N-(Pivaloyloxy)alkoxy-carbonyl Prodrugs of the Glutamine Antagonist 6-Diazo-5-oxo- l -norleucine (DON) as a Potential Treatment for HIV Associated Neurocognitive Disorders

Article abstract of DOI:10.1021/acs.jmedchem.7b00966

Aberrant excitatory neurotransmission associated with overproduction of glutamate has been implicated in the development of HIV-associated neurocognitive disorders (HAND). The glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON, 14) attenuates glutamate

Full text of DOI:10.1021/acs.jmedchem.7b00966

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Article
N-(Pivaloyloxy)alkoxy-carbonyl Prodrugs of the Glutamine
Antagonist 6-Diazo-5-oxo-L-norleucine (DON) as a Potential
Treatment for HIV Associated Neurocognitive Disorders
Michael T. Nedelcovych, Lukáš Tenora, Boe-Hyun Kim, Jennifer Kelschenbach, Wei
Chao, Eran Hadas, Andrej Jan#a#ík, Eva Prchalová, Sarah C. Zimmermann, Ranjeet P.
Dash, Alexandra J. Gadiano, Caroline Garrett, Georg Furtmüller, Byoungchol Oh, Gerald
Brandacher, Jesse Alt, Pavel Majer, David J. Volsky, Rana Rais, and Barbara S. Slusher
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00966 • Publication Date (Web): 31 Jul 2017
Downloaded from http://pubs.acs.org on July 31, 2017
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N-(Pivaloyloxy)alkoxy-carbonyl Prodrugs of the Glutamine Antagonist 6-Diazo-5-oxo-L-
norleucine (DON) as a Potential Treatment for HIV Associated Neurocognitive Disorders
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1,2#
9#
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Michael T. Nedelcovych , Lukáš Tenora , BoeꢀHyun Kim , Jennifer Kelschenbach , Wei
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Chao , Eran Hadas , Andrej Jančařík , Eva Prchalová , Sarah C. Zimmermann , Ranjeet P.
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Dash , Alexandra J. Gadiano , Caroline Garrett , Georg Furtmüller , Byoungchol Oh , Gerald
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10*
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Brandacher , Jesse Alt , Pavel Majer , David J. Volsky , Rana Rais ,Barbara S.
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,2,3,4,5,6*
Slusher
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Johns Hopkins Drug Discovery, Departments of Neurology, Medicine, Oncology,
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Psychiatry, Neuroscience, Molecular and Comparative Pathobiology, and Vascularized
Composite Allotransplantation Laboratory, Department of Plastic and Reconstructive Surgery
Johns Hopkins School of Medicine, Baltimore, MD 21205, U.S.A.
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic
v.v.i., Prague, Czech Republic
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Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, 10029,
U.S.A.
#
These authors contributed equally
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ABSTRACT
Aberrant excitatory neurotransmission associated with overproduction of glutamate has been
implicated in the development of HIVꢀassociated neurocognitive disorders (HAND). The
glutamine antagonist 6ꢀdiazoꢀ5ꢀoxoꢀLꢀnorleucine (DON, 14) attenuates glutamate synthesis in
HIVꢀinfected microglia/macrophages, offering therapeutic potential for HAND. We show that 14
prevents manifestation of spatial memory deficits in chimeric EcoHIVꢀinfected mice, a model of
HAND. 14 is not clinically available, however, because its development was hampered by
peripheral toxicities. We describe the synthesis of several substituted Nꢀ(pivaloyloxy)alkoxyꢀ
carbonyl prodrugs of 14 designed to circulate inert in plasma and be taken up and biotransformed
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to
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The
lead
prodrug,
isopropyl
6ꢀdiazoꢀ5ꢀoxoꢀ2ꢀ
(((phenyl(pivaloyloxy)methoxy)carbonyl)amino)hexanoate (13d), was stable in swine and
human plasma, but liberated 14 in swine brain homogenate. When dosed systemically in swine,
13d provided a 15ꢀfold enhanced CSFꢀtoꢀplasma ratio and a 9ꢀfold enhanced brainꢀtoꢀplasma
ratio relative to 14, opening a possible clinical path for the treatment of HAND.
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INTRODUCTION
HIVꢀassociated neurocognitive disorders (HAND) remain one of the central health issues
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in patients with chronic HIV infection despite viremic control by combined antiretroviral
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therapy (cART). While broad use of cART has markedly reduced the prevalence of HIVꢀ
associated dementia (HAD), it has had limited effects on milder forms of HAND which now
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represent the majority of new cases of HIV neurological disease.
Although the mechanisms of HAND pathogenesis are unclear, accumulating evidence
suggests that HAND is associated with aberrant glutamate metabolism in the central nervous
system (CNS) culminating in elevated extracellular glutamate and disrupted excitatory
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, 7
neurotransmission.
In cultured human macrophages and microglia, HIV infection induces
elevated expression of the glutamateꢀsynthesizing enzyme glutaminase which directly
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contributes to the toxic overproduction of glutamate.
Activated microglia and macrophages
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also release viral proteins and proꢀinflammatory cytokines that independently promote further
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glutamate synthesis and release and inhibit glutamate reuptake in astrocytes and neurons.
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These cumulative insults result in impaired glutamateꢀdependent synaptic function
and
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cognition.
Studies of HAND patients corroborate preclinical findings of disrupted glutamate
metabolism in the CNS. Recent examination of cARTꢀtreated HIV patients revealed increased
cerebrospinal fluid (CSF) glutamate in patients with HAND compared to those without
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neurocognitive impairment. Several magnetic resonance spectroscopy (MRS) studies have also
shown dysregulated glutamate homeostasis in HIV patients that correlate with neurocognitive
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impairment including alterations in white and grey matter glutamate and glutamine.
In
addition, microarray analyses in postꢀmortem brain samples from HAND patients show frontal
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cortical alterations in the expression of genes critical for the regulation of glutamatergic
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signaling and glutamateꢀdependent synaptic plasticity, including glutaminase.
Collectively, these studies suggest that interventions aimed at normalizing glutamate
homeostasis offer therapeutic potential for HAND treatment. The glutamine antagonist 6ꢀdiazoꢀ
5ꢀoxoꢀLꢀnorleucine (DON, 14), which is known to inhibit glutaminase, has been shown to block
glutamate overproduction induced by HIV infection or immune challenge, and to mitigate
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excitotoxic neuronal damage both in vitro and in vivo,
but has not yet been tested in an
animal model of HAND.
In the present work, we tested the ability of 14 to prevent cognitive decline in EcoHIVꢀ
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infected wild type mice which serve as a model of HAND.
EcoHIV is a chimeric HIV in
which the coding region for envelope protein gp120 has been replaced with gp80 from murine
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leukemia virus, rendering EcoHIV capable of infecting conventional mice. EcoHIV infection
results in expression of HIV viral proteins in relevant tissues including microglial cells in the
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brain, and recapitulates many of the neuropathological features of HAND, bolstering the use of
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these mice as a model system for testing possible HAND therapeutics. We report that when 14
was administered to the mice prior to and during EcoHIV infection it was able to completely
prevent their cognitive decline.
Although exciting, the clinical utility of 14 is limited by its peripheral toxicities mediated
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primarily by the gastrointestinal (GI) system
which is known to be highly glutamineꢀ
utilizing. Recently, we described the synthesis of several Nꢀ(pivaloyloxy)alkoxyꢀcarbonyl 14
prodrugs designed to enhance CNS penetration and brain delivery of 14 while reducing
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peripheral exposure and thus GI toxicity. Systemic administration in nonꢀhuman primate of one
such prodrug, compound 13b with isopropyl ester on the carboxylate and Nꢀ
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(
pivaloyloxy)ethoxyꢀcarbonyl on the amine, resulted in about 10ꢀfold enhancement in the CSFꢀ
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toꢀplasma ratio of 14 exposure relative to administration of equimolar 14. These findings
provided the first evidence that a prodrug strategy could significantly alter the tissue distribution
of 14.
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In the current study, we hypothesized that modification of the Nꢀ(pivaloyloxy)methoxyꢀ
carbonyl proꢀmoiety with additional steric bulk on the methylene bridge could slow peripheral
bioconversion of 14 prodrugs and confer further improvement in CNS penetration, as has been
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shown for other prodrugs utilizing carboxylesteraseꢀmediated hydrolyses.
In addition to
increasing steric hindrance and metabolic stability, our modification approach could also
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enhance lipophilicity.
The new 14 prodrugs, compounds 13b-e with addition of methyl,
isopropyl, phenyl, and dimethyl groups to the Nꢀ(pivaloyloxy)methoxyꢀcarbonyl proꢀmoiety,
respectively, had substantially increased calculated partition coefficients (cLogP) versus 14
(1.50, 2.42, 2.75, and 1.81 vs. ꢀ2.50, respectively). However, when evaluated in mouse plasma,
all of the prodrugs were rapidly and completely metabolized to 14, precluding attempts to
meaningfully differentiate their efficacy/toxicity from 14 in the EcoHIV murine model.
Importantly, this conundrum is not uncommon in prodrug discovery, where increased
metabolism in rodents is well documented and higher species are necessary to mimic human
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metabolism.
Thus, these prodrugs were subsequently tested for stability in human plasma as
well as swine, a model organism that closely recapitulates human drug metabolism and
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pharmacokinetics. Although the Nꢀ(pivaloyloxy)methoxyꢀcarbonyl derivative of 14, compound
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3a, was also labile in human and swine plasma, all of the substituted analogues, compounds
3b-e, were found to be metabolically stable. Furthermore, when incubated in homogenates of
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swine brain, the target tissue for 14 release, compounds 13b and 13d were readily converted.
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These compounds were then tested in vivo in swine. Relative to administration of equimolar 14,
systemic administration of 13b and 13d resulted in lower 14 plasma exposure, higher CSF
exposure, and a greater than 7 and 15ꢀfold increased CSFꢀtoꢀplasma ratio, respectively. Having
shown the best profile, 13d and 14 were subsequently assessed in terminal swine studies in
which brain levels were directly measured. 13d afforded a 9ꢀfold enhancement in brainꢀtoꢀ
plasma ratio relative to equimolar 14.
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CHEMISTRY
Scheme 1. Synthesis of N-hydroxysuccinimide Esters of Pivaloyloxy-alkoxycarbonates
Intermediates 4a-c
As shown in Scheme 1, the intermediates 4a-c were synthesized by a three step procedure
starting from the appropriate 1ꢀchloroalkyl carbonochloridate (1a-c) which was reacted with
ethanthiol in presence of triethylamine to yield corresponding Oꢀ(1ꢀchloroalkyl)ꢀSꢀethyl
carbonothioates
(2a-c).
These
intermediates
were
transformed
to
1ꢀ
(((ethylthio)carbonyl)oxy)alkyl pivalate derivatives (3a-c) by reaction with pivalic acid in
presence of base (DIPEA). Oxidation of the thioester moiety by peracetic acid followed by
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reaction with Nꢀhydroxysuccinimide
provided 4a-c in a yield of 56ꢀ64% after column
chromatography (total over 3 steps). The attempt to use the same methodology for phenyl
analogue 13d in the last step resulted in a complex mixture likely due to reactivity of the
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corresponding benzyl cation intermediate. The attempt to prepare the analogous intermediate
with a phenyl was unsuccessful. Although it was not confirmed, this failure could be due to the
oxidation of the benzoacetal group by peracetic acid in the third step.
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Scheme 2. Synthesis of 4-Nitrophenyl Esters of Pivaloyloxy-alkoxycarbonate Intermediate
8
We thus chose to explore the paraꢀnitrophenoxy derivative 8, which can be synthesized
via a route devoid of an oxidant as shown in Scheme 2. First, the chloro(phenyl)methyl
carbonochloridate (6) was prepared by reaction of benzaldehyde (5) with triphosgene in presence
of base (pyridine). Intermediate 6 was reacted with 4ꢀnitrophenol to give the
chloro(phenyl)methyl (4ꢀnitrophenyl) carbonate (7) which was then subjected to reaction with
freshly prepared mercury (II) salt of pivalic acid to yield analog 8. Pivalate 8 was then used to
prepare 13d (Scheme 4). Despite several attempts with different pivalates (potassium, silver,
palladium) we were not successful in eliminating the use of mercury in this step.
Scheme 3. Synthesis of Intermediate 11
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As shown in Scheme 3, compound 11 was prepared in an analogous way. 4ꢀNitrophenyl
propꢀ1ꢀenꢀ2ꢀyl carbonate (9) was prepared from commercially available 2ꢀpropenyl
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chloroformate as previously reported and reacted with hydrogen chloride. The obtained 2ꢀ
chloropropanꢀ2ꢀyl (4ꢀnitrophenyl) carbonate (10) was then reacted with mercury (II) pivalate to
yield the desired intermediate 11, which was then used to prepare analog 13e.
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Scheme 4. Synthesis of 14 Prodrugs 13a-e
O
O
O
O
N
O
R
O
O
O
4
a; R=H
O
N
H
N
N
H N
N
4b; R=CH3
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O
O
N
c; R=isopropyl
a
R R
2 O
O
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O
+
O
O
or
a; R =R =H
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b; R =H; R =CH
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12
13a-e
c
; R =H; R =isopropyl
O
O
O
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2
d; R =H; R =Ph
1
2
O
R1 R2 O
e; R1=R2=CH3
NO2
8
: R =H, R =Ph
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2
11: R =R =CH
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3
Reagents and Conditions: a. DCM or DMF, 0 °C to rt, 2-5 h.
As shown in Scheme 4, the prodrugs 13a-e were prepared by reaction of 14 isopropyl
ester (12) with 4a-c, 8 or 11, respectively, in dichloromethane (DCM) or dimethylformamide
(DMF) at 0 °C or room temperature. Compound 12 was prepared as previously described in
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detail with yield and purity in agreement with the published data. The compounds 13a-e were
obtained in 40ꢀ83% yield after purification by liquid chromatography (LC). To improve yields of
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3d and 13e, an excess of compound 12 and longer reaction time were necessary (see
Experimental section).
RESULTS AND DISCUSSION
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4 Prevented Cognitive Decline, a Major Manifestation of HAND, in EcoHIV-Infected
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Mice. Similar to HAND patients, mice inoculated with EcoHIV exhibited impaired spatial
learning and memory as measured by radial arm water maze (RAWM) 30 days postꢀinfection
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(
Fig. 1). EcoHIV infection resulted in a significant increase in the number of errors (Fig. 1A) and
latency to escape (Fig. 1B) onto a hidden platform in the maze. 14 treatment (1mg/kg, i.p.,
q.o.d.) beginning one day prior to EcoHIV or sham inoculation, and continued throughout the
infection period and RAWM testing, fully normalized cognitive performance as measured by
both errors (main effect of treatment [F(3,140) = 261.8, p < 0.0001], trial [F(4,140) = 146.2, p <
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.0001], interaction [F(12,140) = 1.93, p = 0.0355]) and latency to escape (main effect of
treatment [F(3,140) = 37.00, p < 0.0001], trial [F(4,140) = 56.03, p < 0.0001], interaction
[F(12,140) = 1.832, p = 0.0484]). Neither EcoHIV infection nor 14 treatment affected latency to
escape to a visible platform (Fig. 1C; main effect of trial [F(3,112) = 34.44, p < 0.0001])
indicating no impairment in visual or motor function. Additionally, no mice exhibited overt signs
of toxicity (i.e. diarrhea, weight loss). The EcoHIVꢀinfected mice exhibited measurable viral
loads in the spleen and brain (Fig. 1D). 14 treatment actually caused an increase in peripheral
viral load (t(14) = 2.58, p = 0.022) likely due to the well described 14ꢀmediated inhibition of T
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cell activity/proliferation
which is known to be required for endogenous suppression of
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EcoHIV replication. Therefore, despite enabling a modest increase in EcoHIV replication, 14
still prevented cognitive decline, suggesting its mechanism of action to be secondary to the
infection itself. These findings suggest that glutaminase inhibition through 14 delivery to the
CNS prior to or during HIV infection may prevent the development of cognitive impairment in
HAND patients.
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N-(Pivaloyloxy)alkoxy-carbonyl Prodrugs of 14 Were Synthesized with Improved
Lipophilicity. Despite robust efficacy in the EcoHIV model of HAND, the GI toxicity induced
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by 14 in humans has hampered its clinical development.
Previous efforts by our group to
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develop more tolerable prodrugs of 14 yielded compound 13a (Table 1) through coverage of 14’s
carboxylate and amine groups with isopropyl ester and a primary Nꢀ(pivaloyloxy)methoxyꢀ
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carbonyl group, respectively. We rationalized that additional bulky, lipophilic substituents
would increase cLogP and further improve CNS penetration. Thus, methylꢀ, isopropylꢀ, phenylꢀ,
and dimethylꢀ analogues of 13a (compounds 13b-e) were synthesized as described above. cLogP
for each of these prodrugs was incrementally and substantially increased relative to 14 (Table 1).
We then initiated testing of their ex vivo metabolic stability.
N-(Pivaloyloxy)alkoxy-carbonyl Prodrugs of 14 Were Completely Unstable in Mouse
Plasma, Restricting Their Evaluation in Mouse Models. All prodrugs 13a-e were found to be
completely metabolized during a 60 minute incubation in mouse plasma (Table 1). A
representative chromatogram for the rapid metabolism of 13d in mouse plasma is provided in
Fig. S1 showing complete conversion to 14 within 10 min. The rapid metabolism of the 14
prodrugs precluded the examination of their efficacy and toxicity in mice as they would be
immediately converted to 14. As mentioned above, enhanced metabolism in rodents is a common
5
4, 55
issue with prodrugs of various classes,
necessitating the use of higher species, such as swine,
56
that more closely model human metabolism.
N-(Pivaloyloxy)alkoxy-carbonyl Prodrugs of 14 Were Stable in Human and Swine Plasma
but Labile in Brain Homogenate. Consistent with previous reports of the metabolic lability of
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Nꢀ(pivaloyloxy)methoxyꢀcarbonyl groupꢀcontaining prodrugs,
compound 13a was found
to be rapidly metabolized during a 60 minute incubation in human plasma (Table 1). In contrast,
we found that modification of 13a with methylꢀ, isopropylꢀ, phenylꢀ, and dimethyl substitutions
10
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(
13b-e) resulted in a significant improvement in human plasma stability (Table 1). As our goal
was to evaluate these prodrugs in an animal model which mimicked human metabolism, we next
tested the stability of compounds 13b-e in swine plasma. We found that all of the prodrugs,
similar to human plasma, were stable in swine plasma (Table 1). Given that the target
compartment for 14 activity in HAND is the brain, 13b-e were then tested for metabolic lability
in swine brain homogenate. Compounds 13c and 13d were found to be readily biotransformed in
swine brain homogenate, whereas 13b was moderately labile and 13e was mostly stable (Fig. 2).
Swine brain homogenate halfꢀlives were calculated for each compound and found to be
significantly different from each other [F(3,8) = 31.42, p < 0.0001].
When Tested in Swine, Compounds 13b and 13d Resulted in Enhanced 14 CSF-to-Plasma
Ratios; Compound 13d Showed an Optimal Profile. To determine if the ex vivo metabolism
data translated in vivo, 14, 13b and 13d were selected for pharmacokinetic evaluation in swine.
Consistent with their observed metabolic stability, i.v. infusion of 13b and 13d (1.6 mg/kg 14
equivalent dose) resulted in 3 to 5ꢀfold lower 14 plasma exposures relative to an equimolar dose
of 14 (Fig. 3A). Plasma AUC0ꢀt for 14, 13b, and 13d were 29.9, 8.00, and 5.70 nmol·hr/ml,
respectively. The opposite trend occurred in CSF, where the prodrugs delivered substantially
higher amounts of 14 to the CSF (Fig. 3B; Table 2), resulting in significantly increased CSFꢀtoꢀ
plasma ratios (Fig. 3C; Table 3). The improvement over 14 in CNS penetration correlated with
the lipophilicity of each prodrug. The cLogP for 13d was nearly twice that calculated for 13b
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(
2.75 vs. 1.50) and was associated with a doubling of the improvement in CSFꢀtoꢀplasma ratio in
vivo (approximate 15ꢀfold increase for 13d vs. 7ꢀfold increase for 13b relative to equimolar 14).
These experiments confirm that POMꢀbased ester prodrugs substantially improve CNS delivery
of 14, and support the hypothesis that iterative derivatization of this proꢀmoiety with sterically
hindering, lipophilic residues promotes better CNS targeting. It should be noted that the 13b
0
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diastereomers were separable by standard column chromatography, whereas the 13d
diastereomers were not readily separable by the same method and thus were administered as a
mixture. This may not influence the clinical relevance of these findings as human plasma
stabilities of the 13b diastereomers were comparable (91% vs 89% remaining at 60 min for 13b-
4
7
1
and 13b-2, respectively). However, as 13d moves towards clinical development,
stereochemical purity will be prioritized.
In Terminal Swine Studies, Compound 13d Resulted in Enhanced 14 Brain-to-Plasma
Ratio. Because CSF is not always an accurate surrogate for brain concentrations, we conducted
terminal studies in swine with the lead compound 13d where plasma and brain tissue were
collected. At 60 min postꢀinfusion of 13d (1.6 mg/kg 14 equivalent dose), 14 delivery to the
plasma was substantially lower than equimolar 14 (Fig. 4A) while brain levels were comparable
(Fig. 4B; Table 2). This resulted in a 9ꢀfold enhancement in the brainꢀtoꢀplasma ratio of 13d
relative to equimolar 14 (Fig. 4C), similar to its enhanced CSFꢀtoꢀplasma ratio (Table 3). Given
that the 14 CSF levels were significantly lower than that observed in brain tissues, we next
conducted protein binding studies in an attempt to elucidate the reason for these differences.
4
6
Plasma protein binding of 14 was low (F =21%), consistent with previous reports. However,
b
higher brain protein binding of 14 was observed (F = 91%) providing a possible explanation for
b
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the CSF and brain level discrepancies. Based on this value, free levels of 14 in the swine brain
were calculated to be 0.531 nmol/g at 60 min postꢀadministration of 13d, closer to observed
levels of 14 delivered to the CSF by 13d at the same time point (~1.0 nmol/mL; Table 2).
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CONCLUSION
1
4 was robustly efficacious in preventing cognitive decline in EcoHIVꢀinfected mice
used as a model of HAND. However, 14 is not suitable for clinical use due to its doseꢀlimiting
peripheral toxicities. In order to translate these findings to the clinic, we describe the synthesis of
several substituted Nꢀ(pivaloyloxy)alkoxyꢀcarbonyl prodrugs of 14 that improve CNS
penetration. Rational design of 14 prodrugs by addition of lipophilic substituents to the Nꢀ
(pivaloyloxy)alkoxyꢀcarbonyl proꢀmoiety yielded sterically hindered compounds with increased
metabolic stability in swine and human plasma, favorable conversion in brain homogenate, and
substantially improved CNS delivery in vivo in swine, exemplified by compound 13d. Metabolic
stability of these prodrugs in human plasma was very similar to that observed in swine,
suggesting strong translational potential for this strategy. This approach may therefore enable the
clinical use of brain glutaminase inhibition for the treatment of HAND and other
neuropsychiatric disorders characterized by aberrant glutamate metabolism.
EXPERIMENTAL SECTION
The commercially available HPLC grade acetonitrile, catalysts and reagent grade
materials were used as received. TLC was performed on Silica gel 60 F254‐coated aluminum
sheets (Merck) and spots were detected by the solution of Ce(SO ) 4 H O (1%) and
4
2·
2
H P(Mo O ) (2%) in sulfuric acid (10%). Flash chromatography was performed on Silica gel
3
3
10 4
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0 (0.040‐0.063 mm, Fluka) or on Biotage® KP‐C18‐HS or KP‐Sil® SNAP cartridges using the
Isolera One HPFC system (Biotage, Inc.). All chemicals were purchased from SigmaꢀAldrich or
1
TCI and were used without further purification. The H NMR spectra were measured at 400.1
0
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13
1
MHz, C NMR spectra at 100.8 MHz. For standardization of H NMR spectra the internal signal
1
3
of TMS (δ 0.0, CDCl ) or residual signals of CDCl (δ 7.26) were used. In the case of C spectra
3
3
the residual signal of CDCl (δ 77.00) was used. The chemical shifts are given in δ scale; the
3
coupling constants J are given in Hz. The ESI mass spectra were recorded using ZQ micromass
mass spectrometer (Waters) equipped with an ESCi multimode ion source and controlled by
MassLynx software. Alternatively, the low resolution ESI mass spectra were recorded using a
quadrupole orthogonal acceleration time‐of‐flight tandem mass spectrometer (Q‐Tof micro,
Waters) and high resolution ESI mass spectra using a hybrid FT mass spectrometer combining a
linear ion trap MS and the Orbitrap mass analyzer (LTQ Orbitrap XL, Thermo Fisher Scientific).
The conditions were optimized for suitable ionization in the ESI Orbitrap source (sheat gas flow
rate 35 a.u., aux gas flow rate 10 a.u. of nitrogen, source voltage 4.3 kV, capillary voltage 40 V,
capillary temperature 275 °C, tube lens voltage 155 V). The samples were dissolved in methanol
and applied by direct injection. The purity of all compounds subjected to biological testing was
established using HPLC (Jasco Inc.) equipped with a Reprosil 100 C18, 5 ꢁm, 250 × 4 mm
column. The analysis was performed using a gradient of 2% CH CN / 98% H O with 0.1% TFA
3
2
→ 100% CH CN, with UV detection, λ = 210 nm. Purity of all compounds subjected to
3
biological testing was over 95%. Optical rotations were measured in CHCl using an Autopol IV
3
instrument (Rudolph Research Analytical). IR spectra were measured in CHCl on an FTꢀIR
3
spectrometer.
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(
(((2,5-Dioxopyrrolidin-1-yl)oxy)carbonyl)oxy)methyl
pivalate
(4a).
Chloromethyl
carbonochloridate 1a (2.00 g, 1.38 mL, 15.5 mmol) was dissolved in anhydrous Et O (20 mL).
2
The reaction mixture was cooled to 0 °C and the mixture of Et N (1.57 g, 2.16 mL, 15.5 mmol, 1
3
10
11
12
13
14
15
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54
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58
59
60
equiv.) and EtSH (964 mg, 1.15 mL, 15.5 mmol, 1 equiv.) in anhydrous Et O (5 mL) was added
2
dropwise over 5 minutes. The resulting mixture was then stirred overnight (25 h) at rt. Precipitate
was filtered through a pad of celite and solvent was removed under reduced pressure. The crude
Oꢀ(chloromethyl) Sꢀethyl carbonothioate 2a (2.4 g, colorless liquid) was used in the next step
without purification. 2a (2.40 g, 15.5 mmol) was dissolved in pivalic acid (9.51 g, 93.1 mmol, 6
equiv.) and freshly prepared salt of pivalic acid (4.76 g, 46.6 mmol, 3 equiv.) and DIPEA (6.02
g, 8.10 mL, 46.6 mmol, 3 equiv.) was added in few portions. The reaction mixture was heated to
60 °C for 22 h. EtOAc (100 mL) was added and the organic phase was extracted with water (100
mL), sat. NaHCO (3×100 mL), sat. NaCl (100 mL), dried over MgSO , solvent was removed
3
4
under reduced pressure and the crude product 3a (3.30 g, 97%, light yellow liquid) was used in
the next step without purification. (((Ethylthio)carbonyl)oxy)methyl pivalate 3a (3.20 g, 14.5
mmol) was dissolved in anhydrous DCM (40 mL), Nꢀhydroxysuccinimide (3.34 g, 29.1 mmol, 2
equiv.) was added and the suspension was cooled to 0 °C. Peracetic acid (3.31 g (100%), 9.21 g
(36%), 43.6 mmol, 3 equiv., 36% solution in acetic acid) was added dropwise over 15 minutes.
The resulting mixture was stirred for 1 h at 0 °C and 2 h at rt. DCM (50 mL) was added and the
organic phase was washed with water (30 mL), sat. NaCl (30 mL), dried over MgSO , solvent
4
was removed under reduced pressure and column chromatography of the residue (EtOAc/hexane
1
1
:2, R 0.27) yielded 4a as a colorless solid (2.54 g, 64% over 3 steps). H NMR (400 MHz,
f
13
CDCl ): 1.24 (9H, s), 2.84 (4H, s), 5.86 (2H, s). C NMR (101 MHz, CDCl ): 25.56 (2C), 26.86
3
3
(
3C), 38.96, 83.67, 150.90, 168.34 (2C), 176.54. IR (CHCl ): 2979 m, 2939 w, 2876 w, 1823 s,
3
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796 vs, 1649 vs, 1481 m, 1463 m, 1456 w, 1431 m, 1398 w, 1371 m, 1367 m, 1280 m, 1199 vs,
ꢀ1
+
1110 vs, 1047 m, 998 s, 986 s, 942 m, sh, 924 s, 853 w, cm . ESI MS: 296 ([M + Na] ). HR
ESI MS: calcd for C H O NNa 296.07407; found 296.07410.
1
1
15
7
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0
((((2,5-Dioxopyrrolidin-1-yl)oxy)carbonyl)oxy)ethyl
pivalate
(4b).
Chloroethyl
carbonochloridate 1b (2.00 g, 1.51 mL, 14.1 mmol) was dissolved in anhydrous Et O (20 mL).
2
The reaction mixture was cooled to 0 °C and the mixture of Et N (1.43 g, 1.97 mL, 14.1 mmol, 1
3
equiv.) and EtSH (876 mg, 1.02 mL, 14.1 mmol, 1 equiv) in anhydrous Et O (5 mL) was added
2
dropwise over 5 minutes. The resulting mixture was then stirred overnight (17 h) at rt. Precipitate
was filtered through a pad of celite and solvent was removed under reduced pressure. The crude
Oꢀ(1ꢀchloroethyl) Sꢀethyl carbonothioate 2b (2.20 g, colorless liquid) was used in the next step
without purification. 2b (2.20 g, 13.1 mmol) was dissolved in pivalic acid (8.02 g, 78.6 mmol, 6
equiv.) and freshly prepared salt of pivalic acid (4.01 g, 39.3 mmol, 3 equiv.) and DIPEA (5.08
g, 6.85 mL, 39.3 mmol, 3 equiv.) was added in few portions. The reaction mixture was heated to
70 °C for 20 h. EtOAc (100 mL) was added and the organic phase was extracted with water (100
mL), sat. NaHCO (3×100 mL), sat. NaCl (100 mL), dried over MgSO , solvent was removed
3
4
under reduced pressure and the crude product 3b (2.82 g, 92%, light yellow liquid) was used in
the next step without purification. (((Ethylthio)carbonyl)oxy)methyl pivalate 3b (2.82 g, 12.1
mmol) was dissolved in anhydrous DCM (40 mL), Nꢀhydroxysuccinimide (2.77 g, 24.1 mmol, 2
equiv.) was added and the suspension was cooled to 0 °C. Peracetic acid (2.76 g (100%), 7.67 g
(36%), 36.3 mmol, 3 equiv., 36% solution in acetic acid) was added dropwise over 15 minutes.
The resulting mixture was stirred for 1 h at 0 °C and 2 h at rt. DCM (50 mL) was added and the
organic phase was washed with water (30 mL), sat. NaCl (30 mL), dried over MgSO , solvent
4
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was removed under reduced pressure and column chromatography of the residue (EtOAc/hexane
1
1
:2, R 0.27) yielded 4b as a colorless oil (2.02 g, 50% over 3 steps). H NMR (400 MHz,
f
1
3
CDCl ): 1.21 (9H, s), 1.60 (3H, d, J = 5.4), 2.83 (4H, s), 6.81 (1H, q, J = 5.4). C NMR (101
3
10
11
12
13
14
15
16
17
18
19
20
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22
23
24
25
26
27
28
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MHz, CDCl ): 19.43, 25.58 (2C), 26.88 (3C), 38.89, 93.83, 150.01, 168.43, 176.10. IR (CHCl ):
3
3
2978 m, 2939 w, 2875 w, 1821 s, 1795 s, 1748 vs, br, 1480 m, 1432 m, 1396 m, 1371 m, 1365
ꢀ1
m, sh, 1298 w, sh, 1280 s, 1199 s, 1054 vs, 1028 s, 944 vw, sh, 935 w, sh, 811 w, cm . ESI MS:
+
3
10 ([M + Na] ). HR ESI MS: calcd for C H O NNa 310.08972; found 310.08979.
1
2
17
7
1-((((2,5-Dioxopyrrolidin-1-yl)oxy)carbonyl)oxy)-2-methylpropyl pivalate (4c). 1ꢀChloroꢀ2ꢀ
methylpropyl carbonochloridate 1c (2.00 g, 1.71 mL, 11.7 mmol) was dissolved in anhydrous
Et O (20 mL). The reaction mixture was cooled to 0 °C and the mixture of Et N (1.18 g, 1.63
2
3
mL, 11.7 mmol) and EtSH (727 mg, 866 µL, 11.7 mmol) in anhydrous Et O (10 mL) was added
2
dropwise over 10 minutes. The resulting mixture was stirred overnight (23 h) at rt. Precipitate
was filtered through a pad of celite and solvent was removed under reduced pressure. The crude
product Oꢀ(1ꢀchloroꢀ2ꢀmethylpropyl) Sꢀethyl carbonothioate 2c (2.20 g, 96%, colorless liquid)
was used in the next step without purification. 2c (1.20 g, 6.10 mmol) was dissolved in pivalic
acid (3.74 g, 4.20 mL, 36.6 mmol, 6 equiv.) and freshly prepared salt of pivalic acid (1.87 g, 2.10
mL, 18.3 mmol, 3 equiv.) and DIPEA (2.37 g, 3.19 mL, 18.3 mmol, 3 equiv.) was added in few
portions. The reaction mixture was heated to 60 °C for 70 h. EtOAc (100 mL) was added and the
organic phase was extracted with water (50 mL), sat. NaHCO (3×50 mL), sat. NaCl (50 mL),
3
dried over MgSO , solvent was removed under reduced pressure and the crude product 1ꢀ
4
(((ethylthio)carbonyl)oxy)ꢀ2ꢀmethylpropyl pivalate 3c (1.32 g, 83%, light yellow liquid) was
used in the next step without purification. 3c (1.28 g, 4.88 mmol) was dissolved in anhydrous
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DCM (13 mL), Nꢀhydroxysuccinimide (1.12 g, 9.76 mmol, 2 equiv.) was added and the
suspension was cooled to 0 °C. Peracetic acid (1.11 g (100%), 3.09 g (36%), 14.6 mmol, 3
equiv., 36% solution in acetic acid) was added dropwise over 10 minutes. The resulting mixture
was stirred for 1 h at 0 °C and for 2 h at rt. DCM (40 mL) was added and the organic phase was
0
1
2
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8
9
0
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0
washed with water (20 mL), sat. NaCl (20 mL) dried over MgSO , solvent was removed under
4
reduced pressure and column chromatography of the residue (EtOAc/hexane 3:5, R 0.26)
f
1
yielded 4c as a light yellow oil (863 mg, 56% over 3 steps). H NMR (400 MHz, CDCl ): 1.00
3
1
3
(6H, d, J = 6.9), 1.21 (9H, s), 2.08–2.19 (1H, m), 2.81 (4H, s), 6.55 (1H, d, J = 5.0). C NMR
(101 MHz, CDCl ): 16.06, 16.38, 25.55, 26.89, 31.84, 39.05, 98.19, 150.37, 168.48, 176.14. IR
3
(
CHCl ): 2978 m, 2938 w, 2878 w, 1821 s, 1795 s, 1748 vs, br, 1481 m, 1463 w, 1432 m, 1396
3
ꢀ1
w, 1373 m, 1366 m, sh, 1279 m, 1199 s, 1046 m, 998 m, sh, 987 m, 932 s cm . ESI MS: 338
+
(
[M + Na] ). HR ESI MS: calcd for C H O NNa 338.12102; found 338.12115.
1
4
21
7
Chloro(phenyl)methyl
(4-nitrophenyl)
carbonate
(7).
Chloro(phenyl)methyl
64
carbonochloridate (6) was prepared from benzaldehyde (5) by a previously reported method.
Compound 6 (900 mg, 4.39 mmol) was dissolved in anhydrous DCM (20 mL), 4ꢀNitrophenol
(611 mg, 4.39 mmol, 1 equiv.) was added and the mixture was cooled to 0 °C. Pyridine (347 mg,
355 µL, 4.39 mmol, 1 equiv.) in anhydrous DCM (5 mL) was added dropwise over 5 minutes.
Reaction mixture was stirred for 2 h at rt. DCM was evaporated and the crude product was
purified by column chromatography (DCM/hexane 1:1). Compound 7 was obtained as a
1
colorless solid (520 mg, 39%). H NMR (400 MHz, CDCl ): 7.33 (1H, s), 7.41–7.50 (5H, m),
3
1
3
7
.58–7.63 (2H, m), 8.28–8.34 (2H, m). C NMR (101 MHz, CDCl ): 87.37, 121.83 (2C),
3
125.59 (2C), 126.41 (2C), 129.07 (2C), 130.56, 136.35, 145.91, 150.50, 155.07. IR (CHCl3):
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119 w, 3088 w, 3071 vw, 3032 w, 1788 vs, 1772 s, sh, 1619 m, 1595 m, 1530 vs, 1492 s, 1456
m, 1349 vs, 1317 m, 1296 m, 1232 vs, sh, 1178 m, sh, 1165 m, 1111 m, 1105 w, sh, 1078 m,
1054 s, 1029 m, 1014 m, 1002 w, 978 s, 920 w, 872 s, 854 s, 830 vw, 708 s, 695 m, sh, 680 w,
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ꢀ1
+
6
26 vw, 618 vw, 530 vw, 495 w, 403 w cm . ESI MS: 329 ([M + Na] ). HR ESI MS: calcd for
C H O NClNa 330.01397; found 330.01367.
14
10
5
(
((4-Nitrophenyloxy)carbonyl)oxy)(phenyl)methyl pivalate (8). Compound 7 (100 mg, 0.325
mmol) and mercury pivalate (157 mg, 0.390 mmol, 1.2 equiv.) were dissolved in anhydrous
DCM (6 mL). Reaction mixture was stirred under inert atmosphere at rt overnight (16 h). DCM
(
10 mL) was added and reaction mixture was washed with sat. NaHCO (10 mL), brine (10 mL),
3
organic phase was dried over MgSO and DCM was evaporated. The product 8 (115 mg, 95%)
4
1
was used in the next step without purification. H NMR (400 MHz, CDCl ): 1.28 (9H, s), 7.38–
3
1
3
7
.43 (2H, m), 7.44–7.50 (3H, m), 7.57–7.60 (1H, m), 7.61 (1H, s), 8.23–8.33 (2H, m). C NMR
(
101 MHz, CDCl ): 27.02 (3C), 39.11, 93.80, 121.86 (2C), 125.47 (2C), 126.84 (2C), 128.97
3
(
2C), 130.48, 134.39, 145.69, 150.73, 155.32, 176.44. IR (CHCl ): 3118 w, 3087 w, 3072 w,
3
3031 m, 2980 m, 2875 w, 1775 vs, 1747 s, 1618 m, 1595 m, 1529 vs, 1493 s, 1480 m, 1459 m,
399 m, 1365 m, 1349 vs, 1279 vs, 1248 vs, 1165 s, 1123 vs, 1112 s, sh, 1030 s, 1014 m, 1003
m, 970 s, br, 943 s, 918 m, 865 s, 860 s, 832 w, 697 s, 682 w, 633 w, 619 vw, 530 vw, 495 w,
1
ꢀ1
+
403 vw cm . ESI MS: 396 ([M + Na] ). HR ESI MS: calcd for C H O NNa 396.10537; found
19
19
7
396.10546.
2
-(((4-Nitrophenyloxy)carbonyl)oxy)propan-2-yl pivalate (11). 2ꢀChloropropanꢀ2ꢀyl (4ꢀ
59
nitrophenyl) carbonate 10 (300 mg, 1.16 mmol) was dissolved in anhydrous DCM (15 mL).
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Mercury pivalate (559 mg, 1.39 mmol, 1.2 equiv.) was added and the reaction mixture was
stirred overnight (20 h) at rt under inert atmosphere. The solid precipitate (HgCl ) was removed
2
by filtration, DCM (15 mL) was added and organic phase was washed with sat. NaHCO (15
3
0
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mL), sat NaCl (15 mL), dried over MgSO , solvent was removed under reduced pressure and the
4
1
product 11 was obtained as a light yellow oil (301 mg, 80%). H NMR (400 MHz, CDCl ): 1.20
3
1
3
(
9H, s), 1.91 (6H, s), 7.34–7.40 (2H, m), 8.24–8.30 (2H, m). C NMR (101 MHz, CDCl ):
3
25.28 (2C), 27.02 (3C), 39.59, 107.71, 121.98 (2C), 125.40 (2C), 145.50, 149.07, 155.41,
175.97. IR (CHCl ): 3031 w, 2976 w, 2875 w, 1777 m, 1736 m, 1618 w, 1595 w, 1528 mꢀs,
3
1493 m, 1481 w, 1439 w, 1396 w, 1376 w, 1349 m, 1322 w, 1264 m, 1191 m, 1112 vs, 1094 s,
ꢀ1
+
sh, 1030 w, 980 w, 859 m, 682 vw, 491 vw cm . ESI MS: 348 ([M + Na] ). HR ESI MS: calcd
for C H O NNa 348.10537; found 348.10543.
1
5
19
7
Isopropyl
6-diazo-5-oxo-2-((((pivaloyloxy)methoxy)carbonyl)amino)hexanoate
(13a).
Compound 4a (320 mg, 1.17 mmol) was suspended in anhydrous DCM (6 mL). The reaction
47
mixture was cooled to 0 °C and compound 12 (250 mg, 1.17 mmol, 1 equiv.) in anhydrous
DCM (3 mL) was added dropwise. The mixture was stirred for 15 minutes at 0 °C and then 2 h at
rt. Solvent was removed under reduced pressure and column chromatography of the residue
1
(
EtOAc/hexane 1:2, R 0.21) yielded the desired compound 13a (175 mg, 40%) as yellow oil. H
f
NMR (400 MHz, CDCl ): 1.19 (9H, s), 1.23 (3H, d, J = 6.2), 1.24 (3H, d, J = 6.2), 1.90–2.05
3
(1H, m), 2.14–2.25 (1H, m), 2.31–2.51 (2H, m), 4.28 (1H, td, J = 8.2, 4.7), 5.03 (1H, hept, J =
1
3
6
.2), 5.27 (1H, bs), 5.65 (1H, d, J = 8.1), 5.69 (1H, d, J = 5.7), 5.73 (1H, d, J = 5.7). C NMR
(
101 MHz, CDCl ): 21.79, 21.81, 26.97, 36.34, 38.86, 53.63, 54.90, 69.75, 80.33, 154.40,
3
22
1
71.01, 177.51, 193.43. Optical rotation: [α] + 13.0° (c 0.184, CHCl ). IR (CHCl ): 3424 m,
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354 w, br, 3116 w, 2984 s, 2937 m, 2875 s, 2110 vs, 1747 vs, 1730 vs, sh, 1642 s, 1512 s, 1481
ꢀ1
m, 1466 m, 1453 m, 1377 s, 1282 s, 1182 m, 1145 s, 1105 s, 994 s, 942 m, cm . ESI MS: 394
+
([M + Na] ). HR ESI MS: calcd for C H O N Na 394.15847; found 394.15855.
1
6
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Isopropyl 6-diazo-5-oxo-2-(((1-(pivaloyloxy)ethoxy)carbonyl)amino)hexanoate (13b). This
4
7
1
compound was prepared according to a procedure previously described in detail. Yields and H
1
3
NMR and C NMR spectra were in agreement with the published data.
Isopropyl 6-diazo-2-(((2-methyl-1-(pivaloyloxy)propoxy)carbonyl)amino)-5-oxohexanoate
(13c). Compound 4c (399 mg, 1.27 mmol, 0.9 equiv.) was suspended in anhydrous DCM (7 mL).
47
The reaction mixture was cooled to 0 °C and compound 12 (300 mg, 1.41 mmol, 1 equiv.) in
anhydrous DCM (3 mL) was added dropwise. The mixture was stirred for 15 minutes at 0 °C and
then 2 h at rt. Solvent was removed under reduced pressure and column chromatography of the
residue (EtOAc/hexane 1:2, R 0.30) yielded 13c (285 mg, 54%) as yellow oil (mixture of two
f
1
st
stereoisomers 1:1). H NMR (400 MHz, CDCl , 1 stereoisomer): 0.94 (6H, d, J = 6.8), 1.16
3
(9H, s), 1.23 (6H, t, J = 6.3), 1.83–2.50 (4H, m), 4.22–4.31 (1H, m), 5.02 (1H, hept, J = 6.8),
13
st
5
.29 (1H, bs), 5.48 (1H, d, J = 8.3), 6.52 (1H, d, J = 4.9). C NMR (101 MHz, CDCl , 1
3
stereoisomer): 16.40, 16.54, 27.00, 28.05, 31.87, 36.29, 38.96, 53.38, 54.82, 69.64, 94.21,
1
nd
154.28, 171.31, 176.56, 193.87. H NMR (400 MHz, CDCl , 2 stereoisomer): 0.93 (6H, d, J =
3
6.8), 1.18 (9H, s), 1.22 (6H, t, J = 6.3), 1.83–2.50 (4H, m), 4.22–4.31 (1H, m), 5.00 (1H, sept, J
1
3
=
6.8), 5.37 (1H, bs), 5.45 (1H, d, J = 8.3), 6.48 (1H, d, J = 4.9). C NMR (101 MHz, CDCl ,
3
nd
2
1
stereoisomer): 16.37, 16.54, 26.98, 27.74, 31.91, 36.55, 38.92, 53.54, 54.82, 69.66, 93.87,
22
54.22, 171.17, 176.81, 193.58. Optical rotation: [α] + 11.5° (c 0.261, CHCl ). IR (CHCl ):
D
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428 m, 3116 w, 2982 s, 2936 m, 2878 m, 2110 vs, 1741 vs, br, 1731 vs, sh, 1641 s, 1508 s,
480 m, 1463 m, 1400 m, sh, 1385 s, sh, 1377 s, 1365 s, sh, 1281 s, 1231 s, 1183 m, 1146 s,
ꢀ1
+
105 s, 990 s, 941 m cm . ESI MS: 436 ([M + Na] ). HR ESI MS: calcd for C H O N Na
1
9
31
7
3
0
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4
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7
8
9
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36.20542; found 436.20553.
Isopropyl 6-diazo-5-oxo-2-(((phenyl(pivaloyloxy)methoxy)carbonyl)amino)hexanoate (13d).
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Compound 8 (817 mg, 2.19 mmol) was dissolved in anhydrous DMF (15 mL). Compound 12
(700 mg, 3.18 mmol, 1.5 equiv.) in anhydrous DMF (7 mL) was added dropwise. Reaction
mixture was stirred at rt under inert atmosphere for 5 h. DMF was evaporated and column
chromatography of the residue (EtOAc/hexane 1:2, R 0.18) yielded product 13d (648 mg, 66%)
f
1
as a light yellow oil (mixture of two stereoisomers 1:1). H NMR (400 MHz, CDCl ): 1.17–1.31
3
(15H, m), 1.90–2.06 (1H, m), 2.12–2.31 (1H, m), 2.31–2.54 (2H, m), 4.27–4.36 (1H, m), 5.03
(1H, hept, J = 6.3), 5.29 (1H, bs), 5.59 (1H, d, J = 8.1), 7.36–7.42 (3H, m), 7.46–7.51 (2H, m),
1
3
7.61 (s, 1H). C NMR (101 MHz, CDCl ): 21.82 (2C), 26.99 (3C), 27.70, 36.35, 38.99, 53.55,
3
5
4.90, 69.79, 90.93, 126.57 (2C), 128.66 (2C), 129.64, 135.95, 153.82, 171.10, 176.33, 193.51.
22
Optical rotation: [α] + 12.5° (c 0.246, CHCl ). IR (CHCl ): 3425 w, 3116 w, 3098 vw, 3070
D
3
3
vw, 3029 m, 2984 m, 2937 m, 2875 w, 2110 s, 1735 vs, br, 1641 s, 1590 w, 1507 s, 1480 m,
457 m, 1398 m, 1377 s, 1367 s, sh, 1366 s, sh, 1280 s, 1182 m, 1146 s, sh, 1133 s, 1105 s, 1085
1
ꢀ1
+
m, 1057 s, 1027 s, 1003 m, 942 m, 918 w, 697 m, 619 vw cm . ESI MS: 470 ([M + Na] ). HR
ESI MS: calcd for C H O N Na 470.18977; found 470.18985.
2
2
29
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Isopropyl 6-diazo-5-oxo-2-((((2-(pivaloyloxy)propan-2-yl)oxy)carbonyl)amino) hexanoate
(13e). Compound 11 (936 mg, 2.88 mmol) was dissolved in anhydrous DMF (18 mL) and the
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reaction mixture was cooled to 0 °C. Compound 12 (920 mg, 4.32 mmol, 1.5 equiv.) in
anhydrous DMF (6 mL) was added dropwise. Reaction mixture was stirred at 0 °C under inert
atmosphere for 5 h. DMF was evaporated and column chromatography of the residue
10
11
12
13
14
15
16
17
18
19
20
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60
1
(
EtOAc/hexane 1:2, R 0.22) yielded product 13e (955 mg, 83%) as a light yellow oil . H NMR
f
(400 MHz, CDCl ): 1.19 (9H, s), 1.23 (3H, d, J = 6.3), 1.24 (3H, d, J = 6.3), 1.80 (3H, s), 1.83
3
(
3H, s), 1.90–2.01 (1H, m), 2.14–2.27 (1H, m), 2.29–2.51 (2H, m), 4.24 (1H, dt, J = 8.3, 4.7),
1
3
5
.05 (1H, hept, J = 6.3), 5.31 (1H, bs), 5.44 (1H, d, J = 8.2). C NMR (101 MHz, CDCl ):
3
21.80, 21.82, 25.76, 25.91, 27.07 (3C), 27.79, 36.46, 39.48, 53.26, 54.87, 69.59, 105.44, 153.16,
22
1
71.31, 176.21, 193.63. Optical rotation: [α] + 12.8° (c 0.133, CHCl ). IR (CHCl ): 3430 w,
D
3
3
3
116 w, 2984 m, 2936 m, 2874 m, 2110 s, 1732 vs, br, 1641 m, 1502 s, 1481 m, 1466 m, 1462
m, 1455 m, 1452 m, 1397 m, sh, 1384 s, 1374 s, 1365 s, sh, 1198 s, 1184 s, 1147 m, sh, 1128 s,
ꢀ1
+
1
112 s, 1105 s, 1045 m, 942 w cm . ESI MS: 329 ([M + Na] ). HR ESI MS: calcd for
C H O N Na 422.18977; found 422.18982.
18 29
7
3
EcoHIV-Infected Mice. All mouse efficacy studies were conducted in full compliance with NIH
guidelines and with the approval of the Institutional Animal Care and Use Committee at the
Icahn School of Medicine at Mount Sinai. Male C57BL/6J mice at 5 weeks old (The Jackson
Laboratory, Bar Harbor, ME) were maintained on a 12 h light−dark cycle with ad libitum access
to food and water. EcoHIV chimeric virus was generated by transfecting HEK293T cells with
plasmid DNA containing the EcoHIV construct, specifically EcoNDK containing the V5C5
4
2
fragment of gp120. EcoHIV virus was then collected from the culture media, concentrated by
centrifugation, and titred by p24 ELISA (Advanced Biosciences Laboratory, Rockville, MD).
Each mouse was randomized to begin 14 (1mg/kg, i.p., q.o.d.) or saline treatment 1 day prior to
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inoculation with either EcoHIV (2x10 pg p24, i.p.) or sham inoculation with PBS (n=8/group)
6
5
as previously described. The dose of 14 was chosen based on several published efficacy studies
6
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62, 67, 68
in CNS models including multiple sclerosis, brain cancer, sindbus virus
as well as our
0
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lab’s mouse pharmacokinetic studies showing µM brain levels of 14 after 1 mg/kg systemic
6
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dosing. On day 30 postꢀinoculation (p.i.), having allowed time for stable virus propagation and
emergence of cognitive deficits, all mice began behavioral training and testing.
Radial Arm Water Maze. The radial arm water maze (RAWM) test was administered in a pool
of opaque water containing 6 swimming lanes and a hidden platform with visual cues essentially
7
0
as described. The visual cues consisted of both two and threeꢀdimensional objects that were
affixed to the side of the maze pool using clear packing tape. The twoꢀdimensional cues were 6
different brightly colored 8 ½ x 11 sheets of paper each printed with a different black shape
(
square, triangle, star, etc), and the threeꢀdimensional cues were commonly found laboratory
items (funnel, culture flask, pipet tip box, etc). Briefly, RAWM consisted of 4 learning trials
LT) of 60s and 1 postꢀtraining 60s retention test (RT) administered after 30 min rest every day
(
until test completion. Testing was considered complete when control mice reached asymptotic
performance of one error or fewer in finding the hidden platform on trials LT4 and RT. Errors
for the last three days of testing for all groups were then averaged and used for statistical
analysis. The hidden platform was rotated randomly to a different arm each test day to ensure
that mice used working memory to locate the platform. Each of the training trials began by
placing a mouse randomly into one of the 6 swimming arms and allowing the mouse to swim for
6
0s to find the hidden platform, during which time the number of errors (entering an arm without
the platform and/or 20s of immobility) and latency to locate the hidden platform were recorded.
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The retention test was performed in the same manner as the learning trials. The hidden platform
tests were followed by measuring the latency it took for treated and control mice to find a visible
platform in the same context, as a control for possible effects of treatment on animal vision,
motivation, or ability to swim to the platform.
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Viral Load. After completion of behavioral testing, all mice were sacrificed by carbon dioxide
asphyxiation after brief isoflurane anesthesia. Spleen and brain were harvested, DNA was
6
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isolated, and viral load was measured as previously described.
Briefly, total DNA was
isolated from mouse tissues by sequentially homogenizing in Trizol, mixing with DNAzol,
precipitating with ethanol, washing, and treating with NaOH. Quantitative PCR (qPCR) was then
conducted to detect EcoHIV/NDK DNA from the gag gene region using Taqman chemistry with
MGB probes with forward primer 5'–TGGGACCACAGGCTACACTAGA–3', reverse primer
5'–CAGCCAAAACTCTTGCTTTATGG–3'
and
probe
5'–
TGATGACAGCATGCCAGGGAGTGG–3' (ThermoFisher Scientific). A standard curve for
quantitation of copy number was constructed using graded numbers of a plasmid containing the
EcoNDK gag amplicon. Copy number was then normalized to cell count by amplification of
murine gapdh (Mm99999915_g1). All qPCR amplification was performed in an Applied
Biosystems 7500 instrument.
In Vitro Metabolic Stability. Metabolic stability of prodrugs were evaluated as previously
4
7
described. Briefly, prodrugs (10 ꢁM) were spiked in mouse, swine and human plasma, or swine
brain homogenate and incubated in each matrix in an orbital shaker at 37 °C. Relative prodrug
amounts at 0 and 60 min for plasma and 0, 30, and 60 min for brain were measured using liquid
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chromatography and tandem mass spectrometry (LC−MS/MS) by removing 100 ꢁL aliquots of
each mixture in triplicate, and quenching each reaction by addition of three times the volume of
ice cold acetonitrile spiked with the internal standard (losartan 5 ꢁM). The samples were
vortexed for 30 s and centrifuged at 12000g for 10 min. 50 ꢁL of each supernatant was diluted
with 50 ꢁL of water and transferred to a 250 ꢁL polypropylene vial sealed with a Teflon cap.
Prodrug detection was performed on a Thermo Scientific Accela UPLC system coupled to
Accela open autosampler on an Agilent C18 (100 × 2.1 mm id) UPLC column. The autosampler
was temperature controlled and was operated at 10 °C. The mobile phase used for the
chromatographic separation was composed of acetonitrile/water containing 0.1% formic acid and
flow rate of 0.5 mL/min for 4.5 min using gradient elution. The column effluent was monitored
using TSQ Vantage tripleꢀquadrupole massꢀpectrometric detector, equipped with an electrospray
probe set in the positive ionization mode. Samples were introduced into the ionization source
through a heated nebulized probe (350 °C). Relative prodrug amounts were measured from ratio
of peak areas of analyte to IS; percent remaining was calculated by normalizing this value at 60
min to the value obtained at 0 min.
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Protein Binding. The free 14 fraction (fu) in swine plasma and swine brain homogenate was
74
determined using a previously described ultrafiltration method. Briefly, the 14 (10 µM) spiked
plasma and/or brain homogenate and was incubated at 37°C for 30 min, following which the
samples were loaded onto an ultrafiltration column tube (Corning® SpinꢀX® UF with a MW cut
off of 30 KDa), whose base is impermeable to the plasma and brain proteins. The samples were
centrifuged for 45 minutes at 37°C and 4,000 rpm (1800 × g). The concentrations were
determined in both the filtrate and plasma before filtration, following extraction and quantified
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via LCꢀMS/MS. Fraction unbound was calculated from the equation Cu/Ct*100, where Cu was
the concentration in the ultrafiltrate and Ct was the concentration in the plasma or brain
homogenate prior to filtration.
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In Vivo Pharmacokinetics. Swine studies were conducted under a protocol approved by the
Johns Hopkins Animal Care and Use Committee. Adult, female Göttingen x Yucutan miniature
swine (Massachusetts General Hospital, MA) were housed in Johns Hopkins University facilities
accredited by the Association for Assessment and Accreditation of Laboratory Animal Care
International in compliance with the Animal Welfare Act, Animal Welfare Regulations, and the
Public Health Service Policy on the Humane Care and Use of Laboratory Animals. Animals
were maintained on a 14ꢀh light and 10ꢀh dark schedule, provided ad libitum water and a
commercial miniswine diet (Teklad, Madison, WI) with environmental enrichment
(fruit/vegetables) twice daily. Animals were individually housed while on study in order to
monitor behavior and clinical health following drug administration. Whole blood for drug
pharmacokinetic evaluation was collected from a dual lumen central venous catheter (CVC)
implanted in the external jugular vein prior to study initiation. Animals were anesthetized with a
combination of ketamine hydrochloride (20ꢀ30 mg/kg, i.m.) and xylazine (2 mg/kg, i.m.),
intubated, and maintained under isoflurane (1ꢀ2%) inhalant anesthesia. A temporary peripheral
saphenous vein catheter was placed in the hind limb to allow for anatomical separation of drug
infusion and whole blood sampling via CVC. 14, 13b, or 13d were dissolved in a sterile saline
solution containing 5% ethanol and 5% Tween 80 prior to i.v. infusion via saphenous vein
catheter over 1 hour (1 ml/min) for a final dose of 1.6 mg/kg or molar equivalent administered at
1
ml/kg (n=1/dose). Blood samples (1mL) were taken from CVC at predose, 5, 15, 30, 45, and
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0 min. Plasma was separated by low speed centrifugation at 3000g for 10 min at 4°C. CSF was
obtained from the cisterna magna using a 3.5 in x 22 gauge spinal needle (Becton Dickinson
Health Care, Franklin Lakes, New Jersey, USA) at 60 min postꢀdose. All samples were flash
frozen upon harvest and stored at ꢀ80C until bioanalysis.
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In a separate study, the above dosing procedure was repeated except that the animals
(n=2/dose) were euthanized at 60 min postꢀinfusion using Euthasol® (Virbac Animal Health,
Fort Worth, TX, USA) containing pentobarbital sodium and phenytoin sodium at a dose of 1mL
per 10 lbs of body weight and plasma and brain tissues were harvested. To obtain brain tissues, a
horizontal incision connecting both orbits was extended towards the base of the ears and skin and
subcutaneous tissue over the swine skull were removed. A reciprocating saw was used to cut
through the frontal aspect of the calvarium to create a window for intracranial access. Next, the
dura mater was cut and a 2 by 2 cm sample of full thickness frontal cortex was obtained. All
samples were flash frozen and stored at ꢀ80C until bioanalysis.
Bioanalysis. Quantitation of 14 in plasma, CSF, and brain homogenate by LCꢀMS/MS was
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performed as previously described. Briefly, 14 was extracted from plasma, CSF, and brain
samples with methanol containing glutamateꢀd (10µM ISTD) by vortexing followed by
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centrifugation 16000g for 5 min. Supernatants were aliquoted and dried at 45°C for under
vacuum for 1h. Sodium bicarbonate buffer (0.2M, pH 9.0) and dabsyl chloride (10mM) in
acetone were added to each tube, mixed, and incubated for 15 min at 60°C to derivatize. Samples
were then injected and separated on an Agilent 1290 equipped with an Agilent Eclipse plus C18
RRHD 2.1 X 100mm column over a 2.5 min gradient from 20 to 95% acetonitrile + 0.1% formic
acid and quantified on an Agilent 6520 QTOF mass spectrometer. Peak area ratio of the analyte
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to the internal standard was plotted against a 14 standard curve to yield 14 concentrations for
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Pharmacokinetic and Statistical Analysis. Area under the curve (AUC) was calculated by logꢀ
linear trapezoidal rule to the end of sample collection by noncompartmental analysis module in
WinNonlin (version 5.3, Certara, St. Louis, MO). cLogPs were calculated using ChemDraw
Professional (PerkinElmer, Waltham, MA). The halfꢀlife (t ) of prodrug metabolic stability
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from brain homogenates was estimated using the firstꢀorder equation t = 0.693/K ,
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el
where K (elimination rate constant) is the slope of linear regression from natural log percentage
el
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substrate remaining versus incubation time. For all studies, group means and standard errors
were calculated and used for statistical comparisons where appropriate. Differences in radial arm
water maze errors and latencies between groups were assessed by twoꢀway ANOVA (treatment x
trial) with postꢀhoc Tukey test. Viral load in each tissue was compared between treatment groups
by t test. Prodrug halfꢀlives were compared by oneꢀway ANOVA. For all tests, significance was
defined as p < 0.05.
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