Tunable pH-Sensitive Linker for Controlled Release

Article abstract of DOI:10.1021/acs.bioconjchem.6b00027

We have developed a novel pH-sensitive linker based on a phosphoramidate scaffold that can be tuned to release amine-containing drug molecules at various pH values. The pH-triggered phosphoramidate-based linkers are responsive to pH alone and do not require intracellular enzymatic action to initiate drug release. Key to the pH-triggered amine release from these linkers is a proximal acidic group (e.g., pyridinium or carboxylic acid) to promote the hydrolysis of the phosphoramidate P-N bond, presumably through an intramolecular general-acid type mechanism. Phosphoramidate hydrolysis is largely governed by the pK_a of the leaving amine (e.g., primary, secondary, aniline). However, the proximity of the neighboring pyridine group attenuates the stability of the P-N bond to hydrolysis, thus allowing for control over the release of an amine from the phosphoramidate center. Based on the model scaffolds examined, phosphoramidate-based linkers could be selected for particular properties for controlled-release applications such as amine type, stability under physiological conditions, or release rates at various pH values such as intracellular endosomal conditions. The tunability of the phosphoramidate scaffold is expected to find broad applicability in various controlled drug-release applications such as antibody or small-molecule drug conjugates, drug-eluting stents, prodrug activation, as well as intracellular trafficking studies in which pH changes can trigger the release of turn-on dyes.

Full text of DOI:10.1021/acs.bioconjchem.6b00027

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Article
A Tunable pH-Sensitive Linker for Controlled Release
Cindy Jan Choy, Jonathan James Geruntho, Austen Lambert Davis, and Clifford E. Berkman
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00027 • Publication Date (Web): 17 Feb 2016
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Stabilities of phosphoramidyl serine homologs at 25 C.
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Synthesis of model drug-linker phosphoramidates.
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(
A) Raw stacked NMR data for 2a (1.47 ppm) at pH 5.5. The peak at 2.58 ppm is the product of
hydrolysis. (B) Compiled and fitted data for the area of 2a over the standard (Blue) and the hydrolytic
product of 2a (red).
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Summary of stability of model drug-linker phosphoramidates at pH values 3.0, 5.5, and 7.4 at 37 C. * No
detectable hydrolysis observed over 8 hrs.
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Intramolecular general-acid mechanism of phosphoramidate hydrolysis.
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A Tunable pH-Sensitive Linker for Controlled Release
Cindy J. Choy, Jonathan J. Geruntho, Austen L. Davis, and Clifford E. Berkman*
Department of Chemistry, Washington State University, Pullman WA 99164-4630
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Corresponding author:
Corresponding author address:
Clifford E. Berkman, Ph.D.
Washington State University
Department of Chemistry
Pullman, WA 99164ꢀ4630
Tel: 509ꢀ335ꢀ7613
Fax: 509ꢀ335ꢀ8389
Email: cberkman@wsu.edu
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Abstract
We have developed a novel pHꢀsensitive linker based on a phosphoramidate scaffold that
can be tuned to release amineꢀcontaining drug molecules at various pH values. The pHꢀtriggered
phosphoramidateꢀbased linkers are responsive to pH alone and do not require intracellular
enzymatic action to initiate drug release. Key to the pHꢀtriggered amine release from these
linker is a proximal acidic group (e.g., pyridinium or carboxylic acid) to promote the hydrolysis
of the phosphoramidate PꢀN bond, presumably through an intramolecular generalꢀacid type
mechanism. Phosphoramidate hydrolysis is largely governed by the pKa of the leaving amine
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(e.g., primary, secondary, aniline). However, the proximity of the neighboring pyridine group
attenuates the stability of the PꢀN bond to hydrolysis, thus allowing for control over the release
of an amine from the phosphoramidate center. Based on the model scaffolds examined,
phosphoramidateꢀbased linkers could be selected for particular properties for controlledꢀrelease
applications such as amine type, stability under physiological conditions, or release rates at
various pH values such as intracellular endosomal conditions. The tunability of the
phosphoramidate scaffold is expected to find broad applicability in various controlled drugꢀ
release applications such as antibody or smallꢀmolecule drug conjugates, drugꢀeluting stents,
prodrug activation, as well as intracellular trafficking studies in which pH changes can trigger
the release of turnꢀon dyes.
Keywords: Controlled drug release; antibody drug conjugates; ADC; smallꢀmolecule drug
conjugates; drugꢀeluting stents
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Introduction
The narrow therapeutic window and lack of tumor selectivity of cytotoxic chemotherapy
has prompted the emergence of targeted therapeutics that combine the toxicity of a highlyꢀpotent
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chemotherapeutic drug with the specificity of a chemical or biological targeting molecule. At
present, the most successful example of targeted drug delivery is represented by antibody drug
conjugates (ADCs). Currently, there are over 250 clinical trials in which over 40 unique ADC
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agents are being developed. An essential aspect for many effective targeted drug therapies is
the controlled coupling and release of a cytotoxic drug from the targeting molecule (e.g.,
antibody) upon cellular internalization or localization. While much attention within the ADC
field has been focused on consistently and reliably coupling the drug to the targeting
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biomolecule,
there has been considerably less effort to develop controlled, siteꢀspecific
chemical release of the cytotoxic drug. The two most commonly employed cleavable linkers in
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the field of ADC is the valineꢀcitrulline linker
used in the Seattle Genetics' brentuxumab
vendotin, which relies on enzymatic cleavage by cathepsin B in lysosomes, and ImmunoGen’s
succinimidyl 4ꢀ[N-maleimidomethyl]cyclohexaneꢀ1ꢀcarboxylate (SMCC) linker technology.
ImmunoGen’s SMCC linker technology is not cleavable and therefore limits its use to the few
drugs that are active as a SMCCꢀdrug conjugate once the antibody has been completely digested
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in the lysosome. In short, linker technologies that have been developed for ADCs necessitate
that they be both internalized and processed in the lysosome to release the cytotoxic drug. Due
to these requirements, the utility of these linkers is limited to diseases for which the drug
conjugate is both internalized by endocytosis and is transported to lysosomes. Furthermore, such
linkers do not allow for release under microenvironments of tumor cells to take advantage of the
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bystander effect.
Moreover, these linkers cannot be more broadly applied for general
controlled release applications such as drug eluting stents.
Other nonꢀproprietary cleavable linker technologies do exist that rely on either
glutathione reduction of a disulfide linkage or hydrolytic degradation under acidic conditions.
The disulfide linker relies on the higher intracellular glutathione concentration compared to
extracellular glutathione level, however, it has been shown to be unstable in circulation due to
the presence of glutathione in blood. The most well known acidꢀlabile linker is the hydrazide
linker. Pfizer’s ADC, Mylotarg, was developed using the hydrazide linkage but the drug was
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withdrawn from the market, presumably due to premature release prior to full internalization.
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In summary, the current cleavable linker technology for selective drug release is not
broadly applicable for targeted therapeutic and controlledꢀdrug release applications. As
previously stated, the present approach to cleavable linker technology relies on one of three basic
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modes of activation
: 1) proteolytic action by lysosomal enzymes, 2) glutathione reduction of
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a disulfide linkage, or 3) hydrolytic degradation under acidic conditions. All three chemistries
are designed around very specific applications and offer very limited control over the release
characteristics; tunability of the cleavage kinetics often is dependent only upon steric bulk
incorporated into the linker. As a consequence, these linkers have significant limitations,
particularly in controlled release applications such as drugꢀeluting stents.
In our ongoing studies with Nꢀphosphorylated amino acids, we recently discovered a
novel scaffold that could serve as a tunable pHꢀsensitive linker (Figure 1) that can be employed
for the triggered release of an amineꢀcontaining drug. This pHꢀprogrammable phosphoramidate
linker scaffold represents the first chemical architecture that is responsive to structural
modifications to provide a broad range of drug release profiles that can be matched to specific
application needs; an attribute that is absent in current linker
technologies. The ability to “tune” the drug release kinetics of a
pHꢀtriggered phosphoramidate linker is advantageous for
intracellular (e.g., endosomal and lysosomal) drug release to
advance the field of targeted drug therapy as well as controlled
release applications in general.
Specifically, preliminary work in our laboratory
demonstrated that the hydrolytic stability of a phosphoramidate
scaffold could be tuned by altering the distance between a
neighboring weakly acidic moiety and the phosphorus center.
During the development and evaluation of various
phosphoramidate scaffolds (Figure 1), we found that
phosphoramidates linked to the sideꢀchain hydroxyl group of
serine were unusually labile below pH 6.5 (Figure 1C) but stable
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at physiological pH and above.
Based upon the greater
observed acid stability of phosphoryl homoserine (Figure 1B)
and phosphoryl hydroxypropylglycine (Figure 1A) homologs, it
Figure 1. Stabilities of
phosphoramidyl serine
o
homologs at 25 C.
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was concluded that the alphaꢀcarboxylate of serine promoted the acid lability of O-phosphoryl
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serine phosphoramidates (Figure 1C).
From these observations, we hypothesized that any
weakly acidic group proximal to the central phosphoramidate core could activate the linker
through internal assistance. This assertion led us to investigate whether analogs and homologs
that contained a proximal protonated pyridine group (similar pKa to the carboxylic acid) would
demonstrate similar trends in hydrolytic stability associated with structural adjustments such as
proximity. The focus of this study was aimed at preparing and evaluating a series of cleavable
linkers based on phosphoramidates of 2ꢀ(2ꢀpyridinyl)ethanol (2a-d), 2ꢀ(3ꢀpyridinyl)ethanol (3a-
d), 3ꢀ(2ꢀpyridinyl)propanol (4a-d), and 3ꢀ(3ꢀpyridinyl)propanol (5aꢀd), and monitoring the rate
of hydrolysis of the PꢀN bond of model amineꢀcontaining drugs at various pH conditions at 37
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o
C.
Results and Discussion
A library of representative model drugꢀlinker conjugates based on an acidꢀsensitive
phosphoramidate core was designed, prepared, and evaluated for stability at various pH values
(3.0 – 7.4). The rationale for the library design was to determine whether phosphoramidate
analogs that contained a proximal pyridine group was susceptible to similar hydrolytic stability
as previously observed for phosphoramidate analogs consisting of a carboxylate group (Figure
1
). Pyridine alcohols 7-10 were chosen to test the tunability via the proximity of the pyridinyl
nitrogen to the phosphorus center. Amines a-d were selected as representatives of
o
o
chemotherapeutic drugs typical found in ADCs (e.g., 1 amine, 2 amine, and aniline
approximating doxorubincin, MMAE, and a selfꢀimmolating spacer, respectively).
A simple parallel synthetic method, amendable to lateꢀstage diversification, was
employed to synthesize the model drugꢀlinker conjugates (Scheme 1). The common phosphites
and phosphoramidate intermediates were obtained as previously described with minor
2
0
modifications. Briefly, transesterification of diphenyl phosphite was accomplished by treating
it with commercially or readily available pyridinyl alcohols (6-10), followed by benzyl alcohol to
yield phosphites 12ꢀ15. Phosphites 12ꢀ15 were subjected to AthertonꢀTodd conditions with the
appropriate amines (aꢀd) to yield phosphoramidates (17aꢀd – 20aꢀd). Hydrogenolytic
deprotection yielded the final compounds (2aꢀd – 5aꢀd) in quantitative yield. The control
compounds 1 and 1’ were synthesized using similar methodology with the exception that
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fluorenylmethanol (Fm) was used in place of benzyl alcohol; deprotection was achieved by mild
basic hydrolysis with 1M LiOH (aq).
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Scheme 1: Synthesis of model drugꢀlinker phosphoramidates.
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Once prepared, the stability of the model drugꢀlinker conjugates were monitored by P
NMR at pH values of 3.0, 5.5, and 7.4. The NMR array data was compiled using Mnova 10.0
software (Mestrelab Research, Escondido, CA) and the hydrolytic release of the model drug
from the conjugate platform was analyzed in comparison to a constant concentration of the
internal standard triphenylphosphine oxide (Figure 2A). The peak area for each compound was
normalized to the internal standard over 8 h and the rate of hydrolysis followed first order
kinetics (Figure 2B).
As we had initially hypothesized, the pyridinium group, like that of the carboxylic acid,
in the model drugꢀlinker phosphoramidates can serve as a neighboring proton donor (Figure 1)
to assist hydrolysis and release an amine from the phosphoramidate core. Analysis of the
hydrolytic stability of the model drugꢀlinker compounds (Figure 3) reveals that there are two key
factors that influence the hydrolysis rate: 1) the amine type (primary, secondary, aniline, etc.)
and 2) the proximity of the pyridine nitrogen to the phosphorous center. With respect to the first
A
B
Figure 2. (A) Raw stacked NMR data for 2a (1.47 ppm) at pH 5.5. The peak at 2.58 ppm is the
product of hydrolysis. (B) Compiled and fitted data for the area of 2a over the standard (Blue) and
the hydrolytic product of 2a (red).
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factor, we noticed that the amine type has a dominating influence on the hydrolytic stability. For
example, when comparing compounds 2a-d in which the linker is constant (nꢀ1; Y=N; Z=C) but
the amine varied from aniline (2a), secondary aniline (2b), primary amine (2c), and secondary
amine (2d), the trend for hydrolysis rates of the phosphoramidate PꢀN bond was Nꢀ
methylphenethylamine > Nꢀmethylaniline > phenethyl amine > aniline (Figure 3). This trend
held true across the different model drugꢀlinker molecules (Figure 3), which led us to conclude
that the type of the leaving amine is a contributing factor to the hydrolytic rate.
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A neighboring acidic group is essential for the unusual instability of the phophoramidate
PꢀN bond at mild pH conditions, presumably near the pKa of the neighboring acidic group. This
is evident when comparing compounds 2d and 4d to the respective control compounds 1 and 1’,
which lack pyridine nitrogen atoms. An extreme example of this stability difference owing to
the presence of the neighboring acidic group is found when comparing 2d and 1; 2d rapidly
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hydrolyzes (<5 min; within the time to prepare the sample and acquire initial FID scans in P
NMR) in pH 7.4 buffer while 1 is stable (~41 h).
The proximity of the neighboring acidic group (e.g., pyridinium) to the phosphoramidate
center influences the rate hydrolysis of the PꢀN bond. In the series of compounds examined, this
is observed for both the position of the nitrogen in the pyridine ring as well as the number of
methylene units separating the pyridine and the phosphoramidate center. For example, a
representative trend stability at various pH conditions is illustrated by comparing the series of
compounds 2cꢀ5c. This trend is also consistent with the hydrolytic stability data for the
remaining series of compounds (Figure 3).
The data herein appear to be consistant with an intramolecular generalꢀacid mechanism to
explain the influence that the neighboring acidic group exerts on the hydrolysis rate of the
phosphoramidate PꢀN bond (Scheme 2). While intramolecular generalꢀacid mechanisms are
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known to facilitate various chemical transformations
, none have been reported for the
hydrolysis of the PꢀN bond in phosphoramidates. It is understood that at physiological
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conditions, phosphoramidates have zwitterionic character.
In buffered conditions near the
pKa of the neighboring pyridinium group, it is reasonable that this group transfers a proton to the
phosphoramidate resulting in a more electrophilic phosphorus center susceptible to nucleophilic
attack by water. Based on our data, it generally appears that the stability of the
phosphoramidates toward hydrolysis under mild pH conditions is correlated to the proximity of
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the neighboring pyridinium group for efficient proton transfer through a transient cyclic species.
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Figure 3: Summary of stability of model drugꢀlinker phosphoramidates at pH values 3.0, 5.5, and 7.4 at 37 C.
*
No detectable hydrolysis observed over 8 hrs.
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While it is generally assumed that phosphoramidates are inherently unstable to acidic
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conditions,
our observations suggest that there is a spectrum of stability that is dependent on
3
0
both the nature of the amine ligand of the phosphoramidate, and the presence and proximity of
an intermolecular acidic group. With the models examined in this study, which were designed
explore this spectrum of hydrolytic instability, we found that the aniline based phosphoramidates
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(
2a, 3a, 4a, and 5a) in particular, were very stable. In general, the anilineꢀbased compounds
exhibited little to no detectable hydrolysis at pH 7.4 over 8 h, the most stable of which was 5a as
expected. The exception in the aniline series was 2a, however its hydrolytic susceptibility is
believed to be due to the generally observed pronounced effects of intramolecular general acid
assistance from the pyridinium group of the 2ꢀ(2ꢀpyridinyl)ethyl phosphoramidate scaffold.
Indeed, all models based on this scaffold (2aꢀ2d) were uniquely unstable at all three pH
conditions examined.
Scheme 2: Intramolecular generalꢀacid mechanism of phosphoramidate hydrolysis.
Conclusions
We have observed that a proximal pyridine group promotes hydrolysis of a
phosphoramidate PꢀN bond under mildly acidic conditions, presumably through an
intramolecular generalꢀacid type mechanism. In comparison to our preliminary data with
phosphoramidyl serine homologs (Figure 1), the data also suggest that both a carboxylic acid
and a pyridinium group can function as a neighboring proton donor to promote phosphoramidate
hydrolysis. To the extent to which our models allow us to surmise, it appears that the
phosphoramidate hydrolysis observed proceeds through an intramolecular generalꢀacid
mechanism. It should be noted that phosphoramidate hydrolysis is largely governed by the pKa
of the leaving amine. However, the proximity of the neighboring pyridine group can
significantly attenuate the stability of the PꢀN bond to hydrolysis, thus allowing for control over
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the release of an amine from the phosphoramidate center.
In summary, we have demonstrated that the phosphoramidate scaffold displays a wide
range of stability at different pH values. In this study, we examined amines linked to the
phosphoramidate core as representative models for drugs or selfꢀimmolating spacers that could
be released from the phosphoramidate scaffold for relevant controlledꢀrelease applications.
Based on the data presented, candidate linkers could be selected for particular properties for
controlledꢀrelease applications such as amine type, stability under physiological conditions, or
release rates at various pH values. For example 3d could be a candidate linker scaffold for an
ADC application if the desired drug is a secondary amine (e.g., MMAE). For 3d, we did not
detect any hydrolysis at pH=7.4 over the course of 8 h but at approximate endosomal pH (pH =
0
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.5), the t for hydrolysis of the phosphoramidate PꢀN bond (and release of the amine) was 4.3
1/2
h, and would be expected to release faster once endosomalꢀlysosomal fusion occurs. However,
for smallꢀmolecule drug conjugate (SMDC) applications in which the clearance kinetics are
generally rapid in comparison to ADCs, 4d would be suitable (pH 7.4 t = 24 h; pH 5.5 t = 1.6
1
/2
1/2
h) for a secondary amine, while 2a would be a reasonable candidate linker for a SMDC
employing a conventional selfꢀimmolating spacer (pH 7.4 t = 9.2 h; pH 5.5 t = 0.7 h).
1
/2
1/2
In conclusion, the apparent tunability of our phosphoramidate scaffold is expected to find
broad applicability in various controlled drugꢀrelease applications such as antibody or smallꢀ
molecule drug conjugates, drugꢀeluting stents, prodrug activation, as well as intracellular
trafficking studies in which pH changes can trigger the release of turnꢀon dyes. While both the
carboxylic acid and a pyridinium group can function as a neighboring proton donor to promote
phosphoramidate hydrolysis, it is envisioned that the pyridineꢀbased scaffolds offer the
advantage of subtle fineꢀtuning of amineꢀrelease kinetics through substitution effects on the
pyridine ring.
Material and methods
pH Stability Studies. Samples were prepared using 5 mg of compound dissolved in 100
µL MilliꢀQ water, which was mixed with 400 µL of a 1M buffer. Buffers were chosen based for
specific pH values; 4ꢀ(2ꢀhydroxyethyl)ꢀ1ꢀpiperazineethanesulfonic acid (HEPES, pH 7.4),
succinic acid (pH 5.5), and citric acid (pH 3.0). Each buffer was prepared and equilibrated to
±
0.01 pH with 1M NaOH or 1M HCl on an AB15 Accumet Basic pH Meter equipped with an
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accuTupH Ag/AgCl pH probe (Fisher Scientific, Sommerville, NJ).
3
1
Stability studies were performed using P NMR on a Varian 400 MHz (Agilent
o
Technologies, Santa Clara, CA). The instrument was equilibrated to 37 C, with an acquisition of
o
64 scans, a relaxation delay of 1s, an observed pulse of 3.41 µs at 45 , and a preꢀacquisition
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delay of either 34 s or 184 s. The instrument was locked to an internal standard,
triphenylphosphine oxide (TPPO, 40 mM in DMSOꢀd ), in an axial capillary positioned within
6
the sample solution. Stability data was collected for 8 h, or until complete decay was observed.
3
1
After data acquisition was complete, all P chemical shifts were referenced to the internal
standard (TPPO, 27 ppm) and the phase adjusted accordingly.
NMR experiment files were loaded into the software MestReNova v10.0. Phase
correction, baselineꢀfitting, and SavitzyꢀGolay smoothing were performed in the NMR software.
Regions corresponding to the control, starting material, and the product peak were then exported
into the software IGOR Pro 6.37 (Wavemetrics, Lake Oswego, OR, USA). The intensities of
each spectra were normalized to the intensity of the control compound. A first order decaying
3
1,32
exponential fitting function (Equation 1
), using the LevenbergꢀMarquardt algorithm, was
applied to a plot of intensity versus time of the decaying parent compound. If the function
returned a successful fit, the halfꢀlife ꢀꢁ ꢄ
ꢂ
ꢃꢅ of each compound was then calculated by
multiplying the returned coefficient tau ( ) by ln 2.
τ
ꢍꢎꢍ
ꢐ
ꢏꢅ
ꢌꢀ
ꢆ(ꢇ) = ꢆ + ꢉꢊꢋ
Equation 1
ꢈ
Synthesis. All solvents used in reactions were anhydrous and obtained from commercial
1
13
31
sources or freshly distilled over calcium hydride. H, C, and P NMR spectra were recorded
1
on a Bruker 400 MHz spectrometer or a Varian 300 MHz. H NMR chemical shifts are relative
1
3
to TMS (δ=0.00 ppm), CDCl (δ=7.26 ppm), CD OD (δ=3.31 ppm) or D O (δ=4.79 ppm).
C
3
3
2
NMR chemical shifts were relative to CDCl (δ=77.23 ppm) or CD OD (δ=49.00 ppm). High
3
3
Resolution Mass spectrometry (HRMS) spectra were obtained on an Applied Biosystems 4800
MALDIꢀ TOF/TOF mass spectrometer (Applied Biosystems, Foster City, CA).
General procedure for synthesis of Phosphites (11-15): Alcohol (6-10) (0.529 mmol) was
dissolved in 2 mL of freshly distilled pyridine and added dropwise via cannula to a stirring
solution of diphenylphosphite (0.634 mmol) in 3 mL of freshly distilled pyridine. The resulting
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solution was stirred for 2 h under a stream of Ar(g) followed by the dropwise addition of benzyl
alcohol (1.587 mmol) via syringe and stirred overnight. After stirring 15 h, the crude reaction
mixture was concentrated to yield an oil. The phosphite (6-10) was isolated by silica column
chromatography using 10ꢀ30% MeOH:CH Cl as the eluent (40ꢀ67% yield).
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General Atherton-Todd procedure for synthesis of Phosphoramidates (16-20d): The
phosphite 6-10 (0.119 mmol) was dissolved in in a mixture of distilled MeCN (2 mL) and
o
CCl Br (1 mL) and stirred for 15 min at ꢀ15 C. Amine a-d (0.125 mmol) was dissolved in
3
distilled MeCN and Et N (0.369 mmol) was added dropwise to the phosphite solution and stirred
3
overnight. Upon completion of the reaction, the mixture was concentrated to yield a crude
oil. The product 17a-20d was obtained by silica column chromatography using 10ꢀ30%
MeOH:DCM as the eluent (>85%).
Deprotection procedure (1-5d): Method A (for compounds 2a-5d): Potassium
bicarbonate (1 equiv. per benzyl ester) was added to a stirring solution of phosphoramidate 17a-
2
0d in 1:2 dioxane:MQ H O containing 10% Pd/C (0.05 eq). The resulting solution was purged
2
o
and subjected to hydrogen atmosphere (balloon pressure) and stirred overnight at 4 C. The
mixture was filtered through a 0.2 micron filter and lyophilized down to yield the final
compound (2a-5c) in quantitative yield. Method B (for compounds 1 and 1’): 1M LiOH (aq)
(1.6 equiv. per protecting group) was added to a stirring solution of phosphoramidate 16 or 16’ in
methanol. The resulting solution was stirred 16 h at room temperature. The mixture was filtered
through a 0.2 micron filter, concentrated by rotary evaporation, and dried overnight in vacuo to
yield a crude solid. Excess LiOH was removed by redissolving the crude solid in 1 mL of
anhydrous methanol. The resultant suspension was filtered again through a 0.2 micron filter.
The filtrate was concentrated by rotary evaporation and dried under vacuum overnight to yield
the final compound 1 or 1’ as a white solid.
Acknowledgements
This work was supported in part by the National Institutes of Health (R01 CA140617)
and the Washington State University Commercialization Gap Fund (CGF). The authors extend
their gratitude for technical assistance to G. Helms and W. Hiscox (WSU Center for NMR
Spectroscopy) and Gerhard Munske (WSU lab Bioanalysis and Biotechnology for Mass
Spectrometry).
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Supporting Information
NMR characterization data and table of halfꢀlives for compounds 1-5d is available in the
supporting information.
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