Bioorganic & Medicinal Chemistry Letters 29 (2019) 1454–1458

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Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier.com/locate/bmcl

Structure guided drug design to develop kallikrein 5 inhibitors to treat T

Netherton syndrome
Ann L. Walkera, , Ryan P. Binghama, Emma V. Edgara, Alan Ferriea, Duncan S. Holmesa,
⁎

John Liddlea, Oxana Polyakovaa, Monika Rellaa, Kathrine J. Smitha, James H. Thorpea,
Yichen Wangb, Gemma V. Whitea, Robert J. Younga, Alain Hovnanianb
a
GlaxoSmithKline R&D, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK
b
INSERM UMR1163 Laboratory of Genetic Skin Diseases, Imagine Institute and Université Paris Descarte – Sorbonne Paris Cité, Paris, France

ARTICLE INFO ABSTRACT

Keywords: The connection between Netherton syndrome and overactivation of epidermal/dermal proteases particularly
KLK1 KLK5 has been well established. To treat sufferers of this severe condition we wished to develop a topical KLK5
KLK5 inhibitor in order to normalise epidermal shedding and reduce the associated inflammation and itching. In this
KLKB1 paper we describe structure-based optimisation of a series of brightly coloured weak KLK5 inhibitors into col-
LEKTI
ourless, non-irritant molecules with good KLK5 activity and selectivity over a range of serine proteases.
Netherton syndrome
SPINK5

Netherton syndrome (NS) was first described in 1964 by Wilkinson,
Curtis and Hawk1 as being characterised by trichorrhexis invaginata
(bamboo hair), congenital ichthyosiform erythroderma and atopic
diathesis. The cause of this debilitating syndrome has since been
identified as mutations in serine protease inhibitor, Kazal-type 5
(SPINK5),2 which have premature termination codons inserted into the
gene. When homozygous, these mutations result in reduced levels or
absence of serine protease inhibitor lympho-epithelial Kazal-type re-
lated inhibitor (LEKTI) in the skin and other lympho-epithelial tissues.3
The activity of serine protease Kallikrein 5 (KLK5) in the stratum cor-
neum, which is normally tightly regulated by LEKTI,4 is increased
leading to abnormal desquamation.5,6
To treat Netherton syndrome patients we wished to identify potent
KLK5 inhibitors which were suitable for topical application. When we
started this work only a few weak examples of non-peptidic KLK5 in-
hibitors7 had been published although additional molecules have since
been identified.8–11
In our previous paper12 we described optimisation of an initial
phenolic hit against KLK5 to a benzimidazole 1 (Fig. 1) with significant
potency and some selectivity over a related protease Kallikrein 1
(KLK1). However, both 1 and the other examples of this series were
brightly coloured due to the extended chromophore and were therefore
unsuitable for topical application. We were interested in identifying
alternatives to the carboxamide linker, with the intention of obtaining
colourless KLK5 inhibitors. Moving from 1 to an methylamine linker 2
(Fig. 1) gave a colourless molecule with equivalent KLK5 inhibition
(Table 1),13 although we lost the selectivity over KLK1 (Table 1)13
Crystallography of 2 bound to a KLK5 surrogate KLK6 protein con-
struct14 (Fig. 2) showed that the benzamidine bound into the KLK5 S1
pocket as we had seen with other molecules.12 However the benzimi-
dazole now occupied part of the S2 binding site instead of S1′, forming a
face to face π-stacking interaction with His57 on the side distal from the
oxyanion hole. The N-methyl of 2 occupied a space between His99 and
the methylene of Trp215 and the anilinic NH is solvent exposed. Two
positions for increasing KLK5 inhibition were suggested by this binding
mode, substitution or modification of the fused phenyl of the benzi-
midazole and optimisation of the N-methyl group.
Replacement of the benzimidazole N-methyl with larger groups
gave compounds of little interest as these had higher levels of KLK1
than KLK5 inhibition (data not shown); however, removal of the N-
methyl 3, somewhat surprisingly, resulted in increased potency and
ligand efficiency (Table 1). By crystallography, density could be seen
for two slightly different binding modes (Fig. 3). In both, the benzi-
midazole had rotated relative to His57 moving deeper into the S2
pocket and allowing formation of a hydrogen bond between the back-
bone carbonyl of Ser214 and the benzimidazole NH (Fig. 3). From the

Abbreviations: KLK1, kallikrein 1; KLKB1, kallikrein B1; KLK5, kallikrein 5; KLK7, kallikrein 7; KLK8, kallikrein 8; LEKTI, lympho-epithelial kazal-type-related
inhibitor; NS, Netherton syndrome; SPINK5, serine protease inhibitor kazal-type 5 gene
⁎
Corresponding author.
E-mail address: Ann.L.Walker@gsk.com (A.L. Walker).

https://doi.org/10.1016/j.bmcl.2019.04.022
Received 18 February 2019; Received in revised form 10 April 2019; Accepted 12 April 2019
Available online 12 April 2019
0960-894X/ © 2019 Elsevier Ltd. All rights reserved.
A.L. Walker, et al. Bioorganic & Medicinal Chemistry Letters 29 (2019) 1454–1458

Fig. 1. Initial modifications to previously published KLK5 inhibitor 1, gave colourless molecules with an altered KLK5 binding mode (2–9).

Table 1
KLK5 and KLK1 inhibition.
Example number KLK5 pIC5013 KLK5 KLK1 pIC5013 KLK1
(n) LE/LLEa (n) LE/LLEa

1 4.9 (10) 0.30/3.2 3.7 (5) 0.23/2.0

2 4.8 (12) 0.31/3.7 5.2 (11) 0.34/4.1
3 4.7 (8) 0.32/3.6 4.4 (8) 0.30/3.3
4 4.9 (7) 0.30/3.5 4.5 (7) 0.28/3.1
5c 5.0 (18) 0.32/3.1 5.7 (10) 0.37/3.8
6d 5.0 (7) 0.32/3.4 5.2 (7) 0.34/3.6
7c 3.9 (8) 0.31/4.4 3.4 (8) 0.27/3.9
8c 4.6 (9) 0.39/5.1 4.2 (8) 0.36/4.7
9 5.2 (9) 0.32/3.6 4.6 (4) 0.28/3.0

a
LE = pIC50 * 1.37/nheavy and LLE = pIC50 − ChromLogD.
c
HCl salt.
d
Formic acid salt.

Fig. 3. 3 bound to the active site of the KLK6. Removal of the N methyl allows 3
to adopt conformations where the imidazole NH can form a hydrogen bond to
the backbone carbonyl of Ser214. Two conformations of the linker are evident
with the NH either H-bonded to Ser195 or solvent exposed.

Fig. 2. 2 bound to the KLK6 single mutant (I218Y) active site with the ben-
zamidine bound to Asp189, Ser190 sidechains and also to the backbone amide
of Ser190 in the bottom of the S1 pocket. The benzimidazole π-stacks against
His57 and occupies parts of the S2 pocket. All residue numbering corresponds
to the targeted KLK5 enzyme.
density, the phenyl of the benzamidine and linker occupy two slightly
different positions, one with the anilinic NH H-bonded to the hydroxyl
of Ser195 at the rear of the binding pocket and the other with the NH
rotated 180° and pointing out into solvent.
Substitution of the 5-position of the benzimadole was tolerated for a
range of substituents including electron donating and withdrawing
groups 4, 5 and 6 (Fig. 1), but there was little evidence of SAR in this
region for KLK5 (Table 1), which was consistent the crystallographic
data where this area is largely solvent exposed.
Conversion of the N-methyl benzimidazole 2 to an N-methyl imi-
dazole 7 resulted in a reduction in potency with ligand efficiency (LE)
maintained and an increase in LLE confirming the largely lipophilic
nature of the interaction of the fused phenyl. However, the NH imi-
dazole 8, resulted in a molecule with similar potency to 3 and therefore
an increase in both LE and LLE, suggesting that any potency loss from
removal of the fused phenyl is outweighed by formation of a hydrogen
bond to Ser214.
Moving to the 5-phenylimidazo-2-yl 9 resulted in a ∼3-fold im-
provement in KLK5 inhibition compared to 3 (Table 1). In

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A.L. Walker, et al. Bioorganic & Medicinal Chemistry Letters 29 (2019) 1454–1458

a significant proportion of the skin of NS patients and therefore parti-

Fig. 4. 9 bound to the KLK6. In contrast to 3 only a single binding mode is now
seen, with the anilinic NH forming a hydrogen bond to the hydroxyl of Ser195.
crystallography, only a single binding mode was observed for 9 with the
anilinic NH H-bonded to the hydroxyl of Ser195, the imidazole NH
formed an H-bond with the carbonyl of Ser214 and the phenyl in
contact with His57 (Fig. 4).
Examples 2 to 9 all showed similar levels of inhibition at both KLK5
and at KLK1. We had used the related serine protease KLK1 as a primary
selectivity assay in order to have an indicator of potential protease
selectivity issues. Inhibition of KLK1 would be undesirable due to its
role in blood pressure regulation.15 Although topical use of a KLK5
protease inhibitor might be considered unlikely to cause systemic off-
target toxicity; we were aware that any drug is likely to be applied over
cularly in neonates there is potential for significant systemic exposure;
on this basis, we desired to have as selective a profile as possible.
In order to improve KLK5 inhibition and selectivity, we decided to
re-explore the S3 binding pocket (Table 2). From the crystal structures
this looked to be accessible from substitution of the phenyl ring ad-
jacent to the amidine. An ether linker was chosen, both for ease of
synthesis and to avoid the amidine being twisted further out of the
plane of the phenyl ring (9 torsion angle ∼28°). The S3 binding cleft is
bounded on one side by Tyr218 and on the other by the amide side-
chain of Gln192, so we reasoned that groups able to form π/π inter-
actions and those with a polar functionality might both be accom-
modated. Gratifyingly, addition of an ethoxy group 10 led to an
approximately 20-fold increase in KLK5 potency compared with 9 and a
20-fold selectivity window over KLK1. Further expansion to an isobutyl
11 resulted in reduced inhibition, possibly due to the extra bulk, as a
smaller methylcyclopropyl 12 retained better potency and demon-
strated 60-fold selectivity over KLK1. Homologation of 12 to the
ethylcyclopropyl 13 was associated with a gain in potency, back to a
similar level to 10 and this molecule also showed improved selectivity
over KLK1 of ∼250-fold. Polar sidechains 14 and 15 were significantly
weaker than 10; however moving to a benzyloxy group 16 gave a
compound with an IC50 < 10 nM and ∼300-fold selectivity over KLK1
whist retaining good ligand efficiencies. Substitution of the benzyloxy
(17, 18 and 19) made little difference to the activity or selectivity over
KLK1. Heteroaryl replacements of the phenyl (20–23) were well tol-
erated, with the ring size and positioning of the heteroatoms making
little difference to KLK5 binding. The pyrid-3-yl 21 appeared to have a
slight advantage in terms of selectivity over KLK1. Replacing the cy-
clopropyl of 12 or benzylic moiety with a primary amide gave 25,
which showed weaker KLK5 inhibition with unchanged LE and increase
in lipophilic ligand efficiency (LLE). N-substitution increased KLK5 in-
hibition with the N-methyl 25 gaining about 3-fold and the N-

Table 2
Effect of modification to the S3 binding group on KLK5 potency and selectivity over KLK1.

Example number R KLK5 pIC50 (n) KLK5 LE/LLE KLK1 pIC50 (n) KLK1 LE/LLE

a
10 Et 6.5 (7) 0.35/4.3 5.2 (6) 0.28/3.0
11a iBu 5.2 (9) 0.26/2.0 4.1 (6) 0.21/0.9
12a CH2cPr 6.1 (9) 0.31/3.4 4.3 (6) 0.22/1.6
13 (CH2)2cPr 6.7 (10) 0.33/3.5 4.3 (3) 0.21/1.1
14b (CH2)2OH 5.4 (9) 0.28/4.6 4.1 (5) 0.21/3.3
15 (CH2)2NMe2 4.8 (7) 0.23/3.0 4.1 (6) 0.20/2.3
16 Bn 7.1 (4) 0.32/3.6 4.6 (2) 0.21/1.1
17 CH2(2-Cl-phenyl) 7.5 (9) 0.33/3.3 < 5.6 (8)
18 CH2(3-Cl-phenyl) 7.1 (9) 0.31/2.9 < 5.3 (8)
19 CH2(4-Cl-phenyl) 7.2 (9) 0.32/3.0 < 5.3(8)
20b CH2(pyrid-2-yl) 6.9 (9) 0.31/4.9 4.7 (6) 0.21/2.7
21 CH2(pyrid-3-yl) 7.1 (10) 0.32/5.1 4.2 (5) 0.19/2.2
22b CH2(pyrimid-2-yl) 7.1 (9) 0.32/6.1 4.4 (6) 0.20/3.4
23 CH2(oxazol-2-yl) 7.0 (9) 0.33/5.9 4.9 (6) 0.23/3.8
24a (CH2)CONH2 6.1 (9) 0.31/5.9 < 4.3 (6)
25a (CH2)CONHMe 6.6 (9) 0.32/6.1 4.5 (6) 0.22/4.0
26 (CH2)CONHcPr 7.1 (8) 0.32/6.1 4.8 (4) 0.22/3.8
27 (CH2)CONH(CH2)cPr 7.4 (10) 0.32/6.0 < 5.2 (10)

a
Formic acid salt.

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A.L. Walker, et al. Bioorganic & Medicinal Chemistry Letters 29 (2019) 1454–1458

front of the binding site (Fig. 5).

In order to better understand the impact of the S3 binding group on
serine protease selectivity, 21 was profiled against a set of proteases
which included both members of the kallikrein family and other serine
proteases16 which could have potential to cause undesirable side ef-
fects. A ∼100-fold or greater selectivity window was seen against all
the panel proteases except kallikrein 8 (KLK8) which is also part of the
protease cascade in skin and where 10-fold selectivity was achieved
(Table 3).
Although all molecules from this series were colourless, in order to
confirm their suitability of for topical dosing, we measured the ultra-
violet spectra of 21 and 27 in methanol. Both compounds had ab-
sorption with maxima > 290 nm and with molar extinction
coefficients > 1000 a.u.L mol−1 cm−1, which is considered a toxicity
risk, due to the potential for skin damage on exposure to sunlight. While
this uv absorption could potentially be managed by addition of a
sunscreen to the final formulation we decided to progress both com-
pounds to a cellular uv toxicity assay to assess whether the uv ab-
sorption profile was truly indicative of phototoxicity. In this experi-
ment, which measures induction of a phototoxic effect in vitro in a
Neutral Red uptake assay using Balb/c 3T3 fibroblasts +/− UV-A (5 J/
cm2) for 1 h, neither 21 or 27 showed toxicity in excess of 50% of the
vehicle control at concentrations up to 100 μg/mL (maximum re-
commended concentration according to current regulatory guidelines)
and therefore these molecules are considered to be non-phototoxic.
From the start we had been concerned about the highly basic ben-
zamidine moiety (pKa > 11.5) for two reasons, firstly that this might
affect skin permeation, but secondly that this might cause irritation to
the already damaged skin of NS patients. All attempts to replace the
benzamidine functionality with less basic moieties had, however, given
molecules with significantly lower levels of KLK5 binding (data not
shown). We were able to assess the potential for irritation by profiling
21 using an EpiDerm RHE culture (MatTek). This assay measures cell
viability over 24 h and also the levels of IL-1α which is used as a marker
of inflammation. Compared with assay standard 1% Triton X-100 which
is a moderate to mild irritant, 21 was classed as a non-irritant.
Fig. 5. (a) 21 bound to the KLK6 triple mutant surrogate for KLK5. The pyridine In order to be able to formulate and store our KLK5 inhibitor as a
is located in the S1/S3 groove between Tyr218 and Gln192 making an edge to cream or a gel for topical application, we wished to understand the
face π-π stacking interaction with Tyr218. Orientation of the pyridine nitrogen solubility and stability profiles. 21 and 27 were selected as diverse and
is not defined. (b) 27 bound to the KLK6 triple mutant surrogate for KLK5. 27 potent examples of the series. The aqueous solubility of these two
adopts two binding modes, in one (white) the amide functionality of 27 is or-
compounds was assessed at 24 h over the same pH range. The solubility
ientated to form an H-bond between the carbonyl and the side chain of Glu192
of 21 was good with almost 1 mg/ml in water and in Britton-Robinson
which has moved forwards to obscure the S3 pocket. The cyclopropyl methyl
folds across the front of the binding pocket and makes contact with His99.
buffer at pH4, but lower in Britton-Robinson buffer at pH6 (0.29 mg/
mL) and pH8 (0.18 mg/mL). 27 had higher aqueous solubility at all
pHs, with an aqueous concentration of ∼3 mg/mL in water and 0.7 mg/
Table 3 mL in Britton-Robinson buffer at pH 8. The stability of 21 and 27 was
Protease selectivity panel.16
profiled at 40 °C across a range of pH suitable for application to skin
Protease 21 pIC50 (n = 2) (pH 4–pH 8) and under standard and oxidative conditions17 21 and 27
both showed good stability at all pHs under non-oxidative conditions
KLK5 7.1
KLK1 4.2
(T1/2 > 1000 h). Although degradation was observed under oxidative
KLKB1 5.2 conditions at pH 6 and pH 8 (21 T1/2 27.8 h and 10.1 h, 27 T1/2 26.7 h
KLK7 < 4.0 and 14.0 h respectively) this was not considered to problematic as
KLK8 6.1 control of any potential oxidative degradants in a product could be
KLK14 5.4
achieved through the use of protective packaging and antioxidant ex-
Factor Xa < 4.0
Matriptase 4.6 cipients. Further studies will be required to understand the solubility
Neutrophil elastase < 4.0 and stability of the chosen compound in a variety of excipients in order
Thrombin 4.2 to choose the most appropriate formulation.
Urokinase < 4.0 The synthesis of the molecules described was achieved through well
understood synthetic transformations and typical conditions are given
in the schemes below for synthesis of 21 (Scheme 1) and 27 (Scheme 2).
cyclopropyl 26 and N-methylcyclopropyl 27 achieving similar potency
Initial introduction of the S3 binding group was accomplished either
to the benzyloxy derivatives and maintaining a similar LE and improved
through either SnAr displacement with the relevant alcohol on 2-fluoro-
LLE. Interestingly although 27 has similar potency and KLK1 selectivity
4-nitrobenzonitrile or alkylation chemistry using the bromide with 2-
to other S3 binding groups which had been confirmed to bind in the S3
hydroxy-4-nitrobenzonitrile. This was followed by reduction of the
pocket; crystallography shows that the amide substituent doesn’t oc-
nitro group with either palladium on carbon and hydrogen gas or iron
cupy the S3 pocket, but instead folds back to contact His99 across the
and ammonium chloride or Zinc dust and ammonium chloride to give

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A.L. Walker, et al.
Scheme 1. (i) K2CO3, acetone. 51% yield. (ii) Fe, NH4Cl, EtOH, H2O. 70% yield
(iii) NaCNBH3, AcOH, MeOH. 23% yield. (iv) MeOH, HCl. Product not purified.
(v) NH3, MeOH. 57% yield over 2 steps.
Scheme 2. (i) K2CO3, DMF. 98% yield. (ii) Pd/C, H2, MeOH. 46% yield or Zn
dust, NH4Cl, H2O/MeOH, 0 °C. 97% yield. (iii) NaCNBH3, AcOH, MeOH. 47%
yield. (iv) cyclopropylmethylamine, MeOH. 92% yield. (v) NH2OH·HCl,
NaHCO3, MeOH. 73% yield. (vi) Zn, NH4Cl, MeOH. 7% yield.
the aniline. The imidazole group was introduced using the appropriate
aldehyde and the aniline formed previously, by reductive amination
using sodium cyanoborohydride. In the case of 27 and analogues, the
ester had been introduced in step 1 instead of the amide to enable
synthetic diversity and was converted to the target amide at this stage.
Conversion of the nitrile into the amidine, which may be performed
either as a one-pot or two-pot conversion using standard conditions
yielded the target compounds. The reaction can be slow due to steric
hinderance by the adjacent group. In each case the amidine was in-
troduced at the final step due to its polarity and the challenge of per-
forming other chemistry with this group present.
Synthesis of 2118–20
Scheme 1
Synthesis of 2721
Scheme 2
In conclusion, from an initial low potency and highly coloured lead
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Bioorganic & Medicinal Chemistry Letters 29 (2019) 1454–1458
1, we have identified a series of compounds with IC50’s of 100 nM or
less at KLK5 and > 100-fold selectivity over a panel other serine pro-
teases, except for epidermal protease KLK8. Examples of the series have
been further profiled in developability studies, shown to have low risk
of phototoxicity and irritancy and to have stability and solubility pro-
files suitable for further development towards a topical formulation.
Acknowledgements
The authors thank Pam Nassau for preparation of the KLK5 and
KLK1 proteins and the chemistry teams who prepared the majority of
the compounds.
This work was funded by GlaxoSmithKline.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2019.04.022.
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inhibitor, cause Netherton syndrome. Nat Genet. 2000;25:141–142.
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Dermatol. 2004;122:1235–1244.
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13. Two functional protease assays (KLK1 and KLK5), utilising fluorogenic tri-peptide sub-
strates, were run in a kinetic format, at substrate kM measuring inhibitor potency (pIC50).
IC50 determinations performed as a minimum in duplicate against recombinant human
KLK5 using an 11 point dose response curve. IC50 values are reported as an average of two
or more experiments, from hard values where available. Full assay details available in the
Supplementary data.
14. Thorpe JH, Edgar EV, Smith KJ, et al.. Evaluation of a crystallographic surrogate for
Kallikrein 5 in the discovery of novel inhibitors for Netherton Syndrome (submitted for
manuscript).
15. Madeddu P, Emanueli C, El-Dahr S. Mechanisms of disease: the tissue kallikrein-kinin
system in hypertension and vascular remodeling. Nat. Clin. Pract. 2007;3:208.
16. Protease panel assays – details available in Supplementary data.
17. Compound samples were prepared in each buffer (Britton Robinson, pH4, 6 and 8 and SGF)
and 1:1 water/acetonitrile at concentrations of 0.05 mg/ml. Hydrogen peroxide was added
to a set of samples at 20 molar% for each pH condition. The samples were analysed 6 times
over a period of 24 h to check for degradation and calculate half life.
18. General experimental details: All commercial reagents and solvents were obtained from
commercial sources and used without further purification. 1H NMR spectra, chemical shifts
are given in ppm (δ) relative to tetramethylsilane (TMS) as an internal standard.
19. Compound purity: Compounds are > 95% purity by LCMS and NMR unless otherwise
stated. Compound 15: 92% by LCMS, ≥95% by NMR; Compound 20: 93% by LCMS, ≥95%
by NMR.
20. Characterisation for 21: LCMS MH+ 399, retention time 4.08 min, 96.4%. NMR D6 DMSO
includes δH 8.71 (m, 1H), 8.55 (dd, J = 4.8 Hz, J = 1.3 Hz, 1H), 7.89 (d, J = 7.9 Hz, 1H),
7.74 (d, J = 7.5 Hz, 2H), 7.50 (br s, 1H), 7.43–7.37 (m, 2H), 7.32 (t, J = 7.7 Hz, 2H), 7.24
(br m, 1H), 7.17 (m, 1H), 6.65 (s, 1H), 6.43 (dd, J = 8.7 Hz, J = 1.6 Hz, 2H), 5.20 (s, 2H),
4.40 (d, J = 5.0 Hz, 2H).
21. Characterisation for 27: LCMS MH+ 419, retention time 3.90 min, 97.2%. NMR D6 DMSO
includes δH 8.31 (s, 1H), 7.73 (m, 2H), 7.47 (s, 1H), 7.33 (m, 3H), 7.17 (m, 1H), 6.71 (br s,
1H), 6.39 (d, J = 1.8 Hz, 1H), 6.34 (dd, J = 8.6 Hz, J = 2.0 Hz, 1H), 4.56 (s, 2H), 4.33 (d,
J = 3.1 Hz, 2H), 2.99 (d, J = 6.6 Hz, 2H), 0.89 (m, 1H), 0.39 (m, 2H), 0.15 (m, 2H).