Chemistry of ring-substituted 4-(benzothiazol-2-yl)phenylnitrenium ions from antitumor 2-(4-aminophenyl)benzothiazoles

Article abstract of DOI:10.1021/jo400826f

Ring-substituted derivatives of 2-(4-aminophenyl)benzothiazole, 1a, 1b-g, are under development as antitumor agents. One derivative, 1f, has reached phase 1 clinical trials as the prodrug 2f, Phortress (NSC 710305). These amines are activated by CYP450 1A

Full text of DOI:10.1021/jo400826f

Article
pubs.acs.org/joc
Chemistry of Ring-Substituted 4‑(Benzothiazol-2-yl)phenylnitrenium
Ions from Antitumor 2‑(4-Aminophenyl)benzothiazoles
Yang Zhang, Mrinal Chakraborty, Christian G. Cerda-Smith, Ryan N. Bratton, Natalie E. Maurer,
Ethan M. Senser, and Michael Novak*
Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056, United States
S
* Supporting Information
ABSTRACT: Ring-substituted derivatives of 2-(4-
aminophenyl)benzothiazole, 1a, 1b−g, are under development
as antitumor agents. One derivative, 1f, has reached phase 1
clinical trials as the prodrug 2f, Phortress (NSC 710305). These
amines are activated by CYP450 1A1, apparently into hydroxyl-
amines 8a−g that are likely metabolized into esters that ionize
into nitrenium ions responsible for cellular damage. Previously we
showed that 9a, the acetic acid ester of 8a, generates the long-
lived (530 ns) nitrenium ion 11a by hydrolysis or photolysis in
water. In this study, azide trapping shows that 9b−g generate 11b−g via rate-limiting N−O heterolysis. Ion lifetimes, estimated
from azide/solvent selectivities, range from 250 to 1150 ns with identical lifetimes for 11a and 11f. Differences in biological
activity of the amines are likely not due to differences in the chemistry of the cations but to differences in metabolic activation/
deactivation of individual amines. Unlike the nitrenium ions, lifetimes of the esters are strongly dependent on the 3′-Me
substituent. Esters containing 3′-Me (9b, 9f, 9g) have lifetimes of 5−10 s compared to 400−800 s for esters without 3′-Me (9a,
9c, 9d, 9e). This restricts 3′-Me esters to cells/tissues in which activation occurs, concentrating their effects in tumor cells if
metabolism is restricted to those cells.
(1b) or halogens, including Cl (1c), led to enhanced activity
against the original cell lines and a broader spectrum of activity
against certain colon, ovarian, and renal cancer cell lines,
although other cell lines were insensitive to the drugs.1−3
Studies with fluorinated analogues including 1d−g showed that
fluorination at C-5 or C-7 prevented metabolic deactivation of
the drug without compromising antitumor activity.4 The
fluorinated water-soluble prodrug 2f, Phortress (NSC
710305), was approved for phase 1 clinical trials in Britain in
2004.5,6
INTRODUCTION
■
Since 1996 a series of benzothiazole derivatives based on the
lead compound 2-(4-aminophenyl)benzothiazole, 1a (Chart 1),
have been under development as antitumor agents.1 Unsub-
stituted 1a was found to have activity against the human breast
cancer cell lines MCF-7 (IC50 = 0.3−0.8 nM) and MDA 468
(IC50 = 1.6 nM).1 Substitution at the 3′-position by methyl
Chart 1
Initial reports from the preclinical and clinical trials on
Phortress were favorable,7 but very recently it was reported that
a 50-patient study was terminated due to a combination of
pulmonary and liver toxicity and marginal efficacy.8 Never-
theless, 1f and 2f continue to be evaluated in preclinical
studies,9 and other drug candidates containing the 2-(4-
aminophenyl)benzothiazole moiety continue to be investi-
gated.10−21 The complexes 3 are being developed as
radiopharmaceuticals for imaging (3a) and targeted radio-
therapy (3b,186Re, 188Re) of breast cancer.10,11 Other derivatives
of 1a, including 4−6 and 7 that has the same ring substitution
pattern as 1f, are being investigated as radiopharmaceuticals for
binding and in vivo imaging of Aβ-plaques that are associated
with the early stages of Alzheimer’s disease.12−16 Still other
derivatives of 1a are under investigation as antimicrobial and
Received: April 17, 2013
© XXXX American Chemical Society
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antifungal agents.17−20 The 2-(4-aminophenyl)benzothiazole
moiety is appearing in a significant number of drug
candidates.21
Chart 2
Metabolism of 2-(4-aminophenyl)benzothiazoles into the
active metabolite in sensitive cell lines requires the constitutive
presence of the 1A1 isoform of cytochrome P450, CYP1A1,
that is also induced by the drug.22 Activation also leads to
detoxification because CYP1A1 hydroxylates these drugs,
except those fluorinated at C-5 or C-7, leading to an inactive
C-6-hydroxylated metabolite.22,23 Metabolism is associated with
translocation of the aryl hydrocarbon receptor (AhR) to the
nucleus.24,25 The mechanism of action of 1a and its simple ring-
substituted derivatives is thought to involve selective uptake
into sensitive cells followed by AhR binding and translocation
into the nucleus, induction of CYP1A1, oxidation and
conversion of the drug into an electrophilic reactive
intermediate, and formation of extensive DNA adducts
resulting in cell death.6,21 It is suspected that the active
metabolite of 1 is the hydroxylamine 8 or, more likely, an ester
derivative, 9 or 10 (Scheme 1).6,26 It was proposed that the
arylnitrenium ions.30 The hydrolysis product formed in the
absence of other nucleophiles, 13a, was shown to arise from
slow hydrolysis of the initially rapidly formed quinolimine,
14a.29,30 It was also shown that 11a is efficiently trapped by 2′-
deoxyguanosine (dG) to form a C-8 adduct, 15a, analogous to
the major adduct formed by the reaction of nitrenium ions
derived from carcinogenic N-arylamines with dG and dG
residues in DNA.30 Reactions of 11a with adenosine and
cytosine were also demonstrated, but, consistent with the
pattern previously observed for nitrenium ions derived from
carcinogenic N-arylamines, these were much less efficient than
reaction with dG.31
Scheme 1. Probable Metabolism of 1
Because of continued interest in ring-substituted derivatives
of 1a in various fields of medicine we have investigated the
chemistry of the ring-substituted esters 9b−g and characterized
the resulting nitrenium ions 11b−g. The 3′-substituted esters
were chosen because of the reported effects of 3′-substituents
on the antitumor activities of the corresponding amines. The 5-
F- and 6-F-substituted esters were chosen because substitution
by F in that part of the ring system is a common tactic used to
reduce metabolic inactivation of the drug. The choice of mono-
and disubstituted esters allows both substituent effects to be
examined independently and in combination. The ester 9f was
chosen because the ring-substitution pattern of that material is
identical to that of Phortress, 2f. The particular fluorinated
compounds were also chosen because the 6-F substituent is in
resonance contact with the positive charge of 11, while the 5-F
substituent is not. Results of our studies are reported herein.
nitrenium ion 11 (Scheme 2) is responsible for the antitumor
activity of 1, but, until recently, no direct evidence supporting
this proposal was available.26−28
RESULTS AND DISCUSSION
Scheme 2. Proposed Nitrenium Ion 11 Generated from 9
■
Synthesis and Product Characterization. Synthesis of
the esters 9b−g followed a path starting from the
corresponding 2-(4-nitrophenyl)benzothiazoles 16b−g that
was previously published for 9a (Scheme 3).29 The nitro
compounds 16b−g are known materials generated by standard
procedures.4,23,32,33 Pd/C-catalyzed reduction of 16b−g with
hydrazine hydrate led to the hydroxylamines 8b−g in good
yield. These are stable materials that can be purified by
chromatography. The esters were generated by the reaction of
the hydroxylamines with pyruvonitrile in the presence of the
tertiary amine catalyst N-ethylmorpholine. Pyruvonitrile reliably
acetylates hydroxylamines, including the benzothiazole deriva-
tives used here, at O rather than N.29,34 The esters are much
less stable than the hydroxylamines. Only 9c−e could be
isolated and characterized. All esters containing a 3′-Me
substituent (9b, 9f, 9g) were too reactive to isolate. Instead,
these compounds were generated in situ in dry CH3CN
solutions that were stable for several days at −40 °C. These
compounds were characterized by their decomposition kinetics
in aqueous solution, by UV−vis spectroscopy, and by their
characteristic reaction products.
We recently reported the synthesis of 9a and demonstrated
that hydrolysis or photolysis of this material (Scheme 2) led to
29,30
−
an N3 -trappable reactive intermediate identified as 11a.
Laser flash photolysis (lfp) of 9a led to the direct detection of
11a with λmax 570 nm and an aqueous solution lifetime of 530
ns.29 The effect of N3 on the kinetics of decay of 11a
−
−
monitored at 570 nm was consistent with N3 trapping data
obtained during hydrolysis of 9a, demonstrating that the same
intermediate was generated during photolysis and hydrolysis.29
−
The structure of the major N3 adduct, 12a (Chart 2), is
−
consistent with that of N3 adducts derived from other N-
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Scheme 3. Synthesis of 9b−g and Isolation of Characteristic
Reaction Products
Scheme 4. General Kinetic Scheme for Decomposition of 9
order processes. Representative repetitive wavelength scans that
show the biphasic transition from ester to quinol are shown in
Figure S3 in the Supporting Information. Figure 1 shows a
The only isolated products of hydrolysis of all six esters in
pH 7.0 phosphate buffer were the corresponding quinols 13b−
g. At the concentrations employed in kinetic and product
analysis experiments (2.5 × 10−5 M), HPLC data indicate that
the quinols are generated in 85−95% yield with no other
significant products detected. Representative HPLC chromato-
grams are shown in Figures S1 and S2 in the Supporting
Information. At the higher concentrations used in product
isolation experiments (ca. 5 × 10−4 M) HPLC yields appear to
be somewhat lower (60−70%), but no other significant
products are detected by HPLC. Isolated yields of the pure
quinols are in the range of 20−30% after several rounds of
chromatography. The quinols are stable materials that are
analogous to 13a derived from hydrolysis of 9a.29,30 Similar
quinols are the major hydrolysis products of esters of
carcinogenic polycyclic N-arylhydroxylamines that decompose
via a nitrenium ion intermediate.35 Hydrolysis of all the esters
at pH 7.0 in the presence of NaN3 led to azide adducts,
detected by HPLC as characteristically long retention time
products, that were formed at the expense of the quinols.
Representative HPLC chromatograms for the azide reactions
are shown in Figures S1 and S2 in the Supporting Information.
Three of these adducts (12b, 12c, and 12f) were isolated and
fully characterized. These are completely analogous to 12a
Figure 1. Absorbance vs time for hydrolysis of 9d at pH 7.0 with fitted
lines and rate constants.
representative absorbance vs time plot for decay of 9d.
Depending on the wavelength chosen to monitor the reaction,
the data fit either a standard first-order rate equation or a
consecutive first-order rate equation governed by two rate
constants. The larger rate constant, observed at all wavelengths,
was pH independent within the pH range 5.6 to 7.4 for 9b−d
and 9f (kinetics for decay of 9e and 9g were only monitored at
pH 7.0). This rate constant has characteristics equivalent to ko′
previously observed for 9a and was identified as such. The
smaller rate constant, required for fitting of the data at some
wavelengths, is moderately pH-dependent in these buffers as
was previously observed for 9a.30 HPLC data taken during the
hydrolysis of 9c confirmed that the appearance of the peak for
13c was governed by the slow decay of a transient HPLC peak
identified as that of 14c. This result is similar to that previously
observed by HPLC analysis of the formation of 13a during the
decomposition of 9a.29 The smaller pH-dependent rate
constant was identified as k1.
The average values for ko′, as well as the value of k1 at pH 7.0,
for 9a−g are provided in Table 1. Average values for rate
constants were obtained at each pH from duplicate runs
monitored at a single wavelength for 9b, 9f, and 9g and single
runs monitored at multiple wavelengths for 9c, 9d, and 9e. The
rate constant ko′ reported in Table 1 was then obtained by
averaging the values for ko′ obtained at each pH except for 9e
and 9g that were monitored only at pH 7.0. All individual rate
constants obtained in this study are provided in Tables S1
through S6 in the Supporting Information. The most significant
rate effect is caused by the 3′-Me substituent in 9b, 9f, and 9g
that causes a 60- to 100-fold increase in ko′ compared to the
corresponding compounds without the 3′-Me substituent (9a,
−
formed by trapping of 11a with N3 and with similar azide
adducts obtained by trapping other nitrenium ions.30,35 The
formation of azide adducts predominately at the 2-position and
water adducts predominately at the 4-position has been noted
previously for 4-aryl- or 4-alkyl-substituted nitrenium ions and
has been attributed to reversible formation of an unstable initial
4-azido adduct that rearranges to the observed adduct.30,35,36
Reaction Kinetics. Previously, hydrolysis of 9a was shown
to follow the kinetic pathway of Scheme 4.30 At pH > pKa (1.45
for 9aH+), hydrolysis proceeded at a pH-independent rate
governed by ko′. Because ko′ > k1, it was possible to detect the
formation and decay of the quinol imine 14a during kinetics
experiments.29,30 Hydrolysis kinetics of 9b−g were monitored
by UV−vis spectroscopic methods in phosphate buffers (30 °C,
5 vol% CH3CN/H2O, 0.02 M total buffer, μ = 0.5 (NaClO4)).
For all compounds it was possible to detect two pseudo-first-
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Table 1. Kinetic parameters for 9a−g and 11a−g
compound/cation
103 ko′ (s−1
)
a
104 k1 (s−1) at pH 7.0
10−3 kaz/ks (M−1
)
1/ks (ns)
a
b
b,c
9a/11a
9b/11b
9c/11c
9d/11d
9e/11e
9f/11f
2.31 0.05
147 26
5.3 0.1
2.6 0.3
535 20
850 80
830 80
250 20
770 40
530 80
1150 40
d
0.92 0.02
0.58 0.01
3.7 0.1
4.2 0.4
4.1 0.4
1.3 0.1
3.8 0.2
2.6 0.4
5.7 0.2
d
d
d
d
d
2.67 0.10
1.23 0.21
2.19 0.10
4.7 0.1
122
2
0.27 0.02
0.58 0.01
9g/11g
155 18
a
b
c
d
See ref 30. See ref 29. Determined by direct measurement of ks for 11a generated by laser flash photolysis (ref 29). Estimated assuming that kaz =
4.94 × 109 M−1 s−1 (ref 29).
9d, 9e). Effects of the halogen substituents 3′-Cl, 5-F, and 6-F
on ko′ are relatively small. The magnitude of ko′ is nearly
equivalent within experimental error for 9a, 9c, and 9e. The 5-F
substituent of 9d causes a ca. 2-fold decrease in ko′ compared to
9a.
range of reactivity found in this study,29,35,37 so the “azide
clock” method was employed here. Hydrolysis of 9b−g in pH
−
−
7.0 phosphate buffers containing N3 leads to an [N3 ]-
dependent decrease in the yield of 13b−g with no increase in
the hydrolysis rate constant. The rate constants ko′ measured in
−
The rate of hydrolysis of 14a into 13a is dependent on
relative rate constants for hydrolysis of diprotonated,
monoprotonated, and neutral 14a and the pKas of the
diprotonated and monoprotonated species.30 Because the
kinetics of hydrolysis of 14b−g were not followed over a
wide enough pH range to determine detailed kinetic
parameters, it is not possible to ascertain substituent effects
on individual ionization and rate constants for 14b−g. It is clear
from the data in Table 1 that the 3′-Me and 3′-Cl substituents
cause about a 10-fold decrease in the rate of hydrolysis of the
quinol imine at pH 7.0.
the presence and absence of N3 are summarized in Tables S1
through S6, and representative repetitive wavelength scans are
shown in Figure S4 in the Supporting Information. The azide
adduct is detected by HPLC as a long retention time product
with a characteristically long wavelength λmax at ca. 330−350
nm. The quinols 13a−g have shorter HPLC retention times
and λmax at ca. 210−230 nm (Figures S1−S4). The product was
isolated and fully characterized in three cases (12b, 12c, and
12f). Figure 2 shows a representative plot of HPLC peak area
−
vs time for 12b and 13b as a function of [N3 ] determined
from hydrolysis of 9b.
−
Azide Trapping. Hydrolysis of 9a in buffers containing N3
leads to formation of an azide adduct without an increase in the
rate of hydrolysis of 9a, leading to the conclusion that
ionization of 9a generates the nitrenium ion 11a (Scheme 5).29
Scheme 5. Kinetics of Azide Trapping of 11
−
Figure 2. Determination of kaz/ks for 11b from the [N3 ]-dependent
yields of 12b and 13b monitored at the indicated wavelengths.
The selectivity ratio kaz/ks is determined by application of the
standard “azide clock” formulas to the product yields.29,30,35,38
The azide/solvent selectivities for 11b−g are summarized and
compared to the value previously determined for 11a in Table
1. The data show remarkably little dependence on the
substituents within the ions that were under investigation.
The ratios range over a factor of ca. 5 for all seven ions.
The rate constant kaz has been directly measured for 11a at
the apparently diffusion limited value of (4.94 0.17) × 109
M−1 s−1.29 If it is assumed that all the cations react with N3− at
the same diffusion limited rate, the lifetime, 1/ks, of each cation
can be estimated. The estimated lifetimes for 11b−g are
gathered in Table 1 with a comparison to the directly measured
lifetime for 11a. The lifetime of 11a estimated from product
yield data is identical to the directly measured value.29 Lifetimes
range from a low of 250 ns for 11d to a high of 1150 ns for 11g.
−
The N3 /solvent selectivity, kaz/ks, was determined by the
−
[N3 ] dependence of the yields of 12a and 13a (“azide clock”
method).29 The selectivity ratio was confirmed and both rate
constants were measured directly from kinetic studies on 11a
generated by lfp.29 Laser flash photolysis cannot be
implemented for 9b, 9f, and 9g because of their short lifetimes
in water, but rate constants determined by lfp and the “azide
clock” method are in good agreement for nitrenium ions in the
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Both 3′-Me (11b) and 3′-Cl (11c) substituents kinetically
stabilize the cation by a factor of about 1.6 compared to 11a.
The effect of 3′-Me is not surprising because Me is a weak EDG
in aromatic linear free energy correlations and has been shown
to kinetically stabilize arylnitrenium ions in aqueous solution
when it is substituted ortho or para to the nitrenium
center.36,39,40 The effect of 3′-Cl is somewhat surprising, but
there is precedent for moderate kinetic stabilization of
arylnitrenium ions by Cl substituents that can interact with
the positive charge via resonance.36 Substituent effects for 5-F
(11d) and 6-F (11e) differ. The 5-F substituent that is not in
resonance contact with the positive charge moderately
destabilizes the ion by a factor of ca. 2, while the 6-F
substituent that can interact with the positive charge via
resonance stabilizes the ion by a factor of ca. 1.4 compared to
11a. It has previously been shown that the 4′-F substituent in a
4-biphenylylnitrenium ion kinetically stabilizes that cation by a
factor of 2 in aqueous solution.41 The effects of multiple
substituents are approximately additive so that the lifetime of
the 5-F, 3′-Me cation (11f) is approximately the same as 11a,
while the lifetime of the 6-F, 3′-Me cation (11g) is ca. 2-fold
longer than that of 11a. The cation that would be generated
from Phortress, 2f, is 11f, so the lifetime of this ion is in the
same range as the others and is remarkably similar to that of the
unsubstituted ion.
acceleration of hydrolysis of the 3′-Me-substituted esters 9b, 9f,
and 9g.
Comparison of 4-(Benzothiazol-2-yl)phenylnitrenium
Ions and Other Arylnitrenium Ions. We have previously
noted that the aqueous solution lifetime and overall chemistry
of 11a is very similar to that of the 4-biphenylylnitrenium
ion.29−31 Experimental data and calculations agree that the 4-
(benzothiazol-2-yl) substituent is as effective as a 4-phenyl
substituent at stabilizing a fully formed arylnitrenium ion, but
comparisons of ionization rate constants for the ester
precursors show that this substituent destabilizes the transition
state for formation of the cation in comparison to the 4-phenyl
substituent.29,30 The rate constant ko′ is ca. 100-fold smaller for
9a than for the analogous ester precursor of the 4-
biphenylylnitrenium ion, demonstrating that the inductive
electron-withdrawing effect of this substituent dominates the
electron-donating resonance effect in the transition state for
ionization.30 The substituted ions 11b−g and their precursors
9b−9g extend this comparison. These cations have lifetimes in
water that are consistent with substituent effects on lifetimes
observed for other arylnitrenium ions, and they generate similar
−
reaction products with water and N3 . The magnitudes of the
rate constants ko′ for ionization of 9b−g are consistent with the
inductive destabilization of the transition state for ionization
that was previously observed for the parent ester, 9a. Even the
60- to 100-fold increase in ko′ observed for the 3′-Me-
substituted esters 9b, 9f, and 9g in comparison to the
corresponding esters without the 3′-Me substituent (9a, 9d,
9e) has quantitative precedent in the rate constants for the
generation of other arylnitrenium ions in water.36,42 The
remarkable consistency in the properties of these ions in
comparison to other arylnitrenium ions and the divergent
inductive and resonance effects of the 4-(benzothiazol-2-yl)
substituent both play a potential role in the biological activity of
the parent amines as discussed below.
Substituent Effects on Ionization of 9a−g. The most
significant rate effect seen in these reactions is the large
acceleration of ionization (ko′) of the 3′-Me-substituted esters
9b, 9f, and 9g to form the corresponding cations. This appears
to be predominately an electronic effect, although steric effects
may also play a role in moderating the effect. It has previously
been shown that the rate constants for hydrolysis of the N-
sulfonatooxyacetanilides 15a−c (Scheme 6) at 30 °C, the rate-
Scheme 6. Substituent Effects for Aqueous Solvolysis of
15a−c
Implications for Antitumor Activity. None of the
substituents have a major effect on the lifetimes of the
nitrenium ion intermediates 11a−g. Observed differences in the
antitumor activity of the parent amines 1a−g cannot be
ascribed to differences in the stability of these ions. These
differences are more readily attributable to differential
metabolism of the amines. It has already been shown that 5-
F (and 7-F) substituted amines such as 1d and 1f are less
subject to metabolic deactivation through hydroxylation at C-
6.4 Suppression of metabolic deactivation coupled with a lack of
significant change to the formation and reactivity of the
nitrenium ion could account for the improved antitumor
activity of these amines. It is important to note that the
lifetimes of the nitrenium ions 11a−g are in the same range
(200−3000 ns) as those of nitrenium ions derived from well-
known carcinogenic amines such as 2-aminofluorene and 4-
aminobiphenyl.35,37,41 The remarkable similarity of the lifetimes
and chemistry of 11a and the 4-biphenylylnitrenium ion has
already been described.29,30 Unless very carefully tuned for
metabolic activation only in tumor cells, antitumor compounds
that rely on nitrenium ion “warheads” are likely to cause
unintended damage beyond the targeted tumor cells. The
recent reports of pulmonary and liver toxicity encountered in
clinical trials with Phortress suggest that the currently
employed 2-(4-aminophenyl)benzothiazoles do not exhibit
sufficiently selective metabolic activation.8
determining step of which is ionization to form a nitrenium ion,
occurs with rate constants, ko, of 4.5 × 10−6 s−1 for 15a, 1.3 ×
10−3 s−1 for 15b, and 9.5 × 10−6 for 15c.42 The 4-Me
substituent stabilizes the transition state for ionization of these
compounds much more than it stabilizes the nitrenium ion
itself.36 The 290-fold increase in ko observed for 15b compared
to 15a is 3- to 5-fold larger than the rate increase caused by the
3′-Me substituent observed in Table 1. Analysis of ortho-
substituent effects for solvolysis of ArC(Me)2Cl in 90%
aqueous acetone suggested that a modest 6-fold decrease in
the solvolysis rate constant of the 2-Me-substituted compound
compared to 4-Me is attributable to steric hindrance to
formation of the coplanar carbocation.43 The nitrenium ion 11
is also apparently planar30 and would be expected to be subject
to steric destabilization by ortho-substituents, although
+
probably to a smaller extent than ArC(Me)2 . The expected
combined magnitudes of the electronic acceleration and smaller
steric destabilization would appear to explain the observed
The most significant difference in the chemistry of derivatives
of 1a−g is the discovery in this study that the 3′-Me substituent
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g (1.0 mmol) was added to a three-neck 100 mL round-bottom flask.
Dry, freshly distilled THF (50 mL) was added, and the mixture was
stirred under nitrogen while 0.314 g of 5% Pd/C was added. The flask
was then put into an ethylene glycol−dry ice bath at −30 °C. After the
flask had cooled for several minutes, 50 μL of hydrazine monohydrate
was added via syringe and the mixture was stirred for 15 min. The flask
was removed from the bath and allowed to reach room temperature.
Reaction progress was monitored by TLC (20/80 EtOAc/hexanes for
16c, 16d, and 16e; 10/90 EtOAc/CH2Cl2 for 16b, 16f, and 16g). An
additional 50 μL of hydrazine monohydrate was added after the
reaction flask was immersed into the −30 °C bath again. The reaction
mixture was monitored periodically by TLC until there was no sign of
starting material. Occasionally, additional small amounts of hydrazine
hydrate (in 10 μL aliquots) were added to completely remove the
nitro compound before quenching the reaction mixture. The reaction
mixture was then filtered through Celite and rotary evaporated. If
necessary, the product was purified by flash chromatography on silica
gel (10/90 EtOAc/CH2Cl2). Isolated yields ranged from 60% to 90%.
N-(4-(Benzo[d]thiazol-2-yl)-2-methylphenyl)hydroxylamine (8b).
153 mg (60%); mp 158−160 °C (decomp); IR 3200, 1600, 1460,
has a remarkable effect on the reactivity of the esters 9b, 9f, and
9g. In general, the 4-(benzothiazol-2-yl) substituent inhibits
ionization of the esters 9a−g in comparison to other nitrenium
ion precursors, but the 3′-Me substituent accelerates ionization.
The 3′-Me-substituted esters have aqueous solution lifetimes at
30 °C in the range of 5 to 10 s compared to 400 to 800 s for
their counterparts without the 3′-Me substituent. This would
likely limit the mobility of the 3′-Me-substituted esters to the
cells in which metabolic activation occurs and would
concentrate their effects within such cells. Two-photon flash
photolysis studies of photorelease and diffusion through cells of
caged fluorescein, a dye about the same size as 9a−g with a
similar, largely planar aromatic structure, has shown that
diffusion from one lens fiber cell to adjoining cells is isotropic
within deep regions of the lens and has a time constant of ca. 10
s at 21 °C.44 Fluorescien has a pKa of 6.4 and would be largely
present in its anionic carboxylate form at physiological pH. The
esters 9a−g are uncharged at that pH. It appears that
fluorescein diffuses across cell membranes via an unassisted
process through gap junction channels.44 However, if a specific
ion channel-assisted mechanism enhances the diffusion of
fluorescein anion across membranes, that mechanism would
not be available to 9a−g, so the fluorescein result would be an
upper limit for diffusion of these uncharged esters. Diffusion of
an uncharged chemical species with a lifetime of ca. 5−10 s
from a tumor cell to nonadjoining cells more than a few cells
removed would be unlikely. The reduced lifetime of the 3′-Me
esters may improve the selectivity of the antitumor drug as long
as the metabolic activation is confined to tumor cells.
1
1422, 1273 cm−1; H NMR (500 MHz, DMSO-d6) δ 2.16 (3H, s),
7.17 (1H, d, J = 8.5 Hz), 7.37 (1H, t, J = 8.0 Hz), 7.48 (1H, t, J =8.0
Hz), 7.72, (1H, d, J = 1.5 Hz), 7.83 (1H, dd, J = 8.5, 1.5 Hz), 7.94
(1H, d J = 8.0 Hz), 8.04 (1H, d, J = 8.0 Hz), 8.49 (1H, s), 8.61 (1H,
s); 13C NMR (125.8 MHz, DMSO-d6) δ 16.9, 111.5, 121.9, 122.07,
122.11, 123.2, 124.8, 126.3, 126.4, 128.4, 134.0, 152.7, 153.8, 168.0;
high-resolution MS (ES, positive) C14H13N2OS (M + H) calcd
257.0749, found 257.0746.
N-(4-(Benzo[d]thiazol-2-yl)-2-chlorophenyl)hydroxylamine (8c).
249 mg (90%); mp 164−167 °C (decomp); IR 3268, 1600, 1469,
1435, 1219 cm−1; 1H NMR (500 MHz, DMSO-d6) δ 7.27 (1H, d, J =
8.3 Hz), 7.40 (1H, t, J = 7.5 Hz), 7.50 (1H, t, J = 7.5 Hz), 7.91−7.98
(3H, m), 8.07 (1H, d, J = 7.8 Hz), 8.89 (1H, s) 8.95 (1H, s); 13C
NMR (125.8 MHz, DMSO-d6) δ 114.0, 117.2, 122.6, 122.8, 124.5,
125.5, 127.0, 127.6, 127.8, 134.5, 150.4, 154.0, 166.8; high-resolution
MS (ES, positive) C13H10ClN2OS (M + H) calcd 277.0202, 279.0172,
found 277.0196, 279.0182.
EXPERIMENTAL SECTION
■
Kinetics and Azide Trapping Experiments. Kinetics experi-
ments were carried out by UV−vis spectroscopy at 30 °C in aqueous
0.02 M phosphate buffers, pH 5.6 to 7.4, (5 vol% CH3CN−H2O, μ =
0.5 (NaClO4)). Solutions were incubated in the thermostatted cell
holder for ca. 20 min before kinetic runs were initiated by injecting 15
μL of ca. 5.0 mM solutions of 9b−g in CH3CN into 3 mL of the buffer
to obtain a final concentration of ca. 2.5 × 10−5 M. The 3′-Me esters
9b, 9f, and 9g were generated in situ (see below) in dry CH3CN at ca.
5.0 mM concentration and were used for kinetic and product studies
without isolation. Absorbance vs time data were collected at 327 nm
for 9b, 319, 250, 220, and 209 nm for 9c, 319, 244, 222, and 209 nm
for 9d, 322, 300, 245, and 224 nm for 9e, 330 nm for 9f, and 320 nm
for 9g. Absorbance vs time data at each wavelength were fit to either a
first-order or consecutive first-order rate equation.
N-(4-(5-Fluorobenzo[d]thiazol-2-yl)phenyl)hydroxylamine (8d).
237 mg (91%); mp 184−186 °C (decomp); IR 3205, 3133, 1602,
1567, 1471, 1450, 1244 cm−1; 1H NMR (500 MHz, DMSO-d6) δ 6.91
(2H, d, J = 8.5 Hz), 7.25 (1H, dt, J = 8.9, 2.3 Hz), 7.75 (1H, dd, J =
9.9, 2.2 Hz), 7.87 (2H, d, J = 8.6 Hz), 8.07 (1H, m), 8.64 (1H, s), 8.91
19
(1H, s); 13C NMR (125.8 MHz, DMSO-d6) δ 108.6 (1C, d, 2J13
=
C‑
F
2 13 19
23.9 Hz), 112.7 (2C, s), 113.3 (1C, d, J
= 24.6 Hz), 123.5,
= 10.1 Hz), 128.7 (2C, s), 130.1, 155.2 (1C, d,
C‑
F
3 13 19
123.7 (1C, d, J
C‑
F
19
1 13 19
3J13
= 12.4 Hz), 155.5, 161.8 (1C, d, J
= 240.5 Hz), 171.0;
F
C‑
F
C‑
high-resolution MS (ES, positive) C13H10FN2OS (M + H) calcd
261.0498, found 261.0492.
N-(4-(6-Fluorobenzo[d]thiazol-2-yl)phenyl)hydroxylamine (8e).
227 mg (87%); mp 161−163 °C (decomp); IR 3290, 3132, 1601,
1567, 1479, 1452, 1245 cm−1; 1H NMR (500 MHz, DMSO-d6) δ 6.92
(2H, d, J = 8.3 Hz), 7.33 (1H, m), 7.87 (2H, d, J = 8.3 Hz), 7.93−7.99
(2H, m), 8.63 (1H, s), 8.88 (1H,s); 13C NMR (125.8 MHz, DMSO-
−
A series of NaN3 solutions from ca. 0.1 mM to 10 mM in [N3 ]
(exact concentration range depended on the ester) were prepared in
0.02 M phosphate buffer, pH 7.04, (5 vol% CH3CN-H2O, μ = 0.5
(NaClO4)). Solutions were incubated at 30 °C in a water bath for ca.
20 min in a Teflon capped round-bottom reaction vial before 15 μL of
a 5.0 mM solution of 9b−g in CH3CN was added to 3 mL of the
buffer. A control experiment in phosphate buffer in the absence of
NaN3 was also included in each run. Reaction mixtures were analyzed
in duplicate after completion of the reaction by 20 μL injections of the
reaction mixture from each vial onto an HPLC (C-18 reverse phase
column, 70/30 MeOH/H2O eluent, 1 mL/min, monitored by UV
absorbance). Wavelengths monitored were 340 and 212 nm for 9b, 9c,
9f, and 9g, and 338 and 220 nm for 9d and 9e. HPLC peak areas were
plotted against [N3−] and fit to standard azide clock equa-
tions.29,30,35,38 Kinetics of the decomposition of 9b−9g were
2 13 19
3 13 19
d6) δ 108.9 (1C, d, J
= 27.2 Hz), 112.8 (1C, d, J
= 16.4
C‑
F
C‑
F
2 13 19
Hz), 115.0 (1C, d, J
= 26.2 Hz), 123.6 (2C, s), 123.7, 128.6
F
C‑
(2C, s), 135.6 (1C, d, 3J13 F = 12.3 Hz), 151.1, 155.3, 159.9 (1C, d,
19
C‑
2J13
= 242.3 Hz), 168.3; high-resolution MS (ES, positive)
19
F
C‑
C13H10FN2OS (M + H) calcd 261.0498, found 261.0500.
N-(4-(5-Fluorobenzo[d]thiazol-2-yl)-2-methylphenyl)-
hydroxylamine (8f). 235 mg (86%); mp 155−158 °C (decomp); IR
3179, 2900, 1603, 1570, 1524 cm−1; 1H NMR (500 MHz, DMSO-d6)
δ 2.17 (3H, s), 7.17 (1H, d, J = 8.5 Hz), 7.28 (1H, t, J = 9.0 Hz), 7.73
(1H, s), 7.78 (1H, d, J = 10.0 Hz), 7.83 (1H, d, J = 8.0 Hz), 8.10 (1H,
m), 8.56 (1H, s), 8.62 (1H, s); 13C NMR (125.8 MHz, DMSO-d6) δ
−
−
monitored in N3 containing buffers (1 to 50 mM [N3 ]) at 327
nm for 9b, 345, 319, and 268 nm for 9c, 350, 326, and 260 nm for 9d,
350, 321, 290, and 260 nm for 9e, 330 nm for 9f, and 324 nm for 9g.
Synthesis. Synthesis of the Hydroxylamines 8b−g. The nitro
starting materials 16b−g are known compounds that were prepared by
published procedures.4,23,32,33 The appropriate nitro compound 16b−
19
19
17.3, 108.5 (1C, d, 2J13 F = 22.5 Hz), 111.8, 113.3 (1C, d, 2J13
=
C‑
C‑ F
3 13 19
23.75 Hz), 122.3, 123.3, 123.7 (1C, d, J
128.8, 130.1, 153.3, 155.2 (1C, d, J
= 10.0 Hz), 126.8,
= 12.5 Hz), 161.8 (1C, d,
C‑
F
3 13 19
C‑
F
19
F
1J13
= 238.8 Hz), 171.2; high-resolution MS (ES, positive)
C‑
C14H12FN2OS (M + H) calcd 275.0654, found 275.0656.
F
dx.doi.org/10.1021/jo400826f | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
N-(4-(6-Fluorobenzo[d]thiazol-2-yl)-2-methylphenyl)-
−40 °C for no more than one to two days for use in kinetics or
product isolation studies.
hydroxylamine (8g). 203 mg (74%); mp 178−182 °C (decomp); IR
1
3288, 3200 (br), 1608, 1570, 1455, 1265 cm−1; H NMR (500 MHz,
Product Isolation. Quinols 13b−g. For 13c, the corresponding
ester 9c (0.25 mmol) was dissolved in 20 mL of CH3CN. That
solution was added in 1 mL aliquots at 4 h intervals to a flask
containing 500 mL of pH 7.0 phosphate buffer, identical to that used
in the kinetic studies, incubated at 30 °C in a constant temperature
water bath. After the last addition, the reaction flask was incubated for
an additional 12 h at 30 °C prior to extraction with 3 × 100 mL of
CH2Cl2. The CH2Cl2 extract was dried over Na2SO4 and rotary
evaporated to obtain a dark green solid that was purified by radial
chromatography on silica gel (60/40 EtOAc/hexanes) to obtain 21 mg
(30%) of 13c.
For 13d, the 20 mL solution of 9d was added to the buffer in 1 mL
aliquots every 15 min, and the mixture was incubated for 3 h before
extraction. The dark yellow-brown solid obtained after rotary
evaporation was purified by column chromatography on silica gel
(20/80 EtOAc/hexanes), followed by radial chromatography on silica
gel (15/85 EtOAc/CH2Cl2) to obtain 21 mg (33%) of 13d.
For 13e, the 20 mL solution of 9e was added to the buffer in 1 mL
aliquots every 30 min, and the mixture was incubated for 5 h before
extraction. The dark yellow-brown solid was purified in the same way
as 13d to obtain 19 mg (30%) of 13e.
For 13b, 13f, or 13g the 50 mL CH3CN solution of 9b, 9f, or 9g
described above was added in 3 mL aliquots at 10 min intervals to a
flask containing 500 mL of pH 7.0 phosphate buffer incubated at 30
°C in a constant temperature water bath. After the last addition, the
reaction flask was incubated for an additional 24 h at 30 °C. The
reaction mixture was extracted with 3 × 100 mL of CH2Cl2. The
CH2Cl2 extract was dried over Na2SO4 and rotary evaporated to obtain
a yellowish solid that was purified by radial chromatography on silica
gel (10/90 EtOAc/CH2Cl2) to obtain 15 mg (23%) of 13b, 15 mg
(22%) of 13f, and 14 mg (20%) of 13g.
DMSO-d6) δ 2.16 (3H, s), 7.18 (1H, d, J = 8.0 Hz), 7.35 (1H, t, J= 8.0
Hz), 7.71 (1H, s), 7.80 (1H, d, J= 8.5 Hz), 7.94−7.98 (2H, m), 8.51
(1H, s), 8.62 (1H, s). 13C NMR (125.8 MHz, DMSO-d6) δ 17.3, 109.0
19
19
(1C, d, 2J13 F = 25.6 Hz) (7.98), 111.9 (7.18), 115.1 (1C, d, 2J13
C‑
C‑ F
3 13 19
= 25.0 Hz) (7.35), 122.3, 123.4, 123.6 (1C, d, J
= 8.8 Hz)
= 12.1 Hz),
F
C‑
F
3 13 19
(7.94), 126.7 (7.80), 128.7 (7.71), 135.6 (1C, d, J
C‑
151.1, 153.1, 159.8 (1C, d, 1J13 F = 241.8 Hz), 168.5; high-resolution
19
C‑
MS (ES, positive) C14H12FN2OS (M + H) calcd 275.0654, found
275.0652.
Synthesis of the Esters 9b−g. The stable esters 9c, 9d, and 9e were
made as previously described for 9a.29 The appropriate hydroxylamine
(0.21 mmol) was added to 30 mL of dry, freshly distilled benzene
containing 29 μL of N-ethylmorpholine (0.26 mmol). The suspension
was stirred at room temperature for 15 min to maximize dissolution of
the hydroxylamine. Pyruvonitrile (16.5 μL, 0.23 mmol) was added via
syringe in 3−4 μL portions over a period of 10 min. As the
pyruvonitrile was added, the hydroxylamine dissolved completely.
After the last addition, the reaction mixture was stirred for another 15
min. The reaction mixture was transferred to a small separatory funnel
and washed consecutively with 2 × 7 mL of ice cold 0.5 M NaOH, 2 ×
7 mL of ice cold 5% aq NaHCO3, and 2 × 7 mL of ice cold distilled
water. The solution was dried over Na2SO4 and concentrated by rotary
evaporation to obtain the ester in 50−70% isolated yield. Esters were
purified by column or radial chromatography on silica gel using 20/80
EtOAc/hexanes. Purified compounds were stored at −40 °C and
periodically repurified if necessary.
O-Acetyl-N-(4-(benzo[d]thiazol-2-yl)-2-chlorophenyl)-
hydroxylamine (9c). 47 mg (70%) mp 93−96 °C (decomp); IR 3227,
1
1751, 1602, 1472, 1218 cm−1; H NMR (500 MHz, CD3CN) δ 2.23
(3H, s), 7.20 (1H, d, J = 8.0 Hz), 7.41 (1H, t, J = 7.5 Hz), 7.51 (1H, t,
J = 7.5 Hz), 7.98 (3H, m), 8.07 (1H, s), 8.92 (1H, s); 13C NMR
(125.8 MHz, CD3CN) δ 18.9, 116.9, 120.4, 122.9, 123.7, 126.4, 127.5,
128.1, 128.8, 129.5, 135.8, 146.8, 154.9, 166.9, 170.9; high-resolution
MS (ES, positive) C15H11ClNaN2O2S (M + Na) calcd 341.0127,
343.0097, found 341.0129, 343.0060.
4-(Benzo[d]thiazol-2-yl)-4-hydroxy-2-methylcyclohexa-2,5-dien-
one (13b). mp 175−176 °C; IR 3320 (br), 1672, 1641, 1503, 1436,
1052 cm−1; 1H NMR (500 MHz, CD2Cl2) δ 1.93 (3H, d, J = 1.5 Hz),
4.24 (1H, s (br)), 6.30 (1H, d, J = 9.5 Hz), 6.77 (1H, m), 6.98 (1H,
dd, J = 9.8, 3.1 Hz), 7.43 (1H, td, J = 7.1, 1.2 Hz), 7.52 (1H, td, J = 7.3,
1.2 Hz), 7.91 (1H, d, J = 7.7 Hz), 7.99 (1H, d, J = 8.2 Hz); 13C NMR
(125.8 MHz, CD2Cl2) δ 15.3 (1.93), 71.3, 122.0 (7.91), 123.1 (7.99),
125.7 (7.43), 126.5 (7.52), 128.4 (6.30), 135.5, 135.8, 142.8 (6.77),
147.3 (6.98), 152.5, 171.4, 185.3; high-resolution MS (ES, positive)
C14H11NaNO2S (M + Na) calcd 280.0408, found 280.0406.
4-(Benzo[d]thiazol-2-yl)-2-chloro-4-hydroxycyclohexa-2,5-dien-
one (13c). mp 139−141 °C; IR 3270 (br), 1678. 1620, 1471, 1436,
O-Acetyl-N-(4-(5-fluorobenzo[d]thiazol-2-yl)phenyl)-
hydroxylamine (9d). 39 mg (61%); mp 68−70 °C (decomp); IR
1
3248, 1739, 1608, 1453, 1246 cm−1; H NMR (500 MHz, CD2Cl2) δ
2.23 (3H, s), 7.10 (2H, d, J = 8.7 Hz), 7.14 (1H, td J = 8.9, 2.4 Hz),
7.68 (1H, dd, J = 9.7, 2.4 Hz), 7.83 (1H, dd, J = 8.8, 5.2 Hz), 8.01 (2H,
d, J = 8.6 Hz), 8.87 (1H, s); 13C NMR (125.8 MHz, CD2Cl2) δ 18.8,
2 13 19
2 13 19
108.9 (1C, d, J
= 23.9 Hz), 113.4 (1C, d, J
= 25.1 Hz),
C‑
F
C‑
F
115.4 (2C, s), 122.4 (1C, d, 3J13 F = 10.0 Hz), 128.4, 128.5 (2C, s),
130.4, 149.6, 155.2 (1C, d, J
19
1
1286 cm−1; H NMR (500 MHz, CDCl3) δ 4.43 (1H, s (br)), 6.45
C‑
3 13 19
F = 12.0 Hz), 162.0 (1C, d, 2J13
19
(1H, d, J = 9.9 Hz), 7.07 (1H, dd, J = 9.9, 2.9 Hz), 7.20 (1H, d, J = 2.9
Hz), 7.46 (1H, td, J = 8.2, 1.1 Hz), 7.54 (1H, td, J = 8.3, 1.2 Hz), 7.91
(1H, d, J = 8.0 Hz), 8.04 (1H, d, J = 8.2 Hz); 13C NMR (125.8 MHz,
CDCl3) δ 72.6, 122.1 (7.91), 123.3 (8.04), 126.2 (7.46), 126.8 (7.54),
127.7 (6.45), 133.3, 135.6, 143.4(7.20), 147.7 (7.07), 152.0, 169.5,
177.9; high-resolution MS (ES, positive) C13H8ClNaNO2S (M + Na)
calcd 299.9862, 301.9832, found 299.9855, 301.9833.
C‑
C‑
F
= 242.4 Hz), 169.9, 170.2; high-resolution MS (ES, positive)
C15H11FNaN2O2S (M + Na) calcd 325.0423, found 325.0420.
O-Acetyl-N-(4-(6-fluorobenzo[d]thiazol-2-yl)phenyl)-
hydroxylamine (9e). 35 mg (55%); mp 99−101 °C (decomp); IR
3239, 1766, 1609, 1455, 1223 cm−1; 1H NMR (500 MHz, CD2Cl2-d2)
δ 2.29 (3H, s), 7.15 (2H, d, J = 8.5 Hz), 7.26 (1H, td, J = 9.0, 2.6 Hz),
7.64 (1H, d, J = 8.2, 2.6 Hz), 7.99 (1H, dd, J = 8.8, 4.8 Hz), 8.04 (2H,
d, J = 8.6 Hz), 8.92 (1H, s); 13C NMR (125.8 MHz, CD2Cl2-d2) δ
4-(5-Fluorobenzo[d]thiazol-2-yl)-4-hydroxycyclohexa-2,5-dien-
one (13d). mp 128−130 °C; IR 3370 (br), 1663, 1617, 1453, 1140
2 13 19
2 13 19
1
C‑
F
C‑
F
cm−1; H NMR (500 MHz, CD2Cl2-d2) δ 4.2 (1H, s (br)), 6.32 (2H,
18.8, 107.8 (1C, d, J
= 27.0 Hz), 114.7 (1C, d, J
= 24.6
3 13 19
Hz), 115.5 (2C,s), 123.8 (1C, d, J
= 9.3 Hz), 128.4 (2C,s),
= 11.3 Hz), 149.4, 150.9, 160.4 (1C, d,
C‑
F
d, J = 10.0 Hz), 6.99 (2H, d, J = 10.0 Hz), 7.23 (1H, td, 8.8, 2.5 Hz),
3 13 19
C‑
F
7.69 (1H, dd, J = 9.4, 2.4 Hz), 7.87 (1H, dd, 8.5, 5.0 Hz); 13C NMR
128.5, 135.9 (1C, d, J
2J13
= 244.6 Hz), 167.1, 170.2; high-resolution MS (ES, positive)
19
2 13 19
(125.8 MHz, CD2Cl2-d2) δ 71.0, 109.3 (1C, d, J
= 23.7 Hz),
= 10.0 Hz),
F
C‑
F
C‑
F
2 13 19
3 13 19
C15H11FNaN2O2S (M + Na) calcd 325.0423, found 325.0420.
The esters 9b, 9f, and 9g were too reactive to isolate. Instead they
were prepared in dry CH3CN. A general procedure follows. The
appropriate hydroxylamine (0.25 mmol) was dissolved in 50 mL of dry
CH3CN and stirred under N2 at 0 to −10 °C for 10 min followed by
addition of 33 μL (0.30 mmol) of N-ethylmorpholine. Pyruvonitrile
(18.0 μL, 0.25 mmol) was added in 6.0 μL fractions every 10 min over
a period of 20 min. The solution was left under N2 at 0 °C for 1 h after
the last addition of pyruvonitrile. Disappearance of the hydroxylamine
and appearance of a new spot was detected by TLC of the reaction
mixture on silica gel (20/80 EtOAc/hexanes). Solutions were stored at
114.6 (1C, d, J
= 25.2 Hz), 123.0 (1C, d, J
C‑
F
C‑
19
128.7 (2C, s), 131.4, 147.1 (2C, s), 153.5 (1C, d, 3J13 F = 12.1 Hz),
C‑
1 13 19
161.9 (1C, d, J
= 243.9 Hz), 173.4, 184.5; high-resolution MS
C‑
F
(ES, positive) C13H8FNaNO2S (M + Na) calcd 284.0157, found
284.0157.
4-(6-Fluorobenzo[d]thiazol-2-yl)-4-hydroxycyclohexa-2,5-dien-
one (13e). mp 168−170 °C; IR 3333 (br), 1669, 1626, 1505, 1455,
1
1251, 1198 cm−1; H NMR (500 MHz, CD2Cl2-d2) δ 4.08 (1H, s),
6.31 (2H, d, J = 10.5 Hz), 6.98 (2H, d, J = 10.0 Hz), 7.26 (1H, td, J =
9.0, 2.5 Hz)), 7.60 (1H, dd, J = 8.2, 2.6 Hz), 7.95 (1H, dd, J = 9.0, 5.0
19
C‑
Hz); 13C NMR (125.8 MHz, CD2Cl2-d2) δ 70.9, 108.2 (1C, d, 2J13
F
G
dx.doi.org/10.1021/jo400826f | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
19
19
= 26.9 Hz), 115.3 (1C, d, 2J13 F = 24.8 Hz), 124.3 (1C, d, 3J13
=
C‑ F
chromatography (30/70 hexanes/CH2Cl2) to obtain 14 mg (18%) of
C‑
12f: mp 86−88 °C; IR 3385, 2111, 1602, 1567, 1453, 1122 cm−1; H
3 13 19
1
9.5 Hz), 128.7 (2C, s), 136.9 (1C, d, J
= 11.1 Hz), 147.1 (2C,
= 246.2 Hz), 170.3, 184.4; high-
C‑
F
19
s), 149.3, 160.7 (1C, d, 1J13
NMR (500 MHz, CDCl3) δ 2.27 (3H, s), 4.26 (2H, s(br)), 7.15 (1H,
td, J = 9.0, 2.5 Hz), 7.60 (1H, s), 7.67 (1H, dd, J = 10.0, 2.5 Hz), 7.75
C‑
F
resolution MS (ES, positive) C13H8FNaNO2S (M + Na) calcd
284.0157, found 284.0147.
(1H, d, J=2.0 Hz), 7.85 (1H, dd, J=8.5, 5.0 Hz); 13C NMR (125.8
MHz, CD2Cl2) δ 17.1, 108.5 (1C, d, 2J13 F = 26.4 Hz), 112.9 (1C, d,
19
4-(5-Fluorobenzo[d]thiazol-2-yl)-4-hydroxy-2-methylcyclohexa-
2,5-dienone (13f). mp 99−100 °C; IR: 3276 (br), 1670, 1626, 1602,
1455, 1147, 1125 cm−1; 1H NMR (500 MHz, CD2Cl2) δ 1.98 (3H, s),
4.00 (1H, s(br)), 6.35 (1H, d, J = 10.0 Hz), 6.79 (1H, s), 7.00 (dd, J =
9.5, 3.0 Hz), 7.30 (1H, m), 7.64 (1H, m), 8.00 (1H, dd, J = 9.0, 5.0
Hz); 13C NMR (125.8 MHz, CD2Cl2) δ 15.4, 71.3, 109.2 (1C, d,
C‑
19
3 13 19
2J13
= 26.4 Hz), 114.9, 122.2 (1C, d, J
= 9.9 Hz), 123.0,
C‑
F
C‑
F
3 13 19
123.2, 125.0, 126.5, 130.2, 139.8, 155.2 (1C, d, J
161.9 (1C, d, J
= 13.2 Hz),
F
C‑
1 13 19
= 241.5 Hz), 170.1; high-resolution MS (ES,
C‑
F
positive) C14H10FNaN5S (M + Na) calcd 322.0539, found 322.0531.
19
19
2 13 19
2J13
=23.9 Hz), 114.4 (1C, d, J
=25.2 Hz), 122.9 (1C, d,
C‑
F
C‑
F
ASSOCIATED CONTENT
* Supporting Information
19
■
3J13 F = 10.0 Hz), 128.6, 135.7, 142.4, 150.6, 153.5 (1C, d, 3J13
=
C‑ F
C‑
S
19
12.6 Hz), 161.9 (1C, d, 1J13
=244.1 Hz), 174.1, 185.2; high-
C‑
F
Tables S1 through S6, containing a summary of individual rate
constants obtained in this study; Figures S1 and S2, HPLC
chromatograms of the kinetic reaction mixtures for 9c and 9g;
Figure S3, repetitive wavelength scans for the hydrolysis of 9d
and 9e; Figure S4, repetitive wavelength scans for the
resolution MS (ES, positive) C14H10FNaNO2S (M + Na) calcd
298.0314, found 298.0312.
4-(6-Fluorobenzo[d]thiazol-2-yl)-4-hydroxy-2-methylcyclohexa-
2,5-dienone (13g). mp 120−122 °C; IR 3240 (br), 1671, 1627, 1603,
1456, 1148, 1125 cm−1; 1H NMR (500 MHz, CD2Cl2) δ 1.97 (3H, s),
3.99 (1H, s(br)), 6.34 (1H, d, J = 9.5 Hz), 6.79 (1H, s), 6.99 (1H, m),
−
1
decomposition of 9d and 9e in the presence of N3 ; H and
13C NMR data for 8b−g, 9c−e, 12b, 12c, 12f, and 13b−g. This
material is available free of charge via the Internet at http://
pubs.acs.org.
7.29 (1H, t, J = 7.8 Hz), 7.64 (1H, d, J = 6.0 Hz), 7.99 (1H, m); 13C
NMR (125.8 MHz, CD2Cl2) δ 15.4, 71.3, 108.1 (1C, d, 2J13 F = 26.5
19
C‑
2 13 19
3 13 19
Hz), 115.1 (1C, d, J
= 25.3 Hz), 124.2 (1C, d, J
= 9.9
C‑
F
C‑
F
3 13 19
Hz), 128.6, 135.7, 136.9 (1C, d, J
=11.5 Hz), 142.4, 147.0,
C‑
F
149.3, 160.6 (1C, d, 1J13 F =245.0 Hz), 171.2, 185.1; high-resolution
19
C‑
AUTHOR INFORMATION
Corresponding Author
*E-mail: novakm@miamioh.edu.
Notes
MS (ES, positive) C14H10FNaNO2S (M + Na) calcd 298.0314, found
298.0311.
■
Azide Adducts 12b, 12c, and 12f. 2-Azido-4-(benzo[d]thiazol-2-
yl)-6-methylaniline (12b). The 50 mL CH3CN solution of 9b
described above was injected in 1 mL aliquots at 5 s intervals into 500
mL of pH 7.0 phosphate buffer containing 10 mM NaN3, identical to
that used in the kinetics, incubated at 30 °C in a constant temperature
water bath. After the last addition, the reaction mixture was kept at 30
°C for 3 h prior to extraction with 3 × 100 mL of CH2Cl2. The
CH2Cl2 extract was dried over anhydrous Na2SO4 and then rotary
evaporated to get crude solid which was purified by radial
chromatography on silica gel (20/80 EtOAc/hexanes) to obtain 22
mg (31%) of 12b: mp 86−89 °C; IR 3390, 2111, 1620, 1475, 1436,
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the NIH/NIGMS (grant no. R15 GM088751-01) for
support of this work. C.G.C.-S. thanks Miami University and
the Department of Chemistry and Biochemistry for a Beckman
Scholars Award provided by a grant from the Arnold and Mabel
Beckman Foundation. High resolution mass spectra were
obtained at the Campus Chemical Instrument Center at Ohio
State University.
1
1420, 1328 cm−1; H NMR (500 MHz, CD2Cl2) δ 2.23 (3H, s), 4.19
(2H, s (br)), 7.35 (1H, td, J = 7.3, 1.2 Hz), 7.46 (1H, td, J = 7.3, 1.2
Hz), 7.57 (1H, m), 7.73 (1H, d, J = 1.8 Hz), 7.89 (1H, dt, J = 8.0, 0.5
Hz), 7.97 (1H, dt, J = 8.0, 0.5 Hz); 13C NMR (125.8 MHz, CD2Cl2) δ
17.5 (2.23), 115.3 (7.73), 121.9 (7.89), 122.8 (7.97), 123.4, 123.9,
125.0 (7.35), 125.4, 126.5 (7.46), 126.8 (7.57), 135.1, 139.9, 154.6,
167.9; high-resolution MS (ES, positive) C14H11NaN5S (M + Na)
calcd 304.0633, found 304.0624.
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