Optimizing Protonation States for Selective Double-Strand DNA Photocleavage in Hypoxic Tumors: PH-Gated Transitions of Lysine Dipeptides

Article abstract of DOI:10.1021/acs.jmedchem.6b01164

We report pH-switching properties of the new family of dipeptide-acetylene conjugates where pH-gated light-activated double-strand (ds) DNA cleavage is controlled by variations in electronic and geometric parameters. The conjugates have higher activities at the slightly acidic pH values that separate normal and cancerous tissue (pH < 7). This favorable pH dependence originates from several elements of structural design. Basicities of the two amines determine the threshold pH range where the changes in binding and reactivity are observed, whereas the distance between the two amino groups and the hydrophobic aryl alkyne moiety can further modulate DNA binding. The changes of the protonation state from a neutral molecule to a dication results in dramatically increased efficiency of ds DNA photocleavage, the most therapeutically valuable type of DNA cleavage.

Full text of DOI:10.1021/acs.jmedchem.6b01164

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Article
Optimizing Protonation States for Selective Double Strand DNA
Photocleavage in Hypoxic Tumors: pH-Gated Transitions of Lysine Dipeptides
Kemal Kaya, Saumya Roy, Juan Carlos Nogues, Juan Camilo
Rojas, Zlatko Sokolikj, Diego A. R. Zorio, and Igor V. Alabugin
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01164 • Publication Date (Web): 15 Aug 2016
Downloaded from http://pubs.acs.org on August 19, 2016
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Optimizing Protonation States for Selective Double Strand DNA Photocleavage in
Hypoxic Tumors: pHꢀGated Transitions of Lysine Dipeptides
†
,‡
†
†
†
Kemal Kaya , Saumya Roy , Juan Carlos Nogues , Juan Camilo Rojas , Zlatko
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†
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†∗
Sokolikj , Diego A. R. Zorio , Igor V. Alabugin
†Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL
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‡
2306ꢀ4390, USA.
Department of Chemistry, Dumlupınar University, Kütahya, 43100 Turkey
§Department of Biomedical Sciences, College of Medicine, Florida State University,
Tallahassee, FL 32306, USA
Abstract:
We report pHꢀswitching properties of the new family of dipeptideꢀacetylene conjugates
where pHꢀgated lightꢀactivated double strand (ds) DNA cleavage is controlled by
variations in electronic and geometric parameters. The conjugates have higher activities
at the slightly acidic pH values that separate normal and cancerous tissue (pH<7). This
favorable pH dependence originates from several elements of structural design. Basicities
of the two amines determine the threshold pH range where the changes in binding and
reactivity are observed whereas the distance between the two amino groups and the
hydrophobic aryl alkyne moiety can further modulate DNA binding. The changes of the
protonation state from a neutral molecule to a dication results in dramatically increased
efficiency of ds DNA photocleavage, the most therapeutically valuable type of DNA
cleavage.
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Introduction
The 2015 Nobel Prize in Chemistry illustrates the importance of mechanistic
studies of DNA damage and repair. In human cells, a single doubleꢀstrand (ds) DNA
cleavage can potentially cause the loss of more than 100 million base pairs of genetic
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information. Double stranded DNA cleavage, which is much more difficult to repair than
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singleꢀstrand (ss) DNA cleavage, is particularly beneficial for therapeutic purposes since
it leads to efficient selfꢀprogrammed cell death (apoptosis). DNA damage can be
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achieved chemically or photochemically for cancer therapy.
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Natural enediynes, are selfꢀdefense tools for microorganisms that target DNA of
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competing species with astonishing efficiency. The key feature of the enediyne
molecules is ability to cause double stranded DNA (ds DNA) cleavage. For example,
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calicheamicin leads about 25% of ds DNA cleavage (~3:1 ss:ds ratio), significantly
overperforming such common dsꢀcleaving agents as bleomycins (ss:ds ratios of 6:1ꢀ
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0:1 ). Upon activation of enediyne moiety, pꢀbenzyne diradical is generated via
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Bergman cyclization. This highly reactive diradical abstracts hydrogen atoms from
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DNA, initiating a sequence of further reactions that can culminate in ds DNA cleavage.
Unfortunately, the selectivity of these natural antibiotics towards cancer cells is low,
rendering such compounds highly toxic towards healthy cells as well. Such
indiscriminate toxicity calls for the incorporation of additional structural elements that
introduce the required selectivity in this class of cytotoxic reagents. We have shown
earlier that photochemical activation can be the source of such selectivity and that the
DNAꢀdamaging ability of enediynes can be replicated by photochemically activated
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alkynes. Furthermore, we found that the efficiency of such photodamage can be
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regulated by acidity of the media.
Warburg has shown that cancer cells increase their production of lactic acid due
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to their faster metabolism. The cell has a number of mechanisms for exporting H ions
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to extracellular environment. However, drugs like amiloride,
nigericin and
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hydralyzine can equilibrate the extracellular and extracellular H ions concentration.
This process lowers the intracellular pH of cancer cells, thus differentiating tumors from
the healthy tissues. One can take advantage of the acidic environment of cancer tissues by
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designing new chemical agents that selectively react at the lower pH of the tumors.
An attractive approach for optimizing the reactivity and selectivity of such compounds
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involves incorporation of basic amino acids.
Lysine is an attractive amino acid for pH regulation due to the presence of two
amino groups. When lysine’s carboxylic group is used to connect this amino acid to a
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DNAꢀcleaving agent, both lysine’s amino groups are preserved. The first
and secondꢀ
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generation of lysine enediynes, lysine mono acetylenes and metaꢀdiyne lysine
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conjugates were synthesized to make pHꢀgated DNAꢀcleavage applicable to cancer
therapy. The first generation of lysine conjugates was also shown to damage intracellular
DNA and to display interesting selectivity for ds DNA cleavage, targeting Gꢀsites
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flanking ATꢀtracks. The two amino groups of all lysine conjugates have drastically
different basicity – whereas the εꢀamino group is protonated throughout the entire
biologically relevant pHꢀregion, αꢀamino group undergoes protonation at ~pH 7, the
threshold that separates cancer from healthy tissues. The second generation probed for
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the further control of efficiency by using different connections between two lysine
residues.
Because the selectivity of DNA cleavage is controlled by the αꢀamino group of
lysine, we decided to test whether DNAꢀcleavers with even higher selectivity toward
cancer cells can be developed by redesigning the conjugates to possess only αꢀamino
groups. For these new conjugates, the transition to acidic pH will transform neutral
species directly to the dicationic form, potentially increasing the selectivity of the
conjugates toward acidic cancer cells. We have also suggested that we can fineꢀtune the
pHꢀdependence further by varying the distance between the two amino groups (Figure 1).
Figure 1: Left: Design of new amino acidꢀacetylene hybrids with variable distances
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between the two αꢀNH groups. Right: the concept of pHꢀregulation of the DNAꢀcleaving
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activity of lightꢀactivated DNA cleaving agents.
If two amino groups are sufficiently close, they should affect each other’s
basicity. However, the degree of such interaction and its dependence on the spatial
separation between the two bases are unknown. In order to probe this effect, we designed
new dipeptides where the distance between αꢀamino groups is changed in a systematic
way. We have also synthesized dipeptide conjugates where the two αꢀamino groups
mimic the distance between the α ꢀ and εꢀ amino groups of lysine.
Results and Discussion
Synthesis
Peptide bond formation upon condensation with the carboxyl group of other
amino acids converted the εꢀamino group of lysine (11), δꢀamino group of ornithine (14),
and the βꢀamino group of alanine (17), into respective amides. Because these amides
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were derived from amino acids, each of such transformation was equivalent to trading the
terminal amino groups of the intermediate structure for an αꢀamino group in the final
product, providing access to a family of alkyne dipeptide conjugates containing two α ꢀ
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NH groups with the different degrees of spatial separation.
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Scheme 1. Synthesis of designed hybrid molecules.
Bocꢀprotected peptide conjugates (12, 15 and 18 ) were synthesized by coupling
respective amines (11, 14 and 17) with αꢀamino Bocꢀprotected amino acids (9) (glysine,
alanine, phenylalanine and tryptophan) using dicyclohexylcarbodiimide (DCC) or 1ꢀ
ethylꢀ3ꢀ(3ꢀdimethylaminopropyl) carbodiimide (EDCI) / 1ꢀhydroxybenzotriazole (HOBt)
as coupling reagents. These peptides are then coupled to the DNA cleaver (10) after
deprotection of methyl ester. Removal of the Bocꢀgroups with trifluoroacetic acid (TFA)
in CH Cl produces the target compounds 1 – 8 (Scheme 1). The final products were fully
2
2
characterized by spectroscopic methods and used in the DNAꢀphotocleavage studies as
TFA salts. The full list of compounds investigated in this work is given in Scheme 2.
Scheme 2. The structures of dipeptideꢀacetylene conjugates. A: Lysine bridge. B:
Ornithine bridge. C: α ꢀAmino alanine bridge.
Determination of pKa Values
NMR Titrations:
The αꢀamino groups control the selectivity of DNAꢀbinding and cleaving by
creating a number of available protonation states. Formation and interconversion of these
1
states can be readily monitored by HꢀNMR because protonation of amino groups
strongly effects the chemical shift of α–CꢀH groups. By following the chemical shifts of
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the respective protons, pKa values of the individual amino group could be determined
(Figure 2).
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Scheme 3. The pK values obtained from H NMR titration.
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For the lysineꢀbridged conjugates 1 and 2 where the bridge between the two
amino groups is relatively long (8 atoms), the difference between the basicity of these
groups is relatively small. The pK differences of only 0.1 suggest that the two amines
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can be considered as independent functional groups. However, this model is likely to be
an oversimplification because the titration curves clearly show that two αꢀamino groups
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are more basic (pK = 7.3 and 7.4) in 1 than in 2 (pK =6.6 and 6.7). In other words, the
a
a
terminal Ph group of 2 may impose an effect even at the remote amino group positioned
at the center of the molecule. The origin of this interesting observation is unclear at the
moment but one can suggest that it may be an indication of the contribution of a coiled
conformation where the terminal Ph ring is brought to the proximity of the electronꢀ
deficient aromatic πꢀsystem of the DNAꢀphotocleaving part by a combination of
hydrophobic effect and π/πꢀinteractions. If such conformational effect operates, it should
have a consequence for the DNA binding where uncoiling of the more stable
conformation will impose an additional penalty. If this is true, the protonation effect on
binding of compound 2 (and its smaller analogue 6) will be less pronounced in
comparison to that in their analogues without the terminal Ph group. Interestingly, such
behavior is indeed observed (vide infra).
In contrast, the two pK values of the ornithineꢀbridged compounds 4 (pK = 7.2,
a
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.7) and 5 (pK = 7.0, 7.8) are noticeably different (0.5ꢀ0.8 pK unit). This observation
a a
suggests that the two groups are sufficiently close in space, so the first group protonation
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makes the second group protonation more difficult. However, behavior of the Phꢀ
containing compound 6 is once again peculiar. Identical pK s of 7.0 are found for both
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amines (Table 1). Again, this observation suggests that the conformational profile of the
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Phꢀsubstituted conjugate is quite different from that of its cousins 4 and 5. We could not
determine the pK values of compound 3, 7 and 8 by NMR due to their lack of sufficient
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solubility in D O (Scheme 3). For these compounds, we had to turn to the significantly
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more sensitive measurements based on fluorescence quenching.
Figure 2. Chemical shiftꢀpD titration plots for αꢀhydrogens in conjugates 2 and 3 (1 mM)
in D O.
2
Fluorescence Titrations:
Fluorescence provides additional insight in the protonation states of αꢀamino groups
because, upon excitation of the chromophore, the free lone pair of the amino group can
quench excited state via intramolecular photoinduced electron transfer (PET). The
quenching process is blocked when the amino groups are protonated and the donor lone
pair of nitrogen is inaccessible. Changes in fluorescence intensity as a function of pH fit
to the HendersonꢀHasselbalch equation for acidꢀbase equilibrium. Only amino groups
that are spatially close to the chromophore quench its fluorescence efficiently. This
spatial dependence accounts for the observation of only one pKa value for the conjugates
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ꢀ6 (Figure 3).
Figure 3. Selected pKa values obtained from fluorescence.
The difference between pK s measured by NMR and fluorescence stems from the
a
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a
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NMR is the acid dissociation constant of the ground state. However, the pK values from
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fluorescence reflect the excited state properties of the molecules. For most compounds,
the pKas determined by the two methods are quite close, with the excited state pKa being
slightly lower (i.e., amine is slightly less basic). The similarity is expected because the
excitation is concentrated at the conjugated chromophore. The Phꢀsubstituted conjugate 2
is again anomalous. Generally, only one pKa is observed corresponding to the spatially
close ammonium moiety (excited states are short lived and less sensitive to the electronic
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properties of remote parts of the molecule). However, for the βꢀalanine conjugates 7 and
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where the 2 amino group is much closer to the chromophore, its protonation state can
also be gauged by the fluorescence intensity change.
Table 1. Summary of pK values of the compounds.
a
The two acidities play complementary roles in achieving pHꢀgated selectivity of
DNA cleavage. In particular, the ground state acidities are more important in DNA
binding. When the cell has more acidic intracellular pH (cancer cells), the greater fraction
of the compound will be bound to DNA. Thus, the acidic cancer tissues may be targeted
more efficiently. On the other hand, excited state acidities are important for pHꢀgating by
opening or shutting down the photochemical activation pathway responsible for the DNA
damage. This is why we report both the ground state and the excited state acidities.
DNA Binding
(UV)
The interactions between DNA and our conjugates were analyzed by UVꢀVis
spectroscopy. The intrinsic DNA binding constants were calculated using the equation
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[
DNA]/(ε ꢀ ε ) = [DNA]/ (ε ꢀ ε ) + 1/K (ε ꢀ ε )
Equation 1
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where ε is the apparent extinction coefficient of complex with DNA, ε is the extinction
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coefficient of the fully bound complex, ε is the extinction coefficient of unbound
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compound, and K is the DNA binding constant of the compound. [DNA]/(ε ꢀ ε ) is
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plotted vs. [DNA] and slope is divided by yꢀintercept gave the (K ) binding constant.
b
The results are summarized in Table 2. The measured DNA binding constants are
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ꢀ
comparable with those for the known DNA intercalators from the literature (10 ꢀ10 M
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). In every case, the binding constants slightly increase when pH increases ꢀ the trend
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similar to that in other lysine conjugates. Although the increase in DNA binding at the
lower pH was general, the magnitude of this increase varied for compounds of different
nature. When pH increases, ammonium groups lose their charges and as a result, different
binding interactions may become more important than electrostatic interactions with the
backbone. For example, the hydrophobic part of the conjugates (the photocleaver) may
approach one of the DNA grooves in order to get away from the aqueous environment.
Indeed, the spectral information is consistent with changes of the binding modes.
The conjugates show strong absorption at ~310 nm. With the addition of DNA,
the absorbance of the conjugates decreased with a concomitant small red shift, suggesting
intercalative binding between DNA and conjugates. Whereas both a distinct isosbestic
point and a red shift are clearly observed at pH 6, these spectral features disappear at pH
8
(Figure 5), suggesting switch to multiple binding modes at the higher pHs.
Table 2. DNA Binding constants from UV
The magnitudes of the pHꢀinduced variations in binding depend on the substitution.
Again, the Phꢀsubstituted (PhꢀAla) conjugates 2, 6, 8 are anomalous ꢀ they show slightly
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greater binding at pH 6 but also display a noticeably smaller increase at binding (5.7ꢀ,
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.0ꢀ, 1.1ꢀfold, respectively) in comparison the Meꢀsubstituted (Ala) analogues 1,5,7
(10.5, 4.6ꢀ, 3.5ꢀfold, respectively). The glycine analogue shows a >4ꢀfold increase
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comparable to that for the alanine counterpart 5, albeit with a slightly sharper increase at
the pH 6/7 threshold. On the other hand, the tryptophane analogue 3 shows only a small
1
.7 increase, even smaller than the increase in the related PhꢀAla compound 2.
Figure 4. Absorption spectral changes of 1 (15 ꢁM) on titration of CT DNA (0– 40 ꢁM)
in phosphate buffer (pH 6, pH 7 and pH 8).
EtBr Displacement:
The binding constants of the conjugates are comparable with the reported values
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for DNA intercalators in literature . In order to better understand the DNA binding
modes of the conjugates and the effects of varying pH on intercalation, we performed
ethidium bromide (EtBr) displacement experiments. The results summarized in Table 3,
show that all of the conjugates exhibited the most EtBr displacement capabilities at pH 6,
decreasing displacement with increasing pH. The presence of the aromatic systems
(phenyl ring on conjugate 2 and 6 and indole moiety on compouınd 3) can lead to
intercalation with DNA. Furthermore, Schneider et al. described earlier that such
intercalation can be augmented by presence of a positive charge in the vicinity of an
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aromatic moiety assisted by the positively charged amino groups. This structural feature
suggests that these compounds can intercalate between DNA base pairs and displace the
competitive DNA intercalator (EtBr).
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Compounds 2 and 3 exhibited the largest drop in displacement when going from
pH6 to pH7. This pH dependence is constant across all the conjugates and suggests
interactions between our molecules and DNA increases at slightly acidic conditions,
correlating with the observed increase in selectivity for double stranded cleavage of DNA
by the conjugates under acidic conditions discussed below. It is important to consider the
decrease in displacement and interactions at the neutral (pH7) and basic (pH8) conditions
is influenced by the conjugate’s transitions from diꢀcationic to neutral which partially
dictates the affinity for the hydrophilic backbone of DNA vs the hydrophobic grooves, as
well as their solubility. These results are consistent with findings in previous literature
which correlate the increased displacement of the conjugates at pH 6 to protonated amino
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1
3
groups having increased interactions with the negative phosphate backbone of DNA .
Table 3. SternꢀVolmer quenching constants of the compounds (1ꢀ8) from EtBr
displacement.
DNA Photocleavage
The ability of dipeptide conjugates to cleave DNA upon irradiation was
investigated using conversion of supercoiled plasmid DNA (Form I) into the respective
circular (ss cleavage) and linear (ds cleavage) forms (Forms II and III, respectively). The
relative amounts of the three DNA forms were determined by densitometric analysis of
the gel electrophoresis bands. The lysine conjugates at 15 ꢁM concentrations have shown
a higher and more selective DNAꢀcleaving activity than conjugates that mimic lysine
amino groups; With this in mind, we will focus the discussion below on compounds 1, 2
and 3.
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6
Figure 5. Quantified cleavage data for plasmid relaxation assay for DNA photocleavage
with 15 µM of acetylenic conjugates 1 (left), 2 (middle), 3 (right) and 38 µM (b.p.) of
pBR322 plasmid DNA at pH range of 6 – 8 after 10 min. of UV (>300 nm) irradiation.
Reported values represent the average of three experiments. Code: Form I, Blue; Form II,
Red; Form III, Green.
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The most remarkable observation is the selectivity of ds DNA cleavage induced
by compound 2 when the pH changes from neutral to slightly acidic (pH 6). Even though
the reactivity of compounds 1 and 3 is higher than compound 2, the selectivity of
compound 2 between pH 6 and pH 7 is much better, showing no ds DNA cleavage at pH
7
or pH 8. This result correlates with the basicity of the αꢀamino groups of compound 2
(pKa1=6.7 and pKa2=6.8). At pH 6, the dominant form of compound 3 is dicationic. At
pH 7, the dominant form of compound 2 is likely nonprotonated. Considering
electrostatic interactions, the binding affinity of compound 2 and DNA is stronger if these
two αꢀamino groups are protonated.
Figure 6. Quantified cleavage data for plasmid relaxation assay for DNA photocleavage
with 15 µM of acetylenic conjugates 4 (left), 5 (middle), 6 (right) and 38 µM (b.p.) of
pBR322 plasmid DNA at pH range of 6 – 8 after 10 min. of UV (>300 nm) irradiation.
Reported values represent the average of three experiments. Code: Form I, Blue; Form II,
Red; Form III, Green.
In the case of ornithine conjugates 5, 6 and 7, the ability of the ds DNA cleavage
is active at lower pH conditions (pH 6). However; the DNA cleavage at neutral pH (pH
7
) and basic conditions (pH 8) is less pronounced. With alanine conjugates, the ds DNA
photocleavage activity is lost.
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Figure 7. Quantified cleavage data for plasmid relaxation assay for DNA photocleavage
with 15 µM of acetylenic conjugates 7 (left) and 8 (right) and 30 µM (b.p.) of pBR322
plasmid DNA at pH range of 6 – 8 after 10 min. of UV (>300 nm) irradiation. Reported
values represent the average of three experiments. Code: Form I, Blue; Form II, Red.
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Statistical Studies
Further investigation on the origin of strand breaks prompts the question: Are ds
DNA breaks coordinated or do they occur random due to multiple ss DNA breaks? To
33
2
1
answer this question, we used the Freifelder–Trubo (n = n (2h+1)/4L) equation. From
2
this equation, n (double strand breaks) values can be calculated by using typical n
2
1
(
single strand breaks) values obtained from the each experiments. Assuming that the
34
distribution of ds DNA breaks between individual follows a Poisson distribution, we
also calculated the Poisson distribution of strand cuts and average number of ssꢀ(n ) and
1
dsꢀbreaks (n ) per DNA molecule using the equation n = 1/[( f + f + f )/ f ꢀ 1] and –
2
2
I
II
III
III
ln f = n + n = n .
I
1
2
Tot
The DNAꢀphotocleavage experiments were performed as a function of irradiation
time at pH 6 for conjugates 1, 2 and 3. Quantified values from densitometry are shown in
Table 4.
Figure 8. Quantified cleavage data for plasmid relaxation assay for DNA photocleavage
with 15 µM of conjugates 1 (left), 2 (middle), 3 (right) and 30 µM (b.p.) of pBR322
plasmid DNA at pH range of 6 – 8 after 10 min. of UV (>300 nm) irradiation. Code:
Form I, Blue; Form II, Red; Form III, Green.
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Table 4. Statistical analysis of the singleꢀstrand and doubleꢀstrand break formation by 1,
2
and 3 as a function of irradiation time at pH 6.0.
Under these assumptions, n /n is a good indicator for determining if cleavage
1
2
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occurs randomly or in a coordinated way. The observed n /n ratio is at least 41 times
1
2
greater (up to 140 times greater) than random Poisson distribution. Taking the average
numbers, we can conclude that about 90% of ds DNA breaks produced by these
compounds corresponds to the true dsꢀdamage rather than to a combination of random ss
DNA breaks.
Mechanistic Studies
In the absence of light, the conjugates do not induce ds DNA cleavage. Hence,
DNA damage originates from the formation of excited state of the alkyne “warhead”.
3
5
Possible mechanisms responsible for the observed photodamage include a variety of
possibilities such as crossꢀlink formation between dipeptides and DNA, oxidative damage
36
45
through electron transfer from nucleobases,
alkylation of DNA,
hydrogen
3
7,38
39
abstraction,
abasic site generation, and reactive oxygen species (ROS).
Figure 9. Effect of hydroxyl radical/singlet oxygen scavengers (20 mM) upon the
efficiency of DNA cleavage of compounds 1 (left), 2 (middle) and 3 (right) at pH 6 at 15
µM of compounds.
To investigate the effect of reactive oxygen species (ROS), we used the plasmid
relaxation assays for the cleavage with compounds 1, 2, 3 (Figure 9), 4, 5 and 6 (Figure
4
0,41
1
0) in the presence of hydroxyl radical (glycerol, DMSO)
and singlet oxygen
42
(
NaN ) scavengers.
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Figure 10. Effect of hydroxyl radical/singlet oxygen scavengers (20 mM) upon the
efficiency of DNA (30 µM b.p.) cleavage of compounds 4 (left), 5 (middle) and 6 (right)
at pH 6 (20mM sodium phosphate buffer) at 15 µM of compounds.
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Singlet oxygen and hydroxyl radical scavengers show no significant effect on the
efficiency of DNA damage by compounds 3, 4 and 5. However, the DNA cleavage
activity of the conjugates 5, 6 and 7 is inhibited by sodium azide (NaN ) and glycerol. In
3
the case of conjugate 6 with DMSO, there is no inhibition from the scavenger on the ds
DNA cleavage.
Abasic Site Generation:
Next question arises whether or not our conjugates are able penetrate the cell
membrane, find the nuclei and break DNA inside the cell. To answer this question, we
used the abasic site determination experiment to understand if our conjugates can
4
3
function as DNAꢀphotocleavers inside of the cell. Apurinic/apyrimidinic (AP) sites
44
result from the cleavage of Nꢀglycosylic bond as a result of DNA damage initiated by
4
5
46
47
alkylation, deamination and the most frequently oxidation of DNA bases. Oxidative
damage is caused by reactive oxygen species (ROS), in particular, the hydroxyl
radical. The results of AP site determination for conjugate 2 (the most selective
compound) are shown in Figure 11. The graph also shows the internal standard points for
certain numbers AP sites. The control experiment, that involves only UV irradiation,
5
generates 10 abasic sites per 10 base pairs. However, conjugate 2 is able generate 17 AP
sites when it is activated with UV light for 2 hrs. In other words, the amount of AP sites
almost doubled in the presence of conjugates activated with light. Furthermore, these
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experiments have also confirmed that our molecules (1, 2 and 3) are able to penetrate cell
membrane, find the nuclei and perform their DNAꢀcleaving function upon photochemical
excitation.
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Figure 11. Abasic Site Generation/Detection by the compound 2.
Conclusion
We have designed and prepared the third generation of acetyleneꢀdipeptide
conjugates. The conjugates change from neutral to dicationic under acidic conditions,
making them selectively more reactive towards ds DNA in acidified conditions typical
for cancerous tissue. Statistical analysis shows that the ds cleavage was caused by a
coordinated ds cleavage event instead of two random ss cleavage events. Scavenger
experiments have shown that there is no significant effect of reactive oxygen species on
DNA damage. Furthermore, the conjugates are also able to penetrate the cell membrane
and generate abasic sites in DNA.
Experimental Section
1
13
General information. H, C NMR spectra were collected on a Bruker 400 MHz and
00 MHz NMR spectrometer. Mass spectrometry data was collected on a Jeol JMSꢀ
6
600H. UV spectra were recorded on a Shimadzu UVꢀ2100. Fluorescence spectra were
obtained with SPEX FluoMax spectrofluorimeter using rightꢀangle geometry. pH was
adjusted with AB 15 plus pH meter (Accument) after standardization at 25 ºC. All buffers
were prepared and pHꢀadjusted at room temperature (25°C). Purity of compounds is
≥
95%.
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Plasmid DNA Photocleavage. pBR322 plasmid DNA (4,361 b/p; from BioLabs Inc.,
1
ꢁg/ꢁL solution in 10 mM TrisꢀHCl (pH 8.0), and 1mM EDTA buffer) was diluted to a
concentration of 0.01 ꢁg/ꢁL. The solution containing cleavage agent, DNA (30 ꢁM/bp) in
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o
20 mM sodium phosphate buffer was incubated for 1 hour at 30 C. Samples were placed
on ice at a distance of 20 cm from 200 W HgꢀXe lamp (SpectraꢀPhysics, Laser &
Photonics Oriel Instruments with long pass filter with 324 nm cutꢀon wavelength).
Electrophoretic Analysis. The gel electrophoresis was carried out in 1x TBE buffer at
8
0 V using Miligel FisherBiotech Horizontal Electrophoresis System. All gels were run
on 1% agarose slab gels. Before loading, the DNA samples were mixed with 0.33 volume
of tracking dye containing bromophenol blue (0.25%) and glycerol (30%) in water. After
staining in ethidium bromide solution (2 ꢁg/ml) for 1 hour, the gel was washed with
water and pictures were taken. The relative quantities of the supercoiled, nicked, and
linear DNA were calculated by integrating the “area” of each spot by the image analyzer
software Total/Lab (Nonlinear Dynamics Ltd., UK). The amount of supercoiled DNA
was multiplied by factor of 1.4 to account for reduced ethidium bromide intercalation into
supercoiled DNA.
Spectrometric determinations of pK . pH of 10 ꢁM of compound solution in H O was
a
2
adjusted with 10, 100 mM HCl (aq) and NaOH (aq) solution under nitrogen purged
condition. Excitation wavelength was 330 nm and polymethacrylate fluorimeter cuvettes
were used.
AP Site Determination. Human malignant melanoma cell line A375 (CRLꢀ1619™) was
obtained from the American Type Culture Collection (ATCC®). The cell line was
cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and
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penicillin/streptomycin (Life Technologies®). Cells were propagated according to
ATCC® guidelines and maintained in a 37 °C incubator with 5% CO atmosphere. Cells
2
5
(
8 ×10 ) were plated in 10 cm cell culture dishes. Twentyꢀfour hours later, 3 ꢁM of
0
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compounds were added to cells and incubated for 1 h. Treated and untreated cells were
exposed to UV radiation for 20 min and harvested at different time points post UV
radiation. DNA from harvested cells was extracted using GeneElute Mammalian
Genomic DNA kit (cat. no. G1N70, SigmaꢀAldrich) and concentration was determined
using NanoDrop™ Lite Spectrophotometer 1000 (Thermo Fisher Scientific). Abasic Sites
(AP sites) counting was determined by following manufacturer’s instructions for the
DNA Damage Quantification KitꢀAP Site Countingꢀ (Cat#DK02) from Dojindo
Molecular Technologies, Inc.
Analytical data of compounds
(S)ꢀ2,6ꢀDiaminoꢀNꢀ((S)ꢀ5ꢀaminoꢀ6ꢀoxoꢀ6ꢀ((4ꢀ((perfluoropyridinꢀ4
yl)ethynyl)phenyl)amino)hexyl)hexanamide bisꢀtrifluoroacetic acid salt (1): Bocꢀ
protected compound 13a (193 mg, 0.29 mmol) was dissolved in CH Cl (4 mL) and
2
2
trifluoroacetic acid (4 mL) added. Reaction mixture was stirred for 4 h and concentrated
in vacuo. Anhydrous diethyl ether (15 mL) were added to the crude product, solid
immediately came out. The solid material was washed with anhydrous diethyl ether (3 ×
1
1
0 mL) then chloroform (3 × 10 mL) to get pure TFAꢀsalt in 53% yield. H NMR (400
MHz, CD OD): 7.78 (d, J = 7.9 Hz, 2H), 7.64 (d, J = 7.9 Hz, 2H), 4.04 (t, J = 6.5 Hz,
3
1H), 3.86 (q, J = 7.3 Hz, 1H), 3.28 – 3.15 (m, 2H), 2.07 – 1.88 (m, 2H), 1.66 – 1.55 (m,
1
3
2
1
H), 1.54 – 1.47 (m, 2H), 1.43 (d, J = 7.3 Hz, 2H); C NMR (150 MHz, CD OD): δ
3
73.7, 171.5, 147.7 (d, J = 250 Hz), 145.7 (d, J = 272 Hz), 144.1, 109.8, 76.5, 57.7, 52.8,
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+
4
2.6, 34.8, 32.4, 25.7, 20.2; HRMS (ESI): calcd for C H F N O [M + Na] 488.16856,
22 23 4 5 2
found 488.16839.
(
S)ꢀ2ꢀaminoꢀ6ꢀ((S)ꢀ2ꢀaminoꢀ3ꢀphenylpropanamido)ꢀNꢀ(4ꢀ((perfluoropyridinꢀ4ꢀ
10
11
12
13
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16
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32
33
34
35
36
37
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60
yl)ethynyl)phenyl)hexanamide Triꢀtrifluoroacetic acid salt (2): The compound 2 was
1
prepared by using the same procedure as synthesis of compound 1 in 82% yield. H NMR
(
400 MHz, CD OD): 7.76 (d, J=8.3 Hz, 2H), 7.58 (d, J=8.3 Hz, 2H), 7.35ꢀ7.27 (m, 3H),
3
7
.23 (d, J=7.4, 2H), 4.01 (t, J=6.2 Hz, 1H), 3.97 (t, J=7.4 Hz, 1H), 3.27ꢀ3.24 (m, 1H),
1
3
3.11ꢀ3.07 (m, 2H), 3.01ꢀ2.97 (m, 1H),1.96ꢀ1.90 (m, 2H), 1.49ꢀ1.36 (m, 4H); C NMR
150 MHz, CD OD): δ 169.9, 168.9, 145.6 (t, J=14.0 Hz), 144.2ꢀ143.9 (m), 142.4 (d,
(
3
J=35.6 Hz) 141.5, 135.7, 134.3, 130.5, 130.1, 128.8, 121.1, 117.4, 107.2, 74.0, 55.9, 55.1,
+
40.0, 38.9, 32.3, 29.9, 23.2; HRMS (ESI+): calcd for C H F N O H [M + H]
2
8
27
4
5
2
5
42.21791, found 542.21994.
(S)ꢀ2,6ꢀDiaminoꢀNꢀ((S)ꢀ5ꢀaminoꢀ6ꢀoxoꢀ6ꢀ((4ꢀ((perfluoropyridinꢀ4ꢀ
yl)ethynyl)phenyl)amino)hexyl)hexanamide Triꢀtrifluoroacetic acid salt (3): The
compound 3 was prepared by using the same procedure as synthesis of compound 1 in
1
41% yield. H NMR (400 MHz, CD OD): 7.73 (d, J = 8.9 Hz, 2H),7.58 (d, J=7.6 Hz,
3
1
H), 7.53 (d, J = 8.9 Hz, 2H), 7.36 ( d, J = 8.4, 1H), 7.16 (s, 1H), 7.11 (dd, J = 8.4, 7.1
Hz, 1H), 7.03 (dd, J = 7.6, 7.1 Hz, 1H), 4.02 – 3.96 (m, 2H), 3.28 – 3.03 (m, 4H), 1.99 –
13
1.82 (m, 2H), 1.48 – 1.32 (m, 4H); C NMR (150 MHz, CD OD): δ 170.4, 169.3, 145.1
3
(d, J = 254 Hz), 143.1 (d, J = 265 Hz), 141.5, 134.2, 128.5 122.9, 121.2, 120.2, 119.2,
1
17.3, 112.6, 108.3, 107.2, 73.9, 66.9, 55.3, 40.1, 32.4, 29.6, 29.1, 23.1, 15.4 ; HRMS
+
(
ESI): calcd for C H F N O Na [M + Na] 603.21076, found 603.20992.
3
0
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2
2
2
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(
S)ꢀ2ꢀaminoꢀ5ꢀ(2ꢀaminoacetamido)ꢀNꢀ(4ꢀ((perfluoropyridinꢀ4ꢀ
yl)ethynyl)phenyl)pentanamide bis(2,2,2ꢀtrifluoroacetate) (4): The compound 4 was
1
prepared by using the same procedure as synthesis of compound 1 in 78% yield. H NMR
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
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0
1
2
3
4
5
6
7
8
9
0
(600 MHz, CD OD): 7.78 (d, J = 8.6 Hz, 2H), 7.64 (d, J=8.6 Hz, 2H), 4.06 (t, J = 6.4 Hz,
3
1
3
1H), 3.66 (s, 2H), 2.05 – 1.92 (m, 2H), 1.71ꢀ1.66 (m, 2H); C NMR (150 MHz,
CD OD): δ 168.7, 167.5, 163.1 (q, J=35 Hz), 144.9 (d, J=215 Hz), 143.3 (d, J=251 Hz),
3
1
41.4, 134.3, 121.0, 118.3, 117.5, 107.1, 74.0, 54.9, 41.4, 39.8, 30.1, 26.0; HRMS (ESI):
+
calcd for C H F N O H [M + H] 438.15531, found 438.15530.
2
0
19
4
5
2
(S)ꢀ2ꢀaminoꢀ5ꢀ((S)ꢀ2ꢀaminopropanamido)ꢀNꢀ(4ꢀ((perfluoropyridinꢀ4ꢀ
yl)ethynyl)phenyl)pentanamide bis(2,2,2ꢀtrifluoroacetate) (5): The compound 5 was
1
prepared by using the same procedure as synthesis of compound 1 in 85% yield. H NMR
(600 MHz, CD OD): 7.78 (d, J = 8.0 Hz, 2H), 7.64 (d, J=8.0 Hz, 2H), 4.06 (t, J = 6.0 Hz,
3
1
3
H), 3.88 (q, J=6.7 Hz, 1H), 2.03 – 1.93 (m, 2H), 1.71ꢀ1.66 (m, 2H), 1.47 (d, J = 6.9 Hz,
1
3
H); C NMR (150 MHz, CD OD): δ 171.3, 168.8, 163.1 (q, J=35 Hz), 144.9 (d, J=215
3
Hz), 144.0 (d, J=264 Hz), 141.5, 134.2, 121.0, 118.8 (d, J=125 Hz), 117.4, 107.2, 74.0,
+
54.8, 50.2, 39.8, 30.0, 25.8, 17.7; HRMS (ESI+): calcd for C H F N O H [M + H]
2
1
21
4
5
2
4
52.17096, found 452.17061.
(S)ꢀ2ꢀaminoꢀ5ꢀ((S)ꢀ2ꢀaminoꢀ3ꢀphenylpropanamido)ꢀNꢀ(4ꢀ((perfluoropyridinꢀ4ꢀ
yl)ethynyl)phenyl)pentanamide bis(2,2,2ꢀtrifluoroacetate) (6): The compound 6 was
1
prepared by using the same procedure as synthesis of compound 1 in 89% yield. H NMR
(600 MHz, CD OD): 7.79 (d, J = 8.6 Hz, 2H), 7.62 (d, J=8.7 Hz, 2H), 7.30ꢀ7.20 (m, 5H),
3
4
.06 (t, J = 6.3 Hz, 1H), 4.02 (t, J = 7.4 Hz, 1H), 3.26ꢀ3.17 (m, 2H), 3.13 (dd, J=13.7Hz,
13
J=7.3 Hz, 1H), 3.03ꢀ2.99 (m, 1H), 1.89 (q, J=7.6 Hz, 2H), 1.63ꢀ1.54 (m, 2H); C NMR
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(
150 MHz, CD OD): δ 169.9, 168.7, 163.1 (q, J=34 Hz), 144.9 (d, J=215 Hz), 144.0 (t,
3
J=263 Hz), 143.3 (d, J=251 Hz), 141.5, 135.6, 134.2, 130.4, 130.0, 128.8, 121.0, 119.2,
1
18.3, 117.4, 107.1, 74.0, 55.9, 54.7, 39.7, 38.8, 29.9, 25.5; HRMS (ESI): calcd for
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
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37
38
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40
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42
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46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
C H F N O H [M + H] 528.20226, found 528.20168.
27
25
4
5
2
(S)ꢀ2ꢀaminoꢀ3ꢀ((S)ꢀ2ꢀaminopropanamido)ꢀNꢀ(4ꢀ((perfluoropyridinꢀ4ꢀ
yl)ethynyl)phenyl)propanamide bis(2,2,2ꢀtrifluoroacetate) (7): The compound 7 was
1
prepared by using the same procedure as synthesis of compound 1 in 88% yield. H NMR
(400 MHz, CD OD): 7.78 (d, J=8.7 Hz, 2H), 7.56 (d, J=8.7 Hz, 2H), 4.20 (t, J=4.8Hz,
3
13
1
H), 3.95 (q, J=7.0Hz, 1H), 3.82 (dq, J=14.6 J=4.0Hz, 2H), 1.47 (d, J=7.0Hz, 3H); C
NMR (150 MHz, CD OD): δ 172.8, 166.7, 163.3, 145.7 (d, J=14 Hz), 144.8 (d, J=208
3
Hz), 143.2 (d, J=231 Hz), 141.4, 134.2, 121.2, 118.3, 117.5, 107.1, 74.0, 55.3, 50.2, 41.2,
+
1
7.4; HRMS: calcd for C H F N O Na [M + Na] 446.12161, found 446.12178.
1
9
17
4
5
2
(
S)ꢀ2ꢀaminoꢀNꢀ((S)ꢀ2ꢀaminoꢀ3ꢀoxoꢀ3ꢀ((4ꢀ((perfluoropyridinꢀ4ꢀ
yl)ethynyl)phenyl)amino)propyl)ꢀ3ꢀphenylpropanamide bis(2,2,2ꢀtrifluoroacetate)
(
8): The compound 8 was prepared by using the same procedure as synthesis of
1
compound 1 in 84% yield. H NMR (600 MHz, CD OD): 7.78 (d, J=8.7 Hz, 2H), 7.63 (d,
3
J=8.6 Hz, 2H), 7.36ꢀ7.26 (m, 5H), 4.17 (t, J=5.5 Hz, 1H), 4.12 (dd, J=9.1 Hz J=5.6 Hz,
1
H), 3.83 (dd, J=14.6 Hz J=5.8 Hz, 1H), 3.74 (dd, J=14.6 J=5.2 Hz, 1H), 3.24 (dd,
1
3
J=14.3 J=5.5 Hz, 1H), 2.96 (dd, J=14.3 J=9.2 Hz, 1H); C NMR (150 MHz, CD OD): δ
3
1
71.5, 166.7, 163.3 (q, J=35 Hz), 144.9 (d, J=217 Hz), 144.1 (t, J=247 Hz), 144.0 (d,
J=26 Hz), 141.4, 135.5, 134.2, 130.4, 130.1, 128.9, 121.2, 116.9, 118.2 (d, J=293 Hz),
+
1
5
17.5, 107.2, 74.0, 55.8, 55.2, 41.2, 38.5; HRMS: calcd for C H F N O Na [M + Na]
25 21 4 5 2
22.15291, found 522.15258.
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(
S)ꢀmethyl2ꢀ((tertꢀbutoxycarbonyl)amino)ꢀ6ꢀ((S)ꢀ2ꢀ((tertꢀ
butoxycarbonyl)amino)propanamido)hexanoate (12a): To a
protected acid, BocꢀLꢀAlaꢀOH (9b) (400 mg, 2.11 mmol) in CH Cl (15 mL) at 0 °C,
solution of Bocꢀ
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
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6
7
8
9
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8
9
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8
9
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2
3
4
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7
8
9
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EDCI (506 mg, 2.64 mmol) and HOBt (356 mg, 2.64 mmol) were added sequentially.
After 20 min, the mixture of amine Lys(NꢀαꢀBoc)ꢀOMe (11) (458 mg, 1.76 mmol) and
DIPEA(0.9 mL, 5.28 mmol) were added to the reaction mixture. After stirring for 12 h at
room temperature, the reaction mixture was extracted with ethyl acetate, washed with
saturated NH Cl solution, water, brine, dried (Na SO ), filtered, and concentrated in
4
2
4
vacuo. Chromatographic purification (SiO , 40 % ethyl acetate in hexane eluant) of the
2
1
residue provided dipeptide compound 12a in 46 % yield. H NMR (400 MHz, CDCl ): δ
3
6.23 (bs,1H), 5.16 (bs, 1H), 5.05(bs,1H), 4.24(bs, 1H), 4.17 – 4.04 (m,1H), 3.73 (s,1H),
3
.33 – 3.14 (m, 2H), 1.85 – 1.72 (m, 1H), 1.71 – 1.48 (m, 5H), 1.46 – 1.41 (m, 18H), 1.34
1
3
(d, J = 7.3 Hz, 3H); C NMR (100 MHz, CDCl ): δ 173.3, 172.9, 155.5, 155.4, 79.4,
3
6
0.2, 53.2, 52.0, 49.8, 38.6, 31.7, 28.2, 22.4, 18.5; HRMS (ESI): calcd for C H N O Na
20 37 3 7
+
[
M + Na] 454.25292, found 454.25280.
(S)ꢀmethyl 2ꢀ((tertꢀbutoxycarbonyl)amino)ꢀ6ꢀ((S)ꢀ2ꢀ((tertꢀbutoxycarbonyl)amino)ꢀ3ꢀ
phenylpropanamido)hexanoate (12b): The compound 12b was prepared by using the
1
same procedure as synthesis of compound 12a in 51% yield. H NMR (600 MHz,
CDCl ): 7.31ꢀ7.18 (m, 5H), 5.81 (bs, 1H), 5.14 (bs, 2H), 4.27 (bs, 1H), 4.22 (bs, 1H),
3
3
9
1
2
.73 (s, 3H), 3.14 (bs, 2H), 3.07ꢀ3.00 (m, 2H), 1.73 (bs, 1H), 1.63ꢀ1.56 (m, 1H), 1.44 (s,
H), 1.40 (s, 9H), 1.37 (bs, 2H), 1.24 (bs, 2H); (150 MHz, CDCl ): δ 173.4, 171.3, 155.6,
3
37.0, 129.4, 128.8, 127.1, 80.3, 80.1, 56.2, 53.4, 52.4, 39.0, 38.8, 32.2, 29.0, 28.5, 28.4,
+
2.6; HRMS (ESI+): calcd for C H N O Na [M + Na] 530.28422, found 530.28421.
2
6
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(
S)ꢀmethyl 2ꢀ((tertꢀbutoxycarbonyl)amino)ꢀ6ꢀ((S)ꢀ2ꢀ((tertꢀbutoxycarbonyl)amino)ꢀ3ꢀ
(1Hꢀindolꢀ3ꢀyl)propanamido)hexanoate (12c): The compound 12c was prepared by
1
using the same procedure as synthesis of compound 12a in 41% yield. H NMR (400
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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37
38
39
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
MHz, CDCl ): δ 8.9 (bs, 1H), 7.67 (d, J = 7.5 Hz, 1H), 7.39 (d, J = 8.1 Hz, 1H), 7.19 (dd,
3
J = 7.5,7.1 Hz, 1H), 7.04(s, 1H), 5.56 (bs, 1H), 5.21 (s, 1H), 5.10 (s, 1H), 4.43 (bs, 1H),
4
.25 – 4.15 (m, 1H), 3.73 (s, 3H), 3.39 (d, J = 9.0 Hz, 1H), 3.15 – 3.02 (m, 3H), 1.73 –
13
1
.55(m, 2H), 1.49(s, 9H), 1.45(s, 9H), 1.32 – 0.97, (m, 4H); C NMR (100 MHz,
CDCl ): 173.1, 171.6, 155.5, 155.3, 136.1, 127.2, 123.2, 121.6, 119.1, 118.4, 111.2,
3
1
09.9, 77.8, 55.2, 53.2, 51.9, 38.6, 31.7, 28.1, 22.3; HRMS (ESI): calcd for
+
C H N O Na [M + Na] 569.29512, found 569.29517.
28 42
4
7
(
[
(
S)ꢀmethyl 2ꢀNꢀ[(1S)ꢀ5ꢀ[[(2S)ꢀ2,6ꢀbis(tertꢀbutoxycarbonylamino)hexanoyl]amino]ꢀ1ꢀ
[4ꢀ[2ꢀ(2,3,5,6ꢀtetrafluoroꢀ4ꢀpyridyl)ethynyl]phenyl]carbamoyl]pentyl]carbamate
13a): The dipeptide 12a (320 mg, 0.74 mmol) was dissolved in THF:MeOH:H O (3:1:1,
2
1
0 mL) at 0 °C. Then, LiOH·H O (93 mg, 2.22 mmol) was added and stirred at the room
2
temperature for 3 h. The reaction mixture was then acidified to pH 2 with 1N HCl. It was
extracted with ethyl acetate, washed with brine, dried (Na SO ), filtered, and
2
4
concentrated in vacuo to get the crude acid and used for further reaction. The prepared
acid was dissolved in CH Cl (10 mL) and cooled to 0 °C. It was sequentially treated
2
2
with DCC (229 mg, 1.11 mmol) and HOBt (149 mg, 1.11 mmol). After 15 min, amine
10), (237 mg, 0.89 mmol) was added to the reaction mixture and stirred for 24 h, raising
(
the temperature to rt. The reaction mixture was extracted with ethyl acetate, washed with
saturated NH Cl solution, saturated NaHCO , 1 N HCl, water, brine, dried (Na SO ),
4
3
2
4
filtered, and concentrated in vacuo. Chromatographic purification (SiO , 60 % ethyl
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1
acetate in hexane eluant) of the residue provided compound 13a in 42 % yield. H NMR
(
400 MHz, CDCl ): δ 9.28 (bs, 1H), 7.64 (d, J = 6.9 Hz, 2H), 7.53 (d, J = 6.9 Hz, 2H),
3
6
.77 (bs, 1H), 5.78 (bs, 1H), 5.31 (bs, 1H), 4.31 – 4.16 (m, 2H), 3.36 (s, 1H), 3.13 (s,
0
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2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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8
9
0
1
2
3
4
5
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7
8
9
0
1
H), 1.92 – 1.78 (m, 1H), 1.77 – 1.67 (m, 1H), 1.58 – 1.39 (m, 20H), 1.38 (d, J = 7.3 Hz,
1
3
2H C NMR (150 MHz, CD OD): δ 173.5, 171.7, 156.3, 155.8, 143.3 (d, J = 250 Hz),
3
1
41.5 (d, J = 280 Hz), 140.3, 133.2, 119.5, 117.3, 115.3, 107.0, 80.3, 73.1, 55.2, 50.0,
+
3
8.5, 31.5, 28.9, 28.3, 22.7, 18.6; HRMS (ESI): calcd for C H F N O Na [M + Na]
3
2
39
4
5
6
688.27342, found 688.27351.
{5ꢀ(2ꢀtertꢀButoxycarbonylaminoꢀ3ꢀphenylꢀpropionylamino)ꢀ1ꢀ[4ꢀ(2,3,5,6ꢀ
tetrafluoroꢀpyridinꢀ4ꢀylethynyl)ꢀphenylcarbamoyl]ꢀpentyl}ꢀcarbamic acid tertꢀbutyl
ester (13b): The compound 13b was prepared by using the same procedure as synthesis
1
of compound 13a in 35% yield. H NMR (400 MHz, CD CN): 8.92 (bs, 1H), 7.71 (d,
3
J=8.6 Hz, 2H), 7.59ꢀ757 (m, 2H), 7.30ꢀ7.19 (m, 5H), 6.72 (t, J= 5.5 Hz, 1H), 5.79 (d,
J=7.1 Hz, 1H), 5.50 (d, J=7.1 Hz, 1H), 4.18 (dd, J=7.4, 1H), 4.04 (bs, 1H), 3.16ꢀ3.04 (m,
13
3
H), 2.78 (dd, J=13.6,8.9, 1H), 1.78ꢀ1.63 (m, 2H), 1.40 (s, 11H), 1.33 (s, 11H); C
NMR (400 MHz, CD CN): δ 172.7, 172.6, 156.8, 156.3, 142.0, 145.8ꢀ143.0 (m), 138.7,
3
1
34.1, 130.2, 129.2, 127.5, 120.4, 115.7, 107.3, 80.1, 79.9, 79.1, 73.6, 56.9, 56.6, 38.9,
+
3
1.9, 29.6, 28.5, 28.4, 23.4; HRMS (ESI+): calcd for C H F N O Na [M + Na]
3
8
43
4
5
6
764.30472, found 764.30374.
(
[
(
S)ꢀmethyl 2ꢀNꢀ[(1S)ꢀ5ꢀ[[(2S)ꢀ2,6ꢀbis(tertꢀbutoxycarbonylamino)hexanoyl]amino]ꢀ1ꢀ
[4ꢀ[2ꢀ(2,3,5,6ꢀtetrafluoroꢀ4ꢀpyridyl)ethynyl]phenyl]carbamoyl]pentyl]carbamate
13c): The compound 13c was prepared by using the same procedure as synthesis of
1
compound 13a in 38% yield. H NMR (400 MHz, CDCl ): δ 9.07 (bs, 1H), 8.79 (d, J =
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.9 Hz, 1H), 7.66 (d, J = 7.7 Hz, 1H), 7.56(d, J = 8.1 Hz, 1H), 7.36 (d, J = 8.1 Hz, 1H),
.19 (dd, J = 7.7, 7.1 Hz, 1H), 7.11 ( dd, J = 8.1, 7.1 Hz, 1H), 6.06 (s, 1H), 5.95 (bs, 1H),
.42 (bs, 1H), 5.27 (bs, 1H), 4.45 (bs, 1H), 4.06 (bs,1H) 3.51 – 3.41 (m, 1H), 3.32 – 2.96
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
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44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13
(
m, 3H), 1.96 – 1.88 (m, 1H), 1.85 – 1.53 (m, 5H), 1.48 (s, 9H), 1.43 (s, 9H); C NMR
(150 MHz, CDCl ): δ 169.2, 168.8, 153.9, 153.2, 141.0 (d, J = 270 Hz), 139.2(d, J = 250
3
Hz), 137.7, 133.8, 130.8, 124.9, 120.9, 119.6, 117.1, 116.2, 115.0, 113.2, 108.8, 107.9,
1
04.3, 78.2, 77.7, 52.9, 52.5, 35.9, 28.9, 26.1, 23.8, 23.0, 22.4, 19.9; HRMS (ESI): calcd
+
for C H F N O Na [M + Na] 803.31561, found 803.31332.
4
0
44
4
6
6
(
S)ꢀmethyl2ꢀ((tertꢀbutoxycarbonyl)amino)ꢀ5ꢀ(2ꢀ((tertꢀ
butoxycarbonyl)amino)acetamido)pentanoate (15a): To a solution of Bocꢀprotected
acid, BocꢀGlyꢀOH (9a) (285 mg, 1.63 mmol) in CH Cl (15 mL) at 0 °C, EDCI (391 mg,
2
2
2
.04 mmol) and HOBt (276 mg, 2.04 mmol) were added sequentially. After 20 min, the
mixture of amine BocꢀOrnꢀOMe (14) (335 mg, 1.36 mmol) and DIPEA (0.7 mL, 4.08
mmol) were added to the reaction mixture. After stirring for 12 h at room temperature,
the reaction mixture was extracted with ethyl acetate, washed with saturated NH Cl
4
solution, water, brine, dried (Na SO ), filtered, and concentrated in vacuo.
2
4
Chromatographic purification (SiO , 50 % ethyl acetate in hexane eluant) of the residue
2
1
provided dipeptide compound 15a in 49% yield. H NMR ( 400 MHz, CDCl ): 6.46 (bs,
3
1H), 5.27 (bs, 1H), 5.18 (d, J = 7.6 Hz, 1H), 4.28 (d, J =5.2 Hz, 1H), 3.76 (d, J= 5.6 Hz,
2
9
8
+
H), 3.72 (s, 3H), 3.29 (q, J=6.2 Hz, 2H), 1.85ꢀ1.76 (m, 1H), 1.67ꢀ1.56 (m, 3H), 1.44 (s,
1
3
H), 1.42 (s, 9H); C NMR (100 MHz, CD OD): δ 173.1, 169.7, 156.3, 155.6, 80.3,
3
0.2, 53.2, 52.5, 44.5, 39.0, 30.3, 28.4, 25.6; HRMS (ESI+): calcd for C H N3O Na [M
1
8
33
7
+
Na] 426.22162, found 426.22159.
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1
1
2
2
2
2
2
2
2
2
2
2
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3
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(
S)ꢀmethyl2ꢀ((tertꢀbutoxycarbonyl)amino)ꢀ5ꢀ((S)ꢀ2ꢀ((tertꢀ
butoxycarbonyl)amino)propanamido)pentanoate (15b): The compound 15b was
1
prepared by using the same procedure as synthesis of compound 15a in 72% yield. H
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NMR (600 MHz, CDCl ): 6.40 (bs, 1H), 5.14 (d, J = 6.7 Hz, 1H), 5.03 (bs, 1H), 4.28 (d,
3
J = 5.2 Hz, 1H), 4.11 (bs, 1H), 3.73 (s, 3H), 3.34ꢀ3.22 (m, 2H), 1.82 (bs, 1H), 1.66ꢀ1.54
(m, 3H), 1.44 (s, 18H), 1.34 (d, J = 7.1 Hz, 3H); (150 MHz, CDCl ): δ 173.1, 173.0,
3
1
55.6, 80.0, 53.2, 52.4, 50.2, 38.9, 30.1, 28.4, 25.6, 18.7; HRMS (ESI+): calcd for
+
C H N O Na [M + Na] 440.23727, found 440.23652.
19
35
3
7
(S)ꢀmethyl 2ꢀ((tertꢀbutoxycarbonyl)amino)ꢀ5ꢀ((S)ꢀ2ꢀ((tertꢀbutoxycarbonyl)amino)ꢀ3ꢀ
phenylpropanamido)pentanoate (15c): The compound 15c was prepared by using the
1
same procedure as synthesis of compound 15a in 75% yield. H NMR (400 MHz,
CDCl ): 7.31ꢀ7.18 (m, 5H), 5.97 (bs, 1H), 5.08 (bs, 1H), 5.07 (bs, 1H), 4.28ꢀ4.19 ( m,
3
2
H), 3.73 (s, 3H), 3.18 (d, J = 3.6 Hz, 2H), 3.07ꢀ2.98 (m, 2H), 1.44 (s,11H), 1.40 (s, 11H;
1
3
); C NMR (100 MHz, CDCl ): δ 173.0, 171.5, 155.5, 136.9, 129.3, 128.5, 126.8, 79.9,
3
5
5.9, 53.1, 52.2, 38.9, 29.9, 29.6, 28.3, 28.2, 25.4; HRMS (ESI+): calcd for
+
C H N O Na [M + Na] 516.26857, found 516.26663.
25
39
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{
4ꢀ(2ꢀtertꢀButoxycarbonylaminoꢀacetylamino)ꢀ1ꢀ[4ꢀ(2,3,5,6ꢀtetrafluoroꢀpyridinꢀ4ꢀ
ylethynyl)ꢀphenylcarbamoyl]ꢀbutyl}ꢀcarbamic acid tertꢀbutylester (16a): The
compound 16a was prepared by using the same procedure as synthesis of compound 13a
1
in 22% yield. H NMR (600 MHz, CDCl ): 9.25 (bs, 1H), 7.71 (d, J = 8.6 Hz, 2H), 7.56
3
(d, J=8.6 Hz, 2H), 6.70 (bs, 1H), 5.38 (d, J=8.6 Hz, 1H), 5.26 (bs, 1H), 4.52 (bs, 1H),
3
.84 (d, J=4.9Hz, 2H), 3.68 (bs, 1H), 3.30 (bs, 1H), 1.81 (bs, 1H), 1.66ꢀ1.59 (m, 3H),
2
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Page 27 of 57
Journal of Medicinal Chemistry
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+
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.45 (s, 9 H), 1.41 (s, 9H); HRMS (ESI+): calcd for C H F N O Na [M + Na]
3
0
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60.24212, found 660.24018.
1ꢀ{4ꢀ(3,3ꢀDimethylꢀbutyrylamino)ꢀ4ꢀ[4ꢀ(2,3,5,6ꢀtetrafluoroꢀpyridinꢀ4ꢀylethynyl)ꢀ
(
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phenylcarbamoyl]ꢀbutylcarbamoyl}ꢀethyl)ꢀcarbamic acid tertꢀbutyl ester (16b): The
compound 16b was prepared by using the same procedure as synthesis of compound 13a
1
in 24% yield. H NMR (600 MHz, CDCl ): 9.31 (bs, 1H), 7.75 (d, J = 8.1 Hz, 2H), 7.57
3
(d, J=8.3 Hz, 2H), 6.67 (bs, 1H), 5.37 (d, J = 7.5 Hz, 1H), 4.90 (bs, 1H), 4.57 (bs, 1H),
4.13 (t, J = 6.6 Hz, 1H), 3.78 (bs, 1H), 3.16 (bs, 1H), 1.72 (bs, 2H), 1.58 (bs, 2H), 1.44 (s,
9
H), 1.38 (d, J = 6.8 Hz, 3H), 1.33 (s, 9H); NMR (150 MHz, CDCl ): δ 174.5, 171.5,
3
1
56.4, 155.9, 143.9 (d, J = 229 Hz), 142.8 (t, J = 262 Hz), 140.8, 133.5, 119.4, 117.8,
115.5, 107.2, 80.7, 80.2, 73.3, 53.3, 50.9, 37.8, 30.7, 28.5, 28.3, 26.1, 18.3; HRMS
+
(
ESI+): calcd for C H F N O Na [M + Na] 674.25777, found 674.25669.
3
1
37
4
5
6
{4ꢀ(2ꢀtertꢀButoxycarbonylaminoꢀ3ꢀphenylꢀpropionylamino)ꢀ1ꢀ[4ꢀ(2,3,5,6ꢀ
tetrafluoroꢀpyridinꢀ4ꢀylethynyl)ꢀphenylcarbamoyl]ꢀbutyl}ꢀcarbamic acid tertꢀbutyl
ester (16c): The compound 16c was prepared by using the same procedure as synthesis
1
of compound 13a in 33% yield. H NMR (600 MHz, CDCl ): 9.28 (bs, 1H), 7.73 (d, J =
3
8
.5 Hz, 2H), 7.58 (d, J=8.5 Hz, 2H), 7.31ꢀ7.18 (m, 5H), 6.23 (bs, 1H), 5.32 (d, J = 7.7
Hz, 1H), 4.96 (bs, 1H), 4.55 (bs, 1H), 4.26 (q, J = 7.2 Hz, 1H), 3.71 (bs, 1H), 3.10ꢀ3.07
(m, 1H), 3.04 (bs, 2H), 1.65ꢀ1.58 (m, 2H), 1.56ꢀ1.49 (m, 2H), 1.45 (s, 9H), 1.29 (s, 9H);
13
C NMR (150 MHz, CDCl ): δ 173.0, 171.4, 156.4, 155.7, 143.6 (d, J=215 Hz), 143.5
3
(d, J=281 Hz), 142.8 (d, J=262 Hz), 140.8, 136.4, 133.5, 129.3, 128.9, 127.3, 119.4,
1
17.8, 115.5, 107.1, 80.7, 80.2, 73.3, 56.6, 53.3, 38.7, 37.9, 30.5, 28.5, 28.2, 26.0; HRMS
+
(
ESI+): calcd for C H F N O Na [M + Na] 750.28907, found 750.28720.
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Journal of Medicinal Chemistry
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(
S)ꢀmethyl2ꢀ((tertꢀbutoxycarbonyl)amino)ꢀ3ꢀ((S)ꢀ2ꢀ((tertꢀ
butoxycarbonyl)amino)propanamido)propanoate (18a): To a solution of Bocꢀ
protected acid, BocꢀAlaꢀOH (9b) (150 mg, 0.79 mmol) in CH Cl (8 mL) at 0 °C, EDCI
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0
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0
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(189 mg, 0.99 mmol) and HOBt (134 mg, 0.99 mmol) were added sequentially. After 20
min, the mixture of amine (NꢀBocꢀβꢀamino)ꢀAlaꢀOMe (17) (144 mg, 0.66 mmol) and
DIPEA (0.3 mL, 1.98 mmol) were added to the reaction mixture. After stirring for 12 h at
room temperature, the reaction mixture was extracted with ethyl acetate, washed with
saturated NH Cl solution, water, brine, dried (Na SO ), filtered, and concentrated in
4
2
4
vacuo. Chromatographic purification (SiO , 50 % ethyl acetate in hexane eluant) of the
2
1
residue provided dipeptide compound 18a in 84% yield. H NMR (400 MHz, CDCl ):
3
6.94 (bs, 1H), 5.68 (d, J=7.6 Hz, 1H), 5.18 (bs, 1H), 4.35 (bs, 1H), 4.10 (q, J=6.9 Hz,
1
3
2
4
H), 3.71 (s, 3H), 3.60 (q, J=5.6 Hz, 2H), 1.42 (s, 9H), 1.40 (s, 9H), 1.30 (d, J=7.0Hz,
1
3
H); ); C NMR (100 MHz, CDCl ): δ 173.7, 171.3, 155.7, 80.2, 53.8, 52.7, 50.3, 41.4,
3
+
8.4, 28.3, 18.3; HRMS: calcd for C H N O Na [M + Na]
412.20597, found
1
7
31
3
7
12.20647.
(S)ꢀmethyl 2ꢀ((tertꢀbutoxycarbonyl)amino)ꢀ3ꢀ((S)ꢀ2ꢀ((tertꢀbutoxycarbonyl)amino)ꢀ3ꢀ
phenylpropanamido)propanoate (18b): The compound 18b was prepared by using the
1
same procedure as synthesis of compound 18a in 81% yield. H NMR (600 MHz,
CDCl ): 7.31ꢀ7.18 (m, 5H), 6.42 (bs, 1H), 5.40 (d, J=6.3Hz 1H), 4.98 (bs, 1H), 4.29 (bs,
3
13
2
H), 3.73 (s, 3H), 3.57 (bs, 2H), 3.08ꢀ2.98 (m, 2H), 1.43 (s, 9H), 1.40 (s, 9H); C NMR
(150 MHz, CD OD): δ 172.2, 171.3, 155.7, 136.7, 129.4, 128.9, 127.2, 80.5, 80.4, 56.1,
3
+
5
4
3.6, 52.8, 41.6, 38.3, 28.4, 28.3; HRMS: calcd for C H N O Na [M + Na]
23 35 3 7
88.23727, found 488.23631.
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Journal of Medicinal Chemistry
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{
2ꢀ(2ꢀtertꢀButoxycarbonylaminoꢀpropionylamino)ꢀ1ꢀ[4ꢀ(2,3,5,6ꢀtetrafluoroꢀpyridinꢀ
4
ꢀylethynyl)ꢀphenylcarbamoyl]ꢀethyl}ꢀcarbamic acid tertꢀbutyl ester (19a): The
compound 19a was prepared by using the same procedure as synthesis of compound 13a
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1
in 19% yield. H NMR (600 MHz, CDCl ): 9.42 (bs, 1H), 7.62 (d, J=8.0 Hz, 2H), 7.56
3
(d, J=8.6 Hz, 2H), 7.11 (bs, 1H), 6.16 (bs, 1H), 5.03 (bs, 1H), 4.38 (bs, 1H), 4.09 (t,
1
3
J=6.6, 1H), 3.92 (bs, 1H), 3.52 (bs, 1H), 1.46 (s, 9H), 1.37 (s, 9H), 1.369 (s, 3H); C
NMR (150 MHz, CDCl ): δ 174.5, 169.6, 157.1, 155.9, 142.7 (t, J=253 Hz), 141.7 (d,
3
J=263 Hz), 140.1, 133.5, 119.8, 117.6 (d, J= 16 Hz), 116.1, 106.9, 81.0, 80.8, 73.5, 55.6,
+
5
6
1.3, 41.1, 28.4, 28.3, 18.1; HRMS (ESI+): calcd for C H F N O Na [M + Na]
29 33 4 5 6
46.22647, found 646.22471.
1ꢀ{2ꢀtertꢀButoxycarbonylaminoꢀ2ꢀ[4ꢀ(2,3,5,6ꢀtetrafluoroꢀpyridinꢀ4ꢀylethynyl)ꢀ
phenylcarbamoyl]ꢀethylcarbamoyl}ꢀ2ꢀphenylꢀethyl)ꢀcarbamic acid tertꢀbutyl ester
(
(
19b): The compound 19b was prepared by using the same procedure as synthesis of
1
compound 13a in 21% yield. H NMR (600 MHz, CDCl ): 9.32 (bs, 1H), 7.60ꢀ7.56 (m,
3
4
H), 7.29ꢀ7.18 (m, 5H), 6.83 (bs, 1H), 5.99 (bs, 1H), 4.94 (d, J=4.1Hz 1H), 4.29 (d,
J=6.1Hz 1H), 4.27 (bs, 1H), 3.92 (bs, 1H), 3.48 (bs, 1H), 3.11 (dd, J=13.8Hz, J=6.1Hz
1
3
1
H), 2.99 (dd, J=13.9Hz, J=7.7Hz 1H), 1.48 (s, 9H), 1.34 (s, 9H); C NMR (150 MHz,
CDCl ): δ 173.2, 169.5, 157.2, 155.8, 144.4 (d, J= 13 Hz), 142.8 (t, J=260 Hz), 142.7 (d,
3
J=47 Hz), 140.9 (d, J=7 Hz), 140.1, 136.2, 133.5, 129.2, 129.1, 127.4, 119.7, 117.7 (d, J=
1
6 Hz), 116.1, 106.9, 81.2, 80.9, 73.5, 56.6, 55.7, 40.8, 38.2, 28.5, 28.3; HRMS: calcd for
+
C H F N O Na [M + Na] 722.25777, found 722.25920.
35 37
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