Metallocene-based inhibitors of cancer-associated carbonic anhydrase enzymes IX and XII

Article abstract of DOI:10.1021/jm300427m

In this study, 20 metallocene-based compounds comprising extensive structural diversity were synthesized and evaluated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. These compounds proved moderate to good CA inhibitors in vitro, with several compounds displaying selectivity for cancer-associated isozymes CA IX and CA XII compared to off-target CA I and CA II. Compound 6 was the most potent ferrocene-based inhibitor with K_is of 5.9 and 6.8 nM at CA IX and XII, respectively. A selection of key drug-like parameters comprising Log P, Log D, solubility, and in vitro metabolic stability and permeability were measured for two of the ferrocene-based compounds, regioisomers 1 and 5. Compounds 1 and 5 were found to have characteristics consistent with lipophilic compounds, however, our findings show that the lipophilicity of the ferrocene moiety is not well modeled by replacement with either a naphthyl or a phenyl moiety in software prediction tools.

Full text of DOI:10.1021/jm300427m

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Article
Metallocene-based inhibitors of cancer-
associated carbonic anhydrase enzymes IX and XII
Adam J Salmon, Michael Lloyd Williams, Quoc K Wu, Julia Morizzi, Daniel Gregg,
Susan A. Charman, Daniela Vullo, Claudiu T Supuran, and Sally-Ann Poulsen
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm300427m • Publication Date (Web): 27 Apr 2012
Downloaded from http://pubs.acs.org on May 17, 2012
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Metallocene-based inhibitors of cancer-associated carbonic anhydrase enzymes IX and
XII
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Adam J. Salmon†, Michael L. Williams†, Quoc K. Wu‡, Julia Morizzi‡, Daniel Gregg‡, Susan
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‡
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A. Charman , Daniela Vullo, Claudiu T. Supuran and Sally-Ann Poulsen
†Eskitis Institute for Cell and Molecular Therapies, Griffith University, Nathan, Queensland
4111, Australia, ‡Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical
Sciences, Monash University (Parkville Campus), 381 Royal Parade, Parkville, Victoria 3052,
§
Australia, and Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Università
degli Studi di Firenze, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy.
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Abstract
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In this study 20 metallocene-based compounds comprising extensive structural diversity were
synthesized and evaluated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. These
compounds proved moderate to good CA inhibitors in vitro, with several compounds
displaying selectivity for cancer-associated isozymes CA IX and CA XII compared to off-
target CA I and CA II. Compound 6 was the most potent ferrocene-based inhibitor with Kis of
5.9 and 6.8 nM at CA IX and XII, respectively. A selection of key drug-like parameters
comprising Log P, Log D, solubility, and in vitro metabolic stability and permeability were
measured for two of the ferrocene-based compounds, regioisomers 1 and 5. Compounds 1 and
5 were found to have characteristics consistent with lipophilic compounds, however our
findings show that the lipophilicity of the ferrocene moiety is not well modelled by
replacement with either a naphthyl or a phenyl moiety in software prediction tools.
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Introduction
Reports of safe and efficacious organometallic inhibitors for a growing number of
therapeutically relevant enzymes have resulted in widened acceptance of organometallic
compounds as viable candidates for targetted therapeutic applications.1 To date numerous
classes of organometallic compounds have found application in medicinal chemistry; these
include metallocenes, half-sandwich metallocenes, metal carbenes, metal carbonyls and metal-
arene compounds.1 The classical metallocenes, ferrocene and ruthenocene, are sandwich
compounds wherein the metal is located between two cyclopentadienyl (Cp) rings. These
compounds are stable in air, kinetically inert and uncharged, with their metal atom in a low
oxidation state. Both ferrocene and ruthenocene are amenable to derivatization reactions such
as Friedel-Craft acylations, formylation, sulfonation and lithiation (to name a few) and this
permits a straightforward synthesis of metallocene-based organometallic compounds.2 The
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toxicology of ferrocene is particularly well studied and this compound may be administered
orally without toxicity.3 Ferrocene is metabolized in the liver by cytochrome P450 enzymes
similarly to benzenes.3 Ferrocenium salts were the first organometallic compounds for which
antiproliferative properties were reported4 and today there are a number of ferrocene-based
compounds that have found use as therapies.3 Notably, the replacement of a benzene ring with
a ferrocene fragment within the structure of two established drugs has led to compounds
wherein the ferrocene chemistry is implicated in the drug mode of action. Hydroxyferrocifen is
a ferrocene analogue of tamoxifen that selectively targets breast cancer5 while ferroquine is a
ferrocene analogue of chloroquine that targets the malaria parasite, both of these ferrocene
analogues are in clinical development.6 An alternate approach, wherein the ferrocene moiety is
appended as a substituent onto a known pharmacophore, has been applied in a number
successful medicinal chemistry campaigns to take advantage of the physicochemical and
structural properties of ferrocene for improved biological activity.3 Ruthenocene and its
derivatives are much less studied than their isostructural ferrocenyl counterparts, and to date
only a small number of ruthenocene-based compounds appear in the literature in the context of
drug discovery.7
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Carbonic anhydrases (CAs) are zinc metalloenzymes that catalyze the reversible
hydration of carbon dioxide to bicarbonate and a proton.8 CA isozymes IX and XII are
overexpressed in cancer cells of many hypoxic tumors where they provide a pH-regulating
system that contributes to hypoxic tumor cell survival and proliferation.9 The significance of
CAs role in cancer has triggered a need to develop novel, drug-like small molecule CA
inhibitors as chemical tools and/or as leads for therapeutic drug discovery.10 Small molecule
CA inhibitors have recently been reported that show promising anticancer properties.11 In this
manuscript we report the design, synthesis, biological activity and selected ADME properties
for a library of novel metallocene-based CA inhibitors. The development of metallocene-based
compounds that can selectively kill tumor cells by inhibiting the validated oncology target CA
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IX, is a new approach with potential to deliver organometallic, drug-like compounds as future
therapies. We recently reported the protein X-ray crystal structures of four of these compounds
in complex with CA II (PDB accession codes 3P55, 3P3H, 3P44 and 3P3J),12 while others
have recently reported structures of piano-stool complexes bound to CA II.13 Our study is the
first wherein isomeric ruthenocene and ferrocene inhibitors have been complexed with the
target protein and a crystal structure obtained.12 The replacement of ferrocene (iron) with
ruthenocene (ruthenium) altered the CA enzyme inhibition of these compounds in a manner
consistent with the subtle but significant difference in structure provided by changing the
metal. We demonstrated that although the metallocene moiety behaves chemically like an
aromatic moiety such as a phenyl group,7a the barrel-shaped sandwich structure of the
metallocene fragment permits access to 3D structural permutations that are not possible with a
flat aromatic ring.12 We identified several hydrophobic interactions with the hydrophobic face
of the CA II binding cavity and hypothesized that this may provide an avenue to continue to
develop new metallocene-based compounds that better occupy the hydrophobic binding pocket
of CAs active site to further inform structure-activity relationships (SAR) of this
organometallic class of CA inhibitors.
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Results and Discussion
Inhibitor design. The ‘tail approach’ has been applied to the design and synthesis of a
growing number of potent and isozyme selective CA inhibitors. The approach involves
covalently tethering a tail fragment onto the established primary sulfonamide CA recognition
pharmacophore (R-SO2NH2 where R = aromatic) to generate the extended pharmacophore:
[tail]-[aromatic]-[ZBG].14 The approach has provided a framework in which CA inhibitor
properties can be readily tuned with respect to structure-property and structure-activity
parameters to deliver CA inhibitors with biopharmaceutical characteristics that are appropriate
for in vivo use. Commonly used covalent attachments between the aromatic group and tail
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fragment are ester, amide, imine, urea and thiourea linkers. Previous work in our research
group has focussed on the 1,3-dipolar cycloaddition reaction (1,3-DCR) between alkynes and
azides to generate novel CA inhibitors with triazole tails.15 Using either 4-azido or 4-ethynyl
benzene sulfonamide as the CA recognition pharmacophore and 1,3-DCR with a
complementary alkyne or azide, we have synthesised compounds that comprise a tail fragment
attached to a CA recognition pharmacophore through an intervening 1,2,3-triazole link.16 We
have previously presented the synthesis and CA enzyme inhibition of four metallocene-based
CA inhibitors prepared by 1,3-DCR, compounds 1-4, Figure 1.7a These compounds comprise
the [tail]-[aromatic]-[ZBG] extended pharmacophore, wherein a triazole-ferrocene or triazole-
ruthenocene is the tail of the CA inhibitor. In the present study we have synthesized 16
additional novel metallocene-based CA inhibitors comprising more extensive structural
diversity with attachment of the tail group, including triazole (5-8), triazole-ester (9-14),
triazole-amide (15-16), amide (17-18) and urea (19-20) linkers, Figure 2. These linkers impart
differing stability and hydrogen bonding attributes to these organometallic CA inhibitors.
Compounds 1-20 comprise two isomeric series, the first series is the para-substituted benzene
sulfonamide [aromatic]-[ZBG] series (1-4, 9-11, 15) and the second series is the meta-
substituted benzene sulfonamide [aromatic]-[ZBG] series (5-8, 12-14, 16). In addition to
metallocenes 1-20, six additional analogues were synthesized wherein the triazole-metallocene
tail fragment was replaced with either a triazole-phenyl tail fragment (21-24) or an
unsubstituted triazole fragment (25-26), Figure 3. Compounds 21-26 were designed as controls
to allow delineation of the metallocene contribution to SAR and structure-property
relationships (SPR).
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Figure 1. Metallocene-based CA inhibitors 1 – 4 synthesized by 1,3-DCR.
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O
O
O
O
N
N
N
N
N
N
N
N
N
S O
NH2
N
S O
NH2
N
S O
NH2
N
S O
NH2
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Ru
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Fe
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Figure 2. Novel metallocene-based CA inhibitors 5-20.
O O
S
O O
S
O O
S
O O
S
NH2
NH2
NH2
NH2
N
N
N
N
N
N
N
N
N
N
N
N
Fe
Ru
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5
Fe
Ru
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O O
S
NH2
O
N
N
Fe
Fe
N
N
N
S
O
NH2
N
O
O
n=1-3
n=1-3
O
O
9-11
12-14
O O
S
NH2
N
O
N
N
Fe
N
Fe
N
N
S
O
NH2
H
N
H
N
O
O
15
16
O O
S
O O
S
NH2
NH2
O
S O
NH2
O
S O
NH2
Fe
HN
HN
Fe
HN
O
HN
HN
HN
O
O
O
Fe
Fe
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20
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18
Figure 3. Control compounds in which the triazole-metallocene tail fragment is replaced by a
triazole-phenyl tail fragment (21-24) or an unsubstituted triazole tail fragment (25, 26).
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O O
S NH2
O O
S NH2
O
O
N
N
N
N
N
N
N
N
N
S O
NH2
N
N
S O
NH2
N
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O O
S NH2
O
N
N
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NH2
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Chemistry. The building blocks for the target compounds 1-20 comprise metallocene-based
alkynes 27-32 and benzenesulfonamide azides 33 and 34, Figure 4. Metallocene-based alkynes
include ethynyl ferrocene 27, ethynyl ruthenocene 28, ethynyl ester substituted ferrocenes 29-
31 and ethynyl amide substituted ferrocene 32. Compound 27 is commercially available,
compound 28 was prepared as reported by us earlier,7a while esters and amide 29-32 were
prepared from fluorocarbonylferrocene 35 and the respective alcohol or amine.17 The acyl
fluoride 35 is less susceptible to hydrolysis than the corresponding acyl chloride, and this
permitted the straightforward handling, purification and storage of 35.17 Azido
benzenesulfonamides (3316d and 34) were synthesized from their corresponding commercially
available amines using neutral conditions reported for the synthesis of aryl azides.18 Compound
34 is novel and this is the first application of click chemistry from this azide. Compounds 1-16
were synthesized by 1,3-DCR of alkynes 27-32 with azides 33 and 34. The 1,4-disubstituted-
1,2,3-triazole inhibitors 1, 3, 5, 7, 9-16 were synthesized by copper-catalyzed azide-alkyne
cycloaddition (CuAAC), while the 1,5-disubstituted-1,2,3-triazole regioisomers 2, 4, 6 and 8
were synthesized by ruthenium-catalyzed azide-alkyne cycloaddition (RuAAC), Scheme 1.
The amide-linked compounds 17 and 18 were prepared by the reaction of acyl fluoride 35 with
commercially available 4–aminobenzenesulfonamide or 3-aminobenzenesulfonamide,
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respectively, Scheme 2. Urea-linked CA inhibitors 19 and 20 were synthesized indirectly from
acyl fluoride 35 according to Scheme 3. Control compounds 21-26 were synthesized from
azides 33 or 34 and commercially available phenyl acetylene (21-24) or TMS acetylene (25-
26), similarly to Scheme 1. Note that removal of the TMS group using TBAF was required for
the synthesis of 25.
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Figure 4. Metallocene-base alkynes (27-32) and [aromatic]-[ZBG] azide (33 and 34) building
blocks.
O O
S NH2
O
O
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Fe
Fe
Ru
O
N3
S O
NH2
N3
n=1-3
N
H
O
34
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29-31
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33
para series
meta series
Scheme 1. Synthesis of metallocene based CA inhibitors 1-16 using either CuAAC or RuAAC.
CuAAC
RuAAC
N
N
SO2NH2
SO2NH2
N
N
SO2NH2
Metallocene
+
N
N3
N
e
n
e
c
27-32
33-34
o
l
l
a
t
e
M
1,3, 5, 7, 9-16
2, 4, 6, 8
Reagents and Conditions: CuAAC: azide 33 or 34 (0.02 – 0.1 M), alkyne 27-32 (1.0 equiv.),
CuSO4.5H2O (0.2 equiv.), sodium ascorbate (0.4 equiv.), tBuOH:H2O (1:1), 40 ºC, 18 h.
RuAAC: azide 33 or 34 (0.02 – 0.15 M), alkyne 27 or 28 (1.0 equiv.), [Cp*RuCl(PPh3)2] or
[Cp*RuCl(cod)] (5 mol%), toluene, N2, 100 ºC, 18 h.
Scheme 2. Synthesis of amide-linked metallocene-based CA inhibitors 17 and 18.
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SO2NH2
SO2NH2
O
+
Fe
H2N
Fe
HN
O
F
35
17 (para series)
18 (meta series)
9
Reagents and Conditions: Compound 35 (1.0 mmol) in DCM (5 mL), 3- or 4-
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aminobenzenesulfonamide (1.1 equiv.) in DMF (0.35 mL) and pyridine (0.1 mL), rt, 5 days.
Scheme 3. Synthesis of urea-linked metallocene-based CA inhibitors 19 and 20.
SO2NH2
HN
HN
O
O
Fe
Fe
(ii), (iii)
(i)
O
Fe
F
N3
19 (para series)
20 (meta series)
35
36
Reagents and Conditions: (i) Compound 35 (4.3 mmol) in THF (2 mL), sodium azide (4.5
equiv.) in water (10 mL), rt, 3 h; (ii) Compound 36 (0.5 mmol), toluene, 100 ºC, 1 h; then (iii)
p- or m-aminobenzenesulfonamide (1.1 equiv.), DMF (1 mL), 50 ºC, 18 h.
Carbonic Anhydrase Inhibition Studies and Structure-Activity Relationships. The enzyme
inhibition data for 1-26 were obtained for the physiologically dominant CA I and II and tumor-
associated transmembrane CA IX and XII, Table 1.
Table 1.CA inhibition data for compounds 1-26 against human CA isozymes I, II, IX and XII.
Ki (nM)a
Ki (nM)a
Compd
Compd
(meta
(para
CA Ib
3900
CA IIb
CA IXc
85
CA XIIc
-
CA Ib
1790
CA IIb CA IXc CA XIIc
series)
series)
1d
80
4165
33.1
18.8
5
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2 d
3 d
4 d
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1600
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36
9.7
12.3
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4
65
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-
-
361
70
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5.9
8.2
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8.1
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186
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69
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7.9
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7
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-
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77
80
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8.0
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57
63
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3.8
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76
81
43
4.0
10
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44
7.9
75.5
76.2
137
72
56
5.2
2580
101
2180
60
415
10.3
2570
47
3680
1890
2290
51
4690
3720
90.5
45
26.2
31.5
21.4
5.8
542
433
41
61
509
618
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68
42
345
596
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58
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aErrors in the range of ± 5-10 % of the reported value, from three determinations. Human
b
c
d
(cloned) isozymes. Catalytic domain of human (cloned) isozymes. Previously reported in
reference 7a.
The regioisomeric compounds 25 and 26 provide a baseline to assess the impact of the
metallocene substituent of triazoles 1-4 and 5-8, respectively, on CA inhibition. These controls
comprise a 1,2,3-triazole that is linked either para (25) or meta (26) to the primary sulfonamide
moiety of the benzenesulfonamide CA pharmacophore. Compound 25 is a weak inhibitor of
CA I, II and IX (Kis 345 – 596 nM) and a moderate inhibitor of CA XII (Ki 63 nM), while the
meta regioisomer 26 has similarly weak inhibition at CA I (Ki = 618 nM) but moderate
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inhibition at CA II, IX and XII (Kis 41 – 68 nM). These inhibition data suggest that meta
substitution relative to the primary sulfonamide functional group of the benzenesulfonamide
CA anchor when compared to an identical para substituent has minimal effect on inhibition of
CA I and XII, however a greater impact is observed with isozymes II and XII, where meta
substitution provided an order of magnitude improvement in CA inhibition. Control
compounds 21-24 are related to compounds 25 and 26 but differ in comprising a phenyl-
triazole tail fragment in place of the unsubstituted triazole tail fragment. Compounds 21-24
comprise both 4- and 5-phenyl substituted 1,2,3-triazoles and provide complex and
informative SAR. The phenyl moiety is a flat and relatively compact aromatic system in
contrast to the barrel-shaped sandwich structure of classical metallocenes. The meta- versus
para- substitution had less impact in these control compounds than for the unsubstituted
triazoles 25 and 26. Compounds 23 and 24, where the phenyl substituent is at the 5-position of
the 1,2,3-triazole, were weak inhibitors of CA I (Kis 509 – 542 nM) similarly to the
unsubstituted triazoles 25 and 26. The phenyl substituent in the para series compounds 21 and
23 improved CA inhibition by an order of magnitude over unsubstituted 25 at CA II, CA IX
and CA I (21 only) with moderate inhibition at CA XII similarly to 25. The phenyl substituent
in the meta series compounds 22 and 24 exhibited a trend closely aligned to unsubstituted
triazole 26 with one exception, compound 22 (the 4-phenyl substituted 1,2,3-triazole) was a
~10-fold more potent CA XII inhibitor (Ki = 5.8 nM) than 26.
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The 20 metallocene-based CA inhibitors, compounds 1-20, comprise significant structural
diversity. SAR is assessed in the context of SAR described above for contol compounds 21-26
as well across a number of different structural groupings:
(i) Ferrocene-triazole and ruthenocene-triazole tails. Compounds 1-8 comprise a metallocene-
triazole tail moiety directly attached to the benzene sulfonamide pharmacophore and so are
direct analogues of control compounds 21-26 described above. These inhibitors were
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synthesized by either CuAAC (1, 3, 5 and 7) or RuAAC (2, 4, 6 and 8) and so are 1,4- and 1,5-
disubstituted triazoles, respectively. The metallocene substituent generally contributed to
improved CA inhibition compared to the unsubstituted triazoles 25 and 26. At CA I the
metallocene moiety exhibited similar SAR to the phenyl moiety (21-24), however at CA II, IX
and XII the metallocene moiety predominantly acted to improve CA inhibition compared to a
phenyl moiety, with Kis for compounds 3 and 6-8 each 10 nM or less at these isozymes. Of the
para substituted compounds 1-4 the ruthenocene analogues 3 and 4 were better CA inhibitors
across all CA isozymes than the ferrocene inhibitors 1 and 2, while of the meta substituted
compounds 5-8 the ruthenocene analogues 7 and 8 were similar in potency to ferrocene-based
inhibitor 6, yet better across all CA isozymes compared to ferrocene-based inhibitor 5. A
notable difference in regioisomers activity was evident with the ferrocene analogues 5 and 6,
with compound 6 significantly more potent than its regioisomer compound 5. Compound 6, the
most potent ferrocene-based inhibitor, had Kis ranging from 3.2 to 6.8 nM at CA II, IX and
XII. Differences in potency across the four ruthenocene analogues was less pronounced, for
example the ruthenocenyl 1,4- and 1,5-disubstituted triazole regioisomers (compounds 7 and 8)
were of similar potency. The SAR we report surrounding the ferrocenyl and ruthenocenyl-
traizole tails for the different CA isozymes is consistent with our SAR findings for recently
reported regioisomeric CA inhibitors with carbohydrate-triazole tails prepared by both
RuAAC16a and CuAAC.16c Here the barrel-shaped metallocene moiety has provided a way to
disciminate the CA isozymes active site when compared to the correposnding phenyl
analogues, further suggestive of a potentially valuable structural role for the organometallic
fragment in continued CA inhibitor development for desired biological activity.
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(ii) Covalent linker to tail fragment. A stand out SAR from inspection of Ki values presented in
Table 1 is the weaker CA inhibition observed for compounds 15-20 compared to other
metallocene inhibitors of this study. Compounds 15-20 comprise an amide or urea covalent
linker between the [aromatic]-[ZBG] CA pharmacophore and metallocene tail fragment and
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while generally weaker as CA inhibitors, inhibition of cancer-associated isozymes IX and XII
is greater than for off-target isozymes I and II, a sought after selectivity profile with CA
inhibitor development. Esters 9 and 12 are bioisosteres of amides 15 and 16, yet the esters
remarkably exhibit two orders of magnitude better CA I and II inhibition than their
corresponding amides. The para-substituted compound 9 (ester) and 15 (amide) have similar
potency at CA IX and XII, while the meta-substituted compound 12 (ester) was a better CA IX
and XII inhibitor than its amide counterpart 16. Increasing the length of the alkyl chain of the
ester linkage (compare 9-11 and 12-14) had minimal effect on CA inhibition with the
exception of the longer chain compound 11 which was a 10-fold better CA IX inhibitor (Ki =
7.9 nM) than the shorter chain ester 9 and 10.
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(iii) Para-substitution and meta-substitution. The ten para-substituted analogues (1-4, 9-11, 15,
17, 19) had CA I inhibition constants that ranged from 9-3900 nM, while the ten meta-
substituted analogues (5-8, 12-14, 16, 18, 20) had CA I inhibition constants that ranged from
42-3680 nM, both trends reflecting the impact of the considerable diversity present across the
metallocene-based CA inhibitor library. Pairwise comparison of para- versus meta-substitution
shows generally similar inhibition at CA I, while at CA II compounds are similar with a few
notable compound pairs as exceptions (1 and 5, 2 and 6, 15 and 16, 17 and 18) all of which
have the para substitution pattern more potent than the meta subtitution pattern. At CA IX the
meta-series compounds are generally good inhibitors, and better than their corresponding para-
series compounds. At CA XII many of the compounds, both para- and meta-, are very potent
inhibitors with low nM inhibition constants. For the control compounds 21-24, the phenyl-
triazole substituent displayed little difference between meta and para relationship across all
isozymes (except for 21 versus 22 at CA XII).
In vitro ADME properties. The metallocene-based compounds in this study are good CA
inhibitors in vitro, and in addition several compounds display selectivity for the cancer-
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associated CAs compared to off-target CAs. The design of useful drugs and/or probes for
medicinal chemistry requires balancing compound activity with a number of different
compound properties, with the knowledge that each impacts on the drugs performance in vivo.
A recent analysis of compounds published in the medicinal chemistry literature demonstrated
that there is a noticeable creep of compound properties outside recognized drug-like
parameters,19 and here we were keen to build an understanding of the impact of the ferrocene
moiety on a selection of key drug-like parameters. We have experimentally measured and
determined Log P, Log D, solubility, metabolism and in vitro permeability for four CA
inhibitors sharing the core structure of a disubstituted triazole benzenesulfonamide: two
ferrocene-based compounds (regioisomers 1 and 5), and two analogues of 1, one where the
ferrocene is replaced by a phenyl moiety (compound 21), and another where the ferrocene is
replaced by a glucosyl moiety (compound 37), Figure 5.16c
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Figure 5. Compounds for ADME studies.
O
O
N
N
O
N
N
N
N
OH
O
N
N
S O
NH2
S O
NH2
N
S O
NH2
Fe
HO
HO
OH
21
37
1
O O
S
O
N
NH2
N
N
S O
NH2
N
N
N
Fe
5
38
Lipophilicity. Log P and Log D describe a compound’s lipophilicity, and values often
correlate with a number of key biopharmaceutical parameters in drug discovery. Table 2 shows
experimental Log P and Log D values for compounds 1, 5, 21 and 37 and calculated Log P
(cLog P) values for compounds 21, 37 and 38. The Log D (Log D at pH 7.4 and pH 3.0) values
were measured using both a RP-HPLC method20 and the traditional shake flask method. As
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expected from the ionization properties of the compounds (expected to be neutral at both pH
values), pH did not impact on the measured partition coefficients, Table 2. Ferrocene-based
regioisomers 1 and 5 had similar Log D7.4 and Log D3.0 values, with values ~0.6 log units
higher than for the phenyl compound 21. This demonstrates that the ferrocene moiety is more
strongly lipophilic than the phenyl moiety. As expected, carbohydrate-based compound (37)
with four hydroxyl groups has a markedly reduced Log D value (Log D7.4 or 3.0 of -0.1)
compared to compounds 1, 5 and 21, this is consistent with the increased hydrophilicity of 37.
The use of software tools to predict Log P (cLog P) is now routine in medicinal
chemistry, however common programs lack a metallocene substructure in their training set.
The experimentally measured octanol-water partition coefficient for ferrocene is 3.46.21 This
was of the same order as that measured for naphthalene (3.30), but much higher than that for
benzene (2.13).21 Using ChemDraw Ultra 12 software the cLog P for the virtual compound 38,
where a naphthyl moiety replaces the ferrocenyl moiety of 1, is 3.7. This value is 0.8 log units
higher than the experimentally determined Log D7.4 value for 1 and 5. The cLog P for the
phenyl compound 21 is 2.5 (close to the measured value) and is 0.4 log units lower than the
experimentally determined Log D7.4 value for 1 and 5. The ferrocene moiety lipophilicity of
compounds 1 and 5 is thus not well modelled by replacement with either a naphthyl or a phenyl
moiety as the measured LogD of ferrocene-based compounds 1 and 5 falls between the clog P
values calculated for these two aromatics.
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Table 2. Partitioning data for test compounds at 25 °C.
a
a
b
b
c
Log D7.4 Log D3.0 Log D7.4 Log D3.0
cLog P
Compd
2.9
2.9
2.3
2.9
2.9
2.3
2.9
3.0
2.4
2.1
2.7
2.3
-
-
1
5
2.5
21
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d
d
-0.1
-
-0.1
-
-
-1.6
3.7
37
38
-
-
-
6
7
8
b
c
aChromatographic estimation method. Shake flask method. Calculated using ChemDraw
Ultra 12. dConcentration values were outside of the linear range of the assay.
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Permeability. The experimental lipophilicity results imply that compounds 1, 5 and 21 should
exhibit good passive diffusion across gastrointestinal epithelial cells. The Caco-2 cell model
was used to measure the in vitro permeability (Papp) of compounds 1, 5, 21 and 37. Typical Papp
values for high permeability compounds are > 2 × 10-5 cm s-1 while for low permeability
compounds are < 2 × 10-6 cm s-1. The experimental values of Papp at pH 7.4 using the Caco-2
assay are presented in Table 2. For all compounds tested, there was good mass balance (>80%)
indicating minimal retention of compounds within the cell monolayer and minimal non-
specific adsorption. Papp values for the control compounds mannitol (low permeability marker)
and propranolol (high permeability marker) were consistent with historical results. Compounds
1, 5 and 21 have Papp values consistent with high permeability and good oral absorption. The
Papp values for 1, 5 and 21 are very similar indicating that the ferrocene and phenyl moieties
similarly contribute to membrane permeability in this model. For the carbohydrate-based
compound (37) no compound was detected in the acceptor chamber (below the analytical lower
limit of quantitation) indicative of a compound with very low permeability. The permeability
results are in agreement with predictions based on experimental lipophilicity.
Table 3. Caco-2 permeability coefficients for test compounds.
Mass Balance
-1
a
Papp (cm s )
Std. Dev.
Compd
1
(%)
83
5.8 × 10-5
1.6 × 10-5
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6.1 × 10-5
5.8 × 10-5
NDb
1.5 × 10-5
8.5 × 10-6
NDb
87
84
5
21
90
37
9
1.7 x 10-6
4.2 x 10-5
7.0 x 10-7
2.9 x 10-6
100
100
mannitol
propranolol
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aAverage of 3-4 determinations. bND = not determined as compound 37 was not detected in the
acceptor chamber. Mannitol and propranolol were included as low and high permeability
markers, respectively.
Solubility. Solubility is another property that can significantly affect oral absorption of a drug.
The kinetic and equilibrium solubility results for the four test compounds (at pH 2.0, pH 6.5
and in water) are presented in Table 4. The kinetic solubility results (presented as a range)
exhibited a similar trend to the equilibrium solubility results, although the absolute values
differ. The glycoconjugate CA inhibitor 37 has moderate kinetic solubility and good
equilibrium solubility, suggesting that the kinetic solubility assay underestimates the actual
solubility of this compound. Metallocene compound 1 and phenyl compound 21 were found to
be sparingly soluble under both kinetic and equilibrium conditions. Interestingly metallocene
compound 5, the regioisomer of compound 1, has moderate solubility, with values reasonably
consistent under both kinetic and equilibrium conditions.
Table 4. Solubility data for the test compounds at 25 °C.
Kinetic
Equilibrium
solubility
Kinetic
Equilibrium
solubility
Equilibrium
solubility
solubility
solubility
Compd
a
b
a
b
b
(pH 2)
(pH 2)
(pH 6.5)
(pH 6.5)
(water)
< 1.6
5.0
< 1.6
1.7
0.7
1
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6.3 - 12.5
1.6 - 3.1
26.0
1.5
6.3 - 12.5
1.6 - 3.1
5.1
1.4
978
21.6
2.2
5
21
37
25.0 - 50.0
905
25.0 - 50.0
1088
aKinetic solubility results determined using the nephelometric screening method, µg/mL.
bEquilibrium solubility results quoted represent 24 h data, µg/mL.
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Metabolic Stability. Next we determined the in vitro metabolic stability of the four
compounds using human liver microsomes as a preliminary indication of the likely in vivo
metabolic clearance and to see if any metabolic products could be detected. The four test
compounds exhibited low to moderate rates of degradation in human liver microsomes, and no
metabolites were detected for any of the test compounds. Based on the in vitro intrinsic
clearance values, these compounds would be expected to be subject to low to intermediate
hepatic clearances in vivo. There was no apparent degradation of the compounds in microsomal
matrix in the absence of cofactors, suggesting that the apparent rates of degradation in the
presence of cofactor were due solely to cofactor-dependent microsomal metabolism. There was
also no major increase in the rate of degradation observed in microsomal samples containing
NADPH and UDPGA (supplemented with the pore-forming peptide, alamethicin) relative to
NADPH alone, suggesting that these compounds were not susceptible to primary
glucuronidation in the microsomal test system, Table 5.
Table 5. Metabolic stability parameters for test compounds based on NADPH-dependent
degradation profiles in human liver microsomes.
Degradation
half-life
In vitro CLint
(ꢀL/min/mg
protein)a
Microsome-
predicted
EHa
Metabolites
detectedb
Compd
(min)a
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212 57.0
123 26.7
354 822
279 124
8.6 2.3
15 3.6
5.1 1.1
7.4 4.1
0.32 0.06
None
None
None
None
1
5
0.44 0.06
0.22 0.04
0.28 0.11
21
37
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aValues are represented as mean SD (n = 3). bThe metabolite search strategy was directed
towards potential products of oxygenation, bis-oxygenation, oxygenation plus glucuronidation,
and N-dealkylation.
Conclusions
In this study 20 metallocene-based CA inhibitors (compounds 1 to 20) comprising extensive
structural diversity were synthesized and evaluated as CA inhibitors. These compounds were
moderate to good CA inhibitors in vitro, and several compounds displayed selectivity for the
cancer-associated CAs compared to off-target CAs. At CA I the metallocene moiety exhibited
similar SAR to the phenyl moiety (compounds 21-24), however at CA II, IX and XII the
metallocene moiety predominantly acted to improve CA inhibition compared to a phenyl
moiety. The SAR surrounding the ferrocenyl and ruthenocenyl-traizole tails for the different
CA isozymes is consistent with our SAR findings for recently reported regioisomeric CA
inhibitors with carbohydrate-triazole tails prepared also by CuAAC or RuAAC. The measured
Log P, Log D, solubility, metabolism and in vitro permeability of two ferrocene-based
compounds (regioisomers 1 and 5) and the phenyl and glucosyl analogues of 1 (compounds 21
and 37, respectively) resulted in values consistent with the general structural features of the
compounds. Compounds, 1, 5 and 21 were found to have characteristics consistent with
lipophilic compounds. Compound 37 is less lipophilic than the other three compounds, this
was reflected in the solubility and partition coefficient values for this compound. A significant
finding with implications for the study of metallocene-based compounds in medicinal
chemistry, is that the ferrocene moiety lipophilicity is not well modelled by replacement with
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either naphthyl or a phenyl standard aromatic rings. The measured LogD of ferrocene-based
compounds 1 and 5 falls between the clog P values calculated for either the naphthyl or the
phenyl analogue. The barrel-shaped metallocene moiety has provided a means to disciminate
the CA isozymes active site when compared to the corresponding phenyl analogues, while
biopharmaceutical properties were typically similar. These compounds may constitute
potentially valuable leads for the development of CA inhibitor-based therapeutics and provide
further support for the application of metallocenes in CA inhibitor development.
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Experimental Section
Chemistry. All starting materials were either purchased from commercial suppliers. Known
building blocks were either commercially available (27), synthesized as reported by us earlier
(287a, 3316d) or synthesized according to literature (3517). All reactions were monitored by TLC
using silica plates with visualization of product bands by UV fluorescence (λ = 254 nm) and
ninhydrin. Silica gel flash chromatography was performed using silica gel 60 Å (230-400
mesh). NMR (1H, 13C {1H}, gCOSY and HSQC) spectra were recorded on a 500 MHz
spectrometer at 30 °C. Chemical shifts for 1H and 13C NMR acquired in DMSO-d6 are reported
in ppm relative to residual solvent proton (δ = 2.50 ppm) and carbon (δ = 39.5 ppm) signals,
1
respectively. Chemical shifts for H NMR acquired in CDCl3 are reported in ppm relative to
residual solvent proton (δ = 7.26 ppm). Multiplicity is indicated as follows: s (singlet); d
(doublet); t (triplet); m (multiplet); dd (doublet of doublet); ddd (doublet of doublet of
doublet); br (broad). Coupling constants are reported in Hertz (Hz). Labelling of compounds
used for NMR assignments is shown below.
O O
S
NH2
A
N
N
B'
N
!
″
B
C
#
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Melting points are uncorrected. High- and low- resolution mass spectra were acquired 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. All compounds were analyzed
for purity by HPLC with both UV (200 to 400 nm) and ELSD (Evaporative Light Scattering
Detection) detection used. Purity of all compounds was ≥ 95%.
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General Procedure 1. Synthesis of 1,4-disubstituted-1,2,3-triazoles by CuAAC: A mixture
of azide (0.02 - 0.1 M) and alkyne (1.0 equiv.) in tert-butyl alcohol and water (1:1) with
CuSO4.5H2O (0.2 equiv.) and sodium ascorbate (0.4 equiv.) was stirred vigorously overnight
(18 h) at 40 °C under a nitrogen atmosphere. The precipitate that formed was collected by
vacuum filtration, washed with water and purified by flash chromatography (1:1 ethyl acetate:
n-hexane then 100 % ethyl acetate) using solid addition from ethyl acetate.
General Procedure 2. Synthesis of 1,5-disubstituted-1,2,3-triazoles by RuAAC: A mixture
of azide (0.02 - 0.15 M) and alkyne (1.0 equiv.) in toluene with [Cp*RuCl(PPh3)2] or
[Cp*RuCl(cod)] (5 mol%) was heated (100 °C) with stirring overnight (18 h). The reaction
solvent was removed in vacuo and the remaining residue purified by flash chromatography (1:1
ethyl acetate:n-hexane, then 100 % ethyl acetate).
General Procedure 3. Synthesis of alkyne building blocks 29-32. A mixture of
fluorocarbonylferrocene 35 (1.0 mmol), corresponding alcohol or amine (1.0-1.5 equiv.) and 4-
dimethylaminopyridine (1.0-1.5 equiv.) were prepared in DCM and stirred at rt for 20 h. The
reaction solvent was removed in vacuo and the remaining residue dissolved in ethyl acetate,
washed with brine (2 × 30 mL), dried (MgSO4) and solvent removed. Compounds were used
as substrates in General Procedure 1 without further purification.
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3-(4-Ferrocenyl-1H-1,2,3-triazol-1-yl)benzenesulfonamide (5). The title compound was
prepared from fragments 27 and 34 according to General Procedure 1 and isolated as an orange
6
7
8
1
solid (154 mg, 0.38 mmol, 75 %). Mp 186-188 °C. H NMR (500 MHz, d6-DMSO): δ 9.02 (s,
9
1H, triazole CH), 8.41 (s, 1H, Ar-HA), 8.16-8.17 (m, 1H, Ar-HB or B’), 7.92-7.94 (m, 1H, Ar-HB’
or B), 7.82-7.85 (m, 1H, Ar-HC), 7.58 (s, 2H, SO2NH2), 4.81-4.82 (m, 2H, Cp-H), 4.36-4.38 (m,
2H, Cp-H), 4.09 (s, 5H, unsubstituted Cp-H); 13C {1H} NMR (125 MHz, d6-DMSO): δ 147.0
(triazole C or Ar-C), 145.7 (Ar-C or triazole C), 136.7 (Ar-C), 130.8 (Ar-CHA), 125.2 (Ar-CHB
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B’), 122.7 (Ar-CHB’ or B), 118.5 (triazole CH), 116.8 (Ar-CHC), 74.9 (Cp-C), 69.3
or
(unsubstituted Cp), 68.6 (Cp-CH), 66.5 (Cp-CH); LRMS (ESI+): m/z 407.3 [M+H]+; HRMS
(ESI) calcd for C18H16FeN4O2SH 407.0463, found 407.0450.
3-(5-Ferrocenyl-1H-1,2,3-triazol-1-yl)benzenesulfonamide (6). The title compound was
prepared from fragments 27 and 34 according to General Procedure 2 and isolated as an orange
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solid (86 mg, 0.21 mmol, 42 %). Mp 184-186 °C. H NMR (500 MHz, d6-DMSO): δ 8.11 (s,
1H, triazole CH), 8.07-8.08 (m, 1H, Ar-HB or B’), 7.98 (s, 1H, Ar-HA), 7.82-7.85 (m, 1H,
Ar-HC), 7.75-7.76 (m, 1H, Ar-HB’ or B), 7.60 (br s, 2H, SO2NH2), 4.35-4.36 (m, 2H, Cp-H), 4.26
(s, 2H, Cp-H), 4.12 (s, 5H, unsubstituted Cp-H); 13C {1H} NMR (125 MHz, d6-DMSO): δ
145.4 (Ar-C), 137.5 (triazole C), 136.8 (Ar-C), 132.1 (triazole CH), 130.4 (Ar-CHC), 129.7
(Ar-CHB
B’), 127.0 (Ar-CHB’
B), 123.8 (Ar-CHA), 69.6 (unsubstituted Cp-CH), 69.4
or
or
(Cp-CH), 67.8 (Cp-CH), Cp-C not detected; LRMS (ESI+): m/z 407.3 [M+H]+; HRMS (ESI)
calcd for C18H16FeN4O2SH 407.0463, found 407.0447.
3-(4-Ruthenocenyl-1H-1,2,3-triazol-1-yl)benzenesulfonamide (7). The title compound was
prepared from fragments 28 and 34 according to General Procedure 1 and isolated as an off-
white solid (10 mg, 0.02 mmol, 22 %). Mp 211-212 °C. 1H NMR (500 MHz, d6-DMSO):
δ 8.89 (s, 1H, triazole CH), 8.36 (s, 1H, Ar-HA), 8.11-8.12 (m, 1H, Ar-HB or B’), 7.90-7.92 (m,
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1H, Ar-HB’ or B), 7.80-7.83 (m, 1H, Ar-HC), 7.57 (s, 2H, SO2NH2), 5.22 (s, 2H, Cp-H), 4.73 (s,
2H, Cp-H), 4.53 (s, 5H, unsubstituted Cp-H); 13C {1H} NMR (125 MHz, d6-DMSO): δ 146.3
(triazole C or Ar-C), 145.7 (Ar-C or triazole C), 136.7 (Ar-C), 130.8 (Ar-CHA), 125.2
(Ar-CHB or B’), 122.7 (Ar-CHB’ or B), 118.7 (triazole CH), 116.8 (Ar-CHC), 78.6 (Cp-C), 71.3
(unsubstituted Cp), 70.7 (Cp-CH), 69.2 (Cp-CH); LRMS (ESI¯ ): m/z 447.2 [M-H]-; HRMS
(ESI) calcd for C18H16RuN4O2SH 449.0143, found 449.0123.
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3-(5-Ruthenocenyl-1H-1,2,3-triazol-1-yl)benzenesulfonamide (8). The title compound was
prepared from fragments 28 and 34 according to General Procedure 2 and isolated as an off-
white solid (58 mg, 0.13 mmol, 26 %). Mp 184-186 °C (decomp). 1H NMR (600 MHz, d6-
DMSO): δ 9.41 (s, 1H, triazole CH), 8.49 (s, 1H, Ar-HA), 8.24-8.26 (m, 1H, Ar-HB or B’), 7.95-
7.97 (m, 1H, Ar-HB’ or B), 7.83-7.85 (m, 1H, Ar-HC), 7.59 (s, 2H, SO2NH2), 5.66 (s, 2H, Cp-H),
5.01 (s, 2H, Cp-H), 4.60 (s, 5H, unsubstituted Cp-H); 13C {1H} NMR (150 MHz, d6-DMSO): δ
147.8 (triazole C), 145.7 (Ar-C), 136.3 (Ar-C), 130.8 (Ar-CHC), 126.1 (triazole CH), 125.9
(Ar-CHB
B’), 123.6 (Ar-CHB’ or B), 117.8 (Ar-CHA), 82.5 (Cp-C), 74.1 (Cp-CH), 72.3
or
(Cp-CH), 72.2 (unsubstituted Cp-CH); LRMS (ESI¯ ): m/z 447.3 [M-H]-; HRMS (ESI) calcd
for C18H16RuN4O2SH 449.0143, found 449.0131.
(1-(4-Sulfamoylphenyl)-1H-1,2,3-triazol-4-yl)methyl ferrocenyl-1-carboxylate (9). The title
compound was prepared from fragments 29 and 33 according to General Procedure 1 and
isolated as an orange solid (51 mg, 0.11 mmol, 44 %). Mp 254-255 °C (decomposed). 1H
NMR (500 MHz, d6-DMSO): δ 9.04 (s, 1H triazole CH), 8.16-8.17 (m, 2H, Ar-H), 8.01-8.03
(m, 2H, Ar-H), 7.50 (s, 2H, SO2NH2), 5.40 (s, 2H, CH2), 4.79 (s, 2H, Cp-H), 4.50 (s, 2H,
Cp-H), 4.17 (s, 5H, unsubstituted Cp-H); 13C {1H} NMR (125 MHz, d6-DMSO): δ 171.1
(C=O), 144.6 (triazole C and Ar-C), 139.2 (Ar-C), 128.2 (Ar-CH), 123.8 (triazole CH), 121.1
(Ar-CH), 72.3 (Cp-CH), 70.9 (Cp-C), 70.6 (Cp-CH), 70.3 (unsubstituted Cp-CH), 57.4 (CH2);
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LRMS (ESI+): m/z 489.0 [M+Na]+; HRMS (ESI) calcd for C20H18FeN4O4SNa 487.0034,
found 487.0314.
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2-(1-(4-Sulfamoylphenyl)-1H-1,2,3-triazol-4-yl)ethyl ferrocenyl-1-carboxylate (10). The
title compound was prepared from fragments 30 and 33 according to General Procedure 1 and
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isolated as an orange solid (108 mg, 0.22 mmol, 90 %). Mp 216-217 °C. H NMR (500 MHz,
d6-DMSO): δ 8.86 (s, 1H, triazole CH), 8.11-8.13 (m, 2H, Ar-H), 8.00-8.02 (m, 2H, Ar-H),
3
7.48 (s, 2H, SO2NH2), 4.73 (s, 2H, Cp-H), 4.49 (t, JCH-CH = 5 Hz, 2H, α-CH2), 4.46 (s, 2H,
3
Cp-H), 3.18 (t, JCH-CH = 5 Hz, 2H, β-CH2); 13C {1H} NMR (100 MHz, d6-DMSO): δ 170.5
(C=O), 145.1 (triazole C), 143.6 (Ar-C), 138.6 (Ar-C), 127.5 (Ar-CH), 121.1 (triazole CH),
119.9 (Ar-CH), 71.3 (Cp-CH), 70.5 (Cp-C), 69.7 (Cp-CH), 69.4 (unsubstituted Cp-CH), 62.4
(α-CH2), 25.0 (β-CH2); LRMS (ESI+): m/z 480.0 [M+H]+; HRMS (ESI) calcd for
C21H20FeN4O4SNa 501.0494, found 501.0480.
3-(1-(4-Sulfamoylphenyl)-1H-1,2,3-triazol-4-yl)propyl ferrocenyl-1-carboxylate (11). The
title compound was prepared from fragments 31 and 33 according to General Procedure 1 and
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isolated as an orange solid (119 mg, 0.24 mmol, 96 %). Mp 175-176 °C. H NMR (500 MHz,
d6-DMSO): δ 8.76 (s, 1H, triazole CH), 8.10-8.12 (m, 2H, Ar-CH), 8.00-8.02 (m, 2H, Ar-CH),
7.49 (s, 2H, SO2NH2), 4.75 (s, 2H, Cp-CH), 4.48 (s, 2H, Cp-CH), 4.27 (t, 3JCH-CH = 7.5 Hz, 2H,
3
α-CH2), 4.24 (s, 5H, unsubstituted Cp-CH), 2.91 (t, JCH-CH = 7.5 Hz, 2H, γ-CH2), 2.11 (dt,
3
3JCH-CH = 7 Hz, JCH-CH = 7 Hz, 2H, β-CH2); 13C {1H} NMR (125 MHz, d6-DMSO): δ 170.5
(C=O), 147.7 (triazole C), 143.5 (Ar-C), 138.7 (Ar-C), 127.4 (Ar-CH), 120.5 (triazole CH),
119.9 (Ar-CH), 71.3 (Cp-CH), 70.8 (Cp-C), 69.6 (Cp-CH), 69.5 (unsubstituted Cp-CH), 62.9
(α-CH2), 27.9 (β-CH2), 21.6 (γ-CH2); LRMS (ESI¯ ): m/z 493.2 [M-H]-; HRMS (ESI) calcd for
C22H22FeN4O4SNa 515.0650, found 515.0662.
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(1-(3-Sulfamoylphenyl)-1H-1,2,3-triazol-4-yl)methyl ferrocenyl-1-carboxylate (12). The
title compound was prepared from fragments 29 and 34 according to General Procedure 1 and
isolated as an orange solid (63 mg, 0.14 mmol, 54 %). Mp 200-201 °C. 1H NMR (500 MHz,
d6-DMSO): δ 9.03 (s, 1H, triazole CH), 8.40 (s, 1H, Ar-HA), 8.15-8.17 (m, 1H, Ar-HB or B’),
7.91-7.93 (m, 1H, Ar-HB’ or B), 7.80-7.83 (m, 1H, Ar-HC), 7.56 (br s, 2H, SO2NH2), 5.40 (s, 2H,
CH2), 4.79 (s, 2H, Cp-H), 4.50 (s, 2H, Cp-H), 4.16 (s, 5H, unsubstituted Cp-H);
13C {1H} NMR (125 MHz, d6-DMSO): δ 170.3 (C=O), 145.7 (triazole C), 143.8 (Ar-C), 136.5
(Ar-C), 130.9 (Ar-CHC), 125.5 (Ar-CHB or B’), 123.1 (Ar-CHB’ or B), 123.1 (triazole CH), 117.2
(Ar-CHA), 71.5 (Cp-CH), 70.1 (Cp-C), 69.8 (Cp-CH), 69.5 (unsubstituted Cp-CH), 56.6 (CH2);
LRMS (ESI¯ ): m/z 465.2 [M-H]-.
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2-(1-(3-Sulfamoylphenyl)-1H-1,2,3-triazol-4-yl)ethyl ferrocenyl-1-carboxylate (13). The
title compound was prepared from fragments 30 and 34 according to General Procedure 1 and
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isolated as an orange solid (100 mg, 0.21 mmol, 84 %). Mp 167-168 °C. H NMR (500 MHz,
d6-DMSO): δ 8.86 (s, 1H, triazole CH), 8.38 (s, 1H, Ar-HA), 8.11-8.12 (m, 1H, Ar-HB or B’),
7.89-7.90 (m, 1H, Ar-HB’ or B), 7.80-7.82 (m, 1H, Ar-HC), 7.56 (s, 2H, SO2NH2), 4.73 (s, 2H,
3
Cp-H), 4.50 (t, JCH-CH = 7.5 Hz, 2H, α-CH2), 4.46 (s, 2H, Cp-H), 4.10 (s, 5H, unsubstituted
3
Cp-H), 3.18 (t, JCH-CH = 7.5 Hz, 2H, β-CH2); 13C {1H} NMR (125 MHz, d6-DMSO): δ 170.5
(C=O), 147.8 (triazole C), 145.1 (Ar-C), 136.8 (Ar-C), 130.9 (Ar-CHC), 125.3 (Ar-CHB or B’),
122.7 (Ar-CHB’ or B), 121.2 (triazole CH), 116.9 (Ar-CHA), 71.3 (Cp-CH), 70.6 (Cp-C), 69.7
(Cp-CH), 69.4 (unsubstituted Cp-CH), 62.5 (α-CH2), 25.1 (β-CH2); LRMS (ESI¯ ): m/z 479.3
[M-H]-.
3-(1-(3-Sulfamoylphenyl)-1H-1,2,3-triazol-4-yl)propyl ferrocenyl-1-carboxylate (14). The
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title compound was prepared from fragments 31 and 34 according to General Procedure 1 and
isolated as an orange solid (111 mg, 0.22 mmol, 90 %). Mp 159-161 °C. 1H NMR (500 MHz,
d6-DMSO): δ 8.76 (s, 1H, triazole CH), 8.37 (s, 1H, Ar-HA), 8.09-8.11 (m, 1H, Ar-HB or B’),
7.89-7.91 (m, 1H, Ar-HB’ or B), 7.78-7.82 (m, 1H, Ar-HC), 7.55 (s, 2H, SO2NH2), 4.75 (s, 2H,
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Cp-H), 4.48 (s, 2H, Cp-H), 4.26 (t, JCH-CH = 7.5 Hz, 2H, α-CH2), 4.23 (s, 5H, unsubstituted
3
3
3
Cp-H), 2.91 (t, JCH-CH = 7.5 Hz, γ-CH2), 2.11 (dt, JCH-CH = 6.9 Hz, JCH-CH = 6.9 Hz, 2H, β-
CH2); 13C {1H} NMR (125 MHz, d6-DMSO): δ 170.6 (C=O), 147.7 (triazole C), 145.7 (Ar-C),
136.8 (Ar-C), 130.8 (Ar-CHC), 125.1 (Ar-CHB or B’), 122.7 (Ar-CHB’ or B), 120.5 (triazole CH),
116.9 (Ar-CHA), 71.3 (Cp-CH), 70.8 (Cp-C), 69.7 (Cp-CH), 69.5 (unsubstituted Cp-CH), 63.0
(α-CH2), 27.9 (β-CH2), 21.7 (γ-CH2); LRMS (ESI¯ ): m/z 493.2 [M-H]-.
N-(1-(4-Sulfamoylphenyl)-1H-1,2,3-triazol-4-yl)methyl ferrocenyl-1-carboxamide (15).
The title compound was prepared from fragments 32 and 33 according to General Procedure 1
and isolated as an orange solid (93 mg, 0.20 mmol, 80 %). Mp 181-183 °C. 1H NMR (500
MHZ, d6-DMSO): δ 8.76 (s, 1H, triazole CH), 8.40 (br s, 1H, NH), 8.14-8.16 (m, 2H, Ar-H),
3
8.00-8.02 (m, 2H, Ar-H), 7.50 (s, 2H, SO2NH2), 4.84 (s, 2H, Cp-H), 4.55 (d, JCH-NH = 6 Hz,
2H, CH2), 4.36 (s, 2H, Cp-H), 4.14 (s, 5H, unsubstituted Cp-H); 13C {1H} NMR (125 MHz,
d6-DMSO): δ 169.8 (C=O), 147.6 (triazole C), 144.4 (Ar-C), 139.4 (Ar-C), 128.2 (Ar-CH),
122.0 (triazole CH), 120.8 (Ar-CH), 76.9 (Cp-C), 70.7 (Cp-CH), 70.0 (unsubstituted Cp-CH),
69.0 (Cp-CH), 35.0 (CH2); LRMS (ESI¯ ): m/z 464.3 [M-H]-.
N-(1-(3-Sulfamoylphenyl)-1H-1,2,3-triazol-4-yl)methyl ferrocenyl-1-carboxamide (16).
The title compound was prepared from fragments 32 and 34 according to General Procedure 1
and isolated as an orange solid (90 mg, 0.19 mmol, 19 %). Mp 175-177 °C. 1H NMR (500
MHz, d6-DMSO): δ 8.73 (s, 1H, triazole CH), 8.38-8.39 (m, 2H, Ar-HA and NH), 8.13-8.14 (m,
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1H, Ar-HB or B’), 7.89-7.90 (m, 1H, Ar-HB’ or B) 7.78-7.81 (m, 1H, Ar-HC), 7.55 (s, 2H,
4
5
3
SO2NH2), 4.83 (s, 2H, Cp-H), 4.54 (d, JCH-NH = 5 Hz), 4.35 (s, 2H, Cp-H), 4.13 (s, 5H,
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8
13
unsubstituted Cp-H); C {1H} NMR (125 MHz, d6-DMSO): δ 169.1 (C=O), 146.8 (triazole C
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or Ar-C), 145.7 (Ar-C or triazole C), 136.7 (Ar-C), 130.9 (Ar-CHC), 125.3 (Ar-CHB or B’), 122.9
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(Ar-CHB’
B), 121.2 (triazole CH), 117.0 (Ar-CHA), 76.1 (Cp-C), 70.0 (Cp-CH), 69.3
or
(unsubstituted Cp-CH), 68.3 (Cp-CH), 34.2 (CH2); LRMS (ESI¯ ): m/z 464.3 [M-H]-.
N-(4-Sulfamoylphenyl) ferrocenyl-1-carboxamide (17). A solution of 35 (232 mg, 1.0
mmol) in DCM (5 mL) was added to a solution of 4-aminobenzenesulfonamide (1.1 equiv.,
190 mg, 1.1 mmol) in DMF (0.35 mL) and pyridine (0.1 mL) and the reaction stirred for 5 days
at rt. The reaction mixture was next diluted with ethyl acetate (20 mL) and washed with 2M
HCl (20 mL) and brine (2 × 20 mL). The organic fraction was dried over MgSO4 and
evaporated in vacuo before purification by flash chromatography (1:9 methanol:DCM to give
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the title compound as an orange solid (377 mg, 0.98 mmol, 98 %). Mp 256-258 °C. H NMR
(500 MHz, d6-DMSO): δ 11.26 (s, 1H, NH), 7.58-7.61 (m, 2H, Ar-H), 6.59-6.62 (m, 2H,
Ar-H), 6.08 (s, 2H, SO2NH2), 4.92 (s, 2H, Cp-H), 4.43 (s, 2H, Cp-H), 4.00 (s, 5H,
unsubstituted Cp-H); 13C {1H} NMR (125 MHz, d6-DMSO): δ 168.0 (C=O), 153.5 (Ar-C),
129.9 (Ar-CH), 112.2 (Ar-CH), 109.5 (Ar-C), 73.0 (Cp-C), 71.5 (Cp-CH), 69.5 (unsubstituted
Cp-CH), 69.1 (Cp-CH); LRMS (ESI¯ ): m/z 383.2 [M-H]-.
N-(3-Sulfamoylphenyl) ferrocenyl-1-carboxamide (18). The title compound was synthesized
from 35 and 3-aminobenzenesulfonamide similarly to the method described for 17 (orange
1
solid, 266 mg, 0.69 mmol, 69 %). Mp 183-185 °C. H NMR (500 MHz, d6-DMSO): δ 9.73 (s,
1H, NH), 8.26 (s, 1H, Ar-HA), 7.95 (m, 1H Ar-HC), 7.51 (s, 2H, Ar-HB and Ar-HB’), 7.37 (s,
2H, SO2NH2), 5.03 (apparent s, 2H, Cp-H), 4.48 (apparent s, 2H, Cp-H), 4.22 (s, 5H,
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unsubstituted Cp-H); 13C {1H} NMR (125 MHz, d6-DMSO): δ 169.1 (C=O), 145.0 (Ar-C),
140.2 (Ar-C), 130.0 (Ar-CHC), 123.4 (Ar-CHB or B’), 120.6 (Ar-CHB’ or B), 117.6 (Ar-CHA), 76.3
(Cp-C), 71.2 (Cp-CH), 70.0 (unsubstituted Cp-CH), 69.2 (Cp-CH); LRMS (ESI¯ ): m/z 383.5
[M-H]-.
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4-(3-Ferrocenylureido)benzenesulfonamide (19). Compound 36 (128 mg, 0.5 mmol) was
heated (100 °C) in toluene (5 mL) for 1 h then evaporated in vacuo. The resulting oil was
redissolved in DCM, and a solution of 4-aminobenzenesulfonamide (1.1 equiv., 95 mg, 0.55
mmol) in DMF (1 mL) added before stirring at 50 °C for 18 h. The reaction mixture was then
diluted in ethyl acetate and washed using HCl (2.0 M, 20 mL), aqueous NaHCO3(sat) (20 mL),
and brine (2 × 20 mL). The organic phase was dried (MgSO4) and evaporated in vacuo before
further purification by flash chromatography on silica gel (3:2 ethyl acetate:n-hexane).
Evaporation of the eluant resulted in the isolation of the title compound as an orange solid (30
1
mg, 0.075 mmol, 15 %). Mp 268-269 °C (decomp.). H NMR (500 MHz, d6-DMSO): δ 8.85
(s, 1H, NH), 7.96 (s, 1H, NH), 7.71-7.72 (m, 2H, Ar-H), 7.58-7.60 (m, 2H, Ar-H), 7.17 (s, 2H,
NH2), 4.53 (s, 2H, Cp-H), 4.15 (s, 4H, Cp-H), 3.97-3.98 (m, 2H, Cp-H); 13C {1H} NMR (125
MHz, d6-DMSO): δ 152.5 (C=O), 143.0 (Ar-C), 136.6 (Ar-C), 126.7 (Ar-CH), 117.0 (Ar-CH),
96.1 (Cp-C), 68.6 (unsubstituted Cp-CH), 63.6 (Cp-CH), 60.7 (Cp-CH); LRMS (ESI¯ ): m/z
398.2 [M-H]-.
3-(3-Ferrocenylureido)benzenesulfonamide (20). Compound 36 (179 mg, 0.7 mmol) was
heated (100 °C) in toluene (5 mL) for 1 h then evaporated in vacuo. The resulting oil was
dissolved in DCM, and a solution of 3-aminobenzenesulfonamide (1.1 equiv., 132 mg, 0.77
mmol) in DMF (1 mL) added before stirring at 50 °C for 18 h. The reaction mixture was then
diluted in ethyl acetate and washed using HCl (2.0 M, 20 mL), aqueous NaHCO3(sat) (20 mL),
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and brine (2 × 20 mL). The organic phase was dried (MgSO4) and evaporated in vacuo before
further purification by flash chromatography on silica gel (3:2 ethyl acetate:n-hexane).
Evaporation of the eluant resulted in the isolation of the title product as an orange solid (74 mg,
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0.19 mmol, 26 %). Mp 201-202 °C. H NMR (500 MHz, d6-DMSO): δ 8.78 (s, 1H, NH), 8.08
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(s, 1H, Ar-HA), 7.88 (s, 1H, NH), 7.51-7.52 (m, 1H, Ar-HB or B’), 7.43-7.46 (m, 2H, Ar-HC),
7.39-7.41(m, 1H, Ar-HB’ or B), 4.54 (apparent s, 2H, Cp-H), 4.15 (s, 5H, unsubstituted Cp-H),
3.97 (s, 2H, Cp-H); 13C {1H} NMR (125 MHz, d6-DMSO): δ 152.6 (C=O), 144.6 (Ar-C), 140.3
(Ar-C), 129.2 (Ar-CHC), 120.7 (Ar-CHB or B’), 118.4 (Ar-CHB’ or B), 114.7 (Ar-CHα), 96.4
(Cp-C), 68.6 (unsubstituted Cp-CH), 63.6 (Cp-CH), 60.5 (Cp-CH); LRMS (ESI¯ ): m/z 398.3
[M-H]-.
4-(4-Phenyl-1H-1,2,3-triazol-1-yl)benzenesulfonamide (21). The title compound was
prepared from phenyl acetylene and azide 33 according to General Procedure 1 and isolated as
a bright yellow solid (251mg, 0.84 mmol, 84 %). Mp 284-285 °C. 1H NMR (500 MHz,
d6-DMSO): δ 9.41 (s, 1H triazole CH), 8.17-8.19 (m, 2H, Ar-H), 8.06-8.08 (m, 2H, Ar-H),
7.95-7.97 (m, 2H, Ar-Hα), 7.52 (br s, 4H, Ar-Hβ and SO2NH2), 7.39-7.42 (m, 1H, Ar-Hγ); 13C
{1H} NMR (125 MHz, d6-DMSO): δ 147.6 (Ar-C), 143.8 (triazole C), 138.6 (Ar-C), 129.9
(Ar-C), 129.0 (Ar-CHβ), 128.4 (Ar-CHα), 127.5 (Ar-CH), 125.4 (Ar-CHγ), 120.2 (Ar-CH),
119.8 (triazole CH); LRMS (ESI¯ ): m/z 299.4 [M-H]-.
3-(4-Phenyl-1H-1,2,3-triazol-1-yl)benzenesulfonamide (22). The title compound was
prepared from phenyl acetylene and azide 34 according to General Procedure 1 and isolated as
bright yellow solid (254 mg, 0.85 mmol, 85 %). Mp 283-284 °C. 1H NMR (500 MHz, d6-
DMSO): δ 9.43 (s, 1H, triazole CH), 8.44 (s, 1H, Ar-HA), 8.17-8.19 (m, 1H, Ar-HB or B’), 7.94-
7.98 (m, 3H, Ar-Hα and Ar-HB’ or B), 7.84-7.87 (m, 1H, Ar-HC), 7.58 (br s, 2H, SO2NH2),
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