Carbonic Anhydrase Activators
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 289
4-Flu or oph en ylsu lfon ylu r eido-glycyl-h istidyl-â-alan yl-
was added in one portion, with energetic stirring and eventual
cooling of the reaction mixture. The mixture was then stirred
for 1-2 h at 4 °C, the solvent was evaporated in vacuo, and
the product was purified either by recrystallization from
water-ethanol (1:1, v/v) or by preparative HPLC (in the case
of fpu-GlyGly, ots-His, fpu-Val, fpu-Trp, and ots-Phe, when the
arylsulfonylureido-amino acid/dipeptide contained variable
amounts of unreacted amino acid and substituted benzene-
sulfonamide). Conditions were as follows: C18 reversed-phase
Bondapack or Dynamax-60A (25 × 250 mm) columns; 90%
acetonitrile/8% ethanol/2% water, 30 mL/min. Remarkably, the
reaction of L-Lys monohydrochloride or L-Arg monohydrochlo-
ride with the two arylsulfonyl isocyanates in the conditions
mentioned above led to the formation of only one very pure
product, i.e., the R-derivatized compound, without derivatiza-
tion of the ꢀ-amino moiety in the case of Lys or the guanidino
one in the case of Arg. This is probably due to the fact that
H+ acts in this case as a very good side chain protecting group
for these two amino acids. This has been further confirmed
by the synthesis of R-fpu-Lys and R-fpu-Arg from the ap-
propriately protected amino acid derivatives (N-ꢀ-acetyl-L-Lys
and ω-N-tritylsulfenyl-L-Arg) and 4-fluorophenylsulfonyl iso-
cyanate, followed by deprotection of the side chain in standard
conditions (data not shown).
h istid in e A21: mp 239-40 °C; IR (KBr, cm-1
) 1151 (SO2sym),
1283 (amide III), 1369 (SO2as), 1596 (amide II), 1715 (amide
I), 3063 (NH); 1H NMR (DMSO-d6, δ, ppm) 2.77-2.89 (m, 2H,
CH2 of â-Ala), 3.06-3.23 (m, 2H, CH2 of â-Ala), 3.35-3.47 (m,
4H, CHCH2 of 2 His), 3.61 (s, 2H, CH2 of Gly), 4.52-4.66 (m,
3
1H, CHCH2 of His), 7.33 (s, 2H, CH-5 of 2 His), 7.60 (d, J HH
3
) 8.1, 2H, Hortho of FC6H4), 7.93 (d, J HH ) 8.1, 2H, Hmeta of
FC6H4), 8.39 (br s, 5H, 3CONH + NHCONH), 8.47 (s, 2H, CH-2
of 2 His), 8.84 (s, 2H, imidazole NH from 2 His), 10.23 (br s,
1H, COOH); 13C NMR (DMSO-d6, δ, ppm) 33.5 (s, CH2 of His),
37.8 (s, NHCH2CH2 of â-Ala), 40.9 (s, CH2 of Gly), 41.3 (s,
CH2CH2CO of â-Ala), 59.6 (s, CHCH2 of His), 122.7 (s, C-4 of
His), 130.1 (s, Cmeta of FC6H4), 132.3 (s, C-5 of His), 133.5 (s,
NHCONH), 134.7 (s, Cortho of FC6H4), 137.3 (s, C-2 of His),
139.9 (s, Cpara of FC6H4), 145.0 (s, Cipso of FC6H4), 175.4 (s,
CH2CO of Gly), 175.6 (s, CONH of His-â-Ala), 175.9 (s, CH2CO
of â-Ala), 180.9 (s, CO2H of carboxyterminal His). Anal. (C24H28
FN9O8S) C, H, N.
-
2-Meth ylp h en ylsu lfon ylu r eid o-a r gin yl-â-a la n yl-h isti-
d in e B11: white crystals, mp 237-8 °C (dec); IR (KBr, cm-1
)
1157 (SO2sym), 1284 (amide III), 1370 (SO2as), 1585 (amide II),
1718 (amide I), 3060 (NH); 1H NMR (DMSO-d6, δ, ppm) 1.71-
2.04 (m, 2H, CHCH2CH2 of Arg), 2.51-2.65 (m, 2H, CHCH2-
3
CH2 of Arg), 2.72 (s, 3H, Me), 2.78 (t, J HH ) 6.5, 1H,
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
A1-A24 a n d B1-B24. An amount of 10 mmol of 3 was
dissolved in 50 mL of anhydrous acetonitrile and treated with
a solution obtained from 10 mmol of arylsulfonyl-ureido amino
acid/dipeptide (10 mmol) dissolved in 10 mL of the same
solvent, followed by 10 mmol of diisopropyl-carbodiimide (or
EDCI‚HCl + Et3N) and 10 mmol of 1-hydroxybenzotriazole in
anhydrous acetonitrile as solvent. The reaction mixture was
stirred at 4 °C for 3-9 h (TLC control). The solvent was then
evaporated in vacuo and the residue taken up in ethyl acetate
(50 mL), poured into a 5% solution of sodium bicarbonate (50
mL), and extracted with ethyl acetate. The combined organic
layers were dried over sodium sulfate and filtered, and the
solvent was removed in vacuo. In many cases the compounds
of type 4 or 7 were precipitated, filtered, dried, and deprotected
at the N-1 imidazolic moiety in the following way. The crude
4/7 was dissolved in 20 mL of dioxane and treated with 25
mL of a 4 M HCl solution in dioxane, followed by heating at
40 °C for 6-8 h (TLC control). The solvent was then evapo-
rated under reduced pressure, the residue was taken up in 50
mL of a 5% solution of sodium bicarbonate, and the trityl
sulfenyl chloride formed during the deprotection step was
extracted in 2 × 50 mL of Et2O. The water phase was
evaporated in vacuo to a small volume, when generally
compounds (A,B)1-24 precipitated by letting the mixture
stand at 4 °C overnight. The pure compounds were obtained
after recrystallization from ethanol-water (1:1, v/v). In some
cases, preparative HPLC was done (C18 reversed-phase Bonda-
pack or Dynamax-60A (25 × 250 mm) columns; 90% acetoni-
trile/8% methanol/2% water, 30 mL/min) in order to obtain the
pure title derivatives.
(CH2)2CH2CO of Arg), 2.79-2.87 (m, 2H, CH2 of â-Ala), 3.11-
3.26 (m, 2H, CH2 of â-Ala), 3.30-3.46 (m, 2H, CH2CH2NH of
Arg), 3.36-3.48 (m, 2H, CHCH2 of His), 3.51-3.65 (m, 1H,
CH2CH(NH)CO of Arg), 4.57-4.68 (m, 1H, CHCH2 of His), 7.34
(s, 1H, CH-5 of His), 7.50-7.98 (m, 4H, 2-MeC6H4), 8.27 (br s,
4H, 2CONH + NHCONH), 8.38 (s, 1H, CH-2 of His), 8.81 (s,
1H, imidazole NH), 10.12 (br s, 1H, COOH); 13C NMR (DMSO-
d6, δ, ppm) 26.0 (s, Me of tosyl), 29.5 (s, CH2CH2CH2 of Arg),
33.3 (s, CH2 of His), 35.4 (s, CHCH2CH2 of Arg), 37.4 (s,
NHCH2CH2 of â-Ala), 40.8 (s, CH2CH2CO of â-Ala), 45.6 (s,
CH2CH2NH of Arg), 59.5 (s, CHCH2 of His), 59.8 (s, CH2CH-
(NH)CO2H of Arg), 122.7 (s, C-4 of His), 130.4 (s, Cmeta of
MeC6H4), 132.0 (s, NHCONH), 134.3 (s, C-5 of His), 135.2 (s,
Cortho of MeC6H4), 137.5 (s, C-2 of His), 144.8 (s, Cipso of
MeC6H4), 148.9 (s, Cpara of ClC6H4), 161.6 (s, NHC(dNH)NH2
of Arg), 170.8 (CONH from Arg), 175.9 (s, CONH of â-Ala),
180.3 (s, CO2H of carboxyterminal His). Anal. (C23H33N9O7S)
C, H, N.
2-Meth ylp h en ylsu lfon ylu r eid o-ison ip ecotyl-â-a la n yl-
h istid in e B20: mp 253-5 °C (dec); IR (KBr, cm-1) 1158
(SO2sym), 1284 (amide III), 1361 (SO2as), 1584 (amide II), 1720
(amide I), 3065 (NH); 1H NMR (DMSO-d6, δ, ppm) 1.86-2.30
(m, 8H, 2 CH2CH2 of Inp), 2.61 (s, 3H, Me), 2.79-2.88 (m, 2H,
CH2 of â-Ala), 3.11-3.20 (m, 2H, CH2 of â-Ala), 3.24-3.59 (m,
3H, CHCO of Inp + CHCH2 of His), 4.57-4.63 (m, 1H, CHCH2
of His), 7.32 (s, 1H, CH-5 of His), 7.56-7.99 (m, 4H, MeC6H4),
8.27 (br s, 3H, 2CONH + Inp-NCONH), 8.35 (s, 1H, CH-2 of
His), 8.81 (s, 1H, imidazole NH), 10.10 (br s, 1H, COOH); 13
C
NMR (DMSO-d6, δ, ppm) 21.2 (s, CH2 of Inp), 26.4 (s, Me of
tosyl), 33.0 (s, CH2 of His), 37.3 (s, NHCH2CH2 of â-Ala), 40.5
(s, CH2CH2CO of â-Ala), 47.3 (s, NCH2 of Inp), 53.6 (s, CHCO
of Inp), 59.8 (s, CHCH2 of His), 122.5 (s, C-4 of His), 132.3 (s,
NHCON), 132.8 (s, Cmeta of MeC6H4), 134.0 (s, C-5 of His), 135.6
(s, Cortho of MeC6H4), 137.6 (s, C-2 of His), 145.3 (s, Cpara of
MeC6H4), 148.4 (s, Cipso of MeC6H4), 173.8 (s, CONH of Inp),
175.0 (s, CONH of â-Ala), 179.7 (s, CO2H of His). Anal.
(C23H30N6O7S) C, H, N.
En zym e P r ep a r a tion s. Human CA I and CA II cDNAs
were expressed in Escherichia coli strain BL21 (DE3) from the
plasmids pACA/hCA I and pACA/hCA II described by Lindskog
et al.27 (The two plasmids were a gift from Prof. Sven Lindskog,
Umea University, Sweden). Cell growth conditions were those
described by this group,28 and enzymes were purified by
affinity chromatography according to the method of Khalifah
et al.29 Enzyme concentrations were determined spectropho-
tometrically at 280 nm, utilizing a molar absorptivity of 49
mM-1 cm-1 for CA I and 54 mM-1 cm-1 for CA II, respectively,
based on Mr ) 28.85 kDa for CA I and 29.30 kDa for CA II,
respectively.30 CA IV was isolated from bovine lung mi-
4-F lu or op h en ylsu lfon ylu r eid o-glycyl-â-a la n yl-h ist i-
d in e A1: tan crystals, mp 202-4 °C (dec); IR (KBr, cm-1) 1147
(SO2sym), 1285 (amide III), 1362 (SO2as), 1580 (amide II), 1715
(amide I), 3060 (NH); 1H NMR (DMSO-d6, δ, ppm) 2.79-2.88
(m, 2H, CH2 of â-Ala), 3.11-3.26 (m, 2H, CH2 of â-Ala), 3.34-
3.45 (m, 2H, CHCH2 of His), 3.65 (s, 2H, CH2 of Gly), 4.57-
4.63 (m, 1H, CHCH2 of His), 7.32 (s, 1H, CH-5 of His), 7.62 (d,
3
3J HH ) 8.1, 2H, Hortho of FC6H4), 7.91 (d, J HH ) 8.1, 2H, Hmeta
of FC6H4), 8.28 (br s, 4H, 2CONH + NHCONH), 8.35 (s, 1H,
CH-2 of His), 8.84 (s, 1H, imidazole NH), 10.23 (br s, 1H,
COOH); 13C NMR (DMSO-d6, δ, ppm) 33.3 (s, CH2 of His), 37.4
(s, NHCH2CH2 of â-Ala), 40.6 (s, CH2 of Gly); 40.8 (s, CH2CH2-
CO of â-Ala), 59.6 (s, CHCH2 of His), 122.2 (s, C-4 of His), 130.5
(s, Cmeta of FC6H4), 132.9 (s, NHCONH), 134.2 (s, C-5 of His),
135.3 (s, Cortho of FC6H4), 137.2 (s, C-2 of His), 145.9 (s, Cipso of
FC6H4), 148.4 (s, Cpara of FC6H4), 167.6 (CONH of Gly), 175.6
(s, CH2CO of â-Ala), 180.4 (s, CO2H of His). Anal. (C18H21
FN6O7S) C, H, N.
-