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Design and development of a series of borocycles as selective, covalent kallikrein 5 inhibitors
Bioorganic & Medicinal Chemistry Letters 29 (2019) 126675
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Design and development of a series of borocycles as selective, covalent kallikrein 5 inhibitors
T
Ann L. Walkera, Alexis Denisa, Ryan P. Binghama, Anne Bouillota, Emma V. Edgara, Alan Ferriea, ⁎ Duncan S. Holmesa, Alain Larozea, John Liddlea, , Marie-Helene Foucheta, Alexandre Moquettea, Pam Nassaua, Andrew C. Pearcea, Oxana Polyakovaa, Kathrine J. Smitha, Pamela Thomasa, James H. Thorpea, Lionel Trotteta, Yichen Wangb, Alain Hovnanianb a b
GlaxoSmithKline R&D, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK INSERM UMR1163 Laboratory of Genetic Skin Diseases, Imagine Institute and Université Paris Descarte-Sorbonne Paris Cité, Paris, France
ARTICLE INFO
ABSTRACT
Keywords: KLK1 KLK5 KLKB1 LEKTI Netherton syndrome SPINK5
The connection between Netherton syndrome and overactivation of epidermal/dermal proteases, particularly Kallikrein 5 (KLK5) has been well established and it is expected that a KLK5 inhibitor would improve the dermal barrier and also reduce the pain and itch that afflict Netherton syndrome patients. One of the challenges of covalent protease inhibitors has been achieving selectivity over closely related targets. In this paper we describe the use of structural insight to design and develop a selective and highly potent reversibly covalent KLK5 inhibitor from an initial weakly binding fragment.
Netherton Syndrome (NS) is a devastating rare congenital skin disease characterised by ichthyosis, bamboo hair and failure to thrive early in life.1 Newborns afflicted by this condition are at a significant risk of death due a leaky skin barrier which can lead to dehydration and infection and older children and adults suffer from red and scaly skin, itching and allergic manifestations. The cause of NS has been identified as loss-of-function mutations of the SPINK5 gene.2 Bi-allelic SPINK5 mutations result in reduced or no expression of serine protease inhibitor lympho-epithelial Kazla-typerelated inhibitor (LEKTI). LEKTI is normally expressed in the most differentiated layer of the epidermis and regulates the activity of several serine proteases including Kallikrein 5 (KLK5); its ability to inhibit KLK5 is pH dependant and is lost in the more acidic outer layers of the stratum corneum allowing KLK5 driven cleavage of desmosomal proteins and loss of the outer-most skin layer.3 In NS individuals, the unregulated activity of KLK5 in the lower layers of the stratum corneum results in premature desquamation. The unregulated protease activity within the epidermis also activates PAR-2 and triggers inflammatory reactions, leading to erythroderma and to the profound itching which is characteristic of NS.4
There is no curative therapy for NS and current treatment is limited to intensive nursing care of new-borns, frequent application of emollients and treatment with anti-infectives when necessary. We therefore desired to identify topical inhibitors of KLK5 as these would be expected to reduce the abnormal protease activity leading to improved skin barrier function and reduced allergy for NS individuals. A number of KLK5 inhibitors have been published,5,6 including both covalent examples7,8 and non-covalent series,9–12 however, we wished to identify more potent and soluble examples which would be suitable for topical application in a gel or cream. Our KLK5 screening approach had previously identified benzamidine hydrochloride as a weak inhibitor of KLK5 (pIC50 3.3, n = 2) and using our triple mutant KLK6 crystallography system13 we had shown that this molecule bound in the S1 pocket forming hydrogen bonding interactions with Asp189. This site is normally occupied by an arginine of LEKTI, explaining the preference for a strongly basic functionality in this pocket. We have previously described molecules inhibiting KLK5 through occupation of the S1 and S1′ pocket11 and through occupation of S1 and S2 pockets,12 but in both cases these were non-covalent inhibitors. We
Abbreviations: KLK1, kallikrein 1; KLKB1, kallikrein B1; KLK5, kallikrein 5; KLK7, kallikrein 7; KLK8, kallikrein 8; KLK14, kallikrein 14; LCMS, liquid chromatography-mass spectrometry; LEKTI, lympho-epithelial kazal-type-related inhibitor; NS, Netherton Syndrome; NMR, nuclear magnetic resonance; SPINK5, serine protease inhibitor kazal-type 5 gene ⁎ Corresponding author. E-mail address: [email protected] (J. Liddle). https://doi.org/10.1016/j.bmcl.2019.126675 Received 23 July 2019; Received in revised form 4 September 2019; Accepted 5 September 2019 Available online 07 September 2019 0960-894X/ © 2019 Elsevier Ltd. All rights reserved.
Bioorganic & Medicinal Chemistry Letters 29 (2019) 126675
A.L. Walker, et al.
Table 1 Borocycle modifications.
KLK5 mean pIC50 (n)20
KLK5 LEc/LLEc
KLK1 Mean pIC50 (n)21
–CH2NH–
3.7 (2)
0.24/2.4
5.1 (8)
4-
–CH2O–
4.5 (7)
0.29/3.2
3.3 (4)
3a
3-
–CH2O–
4.9 (6)
0.32/3.7
3.8 (2)
4a
4-
–CH2O–
4.4 (4)
0.28/3.3
4.4 (4)
5a
4-
–OCH2–
5.2 (13)
6a
4-
–O–
4.3 (2)
7a
4-
–CH2O–
5.2 (13)
8a,b
4-
–
3.4 (3)
0.25/2.6
4.1 (4)
9a,b
3-
–
5.4 (13)
0.37/3.9
6.3 (8)
10a,b
4-
–
7.1 (19)
0.49/5.5
5.0 (16)
11a,b
4-
–CH2–
6.8 (17)
0.47/5.5
5.3 (12)
12a,b
4-
–NHCH2–
5.8 (9)
0.36/4.0
< 5.0 (6) 35%I (10 μM)
13a,b
4-
–
4.4 (13)
Eg. No.
Benzamidine linker position
Linker
1a
4-
2a
a b c
Borocycle
HCl salt. Racemic. LLE = 1.36 (pIC50 − clogP)/Heavy atom count + 0.11. LE = 1.37 × pIC50/Heavy atom count.
2
5.0 (8)
0.29/3.3
4.6 (2)
3.8 (2)
4.2 (2)
Bioorganic & Medicinal Chemistry Letters 29 (2019) 126675
A.L. Walker, et al.
Fig. 2. The R-enantiomer of 14 bound to KLK5 surrogate. The pyridyl sidechain occupies the S3 pocket, stacking against the edge of Tyr218. A shell of water runs along the peptide chain and interlinks with the pyridine N. Both enantiomers of the racemate are observed in the crystal structure. Table 2 S3 binding groups.
Fig. 1. a) Compound 10 bound to the KLK6 surrogate construct of KLK5 illustrating the covalent link between the boron and catalytic Ser195 and the hydroxyl occupying the oxyanion hole. b) Compound 12 bound to the KLK6 surrogate construct of KLK5. The amidine is hydrogen bonded to Asp189 in the bottom of the S1 pocket and the boron catalytic serine has coordinated to the boron forming a tetrahedral complex. All residue numbering corresponds to the targeted KLK5 enzyme.
Benzoxaborin
also decided to pursue covalent inhibitors based on the S1 binding benzamidine, taking the approach of growing this benzamidine from the S1 into the S1′ pocket, and attempting to place an appropriate functionality in a position to react with the catalytic serine. We thought that this was more likely to deliver selective KLK5 inhibitors than solely targeting the catalytic serine. There are some challenges with this approach as the reactive functionality is required to be stable in the presence of a highly basic amidine and for topical application the molecule must have stability suitable for formulation in a gel or cream with an appropriate shelf life for the product. We quickly ruled out lactams as an approach as these were incompatible with amidine formation. Boron containing compounds are gradually gaining acceptance as potential drug molecules14,15 and four examples have now been approved; tavaborole for antifungal use,16 bortezomib17 and ixazomib18 for treatment of multiple myeloma and mantle cell lymphoma and crisaborole19 as a topical treatment for atopic dermatitis. We felt that a benzoxaborole (6/5) or benzoxaborin (6/6) functionality might offer an appropriate stability profile for a topical KLK5 inhibitor and chose to target these as the reactive functionality. In our crystallographic system the catalytic serine has been shown to occupy a range of positions, so our initial focus was to prepare a range of benzoxaboroles and benzoxaborins linked to either the 3- or 4-position of the
Benzoxaborole
Eg. No.
R
Template
KLK5 mean pIC50 (n)
KLK5 LEc/LLEc
KLK1 Mean pIC50 (n)
14a,b 15a,b 16a,b 17a,b
Pyridin-3-yl 2-Chlorophenyl 3-Chlorophenyl Pyridin-3-yl
Benzoxaborin Benzoxaborin Benzoxaborin Benzoxaborole
8.2 8.0 8.1 7.7
0.40/6.3 0.38/4.1 0.38/4.1 0.38/5.9
4.5 6.2 5.8 3.5
(12) (9) (9) (15)
(4) (6) (6) (2)
a
HCl salt. Racemic. c LLE = 1.36 (pIC50 − clogP)/Heavy atom count + 0.11. LE = 1.37 × pIC50/ Heavy atom count. b
S1 binding benzamidine by a variety of types and lengths of linking functionality (Table 1). The linkers were attached to a variety of positions on the boron heterocycles and their fused aromatic groups to explore a range of binding positions for the boron. The compounds linked to the S1 benzamidine through the fused aromatic of the benzoxaborole (1–7), were either inactive or very weak inhibitors of KLK5,20 leading to the conclusion that even if there is binding of the benzamidine into S1 as desired, the boron and catalytic serine are not located in close proximity. The early examples suggested that the reactive functionality needed to be positioned closer to the S1 3
Bioorganic & Medicinal Chemistry Letters 29 (2019) 126675
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Table 3 Addition of S1′ substituent.
Benzoxaborin
Benzoxaborole
Eg. No.
Phenyl substitution
Template
KLK5 mean pIC50 (n)
KLK5 LEc/LLEc
Borocycle pKa
KLK1 mean pIC50 (n)
14a,b 17a,b 18a,b
H H 3′-Me
Benzoxaborin Benzoxaborole Benzoxaborin
8.2 (12) 7.7 (15) 7.6 (9)
0.40/6.3 0.38/5.9 0.36/4.6
4.83 4.80 nt
19a,b 20a,b
4′-F 4′-F
Benzoxaborin Benzoxaborole
8.2 (10) 7.9 (9)
0.39/6.1 0.37/5.7
4.66 4.77
21a,b 22a,b
4′-CF3 4′-CF3
Benzoxaborin Benzoxaborole
8.5 (11) 7.9 (10)
0.36/5.4 0.34/5.0
4.54 4.51
23a,b 24a,b
5′-F 5′-Me
Benzoxaborole Benzoxaborole
7.8 (9) 7.7 (9)
0.37/5.6 0.36/5.3
4.66 4.73
25a,b
5′-OMe
Benzoxaborole
7.4 (9)
0.34/5.5
4.71
26a,b
4′-F, 5′-Cl
Benzoxaborin
8.1 (14)
0.37/5.0
4.26
4.5 (4) 3.5 (2) < 5.0 (6) 27%I (10 μM) 5.4 (6) < 5.0 (6) 16%I (10 μM) 6.4 (10) < 5.0 (5) 24%I (10 μM) < 5.0 (6) 10%I (10 μM) < 5.0 (6) 10%I (10 μM) < 5.0 (6) 13%I (10 μM) 5.2 (10)
a b c
HCl salt. Racemic. LLE = 1.36 (pIC50 − clogP)/Heavy atom count + 0.11. LE = 1.37 × pIC50/Heavy atom count.
Table 4 Protease selectivity profiles. Data n = 2 unless otherwise annotated.
14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
KLK5
KLK5 low enzyme (n)
KLK1
KLKB1
KLK7
KLK8
KLK14
Factor Xa
Matriptase
Thrombin
Neutrophil elastase
Urokinase
8.2 8.0 8.1 7.7 7.6 8.2 7.9 8.5 7.9 7.8 7.7 7.4 8.1 8.3 8.3
8.4 (8) 8.1 (7) 8.3 (4) 8.2 (4) 7.6 (3) 8.8 (3) 8.1 (4) 9.3 (11) 8.6 (4) 8.0 (4) 8.1 (3) 7.6 (3) 8.9 (4) > 9.5 (13) > 9.1 (7)
4.5 (4) 6.2 (6) 5.8 (6) 3.5 (2) < 5.0 (6) < 6.5 (6) < 5.0 (6) 6.4 (10) < 5.0 (5) < 5.0 (6) < 5.0 (6) < 5.0 (6) < 5.4 (10) 6.6 (12) 5.7 (13)
7.0 7.3 8.3 7.1 7.1 7.1 6.1 8.0 6.7 6.1 6.1 5.9 6.7 7.7 6.4
< 4.0 (2) 4.4 (2) 4.4 (4) < 4.0 < 4.0 < 4.0 < 4.0 4.4 < 4.0 < 4.0 < 4.0 < 4.0 < 4.0 4.1 < 4.0
6.4 6.6 7.0 6.7 5.8 6.5 5.1 7.6 5.7 4.9 5.0 4.9 6.6 8.2 7.5
6.1 5.8 6.6 5.7 6.4 6.5 4.9 6.9 4.9 4.5 4.6 4.4 6.7 7.5 6.4
5.1 5.0 5.7 < 4.0 4.3 5.3 4.2 5.8 4.5 4.2 4.5 4.1 5.3 6.4 5.4
5.2 5.1 7.9 5.1 5.9 5.0 4.2 5.7 4.5 4.4 4.6 4.6 4.8 5.9 5.7
5.6 5.5 6.4 5.0 5.5 5.7 5.0 5.4 4.7 4.8 5.2 4.5 5.7 5.9 4.7
5.3 5.2 4.8 4.9 6.1 5.8 4.5 < 4.0 < 4.0 4.0 4.6 < 4.0 5.9 < 4.0 < 4.0
4.3 4.3 7.2 4.1 5.3 4.1 < 4.0 4.9 < 4.0 < 4.0 < 4.0 < 4.0 4.1 5.1 < 4.0
(12) (9) (9) (15) (9) (10) (9) (11) (10) (9) (9) (9) (14) (13) (17)
(1) (4) (3) (4) (3) (3) (3)
binding pocket, so the linkage to the benzamidine was modified to either a direct link to the boron containing ring (8, 9, 10) or via a 1–2 atom linker (11, 12). These examples overall had improved KLK5 inhibitory activities. Linking from the 4-position of the benzamidine give more active inhibitors than linking from the 3-position (compare 9 and 10) and the borolane ring sizes were also important (8 and 10) with a preference for the 6-membered benzoxaborin. However, the methylene linked 5-membered benzoxaborole (11) showed similar activity to benzoxaborin 10, while the aminomethyl linker example (12) was a less potent inhibitor of KLK5. Removal of the fused phenyl from the benzoxaborin to give a 1,2oxaborinan-2-ol 13 resulted in a loss of KLK5 inhibition of almost 1000fold compared with 10, possibly due to the loss of an edge to face π-
stacking interaction with His57 and changes in the reactivity of the borane. We were able to confirm the binding mode for a selection of the compounds described above. As expected from its potency, 10 bound to our KLK5 surrogate construct in a covalent manner with clear engagement of the catalytic serine to form the expected tetracoordinate boronate complex which mimics the transition state of the peptide bond hydrolysis.22 The boronate hydroxyl occupies the oxyanion hole and the amidine forms an interaction with Asp189 at the bottom of the S1 pocket in a similar manner to our previously published non-covalent inhibitors. 12 also engaged the catalytic serine and with the benzamidine located in the S1 pocket and H-bonded to Asp189. The boron is located higher in the oxyanion hole than we see in 10 (Fig. 1b). 4
Bioorganic & Medicinal Chemistry Letters 29 (2019) 126675
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profiles;12 so we decided to implement the same strategy in this covalent series focusing efforts on the two templates exemplified by benzoxaborin 10 and benzoxaborole 11. Based on our previous work12 we decided to incorporate pyridin-3ylmethyleneoxy (14 and 17) and 2-chlorobenzyloxy substituents (15, 16), these resulted in an increase in potency of approximately 10-fold at KLK5 consistent with them occupying the S3 pocket as we had seen for our non-covalent series and forming an edge to face π-stacking interaction with Tyr218. We were able to confirm this binding mode in our KLK5 surrogate crystallography system (Fig. 2). Gratifyingly these examples demonstrated a similar or improved selectivity window over KLK1 of 100 to > 1000-fold (Table 2), with similar levels of selectivity for both templates for KLK5 over KLK1 (compare 14 and 17). To understand the broader impact of the S3 binding substituent on selectivity, we submitted a number of examples to a panel of 8 serine proteases24 (Table 4) which included some where we had safety concerns about their inhibition and also some other members of the kallikrein protease family including Kallikreins 7, 8 and 14 (KLK7, KLK8, KLK14). KLK7, KLK8 and KLK14 are believed to be involved in skin desquamation and in the cases of KLK7 and KLK14, their activity is largely modulated by KLK5 so inhibition may be beneficial in NS. However, none of the compounds tested showed any significant inhibition of KLK7 and only modest activity at KLK14. Gratifyingly selectivity over the other proteases was generally good with the exception of KLKB1 where the compounds were at best ∼10-fold selective. A second approach to increasing potency and selectivity was to optimise the S1′ binding group and particularly the substitution of the fused phenyl of the benzoxaborole/benzoxaborin. A substituent not only has the potential to form additional interactions with the S1′ pocket, but may modulate the strength of the π-stacking interaction between the fused phenyl and His57, and potentially also influences the pKa and reactivity of the borocycle. The 3′-position is close to the wall of the binding site and substitution with a methyl 18 was associated with a reduction in activity (Table 3). A 4′-position substituent potentially fills a space bounded by His57, Lys60, Leu41, Cys58 and Ser105 but we initially saw little change in KLK5 inhibition potencies although there was a weak preference for electron withdrawing substituents possibly due to the presence of His57 and Lys60. The 5′-position substituent is para to the boron and might be expected to have the greatest effect on pKa and reactivity of the borane. Literature suggested that the two templates should have different pKa ranges,25 however measured pKa’s for a number of examples showed little difference and there was no evidence of correlation of pKa with KLK5 inhibitory potency. The 6′position is water exposed and little work was done in this area. Although we saw only minor increases in activity, a few compounds were approaching the theoretical tight binding limit of the KLK5 assay (pIC50 = 8.4) and so were retested under a low enzyme concentration protocol (theoretical tight binding limit pIC50 = 10; 50% of the enzyme concentration 2 × 10−10 M). Most of the compounds retained a similar KLK5 activity between the two assays, however benzoxaborins 21, 22 and 26 showed significant potency increases moving to the low enzyme KLK5 assay (Table 4). This confirmed the trend for preference of electron withdrawing substituents and in particular for a 4′-CF3 group in this template. The differences in KLK5 inhibition between the templates was not readily explained as the substituents occupy almost exactly the same position by crystallography in our KLK6 mutant systems. Broader selectivity profiles for these examples followed earlier trends, but 21 and 26 now had a significant selectivity window over KLKB1 (theoretical tight binding limit pIC50 8.5) (Table 4). Preparation of the single enantiomers of 21 (R enantiomer 27 and S enantiomer 28) resulted in compounds with similar KLK5 activity, as was predicted from the observation of both enantiomers of 17 in the KLK5 crystal structure (Fig. 2) and the overall selectivity profiles are also similar. These benzoxaborins and benzoxaboroles are reversible covalent inhibitors in that the coordination of the hydroxyl of the catalytic serine
Scheme 1. (i) Zn(CN)2, TMEDA, Xantphos, Pd2(dba)3, DMF, 120 °C, sealed tube. 70% yield. (ii) 3-hydroxymethylpyridine, NaH, DMF, rt. 47% yield. (iii) 2Bromo-1-iodo-4-(trifluoromethyl)benzene, Xantphos, Pd2(dba)3, Cs2CO3, 1,4dioxane, 90 °C. 64% yield. (iv) NaBH4, THF/MeOH, 0 °C. (v) bisneopentylglycolatodiborane, Pd(OAc)2, tBuPh2P, KOAc, 1,4-dioxane, 75 °C. 88% yield. (vi) HCl(g), MeOH, rt. Product not purified. (vii) NH3.MeOH, 0 °C to rt. 44% yield over 2 steps.
Scheme 2. (i) DIPEA, nBuLi, HMPA, THF, −78 °C to rt to −78 °C. (ii) 2-bromo4-(trifluoromethyl)benzaldehyde, −78 °C. 32% yield (iii) bisneopentylglycolatodiborane, Pd(OAc)2, tBuPh2P, KOAc, 1,4-dioxane, 80 °C. 61% yield. (iv) 3hydroxymethylpyridine, NaH, DMF, rt. 63% yield.(v) HCl(g), MeOH, rt. Product not purified. (vi) NH3.MeOH, 0 °C to rt. 65% yield over 2 steps.
Although aiming for a topical medicine we desired a good selectivity profile over other serine proteases. Unlike many topical medicines, for NS we expect to have to treat a significant percentage of a damaged skin surface which could result in systemic drug levels sufficient to engage off-target proteases, particularly in NS neonates where the surface to volume ratio is much higher than in adults. Encouragingly 10 and 11 both showed 30–100-fold selectivity over KLK1, a blood serine protease with a role in vasoconstriction,23 that we used as a primary selectivity assay. Previous efforts on our S2 binding series had shown that 2-substitution of the benzamidine resulted in molecules with increased KLK5 inhibitory activity and improved broad serine protease selectivity 5
Bioorganic & Medicinal Chemistry Letters 29 (2019) 126675
A.L. Walker, et al.
to the boron is a dynamic process, so we were keen to understand the binding kinetics of our preferred molecules. Interestingly, despite having similar KLK5 potency 28 had a fast off-rate in jump dilution experiments (K-off apparent is 0.1284/min, T1/2 5 min) and 27 had a relatively slow T1/2 in the order of 70 min.26 This result isn’t readily explained by differences in potency and we assume that this is the result of the subtle differences in binding geometries. Based on the activity, extended Toff and selectivity of 27, this molecule was chosen for further profiling. 27 had a 1000-fold selectivity window against all targets in GSK’s liability panel (57 targets) and was negative in mini-Ames despite our earlier concerns around the potential for genotoxicity of borane species.27 For topical formulation we wished to understand the uv absorbance, stability and solubility of 27, initially in aqueous buffers before engaging in further formulation studies. 27, dissolved in methanol, didn’t show absorbance of uv in the critical range above 290 nm giving this molecule a low risk for light mediated skin toxicity. The solubility of 27 in Britton-Robinson buffers between pH2 and pH8 was measured at 12 h and 24 h, the solubility was excellent, ranging from 3.2 mg/mL at pH6/12 h to > 9 mg/mL at pH 4/24 h. In initial stability studies28 at pHs between 4 and 10, 27 had a half-life of > 1000 h. Due to the highly polar and basic nature of the amidine, examples (1–26) were synthesised with the formation of the amidine from the analogous nitrile as the final step in either a one pot or two pot conversion. To prepare the nitriles a variety of routes were used, particularly for examples in Table 1, with preparation of the linker or borocycle as dictated by the available starting materials. For the preferred benzoxaborins the benzyl phenyl ketone intermediate could be prepared by reaction of the methyl aryl ketone with a 1-bromo-2-iodobenzene (Scheme 1). Reduction of the ketone with sodium borohydride and formation of the borocycle under palladium catalysed conditions gave the desired nitrile intermediate. The 2-substituent on the benzamidine could be introduced either early in the synthesis for example at one of the ketone intermediates or introduced just prior to conversion to the amidine. The benzoxaboroles were prepared in an analogous manner (Scheme 2), but in this case the hydroxy bromo precursor to the benzoxaborole could be obtained directly from lithiation of the 4-methylbenzonitrile and reaction with an appropriate 2-bromobenzaldehyde. Synthesis of 2129–31 Scheme 1 Synthesis of 2232 Scheme 2 In conclusion, we show here the development and optimisation of a series of KLK5 inhibitors arising from a collaboration between the Imagine Institute and GSK. Using structure based design with a KLK6 mutant protein as a surrogate for KLK5, we have been able to design and optimise a series of highly potent reversible covalent KLK5 inhibitors. 27 shows excellent selectivity against both protease and nonprotease targets and has stability and solubility suitable for further development based on our initial developability studies.
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Kallikrein 5 inhibitors identified through structure based drug design in search for a treatment for Netherton syndrome. Bioorg Med Chem Lett. 2019;29:821–825. 12. Walker AL, Bingham R, Edgar EV, et al. Structure guided drug design to develop kallikrein 5 inhibitors to treat Netherton syndrome. Bioorg Med Chem Lett. 2019;29:1454–1458. 13. 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. Acta Cryst F75. 2019;75:385–391. 14. Mereddy GR, Chakradhar A, Rutkoski RM, Jonnalagadda SC. Benzoboroxoles: synthesis and applications in medicinal chemistry. J Organomet Chem. 2018;865:12–22. 15. Nocentini A, Supuran CT, Winum J-Y. Benzoxaborole compounds for therapeutic uses: a patent review (2010–2018). Expert Opin Ther Pat. 2018;26:493–504. 16. Markham A. Tavaborole: first global approval. Drugs. 2014;74:1555–1558. 17. Kane RC, Bross PF, Farrell AT, Richard Pazdur R. Velcade®: U.S. FDA approval for the treatment of multiple myeloma progressing on prior therapy. Oncologist. 2003;8:508–513. 18. Shirley M. Ixazomib: first global approval. Drugs. 2016;76:405–411. 19. FDA approves Eucrisa for eczema. www.fda.gov/newsevents/newsroom/ pressannouncements/ucm533371.htm. 20. KLK5 assay – details available in supplementary data. 21. KLK1 assay – details available in supplementary data. 22. Smoum R, Rubinstein A, Dembitsky VM, Srebnik M. Boron containing compounds as protease inhibitors. Chem Rev. 2012;112:4156–4220. 23. Madeddu P, Emanueli C, El-Dahr S. Mechanisms of disease: the tissue kallikrein-kinin system in hypertension and vascular remodeling. Nature Clin Pract. 2007;3:208. 24. Serine protease panel – details available in supplementary data. 25. Tomsho JW, Pal A, Hall DG, Benkovic SJ. ACS Med Chem Lett. 2012;3:48–52. 26. Rate curves were generated from the mean of the 3 samples at each time point and data were fitted in Grafit using a single exponential decay to define the off-rate, from which the t1/2 was calculated (t1/2 = Ln 2 (0.69)/K-off apparent). 27. O’Donovan MR, Mee CD, Fenner S, Teasdale A, Philips DH. Boronic acids – a novel class of bacterial mutagen. Mutat Res. 2011;724:1. 28. Compound samples were prepared in each buffer (Britton Robinson, pH4, 6 and 8 and SGF) and 1:1 water/acetonitrile at concentrations of 0.05mg/ml. Hydrogen peroxide was added to a set of samples at 20 M% for each pH condition. The samples were analysed 6 times over a period of 24h to check for degradation and calculate half life. 29. 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. 30. Compound purity: Compounds are > 95% purity by LCMS and NMR unless otherwise stated. 31. Characterisation for 21: LCMS MH+ 442, retention time 1.61min, 99.0%. NMR D6 DMSO includes δH 9.30 (s, 2H), 9.23 (s, 2H), 9.01 (br. s, 1H), 8.86 (br. s, 1H), 8.45 (d, J = 7.7 Hz, 1H), 8.10 (s, 1H), 7.92 (m, 1H), 7.81 (m, 1H), 7.61 (d, J = 7.9 Hz, 1H), 7.50 (m, 2H), 7.30 (d, J = 8.1 Hz, 1H), 5.43 (m, 3H), 3.29 (dd, J = 16.2, 3.3 Hz, 1H), 3.18 (dd, J = 16.2, 10.3 Hz, 1H). 32. Characterisation for 22: LCMS MH+ 442, retention time 1.55min, 98.0%. NMR D6 DMSO includes δH 9.22 (s, 2H), 9.15 (s, 2H), 8.94 (d, J = 1.5 Hz, 1H), 8.79 (dd, J = 5.4 Hz, J = 1.4 Hz, 1H), 8.39 (d, J = 8.1 Hz, 1H), 8.11 (s, 1H), 7.86 (m, 2H), 7.72 (d, J = 7.9 Hz, 1H), 7.49 (d, J = 7.9 Hz, 1H), 7.33 (s, 1H), 7.09 (m, 1H), 5.58 (dd, J = 8.4, 3.8 Hz, 1H), 5.33 (m, 2H), 3.44 (dd, J = 14.1, 3.8 Hz, 1H), 2.89 (dd, J = 14.0, 8.8 Hz, 1H).
Acknowledgements The authors thank the chemistry teams who prepared 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.2019.126675.
6
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Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Design and development of a series of borocycles as selective, covalent kallikrein 5 inhibitors
T
Ann L. Walkera, Alexis Denisa, Ryan P. Binghama, Anne Bouillota, Emma V. Edgara, Alan Ferriea, ⁎ Duncan S. Holmesa, Alain Larozea, John Liddlea, , Marie-Helene Foucheta, Alexandre Moquettea, Pam Nassaua, Andrew C. Pearcea, Oxana Polyakovaa, Kathrine J. Smitha, Pamela Thomasa, James H. Thorpea, Lionel Trotteta, Yichen Wangb, Alain Hovnanianb a b
GlaxoSmithKline R&D, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK INSERM UMR1163 Laboratory of Genetic Skin Diseases, Imagine Institute and Université Paris Descarte-Sorbonne Paris Cité, Paris, France
ARTICLE INFO
ABSTRACT
Keywords: KLK1 KLK5 KLKB1 LEKTI Netherton syndrome SPINK5
The connection between Netherton syndrome and overactivation of epidermal/dermal proteases, particularly Kallikrein 5 (KLK5) has been well established and it is expected that a KLK5 inhibitor would improve the dermal barrier and also reduce the pain and itch that afflict Netherton syndrome patients. One of the challenges of covalent protease inhibitors has been achieving selectivity over closely related targets. In this paper we describe the use of structural insight to design and develop a selective and highly potent reversibly covalent KLK5 inhibitor from an initial weakly binding fragment.
Netherton Syndrome (NS) is a devastating rare congenital skin disease characterised by ichthyosis, bamboo hair and failure to thrive early in life.1 Newborns afflicted by this condition are at a significant risk of death due a leaky skin barrier which can lead to dehydration and infection and older children and adults suffer from red and scaly skin, itching and allergic manifestations. The cause of NS has been identified as loss-of-function mutations of the SPINK5 gene.2 Bi-allelic SPINK5 mutations result in reduced or no expression of serine protease inhibitor lympho-epithelial Kazla-typerelated inhibitor (LEKTI). LEKTI is normally expressed in the most differentiated layer of the epidermis and regulates the activity of several serine proteases including Kallikrein 5 (KLK5); its ability to inhibit KLK5 is pH dependant and is lost in the more acidic outer layers of the stratum corneum allowing KLK5 driven cleavage of desmosomal proteins and loss of the outer-most skin layer.3 In NS individuals, the unregulated activity of KLK5 in the lower layers of the stratum corneum results in premature desquamation. The unregulated protease activity within the epidermis also activates PAR-2 and triggers inflammatory reactions, leading to erythroderma and to the profound itching which is characteristic of NS.4
There is no curative therapy for NS and current treatment is limited to intensive nursing care of new-borns, frequent application of emollients and treatment with anti-infectives when necessary. We therefore desired to identify topical inhibitors of KLK5 as these would be expected to reduce the abnormal protease activity leading to improved skin barrier function and reduced allergy for NS individuals. A number of KLK5 inhibitors have been published,5,6 including both covalent examples7,8 and non-covalent series,9–12 however, we wished to identify more potent and soluble examples which would be suitable for topical application in a gel or cream. Our KLK5 screening approach had previously identified benzamidine hydrochloride as a weak inhibitor of KLK5 (pIC50 3.3, n = 2) and using our triple mutant KLK6 crystallography system13 we had shown that this molecule bound in the S1 pocket forming hydrogen bonding interactions with Asp189. This site is normally occupied by an arginine of LEKTI, explaining the preference for a strongly basic functionality in this pocket. We have previously described molecules inhibiting KLK5 through occupation of the S1 and S1′ pocket11 and through occupation of S1 and S2 pockets,12 but in both cases these were non-covalent inhibitors. We
Abbreviations: KLK1, kallikrein 1; KLKB1, kallikrein B1; KLK5, kallikrein 5; KLK7, kallikrein 7; KLK8, kallikrein 8; KLK14, kallikrein 14; LCMS, liquid chromatography-mass spectrometry; LEKTI, lympho-epithelial kazal-type-related inhibitor; NS, Netherton Syndrome; NMR, nuclear magnetic resonance; SPINK5, serine protease inhibitor kazal-type 5 gene ⁎ Corresponding author. E-mail address: [email protected] (J. Liddle). https://doi.org/10.1016/j.bmcl.2019.126675 Received 23 July 2019; Received in revised form 4 September 2019; Accepted 5 September 2019 Available online 07 September 2019 0960-894X/ © 2019 Elsevier Ltd. All rights reserved.
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Table 1 Borocycle modifications.
KLK5 mean pIC50 (n)20
KLK5 LEc/LLEc
KLK1 Mean pIC50 (n)21
–CH2NH–
3.7 (2)
0.24/2.4
5.1 (8)
4-
–CH2O–
4.5 (7)
0.29/3.2
3.3 (4)
3a
3-
–CH2O–
4.9 (6)
0.32/3.7
3.8 (2)
4a
4-
–CH2O–
4.4 (4)
0.28/3.3
4.4 (4)
5a
4-
–OCH2–
5.2 (13)
6a
4-
–O–
4.3 (2)
7a
4-
–CH2O–
5.2 (13)
8a,b
4-
–
3.4 (3)
0.25/2.6
4.1 (4)
9a,b
3-
–
5.4 (13)
0.37/3.9
6.3 (8)
10a,b
4-
–
7.1 (19)
0.49/5.5
5.0 (16)
11a,b
4-
–CH2–
6.8 (17)
0.47/5.5
5.3 (12)
12a,b
4-
–NHCH2–
5.8 (9)
0.36/4.0
< 5.0 (6) 35%I (10 μM)
13a,b
4-
–
4.4 (13)
Eg. No.
Benzamidine linker position
Linker
1a
4-
2a
a b c
Borocycle
HCl salt. Racemic. LLE = 1.36 (pIC50 − clogP)/Heavy atom count + 0.11. LE = 1.37 × pIC50/Heavy atom count.
2
5.0 (8)
0.29/3.3
4.6 (2)
3.8 (2)
4.2 (2)
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Fig. 2. The R-enantiomer of 14 bound to KLK5 surrogate. The pyridyl sidechain occupies the S3 pocket, stacking against the edge of Tyr218. A shell of water runs along the peptide chain and interlinks with the pyridine N. Both enantiomers of the racemate are observed in the crystal structure. Table 2 S3 binding groups.
Fig. 1. a) Compound 10 bound to the KLK6 surrogate construct of KLK5 illustrating the covalent link between the boron and catalytic Ser195 and the hydroxyl occupying the oxyanion hole. b) Compound 12 bound to the KLK6 surrogate construct of KLK5. The amidine is hydrogen bonded to Asp189 in the bottom of the S1 pocket and the boron catalytic serine has coordinated to the boron forming a tetrahedral complex. All residue numbering corresponds to the targeted KLK5 enzyme.
Benzoxaborin
also decided to pursue covalent inhibitors based on the S1 binding benzamidine, taking the approach of growing this benzamidine from the S1 into the S1′ pocket, and attempting to place an appropriate functionality in a position to react with the catalytic serine. We thought that this was more likely to deliver selective KLK5 inhibitors than solely targeting the catalytic serine. There are some challenges with this approach as the reactive functionality is required to be stable in the presence of a highly basic amidine and for topical application the molecule must have stability suitable for formulation in a gel or cream with an appropriate shelf life for the product. We quickly ruled out lactams as an approach as these were incompatible with amidine formation. Boron containing compounds are gradually gaining acceptance as potential drug molecules14,15 and four examples have now been approved; tavaborole for antifungal use,16 bortezomib17 and ixazomib18 for treatment of multiple myeloma and mantle cell lymphoma and crisaborole19 as a topical treatment for atopic dermatitis. We felt that a benzoxaborole (6/5) or benzoxaborin (6/6) functionality might offer an appropriate stability profile for a topical KLK5 inhibitor and chose to target these as the reactive functionality. In our crystallographic system the catalytic serine has been shown to occupy a range of positions, so our initial focus was to prepare a range of benzoxaboroles and benzoxaborins linked to either the 3- or 4-position of the
Benzoxaborole
Eg. No.
R
Template
KLK5 mean pIC50 (n)
KLK5 LEc/LLEc
KLK1 Mean pIC50 (n)
14a,b 15a,b 16a,b 17a,b
Pyridin-3-yl 2-Chlorophenyl 3-Chlorophenyl Pyridin-3-yl
Benzoxaborin Benzoxaborin Benzoxaborin Benzoxaborole
8.2 8.0 8.1 7.7
0.40/6.3 0.38/4.1 0.38/4.1 0.38/5.9
4.5 6.2 5.8 3.5
(12) (9) (9) (15)
(4) (6) (6) (2)
a
HCl salt. Racemic. c LLE = 1.36 (pIC50 − clogP)/Heavy atom count + 0.11. LE = 1.37 × pIC50/ Heavy atom count. b
S1 binding benzamidine by a variety of types and lengths of linking functionality (Table 1). The linkers were attached to a variety of positions on the boron heterocycles and their fused aromatic groups to explore a range of binding positions for the boron. The compounds linked to the S1 benzamidine through the fused aromatic of the benzoxaborole (1–7), were either inactive or very weak inhibitors of KLK5,20 leading to the conclusion that even if there is binding of the benzamidine into S1 as desired, the boron and catalytic serine are not located in close proximity. The early examples suggested that the reactive functionality needed to be positioned closer to the S1 3
Bioorganic & Medicinal Chemistry Letters 29 (2019) 126675
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Table 3 Addition of S1′ substituent.
Benzoxaborin
Benzoxaborole
Eg. No.
Phenyl substitution
Template
KLK5 mean pIC50 (n)
KLK5 LEc/LLEc
Borocycle pKa
KLK1 mean pIC50 (n)
14a,b 17a,b 18a,b
H H 3′-Me
Benzoxaborin Benzoxaborole Benzoxaborin
8.2 (12) 7.7 (15) 7.6 (9)
0.40/6.3 0.38/5.9 0.36/4.6
4.83 4.80 nt
19a,b 20a,b
4′-F 4′-F
Benzoxaborin Benzoxaborole
8.2 (10) 7.9 (9)
0.39/6.1 0.37/5.7
4.66 4.77
21a,b 22a,b
4′-CF3 4′-CF3
Benzoxaborin Benzoxaborole
8.5 (11) 7.9 (10)
0.36/5.4 0.34/5.0
4.54 4.51
23a,b 24a,b
5′-F 5′-Me
Benzoxaborole Benzoxaborole
7.8 (9) 7.7 (9)
0.37/5.6 0.36/5.3
4.66 4.73
25a,b
5′-OMe
Benzoxaborole
7.4 (9)
0.34/5.5
4.71
26a,b
4′-F, 5′-Cl
Benzoxaborin
8.1 (14)
0.37/5.0
4.26
4.5 (4) 3.5 (2) < 5.0 (6) 27%I (10 μM) 5.4 (6) < 5.0 (6) 16%I (10 μM) 6.4 (10) < 5.0 (5) 24%I (10 μM) < 5.0 (6) 10%I (10 μM) < 5.0 (6) 10%I (10 μM) < 5.0 (6) 13%I (10 μM) 5.2 (10)
a b c
HCl salt. Racemic. LLE = 1.36 (pIC50 − clogP)/Heavy atom count + 0.11. LE = 1.37 × pIC50/Heavy atom count.
Table 4 Protease selectivity profiles. Data n = 2 unless otherwise annotated.
14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
KLK5
KLK5 low enzyme (n)
KLK1
KLKB1
KLK7
KLK8
KLK14
Factor Xa
Matriptase
Thrombin
Neutrophil elastase
Urokinase
8.2 8.0 8.1 7.7 7.6 8.2 7.9 8.5 7.9 7.8 7.7 7.4 8.1 8.3 8.3
8.4 (8) 8.1 (7) 8.3 (4) 8.2 (4) 7.6 (3) 8.8 (3) 8.1 (4) 9.3 (11) 8.6 (4) 8.0 (4) 8.1 (3) 7.6 (3) 8.9 (4) > 9.5 (13) > 9.1 (7)
4.5 (4) 6.2 (6) 5.8 (6) 3.5 (2) < 5.0 (6) < 6.5 (6) < 5.0 (6) 6.4 (10) < 5.0 (5) < 5.0 (6) < 5.0 (6) < 5.0 (6) < 5.4 (10) 6.6 (12) 5.7 (13)
7.0 7.3 8.3 7.1 7.1 7.1 6.1 8.0 6.7 6.1 6.1 5.9 6.7 7.7 6.4
< 4.0 (2) 4.4 (2) 4.4 (4) < 4.0 < 4.0 < 4.0 < 4.0 4.4 < 4.0 < 4.0 < 4.0 < 4.0 < 4.0 4.1 < 4.0
6.4 6.6 7.0 6.7 5.8 6.5 5.1 7.6 5.7 4.9 5.0 4.9 6.6 8.2 7.5
6.1 5.8 6.6 5.7 6.4 6.5 4.9 6.9 4.9 4.5 4.6 4.4 6.7 7.5 6.4
5.1 5.0 5.7 < 4.0 4.3 5.3 4.2 5.8 4.5 4.2 4.5 4.1 5.3 6.4 5.4
5.2 5.1 7.9 5.1 5.9 5.0 4.2 5.7 4.5 4.4 4.6 4.6 4.8 5.9 5.7
5.6 5.5 6.4 5.0 5.5 5.7 5.0 5.4 4.7 4.8 5.2 4.5 5.7 5.9 4.7
5.3 5.2 4.8 4.9 6.1 5.8 4.5 < 4.0 < 4.0 4.0 4.6 < 4.0 5.9 < 4.0 < 4.0
4.3 4.3 7.2 4.1 5.3 4.1 < 4.0 4.9 < 4.0 < 4.0 < 4.0 < 4.0 4.1 5.1 < 4.0
(12) (9) (9) (15) (9) (10) (9) (11) (10) (9) (9) (9) (14) (13) (17)
(1) (4) (3) (4) (3) (3) (3)
binding pocket, so the linkage to the benzamidine was modified to either a direct link to the boron containing ring (8, 9, 10) or via a 1–2 atom linker (11, 12). These examples overall had improved KLK5 inhibitory activities. Linking from the 4-position of the benzamidine give more active inhibitors than linking from the 3-position (compare 9 and 10) and the borolane ring sizes were also important (8 and 10) with a preference for the 6-membered benzoxaborin. However, the methylene linked 5-membered benzoxaborole (11) showed similar activity to benzoxaborin 10, while the aminomethyl linker example (12) was a less potent inhibitor of KLK5. Removal of the fused phenyl from the benzoxaborin to give a 1,2oxaborinan-2-ol 13 resulted in a loss of KLK5 inhibition of almost 1000fold compared with 10, possibly due to the loss of an edge to face π-
stacking interaction with His57 and changes in the reactivity of the borane. We were able to confirm the binding mode for a selection of the compounds described above. As expected from its potency, 10 bound to our KLK5 surrogate construct in a covalent manner with clear engagement of the catalytic serine to form the expected tetracoordinate boronate complex which mimics the transition state of the peptide bond hydrolysis.22 The boronate hydroxyl occupies the oxyanion hole and the amidine forms an interaction with Asp189 at the bottom of the S1 pocket in a similar manner to our previously published non-covalent inhibitors. 12 also engaged the catalytic serine and with the benzamidine located in the S1 pocket and H-bonded to Asp189. The boron is located higher in the oxyanion hole than we see in 10 (Fig. 1b). 4
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profiles;12 so we decided to implement the same strategy in this covalent series focusing efforts on the two templates exemplified by benzoxaborin 10 and benzoxaborole 11. Based on our previous work12 we decided to incorporate pyridin-3ylmethyleneoxy (14 and 17) and 2-chlorobenzyloxy substituents (15, 16), these resulted in an increase in potency of approximately 10-fold at KLK5 consistent with them occupying the S3 pocket as we had seen for our non-covalent series and forming an edge to face π-stacking interaction with Tyr218. We were able to confirm this binding mode in our KLK5 surrogate crystallography system (Fig. 2). Gratifyingly these examples demonstrated a similar or improved selectivity window over KLK1 of 100 to > 1000-fold (Table 2), with similar levels of selectivity for both templates for KLK5 over KLK1 (compare 14 and 17). To understand the broader impact of the S3 binding substituent on selectivity, we submitted a number of examples to a panel of 8 serine proteases24 (Table 4) which included some where we had safety concerns about their inhibition and also some other members of the kallikrein protease family including Kallikreins 7, 8 and 14 (KLK7, KLK8, KLK14). KLK7, KLK8 and KLK14 are believed to be involved in skin desquamation and in the cases of KLK7 and KLK14, their activity is largely modulated by KLK5 so inhibition may be beneficial in NS. However, none of the compounds tested showed any significant inhibition of KLK7 and only modest activity at KLK14. Gratifyingly selectivity over the other proteases was generally good with the exception of KLKB1 where the compounds were at best ∼10-fold selective. A second approach to increasing potency and selectivity was to optimise the S1′ binding group and particularly the substitution of the fused phenyl of the benzoxaborole/benzoxaborin. A substituent not only has the potential to form additional interactions with the S1′ pocket, but may modulate the strength of the π-stacking interaction between the fused phenyl and His57, and potentially also influences the pKa and reactivity of the borocycle. The 3′-position is close to the wall of the binding site and substitution with a methyl 18 was associated with a reduction in activity (Table 3). A 4′-position substituent potentially fills a space bounded by His57, Lys60, Leu41, Cys58 and Ser105 but we initially saw little change in KLK5 inhibition potencies although there was a weak preference for electron withdrawing substituents possibly due to the presence of His57 and Lys60. The 5′-position substituent is para to the boron and might be expected to have the greatest effect on pKa and reactivity of the borane. Literature suggested that the two templates should have different pKa ranges,25 however measured pKa’s for a number of examples showed little difference and there was no evidence of correlation of pKa with KLK5 inhibitory potency. The 6′position is water exposed and little work was done in this area. Although we saw only minor increases in activity, a few compounds were approaching the theoretical tight binding limit of the KLK5 assay (pIC50 = 8.4) and so were retested under a low enzyme concentration protocol (theoretical tight binding limit pIC50 = 10; 50% of the enzyme concentration 2 × 10−10 M). Most of the compounds retained a similar KLK5 activity between the two assays, however benzoxaborins 21, 22 and 26 showed significant potency increases moving to the low enzyme KLK5 assay (Table 4). This confirmed the trend for preference of electron withdrawing substituents and in particular for a 4′-CF3 group in this template. The differences in KLK5 inhibition between the templates was not readily explained as the substituents occupy almost exactly the same position by crystallography in our KLK6 mutant systems. Broader selectivity profiles for these examples followed earlier trends, but 21 and 26 now had a significant selectivity window over KLKB1 (theoretical tight binding limit pIC50 8.5) (Table 4). Preparation of the single enantiomers of 21 (R enantiomer 27 and S enantiomer 28) resulted in compounds with similar KLK5 activity, as was predicted from the observation of both enantiomers of 17 in the KLK5 crystal structure (Fig. 2) and the overall selectivity profiles are also similar. These benzoxaborins and benzoxaboroles are reversible covalent inhibitors in that the coordination of the hydroxyl of the catalytic serine
Scheme 1. (i) Zn(CN)2, TMEDA, Xantphos, Pd2(dba)3, DMF, 120 °C, sealed tube. 70% yield. (ii) 3-hydroxymethylpyridine, NaH, DMF, rt. 47% yield. (iii) 2Bromo-1-iodo-4-(trifluoromethyl)benzene, Xantphos, Pd2(dba)3, Cs2CO3, 1,4dioxane, 90 °C. 64% yield. (iv) NaBH4, THF/MeOH, 0 °C. (v) bisneopentylglycolatodiborane, Pd(OAc)2, tBuPh2P, KOAc, 1,4-dioxane, 75 °C. 88% yield. (vi) HCl(g), MeOH, rt. Product not purified. (vii) NH3.MeOH, 0 °C to rt. 44% yield over 2 steps.
Scheme 2. (i) DIPEA, nBuLi, HMPA, THF, −78 °C to rt to −78 °C. (ii) 2-bromo4-(trifluoromethyl)benzaldehyde, −78 °C. 32% yield (iii) bisneopentylglycolatodiborane, Pd(OAc)2, tBuPh2P, KOAc, 1,4-dioxane, 80 °C. 61% yield. (iv) 3hydroxymethylpyridine, NaH, DMF, rt. 63% yield.(v) HCl(g), MeOH, rt. Product not purified. (vi) NH3.MeOH, 0 °C to rt. 65% yield over 2 steps.
Although aiming for a topical medicine we desired a good selectivity profile over other serine proteases. Unlike many topical medicines, for NS we expect to have to treat a significant percentage of a damaged skin surface which could result in systemic drug levels sufficient to engage off-target proteases, particularly in NS neonates where the surface to volume ratio is much higher than in adults. Encouragingly 10 and 11 both showed 30–100-fold selectivity over KLK1, a blood serine protease with a role in vasoconstriction,23 that we used as a primary selectivity assay. Previous efforts on our S2 binding series had shown that 2-substitution of the benzamidine resulted in molecules with increased KLK5 inhibitory activity and improved broad serine protease selectivity 5
Bioorganic & Medicinal Chemistry Letters 29 (2019) 126675
A.L. Walker, et al.
to the boron is a dynamic process, so we were keen to understand the binding kinetics of our preferred molecules. Interestingly, despite having similar KLK5 potency 28 had a fast off-rate in jump dilution experiments (K-off apparent is 0.1284/min, T1/2 5 min) and 27 had a relatively slow T1/2 in the order of 70 min.26 This result isn’t readily explained by differences in potency and we assume that this is the result of the subtle differences in binding geometries. Based on the activity, extended Toff and selectivity of 27, this molecule was chosen for further profiling. 27 had a 1000-fold selectivity window against all targets in GSK’s liability panel (57 targets) and was negative in mini-Ames despite our earlier concerns around the potential for genotoxicity of borane species.27 For topical formulation we wished to understand the uv absorbance, stability and solubility of 27, initially in aqueous buffers before engaging in further formulation studies. 27, dissolved in methanol, didn’t show absorbance of uv in the critical range above 290 nm giving this molecule a low risk for light mediated skin toxicity. The solubility of 27 in Britton-Robinson buffers between pH2 and pH8 was measured at 12 h and 24 h, the solubility was excellent, ranging from 3.2 mg/mL at pH6/12 h to > 9 mg/mL at pH 4/24 h. In initial stability studies28 at pHs between 4 and 10, 27 had a half-life of > 1000 h. Due to the highly polar and basic nature of the amidine, examples (1–26) were synthesised with the formation of the amidine from the analogous nitrile as the final step in either a one pot or two pot conversion. To prepare the nitriles a variety of routes were used, particularly for examples in Table 1, with preparation of the linker or borocycle as dictated by the available starting materials. For the preferred benzoxaborins the benzyl phenyl ketone intermediate could be prepared by reaction of the methyl aryl ketone with a 1-bromo-2-iodobenzene (Scheme 1). Reduction of the ketone with sodium borohydride and formation of the borocycle under palladium catalysed conditions gave the desired nitrile intermediate. The 2-substituent on the benzamidine could be introduced either early in the synthesis for example at one of the ketone intermediates or introduced just prior to conversion to the amidine. The benzoxaboroles were prepared in an analogous manner (Scheme 2), but in this case the hydroxy bromo precursor to the benzoxaborole could be obtained directly from lithiation of the 4-methylbenzonitrile and reaction with an appropriate 2-bromobenzaldehyde. Synthesis of 2129–31 Scheme 1 Synthesis of 2232 Scheme 2 In conclusion, we show here the development and optimisation of a series of KLK5 inhibitors arising from a collaboration between the Imagine Institute and GSK. Using structure based design with a KLK6 mutant protein as a surrogate for KLK5, we have been able to design and optimise a series of highly potent reversible covalent KLK5 inhibitors. 27 shows excellent selectivity against both protease and nonprotease targets and has stability and solubility suitable for further development based on our initial developability studies.
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Rate curves were generated from the mean of the 3 samples at each time point and data were fitted in Grafit using a single exponential decay to define the off-rate, from which the t1/2 was calculated (t1/2 = Ln 2 (0.69)/K-off apparent). 27. O’Donovan MR, Mee CD, Fenner S, Teasdale A, Philips DH. Boronic acids – a novel class of bacterial mutagen. Mutat Res. 2011;724:1. 28. Compound samples were prepared in each buffer (Britton Robinson, pH4, 6 and 8 and SGF) and 1:1 water/acetonitrile at concentrations of 0.05mg/ml. Hydrogen peroxide was added to a set of samples at 20 M% for each pH condition. The samples were analysed 6 times over a period of 24h to check for degradation and calculate half life. 29. 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. 30. Compound purity: Compounds are > 95% purity by LCMS and NMR unless otherwise stated. 31. Characterisation for 21: LCMS MH+ 442, retention time 1.61min, 99.0%. NMR D6 DMSO includes δH 9.30 (s, 2H), 9.23 (s, 2H), 9.01 (br. s, 1H), 8.86 (br. s, 1H), 8.45 (d, J = 7.7 Hz, 1H), 8.10 (s, 1H), 7.92 (m, 1H), 7.81 (m, 1H), 7.61 (d, J = 7.9 Hz, 1H), 7.50 (m, 2H), 7.30 (d, J = 8.1 Hz, 1H), 5.43 (m, 3H), 3.29 (dd, J = 16.2, 3.3 Hz, 1H), 3.18 (dd, J = 16.2, 10.3 Hz, 1H). 32. Characterisation for 22: LCMS MH+ 442, retention time 1.55min, 98.0%. NMR D6 DMSO includes δH 9.22 (s, 2H), 9.15 (s, 2H), 8.94 (d, J = 1.5 Hz, 1H), 8.79 (dd, J = 5.4 Hz, J = 1.4 Hz, 1H), 8.39 (d, J = 8.1 Hz, 1H), 8.11 (s, 1H), 7.86 (m, 2H), 7.72 (d, J = 7.9 Hz, 1H), 7.49 (d, J = 7.9 Hz, 1H), 7.33 (s, 1H), 7.09 (m, 1H), 5.58 (dd, J = 8.4, 3.8 Hz, 1H), 5.33 (m, 2H), 3.44 (dd, J = 14.1, 3.8 Hz, 1H), 2.89 (dd, J = 14.0, 8.8 Hz, 1H).
Acknowledgements The authors thank the chemistry teams who prepared 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.2019.126675.
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