Second-Generation Tunable pH-Sensitive Phosphoramidate-Based Linkers for Controlled Release

Article abstract of DOI:10.1021/acs.bioconjchem.6b00422

We developed a second generation of tunable pH-sensitive linkers based on our phosphoramidate scaffold to release amine-containing drugs under acidic conditions. The pH-triggered phosphoramidate-based linkers are responsive to pH and do not require intracellular enzymatic action to initiate drug release. On the basis of the model scaffolds examined, phosphoramidate-based linkers were 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. Key to the pH-triggered amine release from these linker is a proximal carboxylic acid to promote 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. However, the proximity of the neighboring carboxylic acid attenuates the stability of the P-N bond to hydrolysis, thus allowing for control over the release of an amine from the phosphoramidate center. In addition, we observed that the Thorpe-Ingold effect and rigidification of the scaffold could further enhance the rate of release. Esterification of the neighboring carboxylic acid was found to protect the scaffold from rapid release at low pH. This latter observation is particularly noteworthy as it suggests that the phosphoramidate-based drug-conjugate scaffold can be protected as an ester prodrug for oral administration. While the tunability phosphoramidate linkers is attractive for applications in intracellular trafficking studies in which pH changes can trigger release of turn-on dyes, antibody drug conjugates, small-molecule drug conjugates, and drug eluting stents (DES), the promise of oral delivery of drug conjugates is expected to have broad impact in controlled release applications.

Full text of DOI:10.1021/acs.bioconjchem.6b00422

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Article
Second-Generation of Tunable pH-Sensitive
Phosphoramidate-Based Linkers for Controlled Release
Cindy Jan Choy, Corinne Rose Ley, Austen Lambert Davis, Brian Scott
Backer, Jonathan James Geruntho, Brian H Clowers, and Clifford E. Berkman
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00422 • Publication Date (Web): 26 Aug 2016
Downloaded from http://pubs.acs.org on August 27, 2016
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Second-Generation of Tunable pH-Sensitive Phosphoramidate-Based Linkers for
Controlled Release
Cindy J. Choy†, Corinne R. Ley†, Austen L. Davis, Brian S. Backer, Jonathan J. Geruntho,
Brian H. Clowers, and Clifford E. Berkman*
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Washington State University, Department of Chemistry, PO Box 644630
Pullman, WA 99164-4630
†
Coꢀfirst authors
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 secondꢀgeneration of tunable pHꢀsensitive linkers based on our
phosphoramidate scaffold to release amineꢀcontaining drugs under acidic conditions. The pHꢀ
triggered phosphoramidateꢀbased linkers are responsive to pH and do not require intracellular
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enzymatic action to initiate drug release.
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. Key to the pHꢀtriggered amine
release from these linker is a proximal 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.
However, the proximity of the neighboring carboxylic acid attenuates the stability of the PꢀN
bond to hydrolysis, thus allowing for control over the release of an amine from the
phosphoramidate center. In addition, we observed that the ThorpeꢀIngold effect and
rigidification of the scaffold could further enhance the rate of release. Esterification of the
neighboring carboxylic acid was found to protect the scaffold from rapid release at low pH. This
latter observation is particularly noteworthy as it suggests that the phosphoramidateꢀbased drugꢀ
conjugate scaffold can be protected as an ester prodrug for oral administration. While the
tunability phosphoramidate linkers is attractive for applications in intracellular trafficking studies
in which pH changes can trigger the release of turnꢀon dyes, antibody drug conjugates (ADC),
smallꢀmolecule drug conjugates (SMDC) and drug eluting stents (DES), the promise of oral
delivery of drugꢀconjugates is expected to have broad impact in applications or controlledꢀ
release.
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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 field of targeted drug delivery is dominated by antibody drug conjugates (ADCs).
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Currently, there are over 250 clinical trials representing over 40 unique ADC agents. A critical
aspect to effective targeted drug therapies is the controlled 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
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the targeting biomolecule,
there has been considerably less effort to develop controlled, siteꢀ
specific chemical release of the cytotoxic drug. The two most common cleavable linkers in the
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field of ADC is Seattle Genetics’ valineꢀcitrulline linker,
which relies on enzymatic cleavage
succinimidyl 4ꢀ[N-
by cathepsin in lysosomes, and ImmunoGen’s
B
maleimidomethyl]cyclohexaneꢀ1ꢀcarboxylate (SMCC) linker technology. ImmunoGen’s SMCC
linker technology is not cleavable and therefore restricts its utility to the few drugs that are active
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0
as a SMCCꢀdrug conjugate once the antibody has been digested in the lysosome. These linker
technologies 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 applications for which the drug conjugate is both internalized by endocytosis and is
transported to lysosomes. In addition, such linkers do not allow for release under
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1,12
microenvironments of tumor cells to take advantage of the bystander effect.
Moreover, these
linkers cannot be more broadly applied for general controlled release applications such as drug
eluting stents.
Other nonꢀproprietary cleavable linkers exist that are labile to the action of glutathione on
a disulfide linkage or hydrolysis under acidic conditions. The disulfide linkers rely on 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 withdrawn from the market, presumably
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due to premature release prior to full internalization.
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In summary, the current cleavable linkers for selective drug release are not broadly
applicable for targeted therapeutic and controlledꢀdrug release applications. In general, linker
technology relies on one of three basic modes of activation: 1) proteolytic action by lysosomal
enzymes, 2) glutathione reduction of a disulfide linkage, or 3) hydrolytic degradation under
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acidic conditions.
All three modes are designed around specific applications and offer
limited control over the release characteristics; tunability of the cleavage kinetics often is
dependent only upon steric bulk incorporated into the linker. Consequently, these linkers have
significant limitations, particularly in controlled release applications such as drugꢀeluting stents.
We recently reported a novel phosphoramidate scaffold that can be employed to
selectively release amineꢀcontaining drugs and/or selfꢀimmolating spacers at varying rates
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dependent upon pH (Figure 1, 1ꢀ5). We envision that cleavable linkers based on this scaffold
can be tailored for specific requirements of controlledꢀrelease applications, such as amine type,
stability under physiological conditions, or release rates at various pH conditions.
Representative applications for such tunable pHꢀprogrammable cleavable linkers include
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.
In our first report, we demonstrated that
phosphoramidate hydrolysis was largely governed by the
pKa of the leaving amine (e.g., primary, secondary,
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aniline).
However, the proximity of a neighboring
pyridine group further attenuated the stability of the PꢀN
bond to hydrolysis, thus allowing for control over the
release of an amine from the phosphoramidate center. The
proximal pyridinium group promoted the hydrolysis of the
phosphoramidate PꢀN bond, presumably through an
intramolecular generalꢀacid type mechanism. The focus of
this study was to examine the expected analogous
tunability of the phosphoramide scaffold when the
neighboring proton donor was a carboxylic acid (Figure 1,
Figure 1. Phosphoramidateꢀbased pHꢀ
programmable cleavable linkers.
6
ꢀ11).
Again, we examined amines linked to the
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phosphoramidate core as representative models for drugs or selfꢀimmolating spacers that could
be released from the phosphoramidate scaffold for relevant controlledꢀrelease applications. The
specific series of cleavable linker models for this study were phosphoramidates of serine (6a-d),
threonine (7a-d and 7c´), homoserine (8a-d), 5ꢀhydroxynorvaline (9a-b), 2ꢀaminoethanol (10a-
d), 3ꢀaminopropanol (11a-b), and 2ꢀ(hydroxymethyl)benzoate (12a,c) and monitoring the rate of
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hydrolysis of the PꢀN bond of model amineꢀcontaining drugs at various pH conditions at 37 C.
Results and Discussion
A library of representative model drugꢀlinker conjugates based on an acidꢀsensitive
phosphoramidate core (Figure 1, 6ꢀ12) was prepared and evaluated for stability at various pH
values (3.0 – 7.4). The rationale for the library design was to confirm that phosphoramidates
containing a proximal carboxylic acid were susceptible to similar hydrolytic stability as was
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previously observed for phosphoramidates containing a proximal pyridinium group. Alcohols
4ꢀ17 were chosen to test the tunability via the proximity of the carboxylate to the phosphorus
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center. Alcohols 18ꢀ19 were chosen to monitor the stability of phosphoramidates lacking the
neighboring carboxylate. Amines aꢀd were selected as representatives of chemotherapeutic drugs
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typically found in ADCs (e.g., a aniline, c 1 amine, and d 2 amine to mimic a selfꢀimmolating
spacer, doxorubicin, and MMAE, respectively).
In general, alcohols (14ꢀ19) were readily prepared from commercially available amino
Scheme 1: Synthesis of model drugꢀlinker phosphoramidates.
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acids and/or amines. To prepare the phosphoramidate models for this study, a common method
was employed to install the phosphorus core and enable lateꢀstage diversification with
representative amines (Scheme 1). Transesterification of diphenyl phosphite by sequential
addition of the functionalized alcohol then benzyl alcohol provided Hꢀphosphonates 20ꢀ25,
which were then subjected to AthertonꢀTodd conditions to generate the protected
phosphoramidate intermediates 26aꢀd through 31aꢀd. Global deprotection of these precursors by
hydrogenolysis yielded the model drugꢀlinker conjugates 6ꢀ11. Compounds 12 b-c were
synthesized analogously as 6ꢀ11 starting with ethyl 2ꢀ(hydroxymethyl)benzoate and
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fluorenylmethanol. Deprotection was achieved with 1M LiOH as previously described.
Once prepared, the stability of the model drugꢀlinker conjugates 6-12 was monitored by
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P NMR at pH 3.0, 5.5, and 7.4 at 37 C. The NMR array data was compiled using Mnova 10.0
software (Mestrelab Research, Escondido, CA), and the hydrolytic drug release of the respective
amine was analyzed in comparison to a constant concentration of the external standard
triphenylphosphine oxide (Figure 2A). The peak area for each compound was normalized to the
standard over 8 h, and the rate of hydrolysis followed first order kinetics (Figure 2B).
Figure 2. (A) Raw stacked NMR data for 7a (1.17 ppm) at pH 5.5. The peak at 1.79 ppm is the hydrolytic
product. (B) Compiled and fitted data for the area of 7a (blue) normalized to the standard and its hydrolytic
product (red).
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As expected, the hydrolysis rates of our secondꢀgeneration models (Figure 3) were
largely influenced by two distinct elements: 1) the type of amine (e.g. primary, secondary,
aniline, secondary aniline), and 2) the proximity of the carboxylic acid to the phosphorus center.
Previously we reported that the structure of the amine impacts hydrolysis of the PꢀN bond such
that Nꢀmethylphenethylamine > Nꢀmethylaniline > phenethylamine > aniline. This trend was
again observed for each series of model drugꢀlinker phosphoramidates in this study. For
example, when comparing compounds 8a-d; hydrolysis at pH 5.5 is most rapid for 8d (6.6 h)
while no detectable hydrolysis was observed for 8a over 8 h. The trend was further illustrated by
hydrolysis of control compounds 10a-d, which lack the carboxylic acid, but displayed notable
decomposition at pH 3.0 where 10d > 10b > 10c > 10a.
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O
P
H
N
N
N
O
*
*
*
1
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11.4
OH
O
0.34
0.31
0.26
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O
O
O
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N
H
10d
9d
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OH
HO
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C
O
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O
P
N
H
9.49
OH
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O
H
N
73.4
N
N
O
P
8d
6.22
OH
HO
HO
2
C
O
2
C
O
O
1.54
O
P
N
H
0.08
0.00
6
d
OH
2
HO C
7
.40
H
N
O
P
O
12c
0.36
0.00
OH
O
H
N
H
N
O
*
*
P
1
1c
OH
O
2.23
O
H
N
O
O
*
*
P
N
H
10c
9c
OH
2.48
HO
2
C
O
H
N
O
*
O
P
N
H
30.0
29.8
OH
1.46
H
N
O
H
N
9
4.8*
O
P
8c
OH
HO
2
C
O
0.89
MeO
O
2
C
O
H
N
O
P
*
N
H
7c'
7c
17.1
6.10
OH
HO
2
2
C
C
O
O
H
N
O
2.32
O
0.09
0.00
P
N
H
OH
HO
H
N
O
13.1
O
P
N
H
6c
0.76
0.00
OH
2
HO C
7.17
O
P
N
N
O
12b
11b
10b
8b
0.98
0.24
OH
O
H
N
*
O
P
20.8
OH
O
0.32
0.50
0.23
O
O
57.4
N
O
P
N
H
26.3
OH
O
H
N
48.5
N
N
O
P
16.0
OH
HO
HO
2
C
O
2
C
O
O
O
5.81
P
N
H
0.16
0.00
6
b
OH
*
*
H
N
O
H
N
O
11a
10a
8a
P
29.9
OH
O
O
H
N
O
*
O
P
N
H
OH
37.1
H
N
O
H
N
*
*
O
P
16.5
OH
HO
2
C
O
HO
2
C
O
O
H
N
O
29.1
O
P
N
H
7a
2.02
0.00
OH
HO
2
C
H
N
O
94.0*
O
P
N
H
6a
17.00
OH
0.20
0
.00
12.00
24.00
36.00
48.00
60.00
72.00
84.00
96.00
Half life in Hour (t1/2
)
pH 7.4
pH 5.5
pH 3.0
o
Figure 3: Stability of model drugꢀlinker phosphoramidates at pH 3.0, 5.5, and 7.4 (37 C). *No detectable
hydrolysis observed over 8 hrs.
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While phosphoramidates are generally unstable under highly acidic conditions, the
presence of a neighboring acidic group is essential for the unusual lability of the PꢀN bond under
mild pH conditions. This is evident by the rapid hydrolysis of 6a-d and 8a-d relative to their
respective control compounds 10a-d and 11c-d, which lack a proximal carboxylic acid. The
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serineꢀderived compounds 6c-d were particularly unstable as the P FID could not be acquired
at pH 3.0 due to complete hydrolysis of the phosphoramidates in less than 5 min. Similarly,
compounds 8cꢀd show an approximately 2ꢀfold increase in the rate of hydrolysis relative to the
control compounds 11cꢀd, which lack a proximal carboxylic acid.
The stability profiles of our model drugꢀlinker conjugates support our hypothesis that the
proximity of the neighboring acidic group (e.g., carboxylic acid) to the phosphorus centers
strongly influences the rate of hydrolysis of the P–N bond. As the distance is increased in the
phosphoramidates based on serine (6aꢀd, n=1), homoserine (8aꢀd, n=2), and 5ꢀhydoxynorvaline
(
9cꢀd, n=3), the hydrolytic stability also increases at all pH values examined. This was
previously observed in both our preliminary studies with carboxylateꢀcontaining
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phosphoramidates as well as our firstꢀgeneration pyridinium scaffolds. In fact, extending the
distance of the carboxylate from 1 to 3 methylene units (e.g., 9cꢀd) resulted in a model drugꢀ
linker conjugates with similar stability profile as the control compound that lack a neighboring
carboxylic acid.
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As discussed in our previous report, the data herein appear to be consistant with an
intramolecular generalꢀacid mechanism to explain the influence that the neighboring carboxylate
exerts on the hydrolysis rate of the phosphoramidate PꢀN bond. We initially proposed such a
1
8
mechanism for the pryidiniumꢀbased scaffolds, which could also explain the pHꢀstability trends
of our current carboxylateꢀbased scaffolds (Scheme 2A).
Intramolecular generalꢀacid
1
9-23
mechanisms are known to facilitate various chemical transformations
and we recently
proposed this for the hydrolysis of the PꢀN bond in phosphoramidates possessing a pyridininium
18
group proximal to the central phosphorus atom.
It is understood that at physiological
24,25
conditions, phosphoramidates have zwitterionic character.
In buffered conditions near the
pKa of the neighboring carboxylic acid, 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. However, in some cases (e.g., 6c, 6d, 8d, 12b, and 12c), a transient species was
3
1
observed in the P NMR (at approximately ꢀ5.0 ppm) during the pH stability studies (see
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Supporting Information, Figure S1 as a representative example), which suggested that an
intermediate mixed anhydride may be forming due to an intramolecular substitution at the
phosphorus center (Scheme 2B) before being intercepted by water. Such putative cyclic mixed
2
6,27
anhydrides have been proposed previously
but at this time, we have not determined if one of
0
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these two mechanisms predominates for the carboxylateꢀbased scaffolds of this study in contrast
1
8
to the pryidiniumꢀbased scaffolds previously reported. Regardless of the mechanism (Scheme
2A,B), the data herein suggests that the stability of the phosphoramidates toward hydrolysis
under mild pH conditions is correlated to the proximity of the neighboring carboxylic acid for
efficient proton transfer or substitution through a transient cyclic species.
Scheme 2: Proposed intramolecular mechanisms of phosphoramidate hydrolysis.
A pronounced effect on stability was observed for the threonineꢀbased compounds 7a,c.
Here we invoked the ThorpeꢀIngold effect (commonly known as the gemꢀdimethyl effect), which
28-30
is known to promote intramolecular interactions,
to destabilize the linker at mild pH
conditions for more rapid drug release than the unsubstituted analogs 6a-d. Indeed, the added
methyl group promoted rapid hydrolysis of the model drugꢀlinker conjugates 7a-d. This is most
notable for the aniline conjugate 7a because the aniline conjugates exhibited little to no
hydrolysis over 8 h. However, the added methyl group of the threonine residue in 7a, in
comparison to the serine analog 6a, promoted rapid hydrolysis further demonstrating the
tunability of the phosphoramidateꢀbased drugꢀconjugate scaffold.
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In addition to enhancing the rate of hydrolysis for the phosphoramidates through the
ThorpeꢀIngold effect, we also investigated the potential for a proximity effect by rigidifying the
3
1
scaffold as in 12b,c. Indeed, substituting the homoserine residue of 8aꢀd with the more rigid
benzoate of 12b,c resulted in model drugꢀconjugate scaffolds with a halfꢀlives that were similar
10
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12
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14
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to the shorter serineꢀbased homologs 7b,c. These results, reminiscent of the cis-aconityl linker,
are encouraging and particularly attractive in that the pKa of the benzoate’s carboxylate can be
3
3
predictably tuned based on Hammett sigma constants.
To further demonstrate that the proximal carboxylates were essential in promoting rapid
hydrolysis of the phosphoramidateꢀbased drugꢀconjugate scaffold, and to confirm that the
scaffold could be protected from acidic hydrolysis in the form of a prodrug, we prepared 7c´ as
the protected methyl ester of 7c. Based on the pHꢀstability data for 7c´ compared to 7c, it was
confirmed that the carboxylate is key to promoting rapid hydrolysis of the phosphoramidate PꢀN
bond. Moreover, the pH stability of 7c´ is interesting from a prodrug development perspective as
it suggests that the phosphoramidateꢀbased drugꢀconjugate scaffold can be protected as an ester
prodrug for oral administration after which the scaffold can be activated by plasma or liver
esterases.
Conclusions
In summary, we have again demonstrated that the phosphoramidate scaffold displays a
wide range of stability at different pH values. Evaluation of our secondꢀgeneration
phosphoramidateꢀbased drugꢀlinker scaffolds further supports our hypothesis that a proximal
carboxylic acid can promote hydrolysis of the PꢀN bond under mildly acidic conditions.
18
Together with our pyridiniumꢀbased scaffolds, the data suggests that any neighboring protonꢀ
donor may be employed to manipulate the lability of the PꢀN bond for controlledꢀrelease of an
amineꢀcontaining drug, presumably through an intramolecular generalꢀacid mechanism. In
addition, we have found that the stability of the PꢀN bond can be further attenuated through the
ThorpeꢀIngold effect and rigidification. The benzoateꢀbased scaffold 12 is particularly attractive
in that the pKa of the neighboring carboxylic acid can be predictably modulated allowing for
another degree of tunability. The stability profile of the ester analog 7c´ suggests that the
scaffold could be used in prodrug applications and allow for oral administration.
1
8
Based on the data herein combined with our firstꢀgeneration scaffolds, it is expected
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that cleavable 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, it is envisioned that 8d could serve as a candidate linker
scaffold for ADC applications with a secondary amineꢀcontaining drug (e.g., MMAE). Little to
no detectable hydrolysis of 8d at pH 7.4 was observed over 8 h whereas the t1/2 was 6.22 h at pH
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5.5. In the context of smallꢀmolecule drug conjugates (SMDCs), the more labile scaffolds, such
as 6c (pH 7.4 t1/2 = 13.1 h; pH 5.5 t1/2 = 0.76 h), would be better matched to the in vivo clearance
profiles of smallꢀmolecules.
In conclusion, we expect the modular tunability of our phosphoramidate scaffold to find
broad applicability in various controlled drugꢀrelease applications such as ADCs or SMDCs,
drugꢀeluting stents, prodrugs, as well as intracellular trafficking studies in which pH changes can
1
8
trigger the release of turnꢀon dyes. Having previously demonstrated that the pyridinium group
and now the benzoate group can function as a neighboring proton donor to promote
phosphoramidates hydrolysis, it is envisioned that these scaffolds offer the advantage of subtle
fineꢀtuning of amineꢀrelease kinetics through substitution effects on the aromatic rings.
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 1 M 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
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
6
4 scans, a relaxation delay of 1s, an observed pulse of 3.41 µs at 45 , and a preꢀacquisition
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.
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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
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3
4,35
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 Varian 300 MHz , 400 or 600 MHz spectrometer. H NMR chemical shifts are relative to
1
3
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=49.15 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 H-Phosphonates (20-25). General procedure for
synthesis of H-phosphonates 20-25. The alcohol 14-20 (0.893 g, 2.85 mmol, 1 eq) was
o
dissolved in freshly distilled pyridine (4 mL) under Ar (g) and cooled to ꢀ5 C. A 50% v/v
solution of diphenylphosphite (1.65 mL, 4.23 mmol, 1.5 eq) in freshly distilled pyridine under Ar
(g) was added dropwise via syringe to the cooled alcohol solution. The mixture was allowed to
warm to room temperature until the alcohol was consumed. Benzyl alcohol (0.740 mL, 7.12
mmol, 2.5 eq) was added via syringe, and the solution was stirred at room temperature for 4 h.
The crude mixture was taken up with EtOAc (50 mL) and washed sequentially with 10% HCl
(
3X, 50 mL) to remove the pyridine and brine (50 mL). The organic layer was dried over
anhydrous Na SO (s) and the solvent was removed in vacuo. The product was obtained via
2
4
silica flash chromatography with EtOAc:Hex as the eluent, dried overnight under high vacuo,
then advanced immediately without further characterization (36.4ꢀ72.5% yield).
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General Atherton-Todd method for phosphoramidates 26-31. All phosphoramidates are
obtained as a mixture of enantiomers/diastereomers. Chirality on the phosphorus atom is lost
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1
after removal of benzyl esters. All P and C NMR are decoupled to H. Amines used as the
hydrochloride salt were prepared by adding the amine to a cooled solution of 4 N HCl in 1,4ꢀ
dioxanes and subsequently filtered and washed with dry diethyl ether.
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Method A: A solution of the Hꢀphosphonate (0.390 mmol, 1.0 eq) in dry solvent (CH CN
3
o
or CH Cl ), 2.0 mL, was cooled to ꢀ15 C under Ar (g). Carbon tetrachloride (CCl ) or
2
2
4
bromotrichloromethane (BrCCl ), 2.0 mL, was added via syringe and the solution was stirred for
3
o
1
5 min at ꢀ15 C. A cooled solution of the amine hydrochloride (0.40 mmol, 1.02 eq) and Et N
3
(
0.790 mmol, 2.0 eq) in dry solvent (CH CN or CH Cl ) under Ar (g) was transferred dropwise
3 2 2
via syringe to the Hꢀphosphonate solution. The reaction was allowed to warm to room
temperature and monitored the consumption of the Hꢀphosphonate. The crude solution was
concentrated in vacuo. The residue was diluted with EtOAc (50 mL) and washed sequentially
with 10% HCl (50mL) and brine (50 mL). The organic layer was dried over anhydrous Na SO4
2
(
s) and the solvent was removed in vacuo. The pure phosphoramidate was isolated by silica or
reversedꢀphase flash chromatography if required (25.2ꢀ99.1%).
Method B: A solution of the Hꢀphosphonate (0.720 mmol, 1.0 eq) in dry CH Cl (7.2
2
2
o
mL) was cooled to ꢀ15 C under Ar (g). BrCCl (7.2 mL) was added via syringe and the solution
3
o
was stirred for 15 min at ꢀ15 C. A cooled solution of the amine hydrochloride (0.800 mmol, 1.1
eq) and catalytic amount of 4ꢀDMAP (0.072 mmol, 0.1 eq) in dry CH Cl under Ar (g) was
2
2
transferred dropwise via syringe to the Hꢀphosphonate solution. Et N (1.520 mmol, 2.1 eq) was
3
added dropwise, and the solution was immediately stirred at room temperature until consumption
of the phosphite. The crude solution was diluted with CH Cl (50 mL) and washed sequentially
2
2
with 10% HCl (50 mL) and brine (50 mL). The organic layers were collected and dried over
anhydrous Na SO (s) and the solvent was removed in vacuo. The pure phosphoramidates were
2
4
purified by either silica or reversedꢀphase chromatography (10.1ꢀ33.8%).
General procedure for global deprotection for linkers 6-11. To a solution of the
phosphoramidate (0.09 mmol) in 1,4ꢀdioxanes was added KHCO (s) (0.18 mmol, 1.0 eq per
3
benzyl group) in ddH O and 10% Pd/C (0.019 mmol). The mixture was stirred vigorously,
2
purged with N (g) and then charged with H (g) under balloon pressure at room temperature for
2
2
1ꢀ2 h. The crude reaction mixture was filtered through a 0.2 ꢁm PTFE micropore filtration disk
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Whatman) and the filter was then washed with ddH O. The solvent was lyophilized to yield the
2
pure product as the potassium salt in quantitative yield.
Supporting Information
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A table of halfꢀlives for compounds 6ꢀ12, characterization data for intermediates 14ꢀ31
and final compounds 6ꢀ12 are available in the Supporting Information.
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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Rejniak, S. X., Gordon, K. A., DeBlanc, R., Toki, B. E., Law, C. L., Doronina, S. O.,
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(
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(
(
15) Sassoon, I., and Blanc, V. (2013) Antibodyꢀdrug conjugate (ADC) clinical pipeline: a
review. Methods in molecular biology 1045, 1ꢀ27.
16) Ducry, L., and Stump, B. (2010) Antibodyꢀdrug conjugates: linking cytotoxic payloads to
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(
(
(
20) Kirby, A. J., Lima, M. F., da Silva, D., Roussev, C. D., and Nome, F. (2006) Efficient
Intramolecular General Acid Catalysis of Nucleophilic Attack on a Phosphodiester.
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21) Fersht, A. R., and Kirby, A. J. (1967) Intramolecular general acid catalysis of ester
hydrolysis by the carboxylic acid group. Journal of the American Chemical Society 89,
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24) Benkovic, S. J., and Schray, K. J. (1978) The Mechanism of Phosphoryl Transfer, in
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Graphical Abstract
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Phosphoramidate-based pH-programmable cleavable linkers
Figure 1
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Synthesis of model drug-linker phosphoramidates
Scheme 1
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Bioconjugate Chemistry
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Stability of model drug-linker phosphoramidates at pH 3.0, 5.5, and 7.4 (37 oC). *No detectable hydrolysis
observed over 8 hrs.
Figure 3
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Bioconjugate Chemistry
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Proposed intramolecular mechanisms of phosphoramidate hydrolysis.
Scheme 2
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