An Unusual Natural Product Primary Sulfonamide: Synthesis, Carbonic Anhydrase Inhibition, and Protein X-ray Structures of Psammaplin C

Article abstract of DOI:10.1021/acs.jmedchem.6b00443

Psammaplin C is one of only two described natural product primary sulfonamides. Here we report the synthesis of psammaplin C and evaluate the inhibition profile against therapeutically relevant carbonic anhydrase (CA) zinc metalloenzymes. The compound exhibited unprecedented inhibition of an important cancer-associated isozyme, hCA XII, with a K_i of 0.79 nM. The compound also displayed good isoform selectivity for hCA XII over other CAs. We present the first reported protein X-ray crystal structures of psammaplin C in complex with human CAs. We engineered the easily crystallized hCA II enzyme to mimic both the hCA IX and hCA XII binding sites and then utilized protein X-ray crystallography to determine the binding pose of psammaplin C within the hCA II, hCA IX, and hCA XII mimic active sites, all to high resolution. This is the first time a natural product primary sulfonamide inhibitor has been assessed for inhibition and binding to CAs.

Full text of DOI:10.1021/acs.jmedchem.6b00443

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Article
An Unusual Natural Product Primary Sulfonamide: Synthesis, Carbonic
Anhydrase Inhibition and Protein X-ray Structures of Psammaplin C
Prashant Mujumdar, Kanae Teruya, Kathryn F. Tonissen, Daniela
Vullo, Claudiu T Supuran, Thomas S. Peat, and Sally-Ann Poulsen
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00443 • Publication Date (Web): 12 May 2016
Downloaded from http://pubs.acs.org on May 15, 2016
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An Unusual Natural Product Primary Sulfonamide: Synthesis, Carbonic Anhydrase
Inhibition and Protein X-ray Structures of Psammaplin C
†
†
†
‡
Prashant Mujumdar, Kanae Teruya, Kathryn F. Tonissen, Daniela Vullo, Claudiu T.
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‡
§
*,†
Supuran, Thomas S. Peat, and SallyꢀAnn Poulsen
†
Eskitis Institute for Drug Discovery, Griffith University, Don Young Road, Nathan,
Queensland 4111, Australia
‡
Polo Scientifico, Neurofarba Department and Laboratorio di Chimica Bioinorganica,
Universitàdegli Studi di Firenze, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence,
Italy
§
CSIRO, 343 Royal Parade, Parkville, Victoria 3052, Australia
*
Corresponding author Tel: +61ꢀ7ꢀ37357825; Eꢀmail: s.poulsen@griffith.edu.au
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Abstract
Psammaplin C is one of only two described natural product primary sulfonamides. Here we
report the synthesis of psammaplin C and evaluate the inhibition profile against
therapeutically relevant carbonic anhydrase (CA) zinc metalloenzymes. The compound
exhibited unprecedented inhibition of an important cancerꢀassociated isozyme, hCA XII, with
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a K of 0.79 nM. The compound also displayed good isoform selectivity for hCA XII over
other CAs. We present the first reported protein Xꢀray crystal structures of psammaplin C in
complex with human CAs. We engineered the easily crystallized hCA II enzyme to mimic
both the hCA IX and hCA XII binding sites and then utilized protein Xꢀray crystallography to
determine the binding pose of psammaplin C within the hCA II, hCA IX and hCA XII mimic
active sites, all to high resolution. This is the first time a natural product primary sulfonamide
inhibitor has been assessed for inhibition and binding to CAs.
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Introduction
Carbonic anhydrases (CA, EC 4.2.1.1) are zinc metalloenzymes that catalyze the reversible
ꢀ
+
hydration of carbon dioxide to bicarbonate and a proton: CO
2
+ H
2
3
O ꢀ HCO + H . This
equilibrium is critical for human health, and blocking the endogenous chemistry catalyzed by
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CAs is a current target for therapeutic intervention. Most reported small molecule CA
inhibitors incorporate a primary sulfonamide functional group, this group blocks the enzyme
2
activity by coordination to the catalytic zinc. Natural products (NPs) offer extraordinary
chemical diversity, and in recent times have been explored as a source of novel CA
inhibitors. NPs so far identified that can inhibit CA are however not primary sulfonamides,
instead they have an alternate zinc binding group (e.g. phenol or carboxylate group) within
3
,4
their structure. Given the prominence of the primary sulfonamide group in medicinal
5
chemistry, we conducted a literature search of the Dictionary of Natural Products database
6
and found just two primary sulfonamide NPs. We recently reviewed the discovery, total
synthesis, and known bioactivity for these compounds, which includes psammaplin C
6
(
Figure 1). This unusual NP was isolated in 1991 from the marine sponge Pseudoceratina
7
purpurea. The structure of psammaplin C comprises several structural elements, (i) a 3ꢀ
bromoꢀ4ꢀhydroxy benzylidine moiety, (ii) an oxime group, (iii) an aminoethane sulfonamide
chain. Psammaplin C was assessed for histone deacetylase inhibition activity and had no
8
activity against this metalloenzyme , which has a preference for a hydroxymate moiety as the
9
zinc binding group. Psammaplin C has not before been investigated for interaction with CA
enzymes. Our aim was to synthesize NP psammaplin C and evaluate the bioactivity against a
comprehensive panel of human CA (hCA) isozymes. We were also interested to assess the
binding pose of this unusual NP within the hCA active sites and determine the protein Xꢀray
structure of psammaplin C in complex with hCA II and recently generated mimics of hCA IX
and hCA XII.
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Figure 1. Psammaplin C − an unusual natural product comprising a primary sulfonamide
group.
Results and Discussion
Chemical Synthesis. We encountered two difficulties with the reported synthetic route of
NP psammaplin C (6). Firstly, the synthesis employs a bisꢀOꢀbenzyl protected oxime acid as
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the key intermediate, however the conditions described to generate this intermediate were
ambiguous, and our efforts to optimize this step were not successful. Another drawback of
the known synthetic method was the need for the strong Lewis acid, trimethylsilyl iodide for
Oꢀbenzyl ether deprotection in the final step towards 6. Trimethylsilyl iodide is extremely
moisture sensitive and inconvenient to handle. We sought to develop an alternate synthesis of
6
in order to circumvent the limitations encountered with the reported synthetic route.
Our synthesis, Scheme 1, is an adaptation of the synthesis reported for the related disulfide
11,12
metabolite psammaplin A.
Reaction of commercially available 3ꢀbromoꢀ4ꢀhydroxy
benzaldehyde 1 with Nꢀacetyl glycine under Erlenmeyer conditions generated the
benzylidene oxazolone 2. Compound 2 was converted to the αꢀketo acid 3 (enol form) by
acid hydrolysis. Subsequent oximation of
3
with Oꢀ(Tetrahydroꢀ2Hꢀpyranꢀ2ꢀ
yl)hydroxylamine gave the mono OꢀTetrahydroꢀ2Hꢀpyranꢀ2ꢀyl protected oxime acid 4.
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Treatment of 4 with Nꢀ hydroxysuccinimide (HOSu) and Nꢀ(3ꢀDimethylaminopropyl)ꢀNꞌꢀ
ethylcarbodiimide hydrochloride (EDC.HCl) afforded an activated succinate ester derivative,
which after work up, was immediately reacted with βꢀaminoethanesulfonamide hydrochloride
11 to generate the OꢀTHP protected psammaplin C derivative 5. Finally, the THP group of 5
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1
3
was removed using 4M HCl in 1,4ꢀdioxane to afford the target NP 6 in as white solid. The
key advantage of this new synthetic route rests with the implementation of the THP
protecting group in lieu of the benzyl protecting groups of the earlier reported synthesis.
Introduction of the OꢀTHP protected oxime acid, compound 5, was straightforward and
required mild reaction conditions (4M HCl in 1,4ꢀdioxane) to deprotect the oximeꢀTHP
group. Significantly, only the oxime oxygen requires protection, while the phenol hydroxyl
group does not, a further advantage of this alternative synthetic route. We anticipate that the
mild conditions and good yields associated with this synthesis will facilitate ready access to
further primary sulfonamide analogues of 6.
Scheme 1. Synthesis of NP 6.
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Reagents and Conditions: (i) Nꢀacetyl glycine, NaOAc, Ac
2
O, 120 °C, 3 h, 85%; (ii) Aq.
HCl (10%), reflux, 15 h, 65%; (iii) H NOTHP, dry pyridine, rt, 15 h, 54%; (iv) a) EDC.HCl,
2
HOSu, dry 1,4ꢀdioxane, 2ꢀ3 h, rt; b) betaꢀaminoethanesulfonamide hydrochloride 11, dry
NEt , dry MeOH, rt, 12 h, 46% (over 2 steps); (v) 4.0 M HCl in 1,4ꢀdioxane, 0 °C, 6ꢀ8 h,
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80%.
The oxime geometry of precursor 5 and the target NP 6 was confirmed as E on the basis of
1
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C NMR data reported for the structurally related disulfide metabolites 12 (E) and 13 (Z)
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(Figure 2). The benzylic carbon of 12 resonates at δ26.7, and is upfield of the corresponding
carbon in 13 (δ35.7). This chemical shift difference is consistent with a configuration change
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of the oxime moiety from E (12) to Z (13).
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Figure 2. C NMR chemical shifts (blue) of precursor 5 and the target NP 6 and known
compounds 12 and 13 are consistent with the E configuration of the oxime moiety.
CA Inhibition. The CA inhibition data for 6 and reference compound acetazolamide (AZA),
the par excellence therapeutically established CA inhibitor, was measured against a panel of
hCA isozymes (Table 1). Compared to AZA, the NP 6 showed remarkable variation among
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the different CA isoforms, with a range of inhibition constants (K
i
‘s) that span five orders of
magnitude (K range 0.79 nM – 10630 nM), while for AZA the CA inhibition is more tightly
i
grouped (K range 2.5 – 250 nM). Compound 6 had exceptional inhibition for hCA XII, with
i
a K of 0.79 nM (entry 8, Table 1). Although low nanomolar CA inhibitors are widely known,
i
subnanomolar CA inhibition is uncommon. The hCA XII expression is upregulated in a wide
selection of hypoxic tumors and has recently been identified as a key player in the facilitation
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of Pꢀglycoprotein mediated multidrug resistance in cancer cell models. Compound 6 may
thus provide a valuable probe compound for biology associated with CAs, and with hCA XII
in particular. Compound 6 has been submitted to the Compounds Australia Open Compound
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Collection (Academic) (https://www.griffith.edu.au/scienceꢀaviation/compoundsꢀaustralia).
Table 1. Human CA inhibition profile for NP 6 and the reference CA inhibitor AZA.
#
i
K (nM)
Entry
hCA isoform
6
AZA
250
12
1
2
3
4
5
hCA I
hCA II
48.1
88.0
75.3
154
hCA IV
hCA VA
hCA VI
74.2
63.1
11.0
9680
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hCA VII
hCA IX
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0.79
10630
379
2.5
25.0
5.7
hCA XII
hCA XIII
hCA XIV
16.4
41.3
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#
Errors ± 10% of the reported values (from three separate assays).
Protein X-Ray Crystal Structures. Atomic level structures with the novel NP 6 bound to
different CA isozymes would allow us to look at the differences in interactions between 6 and
the different active sites in detail. AZA is very compact in size relative to 6, and so is not able
to directly interact with the variable outer rim hCA active site residues. We engineered the
hCA II protein to mimic the active sites of hCA IX and hCA XII. This approach has been
used a number of times to facilitate the structural biology of proteins recalcitrant to
18
crystallization, including CA IX, this is however the first CA XII mimic.
The protein sequences for hCA IX and hCA XII were aligned to the hCA II sequence,
highlighting 14 conserved residues between the three sequences found in the active site, these
were retained in the mimic proteins: N62, H64, Q92, E106, V121, L141, V143, L198, T199,
T200, P201, P202, V207 and W209, Figure 3. The threeꢀdimensional structure of hCA II was
analyzed manually with the sequences of hCA IX and hCA XII superposed on all of the
active site residues. Ten substitutions were made to the hCA II sequence to obtain the hCA
IX mimic protein: A65S, N67Q, E69T, I91L, F131V, G132D, V135L, K170E, L204A and
C206G; while nine substitutions were made to obtain the hCA XII mimic protein: A65S,
N67K, E69N, I91T, F131A, G132S, V135S, L204N and C206T, Figure 3.
1
2
3
4
CA2
MSHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSL 44
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CA9
CA12
DQSHWRY---GGDPPWPRVSPACAGRFQSPVDIRPQLAAFCPAL
NGSKWTYFGPDGENSWSKKYPSCGGLLQSPIDLHSDILQYDASL
:* *
*
* : * . * ***:*:
.
:
:*
CA2
CA9
CA12
KPLSVSY---DQATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHWGSLD 101
RPLELLGFQLPPLPELRLRNNGHSVQLTLPPGLEMA----LGPGREYRALQLHLHWGAAG
TPLEFQGYNLSANKQFLLTNNGHSVKLNLPSDMHI-----QGLQSRYSATQLHLHWGNPN
*
*..
.: : ****:.:: :
.
*
*:*:***
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CA2
CA9
CA12
GQ-GSEHTVDKKKYAAELHLVHWNTKY-GDFGKAVQQPDGLAVLGIFLKVGS-AKPGLQK 159
RP-GSEHTVEGHRFPAEIHVVHLSTAF-ARVDEALGRPGGLAVLAAFLEEGPEENSAYEQ
DPHGSEHTVSGQHFAAELHIVHYNSDLYPDASTASNKSEGLAVLAVLIEMGS-FNPSYDK
******. ::: **:*:** .:
*
:
*****. ::: *
: . ::
CA2
CA9
CA12
VVDVLDSIKTKGKSADFTNFDPRGLLPES-LDYWTYPGSLTTPPLLECVTWIVLKEPISV 218
LLSRLEEIAEEGSETQVPGLDISALLPSDFSRYFQYEGSLTTPPCAQGVIWTVFNQTVML
IFSHLQHVKYKGQEAFVPGFNIEELLPERTAEYYRYRGSLTTPPCNPTVLWTVFRNPVQI
:.. *: : :*..: . ::
***.
*: * *******
* * *:.: : :
CA2
CA9
CA12
SSEQVLKFRKLNFNG--EGEPEELMVDNWRPAQPLKNRQIKASFK
SAKQLHTLSDTLWG-----PGDSRLQLNFRATQPLNGRVIEASFP
SQEQLLALETALYCTHMDDPSPREMINNFRQVQKFDERLVYTSFS
261
*
:*:
:
:
:
*:* .* :. * : :**
Figure 3. Amino acid sequence alignment of the catalytic domain of hCA II, IX and XII.
Conserved residues are highlighted in green, while CA II residues highlighted in yellow were
replaced with the corresponding CA IX and CA XII residues, to create the mimic proteins.
Capasso and coꢀworkers recently reported a rapid and facile inꢀgel technique to enable
19
determination of CA activity and the corresponding molecular weight. The utility of this
technique, known as protonography, as a CA activity assay has since been reported for
multiple classes of CAs including bovine CA (αꢀCA), recombinant CA from Vibrio cholerae
19
20
(
VchCA, αꢀCA), Porphyromonas gingivalis (PgiCA, γꢀCA), and Plasmodium falciparum
2
1
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(
PfCA, ηꢀCA), and native CAs in whole cell lysate of Escherichia coli (βꢀ and γꢀCAs). In
this technique, an SDS gel loaded with CAs is stained with bromothymol blue, a commonly
used pH indicator for pH range between 6.0 and 7.6. The stained gel is then suspended in
carbonated water. An immediate color change from blue to yellow is observed at the position
+
of catalytically active CA as a result of local acidification (due to H ions generated by CA
hydratase activity). Here we have applied protonography to assess the activity of hCA IX and
hCA XII. Both mimic proteins generated a strong yellow band, consistent with the mimics
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retaining the enzymatic activity of the parent hCA II (Figure 4, lanes 1ꢀ3). Control proteins
(bovine serum albumin, BSA, and ovalbumin, OVA) did not generate a color change,
confirming the color change observed is dependent on CA activity (Figure 4, lane 4ꢀ6).
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Figure 4. Protonogram (left) and coomassie blue stain (right) of recombinant hCA II, CA IX
mimic, and CA XII mimic (0.5 ꢁg each). The yellow signal at approximately 29 kDa
confirms CA enzymatic activity (lane 1ꢀ3), which was not observed for nonꢀCA proteins
(lane 4ꢀ6).
Xꢀray crystal structures of the apo forms of the hCA IX and hCA XII mimic proteins were
determined to look at the potential differences between the native active sites. There are no
substantial changes (beyond a few sidechain rotamers) that occur between the apo and
compound 6ꢀbound structures of the CA IX and XII mimic proteins, so we focus on the
bound structures. A comparison of the CA XII mimic structure to a previously elucidated
2
2
crystal structure of human CA XII (Figure 5, PDB code 4HT2) shows that the active sites
are very similar, differing only at the rim of the active site where there is a small change in
the position of a loopꢀhelix (residues 127ꢀ139 (124ꢀ137 in 4HT2), an average of ~2 Å in Cα
positions, with a 2.4 Å maximum difference). Overall the CA XII mimic when compared
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with PDB entry 4HT2, shows some regional backbone variations remote from the active site,
with the overall rmsd between the mimic and 4HT2 of 1.24 Å to 1.18 Å (A and B chains,
respectively), with 11 gaps over 242 aligned residues (of 258), with 41% sequence identity.
Figure 6 shows the Xꢀray crystal structures of hCA II and the hCA IX and hCA XII mimics
in complex with compound 6. These data for the [hCA II:6] complex extend to 1.57 Å
resolution, for the [hCA IX:6] complex to 1.35 Å resolution and for the [hCA XII:6] complex
to 1.40 Å resolution (see supporting information, Table 1).
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Figure 5. A comparison of the CA XII mimic with the previously determined CA XII
structure 4HT2 in the PDB. The CA XII mimic is in cyan with 4HT2 in yellow; the grey
sphere represents the zinc atom in the active site. The left panel highlights the mutations
made in the active site of CA II to make the CA XII mimic. The most significant difference is
found at the top (12 o’clock) at the rim of the active site, where a small helix is translated
about 2 Å.
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Figure 6. The Xꢀray crystal structures of hCA II and the hCA IX and hCA XII mimics.
Panels A and B highlight the sequence changes made to create the mimic proteins; CA II is in
grey, CA IX in magenta and CA XII in cyan; the two orientations in A and B are
approximately a 90 degree rotation from each other. Panels C and D show compound 6 as
bound to the three different proteins; C and D are approximately 90 degrees rotated from
each other. The grey sphere represents the zinc atom in the active site of the proteins. It is
clear that compound 6 adopts a different orientation in each of the three structures, although
the sulfonamide moiety is in almost the same position for each structure.
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Except in the [hCA IX:6] complex, where little of the extended structure of compound 6
beyond the sulfonamide is seen, compound 6 makes multiple interactions with the protein as
it extends out of the catalytic pocket (see supporting information). There was clear density for
the sulfonamide moiety of compound 6 adjacent to the zinc atom in the active sites after the
initial rounds of refinement for all of the complexes. The sulfonamide functional group
interacts directly with the zinc and has potential hydrogen bonds to active site residue Thr199
with the same pose as that for >200 sulfonamide/hCA II complexes reported in the PDB. In
the [hCA II:6] complex, the amide nitrogen is 2.95 Å from the Thr200 side chain and the
amide oxygen is bound to a water molecule, which is close to Gln92. The nitrogen of the
oxime is within hydrogen bonding distance to the Thr200 side chain and the hydroxyl of the
oxime is 2.6 Å to the Pro201 carbonyl. In the [hCA XII:6] complex, the nitrogen of 6 in the
amide linker is 3.1 Å from the Thr200 side chain and the amide oxygen is directly bound (2.7
Å) to the side chain of Gln92. In this complex, the oxime oxygen is bound to a water
molecule at 2.7 Å; this water molecule is also coordinated by hydrogen bonds to Tyr7 and
His64. In both complexes, compound 6 is not modelled at full occupancy (about 80%) and
there is some additional positive electron density in the difference maps, suggesting that there
are other conformations of 6 that may be possible in the active site.
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The bromophenol moiety sits in a hydrophobic pocket made up of Phe131, Val135, Pro202
and Leu204 at 3.7 to 4.7 Å with no obvious hydrogen bonds in the [hCA II:6] complex. This
is reminiscent of the interactions made by the hydrophobic tail of brinzolamide in hCA II as
1
8
reported by Pinard et al, but the bromine atom is buried even tighter into this pocket than
the hydrophobic tail of brinzolamide. The ring of the bromophenol moiety of the [hCA XII:6]
complex has close contacts with the Asn62 side chain at 3.1 Å. The bromine’s closest
contact to the hCA XII mimic is 4 Å to the Lys67 side chain, but it makes closer contacts to a
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6
second molecule of 6 found at the top of the active site (about 3.7 Å away). There is also a
second molecule of 6 in the [hCA II:6] complex found nearby which has the bromophenol
moiety bound between the His3 ring and the bromophenol ring of 6 bound to the zinc. This
second molecule of 6 has the hydroxyl moiety making a potential hydrogen bond to the Trp5
ring nitrogen, with the rest of the compound solvent exposed. In the [hCA II:6] complex there
is even a third molecule of 6 found in the structure completely outside the active site and
between crystallographically related monomers (see supporting information). All of the
complex models refined well against the data (see Table 1 of the supporting information).
0
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9
0
Conclusion
In conclusion, psammaplin C 6 occupies a rare place in NP chemical space, as one of only
two primary sulfonamide NPs so far isolated and characterized. Herein we have developed an
improved and facile synthesis of 6 and confirmed the
E stereochemistry about the oxime
center. We have evaluated for the first time the CA inhibition characteristics for this unusual
NP against a panel of ten human CA isoforms. Notably, the NP exhibited a distinctive CA
inhibition profile when compared to the somewhat flat inhibition profile of AZA. Compound
6
is a subnanomolar inhibitor of an important cancerꢀassociated isozyme, hCA XII, and
displays good isoform selectivity for hCA XII. We engineered the easily crystallized hCA II
enzyme to mimic both the hCA IX and hCA XII binding sites, both exhibited catalytic
activity. We utilized protein Xꢀray crystallography to determine the binding pose of 6 within
the hCA II, hCA IX and hCA XII mimic active sites, all to high resolution. The NP engages
in a series of strong interactions with the active site residues of hCA II and hCA XII and
represents the first reported structures of a NP sulfonamide with hCA enzymes.
Experimental Section
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General Chemistry. 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 performed using silica gel 60 Å (230ꢀ400 mesh).
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60
1
13
NMR ( H, C, COSY, HSQC and HMBC) spectra were recorded on the 500 MHz
1
13
6
spectrometer at 25°C. Chemical shifts for H and C NMR obtained in DMSOꢀd are
reported in ppm 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 hertz (Hz). Highꢀ and lowꢀresolution mass spectra were acquired using
electrospray as the ionization technique in positiveꢀion and/or negativeꢀion modes as stated.
All MS analysis samples were prepared as solutions in methanol. Purity of compounds all
compounds was >95% as determined by HPLC (Agilent HPLC 1100 series) with UV
detection. The melting points are uncorrected. Numbering used for NMR assignments is
shown below.
O
OH
OH
OH
1
5
O
14
7
4
4
Br
3
Br
11
Br
3
10
Br
8
9
5
6
5
6
6
5
12
2
2
1
3
9
O
O
O
4
3
1
7
1
7
1
6
10
10
8
O
14
2
8
SO NH
2
2
8
SO NH
2 2
5
7
13
O
1
OH
15
14
O
9
N
H
9
N
H
11
11
4
O1
2
1
2
N
N
N
N
10
O
12
O
HO
3
11
13
6
2
4
5
6
2-Bromo-4-{[(4
E
)-2-methyl-5-oxo-4,5-dihydro-1,3-oxazol-4-ylidene]methyl}phenyl
acetate (2)
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6
To a mixture of anhydrous NaOAc (1.47 g, 17.90 mmol, 1.2 equiv) and
Nꢀacetyl glycine
(
2.01 g, 17.90 mmol, 1.2 equiv) suspended in Ac
temperature under inert atmosphere, was added 3ꢀbromoꢀ4ꢀhydroxybenzaldehyde 1 (3.0 g,
4.92 mmol, 1 equiv). The resulting mixture was stirred at 120 °C for 4 h and then cooled to
room temperature. After complete precipitation, the solid was poured into ice cold water (25ꢀ
0 mL) and vigorously stirred for 15 min. The mixture was then filtered and residual solid
2
O (15.23 g, 149.2 mmol, 10 equiv) at room
1
0
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7
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9
0
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2
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4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
dried under vacuum to obtain title compound 2 (4.11 g, 85.3%, crude) as yellow solid. The
crude product was used for the next step without further purification. A minimal amount of
crude product was purified by flash chromatography (5ꢀ8% acetone in
characterization purposes. Rf = 0.46 (20% acetone in
500 MHz, DMSOꢀ ) δ = 8.55 (s, 1H, Hꢀ9); 8.22 (d,
Hz, 1H, HAr); 7.24 (s, 1H, Hꢀ7); 2.41 (s, 3H, Hꢀ6); 2.35 (s, 3H, Hꢀ15), general assignments
n
ꢀhexane) for
1
n
ꢀhexane). Mp = 196−198°C. H NMR
(
d
6
H
J = 8.5 Hz, 1H, HAr); 7.44 (d, J = 8.5
1
1
13
were confirmed by Hꢀ H gCOSY. C NMR (125 MHz, DMSOꢀ
(Cquat); 166.9 (Cquat); 149.3 (Cquat); 135.7 (Cꢀ9); 133.6 (Cquat); 132.8 (Cquat); 132.3 (CHAr);
26.9 (Cꢀ7); 124.6 (CH ); 116.1 (Cquat); 20.5 (Cꢀ15); 15.4 (Cꢀ6), general assignments were
6 C
d ) δ = 168.0 (Cquat); 167.6
1
Ar
1
13
+
79
81
+
confirmed by Hꢀ C HSQC. LRMS (ESI ): m/z = 324, 326 [M+H; Br, Br] .
(2E)-3-(3-Bromo-4-hydroxyphenyl)-2-hydroxyprop-2-enoic acid (3)
To the crude oxazolone 2 (3.0 g, 9.29 mmol, 1.0 equiv) was added 10% aq. HCl (93 mL, 10
mL/mmol) and the resulting reaction mixture stirred at reflux (102ꢀ104°C) for 12ꢀ14 h. The
reaction mixture was cooled to room temperature and the product extracted in EtOAc (3 × 25
mL). The combined organic fractions were washed with saturated aqueous NaCl (1 × 20
mL/mmol), dried over MgSO
4
and concentrated in vacuo to provide crude aryl pyruvic acid 3
(1.57 g, 65.55%, crude) as a light brown solid. The crude aryl pyruvic acid was used for the
1
next step without further purification. H NMR (500 MHz, DMSOꢀd6) δ = 10.40 (s, 1H);
H
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9.11 (s, 1H); 8.02 (d,
J
= 1.9 Hz, 1H); 7.50 (dd,
J
= 8.5, 1.9 Hz, 1H); 6.93 (d,
J = 8.45 Hz,
1
3
1
H); 6.31 (s, 1H). C NMR (125 MHz, DMSOꢀ
d
6
) δ = 166.3, 153.3, 140.4, 133.3, 130.1,
C
−
79
81
+
127.8, 116.1, 109.3, 108.7; LRMS (ESI ): m/z = 257, 259 [M−H; Br, Br] .
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(2
To a mixture of the arylpyruvic acid 3 (1.5 g, 5.81 mmol, 1.0 equiv) and
pyranꢀ2ꢀyl) hydroxylamine (1.02 g, 8.71 mmol, 1.5 equiv) was added anhydrous pyridine
11.6 mL, 2 mL/mmol) under an argon atmosphere. The resulting reaction mixture was
E
)-3-(3-Bromo-4-hydroxyphenyl)-2-[(oxan-2-yloxy)imino]propanoic acid (4)
Oꢀ(tetrahydroꢀ2H
ꢀ
(
stirred overnight (12ꢀ14 h) at room temperature. Pyridine was removed in vacuo, to the
residue was added 1N HCl (32 mL, 5.5 mL/mmol) and extracted in EtOAc (3 × 25 mL). The
combined organic fractions were washed with saturated aqueous NaCl (1 × 20 mL/mmol),
4
dried over MgSO and concentrated in vacuo. Column chromatography of the crude residue
afforded the title compound 4 (1.12 g, 54.0%) as a pale yellow solid.
f
R = 0.11 (10% MeOH
1
in DCM). Mp = 42−44°C. H NMR (500 MHz, DMSOꢀd6) δ = 13.26 (bs, 1H, ꢀCOOH),
H
1
9
3
1
0.11 (s, 1H, ArꢀOH), 7.36ꢀ7.35 (d,
); 6.88 (d, = 8.3 Hz, 1H, Hꢀ8); 5.36ꢀ5.35 (m, 1H, Hꢀ10); 3.77 (d,
.73 (d, = 13.5 Hz, 1H, CH ); 3.50ꢀ3.48 (m, 2H, HTHP); 1.76ꢀ1.71 (m, 3H, HTHP); 1.61ꢀ
.52 (m, 2H, HTHP); 1.48ꢀ1.45 (m, 1H, HTHP), general assignments were confirmed by Hꢀ H
J
= 2.1 Hz, 1H, Hꢀ5); 7.03 (dd,
J
= 8.35, 2.1 Hz, 1H, Hꢀ
J
J
= 13.5 Hz, 1H, C H);
H
3
J
H
3
1
1
13
gCOSY. C NMR (125 MHz, DMSOꢀd6) δ = 164.5 (Cꢀ1), 152.6 (C ), 151.8 (Cꢀ2), 133.1
C
Ar
(
Cꢀ5), 129.0 (Cꢀ9), 128.1 (C ), 116.3 (Cꢀ8), 109.0 (C ), 100.8 (Cꢀ10), 61.2 (CH ꢀTHP), 29.7
Ar
Ar
2
(
Cꢀ3), 28.1 (CH
2
ꢀTHP), 24.6 (CH
2
ꢀTHP), 18.4 (CH
2
ꢀTHP), general assignments were
1
13
+
79
81
+
confirmed by Hꢀ C HSQC. LRMS (ESI ): m/z = 380, 382 [M+Na; Br, Br] .
(2
E
)-3-(3-Bromo-4-hydroxyphenyl)-2-[(oxan-2-yloxy)imino]-N-(2-
sulfamoylethyl)propanamide (5)
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2
2
2
2
2
2
2
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4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
To a mixture of
O
ꢀTHP protected oximino acid 4 (0.29 g, 0.81 mmol, 1.0 equiv), EDC.HCl
(0.26 g, 1.38 mmol, 1.7 equiv) and HOSu (0.18 g, 1.54 mmol, 1.9 equiv) under Ar
atmosphere was added anhydrous 1,4ꢀdioxane (8 mL, 10 mL/mmol) and the resulting
solution was stirred at room temperature for 3 h. The solvent was evaporated under vacuum
and the residue was dissolved in EtOAc (30ꢀ35 mL), washed with saturated aqueous NaHCO3
0
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8
9
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2
3
4
5
6
7
8
9
0
(2 × 15 mL/mmol), 1N aqueous HCl (2 × 15 mL/mmol) and saturated aqueous NaCl (1 × 20
mL/mmol). The organic fraction was dried over MgSO and concentrated in vacuo to afford
4
crude succinate ester (0.37 g, 0.81 mmol, 1.0 equiv). The succinate ester was dissolved in
anhydrous 1,4ꢀdioxane (8 mL, 10 mL/mmol) under Ar atmosphere and a solution of βꢀ
aminoethanesulfonamide hydrochloride 11 (0.15 g, 0.93 mmol, 1.15 equiv) and NEt (0.15
3
mL, 1.07 mmol, 1.15 equiv of 11) in anhydrous MeOH (5 mL/mmol) was added. The
resulting reaction mixture was stirred at room temperature for overnight (12ꢀ14 h). The
solvent was then evaporated in vacuo to obtain the crude residue. Column chromatography
(gradient 2.5−4% MeOH in DCM) of this crude residue gave title compound 5 (0.173 g,
46.75% over two steps) as a white solid. Rf = 0.21 (10% MeOH in DCM). Mp = 68−70°C.
1
H NMR (500 MHz, DMSOꢀ
d
6
) δ
= 2.05 Hz, 1H, Hꢀ2); 7.04 (dd,
= 8.25 Hz, 1H, Hꢀ5); 5.35ꢀ5.34 (m, 1H, Hꢀ10); 3.77 (d,
= 13.15 Hz, 1H, CHH7); 3.60ꢀ3.51 (m, 2H, Hꢀ10); 3.47ꢀ3.45 (m, 2H, HTHP); 3.16 (t,
H
= 10.08 (s, 1H, ArꢀOH); 8.29 (t,
= 8.3, 2.1 Hz, 1H, Hꢀ6); 6.93 (s, 2H, SO
= 13.15 Hz, 1H, C
J
= 5.85 Hz, 1H, NꢀH);
NH );
H);
7
.38 (d,
.86 (d,
J
J
J
2
2
6
J
H
7
3.70 (d,
J
J
= 7.25 Hz, 2H, Hꢀ11); 1.75ꢀ1.73 (m, 3H, HTHP); 1.63ꢀ1.54 (m, 2H, HTHP); 1.48ꢀ1.45 (m, 1H,
1
1
13
H
THP), general assignments were confirmed by Hꢀ H gCOSY. C NMR (125 MHz, DMSOꢀ
) δ = 162.4 (Cꢀ9), 153.0 (CAr), 152.5 (Cꢀ8), 133.3 (Cꢀ2), 129.1 (Cꢀ6), 128.2 (CAr), 116.2
(Cꢀ5), 108.9 (CAr), 100.6 (CHꢀTHP), 61.1 (CH ꢀTHP), 53.3 (Cꢀ11), 34.4 (Cꢀ10), 28.8 (Cꢀ7),
8.2 (CH ꢀTHP), 24.6 (CH ꢀTHP), 18.3 (CH ꢀTHP), general assignments were confirmed by
d
6
C
2
2
2
2
2
1
13
+
79
81
+
79
Hꢀ C HSQC. LRMS (ESI ): m/z = 464, 466 [M+H; Br, Br] , 486, 488 [M+Na; Br,
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1
+
+
79
+
Br] . HRMS (ESI ): calcd for C16
H
22BrN
3
NaO
6
S [M+Na; Br] 486.0305, found 486.0296;
8
1
+
3 6
calcd C16H22BrN NaO S [M+Na; Br] 488.0291, found 488.0275
(2E)-3-(3-Bromo-4-hydroxyphenyl)-2-(N-hydroxyimino)-N-(2-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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sulfamoylethyl)propanamide (6, Psammaplin C)
To the oxime THP ether 5 (0.125 g, 0.27 mmol, 1.0 equiv) was added 4M HCl in 1,4ꢀdioxane
solution (4.1 mL, 15 mL/mmol) at 0 °C, the reaction mixture was stirred at the same
temperature. After completion of reaction (6−8 h, TLC), the solvent was evaporated in vacuo
to obtain the crude residue. Column chromatography (gradient 4−6% MeOH in DCM) of this
crude residue gave title compound 6 (0.082 g, 80.39%) as a white solid. Rf = 0.11 (10%
1
MeOH in DCM). Mp = 160−162°C. H NMR (500 MHz, DMSOꢀ
d
) δ
= 11.93 (s, 1H, Nꢀ
6
H
OH); 10.03 (s, 1H, ArꢀOH); 8.08 (t,
.01 (dd, = 8.35, 2.1 Hz, 1H, Hꢀ6); 6.93 (s, 2H, SO
3.68 (s, 2H, Hꢀ7); 3.54 (q, = 6.35 Hz, 2H, Hꢀ10); 3.14 (t,
assignments were confirmed by Hꢀ H gCOSY. C NMR (125 MHz, DMSOꢀd6) δ = 163.1
J
= 5.95 Hz, 1H, NꢀH); 7.29 (d,
J
= 2.1 Hz, 1H, Hꢀ2);
7
J
2
NH ); 6.84 (d,
2
J
= 8.3 Hz, 1H, Hꢀ5);
J
J = 6.65 Hz, 2H, Hꢀ11), general
1
1
13
C
(Cꢀ9), 152.3 (CAr), 151.5 (Cꢀ8), 132.8 (Cꢀ2), 129.1 (Cꢀ6), 128.8 (CAr), 116.1 (Cꢀ5), 108.8
1
13
(C
Ar), 53.5 (Cꢀ11), 34.1 (Cꢀ10), 27.7 (Cꢀ7), general assignments were confirmed by Hꢀ C
+
79
81
+
79
HSQC and HMBC. LRMS (ESI ): m/z = 379, 381 [M+H; Br, Br] , 401, 403 [M+Na; Br,
8
1
+
+
79
+
Br] . HRMS (ESI ): calcd for C H BrN NaO S [M+Na; Br] 401.9730, found 401.9734;
11
14
3
5
81
+
calcd for C H BrN NaO S [M+Na; Br] 403.9709, found 403.9711
11
14
3
5
Protein expression and purification. hCA II protein was expressed and purified as
23
previously described. The protein sequences for human CA IX and CA XII were aligned to
the human CA II sequence using ClustalꢀOmega. Clones of the CA IX and CA XII mimic
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6
proteins containing the CA II sequence modified by either 10 or 9 changes (respectively) in
the active site were ordered from Genescript and subsequently subcloned into pET43 using
the BamH1 and Xho1 sites.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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0
1
2
3
4
5
6
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9
0
Competent BL21:DE3 cells were transformed with the respective plasmids and fresh cultures
were grown at the 1 L scale in Teriffic Broth with ampicillin at 100 ꢀg/mL with zinc added to
a final concentration of 50 ꢀM. The cells were grown at 37° C until midꢀlog phase (OD600 0.6
to 0.8) and protein expression was induced using 0.5 mM IPTG. The cells were allowed to
grow for another 4.5 hours, then centrifuged and the pellets were stored in a freezer at ꢀ20° C.
The cell pellets were brought up in Tris buffered saline (TBS: 40 mM Tris at pH 8.0, 150 mM
2
NaCl) with added 150 mM NaCl, 10 mM imidazole, 2 mM MgCl , Benzonase, complete
EDTA free tablets, lysozyme, 1 mM PMSF and the cells were disrupted with three passes
using a cell crusher (Avestin). The resulting slurry was centrifuged to remove cell debris and
the supernatant was loaded onto a HisꢀTrap 1mL FF column in buffer A (TBS plus 150mM
NaCl, 10 mM imidazole). The column was washed with 20 column volumes (CV) of buffer
A, then 20 CV of Buffer A containing 20 mM final imidazole concentration, subsequently the
protein was eluted with 5 CV of buffer A containing 250 mM imidazole. The peak fractions
were pooled and put onto a S200 16/60 gel filtration column in TBS and the peak fraction of
monomeric protein was subsequently pooled and concentrated to 11 mg/mL for the CAꢀIX
mimic and 10.5 mg/mL for the CAꢀXII mimic.
Protein X-ray Crystallography. Concentrated hCAꢀII at about 10 mg/mL was set up in SDꢀ
2
plates (Molecular Dimensions) with the following ratio of protein plus reservoir plus seeds:
50 nL + 225 nL + 25 nL. The plate was incubated at 20º C and the reservoir condition
2
consisted of 2.9 M ammonium sulfate with 0.1 M Tris buffer at pH 8.3. Dry compound was
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added to the crystallization drop after crystals had formed and several days before data were
collected. The CAꢀIX mimic and CAꢀXII mimic proteins were crystallized in sitting drop
plates using 200 nL protein at 5 mg/mL plus 200 nL reservoir solution over 50 ꢀL reservoirs
for the CAꢀXII mimic and 150 nL protein at 5.5 mg/mL plus 120 nL reservoir plus 30 nL
microꢀcrystalline seeds over 50 ꢀL reservoirs for the CAꢀIX mimic. The crystallization plates
were incubated at two temperatures (20º C and 8º C) and large plateꢀlike crystals were found
at both temperatures in optimized ammonium sulfate conditions (2.6 to 2.8 M ammonium
sulfate, 100 mM Tris pH 8.0 to 9.0). Crystals were harvested using nylon loops with the
addition of glycerol as a cryoprotectant (20% final concentration). Data were collected at the
Australian Synchrotron MX1 beamline in 360 one degree oscillations. The data were indexed
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
24
25
26
using XDS and scaled using Aimless . Molecular replacement was done using Phaser
using 4cq0 as the initial starting model for CA II, and the human CAꢀII (PDB code 4NR4) as
a starting model for the CA IXꢀmimic and CA XIIꢀmimic, the mutated residues were built
27
27
into the structures using Coot . All models were manually rebuilt using Coot and refined
28
using Refmac . The initial placement of 6 and the dictionary (cif) file were generated with
28
AFITT (OpenEye Scientific Software) and further refined using Refmac. The structure and
structure factors were deposited in the PDB with accession code 5a6h (CA II with
psammaplin C), 5g01 (CA XII mimic with psammaplin C), 5g03 (CA IX mimic with
psammaplin C), 5g0b (CA IX apo) and 5g0c (CA XII apo).
CA Inhibition Assay. An applied Photophysics stoppedꢀflow instrument was used for
2
9
2
assaying the CAꢀcatalyzed CO hydration activity. IC50 values were obtained from doseꢀ
response curves working at seven different concentrations of the test compound, by fitting the
curves using PRISM (www.graphpad.com) and nonlinear leastꢀsquares methods; values
3
0
represent the mean of at least three different determinations as described by us previously.
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1
1
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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
The inhibition constants (
follows: = IC50/(1 + [
measurement was carried out, and
activity is at halfꢀmaximal. All enzymes used were recombinant, produced in Escherichia coli
K
i
) were then derived by using the ChengꢀPrusoff equation as
K
i
S
]/
K
m
), where [
2
S] represents the CO concentration at which the
K
m
is the concentration of substrate at which the enzyme
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
31,32
as reported earlier.
.5 and 15.0 nM.
The hCA isoform concentrations in the assay system ranged between
7
CA activity assay. The enzymatic activity of CA II, CA IX mimic, and CA XII mimic was
19
determined using protonography. CA protein (0.5 µg each, 1 ꢀL) and control proteins BSA
and OVA (5 µg each, 2 ꢀL each) were prepared in 1× PBS (15 uL). The samples were diluted
with 5× SDS (ꢀ) buffer (5 ꢀL) to give a final sample volume of 25 µL, and separated with
SDSꢀPAGE (12%) at 180 V until the dye front has ran off the gel. After separation, the gel
was incubated in 2.5% Triton Xꢀ100 for 1 h with shaking, followed by two times 10 min
wash with 10% isopropanol (in 100 mM Tris, pH 8.2). The gel was then stained with 0.1%
bromothymol blue (in 100 mM Tris, pH 8.2) for 30 min. The stained gel was then incubated
in carbonated water and color change (from blue to yellow) or color retention (remained blue)
was determined, within 30 s of exposing the gel to carbonated water.
1
13
Supporting Information. H and C NMR spectra for compounds 2-6 and 8-11. COSY and
HMBC spectra for compound 6. HPLC chromatograms for compound 5 and 6.
Crystallography statistics and electron density images, spectral dataꢀimages of all compounds
are provided. This material is available free of charge via the Internet at http://pubs.acs.org.
PDB ID Codes. All of the coordinates and structure factors have been deposited in the PDB
and are available with the following codes 5a6h (CA II with 6), 5g01 (CA XII mimic with 6),
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5g03 (CA IX mimic with 6), 5g0c (CA XII apo), 5g0b (CA IX apo). Authors will release the
atomic coordinates and experimental data upon article publication.
10
11
12
13
14
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22
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Corresponding author
S.ꢀA.P. Telephone: +61 7 3735 7825; eꢀmail: s.poulsen@griffith.edu.au.
Keywords sulfonamide, natural product, carbonic anhydrase XII, protein mimic
Acknowledgement. We thank the Australian Research Council (grant numbers
DP110100071, FT10100185 to S.ꢀA.P.), two EU grants of the 7th framework program and
Griffith University (Ph.D. scholarship to P.M. and K.T.). We thank Professor Emeritus Ian D.
Jenkins for helpful discussions. We thank Dr. Tanja Grkovic for NMR discussions. We thank
Dr. Lucy Woods and Dr. Wendy Loa for HRMS measurements and Anna Raicevic for
expression and purification of the CA IX and CA XII mimics. We thank the CSIRO C3
Crystallisation Centre for all CA crystals, the Australian Synchrotron and beamline scientists
for beam time, and OpenEye Scientific Software for a license to AFITT.
Abbreviations Used.
CA, carbonic anhydrase; hCA, human carbonic anhydrase; AZA, acetazolamide; Ki
inhibition constant; NPs, natural products; EDC.HCl, ꢀ(3ꢀDimethylaminopropyl)ꢀNꞌ
ꢀ hydroxysuccinimide.
,
N
ꢀ
ethylcarbodiimide hydrochloride; HOSu,
N
References
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Table of Contents Graphic
Br
OH
OH
N
First X-ray structures
(
E)
with human CAs
HN
0
1
2
3
4
5
6
7
8
9
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1
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1
2
3
4
5
6
7
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9
0
Inhibitor of cancer
related hCA XII
(Ki = 0.79 nM)
O
O
S
O
H2N
Psammaplin C
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