Carbonic Anhydrase XII Inhibitors Overcome Temozolomide Resistance in Glioblastoma

Article abstract of DOI:10.1021/acs.jmedchem.9b00282

The natural product primary sulfonamide, psammaplin C (1), when used in combination with clinically used chemotherapeutic drugs, including temozolomide, reverses multidrug resistance and increases survival in glioblastoma, a highly aggressive primary brain tumor. We showed previously that the mechanism of action of 1 is novel, acting to indirectly interfere with P-glycoprotein drug efflux activity as a consequence of carbonic anhydrase XII (CA XII) inhibition. To build structure-activity relationships, 45 derivatives of 1 were designed, synthesized, and evaluated against a panel of CA isoforms. Compound 55 was identified as a potent inhibitor of CA XII (K_i = 0.56 nM) and was investigated in vitro and in vivo using samples from glioblastoma patients. The results strengthen the possibility that co-therapy of temozolomide with a CA XII inhibitor may more effectively treat glioblastoma by suppressing an important temozolomide resistance mechanism.

Full text of DOI:10.1021/acs.jmedchem.9b00282

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Article
Carbonic Anhydrase XII Inhibitors Overcome
Temozolomide Resistance in Glioblastoma
Prashant Mujumdar, Joanna Kopecka, Silvia Bua, Claudiu
T Supuran, Chiara Riganti, and Sally-Ann Poulsen
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00282 • Publication Date (Web): 29 Mar 2019
Downloaded from http://pubs.acs.org on March 30, 2019
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Carbonic Anhydrase XII Inhibitors Overcome Temozolomide Resistance in
Glioblastoma
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†

‡
‡
,
Prashant Mujumdar , Joanna Kopecka , Silvia Bua , Claudiu T. Supuran , Chiara Riganti*
and Sally-Ann Poulsen*,†
†Griffith Institute for Drug Discovery, Griffith University, Don Young Road, Nathan, Brisbane,
Queensland 4111, Australia
Department of Oncology, University of Torino, via Santena 5/bis, 10126 Torino, Italy
‡NEUROFARBA Department, Sezione di Scienze Farmaceutiche, Università degli Studi di
Firenze, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence, Italy
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ABSTRACT
The natural product primary sulfonamide, Psammaplin C (1), when used in combination with
clinically used chemotherapeutic drugs, including temozolomide, reverses multidrug resistance
and increases survival in glioblastoma, a highly aggressive primary brain tumor. We showed
previously that the mechanism of action of 1 is novel, acting to indirectly interfere with P-
glycoprotein drug efflux activity as a consequence of carbonic anhydrase XII (CA XII)
inhibition. To build structure-activity relationships, 45 derivatives of 1 were designed,
synthesized and evaluated against a panel of CA isoforms. Compound 55 was identified as a
0
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potent inhibitor of CA XII (K = 0.56 nM) and was investigated in vitro and in vivo using
i
samples from glioblastoma patients. The results strengthen the possibility that co-therapy of
temozolomide with a CA XII inhibitor may more effectively treat glioblastoma by suppressing
an important temozolomide resistance mechanism.
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INTRODUCTION
CA enzymes (CA, EC 4.2.1.1) are involved in maintaining pH homeostasis. This maintenance
is achieved via catalysis of the reversible hydration of carbon dioxide to bicarbonate and a
1
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proton. There are two extracellular facing membrane bound CAs, CA IX and CA XII, that are
highly expressed in the hypoxic core of solid tumors where they contribute to pH homeostasis
2
of the tumor microenvironment, specifically to benefit tumor growth. We recently
demonstrated that CA XII co-localizes with another membrane protein, the drug efflux protein,
P-glycoprotein (Pgp), in a range of drug resistant cancer cells including glioblastoma.3
Furthermore, we showed that CA XII maintains the optimal pH to support the drug efflux
activity of Pgp, leading to drug resistant cells.3, 4 Psammaplin C (1, Figure 1), is one of only
two described natural products comprising a primary sulfonamide functional group in its
5
structure. We recently reported an improved and facile synthesis of compound 1 and
6
subsequently showed that it inhibited CA enzymes. Compound 1 was a particularly potent CA
XII inhibitor (K = 0.79 nM). Using high resolution X-ray crystallography we observed the
i
sulfonamide of 1 directly coordinated to the active site zinc cation in both CA IX and CA XII
related proteins, consistent with the binding pose observed for other primary sulfonamide-CA
2
complexes reported in the protein data bank. Compound 1 was then selected for in vivo studies
in an orthotopic mouse model using primary cells obtained from three glioblastoma patients.
The combination of 1, with both front-line and second-line chemotherapeutics, resensitized
drug resistant glioblastoma to chemotherapy while significantly extending overall survival of
3
the treated animals compared to chemotherapy treatment alone. Notably, compound 1 shows
no toxicity and is only effective if used in combination with a chemotherapy. Given that
compound 1 acts with an unprecedented mechanism of action to reverse multidrug resistance
in aggressive glioblastoma xenografts, the aim of the current study was to build
structure‒activity relationships (SAR) around the core scaffold of 1. To enable this, a series of
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novel target compounds have been designed, synthesized and evaluated for inhibition
properties against the two cancer relevant human CA isoforms, CA IX and XII. We identified
a suitable compound (compound 55) for validation of the in vitro and in vivo action of 1
observed when co-administered with temozolomide in glioblastoma.
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0
OH
Br
O
O
S
NH2
O
N
H
N
HO
Psammaplin C, 1
Figure 1. Psammaplin C 1, a natural product primary sulfonamide, is a subnanomolar inhibitor
6
of the cancer related carbonic anhydrase isozyme, CA XII (K = 0.79 nM). There are three
i
distinct structural components, (a) a 3-bromo-4-hydroxy benzyl group - red, (b) an aminoethyl
primary sulfonamide functional group - blue, and (c) a free oxime moiety – green.
Compound 1 comprises three distinct structural components, (a) a 3-bromo-4-hydroxy benzyl
group, (b) an aminoethyl primary sulfonamide functional group, and (c) a free oxime moiety
(Figure 1). An unhindered primary sulfonamide is a minimum requirement for CA inhibition
and this feature was maintained in the structure of all target compounds (Figure 2). The SAR
around the aminoethyl sulfonamide in 1 was explored by replacement of this component with
variable aliphatic and/or aromatic sulfonamides. The benzyl moiety SAR was explored by
introducing different substitution patterns on the benzyl group. In total, a library of 18 novel
derivatives of 1 were synthesized and evaluated for CA inhibition. Additionally, as the
requirement for the free oxime moiety of 1 on enzyme inhibition was unknown, the 27
intermediate sulfonamide compounds with protected oxime groups were also evaluated for CA
enzyme inhibition in this study (Figure 2). The selection of precursor compounds from which
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to generate the target SAR library compounds 27-68, sulfonamides (2-6) and protected oximes
(7-14), was informed by disconnection of the amide bond of 1 (Figure 2).
1
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R2
R2
SO2NH2
n = 2 2
n = 1 3
n = 0 4
R1
R1
amide
coupling
H2N ( )n
S
O
O
O
x
SO2NH2
H2N
N
H
OH
OH
SO2NH2
SO2NH2
2
H N
N
N
N
N
O
N
Bn O
THP
O
6
5
2
7-42, 59-68, 72-76
R2 = H
8 R1 = Br
11 R2 = H
7
12 R1 = Br
oxime
deprotection
R2 = OH
13 R2 = OH
0 R1 = Br, R2 = OH 14 R1 = Br, R2 = OH
9
1
O
SO2NH2
N
H
N
HO
1, 43-58, 69-71
Figure 2. Library precursor compounds sulfonamides 2-6 and protected oximes 7-14 were used
toward the preparation of psammaplin C derivatives: protected oximes 27-42, 59-68, 72-76 and
free oximes 43-58 and 69-71.
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RESULTS AND DISCUSSION
Chemical Synthesis. Based on our findings when developing a synthetic route to 1 we were
aware that orthogonal oxime protecting groups may be required to accommodate the variable
substituents on the benzyl moiety in the target compounds.6 The synthesis of precursor
compounds in which the oxime hydroxyl group is protected as either a benzyl ether (O-Bn set;
0
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7
-10) or a tetrahydropyran-2-yl ether (O-THP set; 11-14) was carried out. To prepare 7-14
commercially available aldehydes 15-18 were first treated with N-acetyl-glycine under
Erlenmeyer conditions to generate benzylidene oxazolone intermediates 19-22 (Scheme 1).
The oxazolones were then hydrolysed by refluxing in aqueous 10% HCl to generate the
corresponding phenyl pyruvic acids 23-26. Lastly, oximation of crude 23-26 with
NH OBn.HCl or NH OTHP afforded the eight precursor protected oximes 7-10 and 11-14,
2
2
respectively, Scheme 1. 7, 8
Scheme 1. Synthesis of O-Bn and O-THP protected oximes 7-10 and 11-14, respectively, from
commercially available aldehydes 15-18.a,b,c
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R2
R1
(iii)
O
OH
R2
R2
N
R2
R1
BnO
7, 79%
R1
1
1
1
1
1
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1
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8 R = Br, 64%
R1
(i)
(ii)
2
9 R = OH, 61%
O
1
2
10 R = Br, R = OH, 62%
N
O
OH
O
CHO
R2
O
19, 64%
R1
1
1
1
1
5
23
1
1
1
(iv)
6 R = Br
20 R = Br, 73%
24 R = Br
2
2
2
7 R = OH
21 R = OAc, 65%
25 R = OH
O
1
2
1
2
1
2
8 R = Br, R = OH
22 R = Br, R = OAc, 85%
26 R = Br, R = OH
OH
N
THPO
11, 42%
1
12 R = Br, 58%
2
13 R = OH, 44%
1
2
14 R = Br, R = OH, 49%
a
Reagents and conditions: (i) N-acetyl glycine, NaOAc, Ac O, 120 °C, 3 h; (ii) aq. HCl (10%),
2
b
1
reflux, 15 h; (iii) pyridine, H NOBn.HCl, rt, 15 h; (iv) pyridine, H NOTHP, rt, 15 h. R and
2
2
2
c
R = H if not specified otherwise. Yields of 7-14 are over two steps from 19-22.
The O-Bn oxime Psammaplin C derivatives were accessible by the reaction of the amino group
of sulfonamides 2-5 with the carboxylic acid group of the O-Bn protected oximes 7-10 in the
presence of EDC.HCl and HOBt.H O. Amino sulfonamides 2-4 are commercially available
2
9
while thiadiazole sulfonamide 5 was synthesized as reported previously. In principle, 16 O-
Bn oxime Psammaplin C derivatives (27-42) are possible however the subsequent removal of
the O-Bn group using transfer hydrogenation proceeded cleanly only for non-brominated
precursors, while debenzylation occurred with simultaneous debromination for brominated
precursor compounds 28, 32, 36 and 40.10 Our efforts to address the latter side reaction
(
described below, Table 1) were not successful and thus we elected not to synthesise the then
two remaining bromine containing O-Bn compounds (compounds 34 and 38). Although O-Bn
compounds 39-41 were successfully prepared from thiadiazole sulfonamide 5, they were
unreactive to O-debenzylation using transfer hydrogenation. It was hypothesized that the soft
sulfur in the heterocycle of these compounds may poison the Pd catalyst. Furthermore, the
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attempted synthesis of compound 42 from thiadiazole sulfonamide 5 and O-Bn protected oxime
1
0 gave no desired product (evidenced by TLC). In summary, 13 of 16 possible O-Bn protected
oxime derivatives of 1 were prepared (27-33, 35-37 and 39-41) of which six reacted cleanly
toward O-debenzylation in subsequent transfer hydrogenation to provide synthetic access to
the target corresponding free oxime compound 43, 45, 47, 49, 51 and 53.
0
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Scheme 2. Synthesis toward psammaplin C derivatives 27-58 from O-Bn precursors 7-10 and
sulfonamides 2-5.a,b,c
R2
R2
R1
O
R1
O
SO2NH2
7 49%
28 R1 = Br 64%
SO2NH2
2
4
3 63%
N
N
H
H
44
R1 = Br, no reaction
N
N
BnO
2
3
9 R2 = OH 81%
HO
45 R2 = OH 66%
0 R1 = Br, R2 = OH 46%
46 R1 = Br, R2 = OH no reaction
R2
R2
31 41%
R1
R1
O
2 R1 = Br 74%
3
3
3
47 63%
3 R2 = OH 81%
48 R1 = Br, no reaction
O
4, R1 = Br, R2 = OH,
9 R2 = OH 63%
4
5
R2
N
reaction not attemped
N
H
0 R1 = Br, R2 = OH precursor 34 not available
R1
H
N
N
BnO
R2
SO2NH2
HO
SO2NH2
(i)
(ii)
O
R2
OH
R1
O
R1
O
N
BnO
SO2NH2
SO NH2
2
7
8
R1 = Br
35 63%
51 64%
N
N
H
9 R2 = OH
0 R1 = Br, R2 = OH
36 R1 = Br 36%
37 R2 = OH 81%
52 R1 = Br, no reaction
53 R2 = OH 20%
H
N
N
1
BnO
R2
HO
54 R1
= Br, R2 = OH, precursor 38 not available
8, R1 = Br, R2 = OH,
3
reaction not attempted
R2
R1
O
R1
SO2NH2
SO2NH2
S
O
S
39 49%
55 no reaction
N
N
40 R1 = Br 55%
41 R2 = OH 41%
N
56 R1 = Br, no reaction
N
H
N
N
H
7 R2 = OH, no reaction
5
N
N
BnO
HO
58 R1 = Br, R2 = OH, precursor 42 not available
2, R1 = Br, R2 = OH, no reaction
4
a
Reagents and conditions: (i) EDC.HCl, HOBt.H O, 2-5, dry DMF, rt, 24 h; (ii) cyclohexene,
2
b
1
2
c
Pd(OH) , absolute EtOH, reflux, 2-6 h. R , R = H unless specified. Compounds 55-58 were
2
not obtained.
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With the bromo derivatised O-Bn oxime 28 as a model compound we attempted various
alternative conditions to remove the O-Bn moiety to prepare the corresponding free oxime 44,
1
1
Table 1. Formic acid-triethylamine mediated transfer hydrogenation (Table 1, Entry 1) and
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
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3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
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8
9
0
1
2
3
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9
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9
0
Pd-catalysed hydrogenation12, 13 with H (60 mbar) (Table 1, Entry 2) afforded debenzylation
2
and debromination. Pd-catalysed hydrogenation with H (atm) afforded debromination only
2
1
0
(
Table 1, Entry 3). No reaction was observed with either transfer hydrogenation (Table 1,
1
4
15
Entry 4), trimethyl silyl iodide (TMSI) (Table 1, Entry 5), in situ generation of TMSI (Table
1
6
17
1
, Entry 6), use of a Lewis acid AlCl (Table 1, Entry 7) or TFA-toluene (1:1) (Table 1,
3
1
8
Entry 8). In the case of BF .Et O (Table 1, Entry 9) decomposition of 28 was observed
3
2
(evidenced by TLC).
Table 1. Reaction conditions assessed for O-debenzylation of the brominated derivatised O-
Bn oxime 28 as a model compound.
Entry
1
Reaction Conditions
Observation
Pd/C, HCOOH, NEt (cat), EtOH, 60 °C, 3-5
Debenzylation/debromination
3
h
2
H (60 mbar), Pd/C, dioxane-AcOH (1:1), rt,
2
Debenzylation/debromination
1
2 h
3
4
5
6
7
8
H (atm), Pd/C, MeOH-AcOH (1:1), rt, 12 h
Debromination
No reaction
No reaction
No reaction
No reaction
No reaction
2
Pd(OH) , cyclohexene, EtOH, reflux, 12 h
2
TMSI, dry DCM, rt, 3 days
TMSCl, NaI, ACN, 0 °C-rt, 12 h
AlCl , N,N-dimethylaniline, DCM, rt, 12 h
3
TFA:toluene (1:1), rt, overnight
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
9
BF .Et O, NaI, ACN, 0 °C-rt, 12 h
Decomposition (TLC)
3
2
As the brominated and/or thiadiazole containing target free oximes were not accessible using
O-Bn as a protecting group we next investigated the O-THP protected oxime precursors 11-14
as an alternative source for these compounds. Compounds 11-14 were combined with one
equivalent of the corresponding sulfonamide 2-5 in the presence of the amide coupling reagents
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
EDC.HCl and HOBt.H O (Scheme 3). Of the ten target O-THP protected psammaplin C
2
derivatives (59-68) nine formed successfully (59-67). The THP group of 59-67 was then
cleanly removed under acidic conditions (4.0 M HCl in 1,4-dioxane) to afford the
corresponding target psammaplin C free oxime derivatives (44, 46, 48, 50, 52, 54-57) (Scheme
1
9
3
). As neither O-Bn 42 or O-THP 68, (prepared from 5 and 10 or 14, respectively) were
successfully synthesized, this necessarily meant that the free oxime 58 was not accessible.
Scheme 3. Synthesis toward psammaplin C derivatives 44, 46, 48, 50, 52, 54, and 55-68 from
the corresponding O-THP precursor 11-14 and sulfonamide 2-5.a b
1
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R2
R2
R1
O
R1
O
SO2NH2
SO2NH2
4
4
4 R1 = Br 37%
6 R1 = Br, R2 = OH 77%
N
N
59 R1 = Br 76%
60 R1 = Br, R2 = OH 40%
H
H
N
N
THPO
HO
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
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
R2
R2
R1
R1
O
1 R1 = Br 77%
2 R1 = Br, R2 = OH 32%
6
6
8 R1 = Br 48%
50 R1 = Br, R2 = OH 76%
4
O
R2
N
N
H
R1
O
H
N
N
THPO
R2
SO2NH2
HO
SO2NH2
(i)
(ii)
R2
OH
R1
O
R1
O
N
THPO
SO2NH2
SO NH2
2
1
1
1
1
52 R1 = Br, 71%
54 R1 = Br, R2 = OH, 25%
1
N
3 R1 = Br 74%
64 R1 = Br, R2 = OH 20%
2 R = Br
3 R2 = OH
6
N
H
H
N
N
THPO
HO
14 R1 = Br, R2 = OH
R2
R2
R1
O
R1
SO2NH2
SO2NH2
S
O
S
65 55%
66 R1 = Br 58%
67 R2 = OH 43%
8 R1 = Br, R2 = OH, no reaction
55
7
0%
N
N
N
N
N
5
6 R1 = Br 58%
N
H
H
57 R2
58 R1
N
N
= OH 47%
= Br, R2 = OH, not attemped
THPO
HO
6
a
Reagents and conditions: (i) EDC.HCl, HOBt.H O, 2-5, dry DMF, rt, 24 h; (ii) 4M HCl in
2
b
1
2
1
,4-dioxane, 0 °C, 4-10 h. R , R = H unless specified.
The remaining three target derivatives comprise the aminoethyl linked primary sulfonamide
group as found in the natural product 1 (Figure 3). The compounds (69-71)were prepared from
6
and protected oximes oximes 72-75 as as reported by ourselves earlier.3, 4 Compound 1 was
prepared from 76 as reported earlier.6
1
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
R2
R2
R2
R1
R1
R1
O
O
O
SO NH2
SO NH
SO NH
2 2
2
2
2
N
H
N
N
H
H
N
N
N
Bn
O
THP
O
HO
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
1
2
1
1
2
7
2 R = R = H
74 R = Br
69 R = R = H
2
2
1
73 R = OH
75 R = OH
70 R = Br
1
2
2
76 R = Br, R = OH
71 R = OH
1
2
1 R = Br, R = OH
Figure 3. Aminoethyl primary sulfonamides: natural product 1, protected oxime derivatives
2-76 and free oxime derivatives 69, 70 and 71.3, 6
7
Carbonic Anhydrase Inhibition. The CA I, II, IX and XII inhibition and selectivity data for
psammaplin C (1), and the free oxime target compounds (43-58, 69-71) are provided in Table
2
. For comparison, data is also provided for the clinically used CA inhibitor, the related
thiadiazole sulfonamide acetazolamide (AZA). The CA I, II, IX and XII inhibition and
selectivity data for O-Bn or O-THP protected derivatives (27-33, 35-37, 39-41 and 59-67) is
provided in Table 3.
Table 2. Human CA inhibition profile for psammaplin C (1), its derivatives (43-58, 69-71) and
acetazolamide (AZA).
R2
SO NH2
2
SO2NH2
SO2NH2
R1
O
43 - 46
47 - 50
51 - 54
S
N
SO NH2
2
H
SO2NH2
, 69-71
N
HO
N N
5 - 57
1
5
5
8 not synthesised
a
b
K (nM)
K ratios
i
i
1
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Compd R1 R2 CAI CAII CAIX CAXII I/IX I/XII II/IX II/XII
4
3
H
H
H
6.5
3.5
34.1
>50000 0.191
-
0.103
-
44
Br
587
5.6 >50000
775
680
209
471
-
0.757
-
-
-
0.007
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
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
4
4
5
6
H OH 59.3 0.05 12600
0.005 0.087
0.063 0.040
-
-
Br OH 8.3
0.04
140
254
132
47
H
H
H
619
809
3400
0.182 1.31 0.041 0.297
0.091
4
4
5
8
9
0
Br
2800 >50000 0.289
-
-
H OH 81.6 36.8
Br OH 72.6 25.0
353
343
3.5
174
312
7.9
0.231 0.469 0.104 0.211
0.212 0.233 0.073 0.080
8.77 3.89 2.06 0.911
6.88 5.23 0.735 0.559
4.37 0.743 0.129 0.022
0.267 0.130 0.033 0.016
1.69 3.93 0.846 1.96
0.580 1.21 0.079 0.164
2.44 6.43 0.500 1.32
51
H
H
H
30.7
7.2
5
5
2
3
Br
192 20.5
27.9
3.5
36.7
20.6
21.6
0.56
45.2
7.3
H OH 15.3 0.45
54
Br OH 2.8
0.35
1.1
7.4
9.6
-
10.5
1.3
5
5
5
6
H
H
H
2.2
Br
54.5
94.0
19.2
-
57
8c
H OH 46.9
Br OH
5
-
-
-
-
-
-
-
-
-
-
6
9
0
H
H
H
3930 242 >50000 >50000
7
Br
9.1
4.5
3300
3120
12.3
25
43.2
219
0.79
5.7
0.003 0.211 0.001 0.104
0.503 7.17 0.048 0.689
71
1
H OH 1570 151
Br OH 48.1 88.0
3.91 60.9 7.15
111
AZA
-
-
250
12
10.0 43.9 0.480 2.11
a
b
Errors ± 5% of the reported values from three separate assays. The K ratios are indicative of
i
isozyme selectivity for transmembrane CA IX and XII. Where values are <0.0001 no value is
c
shown. Compound 58 not synthesized.
1
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 3. CA I, II, IX and XII inhibition and isozyme selectivity data for intermediate
compounds, O-Bn or O-THP protected derivatives 27-33, 35-37, 39-41, 59-67 and 72-76.
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
SO NH
SO NH
SO NH
2
2
2
2
2
2
R2
R1
O
3
3
3
2
7-30 (R = Bn)
31-33 (R = Bn)
35-37 (R = Bn)
3
3
3
5
9-60 (R = THP)
61-62 (R = THP)
63-64 (R = THP)
S
SO NH
N
2
2
SO NH
2
2
H
N
N N
3
R O
3
3
3
9-41 (R = Bn)
72-73 (R = Bn)
3
3
6
5-67 (R = THP)
74-76 (R = THP)
a
b
K (nM)
i
K ratios
i
Compd R1 R2
CAI
CAII
CAIX CAXII I/IX I/XII II/IX II/XII
2
7
H
H
H
>50000 3780 >50000 >50000
>50000 >50000 >50000 >50000
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
28
Br
-
2
3
3
3
3
3
9
0
1
2
3
5
H OH
669
37.6
>50000 >50000
-
Br OH >50000 6470 >50000 >50000
-
H
H
H
>50000
>50000 1750 >50000 >50000
37.3 2.2
479
395
>50000
1.21
Br
-
-
-
-
H OH
5680 >50000 0.007
H
H
H
>50000 >50000 >50000 >50000
>50000 >50000 >50000 >50000
-
-
36
Br
3
3
7
9
H OH
276
46.5
800
5.8
207
3.2
179
5.1
825
39.1
862
5.5
1.54 0.335 1.16 0.251
9.12 1.19 0.627 0.082
H
H
H
40
Br
160
0.72
2510
2.4
0.320 0.928 0.064 0.186
2.42 1.06 0.300 0.130
4
5
1
9
H OH
Br
H
>50000 4050 >50000 >50000
-
-
-
-
1
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9
6
6
0
1
Br OH 3750
265
18430 16400 0.203 0.229 0.014 0.016
Br
Br OH
Br
Br OH
H
8450
121
1460 >50000 >50000
-
-
-
-
62
6.9
2450
397
0.049 0.305 0.003 0.017
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
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
6
6
3
4
H
173
1950 >50000 >50000
-
-
-
-
28.7
34.6
741
12.6
0.08
122
0.78
2.1
35.9
4.6
35.4
0.90
733
0.799 0.811 0.351 0.356
7.52 38.4 0.017 0.089
0.787 1.01 0.130 0.166
1.84 1.40 0.029 0.022
65
H
H
H
6
6
7
7
7
7
6
7
2
3
4
5
Br
942
27.3
H OH
50.1
60.1
35.8
H
H
>50000 >50000
-
-
-
-
H OH 4130
339
4150 6200 0.995 0.666 0.082 0.055
Br
H
8060
896
4350 >50000 >50000
-
-
-
-
H OH
Br OH
647
592
5400
4600
466
570
0.166 1.92 0.120 1.39
76c
566
0.123 0.993 0.129 1.04
a
b
Errors ± 5% of the reported values from three separate assays. The K ratios are indicative of
i
isozyme selectivity for transmembrane CAs IX and XII. Where values are <0.0001 no value is
c
6
shown. Compound 76 was synthesized as reported by us previously.
Structure-Activity Relationships. The three distinct components of the core structure natural
product 1 were systematically varied, including the (a) the nature of the oxime moiety, (b) the
benzyl group substituents, and (c) spacer to the primary sulfonamide functional group.
Comparing the inhibition data of Table 2 (free oximes) and Table 3 (O-Bn and O-THP
protected oximes) it is apparent that compounds comprising a free oxime group are generally
stronger CA inhibitors than the corresponding compound where the oxime protected.
Furthermore, the less hydrophobic THP protecting group appears generally better tolerated
than the benzyl protecting group of the oximes. The protected oximes of the sulfonamide
1
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
partners 2, 3, 4 and 6 mostly exhibited weak inhibition (K s > 1 M), with many showing no
i
inhibition at the highest test concentration of 50 M. The exceptions to this trend indicate
however that there is a complex interplay between the oxime and the spacer to the essential
primary sulfonamide that impacts on the enzyme inhibition characteristics. For example, in the
subset of thiadiazole sulfonamides 39 (O-Bn), 65 (O-THP) and 55 (free oxime) the presence
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
of an oxime protecting group has almost no impact on activity at CA IX (K s 5.1, 4.6 and 1.3
i
nM, respectively) and minimal impact at CA XII (K s = 39.1, 0.90 and 0.56 nM, respectively).
i
The thiadiazole group is found in the par excellence clinically used sulfonamide, acetazolamide
(AZA) (CA IX = K 25 nM, CA XII K 5.7 nM). Notably, the thiadiazole compounds of our
i
i
study offer stronger cancer associated CA inhibition than AZA. When comparing the
compounds with the natural product lead compound 1 (CA XII K = 0.79 nM, CA IX K = 12.3
i
i
nM), it is noted that while compound 1 has around 10-fold selectivity for CA XII over CA IX,
compounds 65 and 55 also have selectivity for CA XII over CA IX (albeit lessened), while for
compound 39 the selectivity is reversed with 39 ~8-fold selective for CA IX over CA XII. The
profile of these compounds, as strong CA IX and XII inhibitors with varying degrees of
isozyme selectivity, represents a significant finding for the provision of novel lead compounds
with potential for future biological applications to validate the anticancer potential of CA IX
and CA XII. Lastly, when considering the SAR of the benzyl substitution pattern (3-bromo-4-
hydroxy, 4-hydroxy, 3-bromo and unsubstituted) there is no clear SAR, again the linker to the
sulfonamide partner appears to be the dominant factor in driving the SAR profile within the
various subsets of compounds. This finding does however indicate that the benzyl group may
provide a useful handle to fine-tune for drug-like properties without adverse impacts on
bioactivity. This is a reassuring attribute for further developing this novel chemotype if applied
within a drug lead optimization campaign.
1
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9
In vitro efficacy
Glioblastoma stem cells (SC) are a tumor cell subset responsible for tumor recurrence and
relapse owing to their high chemorefractoriness.20 One mechanism by which SC induce
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
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
2
1
chemoresistance is their high level of P-glycoprotein (Pgp) , a surface-associated efflux pump
22
that recognizes a broad spectrum of chemotherapeutic drugs , including temozolomide
2
3
(TMZ), the first-line treatment option in glioblastoma. The co-administration of direct Pgp
inhibitors (eg. tariquidar) with chemotherapy has repeatedly failed in clinical trials because of
high toxicity. This toxicity is attributed to inhibition of Pgp present in healthy tissues and to
unexpected drug-drug interactions.24 We have recently reported an alternative approach,
selective reduction of Pgp activity in glioblastoma SC without the off target toxicity associated
with using direct Pgp inhibitors. Our approach is to instead target CA XII, an enzyme central
to supporting Pgp activity in the hypoxic microenvironment of glioblastoma SC, however is
3
not required for Pgp activity elsewhere. The action of CA XII generates the mild alkaline pH
under which Pgp operates with the highest efficiency, and therefore inhibition of CA XII
renders Pgp significantly less effective.25
Compound 55, a free oxime derived from the thiadiazoyl sulfonamide scaffold, showed very
strong inhibition of all the CA isozymes, in particular CA XII (K = 0.56 nM). Compound 55
i
was stable in Balb/c mice plasma and to mouse microsomes (Supporting Information, Table
1
). This compound was selected to validate the restorative effect on TMZ efficacy found when
combining 1 with TMZ in drug resistant glioblastoma. From primary glioblastoma cells derived
from three patients with a poor clinical response to TMZ (unknown patient numbers, UPN1-3)
we separated and propagated the more differentiated component, termed adherent cells (AC),
and the SC-enriched component, termed neurospheres (NS). Cells in both culture conditions
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were profiled for CA XII and Pgp expression and interaction as reported by ourselves
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previously. Data are summarized in Supporting Information, Table 2.
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Preliminary in vitro assays were carried out to establish that compound 55 reduces Pgp activity
in NS similarly to compound 1. Cells from UPN1-3 were treated with doxorubicin (dox, 5 M)
in the presence or absence of compound 1 (10 nM) or 55 (10 nM). Compound 55 lowered Pgp
ATPase activity (Supporting Information, Figure 1A) and increased the intracellular dox
concentration (Supporting Information, Figure 1B) to a level comparable to the concentration
found in AC. This in turn partially restored dox-induced damage, measured as the extracellular
release of lactate dehydrogenase (LDH; Supporting Information, Figure 1C) while reducing
cell viability (Supporting Information, Figure 1D). Next, studies were extended to the
glioblastoma front-line drug TMZ. In NS, TMZ reduces both Pgp expression and activity
(Figure 4A-B) in line with previous findings indicating that TMZ is both a substrate and a down
regulator of Pgp expression.23, 26 Compound 55 had no effect on Pgp expression (Figure 4A)
but reduced Pgp activity (Figure 4B), this reduction was more pronounced when 55 was used
in combination with TMZ (Figure 4B). Compound 55 enhanced TMZ accumulation (Figure
4
C) and TMZ efficacy to a level comparable with that found in TMZ-treated AC (Figures 4D-
F), where Pgp and CA XII are undetectable, or TMZ-treated NS Pgp knock-out clones (Figures
3
4C-F), where Pgp is undetectable while CA XII levels are unaltered. The efficacy parameters
measured included cell damage (Figure 4D), apoptosis - as indicated by the cleavage of caspase
3
, (Figure 4E) and cell viability (Figure 4F). All findings were consistent with earlier studies
performed using 1 and provide confirmation that CA XII inhibition is likely responsible for the
chemosensitizing effect observed.3
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Figure 4. Compound 55 restores temozolomide efficacy in glioblastoma stem cells.
NS were grown for 48 h (panels A-E) or 72 h (panel F) in fresh medium (-) or in medium
containing 50 μM temozolomide (T) or 10 nM compound 55, alone or in combination. Panels
B-C-D-F: pooled data of patients UPN1-3 are presented as means+SD (n=4 independent
experiments for each patient). AC were included as control cells with undetectable CA XII or
Pgp levels. A. UPN2 NS were lysed and immunoblotted for Pgp and CA XII. The figure is
representative of one out of three experiments. B. Spectrophotometric measure of Pgp ATPase
activity, measured in triplicate in NS. *p<0.01: T/55/T+55-treated vs. untreated (-) cells;
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°
p<0.005: T+55-treated vs T-treated cells. C. Intracellular concentration of temozolomide
(
TMZ), measured in duplicates after cell radiolabelling. NS clones knocked out for Pgp
(KOPgp) and AC were included as control cells where expression of Pgp is undetectable.
p<0.001: 55-treated or KOPgp vs. untreated (-) NS. D. LDH release, measured
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*
spectrophotometrically in duplicates. *p<0.001: treated vs. untreated (-) NS or AC; °p<0.001:
T+55/T+KOPgp vs. T-treated NS. E. UPN2 NS, incubated as reported in A and/or knocked
out for Pgp, were lysed and immunoblotted for procaspase 3 and cleaved caspase 3. The figure
is representative of one out of three experiments. F. Cell viability measured by a
chemiluminescence-based assay in quadruplicates. *p<0.001: treated vs. untreated (-) NS or
AC; °p<0.001: T+55/T+KOPgp vs. T-treated NS.
In vivo efficacy
Orthotopically implanted glioblastoma NS-derived tumors (with co-expression of Pgp and
CAXII) from UPN1-3 (GB-NS-PDX, or patient-derived xenografts) were prepared to assess
the in vivo effect of TMZ (50 mg/kg), compound 55 (3.8 µg/kg) and the combination of TMZ
(50 mg/kg) plus 55 (3.8 µg/kg). The dose selected for 55 was matched to the dosage used
3
previously for 1 , to allow a direct comparison between the two compounds. Consistent with
the cell culture studies above, GB-NS-PDX from UPN1 and UPN2 were resistant to TMZ
(Figures 5A-B). Indeed, the mean tumor volumes at the end of the treatment were
3
3
1
46.50+36.45 mm for untreated animals and 117.67+50.55 mm for TMZ-treated animals
3
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(UPN1); 237.00+92.37 mm for untreated animals and 231.83+68.32 mm for TMZ-treated
animals (UPN2). The GB-NS-PDX from UPN3 showed partial sensitivity to TMZ (Figures
3
5
A-B), with a mean tumor volume at the end of the treatment 249.67+48.19 mm for untreated
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animals and 116.33+40.25 mm for TMZ-treated animals. The response of UPN3 correlates
with the more favorable genetic profile toward TMZ sensitivity, time to recurrence after TMZ
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treatment, and longest overall survival of the three patients, as reported earlier. When GB-NS-
PDX were treated with compound 55 alone there was no reduction of tumor growth. When
compound 55 and TMZ were combined, tumor growth in all GB-NS-PDX was significantly
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decreased with the mean tumor volumes 91.50+13.54 mm 115.17+25.62 mm and
,
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2.00+11.67 mm for UPN1, UPN2 and UPN3, respectively, at the end of the combined
treatment (Figures 5A-B). These volumes are significantly smaller than those of untreated mice
for all the UPNs and of TMZ-treated mice for UPN1 and UPN2.
As expected, the overall survival showed an opposite trend to tumor growth. The median
survival of untreated and TMZ-treated mice was 49 and 42.5 months, respectively, for UPN1,
4
7 and 46.5 months, respectively, for UPN2 and 47.5 and 67.5 months, respectively, for UPN3
(Figure 5C). While treatment with 55 alone had no effect on the overall survival, when 55 is in
combination with TMZ there is a marked increase in the animal survival, with the median
survival 58.5 months for UPN1, 65.5 months for UPN2 and 75.5 months for UPN3. This
corresponds to an increase of 9.5, 18.5 and 28 months over untreated mice, and an increase of
1
6, 19 and 8 months over TMZ treated mice, respectively for UPN1-3. These data suggest that
the use of 55 is particularly effective against the GB more resistant to TMZ. The combination
of TMZ with 55 rescued the antiproliferative and pro-apoptotic effects of TMZ, as verified by
reduced intratumor-positive staining for Ki67 and increased activation of caspase 3 (Figures
5
D-E).
.
Overall, these data suggest that the cytotoxic effects observed with the combination of
compound 55 and TMZ in vitro were recapitulated in vivo. Furthermore, although TMZ alone
reduced Pgp expression and activity in NS, this reduction was not sufficient to yield anti-tumor
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efficacy in vivo. The greater inhibition of Pgp, as achieved by combining the potent CA XII
inhibitor 55 with TMZ, was however able to restore the intracellular cytotoxic levels of TMZ
and facilitate enhanced drug efficacy in GB-NS-PDX. We are aware that Pgp is not the only
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resistance mechanism of TMZ, with genetic background (O -methylguanine DNA
methyltransferase - MGMT - status, isocitrate dehydrogenase 2 - IDH2 - and p53 mutations,
epithelial growth factor receptor – EGFR - amplification) also a predictor of patient response
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to TMZ. As reported previously , the patient samples of this study have different genetic
backgrounds; however, all patient-derived NS co-express Pgp and CA XII. We have observed
significantly increased TMZ resistance in the corresponding patients NS over AC independent
3
of MGMT status or other genetic differences. Collectively, our data imply that targeting this
Pgp/CA XII interaction may be exploited to improve TMZ efficacy in vivo. Our results confirm
this combination therapy strategy was indeed effective in restoring TMZ efficacy in vivo, and
importantly, in increasing overall survival in GB-NS-PDX bearing TMZ-resistant tumors.
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Figure 5. Compound 55 restores temozolomide efficacy in resistant orthtotopic patient-
derived glioblastoma xenografts.
A. Representative in vivo bioluminescence imaging of orthotopically implanted UPN2 NS, in
animals treated with vehicle (ctrl), compound 55 and temozolomide (T), as follows: 1) control
group, treated with 0.2 mL saline solution intravenously (i.v.); 2) 55 group, treated with 3.8
g/kg compound 55 i.v.; 3) T group, treated with 50 mg/kg T per os (p.o.); 4) T+55 group,
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treated with 50 mg/kg T p.o.+ 3.8 g/kg compound 55 i.v. (6 animals/group). B. Tumor volume
at the time of sacrifice. Pooled data of patients UPN1-3-derived tumors are presented as
means+SD. UPN1: *p<0.005: T+55 vs. ctrl; not-significant: T+55 vs. T; UPN2: *p<0.001:
T+55 vs. ctrl; °p< 0.001: T+55 vs. TMZ; UPN3: *p<0.005: T/T+55 vs. ctrl; °p<0.05: T+55 vs.
T. C. Overall survival probability was calculated using the Kaplan-Meier method. UPN1:
p<0.05: T+55 vs. ctrl; p<0.05: T+55 vs. T. UPN2: p<0.001: T+55 vs. ctrl; p<0.001: T+55 vs.
T. UPN3: p<0.01: T+55 vs. ctrl; not significant: T+55 vs. T (log rank test; not reported in the
figure). D. Representative intratumor staining with hematoxylin and eosin (HE) or the indicated
antibodies, from UPN2-derived tumors. The photographs are representative of sections from 5
tumors/group of treatment. Bar=10 µm (10 ocular lens, 20 objective). E. Quantification of
immuno-histochemical images, performed on sections with 111-94 nuclei/field. The
percentage of proliferating cells was determined by the ratio Ki67-positive nuclei/total number
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(hematoxylin-positive) of nuclei using ImageJ software. The ctrl group percentage was
considered 100%. The percentage of caspase 3-positive cells was determined by Photoshop
program. Data are presented as means+SD.*p<0.001: T+55 vs. ctrl; °p<0.02: T+55 vs. T.
CONCLUSION
The inspiration for this study was the unprecedented in vivo activity of the lead compound, the
unusual natural product 1, toward reversal of drug resistance in glioblastoma. The combination
of 1, with chemotherapy, resensitized drug resistant glioblastoma to TMZ while significantly
extending overall survival of treated animals compared to chemotherapy treatment alone. We
have established substantial SAR around 1 in this study, in particular demonstrating that the
ethyl sulfonamide moiety, when replaced with a thiadiazoyl sulfonamide, gives two novel
compounds (55 and 65) with sub-nanomolar CA XII inhibition activity as well as very good
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inhibition of the other cancer-associated CA isozyme, CA IX. Compound 55 was selected for
in vitro and in vivo evaluation in patient derived cell and xenograft models of glioblastoma.
The bioactivity of 55 paralleled that of compound 1 in glioblastoma. This provides more
evidence that specific inhibition of CA XII can resensitize glioblastoma to chemotherapy and
increase overall survival. Our results suggest that exploration of a new combination therapy,
based on a CA XII inhibitor with TMZ, as a potentially viable clinical tool to overcome Pgp-
mediated TMZ-resistance in glioblastoma SC. We propose this combination therapy may be
more effective than current treatment options.
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EXPERIMENTAL SECTION
General Chemistry. Amino sulfonamide compounds 2-4 are commercially available while
amino sulfonamide compounds 5 and 6 were synthesized as reported.6, 9 Compounds 14, 22,
6
3, 4
26, and 76 were synthesized as reported. Compounds 69-75 were synthesized as reported.
All reactions were carried out in dry solvents under anhydrous conditions, unless otherwise
mentioned. All chemicals were purchased commercially and used without further purification.
All reactions were monitored by TLC using silica plates with visualization of product bands
by UV fluorescence (λ = 254 nm) and charring with vanillin (6 g vanillin in 100 mL of EtOH
containing 1% (v/v) concentrated sulfuric acid) stain. Silica gel flash chromatography was
1
13
performed using silica gel 60 Å (230-400 mesh). NMR ( H, C, COSY, and HSQC) spectra
were recorded on Bruker AVANCE III HD 500 MHz NMR spectrometer equipped with a BBO
1
13
probe at 25 °C. Chemical shifts for H and C NMR obtained in DMSO-d are reported in ppm
6
relative to residual solvent proton (δ = 2.50 ppm) and carbon (δ = 39.5 ppm) signals
respectively. Multiplicity is indicated as follows: s (singlet), d (doublet), t (triplet), q (quartet),
m (multiplet), dd (doublet of doublet), bs (broad signal). Coupling constants are reported in
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hertz (Hz). LRMS (ESI) data were recorded on a Waters ZQ ESI mass spectrometer using
electrospray as the ionization technique in positive-ion and/or negative-ion modes as stated.
HRMS (ESI) data were acquired on a 12 T SolariX XR FT-ICR-MS using electrospray as the
ionization technique in positive-ion and/or negative-ion modes as stated. All MS analysis
samples were prepared as solutions in methanol. Purity of all compounds was >95% as
determined by Agilent HPLC 1100 series with UV detection. The melting points are
uncorrected. Proton and carbon atoms for NMR assignments are designated as shown below.
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Ph-OH
OH
1
HAr (in H NMR)
Br
O
CHAr (in 13C NMR) OH
Cquat
O
Ph-OH
Br
O
O
Ph-CH -C
2
Br
O
Ph-CH -C
OH
2
N
O
Cquat
OH
Ph-CH=C
Ph-CH=C
N
O
O
N
CHTHP
Ph-CH -O
2
O
Ph-CH -O
2
1
HTHP (in H NMR)
Methyl proton: CH (in H and 13C NMR)
1
3
CH2-THP (in 13C NMR)
1
Acidic proton: COOH (in H NMR)
Ph-OH
OH
1
OH
2
HN-CH -CH2
HAr (in H NMR)
Br HN-CH -CH2
Br CHAr (in 13C NMR)
2
2
SO NH2
2
SO NH2
O
O
Ph-CH -C
2
Ph-CH -C
N
H
Cquat
2
N
H
N
N
O
O
2
HN-CH -CH2
CHTHP
HN-CH -CH2
O
2
1
Ph-CH -O
HTHP (in H NMR)
2
CH2-THP (in 13C NMR)
Ph-CH -O
2
1
Amide proton: N-H (in H NMR)
1
Oxime proton: N-OH (in H NMR)
1
Sulfonamide proton: SO NH (in H NMR)
2
2
General Procedure 1. Synthesis of benzylidene oxazolones.
To a mixture of anhydrous NaOAc (1.0 equiv) and N-acetyl glycine (1.0 equiv) suspended in
Ac O (10.0 equiv) at rt under inert atmosphere, was added the relevant benzaldehyde 15-18
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(
1.0 equiv). The resulting mixture was stirred at 120 °C for 4 h and then cooled to rt. After
complete precipitation, the solid was filtered, poured into ice cold water (25-30 mL) and
vigorously stirred for 15-20 min. The mixture was then filtered and the residual solid dried
under reduced pressure. The crude product was used in the next step without further
purification. The title compounds were however purified for characterization purposes as
described for each compound of the series 19-22.
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General Procedure 2. Synthesis of arylpyruvic acids followed by oxime group protection.
A mixture of the required oxazolone 19-22 (1 equiv) in 10% aqueous HCl (10 mL/mmol) was
stirred at reflux in water (100−102 °C) for 12-14 h. The reaction mixture was then allowed to
cool to rt over 2-3 h and the product precipitated. After complete precipitation, the solid was
collected by suction filtration, washed with ice cold water (10-15 mL) and dried under reduced
pressure. The filtrate was washed with EtOAc (3 × 20 mL) and the organic fractions combined,
washed with water (1 × 20 mL) and saturated aqueous NaCl (1 × 20 mL), dried over MgSO4
and concentrated under reduced pressure. The oily residue was triturated with a minimum
amount of n-hexane. The n-hexane portion was carefully decanted and the remaining crude
solid was dried under reduced pressure. The crude solids of the aryl pyruvic acids 23-26
collected by filtration and extraction-trituration were combined and used for the next step
without any purification.
To a mixture of the required arylpyruvic acid derivative 23-26 (1 equiv) and benzyloxyamine
hydrochloride (NH OBn.HCl, 1.5 equiv) or O-(tetrahydro-2H-pyran-2-yl) hydroxylamine
2
(NH OTHP, 1.5 equiv) was added anhydrous pyridine (2 mL/mmol) under an argon
2
atmosphere. The reaction mixture was stirred overnight (12-14 h) at rt. Pyridine was removed
under reduced pressure, to the residue was added 1.0 N HCl (5.5 mL/mmol) and extracted in
2
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EtOAc (3 × 25 mL). The combined organic fractions were washed with saturated aqueous NaCl
(
1 × 20 mL/mmol), dried over MgSO and concentrated under reduced pressure. The desired
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protected oximes were purified as described for each compound in the series of 7-14.
General Procedure 3. Amide coupling
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To a mixture of O-Bn or O-THP protected oximino acid 7-14 (1.0 equiv), EDC.HCl (1.5 equiv)
and HOBt.H O (1.5 equiv) under an argon atmosphere, was added anhydrous DMF (10
2
mL/mmol) and the resulting solution was stirred at rt for 45 min. Next, the corresponding
aromatic amine sulfonamide 2-5 (1.0 equiv) was added and the mixture stirred at rt for 24 h.
The solvent was evaporated under high vacuum and water (15 mL), added to the remaining
residue, followed by extraction with EtOAc (3 × 25 mL). The combined organic fractions were
washed with saturated aqueous NaHCO (2 × 15 mL/mmol), saturated aqueous NaCl (1 × 20
3
mL/mmol), dried over MgSO and concentrated under reduced pressure. The desired
4
compounds were purified as described in the series of 27-33, 35-37, 39-41, and 59-67.
General Procedure 4. Deprotection of oxime benzyl ether using transfer hydrogenation
To a solution of the required oxime benzyl ether (27, 29, 31, 33, 35 and 37) (1.0 mmol) in
absolute EtOH (8 mL/mmol) and cyclohexene (4 mL/mmol) was added 20% Pd(OH) and the
2
resulting reaction mixture stirred at reflux in EtOH (78−82 °C). After the reaction was complete
(as evidenced by TLC monitoring), the mixture was filtered through a Celite bed and washed
with MeOH or acetone (2 × 20 mL). The filtrate was evaporated under reduced pressure and
the residue purified as described for the compound series 43, 45, 47, 49, 51 and 53.
General Procedure 5. Deprotection of oxime tetrahydropyran-2-yl ether
To the required oxime tetrahydropyran-2-yl ether 59-67 (1.0 mmol) was added 4.0 M HCl in
1,4-dioxane (15 mL/mmol) at 0 °C. The reaction mixture was stirred at 0 °C until complete (as
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evidenced by TLC monitoring). The solvent was evaporated under reduced pressure and the
residue purified as described for the compound series of 44, 46, 48, 50, 52 and 54-57.
(2E)-2-[(Benzyloxy)imino]-3-phenylpropanoic acid (7)
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Compound 7 was synthesized from crude aryl pyruvic acid 23 (0.5 g, 3.05 mmol) according to
general procedure 2. The crude residue was purified by flash chromatography (0.5−1% MeOH
in DCM) to afford the title compound as a white solid (0.65 g, 79%). R = 0.37 (10% MeOH in
f
1
DCM). Mp = 75−77 °C. H NMR (500 MHz, DMSO-d ) δ = 13.16 (bs, 1H, COOH), 7.36-
6
H
7
.16 (m, 10H, H ), 5.27 (s, 2H, Ph-CH -O), 3.85 (s, 2H, Ph-CH -C), general assignments were
Ar 2 2
1
1
13
confirmed by H- H gCOSY. C NMR (125 MHz, DMSO-d ) δ = 164.3 (Cquat), 151.2 (Cquat),
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C
1
1
36.9 (Cquat), 135.9 (Cquat), 128.5 (2 × CH ), 128.4 (2 × CH ), 128.3 (2 × CH ), 128.0 (CH ),
Ar Ar Ar Ar
27.9 (2 × CH ), 126.4 (CH ), 76.5 (Ph-CH -O), 30.8 (Ph-CH -C), general assignments were
Ar
Ar
2
2
1
13
+
+
confirmed by H- C HSQC. LRMS (ESI): m/z = 270 [M + H] , 292 [M + Na] . HRMS (ESI):
+
calcd for [C H NO − H] 268.0979, found 268.0978
1
6
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3
(2E)-2-[(Benzyloxy)imino]-3-(3-bromophenyl)propanoic acid (8)
Compound 8 was synthesized from crude aryl pyruvic acid 24 (0.7 g, 2.88 mmol) according to
general procedure 2. The crude residue was purified by flash chromatography (1% MeOH in
DCM) to afford the title compound as a white solid (0.64 g, 64%). R = 0.32 (10% MeOH in
f
1
DCM). Mp = 130−132 °C. H NMR (500 MHz, DMSO-d ) δ = 13.2 (bs, 1H, COOH), 7.42
6
H
(
d, J = 7.8 Hz, 1H, H ), 7.38-7.30 (m, 6H, H ), 7.24 (t, J = 7.8 Hz, 1H, H ), 7.17 (d, J = 7.6
Ar Ar Ar
Hz, 1H, H ), 5.27 (s, 2H, Ph-CH -O), 3.84 (s, 2H, Ph-CH -C), general assignments were
Ar
2
2
1
1
13
confirmed by H- H gCOSY. C NMR (125 MHz, DMSO-d ) δ = 164.1 (Cquat), 150.5 (Cquat),
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C
1
38.7 (Cquat), 136.8 (Cquat), 131.2 (CH ), 130.5 (CH ), 129.3 (CH ), 128.4 (2 × CH ), 128.0
Ar Ar Ar Ar
(
CH ), 127.9 (2 × CH ), 127.6 (CH ), 121.6 (C ), 76.7 (Ph-CH -O), 30.4 (Ph-CH -C),
Ar Ar Ar quat 2 2
1
13
general assignments were confirmed by H- C HSQC. LRMS (ESI): m/z = 370, 372 [M + Na,
2
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