Molecular Imaging of Sirtuin1 Expression-Activity in Rat Brain Using Positron-Emission Tomography-Magnetic-Resonance Imaging with [18F]-2-Fluorobenzoylaminohexanoicanilide

Article abstract of DOI:10.1021/acs.jmedchem.8b00253

Sirtuin 1 (SIRT1) is a class III histone deacetylase that plays significant roles in the regulation of lifespan, metabolism, memory, and circadian rhythms and in the mechanisms of many diseases. However, methods of monitoring the pharmacodynamics of SIRT1

Full text of DOI:10.1021/acs.jmedchem.8b00253

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Article
Molecular imaging of Sirtuin1 expression-activity in the rat brain
using Positron Emission Tomography/Magnetic Resonance
Imaging (PET/MRI) with [18F]-2-fluorobenzoylaminohexanoicanilide.
Robin Bonomi, Vadim Popov, Maxwell Thomas Laws, David Gelovani, Anjoy Majhi, Aleksandr Shavrin,
Xin Lu, Otto Muzik, Nashaat Turkman, Renshyan Liu, Thomas Mangner, and Juri George Gelovani
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00253 • Publication Date (Web): 27 Jul 2018
Downloaded from http://pubs.acs.org on July 28, 2018
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Molecular imaging of Sirtuin1 expression-activity in the rat brain using positron emission
tomography/magnetic resonance imaging (PET/MRI) with
[18F]-2-
fluorobenzoylaminohexanoicanilide.
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Robin Bonomia,b, Vadim Popova,b, Maxwell T. Lawsa,b, David Gelovania,b, Anjoy Majhia,b, Aleksandr
Shavrina,b, Xin Lub,c, Otto Muzikb,c, Nashaat Turkmana,b, Renshyan Liud, Thomas Mangnerb,c, Juri G.
Gelovania,b*
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a) Karmanos Cancer Institute, Detroit MI 48202 USA
b) Departments of Biomedical Engineering, Oncology, Neurosurgery, Wayne State University,
Detroit MI 48202 USA
c) Cyclotron/Radiochemistry Facility, Wayne State University, Detroit MI 48202 USA
d) National Taiwan University, Taipei City, Taiwan 10617
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ABSTRACT
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Sirtuin 1 (SIRT1) is a class III histone deacetylase, which plays significant roles in regulation of
lifespan, metabolism, memory, circadian rhythms, and in mechanisms of many diseases. However,
methods of monitoring the pharmacodynamics of SIRT1ꢀtargeted drugs are limited to blood sampling
due to invasive nature of biopsies. For nonꢀinvasive monitoring of spatial and temporal dynamics of
SIRT1 expressionꢀactivity in vivo by PET/CT(MRI), we developed a novel substrateꢀtype radiotracer
[18F]ꢀ2ꢀfluorobenzoylaminohexanoicanilide (2ꢀ[18F]BzAHA). PET/CT/MRI studies in rats demonstrated
increased accumulation of 2ꢀ[18F]BzAHAꢀderived radioactivity in the hypothalamus, hippocampus,
nucleus accucmbens, and locus coueruleus, consistent with autoradiographic and immunofluorescent
(IMF) analyses of brain tissue sections. Pretreatment with SIRT1 specific inhibitor, EXꢀ527 (5 mg/kg,
i.p.), resulted in about 20% reduction of 2ꢀ[18F]BzAHAꢀderived radioactivity accumulation in these
structures. In vivo imaging of SIRT1 expressionꢀactivity should facilitate studies to improve the
understanding of SIRT1ꢀmediated regulation in the brain and aid the development and clinical
translation of SIRT1ꢀtargeted therapies.
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INTRODUCTION
Class III HDAC enzymes are termed “silent information regulators” or “sirtuins” (SIRTs),
originating from the yeast Sir2 homologue, are Zn2+ꢀindependent, but NAD+ꢀdependent1. Seven isotypes
of SIRTs (SIRT 1ꢀ7) have been identified in humans and other mammals2. These enzymes play
important roles in epigenetic regulation of gene expression, cell cycle, metabolism, apoptosis, life span3,
and circadian rhythms4. In particular, SIRT1 has been recognized as a key regulator of several molecular
pathways that are important in the development and progression of cancer5, diabetes6, cardiovascular7,
and neurological diseases8. SIRT1 shuttles between the cell nucleus and cytoplasm and deacetylates both
histone proteins, H3K9, H3K14, and H4K16, as well as multiple nonꢀhistone proteins, including
PPARγ, NFꢀKβ, members of p53 family of proteins, and others9.
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Due to biological importance of SIRT1, several small molecule activators and inhibitors of SIRT1
have been developed and tested for therapy of various diseases. For example, resveratrol, a natural
activator of SIRT1, and SRT2104, a synthetic activator, have undergone clinical trials for treatment of
diabetes and obesity10. Because of the widely recognized role of SIRT1 in cancer, several SIRT1
inhibitors, including Selisistat (EXꢀ527)11, Sirtinol12, and Tenovinꢀ613, have been tested for therapy of
various malignancies14. Also, SIRT1ꢀtargeted drugs have been tested for treatment of various
neurodegenerative diseases15. However, methods for monitoring the pharmacodynamics of SIRT1ꢀ
specific activators and inhibitors are limited due to unacceptable traumatism from invasive biopsies,
especially in CNS. Therefore, in most clinical trials, the pharmacodynamics of various SIRT1ꢀtargeted
drugs has been monitored by measuring SIRT1 activity in blood mononuclear cells or in muscle or liver
tissue biopsies16. Although, measurements of SIRT1 mRNA or protein expression levels in fixed tissues,
reflect the levels of SIRT1 expression, these methods don’t provide the information about SIRT1
enzyme activity. Furthermore, measurements of SIRT1 enzyme activity in tissue lysates are performed
under biochemically optimized conditions and do not accurately reflect the SIRT1 enzyme activity in
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organs and tissues in vivo, because of the differences in intracellular NAD+ levels and absence of other
factors that influence enzymatic activity of SIRT1. Therefore, we aimed to develop a nonꢀinvasive and
quantitative in vivo imaging method using a SIRT1ꢀspecific substrateꢀtype radiotracer, which should
provide valuable information about SIRT1 expression and activity levels in different organs and tissues
under physiologic or pathologic conditions. Moreover, such in vivo imaging methodology should
provide the means for nonꢀinvasive monitoring of pharmacodynamics of novel SIRT1 activators and
inhibitors and facilitate their translation into the clinic.
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Our approach to development of a SIRT1 specific substrateꢀtype radiotracer for PET imaging was
based on a wellꢀestablished catalytic mechanism of SIRT1 for various endogenous and artificial
substrates17. Previously, we successfully used this approach for development of HDAC class IIa specific
substrateꢀtype radiotracers [18F]FAHA18 and [18F]TFAHA19. The current approach was based on
previous reports demonstrating that SIRT1 is able to cleave large aromatic chemical groups, such as the
phenylacetyl20, from the εꢀamine of lysine. Although other SIRT enzymes can cleave long acyl chains
from lysine, they are not capable of cleaving aromatic ringꢀcontaining moieties20. Built on these reports,
we synthesized a focused library of 10 compounds in which the εꢀamine of lysine was derivatized with
aromatic ringꢀcontaining moieties (with or without halogen substitutions), determined their substrate
efficiencies for SIRTs 1ꢀ7, and established structureꢀactivity relationships (SAR). As the result, we
selected 2ꢀfluorobenzoylꢀsubstituted lysine analogue for further radiolabeling with Fꢀ18 and in vivo
imaging studies. The lead compound, [18F]ꢀ2ꢀfluorobenzoylaminohexanoicanilide (2ꢀ[18F]BzAHA),
demonstrated efficacy for PET/CT/MR imaging of SIRT1 expressionꢀactivity in the rat brain. The sites
of increased 2ꢀ[18F]BzAHAꢀderived radioactivity accumulation in the rat brain (i.e., hippocampus, n.
accumbens, hypothalamus, locus coeruleus) exhibited higher levels of SIRT1 expressionꢀactivity, as
demonstrated by comparative autoradiographic and immunofluorescent microscopic (IMF) analyses of
rat brain tissue sections. Treatment of rats with EXꢀ527, a SIRT1ꢀselective inhibitor, resulted in an
inhibition of SIRT1 activity, as manifested by a 20ꢀ22% reduction of 2ꢀ[18F]BzAHA accumulation in the
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n.accumbens, hippocampal CA3, and amygdala, as compared to baseline. Taken together, current
studies demonstrated the feasibility of PET/CT/MR imaging with 2ꢀ[18F]BzAHA for nonꢀinvasive and
quantitative in vivo visualization of spatial distribution of SIRT1 expression and activity in various
structures of the brain, as well as for
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monitoring the pharmacodynamics of
SIRT1 inhibitors.
RESULTS
Chemistry. A focused library of
compounds was developed for in vitro
assessment of SIRT1 catalytic activity
using
the
carbazolꢀlysineꢀ
aminomethylcoumarin backbone (Cbzꢀ
LysꢀAMC) derivatized on lysine ɛꢀ
amine with various benzoyl and
phenylacetyl containing substituents
(Fig. 1, Table S1). Compounds (1-10)
were synthesized using variations of
existing methodologies for synthesis of
peptidomimetics in yields between 50ꢀ
80% with chemical purity of >95%, as
Fig. 1. A) Chemical synthesis for compounds 1-10 using the Cbzꢀ
LysꢀAMC backbone. B) Synthesis of 2ꢀFBzAHA (12), precursor 2ꢀ
NO2BzAHA (13), and radiosynthesis of 2ꢀ[18F]BzAHA (14) from
the aminohexanoicanilide backbone. C) Quality control
chromatogram demonstrating matching elution times for both the
standard compound 12 (blue) and the radiofluorinated compound
14, 2ꢀ[18F]BzAHA (red).
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assessed by TLC, H, 13C, and 19F
NMR, and the identity of compounds verified by HRMS (Supplemental Materials). The 2ꢀ
nitrophenylaminohexanoicanilide (13) was synthesized by coupling 2ꢀnitrobenzoyl acetyl chloride with
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Fig. 2. A-C) Catalytic efficacy (Kcat, secꢀ1 ) for compounds 1-10 with SIRTs 1ꢀ7 determined by enzymatic assay
with fluorescent readout. A) compounds 1-4 and 10 demonstrating differences in Kcat due to aromatic
substitution of fluorine atom at varying positions on the benzoyl moiety, or the addition of a bulkier, ethyl group;
B) Variations in Kcat due to use of either a benzoyl or phenylacetyl moiety with paraꢀpositioning of iodine or
fluorine, demonstrating a significant increase with paraꢀflurobenzoyl (4) as compared to the others; C)
Comparison of orthoꢀ vs. paraꢀ fluorine substitution (6 vs. 7) on the aromatic ring of a phenylacetyl moiety. D)
Catalytic efficacy (Kcat / Km, secꢀ1Mꢀ1) for selected compounds with recombinant SIRT1 as determined by
fluorometric assay demonstrating 2 is cleaved most efficiently by SIRT1. E) Compound 2 is cleaved selectively
by SIRT1 with at least 3ꢀfold greater efficacy as compared to SIRTs 2ꢀ7 and HDACs 1ꢀ11. F) SIRT1 cleavage of
2 with increasing levels of EXꢀ527, a selective SIRT1 inhibitor, to determine an IC50 of 12.60+/ꢀ 2.1ꢁM. Error
bars represent standard deviation for A-D with all experiments performed in triplicate. P<0.01 represented by
**** and P<0.05 represented by *; both denote statistical significance.
phenylhexamide in a 70% yield with >95% chemical purity (14) and used for subsequent
radiofluorination to produce 2ꢀ[18F]BzAHA with high radiochemical purity >99% (Fig. 1).
Enzyme substrate affinity studies. Enzymatic substrate affinity studies in a panel of recombinant
SIRT 1ꢀ7 enzymes demonstrated that CbzꢀLys(R)ꢀAMC derivatized by benzoyl or phenylacetyl moietyꢀ
containing leaving groups (R) were effectively cleaved by SIRT1 with Kcat (0.41±0.014) *10ꢀ3sꢀ1 and
(0.37±0.02)*10ꢀ3sꢀ1, respectively, whereas other SIRT enzymes cleaved these moieties significantly less
effectively (Fig. 2; Tab. S1, S2). As compared to nonꢀsubstituted phenylꢀbased leaving group, fluorine
substitutions in the 2ꢀ and 4ꢀpositions of benzoyl moiety demonstrated about 2ꢀfold increased Kcat for
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SIRT1, whereas fluorine substitution in the 3ꢀposition of benzoyl did not significantly affect the Kcat for
SIRT1, while the 4ꢀethyl substitution increased the Kcat for SIRT1, but only minimally. Unexpectedly,
the 4ꢀiodo substitution of benzoyl moiety resulted in a significant (40ꢀfold) decrease in the Kcat for
SIRT1. As compared to nonꢀsubstituted phenylacetyl moiety containing leaving group, both 4ꢀfluoro
and 4ꢀiodo substitutions resulted in about a 3ꢀfold decrease in Kcat for SIRT1, whereas the 2ꢀfluoro
substation of phenylacetyl moiety resulted in a 7ꢀfold decrease of Kcat for SIRT1. 2ꢀ, 3ꢀ, and 4ꢀfluoro
substituted benzoyl moieties, were cleaved most efficiently by SIRT1, with Kcat/Km of 26.02±7.07,
63.39±4.32, and 16.08±1.32 Mꢀ1s-1, respectively (Fig. 2, Tab. S2). The 2ꢀfluorobenzoyl leaving group
was cleaved 5ꢀfold more effectively by SIRT1 than any other HDAC enzyme (Fig. 2B). The Cbzꢀ
Lys(2FBz)ꢀAMC was effective as a fluorogenic substrate for SIRT1 enzyme activity assay in vitro (Fig.
2C) and for assessment of EXꢀ527 inhibitory activity, yielding 12.60 ꢁM IC50 of EXꢀ527 (Fig. 2D).
Therefore, 2ꢀfluorobenzoyl leaving group was selected for the development of 2ꢀ[18F]fluorobenzoylꢀ
aminohexanoic anilide (2ꢀ[18F]BzAHA) for in vivo PET imaging studies.
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NMR spectroscopy of SIRT1-2F-BzAHA reaction product. An in vitro assay of recombinant
SIRT1 with 2FꢀBzAHA monitored with 19FꢀNMR resulted in two peaks: one at ꢀ114 ppm corresponding
to the parent compound and one at ꢀ231±3 ppm corresponding to a reaction intermediate or product.
(Fig. S1-S2).
PET/CT/MR imaging with 2-[18F]BzAHA. Dynamic PET/CT/MR imaging of the rat brain with 2ꢀ
[18F]BzAHA demonstrated an efficient crossing of blood brain barrier (BBB) with rapid equilibration
between blood and brain compartments within the first 2 minutes post i.v. injection (Fig. S3).
Significant transient accumulation of 2ꢀ[18F]BzAHAꢀderived radioactivity was observed in
n.accumbens, hypothalamus, hippocampus, locus coeruleus, lateral septal nucleus, caudate putamen
and amygdala (Fig. 3, 4 and S4). In contrast, the mesencephalic structures, brainstem, and cerebellum,
showed only minimal accumulation of 2ꢀ[18F]BzAHAꢀderived radioactivity. The highest PET signal
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Fig. 3. A) PET/CT/MR coronal and axial images 20 minutes post i.v. administration of 2ꢀ[18F]BzAHA,
demonstrating increased 2ꢀ[18F]BzAHA derived accumulation within regions of the brain corresponding to
hippocampus (CA1ꢀCA3), lateral septal nucleus (LS), nucleus accumbens (Acb) as seen in B) corresponding
maps of the brain to the PET images. C) Autoradiography performed for matching sections of the brain at 20
minutes post i.v. administration of 2ꢀ[18F]BzAHA demonstrating increased radioactivity within regions of the
hippocampus, Acb, and lateral septal nucleus. D) Immunofluorescence of corresponding slices of brain
demonstrating increased levels of SIRT1 expression in the hippocampus (Alexafluor488ꢀconjugated antiꢀSIRT1,
green), especially CA2 and CA3, overlaid on DAPI nuclear stain (blue).
contrast between different brain structures was observed at 10ꢀ20 min post i.v. administration of 2ꢀ
[18F]BzAHA (Fig. 3, S3). Patlak graphical analysis of timeꢀactivity data in the brain using brainstem as
a reference tissue demonstrated two phases of 2ꢀ[18F]BzAHAꢀderived radioactivity accumulation with
different unidirectional accumulation rate constants (Ki): a) larger Ki during 0ꢀ15 min post injection; and
b) smaller Ki during 15ꢀ60 min post injection (Fig. S5) consistent with time course of metabolic
degradation of 2ꢀ[18F]BzAHA (Fig. S6). Logan plot analysis also demonstrates increased accumulation
in regions of the brain as compared to brainstem (reference tissue), as evidenced by slope >1 (Fig. S7).
Treatment of rats with SIRT1ꢀselective inhibitor EXꢀ527 (5 mg/kg i.p.) 30 min prior to administration of
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2ꢀ[18F]BzAHA resulted in
a
statistically
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significant reduction by 20ꢀ22% of 2ꢀ
[18F]BzAHAꢀderived radioactivity accumulation
in the brain regions exhibiting higher levels of
SIRT1, as compared to baseline (preꢀtreatment)
levels in the same animals (p<0.05, paired
Student tꢀtest) (Fig. 5).
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Fig. 4. A) PET/CT/MR image of the hypothalamus taken
Radiolabeled metabolite analyses. The at 20 minutes post i.v. administration of 2ꢀ[18F]BzAHA,
B) PET image overlaid on atlas map of the corresponding
analyses of timeꢀactivity profiles of 2ꢀ
brain region demonstrating coꢀlocalization between areas
of increased 2ꢀ[18F]BzAHAꢀderived radioactivity and
[18F]BzAHAꢀderived radiolabeled metabolites in
[18F]BzAHA ꢀderived accumulation within regions of the
blood plasma demonstrated a rapid degradation
hypothalamus region as shown on the map. C)
PET/CT/MR image demonstrating increased 2ꢀ
locus coeruleus. D) PET image overlaid on atlas map of
the corresponding brain region demonstrating coꢀ
of 2ꢀ[18F]BzAHA with monoꢀexponential halfꢀ
life of 3.8 minutes, resulting in a complete loss
of the parent compound from plasma by
15 minutes post i.v. injection. The exact
nature of radiolabeled metabolites has
not been determined. Free [18F]fluoride
was not detectable, consistent with the
absence of radioactivity accumulation in
the bones (Fig. S6).
localization between areas of increased 2ꢀ[18F]BzAHAꢀ
derived radioactivity and locus coeruleus region as shown
Comparative
autoradiographic
Fig. 5. A) PET/CT/MR images at 20 minutes post i.v. administration
of 2ꢀ[18F]BzAHA. B) PET/CT/MR images at 20 minutes post i.v.
administration of 2[18F]BzAHA and 50 minutes post preꢀtreatment
with SIRT1 selective inhibitor, EXꢀ527 (i.p.; 5mg/kg). C) SUV
values for ROI at 20 min. post i.v. administration of 2ꢀ[18F]BzAHA
with (gray) and without (black) preꢀtreatment of EXꢀ527. Error bars
and immunofluorescence microscopy
analyses. Comparative analysis of
PET/CT/MR images 2ꢀ[18F]BzAHA represent standard deviation (N=2). * indicates P<0.05
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with autoradiograms of corresponding brain sections, obtained at 20 minutes post i.v. administration of
2ꢀ[18F]BzAHA (Fig. S14), with the corresponding fluorescence microscopy images of SIRT1
expression in adjacent brain tissue sections, demonstrated coꢀlocalization of 2ꢀ[18F]BzAHAꢀderived
radioactivity with brain structures expressing high levels of SIRT1, such as hippocampus,
hypothalamus, lateral septal nucleus (LSN), and nucleus accumbens (NAC) (Fig. 3; Fig. S8-S14).
SIRT1 expression was highest in the hippocampal CA2 and CA3 neurons (Fig. 3; Figs. S8, S9), dentate
gyrus hillus neurons and in the progenitor cells of DG (Fig. S10), whereas lower levels of SIRT1
expression were observed in neurons of hippocampal CA1, granular layer neurons of dentate gyrus
(DG), cerebral cortex, and NAC (Fig. 3; Fig. S8-S12). Additionally, high levels of SIRT1 expression
were observed in the neurons hypothalamic region adjacent to the third ventricle (Fig. 4). The
subcellular localization of SIRT1 in CA2 and CA3 neurons was both nuclear and perinuclear, which
predominantly coꢀlocalized with (phosphoꢀS47)SIRT1 immunofluorescence (Figs. S8). Strong
cytoplasmic staining for SIRT1, but not (phosphoꢀS47)SIRT1, was observed in axons of several neurons
in the stratum pyramidale of hippocampal CA2, traveling deeper into the stratum radiatum, as well as
axons traveling into the stratum oriens (Fig. S9). In DG, SIRT1 expression was observed in perikarya
of neuronal progenitors in the subꢀgranular zone (SGZ), and in the hilus ꢀ in pyramidal neurons,
interneurons, and mossy fiber neurons (Fig. S10). In the cerebral cortex, SIRT1 was predominantly
expressed in large pyramidal neurons (Fig. S12), while the subcellular distribution of SIRT1 in NAC
was more uniform (Fig. S11). It is noteworthy, that strong SIRT1 and (phosphoꢀS47)SIRT1 expression
was observed in pericytes associated with large and mediumꢀsize blood vessels (Fig. S13).
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DISCUSSION AND CONCLUSIONS
Over the past decade, several molecular PET imaging agents have been developed for in vivo
quantitative visualization of enzyme expression and/or activity of different HDAC classes and isoforms,
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including HDAC class IIa specific: [18F]ꢀFAHA21, [18F]ꢀTFAHA19 and HDAC1 specific: 11Cꢀ
Martinostat22, 18FꢀMGS323. Moreover, 11CꢀMartinostat has been already translated into the clinic for
mapping the HDAC1 distribution in human brain in vivo24. PET/CT/MR imaging with these novel
imaging agents enabled monitoring of pharmacokinetics (PK) and BBB penetrance of their nonꢀ
radiolabeled drug analogues and for doseꢀoccupancy studies of HDAC class I enzymes25 and for doseꢀ
dependent inhibition of HDAC class IIa18ꢀ19 enzyme activity in the brain. However, despite the
biological importance of HDAC class III enzymes, especially SIRT1, PET imaging of SIRT1 expression
and activity has not been reported yet.
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Towards the development of a novel substrateꢀtype radiotracer for quantitative molecular imaging of
SIRT1 expression and activity, we studied a focused library of substrates using the CbzꢀLysꢀAMC
backbone and various benzoyl or phenylacetyl leaving groups. This backbone has been widely used for
analysis of SIRT activity26 and was especially useful for preliminary screening of novel potential
inhibitors or activators of SIRT1, due to presence of an AMC moiety enabling high throughput
screening using fluorescenceꢀbased quantitative methods27. Consistent with our current results, others
have also reported that a bulky lipophilic moiety (i.e. CBZ, Boc) in the cap group of the AMCꢀLys(Ac)
backbone aids in SIRT1 catalytic activity28. Furthermore, the catalytic activity and substrate selectivity
of SIRTs depends not only on backbone and cap group, but also on the leaving group9b. In addition to
cleaving an acetyl moiety from the ɛꢀamino terminus of lysine, different SIRT isoforms are capable of
cleaving larger groups, such as malonyl (SIRT5), succinyl (SIRT5), glutaryl (SIRT5), crotonyl (SIRT3),
lipoyl (SIRT4), myristoyl (SIRT2, 6), palmitoyl (SIRT4)20, 29. Most importantly, SIRT1 can cleave ɛꢀ
amino phenylacetylꢀlysine with 56% of the natural deacetylation efficiency20 (Fig. S15).
Current SAR studies demonstrated that the catalytic efficiency of SIRT1 is greatest with a benzoyl,
as opposed to a phenylacetyl leaving group. The highest degree of SIRT1 catalytic efficiency was
observed for 2ꢀfluorobenzoyl (2) and 3ꢀfluorobenzoyl (3) groups with Kcat/Km = 26.02 and 63.39 Mꢀ1sꢀ1,
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respectively (Fig. 2, Tab. S2), which was similar to Kcat/Km = 65.29 Mꢀ1, sꢀ1 of SIRT1ꢀmediated
deacetylation of p53ꢀderived oligo peptide ArgꢀHisꢀLysꢀLys(ac)ꢀAMC (BPS1). Interestingly, the iodine
substitution in the para position of benzoyl and phenylacetyl moieties was more deleterious to the
catalytic efficiency of SIRT1 than the fluorine substitution. This is probably due to a larger Vander
Waals radius of iodine atom causing steric hindrance. These SAR results for SIRT1 catalytic cleavage of
ɛꢀamino derivatized halogenꢀsubstituted benzoylꢀ and phenylacetylꢀ lysine analogues are being reported
herein for the first time.
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Although, the 3ꢀfluorobenzoyl (3) leaving group was cleaved ~60% more efficiently by SIRT1 than
2ꢀfluorobenzoyl (2) leaving group, compound 2 was chosen as the lead substrate for further
development as an imaging agent, because of the increased difficulty of radiolabeling 3. Interestingly,
compound 4 containing the 4ꢀfluorobenzoyl leaving group, was cleaved with the greatest effectiveness
(highest Kcat), but with a much larger Km, thereby decreasing catalytic efficiency, Kcat/Km than 2 (Fig. 2,
Tab. S1, S2). Novel methods for synthesis of 4ꢀfluorobenzoyl groups have been developed, using
iodonium salts30 and copper31 catalysts, which may be useful in the development of 3ꢀflurobenzoyl,
however, these large salt based or ethynyl leaving groups may be less effective for the meta position due
to increased potential for steric hindrance. In a separate study we attempted the radiosynthesis of the 3ꢀ
fluorobenzoyl group using a pinacolꢀderived boronic ester precursor, but the radiolabeling yield was
unacceptably low (results not reported here). To facilitate clinical translation and routine production
using an automated oneꢀstep radiosynthesis method, we decided to proceed with 2ꢀfluorobenzoyl
leaving group for further studies. Furthermore, 2 has demonstrated the highest selectivity as substrate for
SIRT1, when studied in a panel of recombinant SIRT 1ꢀ7 and HDAC 1ꢀ11 isoforms. Additionally, the
SIRT1ꢀmediated enzymatic cleavage of 2 was effectively inhibited by EXꢀ527 (Selisistat) with an IC50
value of 12.6 ± 2.1 ꢁM (Fig. 2), which was 1200ꢀfold higher than the IC50 of 0.0103 ± 0.0025 ꢁM for
fluorogenic acetylated oligoꢀpeptide substrate BPS1. Others have previously reported IC50 of EXꢀ527 for
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BPS1 within the same range (0.098 ꢁM)11a. These results suggest that 2 may also act as a competitive
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To create a pharmacologicallyꢀoptimized substrate radiotracer, we substituted a smaller
aminohexanoicanilide (AHA) for the larger CbzꢀLysꢀAMC backbone in the compound 2, resulting in 2ꢀ
fluorobenzoylꢀacetamidohexanoicanilide (2ꢀFBzAHA). Previous studies (unpublished) have
demonstrated CbzꢀLysꢀAMC backbone unable to effectively cross the BBB due to the large size and
polarity of this moiety, therefore we have substituted a smaller lysineꢀmimetic, AHA, previously used
for HDAC tracers18ꢀ19. Existing methods32 were adapted for radiofluorination on the 2 position of
benzoyl ring for synthesis of 2ꢀ[18F]BzAHA. Initially we developed both, the 2ꢀ
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nitrophenylaminohexanoicanilide
(2ꢀNO2BzAHA)
and
2ꢀtrimethylamonium
salt
phenylaminohexanoicanilide precursors, however, after testing radiofluorination conditions, only 2ꢀ
NO2BzAHA provided high yield 2ꢀ[18F]BzAHA. This is consistent with previous reports on
radiolabeling using a nitroꢀleaving group for radiofluorination of benzoyl rings in the ortho position33.
The enzymatic cleavage of 2FꢀBzAHA by SIRT1 was validated using in vitro SIRT1 enzyme assay
and 19FꢀNMR spectroscopy for readout. SIRT1ꢀmediated catalysis of 2ꢀFPhAHA was confirmed by
19
presence of two peaks in the FꢀNMR spectrum of reaction products: one peak at ꢀ114 ppm (Fig. S1,
S2) corresponded to the parent compound (2FꢀBzAHA) and a second broader peak with the maximum at
ꢀ231ppm (Fig. S2), corresponding to the reaction product (or an intermediate). While it is difficult to
fully characterize this peak, it was presumed an intermediate because it didn’t match the peaks of
potential cleavage products, such as 2Fꢀbenzoic acid (ꢀ113.00 ppm), 2Fꢀbenzaldehyde (ꢀ121.32 ppm), or
2Fꢀbenzamide (ꢀ113.58 ppm) (Fig. S1A-D).
The established mechanism for SIRT1ꢀmediated catalytic cleavage of acetylated lysine is by a mixed
SN1/SN2 reaction resulting in a simultaneous cleavage of nicotinamide from NAD+ and formation of an
oxocarbenium intermediate of acetylꢀlysine and 1’ and 2’ carbons of the ribose. Subsequent nucleophilic
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attack by a water molecule to the tetrahedral carbon of dioxo ring results in cleavage of the acetyl
moiety from the ɛꢀamino terminus of lysine and the formation of 2’ꢀOꢀAcꢀADPR product34. We
hypothesized, that the mechanism of substrate cleavage by SIRT enzymes should allow for 2ꢀ
[18F]benzoyl group to be transferred to adenine dinucleotide (ADN) (Fig. S15) and at least transiently
entrapped in the cell, similar to [18F]FDGꢀ6ꢀphosphate and [18F]FLTꢀ5ꢀphosphate products of [18F]FDG
and [18F]FLT, respectively. Also, it is conceivable, that transient nature of 2ꢀ[18F]BzAHAꢀderived
radioactivity retention in the cells is due to cleavage of removal of 2ꢀ[18F]benzoyl group from the ADN,
similar to previously reported cleavage an acetyl moiety from the ADN group by esterases, such as
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ARH334ꢀ35
.
Because 2ꢀ[18F]BzAHA (cLogP= 4.83) is extremely lipophilic, it freely passes through the BBB, as
evidenced by high levels of 2ꢀ[18F]BzAHA in the brain immediately following the i.v. injection (Fig.
S3). However, as previously noted, following cleavage of 2ꢀ[18F]BzAHA by SIRT1, the radiolabeled
leaving group is only transiently entrapped in the cells following ester linkage to ADPꢀribose. This
allows for differential washout of 2ꢀ[18F]BzAHAꢀderived radioactivity from regions of the brain with
low SIRT1 expressionꢀactivity versus transient retention of 2ꢀ[18F]BzAHAꢀderived radioactivity in areas
with high SIRT1 expressionꢀactivity (Fig. 3, 4, S3).
Analyses of radioactive metabolites in plasma post i.v. administration of 2ꢀ[18F]BzAHA
demonstrated the loss of intact parent compound after 15ꢀminutes of circulation in blood (Fig. S6),
which explains the results of Patlak graphical analysis36, demonstrating a significant change in the
unidirectional influx rate constant (Ki) from 15 minutes post i.v. administration of 2ꢀ[18F]BzAHA. This
is consistent with a loss of effective input function from the intact (unmetabolized) radiotracer beyond
this time point. Patlak plotꢀderived unidirectional influx rate constants (Ki) for regions of the brain with
high SIRT1 expressionꢀactivity were determined using brainstem as the reference tissue37 (Fig. S5),
because brainstem nuclei have similar magnitude of perfusion as thalamic nuclei, but relatively lower
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levels of apparent SIRT1 expressionꢀactivity (except for the locus coeruleus). Although, radioactive
metabolites of 2ꢀ[18F]BzAHA dominate in blood after 20 min (Fig. S6) they don’t contribute
significantly to the accumulation of 2ꢀ[18F]BzAHAꢀderived radioactivity in specific brain regions. This
was demonstrated by dynamic PET imaging studies using potential radiolabeled metabolites: 2ꢀ
[18F]benzaldehyde, and 2ꢀ[18F]ethyl benzoate (metabolized in vivo to 2ꢀ[18F]benzoic acid) (Fig. S16A).
These radiotracers demonstrated significantly different patterns of radioactivity distribution in the brain,
as compared to 2ꢀ[18F]BzAHA (Fig. S16B). Most notably, increased 2ꢀ[18F]benzaldehydeꢀderived
radioactivity accumulation was observed in the brainstem, which was contrary to the absence of 2ꢀ
[18F]BzAHAꢀderived radioactivity accumulation in this region (Fig. S16B).
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Dynamic PET/CT/MR images acquired in normal rats using 2ꢀ[18F]BzAHA demonstrated significant
retention of 2ꢀ[18F]BzAHAꢀderived radioactivity in areas of the brain with relatively higher SIRT1
expression (i.e. hypothalamus, hippocampus, septal nucleus, n. accumbens, locus coeruleus). These in
vivo imaging results have been validated by comparative in situ autoradiographic and
immunofluorescent microscopy (IFM) studies (Fig. 3, 4, S7-S13). Autoradiography provided high
resolution (~100x100 µm) images of 2ꢀ[18F]BzAHA distribution and localization of increased
radioactivity accumulation in the brain, whereas IFM provided confirmation of cellular, and subcellular
localization of total SIRT1 and phsphoꢀSIRT1 expression in the corresponding brain regions. The results
of these comparative in vivo ꢀ in situ validation studies confirmed that nonꢀinvasive PET imaging with ꢀ
2ꢀ[18F]BzAHA accurately reflects the sites of increased SIRT1 expressionꢀactivity in the rat brain, and
are in agreement with previously published reports on the distribution and roles of SIRT1 expression in
the rat brain using ISH and IF38 that are described in greater detail below.
High levels of 2ꢀ[18F]BzAHAꢀderived radioactivity and SIRT1 immunofluorescence observed in the
hippocampus (Fig. 3, S8-S10, S14) are consistent with previous studies using ISH38a, IHC38b, and IF39.
SIRT1 is involved in mechanisms of learning and memory (both shortꢀand longꢀterm); it modulates
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synaptic plasticity through transcriptional regulation of brainꢀderived neurotrophic factor via MeCP2
deacetylation40. Accordingly, animals deficient in SIRT1 demonstrate deficits in memory formation and
related cognitive functions39. Our IFM analyses demonstrated increased levels of SIRT1 expression
particularly in the CA2ꢀCA3 neurons, with lower levels of expression in CA1 neurons (Fig. S8-S10).
Within the neurons, SIRT1 exhibited mixed nuclear and perinuclear localization, although perinuclear
localization was more pronounced. Furthermore, increased levels of SIRT1 expression were observed in
many pyramidal axons traveling deeper into the hippocampal layers, as well as axons of neurons the
inner and outer molecular layers traveling towards the alveus. These Schaffer collateral pathways
demonstrate high levels of SIRT1 expression, consistent with the role of SIRT1 as a potent mediator of
long term potentiation39. Evidence for the role of SIRT1 in hippocampal progenitor cells, within the
dentate gyrus, has been previously reported in mice41 and is consistent with results of our IMF studies
(Fig. S10). Also, SIRT1ꢀmediated regulation plays an important role in the development of agingꢀ
associated neuroꢀcognitive decline as demonstrated by decreased degeneration of pyramidal neurons in
Ammon’s horn in aging rats by treatment with resveratrol, a naturally occurring activator of SIRT142.
The ageꢀrelated differences in SIRT1 activity can be linked to changes in p53 acetylation in the CA2ꢀ3
regions43. Decreases in SIRT1 activity within hippocampus has also been linked to chronic stress,
depressive behaviors44 and Alzheimer’s Disease progression, as decreased SIRT1 expression within the
entorhinal cortex and hippocampus correlates to a higher tau load and amyloidꢀbeta deposits45. Changes
in SIRT1 expression may be quite dynamic in many of these pathological conditions, such as animals
with dietaryꢀinduced obesity exhibit progressive decline in cognitive memory performance with
increasing body mass index; however, treatment with a SIRT1 targeted activator, resveratrol,
ameliorates much of the hippocampal neurodegeneration46. Alternatively, increased SIRT1 expression
levels have been noted in other neurodegenerative diseases, such as Huntington’s disease, which led to
exploratory clinical trials with SIRT1 selective inhibitors, such as Selisistat (EXꢀ527)47 15e. Due to the
pleiotropic role of SIRT1 in neurodegeneration, there is an ongoing debate in the literature regarding the
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effectiveness of SIRT1 activators vs. inhibitors for treatment of neurodegeneration48. Therefore, PET
imaging of SIRT1 activity with 2ꢀ[18F]BzAHA should facilitate the development and clinical translation
of novel potent SIRT1 activators or inhibitors for neuroprotection, prevention and therapy of various
neurodegenerative diseases and age related neuroꢀcognitive decline.
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Another important finding in the current study using PET/CT/MRI with 2ꢀ[18F]BzAHA was high
level of SIRT1 expressionꢀactivity in the n. accumbens (Fig. 3, S11), which is consistent with previous
reports of increased levels of SIRT1 expression demonstrated by ISH38a and IHC38b. Within the n.
accumbens (NAc), SIRT1 was shown to deacetylate FOXO3a and to increase expression of several
target genes that enhance cocaine addiction behaviors49. Furthermore, chronic cocaine usage has been
demonstrated to increase SIRT1 activity within the NAc and mRNA production through an increase in
acetylation of the gene promoter for SIRT1 transcription50. Therefore, PET imaging of SIRT1
expressionꢀactivity with 2ꢀ[18F]BzAHA may advance our understanding of mechanisms of addiction and
aid in development of new therapies of addictive disorders.
Increased levels of 2ꢀ[18F]BzAHAꢀderived radioactivity and SIRT1 immunofluorescence observed
in the hypothalamus (Fig. 4) are also consistent with previous reports using ISH38a, IHC38b, 51 and IF52.
SIRT1 plays the key role in many important functions such as central regulation of metabolism, body
temperature, feeding, and circadian rhythm. In the neurons of arcuate nucleus, SIRT1 is expressed in
proꢀopiomelanocortin (POMC) and agoutiꢀrelated peptide (AgRP) expressing neurons and regulates
energy expenditure40. SIRT1 localization to the POMC neurons in the arcuate nucleus surrounding the
third ventricle was observed using IMF, confirming the involvement of SIRT1 in central regulation of
metabolism52. Additionally, SIRT1 regulates SF1 neurons in the ventromedial hypothalamus to regulate
energy homeostasis53 SIRT1 is crucial to the hypothalamic response to glucose, insulin and leptin54, and
serves as potential target for the treatment of diabetes and obesity. Several studies have demonstrated
that SIRT1 overexpression can rescue obesityꢀinduced insulin resistance through increased levels of
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FoxO1 deacetylation in POMC neurons55. Clinical trials have been conducted to test the ability of
SIRT1ꢀactivators (i.e., resveratrol, SRT2104, nicotinamide riboside) as therapeutic agents for typeꢀ2
diabetes and obesity (i.e., NCT01677611, NCT01150955)56. Thus, imaging of SIRT1 expressionꢀ
activity using PET/CT/MRI with 2ꢀ[18F]BzAHA may advance the understanding of mechanisms of
hypothalamic regulation of metabolism and normal and disease conditions and facilitate the
development and clinical evaluation of novel SIRT1ꢀtargeted therapies of metabolic diseases.
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Furthermore, our observation of high levels of 2ꢀ[18F]BzAHAꢀderived radioactivity in the locus
coeruleus (LC) (Fig. 4) is consistent with previous reports of increased levels of SIRT1 expression as
determined by IHC and IF57. Due to the extremely small size of the LC in the rat brain (~1 mm), partial
volume effect is substantial due to relatively low inꢀplane resolution of microPET R4 (~2 mm), which is
the main reason to low intensity and asymmetry of 2ꢀ[18F]BzAHAꢀderived signals on PET/CT/MR
images of LC (Fig. 4). It has been demonstrated that SIRT1 plays a significant regulatory role within
neurons of the LC, particularly in the sleepꢀwake cycle57ꢀ58. Additionally, increased acetylation of
histone core proteins H3 and H4 within the dorsal raphe nucleus and LC were noted in less resilient rats
exposed to chronic stress, as compared to rats with increased resiliency. Hyperacetylation of H3 and H4
has been linked to decreased level of SIRT1 expression59. Due to the extensive involvement of the LC
within many brain circuitry functions including metabolism, stress, wakefulness, addiction and
neurodegeneration, the ability to nonꢀinvasively image and monitor SIRT1 expressionꢀactivity within
this key structure of the brain may aid in development of novel treatments of various neurological and
metabolic diseases.
The absence of 2ꢀ[18F]BzAHAꢀderived radioactivity accumulation in other brainstem structures, as
seen on PET imaging, autoradiography and IMF, is consistent with reports using ISH38a. Low level of
SIRT1 expression in the brainstem, provides opportunity for accurate quantification of 2ꢀ[18F]BzAHAꢀ
derived radioactivity accumulation in other regions of the brain using reference tissueꢀbased Patlak
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graphical analysis method37 between 0ꢀ20 min post radiotracer administration, while the parent 2ꢀ
[18F]BzAHA is present in the blood (Fig S5). Also, because of the transient (reversible) nature of
entrapment of 2ꢀ[18F]BzAHAꢀderived radioactivity, the apparent volumes of distribution of 2ꢀ
[18F]BzAHA, reflecting SIRT1 expressionꢀactivity product, can be calculated using Logan graphical
analysis60 with brainstem as a reference tissue (Fig S7).
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Current studies also underscore the important role of SIRT1 in brain vasculature. Throughout the
brain there was a strong expression of both SIRT1 and phosphoꢀSIRT1 in pericytes (Fig. S13), which
supports the role of SIRT1 in eNOS–mediated regulation of cerebral blood flow61. Preꢀtreatment of rats
with natural SIRT1ꢀactivator, resveratrol, prior to an ischemic event confers neuroprotection to the
hippocampal pyramidal neurons62. Additionally, in disease conditions SIRT1 is a direct modifier of HIFꢀ
1α, thereby stabilizing the protein under hypoxia, to promote transcription of many inducible gene
targets such as VEGF and MMP163. Therefore, PET imaging SIRT1 expressionꢀactivity in the brain may
advance the understanding of SIRT1 roles in vascular maintenance and remodeling following ischemiaꢀ
reperfusion events (i.e. atherosclerotic and other angiopathies, brain infarcts or trauma).
Also, current studies demonstrated the feasibility of 2ꢀ[18F]BzAHA PET/CT/MRI for nonꢀinvasive
in vivo monitoring of the pharmacodynamics of SIRT1ꢀspecific inhibitors EXꢀ527 at the target level
(inhibition of SIRT1 enzyme activity). A statistically significant decrease in 2ꢀ[18F]BzAHAꢀderived
radioactivity accumulation in the brain was observed after administration of EXꢀ527, as compared to
preꢀtreatment levels, especially in the brain structures exhibiting higher levels of SIRT1 expressionꢀ
activity at baseline (i.e., hippocampus, n.accumbens, amygdala) (Fig. 5). The dose of EXꢀ527 used in
this study was chosen to be similar to doses used in previously published reports11a, however, this dose
demonstrated only a 20% inhibition of 2ꢀ[18F]BzAHAꢀderived radioactivity accumulation. This
observation suggests that higher doses of EXꢀ527 may be required to achieve more inhibition of SIRT1
in these brain structures. Furthermore, the BBB penetrability of EXꢀ527 has not yet been well
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established, and this may be a limiting factor to SIRT1 inhibition in the CNS. Using PET/MRI with 2ꢀ
[18F]BzAHA it should be possible to determine the IC50 doses of EXꢀ527 and other SIRT1ꢀspecific
inhibitors or activators in different structures of the brain by imaging the same subject after
administration of increasing doses of inhibitor, as reported by us previously for SAHA21a.
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CONCLUSIONS
In summary, we have demonstrated that PET/CT/MRI imaging with newly developed first
generation SIRT1ꢀselective radiotracer 2ꢀ[18F]BzAHA can be used for nonꢀinvasive repetitive and
quantitative visualization of SIRT1 expressionꢀactivity in the brain. Molecular imaging with
PET/CT/MRI using SIRT1ꢀspecific substrateꢀtype radiotracers should improve the understanding of
many roles that SIRT1 plays in normal brain physiology, in various brain diseases and pathological
states, and will facilitate the development and clinical translation of novel SIRT1ꢀspecific activators and
inhibitors.
EXPERIMENTAL SECTION
All solvents were purchased from SigmaꢀAldrich Chemicals (Milwaukee, WI) and used without
further purification. BocꢀLys(Ac)ꢀAMC and CbzꢀLysꢀOH were purchased from Bachem (Bubendorf,
Switzerland) and used without further purification. Preparation of 6ꢀaminoꢀ1ꢀhexanoicanilide was
synthesized using a previously published procedure 64. Thin layer chromatography (TLC) was
performed on preꢀcoated Dynamic Absorbance Fꢀ254 silica gel, aluminum backed plates (Norcross,
Georgia); flash chromatography was performed using silica gel pore size 60 Å, 230ꢀ400 mesh particle
size (SigmaꢀAldrich, Milwaukee, WI). NMR spectra including H, 13C and 19F were performed using
1
Mercury 400 MHz, Varian 500 MHz or Agilent 600 MHz spectrometers. Highꢀresolution mass spectra
(HRMS) were obtained using Waters LCT Premier/XE spectrometer (Milford, MA) with an electrospray
ionization (ESI) technique. The analytical high performance liquid chromatography (HPLC) system
included: Ascentis RPꢀAmide 4.4x150 mm column (Supelco, Bellefonte, PA) connected to the 1100
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series pump and UV detector (Agilent Technologies, Stuttgart, Germany), operated at 254 nm, and a
radioactivity detector FC3200 (Eckert and Ziegler Radiopharma, Inc, Berlin, Germany). The semiꢀ
preparative HPLC system included: the Alltima 250x10mm C18 column (Fisher Scientific, Waltham,
MA) connected to the P4.1S pump, Azura 2.1S UV detector (Knauer, Berlin, Germany), operated at 254
nm, and a radioactivity detector FCꢀ3500 (Eckert and Ziegler Radiopharma, Inc, Berlin, Germany). In
general, TLC and HRMS were used to determine the purity of synthesized nonꢀradiolabeled compounds;
radioꢀTLC and combined UV/radioꢀHPLC were used to determine the purity of radiolabeled
compounds. The threshold of acceptable purity was set at ≥95%.
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Chemical Synthesis. Compounds 1-10 were prepared following previously published methods29c, 65 as
shown in the synthetic Fig. 1 with minor modifications. Compounds 1-10 were synthesized from
commercially available Cbzꢀlysine with formation of corresponding acetyl chloride following the
published methods29c which upon reaction with AMC (2) in presence of POCl3 and pyridine provide
desired product in moderate to good yields. To synthesize compound 4, CbzꢀLys (1 equiv.) was
dissolved in a mixture of THF (5 mL) and water (5 mL), then sodium bicarbonate (2 equiv.) and a
solution of Boc2O (2.3 equiv.) in THF (5 mL) was added. After stirring at room temperature overnight,
the THF was removed under reduced pressure and the resulting aqueous solution was neutralized with
2N HCl. The mixture was extracted with ethyl acetate and the combined organic layers were washed
with brine. The organic layer was dried with sodium sulfate and concentrated to clear oil, which was
used for further reaction without any purification with 2 in presence of NMM/isobutyl chloroformate
(Fig 2).
Benzyl (6-benzamido-1-((4-methyl-2-oxo-2H-chromen-7-yl)amino)-1-oxohexan-2-yl)carbamate (1).
Compound 1 was synthesized by method described above using benzoyl chloride (Sigma, Milwaukee,
WI) as reacting R group. The resulting product was chromatograph with 5% methanol in
1
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dichloromethane. H, C and F NMR were used to determine the molecular characterization of this
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8
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compound and TLC and HRMS determined >95% purity. Yield: 63%, H NMR (CDCl3, 600 MHz): δ
9.38 (s, 1H), 7.76 (d, 2H, J = 7.8 Hz), 7.68 (s, 1H), 7.602ꢀ7.46 (m, 3H), 7.39ꢀ7.34 (m, 7H), 6.18 (s, 1H),
5.95 (d, 1H, J = 7.2 Hz), 5.11 (m, 2H), 4.41 (s, 1H), 3.49 (m, 1H), 2.40 (s, 3H), 2.07ꢀ2.05 (m, 1H), 1.88ꢀ
1.80 (m, 1H), 1.70ꢀ1.64 (m, 2H), 1.56ꢀ1.50 (m, 2H). 13C NMR (CDCl3, 600 MHz): δ 171.00, 168.24,
161.26, 156.92, 154.07, 152.46, 141.47, 136.08, 134.25, 134.23, 131.61, 128.61 (2C), 128.25, 128.02,
126.92 (2C), 125.10, 116.04, 115.84, 11.31, 107.30, 67.28, 55.41, 38.73, 38.60, 31.24, 29.71, 28.75,
22.26, 18.56. MS (EI): m/z 542.6 [M+H]. Highꢀresolution MS: Calculated for C31H31N3O6 (M+Na)
564.2111, found 564.2105.
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Benzyl
(6-(2-fluorobenzamido)-1-((4-methyl-2-oxo-2H-chromen-7-yl)amino)-1-oxohexan-2-
yl)carbamate (2). Compound 2 was synthesized by method described above using 2ꢀfluorobenzoyl
chloride (Sigma, Milwaukee, WI) as reacting R group. The resulting product was chromatograph with
1
13
3% methanol/dichloromethane and purified by combiflash with 5% methanol/dichloromethane. H, C
and 19F NMR were used to determine the molecular characterization of this compound and TLC and
1
HRMS determined >95% purity. Yield: 69%, H NMR (DMSOꢀd6, 600 MHz): δ 10.51 (s, 1H), 8.29 (s,
1H), 7.77 (brs, 1H), 7.71 (d, 1H, J = 8.4 Hz), 7.65 (d, 1H, J = 7.8 Hz), 7.54 (t, 1H, J = 7.2 Hz), 7.50ꢀ7.47
(m, 2H), 7.38ꢀ7.31 (m, 4H), 7.24ꢀ7.17 (m, 2H), 6.27 (s, 1H), 5.03 (s, 2H), 4.16 (m, 1H), 3.24 (q, 2H, J =
6.6 Hz), 2.40 (s, 3H), 1.72ꢀ1.66 (m, 2H), 1.56ꢀ1.38 (m, 4H). 13C NMR (DMSOꢀd6, 600 MHz): δ 172.49,
164.15, 160.59, 160.28, 158.63, 156.65, 154.09, 153.68, 142.67, 137.39, 132.66, 130.42, 128.84 (2C),
128.32 (2C), 126.41, 124.85, 116.55, 116.40, 115.77, 115.56, 112.75, 106.19, 65.98, 56.05, 39.33,
31.74, 29.04, 23.39, 18.45.19F (DMSOꢀd6, 300 MHz): δ ꢀ114.49. MS (EI): m/z 560.6 [M+H]. Highꢀ
resolution MS: Calculated for C31H30FN3O6 (M+Na) 582.2016, found 582.1989.
Benzyl
(6-(3-fluorobenzamido)-1-((4-methyl-2-oxo-2H-chromen-7-yl)amino)-1-oxohexan-2-
yl)carbamate (3). Compound 3 was synthesized by method described above using 3ꢀfluorobenzoyl
chloride (Sigma, Milwaukee, WI) as reacting R group. The resulting product was chromatograph with
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1
3% methanol in dichloromethane and purified by combiflash with 5% methanol/dichloromethane. H,
2
3
4
5
13C and 19F NMR were used to determine the molecular characterization of this compound and TLC and
1
6
7
HRMS determined >95% purity. Yield: 66%, H NMR (CDCl3): δ 9.19 (brs, 1H), 7.67 (s, 1H), 7.55ꢀ
8
9
7.49 (m, 4H), 7.46ꢀ7.351 (m, 6H), 7.20 (t, 1H, J = 7.8 Hz), 6.56 (brs, 1H), 6.22 (s, 1H), 5.85 (brs, 1H),
5.14 (m, 2H), 4.40 (s, 1H), 3.51 (m, 2H), 2.43 (s, 3H), 2.07ꢀ2.06 (m, 1H), 1.88ꢀ1.80 (m, 1H), 1.72ꢀ1.60
(m, 2H), 1.56ꢀ1.52 (m, 2H). 13C NMR (CDCl3): δ 170.56, 166.86, 163.54, 161.18, 154.11, 152.21,
141.10, 136.00, 130.32, 128.59 (2C), 128.31, 128.06 (2C), 125.14, 122.43, 118.66, 118.52, 116.15,
10
11
12
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115.80, 114.41, 114.27, 113.41, 107.31, 67.36, 55.34, 38.73, 30.95, 28.61, 22.16, 18.59. F (CDCl3, 300
MHz): δ ꢀ111.60. MS (EI): m/z 560.6 [M+H]. Highꢀresolution MS: Calculated for C31H30FN3O6 (M+Na)
582.2016, found 582.1986.
Benzyl
(6-(4-fluorobenzamido)-1-((4-methyl-2-oxo-2H-chromen-7-yl)amino)-1-oxohexan-2-
yl)carbamate (4). Compound 4 was synthesized by method described above using 4ꢀfluorobenzoyl
chloride (Sigma, Milwaukee, WI) as reacting R group. The resulting product was chromatograph with
1
5% methanol in dichloromethane and purified by combiflash with 5% methanol/dichloromethane. H,
13C and 19F NMR were used to determine the molecular characterization of this compound and TLC and
1
HRMS determined >95% purity. Yield: 72%, H NMR (CDCl3): δ 9.38 (s, 1H), 7.78 (m, 2H), 7.66 (s,
1H), 7.51ꢀ7.45 (m, 2H), 7.39ꢀ7.33 (m, 5H), 6.99 (t, 2H, 6J), 6.65 (t, 1H, J = 6 Hz)), 6.15 (s, 1H), 6.02 (d,
1H, J = 8 Hz)), 5.11 (s, 2H), 4.45 (brs, 1H), 3.49ꢀ3.46 (m, 2H), 2.39 (s, 3H), 2.01ꢀ1.88 (m, 2H), 1.71ꢀ
13
1.67 (m, 2H), 1.55ꢀ1.50 (m, 2H). C NMR (CDCl3): δ 170.91, 167.16, 165.59, 161.17, 154.1, 152.36,
141.37, 136.00, 130.38 (2C), 129.31 (2C), 129.25, 128.60 (2C), 128.32, 128.06, 125.13, 116.12, 115.80,
19
115.69, 115.54, 113.40, 107.30, 67.37, 55.38, 38.66, 30.93, 28.69, 22.17, 18.57. F (CDCl3, 300 MHz):
δ ꢀ107.70. MS (EI): m/z 560.6 [M+H]. Highꢀresolution MS: Calculated for C31H30FN3O6 (M+Na)
582.2016, found 582.1985.
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Benzyl
(6-(4-ethylbenzamido)-1-((4-methyl-2-oxo-2H-chromen-7-yl)amino)-1-oxohexan-2-
3
4
5
yl)carbamate (5). Compound 10 was synthesized by method described above using 4ꢀethylbenzoyl
6
7
chloride (Sigma, Milwaukee, WI) as reacting R group. The resulting product was chromatograph with
8
9
1
5% methanol in dichloromethane and purified by combiflash with 5% methanol/dichloromethane. H,
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11
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13C and 19F NMR were used to determine the molecular characterization of this compound and TLC and
HRMS determined >95% purity. Yield: 77%, 1H NMR (CDCl3): δ 9.71 (s, 1H), 7.68 (m, 3H), 7.44ꢀ7.39
(m, 2H), 7.38ꢀ7.30 (m, 4H), 7.12 (d, 2H, J = 7.8 Hz), 6.87 (s, 1H), 6.26 (d, 1H, J = 7.8 Hz), 6.11 (s, 1H),
5.07 (s, 2H), 4.45 (brs, 1H), 3.42 (m, 2H), 2.62 (q, 2H, J = 7.2 Hz), 2.44 (s, 3H), 1.98ꢀ1.82 (m, 2H),
1.67ꢀ1.48 (m, 4H), 1.19 (t, 3H, J = 7.2 Hz). 13C NMR (CDCl3): δ 171.55, 168.20, 161.40, 156.88,
153.89, 152.75, 148.11, 141.63, 136.17, 131.90, 128.51 (2C), 128.13, 127.97 (2C), 127.89, 127.10 (3C),
125.05, 115.93, 115.84, 113.52, 107.21, 67.14, 55.65, 39.09, 31.78, 28.89, 28.70, 22.55, 18.51, 15.27.
MS (EI): m/z 570.7 [M+H]. Highꢀresolution MS: Calculated for C33H35N3O6 (M+Na) 592.2424, found
592.2433.
Benzyl
(6-(4-iodobenzamido)-1-((4-methyl-2-oxo-2H-chromen-7-yl)amino)-1-oxohexan-2-
yl)carbamate (6). Compound 6 was synthesized by method described above using 4ꢀiodobenzoyl
chloride (Sigma, Milwaukee, WI) as reacting R group. The resulting product was chromatograph with
1
5% methanol in dichloromethane and purified by combiflash with 5% methanol/dichloromethane. H,
13C and 19F NMR were used to determine the molecular characterization of this compound and TLC and
HRMS determined >95% purity. Yield: 60%, 1H NMR (CDCl3): δ 9.17 (s, 1H), 7.69ꢀ7.66 (m, 3H),
7.54ꢀ7.46 (m, 4H), 7.42ꢀ7.34 (m, 5H), 6.21 (s, 1H), 5.86 (brs, 1H), 5.12 (s, 2H), 4.38 (brs, 1H), 3.55ꢀ
3.44 (m, 2H), 2.43 (s, 3H), 2.10ꢀ2.06 (m, 1H), 1.89ꢀ1.84 (m, 1H), 1.71ꢀ1.68 (m, 2H), 1.57ꢀ1.50 (m, 2H).
13C NMR (MeOD): δ 171.50, 166.21, 160.30, 155.42, 152.20, 135.86, 132.30 (2C), 126.97, 126.58
(2C), 126.15, 125.96, 123.76, 122.76, 120.18, 119.09, 118.70, 116.95, 114.96, 114.32, 110.82, 105.17,
96.01, 64.87, 54.25, 37.45, 29.90, 27.00, 21.10, 15.70. MS (EI): m/z 667.5 [M+]. Highꢀresolution MS:
Calculated for C31H30IN3O6 (M+Na) 690.1077, found 690.1075.
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Benzyl
(1-((4-methyl-2-oxo-2H-chromen-7-yl)amino-1-oxo-6-(2-phenylacetamido)hexan-2-
yl)carbamate (7). Compound 7 was prepared following method as described above using phenyl acetyl
chloride (Sigma, Milwaukee, WI) as reacting R group. The resulting product was chromatograph with
2
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5
6
7
8
9
1
3% methanol in dichloromethane and purified by combiflash with 5% methanol/dichloromethane. H,
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13C and 19F NMR were used to determine the molecular characterization of this compound and TLC and
HRMS determined >95% purity. Yield: 59%, This is a known compound 29c, so here we report MS (EI):
m/z 554.6 [MꢀH]. Highꢀresolution MS: Calculated for C32H33N3O6 (M+Na) 578.2267, found 578.2269.
Benzyl
(6-(2-(2-fluorophenyl)acetamido)-1-((4-methyl-2-oxo-2H-chromen-7-yl)amino)-1-
oxohexan-2-yl)carbamate (8). Compound 8 was synthesized by method described above using 2ꢀ
fluorophenyl acetyl chloride (Sigma, Milwaukee, WI) as reacting R group. The resulting product was
chromatograph with 3% methanol in dichloromethane and purified by combiflash with 5%
methanol/dichloromethane. 1H, 13C and 19F NMR were used to determine the molecular characterization
1
of this compound and TLC and HRMS determined >95% purity. Yield: 57%, H NMR (DMSOꢀd6): δ
10.52 (s, 1H), 8.06 (t, 1H, J = 5.4 Hz), 7.78 (s, 1H),7.73ꢀ7.64 (m, 2H), 7.51 (s, 1H), 7.37ꢀ7.24 (m, 7H),
7.12ꢀ7.09 (m, 2H), 6.27 (s, 1H), 5.04 (s, 2H), 4.13 (m, 1H), 3.04 (m, 2H), 2.40 (s, 3H), 1.68ꢀ1.33 (m,
13
6H). C NMR (DMSOꢀd6): δ 172.10, 169.51, 167.22, 160.38, 156.18, 154.00, 153.68, 142.69, 137.39,
132.19, 129.14, 129.01 (2C), 128.96, 128.85 (2C), 126.45, 124.60, 122.62, 115.79, 115.57, 115.48,
112.76, 106.19, 65.99, 55.33, 38.98, 35.74, 31.17, 28.81, 22.85, 18.45. 19F (DMSOꢀd6, 300 MHz): δ ꢀ
117.40. MS (EI): m/z 572.6 [MꢀH]. Highꢀresolution MS: Calculated for C32H32FN3O6 (M+Na)
596.2173, found 596.2180.
Benzyl
(6-(2-(4-fluorophenyl)acetamido)-1-((4-methyl-2-oxo-2H-chromen-7-yl)amino)-1-
oxohexan-2-yl)carbamate (9). Compound 9 was synthesized by method described above using 4ꢀ
fluorophenyl acetyl chloride (Sigma, Milwaukee, WI) as reacting R group. The resulting product was
chromatograph with 3% methanol in dichloromethane and purified by combiflash with 5%
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methanol/dichloromethane. 1H, 13C and 19F NMR were used to determine the molecular characterization
3
4
5
1
of this compound and TLC and HRMS determined >95% purity. Yield: 60%, H NMR (MeOD): δ 7.78
6
7
8
9
(s, 1H), 7.67 (d, 1H, J = 8.2 Hz), 7.46 (d, 1H, J = 2 Hz), 7.39ꢀ7.25 (m, 7H), 7.00ꢀ6.97 (m, 2H), 6.22 (s,
1H), 5.12ꢀ5.11 (m, 2H), 4.26 (t, 1H, J = 5.3 Hz), 3.45 (s, 2H), 3.19 (t, 2H, J = 6.6 Hz), 2.45 (s, 3H),
1.86ꢀ1.74 (m, 2H), 1.56ꢀ1.40 (m, 4H). 13C NMR (MeOD): δ 172.44, 172.36, 162.70, 161.81, 161.08,
157.23, 153.91, 153.82, 141.94, 136.74, 131.58, 130.61, 130.40, 130.35, 128.09, 127.66, 127.48, 125.27,
115.82, 115.79, 114.81, 114.76, 112.22, 106.66, 66.40, 55.83, 42.37, 38.79, 31.46, 28.58, 22.82, 17.12.
19F (MeOD, 300 MHz): δ ꢀ118.40. MS (EI): m/z 574.7 [M+H]. Highꢀresolution MS: Calculated for
C32H32FN3O6 (M+Na) 596.2173, found 596.2173.
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Benzyl (6-(2-(4-iodophenyl)acetamido)-1-((4-methyl-2-oxo-2H-chromen-7-yl)amino)-1-oxohexan-
2-yl)carbamate (10). Compound 10 was synthesized by method described above using 4ꢀiodophenyl
acetyl chloride (Sigma, Milwaukee, WI) as reacting R group. The resulting product was chromatograph
with 3% methanol in dichloromethane and purified by combiflash with 5% methanol/dichloromethane.
1H, 13C and 19F NMR were used to determine the molecular characterization of this compound and TLC
1
and HRMS determined >95% purity. Yield: 72%, H NMR (MeOD): δ 8.11 (brs, 1H), 7.88 (d, 1H, J =
8.4 Hz), 7.82 (s, 1H), 7.76ꢀ7.73 (m, 2H), 7.67 (d, 1H, J = 8.4 Hz), 7.61 (d, 1H, J = 7.8 Hz), 7.54ꢀ7.48 (m,
2H), 7.40ꢀ7.31 (m, 2H), 7.09 (d, 1H, J = 7.8 Hz), 7.04 (d, 1H, J = 8.4 Hz), 6.26 (s, 1H), 5.13 (m, 2H),
4.25 (m, 1H), 3.43 (s, 2H), 3.21 (m, 2H), 2.48 (s, 3H), 1.89ꢀ1.74 (m, 2H), 1.56ꢀ1.48 (m, 4H). 13C NMR
(MeOD): δ 172.04, 171.92, 161.87, 154.00, 153.89, 137.31 (2C), 137.23, 135.45, 131.20, 130.80 (2C),
128.09, 127.67, 127.50, 126.50, 125.35, 115.91, 115.86, 112.26, 106.72, 91.30, 66.41, 55.80, 41.83,
38.53, 31.45, 28.53, 22.78, 17.15. MS (EI): m/z 680.4 [MꢀH]. Highꢀresolution MS: Calculated for
C32H32IN3O6 (M+Na) 704.1233, found 704.1231.
2-fluoro-phenylhexanoicanilide, 2-FPhAHA (12): Compound 11 (0.240g, 1.163 mmol) was dissolved
in 5mL of dichloromethane and 0.5mL triethylamine. The 2ꢀfluoro benzoylchloride (0.2 mL, 1.696
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mmol) was added dropwise at 0°C. The reaction was stirred at 0°C for 30 minutes then gradually
warmed to RT and stirred under argon for 3 hours. The mixture was washed with ethylacetate and 4N
HCl solution. The organic layer was dried over magnesium sulfate and then evaporated to dryness. The
resulting crude mixture was reꢀcrystallized with diethylꢀether overnight and then filtered with vacuum
filtration before being dried under highꢀvacuum. Product 12 (110mg) was made as a white solid in 28%
yield. 1H, 13C and 19F NMR were used to determine the molecular characterization of this compound and
2
3
4
5
6
7
8
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10
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TLC and HRMS determined >95% purity. H NMR (DMSOꢀd6)δ 9.87 (s, 1H), 8.31 (s, 1H) 7.54 (m,
4H), 7.25 (dt, J = 7.8 Hz, 4H), 7.01 (t, 1H), 3.24 (dd, J = 5.9 Hz, 2H), 2.31 (t, J = 7.3 Hz, 2H), 1.62 (m,
4H), 1.35 (m, 2H). 13C NMR (101 MHz, cdcl3) δ 171.33, 163.47, 161.73, 159.28, 138.18, 133.15 (d, J =
9.3 Hz), 131.82, 128.86, 124.75 (d, J = 3.1 Hz), 124.00, 121.21 (d, J = 11.3 Hz), 119.82, 115.98 (d, J =
24.8 Hz), 77.35, 77.03, 76.71, 39.74, 37.32, 29.17, 26.36, 25.02. 19F δ: ꢀ114.64. M/Z [M+Na]+
calculated: 329.1665; M/Z found: 329.1654
2-Nitro-N-(6-oxo-6-(phenylamino)hexyl)benzamide (13): The solution of 2ꢀnitrobenzoyl chloride
(0.12 g, 0.62 mmol) in 0.5 mL of DCM was added dropwise to the mixture of phenylhexanamide (11)
(0.10 g, 0.48 mmol) and diisopropylethylamine (DIPEA) (0.13 mL, 0.73 mmol) in dimethylformamide
(5 mL) at 0°C and then left to stir at room temperature for 12 h. The solvent was then evaporated under
reduced pressure; the crude was dissolved in EtOAc and washed with water. The organic layer was dried
over sodium sulfate. Evaporation and purification with column chromatography (eluent ꢀ EtOAc) gave
the desired precursor, 13, in 70ꢀ72% yield. 1H, C and F NMR were used to determine the molecular
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characterization of this compound and TLC and HRMS determined >95% purity. H NMR (600 MHz,
DMSOꢀd6) δ 9.87 (s, 1H), 8.66 (t, J = 5.5 Hz, 1H), 8.02 (d, J = 8.1 Hz, 1H), 7.75 (t, J = 7.5 Hz, 1H),
7.67 (t, J = 7.8 Hz, 1H), 7.60 (d, J = 8.2 Hz, 2H), 7.57 (d, J = 7.5 Hz, 1H), 7.28 (t, J = 7.8 Hz, 2H), 7.01
(t, J = 7.3 Hz, 1H), 3.22 (dd, J = 12.9, 6.6 Hz, 2H), 2.32 (t, J = 7.4 Hz, 2H), 1.67 – 1.59 (m, 2H), 1.58 –
1.50 (m, 2H), 1.42 – 1.33 (m, 2H). 13C NMR (151 MHz, DMSOꢀd6) δ 171.2, 165.3, 147.0, 139.4, 133.5,
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132.8, 130.5, 129.0, 128.6, 124.0, 122.9, 119.0, 39.0, 36.4, 28.6, 26.1, 24.8. HRMS [M+Na]+ 356.1600;
M/Z found : 356.1598
5
Radiosynthesis of 2-[18F]-fluorobenzoyl-aminohexanoicanilide 2ꢀ[18F]BzAHA (14). The Fꢀ18 was
obtained in kryptofix/K[18F] complex in acetonitrile/water (1 mL) from the PET/Cyclotron Facility,
Wayne State University (Detroit, MI) and transferred into a crimped 2 mL Vꢀvial for azeotropic drying.
Acetonitrile (0.4 mL) was added to the mixture, which was dried under a stream of argon at 90ꢀ100°C.
A solution of 13 (~2 mg) in dry dimethylsulfoxide (0.4 mL) was added to the dried kryptofix/ K[18F]
and heated to 130ꢀ140°C under argon with stirring for 12 minutes. After cooling, the reaction mixture
was passed through the 900 mg Altech silica gel cartridge (Fisher Scientific, Waltham, MA) and eluted
with 2 mL of 10% methanol in dichloromethane (DCM). The solvent was evaporated under a stream of
argon to remove DCM and to reduce the volume to <0.5 mL. The 14 was purified by semiꢀpreparative
HPLC (65% acetonitrile in water) by collecting the peak at 6 min. The decay corrected radiochemical
yield was 10% and the compound purity was >95%, as assessed by analytical HPLC and coꢀelution with
the nonꢀradiolabeled reference standard 12.
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Radiosynthesis of 2-18F-ethylbenzoate (supplemental materials): A solution of kryptofix/ K18F was
received from the cyclotron and transferred into a crimped Vꢀvial for drying. Acetonitrile (0.4 mL) was
added to the mixture and it was dried under a stream of argon at 80°C. A solution of 2ꢀnitro
ethylbenzoic acid (8mg, Sigma, Milwaukee, WI) in dry acetonitrile (0.4mL) was added to the dried
kryptofix/ K18F and the mixture was heated to 120°C and stirred under argon for 20 minutes. The
reaction was cooled and passed through a silica gel cartridge (Alltech, 900 mg) and eluted with 30%
methanol in dichloromethane (2 mL). After evaporation of the solvent under a stream of argon to a
volume <0.5ml and purified by semipreparative HPLC. The desired compound was isolated with 50%
MeCN in water at 20 minutes. This product was injected into analytical HPLC with authentic nonꢀ
radiolabeled (used as purchased from Sigma, Milwaukee, WI) compound to assess identity and purity of
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desired compound. The compound was obtained in 8% radiochemical yield and >95% purity as
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determined by HPLC.
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Radiosynthesis of 2-18F-benzaldehyde (supplemental materials): A solution of kryptofix/ K18F was
received from the cyclotron and transferred into a crimped Vꢀvial for drying. Acetonitrile (0.4 mL) was
added to the mixture and it was dried under a stream of argon at 105°C. A solution 2ꢀnitrobenzaldehyde
(8mg, Sigma, Milwaukee, WI) in dry DMSO (0.4mL) was added to the dried kryptofix/ K18F and the
mixture was heated to 120°C and stirred under argon for 20 minutes. The reaction was cooled and
passed through a silica gel cartridge (Alltech, 900 mg) and eluted with 30% methanol in
dichloromethane (2 mL). After evaporation of the solvent under a stream of argon to a volume <0.5ml
and purified by semipreparative HPLC. The desired compound was isolated with 50% MeCN in water at
20 minutes. This product was injected into analytical HPLC with authentic nonꢀradiolabeled (used as
purchased from Sigma, Milwaukee, WI) compound to assess identity and purity of desired compound.
The compound was obtained in 11% radiochemical yield and >95% purity as determined by HPLC.
SIRT Enzyme Assay. Recombinant SIRT1ꢀ7 enzymes and BPS1 reference substrate for SIRT1, an
oligopeptide corresponding to 379ꢀ382 of p53 (ArgꢀHisꢀLysꢀLys(Ac)ꢀAMC) were purchased from BPS
Bioscience (San Diego, CA). 10 mM solutions of all newly synthesized compounds were prepared in
pure DMSO and then diluted to 100 µM aliquots in DMSO for further use. Enzyme assay reactions were
performed in black, low binding 96ꢀwell microtiter plates (Nagle Nunc International, Rochester, NY).
End point assays were performed by incubation of the appropriate substrate, NAD, a SIRT enzyme, and
inhibitor (if applicable) in enzyme assay buffer (50 µL final volume). Control wells without enzyme
were included in each plate (Table S1). After 40 min at 37 °C, a developer mixture (50 µL) containing
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