the second step by the addition of a developer to produce the fluorescent signal
proportional to the amount of deacetylated substrates. Deacetylation of substrate peptides was used as a read
out of the SIRT activity. All compounds were freshly prepared as 10 mM stock solutions in DMSO and
serially diluted to the indicated concentration in the reaction. All test compounds were preincubated with the
human SIRTs for about 10 minutes before commencing the reaction through the addition of substrate.
Fluorescence was read (λ : 360 nm; λ : 460 nm) using the EnVision Multilabel Plate Reader
ex
em
(PerkinElmer). The percentages of enzyme activity (relative to DMSO controls) and IC50 values were
calculated using the GraphPad Prism 4 program based on a sigmoidal dose–response equation. The number
of replicates is stated in the corresponding text.
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Tubulin acetylation assay. Cell lines and cell culture. Human breast cancer cell line, MCFꢀ7 (Michigan
Cancer Foundationꢀ7) originated from the American Type Culture Collection (ATCC) were obtained from
the Cell Culture Service of Cancer Research UK, where they were tested and authenticated. Cells were
maintained in Dulbecco’s modified eagle’s medium (DMEM, SigmaꢀAldrich, Poole, UK) and supplemented
with 10% fetal calf serum (FCS, First Link Ltd, Birmingham, UK), 100 Unit/ml penicillin/streptomycin
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59
60
(
SigmaꢀAldrich, UK) and 2 mM glutamine under 10 % CO humidified atmosphere at a constant temperature
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of 37°C.
Time course and treatment. MCFꢀ7 cells were cultured in Dulbecco’s modified eagle’s medium and treated
with varied concentrations (0, 1, 10, 20, 30 and 50 µM) of compounds and 50 nM trichostatin A (TSA)
(
T8552, SigmaꢀAldrich, UK) for 24 hours. TSA was used to abolish the activity of nonꢀsirtuin HDAC
members.
Protein preparation. After treatment, MCFꢀ7 cells were harvested by centrifugation at 2000 rpm for 5
minutes, the supernatant was discarded and pellets were stored at ꢀ80°C freezer until lysis was performed.
Cell lysis were performed in 2 volumes of NP40 lysis buffer [1%(v/v) Nonidet Pꢀ40, 150 NaCl , 50 mM
2
TrisꢀHCl (7.6), 5 mM EDTA, 1 mM dithiothreitol (DTT), 1 mM NaF, 2 mM phenylmethylsulfonyl fluoride
(
PMSF), 1 mM sodium orthovanadate and 1× complete protease inhibitor cocktail, as instructed by the
manufacturer (Roche Diagnostics, Burgess Hill, West Sussex, UK)] for 10 min at 4°C. Vortexing and
pipetting were employed to facilitate the lysis. The samples were then centrifuged 13000 rpm at 4°C for 10
min. The supernatant was collected as protein lysate and transferred to a clean Eppendorf prior to
measurement.
To determine protein concentration, the Pierce BCA Protein Assay Reagents A and B (Thermo Scientific)
was employed according to manufacturer’s instruction. Absorbance was read at 562 nm using the Sunrise
spectrophotometer (Tecan, Reading, UK) and absorbance readouts were utilized to assay protein
concentration according to the equation of absorbance ×25=ꢀg/ꢀl.
Western blotting. SDSꢀPAGE gels consisted of an upper stacking and a lower running gel. The running gel
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was made with varying amounts of bisꢀacrylamide solution, according to the molecular size of the protein
being isolated, with percentages of gels used varying from 7 to 14. Lower percentage gels allowed better
examination of higher molecular weight proteins and vice versa. The bisꢀacrylamide with ammonium
persulfate (APS) and tetramethylethylenediamine (TEMED) were used as the catalysts for gel
polymerization.
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Protein separation was obtained by diluting 20 ꢀg of protein lysate with 2 × SDS loading buffer (200 mM
TrisꢀHCl pH6.8, 6% sodium dodecyl sulfate (SDS), 2 mM EDTA, 10% 2ꢀmercaptoethanol, 10% glycerol,
0.02% bromophenol blue) and proteins denatured from 5 min at 100°C.
Samples were then loaded into the SDSꢀPAGE gels and fractionated for 2 hours at 90V using a MiniꢀProtean
III apparatus (BioꢀRad Laboratories, Hemel Hempstead, UK). Once SDSꢀPAGE had finished, proteins were
electroꢀtransferred onto a 0.45 ꢀm Protran nitrocellulose membranes (Schleicher and Schuell, Whatman,
Brentford, UK) in transfer buffer [25 mM tris, 190 mM glycine and 20% (v/v) ethanol] for 90 min at 90 V at
room temperature by using the BioꢀRad TransꢀBlot Cell wet transfer system.
Membranes were blocked in 5% (w/v) biovine serum albumin (BSA) in Tris buffered solution with 0.05%
Tweenꢀ20 (TBST, pH 7.5) for 30 min at room temperature. Primary antibody incubation was performed with
antibodies diluted in a 5% BSAꢀTBST solution overnight at 4°C. The membranes were washed four times
with TBST, every 5 min with 50 ml TBST at room temperature. Membranes were then incubated in their
respective secondary antibodies (SantaꢀCruz Biotech antiꢀrabbit or antiꢀmouse) coupled with horseradish
peroxidase (Dako, Ely, UK) at a 1:2000 dilution for 30 min at room temperature. Subsequent to 5 × 5 min
washes in TBST to remove excess secondary antibodies. Proteins were visualized using enhanced
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2
chemiluminescence (ECL) detection system (GE Healthcare, UK).
Primary antibodies and respective dilutions used were shown as follows: Antiꢀacetyl alpha Tubulin antibody
(
Lys40) (D20G3), 5335s, Rabbit monoclonal, Cell signalling Technology, 1:1000; Antiꢀalpha Tubulin
antibody, DM1A, ab7291, Mouse monoclonal, Abcam, 1:2000; Antiꢀβꢀ Actin antibody, scꢀ1616R, Rabbit
polyclonal, Santa Cruz Biotechnology, 1: 2000.
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Molecular Modeling Methods. All calculations were performed on a Windows 7.0 based Workstation using
the Maestro 10.6 graphical user interface (GUI) of the Schrodinger® software suite (Schrödinger, LLC, New
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York, NY, 2016).
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60
Protein preparation: The Xꢀray structure of SIRT2ꢀ29c was prepared using the ‘Protein Preparation
Wizard’. All water molecules and ions were deleted; atom types and bond orders were corrected, and the
hydrogen atoms were reassigned after deleting the original ones. Missing side chains were added using the
94ꢀ95
Prime 4.1 program.
The protonation states of acidic/basic amino acids were adjusted for pH 7.0.
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Restrained minimization of the protein was performed employing the OPLSꢀ2005 forcefields with the
convergence criteria of RMSD of 0.3 Å for heavy atoms.
Ligand preparation. All molecules were drawn in ChemDraw Pro 13.0 and prepared using the LigPrep
97ꢀ98
wizard of Schrodinger Suite which utilizes Epik 3.6
to generate energetically accessible protonation
states and all possible stereoisomers. No tautomeric forms were generated for the ligands.
Docking. A receptor grid was generated using the centroid of the coꢀcrystallized ligand 29c with default
settings for the size of the enclosing box. All other default settings were used. The ligands were docked into
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the prepared protein using the Glide 7.1 program
implemented in Schrodinger Suite using the standard
100
precision (SP) mode. No constraints were applied to the docking. Ligands were sampled throughout the
docking, including ring conformations. Amide bonds were penalized in their nonplanar conformation. Epik
state penalties were added to the final score. A maximum of 10 poses per ligand were allowed and postꢀ
docking minimization was also allowed.
Cheminformatic Analysis. The cLogP for all molecules was calculated in their neutral state using
102
Datawarrior. All thienopyrimidinone analogues except 9a, 17a and 19a (percentage inhibition at 10 µM
not available) were used for the analysis (Total = 248). The ‘actives’ were defined as analogues with more
than 80% inhibition at 10 µM while ‘inactives’ were defined as those with less than then 50% inhibition at
7
9
10 µM. The structure and activity data for the aminothiazole series was taken from Schiedel et al. For this
series, the ‘actives’ were defined as analogues with IC values less than 5 µM (total = 17) while ‘inactives’
5
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were defined as those which showed inhibition at only 50 µM (total = 20). In case of analogues with chiral
centres activity of the racemate was used.
X-ray Crystallography
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Protein production and crystallization. Sirt2 56ꢀ356 was cloned in a modified pETꢀ19 vector which
expresses the protein of interest with a Nꢀterminal His6ꢀSUMO tag. Expression was carried out in E. coli
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codon+ cells in autoinduction media at 20°C. Purification was performed as described in Di Fruscia et al.,
and included an affinity chromatography step followed by digestion of the expression tag in presence of
Sumo protease (1/100 w/w) and a gel filtration on a S200 16/60 (GE Healthcare) in 20 mM Tris pH 8.0, 150
mM NaCl and 1 mM TCEP. Crystals of the complex between SIRT2 and 29c were obtained by coꢀ
ꢀ1
crystallization at 4°C using vapor diffusion method. Prior to crystallization, the protein at 15 mg.mL was
incubated on ice for at least an hour with 1 mM 29c (final DMSO concentration 5%). Crystals of a maximal
dimension of 50 µm *20 µm *20 µm grew within 2 weeks from drops containing equal volume of the
protein/ligand mix and 2.6M ammonium sulfate, 5% PEG 300, 0.1 M MES buffer pH 5.83.
Diffraction data collection and structure solution. After a short soaking in a cryo protecting solution
composed of the mother liquor supplemented with 25% glycerol (v/v) and 1 mM 29c, crystals were mounted
on a nylon loop and flash frozen in liquid nitrogen. Diffraction data were collected on BL14.1 operated by
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the HelmholtzꢀZentrum Berlin (HZB) at the BESSY II electron storage ring (BerlinꢀAdlershof, Germany)
at 100K using a Pilatus 6M detector (Dectris) and a wavelength of 0.9184 Å. Statistics of the diffraction data
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integrated using XDSAPP can be found in Table 4. The structure was solved by molecular replacement
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77
using Molrep and one molecule of Sirt2 (PDB ID: 4RMG) as a model. Structure refinement was carried
1
06
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out by alternating rounds of positional refinement by phenix.refine and manual rebuilding in Coot.
ASSOCIATED CONTENT
Supporting Information
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Journal of Medicinal Chemistry
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Supporting Information contains Figures SF1ꢀ13, Tables ST1 and ST2, synthetic methods, spectral data and
LCMS methods/data.
ABBREVIATIONS USED
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SIRT, sirtunins; H3K9ac, histone 3 lysine 9 acylation; PKM2, pyruvate kinase 2; DMI, dimethylisoxazole;
DMP, dimethylpyrimidine
AUTHOR INFORMATION
Corresponding author
*
E–mail: m.fuchter@imperial.ac.uk; Fax: +44 (0)2075945805; Tel: +44 (0)2075945815
a
These authors contributed equally
ACKNOWLEDGEMENTS
We thank the Imperial Confidence in Concept scheme and the NIHR Imperial BRC for funding this work.
SS acknowledges the financial support from Cancer Research UK (C33325/A19435). SM and CS thank the
BESSY beamline staff for technical support and CS support by Alzheimer Forschung Initiative eV (grant
#14834). EWFL’s work is supported by MRC (MR/N012097/1), CRUK (A12011) and Breast Cancer Now
(
2012MayPR070; 2012NovPhD016). ZM is funded by a scholarship from the Commonwealth Scholarship
Commission (BDCSꢀ2015ꢀ63).
ANCILLARY INFORMATION
The final model and the structure factors of SIRT2 bound to 29c are deposited to the Protein Databank under
accession code 5MAT. Authors will release the atomic coordinates and experimental data upon article
publication.
REFERENCES AND NOTES
1.
Sauve, A. A. Sirtuin chemical mechanisms. Biochim. Biophys. Acta 2010, 1804, 1591ꢀ603.
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