Design, synthesis and photochemical reactivation of caged prodrugs of 8-hydroxyquinoline-based enzyme inhibitors

Article abstract of DOI:10.1248/cpb.c14-00086

8-Hydroxyquinoline (HQ)-based compounds have recently been proposed as potential candidates for drugs for treating human immunodeficiency virus (HIV), cancer, neurodegenerative diseases (Alzheimer's and Parkinson's disease), and parasitic and bacterial infections. However, HQ itself and its derivatives might be toxic due to their intrinsic affinity for metal cations in living systems. One possible strategy for suppressing the toxicity and side effects of drugs with metal chelation properties, such as HQ, would be masking the critically important moieties with protecting groups that can be subsequently removed under specific conditions. In our previous work, we reported that HQ analogs are potent and selective inhibitors (K_ivalues=0.16-29 μM) of aminopeptidase from Aeromonas proteolytica (AAP) (EC 3.4.11.10), a dinuclear Zn²⁺ peptidase. Based on this background information, HQ sulfonates were synthesized as prodrugs of HQ-based AAP-inhibitors that can be reactivated by photochemical cleavage of the S-O bond in the sulfonate groups. The findings indicate that HQ sulfonates containing methanesulfonyl and 2-aminoethanesulfonyl groups are essentially stable under physiological conditions and undergo photolysis to regenerate the corresponding HQ compounds that function as AAP inhibitors. This methodology could be applied to the design of similar types of Zn²⁺ hydrolase inhibitors and prodrugs.

Full text of DOI:10.1248/cpb.c14-00086

642
Regular Article
Chem. Pharm. Bull. 62(7) 642–648 (2014)
Vol. 62, No. 7
Design, Synthesis and Photochemical Reactivation of Caged Prodrugs of
8-Hydroxyquinoline-Based Enzyme Inhibitors
,a,b
Shinya Ariyasu,a Yuki Mizuseda,b Kengo Hanaya,b and Shin Aoki*
a Center for Technologies against Cancer, Tokyo University of Science; and b Faculty of Pharmaceutical Sciences,
Tokyo University of Science; 2641 Yamazaki, Noda 278–8510 Japan.
Received January 25, 2014; accepted April 10, 2014
8-Hydroxyquinoline (HQ)-based compounds have recently been proposed as potential candidates for
drugs for treating human immunodeficiency virus (HIV), cancer, neurodegenerative diseases (Alzheimer’s
and Parkinson’s disease), and parasitic and bacterial infections. However, HQ itself and its derivatives might
be toxic due to their intrinsic affinity for metal cations in living systems. One possible strategy for suppress-
ing the toxicity and side effects of drugs with metal chelation properties, such as HQ, would be masking
the critically important moieties with protecting groups that can be subsequently removed under specific
conditions. In our previous work, we reported that HQ analogs are potent and selective inhibitors (Ki val-
ues=0.16–29µM) of aminopeptidase from Aeromonas proteolytica (AAP) (EC 3.4.11.10), a dinuclear Zn2+
peptidase. Based on this background information, HQ sulfonates were synthesized as prodrugs of HQ-based
AAP-inhibitors that can be reactivated by photochemical cleavage of the S–O bond in the sulfonate groups.
The findings indicate that HQ sulfonates containing methanesulfonyl and 2-aminoethanesulfonyl groups are
essentially stable under physiological conditions and undergo photolysis to regenerate the corresponding
HQ compounds that function as AAP inhibitors. This methodology could be applied to the design of similar
types of Zn2+ hydrolase inhibitors and prodrugs.
Key words 8-hydroxyquinoline; prodrug; inhibitor; photolysis; Zn2+ peptidase
8-Hydroxyquinoline (HQ)-based compounds have recently alogues that are protected by o-nitrophenyl groups, and can be
been proposed as potent protease-inhibitors1–3) and potential reactivated to form metal complexes, as reported by Kaplan.28)
candidates for drugs for use in the treatment of AIDS, can- Therefore, one of the strategies for suppressing the toxicity
cer, neurodegenerative diseases (Alzheimer’s and Parkinson’s and side effects of metal chelator-based drugs such as HQ
disease), parasitic or bacterial infections.4–13) 5-Chloro-8-hy- would be masking the critically important moieties with pro-
droxy-7-iodoquinoline (clioquinol, Chart 1) has been clini- tecting groups that can be removed under specific conditions.
cally used as an antifungal and antiprotozoal drug prior to the
In our previous work, we reported that HQ 1 and its
1970’s.14) The use of clioquinol was, however, banned after the analogs (2–8) are potent and selective inhibitors (Ki values=
1970’s, because it was suspected that it might cause serious 0.16–29µM) of an aminopeptidase from Aeromonas proteolyti-
side effects called subacute myelo-optico neuropathy, SMON. ca (AAP) (EC 3.4.11.10), a dinuclear Zn2+ peptidase (Chart 2).
Although the mechanism responsible for the development of They have negligible inhibitory activities against leucine ami-
SMON is not fully understood, one of its causes has been at- nopeptidase (LAP), alkaline phosphatase, carboxypeptidase A,
tributed to its ability to form complexes with metal ions such and carbonic anhydrase (CA)31) (Chart 2). The X-ray crystal
as Fe2+ in the human body.15) Recently, however, this situation structure of the AAP–1 complex disclosed that these HQ
seems to be changing somewhat, as mentioned above. As a inhibitors bind to the dinuclear Zn2+ active site via the coor-
typical example,16) 5,7-dichloro-8-hydroxyquinoline (chlorox- dination of 8-O− to two Zn2+ ions and that of a nitrogen atom
ine, Chart 1), a topical antimicrobial agent, has been reported to one Zn2+. On the other hand, 2-methyl-8-hydroxyquinoline
to be an inhibitor of biofilm formation of Candida albicans17) 9 has negligible inhibitory activity against AAP, possibly due
and human platelet type 12-lipoxygenase.18)
It was reported that so-called “caged” drugs, particularly
to the steric hindrance of the methyl group at the 2 position.
We also reported that the HQ sulfonates undergo S–O bond
those containing photocleavable protecting groups, play im- cleavage reactions by photoirradiation in aqueous solutions
portant roles in the control of bioactivities.19–26) As general to afford the corresponding 8-quinolinols and sulfonates with
(not limited to metal chelators) examples of “caged” biologi- negligible byproduct production.32–36)
cally active compounds, Hoffman’s group reported the design
For the purpose of reducing the side effects of HQ-based
and synthesis of “caged ATP,” which is an ATP molecule that agents not only for AAP inhibitors but also for other HQ-
is protected by a photocleavable (2-nitro)phenylethyl group,
to investigate the function of the Na:K pump in human red
blood cells.19) Hess’ group also reported on the use of N-ni-
trobenzyl serotonin, a “caged serotonin,” in a kinetic study of
the serotonin 5-HT3 receptor.23) The advantage of photolabile
groups has been applied to caged-metal chelators27–30) such as
ethylene glycol bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic
acid (EGTA) and ethylenediaminetetraacetic acid (EDTA) an-
The authors declare no conflict of interest.
Chart 1. Structures of Clioquinol and Chloroxine
© 2014 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: shinaoki@rs.noda.tus.ac.jp

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Chart 2. Inhibition of AAP by 8-Quinolinol Analogs
Chart 3. Structures of HQ Methanesulfonates 10–13 and HQ Amino-
ethanesulfonates 14–16
based drugs and homologues, we were prompted to design
and synthesize prodrugs of HQ that can be activated by
photoirradiation as an external trigger.37) Although carboxyl
ester was frequently chosen as protecting group for caged
compounds,38,39) the caboxyl esters of HQ were unstable under
physiological conditions. Recently, only a few papers have
appeared on sulfonates as prodrugs that are more stable than
carboxyl esters and can be reactivated under the condition in
Chart 4. Synthesis of 2-Aminoethanesulfonates 14, 15 and 16
the presence of H2O2 or hypoxia conditions.41)
40)
In this paper, we report on the design and synthesis of HQ were prepared from the corresponding 8-quinolinols and
sulfonates 10–16 (Chart 3) as prodrugs of HQ-type drugs. 1, methanesulfonyl chlorides (see Experimental).42) For 14–16,
3, 6 and 7 were chosen as active forms of these prodrugs, and the tetrabutylammonium salt 18 prepared from 17 according
9 was chosen as the control compound. The methanesulfonyl to a literature procedure43) was converted to the corresponding
group, which has the smallest steric hindrance, was used as sulfonyl chloride, and then condensed with the HQ to afford
a protecting group. 2-Aminoethanesulfonyl group was also 19, 20 and 21 (Chart 4). Treatment of 19–21 with HCl in diox-
introdeced, because 2-aminoethanelsulfonate derivatives ane gave 14–16, respectively, as HCl salts.
should be more hydrophilic than methanesulfonates and the
byproduct of the photoreaction is 2-aminoethanesulfonic acid checked the stability of 10–16 in aqueous solution at pH 8.0
(taurine) that is considered to be nontoxic. The photochemical (10 mM
3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic
Stability of Prodrugs in Aqueous Condition We
unmasking reactions of these prodrugs were evaluated using acid (EPPS) with I=0.1 (NaNO3) containing 2% CH3CN)
AAP and these results are also discussed.
and 37°C, enzymatically. These compounds were incubated
at pH 8.0 for a given period of time (X h) in the absence of
AAP, and the hydrolysis of leucine p-nitroanilide (LeuNA) by
Results and Discussion
Synthesis of Methanesulfonates and 2-Aminoethanesul- AAP at the same pH was followed by monitoring the absorp-
fonate Derivatives of HQ as Prodrugs of AAP Inhibitors tion of p-nitrophenylaniline (405nm) that is produced by the
The preparation of 10 was carried out according to literature hydrolysis of LeuNA44) (Fig. 1). These data indicate that 13,
methods42) and 5-substituted HQ methanesulfonates 11–13 14 and 15 are slowly hydrolyzed, releasing the corresponding

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Vol. 62, No. 7
8-quinolinols, 1, 3 and 7, that have AAP inhibitory activity. hydrolyzed. Although it is likely that 9 is produced from 16,
Because the Ki values of 1, 3 and 6, the original compounds negligible AAP inhibition was observed, possibly due to the
of 10–12, are 0.19–0.68µM, respectively,31) the results shown high Ki value (>100 µM) of 931) (see Chart 2). These results
in Fig. 1 indicate that less than 1% of compounds 10–12 are indicate that the HQ methanesulfonate derivatives 10–12 are
very stable under physiological conditions.
Reactivation of HQ Sulfonate Derivatives by Photoir-
radiation We examined the reactivation of HQ sulfonates,
10–16 by the photochemical cleavage of their S–O bonds.34,35)
First, the generation of the corresponding HQ (1mM) by pho-
toirradiation at >330nm in a 50/50 DMSO-d6/D2O solution
1
(10 mM EPPS (pD 8.0) at 25°C) was monitored by H-NMR.45)
As shown in Fig. 2, 3 is regenerated from 11 with negligible
side reactions.34–36) The photochemical reactivation of 10, 11,
13 and 15 was also followed by UV-Vis spectra (Fig. 3). As
shown in Fig. 3, 13 was activated very slowly by photoirra-
diation, and the photoreactivities of 10, 11 and 15 are almost
identical (the quantum yields for the photolysis (Φ) of 10, 11,
13 and 15 were 7.3×10−5, 9.9×10−5, 5.8×10−6 and 8.3×10−5,
respectively).
We next examined the inhibition of AAP by HQ that was
regenerated by photoirradiation, the experimental protocols
of which are summarized in Chart 5 (Methods A, B, C). In
Method A, reaction mixtures of 10–16 (5 µM) and AAP were
photoirradiated at 313nm at pH 8.0 (10mM EPPS with I=0.1
(NaNO3) containing 2% CH3CN) and 25°C for Ymin (Y=1, 3,
5, 10, 15). LeuNA was then added and its hydrolysis was fol-
lowed by monitoring the change in absorption at 405nm due
Fig. 1. Remaining Activity of AAP after the Incubation of 10, 11, 12
(Solid Circles), 13 (Solid Triangles), 14 (Solid Squares), 15 (Open Cir-
cles), and 16 (Open Squares) at pH 8.0 (10mM EPPS with I=0.1 (NaNO3)
Containing 2% CH3CN) and 37°C for X h
The initial concentrations of these prodrugs were 100µM. Open triangles indicate
the controls (without prodrugs).
Fig. 3. Conversion as the Result of the Photoreaction, as Followed by
UV-Vis Spectra of 10 (Open Circles), 11 (Open Triangles), 13 (Open
Squares) and 15 (Solid Triangles)
Fig. 2. Photolysis of 11 (5 mM) in a Mixture of DMSO-d6 and D2O at
pD 8.0 (10mM EPPS) (50/50) and 25°C Followed by H-NMR (Aromatic
Region)
1
Irradiation at 313nm for Ymin at pH 8.0 (10mM EPPS with I=0.1 (NaNO3) con-
taining 2% CH3CN) and 25°C. Initial concentration of the prodrugs is 50µM.
(a) 11 before photolysis, (b) after photoirradiation with a high-pressure Hg lamp
for 5h, and (c) 3 in DMSO-d6/D2O at pD 8.0 (10mM EPPS) (50/50).
Chart 5. Experimental Protocols for the Photochemical Cleavage Reaction

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645
to the release of p-nitoroaniline, as described above. In Meth- than that of the corresponding HQ methanesulfonates 10 and
ods B and C, the hydrolysis of LeuNA by AAP was measured 11, which could be due to some interactions (electrostatic,
after irradiation of the HQ sulfonates alone and AAP alone to hydrophobic or other interactions) between 14 or 15 and the
evaluate the effect of photoirradiation on AAP.
enzyme. The stronger inhibition of 11 and 15 in comparison
In Figs. 4a–f, it is indicated that the AAP activity in Meth- with the other prodrugs (Fig. 3) is attributable to the fact they
od C (open circle in Fig. 4) negligibly changes after photoir- have higher Φ values than other prodrugs and the smallest Ki
radiation. Besides, the plots in Method A and Method B are values of the parent compound 3 (Chart 2).
almost superimposable in Figs. 4a–f. These data strongly sug-
gest that AAP is essentially not damaged by photoirradiation.
Conclusion
After the photoirradiation of 10, 11, 14, and 15 for 15min, the
We report herein on the design and synthesis of HQ sulfo-
hydrolytic activities of AAP were inhibited by more than 50% nates 10–16 for use as prodrugs of HQ-based AAP-inhibitors
(Figs. 4a, b, d, e). In particular, the AAP-activity in the pres- and their reactivation by photolysis. The findings show that
ence of 11 was decreased to ca. 30% by photoirradiation for HQ methanesulfonates 10 and 11 are stable under physiologi-
only 5min (Fig. 4b). Note that 13 and 16 are exceptions (Figs. cal conditions. The photoirradiation of HQ sulfonates 10, 11,
4c, f), because their photochemical products, 7 and 9, have 14 and 15 promotes the cleavage of the S–O bond of these
larger Ki value than the other HQ’s. Although AAP-inhibition prodrugs, resulting in the regeneration of the corresponding
was observed in the presence of 12 after photoirradiation, HQ derivatives that function as inhibitors of AAP. The most
the results of AAP inhibition were not reproducible (data not efficient inhibition of AAP was observed by photoreactivation
shown), possibly due to slow binding property of 6 to AAP, of 11, possibly due to the fact that it undergoes photocleavage
as we previously reported.31) These results indicate that the the most rapidly and has the smallest Ki values of 3, which
inhibition against AAP after photoirradiation can be directly is the original compound of 11. Although the reactivation ef-
attributed to the corresponding HQ derivatives generated by ficiency of the HQ sulfonates in this paper is somewhat low,
the reactivation of HQ sulfonates. It should also be noted that this “caging” strategy would be useful for the HQ type and re-
the inhibitory activity of the photochemical products from HQ lated inhibitors of not only AAP but also other metal enzymes
2-aminoethanesulfonates 14 and 15 against AAP is less potent and proteins. The substitutions of the HQ derivatives can con-
Fig. 4. Results for the Photochemical Reactivation of 10 (a), 11 (b), 13 (c), 14 (d), 15 (e) and 16 (f) (for Methods A (Open Triangles), B (Open
Squares), and C (Open Circles), See Text and Chart 5)
Irradiation at 313nm for Ymin at pH 8.0 (10mM EPPS with I=0.1 (NaNO3) containing 2% CH3CN) and 25°C. Initial concentration of the prodrugs is 5µM.

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Vol. 62, No. 7
tribute to the acceleration of photochemical S–O bond cleav- 1H-NMR (CDCl3) δ: 3.52 (3H, s), 7.66 (1H, dd, J=4.1, 9.0Hz),
age reaction,35) the red-shift of the irradiation wavelength46) 7.98 (1H, d, J=8.1Hz), 8.57 (1H, d, J=8.7Hz), 9.08 (1H, d,
and the improvement of inhibition of Zn2+ enzymes such as J=3.6Hz). 13C-NMR (CDCl3) δ: 39.6, 121.7, 122.0, 123.3, 125.4
β-lactamases. These results indicate that HQ sulfonates repre- (q, J=5.6Hz), 125.7, 126.2, 133.0 (q, J=2.5Hz), 141.7, 148.5,
sent potential candidates for prodrugs of the inhibitors against 151.4. HR-FAB-MS m/z: 292.0255 (Calcd for C11H9F3NO3S
AAP and that these methods can be applied to the design of [M+H]+: 292.0253).
similar types of Zn2+ hydrolase inhibitors and prodrugs that
13: mp: 117–118°C. IR (neat) cm−1: 3018, 1595, 167, 1500,
could be used in the treatment of HIV, cancer, neurodegenera- 1399, 1333, 1232, 1171, 1150, 1135, 1058, 975, 869, 849, 792,
tive diseases, and parasitic and bacterial infections.
739. 1H-NMR (CDCl3) δ: 3.55 (3H, s), 7.74–7.83 (2H, m),
8.45 (1H, d, J=8.7Hz), 9.08–9.12 (2H, m). 13C-NMR (CDCl3)
δ: 39.7, 121.8, 122.9, 124.8, 125.0, 132.5, 141.3, 143.8, 149.9,
Experimental
General Information All reagents and solvents purchased 151.9. HR-FAB-MS m/z: 269.0233 (Calcd for C10H9N2O5S [M+
were of the highest commercial quality and were used with- H]+: 269.0232). Anal. Calcd for C10H8N2O5S: C, 44.78; H, 3.01;
out further purification. The aminopeptidase from AAP was N, 10.44. Found: C, 44.70; H, 2.76; N, 10.40.
purchased from Sigma-Aldrich. All aqueous solutions were
Synthesis of HQ 2-Aminomethanesulfonates 14, 15 and
prepared using deionized and distilled water. The Good’s 16 HQ 2-Aminoethanesulfonate Hydrochloride Salt (14)
buffer reagents (Dojindo) were commercially obtained: EPPS. A solution of N-Boc-2-aminoethanesulfonate nBu4N+ salt
Melting points were determined on a Yanaco Melting Point 1843) (1877mg, 4.02mmol) in CH2Cl2 (6.0mL) was treated
Apparatus and a Büchi 510 Melting Point Apparatus and are with N,N-dimethylformamide (DMF) (0.031mL, 0.40mmol)
uncorrected. UV-Vis spectra were recorded on a JASCO UV/ and then with triphosgene (477mg, 1.61mmol) at room tem-
VIS spectrophotometer V-550 and V-630 Bio UV-Vis Spec- perature. After stirring at room temperature for 1h, the reac-
trophotometer at 25 0.1°C. IR spectra were recorded on a tion mixture was cooled to 0°C, to which a solution of Et3N
Perkin-Elmer FT-IR spectrophotometer at room temperature. (1.20mL, 8.06mmol), 4-dimethylaminopyridine (DMAP)
1H- (300MHz) and 13C- (75MHz) NMR spectra were recorded (cat.) and 1 (878mg, 6.05mmol) in CH2Cl2 (4mL) was added
on a JEOL Always 300 spectrometer. Tetramethylsilane was dropwise (10min). The resulting solution was stirred over-
1
used as an internal reference for the H and 13C-NMR mea- night at room temperature, the phosgene was then removed by
surements in CDCl3. 3-(Trimethylsilyl)propionic-2,2,3,3-d4 bubbling with Ar, and the solution was diluted with CH2Cl2
acid (TSP) sodium salt was used as an external reference (6mL). The reaction mixture was then washed with a saturat-
for 1H- and 13C-NMR measurements in D2O. MS spectra ed aqueous NH4Cl solution (3×10mL) and brine (10mL). The
were recorded on a JEOL JMS-SX102A and Agilent (Var- organic layer was dried over anhydrous Na2SO4, filtered, and
ian) 910-MS. Elemental analyses were performed on a evaporated. The crude product was purified by column chro-
Perkin-Elmer CHN2410 analyzer at the Research Institute for matography on silica gel with hexanes:AcOEt from 10:1 to
Science and Technology, Tokyo University of Science. Thin- 5:1, followed by reprecipitation from CH2Cl2/hexanes to af-
layer (TLC) and silica gel column chromatographies were ford 19 (96mg, 0.27mmol, 7% yield) as colorless solid, which
performed using a Merck 5554 (silica gel) TLC plates and Fuji was used without further purification in the next step.
Silysia Chemical FL-100D, respectively.
IR (neat) cm−1: 3223, 3052, 2970, 1698, 1557, 1499, 1368,
Synthesis of HQ Methanesulfonates 10, 11, 12 and 1342) 1276, 1165, 1056, 1016, 963, 892, 836, 792, 771, 726. H-NMR
10 was prepared by the reported method (mp: 72.5–74°C).42) (CDCl3) δ: 1.46 (9H, s), 3.69 (2H, t, J=6.0Hz), 3.90 (2H, t,
Methanesulfonyl chloride (1.1eq against HQ) and 4-dimeth- J=6.0Hz), 7.51–7.61 (2H, m), 7.80–7.83 (2H, m), 8.26 (1H,
ylaminopyridine (cat.) were added to a solution of the corre- dd, J=1.5, 8.4Hz), 8.98 (1H, d, J=3.0Hz). HR-FAB-MS m/z:
sponding HQ 3, 6 or 7 (1eq) and Et3N (1.2eq) in CH2Cl2, and 353.1170 (Calcd for C16H21N2O5S [M+H]+: 353.1171).
1
the resulting solution was stirred at room temperature until
4N HCl in dioxane (0.5mL) was added to a solution of 19
the 8-quinolinols disappeared, as confirmed by TLC. After (33mg, 0.093mmol) in 1,4-dioxane (0.5mL). After stirring
concentrating the reaction mixture under reduced pressure, the reaction mixture for 10min, the solvent was removed on a
the remaining residue was dissolved in AcOEt and the organic rotary evaporator to give the crude product, which was puri-
solution was washed with water and then brine. The organic fied by reprecipitation from MeOH/Et2O to give 14 (23mg,
layer was dried over Na2SO4, filtered, and concentrated under 0.071mmol, 76% yield) as colorless solid.
reduced pressure. The resulting residue was purified by col-
umn chromatography on silica gel with hexanes:AcOEt (from 1602, 1549, 1369, 1351, 1297, 1288, 1180, 1165, 1123, 1077,
mp: >200°C. IR (neat) cm−1: 3289, 2830, 2739, 2662, 2085,
1
10:1 to 5:1) and recrystalization from hexanes/AcOEt.
1031, 955, 891, 872, 824, 759, 744. H-NMR (D2O) δ: 3.80
11: mp: 97–98°C. IR (neat) cm−1: 3018, 1591, 1495, 1463, (2H, t, J=5.8Hz), 4.17 (2H, t, J=5.8Hz), 7.70–7.76 (2H, m),
1
1346, 1174, 1143, 1051, 1032, 980, 810, 784. H-NMR (CDCl3) 7.90 (1H, d, J=7.7Hz), 8.08 (1H, d, J=8.1Hz), 8.55 (1H, d,
δ: 3.47 (3H, s), 7.60–7.67 (3H, m), 8.63 (1H, dd, J=1.7, 8.9Hz), J=8.1Hz), 8.97 (1H, d, J=4.6Hz). 13C-NMR (D2O) δ: 34.1,
9.03 (1H, dd, J=1.7, 8.9Hz). 13C-NMR (CDCl3) δ: 36.6, 120.2, 48.5, 122.8, 123.2, 126.7, 127.9, 129.6, 137.2, 140.0, 143.8,
120.9, 123.7, 124.9, 127.6, 130.8, 139.2, 141.8, 148.8. High reso- 151.5. HR-FAB-MS m/z: 253.0647 (Calcd for C11H13N2O3S
lution fast atom bombardment mass spectrum (HR-FAB-MS) [M+H]+: 253.0647). Anal. Calcd for C11H12N2O3S+2HCl+H2O:
m/z: 257.9866 (Calcd for C10H9ClN3OS [M+H]+: 257.9992). C, 38.49; H, 4.70; N, 8.16. Found: C, 38.76; H, 4.41; N, 8.40.
Anal. Calcd for C10H8ClNO3S: C, 46.61; H, 3.13; N, 5.44.
5-Chloro-8-hydroxyquinoline 2-Aminoethanesulfonate
Found: C, 46.69; H, 2.85; N, 5.46.
Hydrochloride Salt (15) 5-Chloro-8-hydroxyquinoline N-
12: mp: 100–102°C. IR (neat) cm−1: 3019, 1579, 1506, Boc-2-aminoethanesulfonate (20) was prepared as yellow
1362, 1321, 1157, 1119, 1060, 1038, 970, 822, 792, 764, 732. solid (160.1mg, 0.6% yield) from 18 (3745mg, 8.0mmol) and

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647
3 (1598mg, 8.9mmol) in a manner similar to that described by UV-Vis. The quantum yield was calculated using the equa-
for 19.
tion Φ=(number of reacted molecules)/(I0×irradiation time). I0
IR (neat) cm−1: 3231, 2976, 1704, 1553, 1484, 1465, 1338, is the number of photon per second at 313nm (1.26×10−6 mol/
1275, 1249, 1162, 1057, 948, 867, 827, 784, 705. H-NMR sec) determined by a ferroxalate actinometry.47)
1
(CDCl3) δ: 1.46 (9H, s), 3.69–3.72 (2H, m), 3.88–3.93 (2H, m),
Reactivation of HQ Sulfonates by Photoirradiation
7.63–7.68 (2H, m), 7.74 (1H, d, J=8.4Hz), 8.65 (1H, dd, J=1.5, In this experiment, a solution of L-leucine-p-nitroanilide
8.7Hz), 9.03 (1H, d, J=4.5Hz). HR-FAB-MS m/z: 387.0825 (LeuNA), a substrate of AAP, and the prodrug was prepared
(Calcd for C16H20ClN2O5S [M+H]+: 387.0781).
using CH3CN. We performed the photoirradiation of AAP
5-Chloro-8-hydroxyquinoline 2-aminoethanesulfonate hy- (0.02 units/mL) alone, the HQ sulfonates alone (5µM), and a
drochloride salt (15) was prepared as yellow crystal (13.1mg, mixture of AAP (0.02 units/mL) and HQ sulfonates (5µM) in
36% yield) from 20 (38.6mg, 0.10mmol) in a manner similar 2.5mL of 10mM EPPS containing 2% CH3CN at pH 8.0 with
to that described for 14.
I=0.1 (NaNO3). These solutions were irradiated at 313nm at
mp: >200°C. IR (neat) cm−1: 3487, 3429, 2887, 2043, 1634, 25°C with a 150W Xe lamp equipped in a spectrofluorometer
1588, 1503, 1468, 1361, 1300, 1223, 1180, 1038, 952, 902, (JASCO FP-6500 with a band-width of 20nm). In the cases of
1
853, 789. H-NMR (D2O) δ: 3.79 (2H, t, J=5.7Hz), 4.18 (2H, AAP alone and the mixture of AAP and HQ sulfonates, 25µL
t, J=5.7Hz), 7.77–7.84 (3H, m), 8.79 (1H, d, J=8.4Hz), 9.00 of substrate (10mM in CH3CN) was added to the reaction
(1H, d, J=4.2Hz). 13C-NMR (D2O) δ: 34.0, 48.5, 123.4, 124.0, mixture after photoirradiation for given periods (Ymin) and
126.7, 126.8, 129.3, 133.4, 140.9, 143.4, 152.4. HR-FAB-MS the change in absorption at 405nm was recorded by means of
m/z: 287.0340 (Calcd for C11H12ClN2O3S [M+H]+: 287.0256). a UV-Vis spectrometer to determine the initial velocity of the
Anal. Calcd for C11H11ClN2O3S+2HCl+H2O: C, 34.98; H, 4.00; hydrolysis. In the case of prodrugs alone, 0.02 units of AAP
N, 7.42. Found: C, 35.36; H, 3.75; N, 7.54.
and 25µL of substrate (10mM in CH3CN) were added and the
2-Methyl-8-hydroxyquinoline 2-Aminoethanesulfonate initial rate of hydrolysis was obtained in the same way.
Hydrochloride Salt (16) 2-Methyl-8-hydroxyquinoline N-
Boc-2-aminoethanesulfonate (21) was prepared as colorless
Acknowledgments This study was supported by Grants-
solid (150.2mg, 5% yield)) from 18 (3745mg, 8.0mmol) and in-Aid from the Ministry of Education, Culture, Sports, Sci-
9 (1528mg, 9.6mmol) in a manner similar to that described ence and Technology (MEXT) of Japan (No. 23790023 for S.
for 19.
Ariyasu and No. 18390009, 19659026, 22390005, 22659005
IR (neat) cm−1: 3216, 2974, 1704, 1558, 1349, 1280, 1160, and 24659058 for S. Aoki) and “Academic Frontier” project
1
1058, 983, 908, 849, 778. H-NMR (CDCl3) δ: 1.42 (9H, s), for private universities: matching fund subsidy from MEXT,
2.79 (3H, s), 3.68–3.71 (2H, m), 3.90–3.95 (2H, m), 7.38 (1H, 2009–2013. We thank Ms. Fukiko Hasegawa (Tokyo Universi-
d, J=8.4Hz), 7.51 (1H, d, J=7.8Hz), 7.42–7.78 (3H, m), 8.12 ty of Science) for mass spectroscopy and Ms. Tomoko Matsuo
(1H, d, J=8.4Hz). HR-FAB-MS m/z: 367.1328 (Calcd for (Tokyo University of Science) for elemental analyses.
C17H23N2O5S [M+H]+: 367.1328).
2-Methyl-8-hydroxyquinoline 2-aminoethanesulfonate hy-
drochloride salt (16) was prepared as colorless needles
(30.1mg, 77% yield) from 21 (42.0mg, 0.11mmol) in a manner
similar to that described for 14.
References and Notes
1) Gilbert A. M., Bursavich M. G., Lombardi S., Georgiadis K. E.,
Reifenberg E., Flannery C. R., Morris E. A., Bioorg. Med. Chem.
Lett., 18, 6454–6457 (2008).
2) Jacobsen J. A., Fullagar J. L., Miller M. T., Cohen S. M., J. Med.
Chem., 54, 591–602 (2011).
3) Rouffet M., de Oliveira C. A. F., Udi Y., Agrawal A., Sagi I.,
McCammon J. A., Cohen S. M., J. Am. Chem. Soc., 132, 8232–8233
(2010).
4) Zheng H., Weiner L. M., Bar-Am O., Epsztejn S., Cabantchik Z. I.,
Warshawsky A., Youdim M. B. H., Fridkin M., Bioorg. Med. Chem.,
13, 773–783 (2005).
5) Musiol R., Jampilek J., Nycz J. E., Pesko M., Carroll J., Kralova K.,
Vejsova M., O’Mahony J., Coffey A., Mrozek A., Polanski J., Mol-
ecules, 15, 288–304 (2010).
6) Musiol R., Tabak D., Niedbala H., Podeszwa B., Jampilek J., Kralo-
va K., Dohnal J., Finster J., Mencel A., Polanski J., Bioorg. Med.
Chem., 16, 4490–4499 (2008).
7) Borchardt R. T., Thakker D. R., Warner V. D., Mirth D. B., Sane J.
N., J. Med. Chem., 19, 558–560 (1976).
8) Zheng H., Youdim M. B. H., Weiner L. M., Fridkin M., Biochem.
Pharmacol., 70, 1642–1652 (2005).
9) Choi S. M., Choi K.-O., Park Y.-K., Cho H., Yang E. G., Park H., J.
Biol. Chem., 281, 34056–34063 (2006).
mp: >200°C. IR (neat, cm−1): 3371, 2886, 2699, 1648, 1600,
1542, 1490, 1418, 1342, 1299, 1221, 1180, 1163, 1069, 1044,
1
910, 845, 798. H-NMR (D2O) δ: 3.82 (2H, t, J=6.3Hz), 4.29
(2H, t, J=6.3Hz), 7.85–7.91 (2H, m), 8.13 (1H, d, J=7.2Hz),
8.21 (1H, d, J=8.1Hz), 8.88 (1H, d, J=8.7Hz). 13C-NMR
(D2O) δ: 22.4, 34.6, 48.5, 125.3, 126.1, 128.5, 129.1, 129.2,
144.0, 144.1, 161.5, 161.6. HR-FAB-MS m/z: 267.0807
(Calcd for C12H15N2O3S [M+H]+: 267.0803). Anal. Calcd for
C12H14N2O3S+2HCl: C, 42.49; H, 4.75; N, 8.26. Found: C,
42.70; H, 4.69; N, 8.21.
Photoreactions Followed by NMR and UV-Vis Experi-
ments In NMR experiments, a 5mM solution of 11 in a 1:1
mixture of DMSO-d6 and D2O (10mM EPPS at pD 8.0) was
irradiated with a high-pressure mercury lamp (USHIO,
USH-500SC (500W)) equipped with an optical filter (Kenko,
Co., Ltd., U-330). The reaction mixtures were analyzed by
1H-NMR.45) In UV-Vis experiments, a 50µM solution of each
of the HQ sulfonates in H2O (10mM EPES at pH 7.4 con-
taining 2% CH3CN) was irradiated with a 150W Xe lamp
equipped in a spectrofluorometer (JASCO FP-6500 with
band-width 20nm). The reaction mixtures were analyzed by
UV-Vis.
10) Innocenti A., Vullo D., Scozzafava A., Supuran C. T., Bioorg. Med.
Chem. Lett., 18, 1583–1587 (2008).
11) Zheng H., Youdim M. B. H., Fridkin M., ACS Chem. Biol., 5,
603–610 (2010).
12) Lee S., Zheng X., Krishnamoorthy J., Savelieff M. G., Park H. M.,
Brender J. R., Kim J. H., Derrick J. S., Kochi A., Lee H. J., Kim
Determination of Quantum Yields of Photolysis The
numbers of reacted molecules after irradiation was estimated

648
Vol. 62, No. 7
31) Hanaya K., Suetsugu M., Saijo S., Yamato I., Aoki S., J. Biol. Inorg.
Chem., 17, 517–529 (2012).
C., Ramamoorthy A., Bowers M. T., Lim M. H., J. Am. Chem. Soc.,
136, 299–310 (2014).
32) Aoki S., Matsuo N., Hanaya K., Yamada Y., Kageyama Y., Bioorg.
Med. Chem., 17, 3405–3413 (2009).
33) Hanaya K., Kageyama Y., Kitamura M., Aoki S., Heterocycles, 82,
1601–1615 (2011).
34) Kageyama Y., Ohshima R., Sakurama K., Fujiwara Y., Tanimoto Y.,
Yamada Y., Aoki S., Chem. Pharm. Bull., 57, 1257–1266 (2009).
35) Ariyasu S., Hanaya K., Tsunoda M., Kitamura M., Hayase M., Abe
R., Aoki S., Chem. Pharm. Bull., 59, 1355–1362 (2011).
36) Ohshima R., Kitamura M., Morita A., Shiro M., Yamada Y., Ikekita
M., Kimura E., Aoki S., Inorg. Chem., 49, 888–899 (2010).
37) Maresca A., Temperini C., Pochet L., Masereel B., Scozzafava A.,
Supuran C. T., J. Med. Chem., 53, 335–344 (2010).
38) Deshmukh M., Chao P., Kutscher H. L., Gao D., Sinko P. J., J. Med.
Chem., 53, 1038–1047 (2010).
39) Di Stefano A., Mosciatti B., Cingolani G. M., Giorgioni G., Ric-
ciutelli M., Cacciatore I., Sozio P., Claudi F., Bioorg. Med. Chem.
Lett., 11, 1085–1088 (2001).
13) Prachayasittikul V., Prachayasittikul V., Prachayasittikul S., Ruchi-
rawat S., Drug Design, Development and Therapy., 7, 1157–1178
(2013).
14) Andersson D. A., Gentry C., Moss S., Bevan S., Proc. Natl. Acad.
Sci. U.S.A., 106, 8374–8379 (2009).
15) Kono R., Jpn. J. Med. Sci. Biol., 24, 195–216 (1971).
16) LeVine H. III, Ding Q., Walker J. A., Voss R. S., Augelli-Szafran C.
E., Neurosci. Lett., 465, 99–103 (2009).
17) Siles S. A., Srinivasan A., Pierce C. G., Lopez-Ribot J. L., Rama-
subramanian A. K., Antimicrob. Agents Chemother., 57, 3681–3687
(2013).
18) Kenyon V., Rai G., Jadhav A., Schultz L., Armstrong M., Jameson
B. II, Perry S., Joshi N., Bougie J. M., Leister W., Taylor-Fishwick
D. A., Nadler J. L., Holinstat M., Simeonov A., Maloney D. J., Hol-
man T. R., J. Med. Chem., 54, 5485–5497 (2011).
19) Kaplan J. H., Forbush B. III, Hoffman J. F., Biochemistry, 17, 1929–
1935 (1978).
40) Daniel K. B., Major Jourden J. L., Negoescu K. E., Cohen S. M., J.
Biol. Inorg. Chem., 16, 313–323 (2011).
41) Stevenson R. J., Denny W. A., Tercel M., Pruijn F. B., Ashoorzadeh
A., J. Med. Chem., 55, 2780–2802 (2012).
42) Leowanawat P., Zhang N., Resmerita A.-N., Rosen B. M., Percec V.,
J. Org. Chem., 76, 9946–9955 (2011).
43) Gennari C., Gude M., Potenza D., Piarulli U., Chemistry, 4, 1924–
1931 (1998).
20) Givens R. S., Kueper L. W. III, Chem. Rev., 93, 55–66 (1993).
21) Ando H., Furuta T., Tsien R. Y., Okamoto H., Nat. Genet., 28,
317–325 (2001).
22) Wieboldt R., Ramesh D., Carpenter B. K., Hess G. P., Biochemistry,
33, 1526–1533 (1994).
23) Breitinger H.-G. A., Wieboldt R., Ramesh D., Carpenter B. K., Hess
G. P., Biochemistry, 39, 5500–5508 (2000).
24) Cui Y., Ulrich H., Hess G. P., J. Membr. Biol., 202, 137–149 (2004).
25) Klán P., Šolomek T., Bochet C. G., Blanc A., Givens R., Rubina M.,
Popik V., Kostikov A., Wirz J., Chem. Rev., 113, 119–191 (2013).
26) Yu H., Li J., Wu D., Qiu Z., Zhang Y., Chem. Soc. Rev., 39, 464–
473 (2010).
44) As reported in our previous publication (ref. 26), the hydrolysis of
LeuNA follows Michaelis–Menten kinetics.
1H-NMR analysis
45) It should be noted that the photoreaction of 11 in
required longer reaction time than that in UV-Vis analysis, because
1
27) Ellis-Davies G. C. R., Kaplan J. H., J. Org. Chem., 53, 1966–1969
(1988).
the concentration of 11 in H-NMR sample (5mM) was much higher
than that in UV-Vis sample (50µM).
28) Kaplan J. H., Annu. Rev. Physiol., 52, 897–914 (1990).
29) Adams S. R., Kao J. P. Y., Tsien R. Y., J. Am. Chem. Soc., 111,
7957–7968 (1989).
30) Bandara H. M. D., Kennedy D. P., Akin E., Incarvito C. D., Bur-
dette S. C., Inorg. Chem., 48, 8445–8455 (2009).
46) Quite recently, we have found HQ derivatives that have absorption
maxima at ca. 380nm. The details will be reported elsewhere.
47) Martínez L. J., Scaiano J. C., J. Phys. Chem. A, 103, 203–208
(1999).