Imidazotriazine derivatives as il-17 modulators

Abstract

N-[(S)-[3-[1-(2,2-difluoropropylcarbamoyl)-3-hydroxy-3-methyl- cyclobutyl]imidazo[1,2-b][1,2,4]triazin-6-yl]-[4-(trifluoromethyl)cyclohexyl]methyl]-4-5 methyl-1,2,5-oxadiazole-3-carboxamide, or a pharmaceutically acceptable salt and / or solvate thereof, being a potent modulator of human IL-17 activity, is accordingly of benefit in the treatment and / or prevention of various human ailments, including inflammatory and autoimmune disorders.

Description

Via Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025 IN THE UNITED STATES PATENT AND TRADEMARK OFFICE SYSTEMS, DEVICES AND METHODS FOR RANDOMLY FRAGMENTING HIGH-MOLECULAR WEIGHT DNA RELATED APPLICATIONS

[0001] This disclosure claims benefit of and priority to U.S. provisional patent application no. 63 / 703,865, filed October 4, 2024, and entitled “SYSTEMS AND METHODS FOR RANDOMLY FRAGMENTING HIGH-MOLECULAR WEIGHT DNA,” the entire disclosure of which is incorporated herein by reference. BACKGROUND

[0002] Many workflows for genomic DNA sequencing and genomic mapping involve preparation of high-molecular-weight (HMW) genomic DNA followed by fragmentation to generate samples with a size distribution that matches the capabilities of the analysis method (i.e., short-read sequencing, long-read sequencing, optical or electronic genome mapping). Popular fragmentation methods include shearing by passage of the sample through a narrow orifice, sonication, and enzymatic treatment with nucleases. Physical fragmentation methods typically require specialized equipment such as the Diagenode Megaruptor, Covaris Sonicators, or automated pipetting devices (for pipette tip shearing).

[0003] Enzymatic methods are attractive because they do not require specialized equipment. However, they can be problematic if the fragmentation enzymes have high sequence specificity, or if they produce samples with single-strand nicks. However, fragmentation enzymes with high sequence specificity can produce DNA products with poor sequence representation in genomic regions resulting in too many or too few enzymatic cleavage sites. To avoid sequence specificity, some commercial “random” fragmentation products use a cocktail of enzymes which include at least one relatively non-specific nickase, and at least one “resolvase,” definedVia Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025 here as an enzyme that cleaves the intact DNA strand opposite from the position of a single- strand nick. Since the cleavage of each strand is not performed in a concerted fashion, such cocktails can produce DNA products with substantial single-stranded nicks, especially if the cocktail is diluted in an effort to produce large products. Nicked DNA inputs will compromise the read length and yield in long-read sequencing experiments. Moreover, randomly-nicked DNA will contain many noisy labeling sites in a mapping workflow that depends on labeling of sequence-specific nicks, such as that currently used by the Nabsys OhmX electronic mapping system (Oliver et al., 2017; Nabsys website accessed July2024). SUMMARY

[0004] Inventions and embodiments thereof according to the present disclosure address issues of the state of the art (including, in some embodiments, those set out above).

[0005] In some embodiments, Cas9 complexed with guide RNAs are used to target common dinucleotide repeats that are abundant and evenly dispersed throughout mammalian genomes, such as, for example, 5’-CA(10)NGG-3’ and 5’-AT(10)NGG-3’. uHMW (i.e.., ultra-high- molecular-weight, samples fragmented with such Cas9 Oxford Nanopore (“Nanopore”) reads with of the genome.

[0006] In some (HMW) genomic DNA (HMW DNA) is , adding one or more reagents to a / the HMW adding an RNA- guided DNA endonuclease mixture, incubatingthe first mixture to RNA-guided DNA endonuclease, and purifying the fragmented DNA sample.

[0007] Such embodiments (as well as other disclosed embodiments) may include at least one of the following additional features, functionality, structure, steps and clarifications (and in some embodiments, a plurality of, and in some embodiments, a majority of, and in some embodiments, substantially all of, and in some embodiments all of): - the one or more reagents are configured to provide a buffer condition suitable for digestion of the HMW DNA sample by the RNA-guided DNA nuclease; - the RNA-guided endonuclease comprises a Cas9 enzyme assembled with one or more guideVia Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025 RNAs molecules (gRNAs); - a / the Cas9 enzyme is configured to target Cas9 cleavage in the HMW DNA sample; - a / the Cas9 cleavage is configured to cleave DNA at a plurality of repeated genomic sequences within the DNA; - incubating the first mixture is performed under conditions and time for producing a desired extent cleavage of the HMW DNA; - a / the conditions (e.g., for incubation) comprise a temperature; o the temperature is selected from the group consisting of: between 20 and 40 deg. C, between 20 and 35 deg. C, between 20 and 30 deg. C, between 20 and 25 deg. C, between 25 and 40 deg. C, between 30 and 40 deg. C, between 35 and 40 deg. C, and ranges therebetween; - a / the time is selected from the group consisting of: between 15 minutes and 2 hours, between 20 minutes and 2 hours, between 30 minutes and 2 hours, between 45 minutes and 2 hours, between 1 and 2 hours, between 1.25 hours and 2 hours, between 1.5 hours and 2 hours, between 1.75 hours and 2 hours, between 15 minutes and 1.75 hours, between 15 minutes and 1.5 hours, between 15 minutes and 1.25 hours, between 15 minutes and 1 hour, between 15 minutes and 45 minutes, between 15 minutes and 30 minutes, between 15 minutes and 20 minutes, between 20 minutes and 1.75 hours, between 20 minutes and 1.5 hours, between 20 minutes and 1.25 hours, between 20 minutes and 1 hour, between 20 minutes and 45 minutes, between 20 minutes and 30 minutes, between 25 minutes and 2 hours, between 25 minutes and 1.75 hours, between 25 minutes and 1.5 hours, between 25 minutes and 1.25 hours, between 25 minutes and 1 hour, between 25 minutes and 45 minutes, between 25 minutes and 30 minutes, between 30 minutes and 1.75 hours, between 30 minutes and 1.5 hours, between 30 minutes and 1.25 hours, between 30 minutes and 1 hour, between 30 minutes and 45 minutes, between 45 minutes and 2 hours, between 45 minutes and 1.75 hours, between 45 minutes and 1.5 hours, between 45 minutes and 1.25 hours, between 45 minutes and 1 hour, between 1 hour and 2 hours, between 1 hour and 1.75 hours, between 1 hour and 1.5 hours, between 1 hour and 1.25 hours, between 1.25 hours and 2 hours, between 1.25 hours and 1.75 hours, between 1.25 hours and 1.5 hours, between 1.5 hours and 2 hours, between 1.5 hours and 1.75 hours, between 1.75 hours and 2 hours, and ranges therebetween; - a / the RNA-guided DNA endonuclease is configured to recognize one or more classes of repeated genomic sequences; andVia Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025 - a / the RNA-guided endonucleases are selected from the of: Streptococcus pyogenes Cas9 thereof.

[0008] In some genomic DNA (HMW DNA) is adding one or more reagents to a / the a buffer condition suitable for a Cas9 preparation to the HMW the Cas9 preparation comprises a the Cas9 preparation configured toare dispersed throughout the genome to produce a fragmented genomic DNA sample, incubating the HMW DNA - Cas9 mixture under conditions and time for producing a desired extent of Cas9 cleavage, inactivating the Cas9 enzyme, and purifying the fragmented DNA.

[0009] In some embodiments, a kit for enzymatic fragmentation of high molecular weight (HMW) genomic DNA is provided which includes one or more RNA-guided DNA endonucleases, and one or more gRNA molecules for recognizing one or more classes of repeated genomic sequences.

[0010] In such or more reagents, an apparatus, the RNA- guided endonucleases sp. Casl2a (AsCasl2a), or

[0011] In some according to the present disclosure.

[0012] These and other are further described with with the subject disclosure.

[0013] FIG. 1 is a SpyCas9 cleavage with to some embodiments of the

[0014] FIGs. 2A-B forperforming methods according to some embodiments of the present disclosure.

[0015] FIGs. 3A1-A6, and 3B1-B8 are schematic diagrams of a DNA extraction andVia Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025 enzymatic fragmentation workflow (using, e.g., cassette / instrument of FIGs.2A-B), according to some embodiments of the present disclosure.

[0016] FIG.4 shows a graph of DNA output from an extraction device / system (see FIGs.2A- B) for extraction using fragmentation with SpyCas9 complexes according to some embodiments of the present disclosure, which targe certain repeat sequences.

[0017] FIG. 5 illustrates an image of a pulsed field gel analysis of the DNA products of resulting from methods according to some embodiments of the disclosure. DETAILED DESCRIPTION

[0018] FIG. 1 is a histogram graph of fragment sizes predicted to be produced by SpyCas9 cleavage with dinucleotide repeat gRNAs, T2T-CHM13 genome. Specifically, CHM13 genome reference sequence produced by the T2T consortium (CHM13v2.0 / hs1 – Assembly accession ID GCA009914755.4) was queried for gRNA target recognition sequences (CA)10NGG, (AC)10NGG, (AT)10NGG, and (TA)10NGG, using the “findMotif” program (available from the Utilities section of the UC Santa Cruz Genome Browser; (see https: / / hgdownload.soe.ucsc.edu / admin / exe / linux.x86_64; accessed July 2024). The histogram shows the distribution of fragment lengths between gRNA recognition sites over the entire CHM13 genome sequence.

[0019] FIGs. 2A-B show photographs of a cassette 200 (FIG. 2A) for use in a system 250 (FIG.2B) (e.g., Sage Science SageHLS cassette and system / instrument. One of skill in the art will appreciate that any quantity of structure listed is merely exemplary, i.e., one or more. The cassette(s) 200 can include two electrophoresis compartments, each containing (1) an agarose gel column (channel 202), (2) a set of six membrane-delimited elution modules 204 in contact with the right side of the gel column, (3) an electrophoresis buffer compartment around the perimeter of the gel columns and the elution modules, (4) a pair electrode ports at the ends of the gel column, and (5) a pair of long electrode ports at the left and right boundaries of theelectrophoresis compartments. The gel column has two input ports at end. The smaller input well 206 (“Sample well”) is used to load a suspension of nuclei for electrophoretic extraction, and the larger input well 208 (“Reagent well”) is used to load an SDS-based lysis reagent. Extraction is a carried out by electrophoresing the SDS lysis reagent down through the sample well, where the nuclei are lysed and the HMW DNA is driven into the agarose wall ofVia Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025 the sample well, where it becomes immobilized due to its large size (typically >>5Mb). After electrophoretic extraction and enzymatic digestion (as described in Example 2), size selection electrophoresis of the digested DNA is carried out, thereby moving the products down the agarose column. After size selection electrophoresis, the products are electroeluted into the elution modules at the right side of the gel column, using long wire electrodes that are inserted into the ports on the left and right boundaries of the electrophoresis compartment. After electroelution, the size-selected DNA products (in electrophoresis buffer) are collected from the elution modules using pipetting devices. See also, e.g., U.S. patent nos. 11,867,661, 11,542,495 and 10,738,298, together “the Patents”, the entire disclosures of which is incorporated herein by reference.

[0020] The instrument 250 includes compartments split between the lid and the base (ref.252 and 254 being arranged in the lid and including electrodes integrated therein (not shown) for applying electrical currents to the gel / wells of the cassettes 200.

[0021] FIGs. 3A-B are schematic diagrams of exemplary DNA extraction and enzymatic fragmentation workflows (e.g., SageHLS DNA extraction), in which more details are provided in Example 2 below. In FIGs.3A1-A6 are high level flow / structure diagrams where each image represents a single lane of an HLS cassette at various stages of the workflow, for obtaining fragmented HMW DNA and cleaving it using SpyCas9 cleavage at genomic repeats (see also, Id., the above-identified incorporated by reference disclosures, including U.S. patent no. 11,867,662, FIGS. 7A-10B, and written description, including col. 5, lines 23-38 for other general characteristics). As shown in FIG. 3A1, a sample can be loaded into the sample well 302a, and a regent(s) added to reagent well 304a. The reagent is driven by electrophoresis into / through the gel 306a and into / through the sample well (see FIG. 3A2). In FIG. 3A3, SpyCas9 cleavage is added to the sample well 302a, and at FIG. 3A4, SDS purification as added to the reagent well 304a. In FIG. 3A5, final purification and size selection via electrophoresis is performed, and the cleaved fragments are then driven laterally via electrophoresis into elution modules 308a.

[0022] FIGs. 3B1-B8 illustrate a cross-sectional / side views of agarose column within a gel cassette (see FIG. 2A). In FIG. 3B1, a regent well 304b (SDS) having at least one reagent is arranged adjacent a cell sample well 302b containing a cell sample. Through electrophoresis, the reagent(s) in 304b are driven into and through well 302b, as shown in FIG.3B2 and 3B3, resulting in lysis without shear force. As shown in FIG.3B4, 3B5, since DNA 306b (i.e., HMWVia Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025 DNA) too large to undergo electrophoresis becomes immobilized in the wall of the sample well, and SDS-coated contaminates 308b are removed.

[0023] Accordingly, as shown in FIG. 3B5-3B6, in the empty sample well (where the HMW DNA is immobilized in the wall thereof), a Cas9 preparation is added to the sample well 302b (which in some embodiments includes a Cas9 enzyme assembled with one or more guide RNA molecules). As shown in FIG.3B7, limited DNA digestion is allowed to occur. Accordingly, thereafter, in FIG. 3B8, electrophoretic purification, size selection and elution are performed. As shown, the fragmented DNA 312b is within the gel, and the cleavase / reagents 310b in the gel as well.

[0024] Figure 4 shows a graph of DNA output from an extraction device / system (e.g., SageHLS) for extraction using fragmentation with SpyCas9 complexes targeting dinucleotide repeat sequences: (CA)10NGG, (AC)10NGG, (AT)10NGG, and (TA)10NGG. Details of the experiment are outlined in Example 2.

[0025] Figure 5 illustrates an image of a pulsed field gel (CHEFMapper, Bio-Rad, Richmond, CA, USA) analysis of the DNA products of the experiment described in Example 2 and Figure 4. “w” =Size markers used were H. wingei chromosome ladder (Bio-Rad). “L” = Phage lambda concatemer ladder (Bio-Rad), “1k” = 1kb Extend ladder (New England Biolabs). Fragmentation via RNA-guided endonuclease cleavage at genomic repeat sequences

[0026] The major classes of human repeat sequences were catalogued in the original draft of the human genome (Lander et al., 2001). A large portion of the human genome is composed of transposon-derived sequences, including SINES, LINES, LTR retrotransposons, and DNA transoposons. Other types of highly repeated sequences in the human genome are microsatellite sequences, also known as simple-sequence repeats, such as mono-, di-, and tri-nucleotide repeating sequences. These repeat types are also observed in virtually all other organisms, procaryotes and eucaryotes, but the abundance of specific repeat types in any given species can vary widely. All of these types of repeats can be used in the disclosed methods (according to some embodiments), the subject disclosure focuses on use of dinucleotide repeats of the human genome as non-limiting examples to describe and exemplify such embodiments.

[0027] Accordingly, in some embodiments, high specificity of RNA-guided endonucleases, such as SpyCas9, are used to cleave at highly repeated sequences in genomic DNA under study. Thus, such embodiments can be adapted to generate genomic DNA product with different size distributions in several ways.Via Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025

[0028] In some embodiments, by targeting repeat elements with a high copy number in the genome, fragmented DNA products with a relatively small size can be produced. Examples of highly repeated sequence elements in the human genome include SINE and LINE elements which are present at several hundred copies per megabase (Lander et al., 2001). Similarly, targeting repeat elements with a lower copy number in the genome can generate a population of larger DNA fragments. Examples of repeated sequence elements in the human genome with a lower copy number include the simple sequence dinucleotide repeats, which are present at about 20 copies per megabase (Lander et al., 2001), as discussed in the next section. In either case, the size of the digestion product can be fine-tuned by adjusting the endonuclease to DNA ratio.

[0029] In general, in some embodiments, complete digestion of specific SpyCas9 target sites can be achieved using a slight molar excess of SpyCas9 to the molar concentration of specific target sites in the input DNA (in some embodiments, approximately 5-10x excess in SpyCas9 concentration over the molar concentration of input DNA target sites, and ranges therebetween). In situations where partial cleavage is desired, an optimum SpyCas9 / input DNA ratio can be determined in test digestions using decreasing enzyme / DNA ratios, since the efficiency of cleavage varies for different gRNA recognition sequences. Fine-tuning product size by limiting amounts of endonuclease is a convenient method when using SpyCas9, since that enzyme does not turn over after cleavage, as it remains tightly bound to the cleaved DNA substrate (Yourik et al., 2019; Jinek et al., 2012).

[0030] In general, in some embodiments, the use of RNA-guided endonucleases that recognize genomic repeat elements to fragment HMW DNA into a size distribution suitable for a particular type of genomic analysis is provided. The desired product size can vary according to such an analysis method. For example, for genomic DNA sequencing using the PacBio Revio platform, a desired size range for genomic sequencing libraries is from approximately 10kb to 25kb, with the peak of DNA mass at approximately 20kb. In contrast, some sequencing applications using the Oxford Nanopore sequencing platforms benefit from using DNA greater than 10kb in size, while other Nanopore methods benefit from using DNA samples greater than 100kb in size. Long range optical or electronic mapping methods such as those marketed by Bionano Genomics and Nabsys benefit from DNA samples with a minimum size of 50kb with the bulk of the fragments greater than 100kb.

[0031] To generate DNA products with the above-noted size ranges, the input HMW for theVia Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025 fragmentation process should be several-fold larger than the desired product size. For example, to generate PacBio libraries with a median 20kb size, the input HMW DNA for fragmentation using the inventive method should ideally be greater than 60kb in size. To generate DNA product for the long-range mapping methods of Bionano Genomics and Nabsys, the input HMW DNA should ideally be greater than 200-300kb in size.

[0032] The catalog of human repeats in the original human genome draft show that the most abundant dinucleotide repeats (with lengths >50bp) were (CA)n and (AT)n, at an abundance of 27.7 and 19.4 copies per 1Mb of genomic sequence, respectively. A fraction of these sites will be bound by a short sequence, the PAM sequence, which can be recognized by an RNA-guided nuclease. For the SpyCas9 enzyme, with a PAM sequence of 5’NGG3’, based on random sequence distribution, it is expected that 6.25% of these repeats would be bound by an NGG PAM (P = 1 x 0.25 x0.25 = 0.0625). On this basis, it can be predicted that gRNAs targeting (CA)n or (AT)n repeats should be present at a frequency of approximately 2.94 copies per 1Mb (see FIG. 1). The predicted average spacing between SpyCas9 cleavage sites using such gRNAs, in some embodiments, is approximately 340,000bp. Genomic DNA samples with this size are recommended for long-read nanopore sequencing (Oxford Nanopore platform) and genomic mapping experiments using optical or electronic sensing (Bionano Genomics and Nabsys platforms).

[0033] To assess this prediction, the CHM13 sequence produced by the T2T consortium (CHM13v2.0 / hs1 – Assembly accession ID GCA009914755.4) was queried for gRNA target recognition sequences (CA)10NGG, (AC)10NGG, (AT)10NGG, and (TA)10NGG, using the “findMotif” program (available from the Utilities section of the UC Santa Cruz Genome Browser; (see, e.g., https: / / hgdownload.soe.ucsc.edu / admin / exe / linux.x86_64; accessed July 2024). The four (4) gRNA target sites divide the CHM13 genome into 2927 fragments. 2847 of the fragments have a size <4Mb and contain approximately 83% of the CHM13 genome sequence. 80 fragments are >4Mb in size and contain about 17% of the genome. The average spacing between all of the dinucleotide target sites was 1Mb, in rough agreement with the statistics of the previous paragraph. However, previous studies on use of SpyCas9 in vitro for targeted isolation of large genomic fragments have demonstrated that off-target cutting typically exceeds on-target cutting by at least a factor of 10 (Shin et al., 2019). Off-target cutting can produce a larger fraction of the genome in fragments of practical size (1Mb or less for long range mapping or Nanopore sequencing) (Zhou et al., 2024).Via Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025

[0034] To achieve smaller average fragment sizes for long-read libraries used by the PacBio platform, i.e., approximately ~20,000 (for example), the gRNA pool can be supplemented with gRNAs that target more abundant repeat families such as the human DNA MER1-Charlie transposons which are present at approximately 182,000 copies per genome (Lander et al., 2001).

[0035] Since repeat sequences are not evenly spaced throughout the genome, it is expected that there will be some genomic regions with closely spaced repeat sequence targets and some regions that lack repeat sequence targets. Accordingly, in some embodiments, two strategies can be blended to address such challenges. In particular, a partial digest strategy can be used to address the problem of sequence loss due to close spacing of repeat targets. Digestion using less specific RNA-guided endonucleases or using reaction conditions or gRNA designs that increase the off-target cutting by the RNA-guided endonuclease, can improve recovery of sequence from genomic regions that lack exact copies of the repeat sequence gRNA targets.

[0036] In some embodiments, an advantage of using RNA-guided endonucleases such SpyCas9 and AsCas12a for genomic fragmentation is that the enzymes make concerted double- stranded breaks with very little production of single-stranded cleavages (Yourik et al. 2019; Jinek et al, 2012, Swartjes et al., 2019). The lack of single-stranded cleavages increases read length and quality in long-read sequencing, and it also decreases background labeling in workflows that introduce labels with site-specific nickases, such as the labeling process used by the Nabsys OhmX electronic mapping workflow (Oliver et al.2017).

[0037] The following examples help detail processes according to some embodiments of the disclosure. Example 1: Preparation of Nuclei from Coriell cell line GM24385 (HG002)

[0038] Cells were decanted from culture flask into 50mL conical centrifuge tubes and washed 2-3 times by centrifugation and resuspension in 25-40mL of Ca-Mg-free phosphate-buffered saline (PBS) to remove media. Centrifugation was conducted at 200xg for 5 minutes at 4C. After washing, cells were resuspended in cold PBS at approximately 1 mL per 1e09 cells. Nine (9) mL of cold nuclear prep buffer (NPB) was added rapidly to the concentrated cells in PBS, and the cells were then vortexed at medium speed (70% of maximum rpm, Fisherbrand benchtop vortex mixer) to rapidly disperse and lyse the cells and liberate the nuclei therefromVia Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025 (NPB = 25 mM Tris, pH 8.0, 5mM EDTA, 0.5% Triton X100, 500 mM sucrose, 4mM spermidine-3 HCl).

[0039] A second 10mL volume aliquot of NPB was added to the tube and it was mixed again thoroughly by medium speed vortexing. After this mixing step, the mixture was centrifuged for 2 minutes at 5,000 x g to pellet the nuclei. The supernatant was decanted, and the bottom of the tube was flicked to loosen the nuclear pellet. The loosened pellet was resuspended in approximately1-2ml NPB. The nuclear suspension was pipetted up and down to using a regular orifice 1000ml pipette tip to ensure complete resuspension. Nine (9) mL of cold NPB was added to the suspension, and it was mixed again by medium speed vortexing. The nuclei were pelleted again by centrifugation (2 minutes at 5,000 x g), the supernatant was decanted, and the final nuclear pellet was resuspended by flicking and pipetting in 1 mL of cold NPB. Nuclei were held at 4C until DNA extraction.

[0040] Measurement of the genomic DNA concentration of the nuclear suspension

[0041] 10uL aliquots or the nuclear suspension were then transferred in duplicate to 1.5 ml microcentrifuge tubes. 190uL of Qubit Lysis Buffer (Sage Science, 51mM Tris base, 29mM TAPS acid, 5mM EDTA, 1% SDS, 50mM NaCl, pH 8.7) was added rapidly and mixed by vigorously pipetting up and down several times. The tubes were vortexed at top speed for 30 seconds.600uL of TE (10 mM TrisHCl, 1mM EDTA, pH 7.5 to 8.0) was added to each tube, and the tubes were vortexed again at maximum speed for 30 seconds. Five (5) μl of the diluted lysed samples were assayed for DNA using the Qubit HS reagent kit (Invitrogen). The “tube concentration” reading obtained from the Qubit fluorimeter was used to calculate the DNA concentration of the original nuclear suspension, using the following dilution factors: - [Qubit tube conc, ng / ml] X (800 / 10) X (200 / 5) = [DNA conc original cell suspension, ng / ml]

[0042] For HMW DNA extraction (see below), the nuclear suspension was diluted to a DNA concentration of 143 ng / μL in NPB. Example 2: Semi-automated HMW DNA extraction using SageHLS instrument with dinucleotide repeat gRNAs and SpyCas9.

[0043] Preparation of the dinucleotide gRNA-SpyCas9 complexes:

[0044] The procedure below provides enough gRNA-SpyCas9 complex for 1 HLS sampleVia Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025 lane. Two-part gRNAs were purchased from IDT (Coralville, IA). The four crRNAs were designed to recognize alternating CA and AT dinucleotide repeats that are common in the human genome: (CA)10NGG, (AC)10NGG, (AT)10NGG, and (TA)10NGG. All gRNA and tracrRNA were dissolved at 100uM concentration in IDT Duplex buffer (IDT, Coralville, IA).

[0045] Two-part gRNA annealing:

[0046] The following components were mixed in a 200ul PCR tube: - 7.7 μl IDT Duplex buffer - 2 μl of a pooled equimolar mixture of all 4 dinucleotide crRNAs. - 1.3 μl of tracrRNA (100 μM).

[0047] The 11uL mixture was vortexed briefly and spun down in a microfuge. Concentrations after this step = 11.8 μM tracrRNA, 18 μM total pooled crRNA. The crRNA-tracrRNA mixture was heated for 5 minutes at 95C in a thermal cycler with a heated lid and allowed to cool on the lab bench at room temperature for 5 minutes. After cooling the tube was centrifuged for 30 seconds to collect condensation.

[0048] Assembly of annealed gRNA with SpyCas9:

[0049] Wild-type Cas9 was obtained from New England Biolabs (Ipswich, MA. Sage 1X Enzyme Buffer is 51mM Tris base, 29mM TAPS acid, 0.1mM EDTA acid, 10mM MgCl2, 32mg / ml Hydroxypropyl-beta-cyclodextrin, 50 ug / ml BSA.). The following components were mixed thoroughly by gentle pipetting (the components are viscous). - 5 μl Sage HLS 4X Enzyme Buffer - 11 μl annealed IDT guide RNAs - 4 μl wild-type S. pyogenes Cas9 enzyme (20uM, New England Biolabs #M0386M).

[0050] The mixture was incubated at 37C for 5 minutes. Concentrations at this stage were 4uM SpyCas9, 6.5uM annealed gRNA. The assembled enzyme mixture (20uL) was diluted with 60uL 1X Sage HLS Enzyme Buffer and mixed well by gentle pipetting. At this point, the Cas9 diluted mixture was stored at 4C until the time to initiate Cas9 digestion. 80ul of the diluted enzyme mixture was used in each HLS lane.

[0051] HMW extraction in the HLS system using SpyCas9 with dinucleotide repeat gRNAs

[0052] HMW DNA was extracted from aliquots of GM24385 nuclei containing 10ug ofVia Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025 genomic DNA (70uL of nuclear suspension in NPB at 143 ng / μl) using the electrophoretic SageHLS system (Sage Science, Inc., Beverly, MA, USA; see also the Patents, herein incorporated by reference) using the manufacturer’s instructions for the HLS-CATCH procedure (see “HLS-CATCH Workflow Guide”, available at https: / / sagescience.com / product- support / sagehls-support / ; accessed July 2024). Pictures of the HLS system which can be used are shown in Figures 2A-B. An HLS-CATCH workflow is also depicted schematically in FIGs.3A-3B.

[0053] Briefly, the procedure was as follows: - An aliquot of the nuclei suspension (70uL, 10ug total DNA) was loaded into the sample well; - 200uL of 1% SDS lysis buffer was loaded into the reagent well, upstream from sample well; and - DNA extraction was performed by electrophoresing the SDS lysis buffer downward through the sample well and gel column for three (3) hours using a constant field of 55V; - The SDS lyses the nuclei as it passes through the sample well, encapsulates non-DNA cellular components into negatively charged particles, and causes them to move down the gel column into lower electrode chamber.

[0054] During the three (3) hour extraction electrophoresis, the HMW genomic DNA was trapped and immobilized just inside the downstream wall of the sample well, due to its large size and the absence of any viscous shear during the extraction process.

[0055] At the end of the 3hr extraction period, the sample well was emptied and refilled with the SpyCas9-gRNA complexes (at a total concentration of 1uM). The complexes were electrophoresed into the sample well wall for 2 minutes at 80V constant field to ensure good mixing of the SpyCas9 complexes with the immobilized genomic DNA. The sample well was emptied and refilled with 1X HLS Enzyme buffer, and the genomic DNA in the sample well wall was digested for 30 minutes (without electrophoresis) at room temperature.

[0056] After digestion, the sample was subjected to size-selection electrophoresis down the agarose column using constant field 55V for 1.5 hours, followed by electroelution for 1.5 hours (at 50V constant field) into the six buffer-filled elution modules to the right side of the gel column.Via Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025

[0057] The eluted samples were evaluated by Qubit DNA assays and pulsed-field agarose gel analysis (Bio-Rad CHEFMapper).

[0058] For the data shown in FIG. 4, HMW extractions on GM24385 nuclei (10ug genomic DNA per lane) in quadruplicate (2 HLS cassettes were used, each cassette has two extraction lanes). SpyCas9-gRNA complexes using all four of the crRNAs recognizing (CA)10NGG, (AC)10NGG, (AT)10NGG, and (TA)10NGG, were used in each lane. The DNA products in the elution modules of lanes 2 and 4 were removed and sheared by 10 cycles of rapid up and down pipetting with a narrow bore 200uL pipette tip, in order to thoroughly homogenize any HMW DNA present. DNA concentration measurements were performed using duplicate 10uL aliquots in the Qubit HS fluorometer system (Invitrogen). The data is shown in FIG. 3. The yield of DNA was greater than 0.5ug per elution module (EM) in EMs 2-4 of lane 2 (total yield EMs 2- 4 = 4.9 ug) and EMs 3-5 of lane 4 (total yield EMs 3-5 = 4.2ug).

[0059] To examine the length of the DNA in the peak fractions, we analyzed the unsheared EM contents from the same cassette (HLS lanes 1 and 2 are located in the same cassette; lanes 3 and 4 are located in the same cassette by pulsed field electrophoresis. As seen in FIG.5, peak fractions of lanes 1 and 3 showed HMW DNA ranging in size from 30kb to just under 1Mb, with maximum fluorescence coming from fractions between 100 and 400kb in size. Such preparations are well suited for long-read Oxford Nanopore sequencing and genomic mapping using optical (Bionano Genomics) or electronic readouts (Nabsys). Example 3: Fragmentation of HMW DNA prepared using the gel-plug method

[0060] The CATCH protocol of Gabrieli et al., 2018 was used to (1) purify human peripheral blood mononuclear cells (PBMCs) from human whole blood, (2) embed the isolated PBMCs in low melting temperature agarose gel plug, and (3) extract and purify HMW DNA in the gel plugs by repetitive washing and incubation with detergent solutions and digestion with Proteinase K. After purification and washing of the gel plugs, they were incubated with SpyCas9 complexed with gRNAs containing crRNAs recognizing (CA)10NGG, (AC)10NGG, (AT)10NGG, and (TA)10NGG. After SpyCas9 digestion, the gel plugs were redigested with Proteinase K to remove the SpyCas9, and then washed in TE buffer to remove the Proteinase K. Finally, the gel plugs were melted by brief incubation at 70C, followed by digestion of the agarose at 43C using agarase (ThermoFisher Scientific). After agarose digestion, the fragmented HMW DNA was precipitated with isopropanol, and the HMW DNA pellet wasVia Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025 washed with 80% ethanol, dried, and resuspended in 10mM Tris-HCl, 1mM EDTA, pH 8. Example 4. method

[0061] Ultra-HMW Monarch HMW DNA Extraction Kit (New England Biolabs, Ipswich, MA, were digested with SpyCas9 (AT)10NGG, and (TA)10NGG recognition 10 ug of HMW DNA were carried out using SpyCas9concentrations ranging between 30nM to 1uM, depending on whether partial or extensive digestion was desired. After digestion, 1ul of Proteinase K (20mg / ml, NEB) is added to the reaction and incubation at 37C is continued another hour to remove the SpyCas9. After Proteinase K digestion, the fragmented HMW DNA was precipitated with isopropanol, and the HMW DNA pellet was washed with 80% ethanol, dried, and resuspended in 10mM Tris-HCl, 1mM EDTA, pH 8. General Considerations

[0062] While various herein, those of ordinary skill in the components, and / or structures for thereof) disclosed herein, and / or described herein, and each of such the scope of the inventive art will readily appreciate that allconfigurations described herein are meant to be merely an example and that the actual parameters, amounts, dimensions, materials, steps, and configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is therefore to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of claims supported by the subject disclosure and equivalents thereto, and inventive embodimentsVia Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025 may be practiced otherwise than as described and claimed. Inventive embodiments of the present disclosure system, article, material, kit, step, In addition, any combination of two or kits, steps, functions / functionality, kits, steps, functions / functionality, within the inventive scope of the

[0063] Embodiments or more features, components, materials, and / or methods, to yield yet other may be distinguishable from the feature disclosed in the particular prior art be distinguishable from the prior art by

[0064] Also, as noted, various inventive concepts may be embodied as one or more methods, of which one or more examples have been provided. The acts performed as part of the method(s) may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include performing some acts simultaneously, even though shown as sequential acts in illustrative embodiments.

[0065] Any and all references to publications or other documents, including but not limited to, patents, patent applications, articles, in the present application, are herein incorporated by all definitions, as defined and used herein, should be definitions, definitions in documents incorporated by defined terms.

[0066] The indefinite articles “a” andand in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” The terms “can” and “may” are used interchangeably in the present disclosure, and indicate that the referred to element, component, structure, function, functionality, objective, advantage, operation, step, process, apparatus, system, device, result, or clarification, has the ability to be used, included, or produced, or otherwise stand for the proposition indicated in the statement for which the term is used (or referred to) for a particular embodiment(s).

[0067] The phrase "and / or" as used herein in the specification and in the claims, should beVia Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025 understood to mean "either or both" of i.e., elements that are conjunctively present in some cases and cases. Multiple elements listed with "and / or" should be construed or more" of the elements so conjoined. Other elements may the elements specifically identified by the "and / or" clause,elements specifically identified. Thus, as a non-limiting example, a reference to "A and / or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0068] As used herein in the be understood to have the same meaning as "and / or" as items in a list, "or" or "and / or" shall be interpreted as of at least one, but also including more than one, of a optionally, additional unlisted items. Only terms clearlyone of' or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e. "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of" "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0069] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and within the list of elements and not excluding any This definition also allows that elements may specifically identified within the list of elements to which related or unrelated to those elements specifically"at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and / or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at leastVia Patent Center Attorney Docket No.: SAGS-024 / 001WO 43649-02120 / WO Date of Deposit: October 3, 2025 one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0070] In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "involving," "holding," "composed of," and the like are to be i.e., to mean including but not limited to. Only the transitional essentially of’ shall be closed or semi-closed transitional forth in the United States Patent Office Manual of Patent ExaminingReferences:

[0071] Lander, et al.2001. Nature 409: 860-921.

[0072] Yourik, et al.2019. RNA 25:35-44.

[0073] Jinek, et al.2012. Science 337:816-

[0074] Swartjes, et al.2019. Biochemical :207-219.

[0075] Oliver et al.2017. bioRxiv.

[0076] Zhou et al.2024. Proc Natl Acad Sci USA. https: / / doi.org / 10.1073 / pnas.2322834121.

[0077] Shin et al.2019. Nucleic Acids Res. https: / / doi:10.1093 / nar / gkz661.

[0078] Gabrieli et al.2018. Nucleic Acids Res. https / / doi:10.1093 / nar / gky411.HPLC; or column chromatography utilising, for example, silica and / or alumina in conjunction with an appropriate solvent system. Where the above-described processes for the preparation of the compounds according to the invention give rise to mixtures of stereoisomers, these isomers may be separated by conventional techniques. In particular, where it is desired to obtain a particular enantiomer of a compound of formula (I) this may be produced from a corresponding mixture of enantiomers using any suitable conventional procedure for resolving enantiomers. Thus, for example, diastereomeric derivatives, e.g. salts, may be produced by reaction of a mixture of enantiomers of formula (I), e.g. a racemate, and an appropriate chiral compound, e.g. a chiral base. The diastereomers may then be separated by any convenient means, for example by crystallisation, and the desired enantiomer recovered, e.g. by treatment with an acid in the instance where the diastereomer is a salt. In another resolution process a racemate of formula (I) may be separated using chiral HPLC. Moreover, if desired, a particular enantiomer may be obtained by using an appropriate chiral intermediate in one of the processes described above. Alternatively, a particular enantiomer may be obtained by performing an enantiomer-specific enzymatic biotransformation, e.g. an ester hydrolysis using an esterase, and then purifying only the enantiomerically pure hydrolysed acid from the unreacted ester antipode. Chromatography, recrystallisation and other conventional separation procedures may also be used with intermediates or final products where it is desired to obtain a particular geometric isomer of the invention. Alternatively the non desired enantiomer may be racemized into the desired enantiomer, in the presence of an acid or a base, according to methods known to the person skilled in the art, or according to methods described in the accompanying Examples. During any of the above synthetic sequences it may be necessary and / or desirable to protect sensitive or reactive groups on any of the molecules concerned. This may be achieved by means of conventional protecting groups, such as those described in Greene’s Protective Groups in Organic Synthesis, ed. P.G.M. Wuts, John Wiley & Sons, 5thedition, 2014. The protecting groups may be removed at any convenient subsequent stage utilising methods known from the art. In another aspect, the present invention provides a synthetic intermediate of formula (II). In a another aspect, the present invention provides compounds of formula (IIA), namely 1-[6-[(S)-amino-[4-(trifluoromethyl)cyclohexyl]methyl]imidazo[1,2- b][1,2,4]triazin-3-yl]-N-(2,2-difluoropropyl)-3-hydroxy-3-methyl- cyclobutanecarboxamide (syn and anti stereoisomers), represented respectively by Formula (IIA’) and (IIA”):. In a particular embodiment according to this aspect, the present invention provides compounds of formula (IIA’), the syn stereoisomer of 1-[6-[(S)-amino-[4- (trifluoromethyl)cyclohexyl]methyl]imidazo[1,2-b][1,2,4]triazin-3-yl]-N-(2,2- difluoropropyl)-3-hydroxy-3-methyl-cyclobutanecarboxamide.It will be appreciated that compounds of formulae (IIA) (specifically (IIA’) and (IIA’’)) are sub-formulae of the compound of formula (II). Any reference to compound of formula (II) contained herein, will therefore include compounds of formulae (IIA). In yet another aspect, the present invention relates to the use of intermediates of formula (II) for the synthesis of compounds of formula (I). Included as an aspect of the invention are salts such as pharmaceutically acceptable salts and / or solvates of compounds of formula (II). BIOLOGICAL ACTIVITY The compound of formula (I) potently inhibits IL-17 induced IL-6 release from human dermal fibroblasts. Therefore, the effect of the compound of formula (I) on IL-17 induced IL-6 from human dermal fibroblasts release was assessed in two independent cell assays. Thus, when tested in either the Primary Human Dermal Fibroblast cell assay or the Primary Th17-Human Dermal Fibroblast Co-culture cell assay as described below, the compound of formula (I) generally exhibits a pIC50 value in excess of 6.0 (pIC50 equals - log10[IC50], in which IC50is expressed as a molar concentration, so the skilled person will appreciate that a higher pIC50 figure denotes a more active compound). Additionally, the compound of formula (I) has advantageous “on-rates” when tested in a relevant assay, such as Surface Plasmon Resonance (SPR). SPR is a technique for analysis of the interactions of two biomolecules with respect to binding kinetics and affinity as well as binding specificity and the "on-rate" is a measure of how quickly a compound can bind its target protein / receptor. EXPERIMENTAL SECTION The following Examples illustrate the preparation of compounds according to the invention. I. ABBREVIATIONS Abbreviations used herein are defined below. Any abbreviations not defined are intended to convey their generally accepted meaning.DCM: dichloromethane MeOH: methanol THF: tetrahydrofuran EtOAc: ethyl acetate DMSO: dimethyl sulfoxide DIPEA: N,N-diisopropylethylamine HCl: hydrochloric acid EDCI.HCl: N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid Hexafluorophosphate TMEDA: N,N,N’,N’-tetra-methylethylene diamine LiHMDS: lithium bis(trimethylsilyl)amide DDQ: 2,3-dichloro-5,6-dicyano-1,4-benzoquinone h: hour r.t.: ambient temperature M: mass; molar RT: retention time HPLC: High Performance Liquid Chromatography LCMS: Liquid Chromatography Mass Spectrometry NMR: Nuclear Magnetic Resonance Naming convention: IUPAC names of chemical reagents, Intermediates and Examples have been generated using (Biovia Draw 2024) version 24.1 (24.1.0.1865). Depending on the Kekule structures of chemical reagents, Intermediates and Examples, Bovia Draw may generate different chemical names. II. ANALYTICAL AND SYNTHETIC METHODS All reactions involving air or moisture-sensitive reagents are performed under a nitrogen atmosphere (inert atmosphere) using dried solvents and glassware. Experiments requiring microwave irradiation are performed on a Biotage Initiator Sixty microwave oven upgraded with version 2.0 of the operating software. Experiments are run to reach the required temperature as quickly as possible (maximum irradiation power: 400 W, noexternal cooling). Commercial solvents and reagents are generally used without further purification, including anhydrous solvents when appropriate (generally Sure-Seal™ products from Aldrich Chemical Company or AcroSeal™ from ACROS Organics). In general, reactions are followed by thin layer chromatography (TLC), high performance liquid chromatography (HPLC) or mass spectrometry (MS) analyses. NMR spectra were recorded on a Bruker Advance III HD 300 MHz or 400 MHz spectrometer. The chemical shifts (6) reported are given in parts per million (ppm), and the coupling constants (J) are in Hertz (Hz). The spin multiplicities are reported as s = singlet, bs = broad singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublet, ddd = doublet of doublet of doublet, dt = doublet of triplet, td = triplet of doublet, and m = multiplet. Mass spectrometric measurements in LC-MS mode are performed as follows: Method 1 Stationary phase: Phenomenex Gemini NX-C182 x 20 mm, 3 μM Mobile Phase A: 10 mM Ammonium formate in water + 0.1% Ammonia solution Mobile Phase B: Acetonitrile + 5% water + 0.1% Ammonia Solution Flow rate: 1 mL / min Gradient program: Time A% B% 0.00 95.00 5.00 1.50 5.00 95.00 2.25 5.00 95.00 2.50 95.00 5.00 Method 2 Stationary phase: Waters Acquity UPLC BEH C182.1 x 50 mm, 1.7 μM column Mobile Phase A: 10 mM Ammonium formate in water + 0.1% Ammonia solution Mobile Phase B: Acetonitrile + 5% water + 0.1% Ammonia Solution Flow rate: 1.5 mL / min Gradient program: Time A% B% 0.00 95.00 5.00 0.10 95.00 5.00 3.50 5.00 95.00 4.00 5.00 95.00 4.05 95.00 5.00When analytical methods are not specified in the below protocols, the methods used were similar to the ones described above. It will be apparent to the person skilled in the art that there are analytical and preparative chromatographic methods analogues to the ones described above can be use for the below procedures. III. INTERMEDIATES INTERMEDIATE 1: Methyl 3-hydroxy-3-methyl-cyclobutanecarboxylateTo a solution of methyl 3-oxocyclobutanecarboxylate (50g, 380 mmol) in anhydrous THF (1.1 L) under nitrogen cooled to -72oC was added dropwise methylmagnesium chloride (3.0M in THF, 150mL, 450 mmol) such that the internal temperature was maintained below -65oC. Following addition, the reaction mixture was stirred at -70oC for 2 hours before being allowed to warm to ambient temperature and stirred for 18 hours.The reaction mixture was cooled to -60°C and quenched with saturated ammonium chloride solution (150 mL). The resulting mixture was diluted with water (500 mL) and iso-hexane (150 mL) and stirred at room temperature for 30 minutes. The aqueous layer was separated, extracted with tert-butyl methyl ether (250 mL x 2), the combined organic extracts dried over sodium sulfate, filtered and concentrated in vacuo to a pale residue which was purified by chromatography on silica with a gradient of 0% to 70% ethyl acetate in iso-hexane to afford the title compound as a colourless oil and a mixture of diastereoisomers.1H NMR (300 MHz, CDCl3) δ 3.69 (s, 3H), 2.77 – 2.60 (m, 1H), 2.36 – 2.28 (m, 4H), 2.27 (s, 1H), 1.37 (s, 3H). INTERMEDIATE 2: Methyl 3-hydroxy-3-methyl-1-(3-methylsulfanyl-4,5-dihydro- 1,2,4-triazin-5-yl)cyclobutanecarboxylateA mixture of 3-methylthio-1,2,4-triazine (24.5g 189 mmol), methyl 3-hydroxy-3-methyl- cyclobutanecarboxylate (prepared according to the procedure of Intermediate 1) (41g, 280 mmol) and TMEDA (145mL, 951 mmol) in anhydrous toluene (200 mL) under nitrogen was cooled to 0oC. To the mixture was added a solution of LiHMDS (1.0M in toluene, 1.1 L, 1100 mmol) via a dropping funnel at a rate such that the internal temperature was maintained below 10oC. Following addition, the mixture was stirred at 0oC for 5 minutes before being warmed to 33oC and stirred for 24 hours. The reaction mixture was cooled to 0°C and quenched with saturated ammonium chloride solution (700 mL) and water (700 mL). The separated aqueous layer was extracted with ethyl acetate (700 mL x 2), the combined organic extracts washed with brine (700 mL), dried over sodium sulfate, filtered and concentrated in vacuo to give a dark brown residue.1H NMR (300 MHz, DMSO) indicated product as an approximate 1:1 mixture of diastereoisomers. LC-MS Method 1 indicated product as a mixture of diastereoisomers at RT: 0.53 minutes (minor desired syn diastereomer) and 0.64 min (major undesired anti diastereomer), 272.0 (M+H), in an approximate 55:45 ratio. The crude product was purified by chromatography on silica eluting with a gradient of 0% to 70% ethyl acetate in iso-hexane to provide the title compound (syn diastereomer with alcohol and ester on the same face of the cyclobutane) (11.5 g, 23%) as a yellow solid.1H NMR (300 MHz, DMSO) δ 10.61 (s, 1H), 6.62 (d, J = 1.9 Hz, 1H), 4.81 (s, 1H), 3.70 - 3.52 (m, 4H), 2.46 - 2.36 (m, 2H), 2.34 – 2.24 (m, 4H), 2.21 - 2.10 (m, 1H), 1.22 (s, 3H).LC-MS (ESI+): Method 1 RT 0.53 minutes m / z = 272.2 (M+H).INTERMEDIATE 3: Methyl 3-hydroxy-3-methyl-1-(3-methylsulfanyl-1,2,4-triazin-5- yl)cyclobutanecarboxylateTo a solution of methyl 3-hydroxy-3-methyl-1-(3-methylsulfanyl-4,5-dihydro-1,2,4- triazin-5-yl)cyclobutanecarboxylate (prepared according to the procedure of Intermediate 2) (11.4 g, 42.0 mmol) in DCM (300 mL) cooled to 0oC was added DDQ (11g, 47 mmol) portion wise. The resultant mixture was stirred at 0oC for 1 hour. The reaction mixture was filtered through Celite® and the filter cake washed with additional DCM (2 x 500 mL). Thefiltrate was washed with saturated sodium bicarbonate solution (2 x 300 mL), dried over anhydrous sodium sulphate, filtered through a short pad of Celite® and concentrated in vacuo to provide the title compound as a dark solid (10.4 g, 92%) which was used without further purification.1H NMR (300 MHz, DMSO) δ 9.28 (s, 1H), 5.25 (s, 1H), 3.63 (s, 3H), 2.81 - 2.71 (m, 4H), 2.63 (s, 3H), 1.01 (s, 3H). LC-MS (ESI+) Method 1 RT 0.70 minutes m / z = 270.0 (M+H)+INTERMEDIATE 4: Methyl 3-methyl-1-(3-methylsulfanyl-1,2,4-triazin-5-yl)-3- trimethylsilyloxy-cyclobutanecarboxylateTo a stirred solution of methyl 3-hydroxy-3-methyl-1-(3-methylsulfanyl-1,2,4-triazin-5- yl)cyclobutanecarboxylate (prepared according to the procedure of Intermediate 3) (8.0 g, 30 mmol) in DCM (150 mL) cooled to 0oC was added di-iso-propylethylamine (13 mL, 75 mmol) followed by trimethylsilyltrifluoromethane sulphonate (8.0 mL, 39 mmol). The resulting solution was stirred at 0oC for 15 minutes before being warmed to ambient temperature and stirred for 17 hours. The resulting deep red reaction mixture was stirred at 0°C for 15 minutes, warmed to room temperature and stirred for 17 hours. The reaction mixture was quenched with saturated sodium bicarbonate solution (200 mL) and extracted with dichloromethane (2 x 150 mL), the combined organic extracts passed through a phase separator and concentrated in vacuo to a dark brown residue which was purified by chromatography on silica eluting with a gradient of 0% to 15% ethyl acetate in iso-hexane to provide the title compound as a yellow oil (8.8 g, 87%).1H NMR (300 MHz, DMSO) δ 9.31 (s, 1H), 3.63 (s, 3H), 2.93 – 2.81 (m, 4H), 2.63 (s, 3H), 1.10 (s, 3H), 0.12 (s, 9H). LC-MS (ESI+) Method 1 RT 1.35 minutes m / z = 342.0 (M+H).INTERMEDIATE 5: Methyl 1-(3-amino-1,2,4-triazin-5-yl)-3-methyl-3- trimethylsilyloxy-cyclobutanecarboxylateTo a stirred solution of methyl 3-methyl-1-(3-methylsulfanyl-1,2,4-triazin-5-yl)-3- trimethylsilyloxy-cyclobutanecarboxylate (prepared according to the procedure of Intermediate 4) (10 g, 29.3 mmol) in DCM (300 mL) cooled to 0oC was added 3- chloroperoxy benzoic acid (70 weight %, 18g, 73 mmol) and the resultant suspension stirred at 0oC for 90 minutes before warming to ambient temperature and stirred for 2 hours. The reaction mixture was diluted with DCM (100 mL) and washed with saturated aqueous sodium bicarbonate solution (2 x 250 mL), 20% aqueous sodium bisulfite solution (250 mL), aqueous sodium bicarbonate solution (250 mL), dried over sodium sulfate and filtered under vacuum. To the filtrate was added anhydrous 1,4-dioxane (100 mL) and the mixture concentrated under vacuum (>250 mbar at 30 °C) to remove residual dichloromethane and afford a solution of the intermediate sulphone in 1,4-dioxane. The resultant solution was cooled to 0oC with stirring under nitrogen and a solution of ammonia (0.5M in 1,4-dioxane, 300mL, 150 mmol) added. The resultant suspension was warmed to ambient temperature and stirred for 15 hours. The reaction mixture was concentrated in vacuo and the resulting crude residue partitioned between ethyl acetate (300 mL) and saturated aqueous potassium carbonate solution (300 mL). The aqueous layer was extracted with additional ethyl acetate (300 mL), the combined organic extracts dried over sodium sulfate, filtered and concentrated in vacuo to afford the title compound (7.5 g, 83%) as a pale-yellow solid.1H NMR (300 MHz, DMSO) δ 8.67 (s, 1H), 7.28 (s, 2H), 3.61 (s, 3H), 2.85 - 2.75 (m, 4H),1.11 (s, 3H), 0.11 (s, 9H). LC-MS (ESI+) Method 1 RT 1.07 minutes m / z = 311.2 (M+H).INTERMEDIATE 6: Methyl 1-(3-amino-1,2,4-triazin-5-yl)-3-hydroxy-3-methyl- cyclobutanecarboxylateTo a stirred solution of methyl 1-(3-amino-1,2,4-triazin-5-yl)-3-methyl-3- trimethylsilyloxy-cyclobutanecarboxylate (prepared according to the procedure of Intermediate 5) (4.0 g, 13 mmol) in DCM (65 mL) was added 4M HCl in 1,4-dioxane (16 mL, 64 mmol). The resulting suspension was stirred at room temperature for 30 minutes before the reaction mixture was concentrated in vacuo. The resulting residue was partitioned between ethyl acetate (200 mL) and saturated aqueous potassium carbonate solution (150 mL). The aqueous phase was extracted with further ethyl acetate (2 x 150 mL), the combined organic extracts dried over sodium sulfate, filtered and concentrated in vacuo to provide the title compound (3.1g, 96%) as an off-white solid.1H NMR (300 MHz, DMSO) δ 8.64 (s, 1H), 7.27 (s, 2H), 5.18 (s, 1H), 3.61 (s, 3H), 2.78 - 2.62 (m, 4H), 1.02 (s,3H). LC-MS (ESI+) Method 1 RT 0.29 minutes m / z = 239.0 (M+H).INTERMEDIATE 7: 1-(3-amino-1,2,4-triazin-5-yl)-N-(2,2-difluoropropyl)-3-hydroxy-3- methyl-cyclobutanecarboxamideA stirred mixture of 2,2-difluoropropylamine hydrochloride (110 mg, 0.803 mmol) and methyl 1-(3-amino-1,2,4-triazin-5-yl)-3-hydroxy-3-methyl-cyclobutanecarboxylate (prepared according to the procedure of Intermediate 6) (120 mg, 0.504 mmol) in anhydrous THF (5 mL) under nitrogen was cooled to 0 °C and a solution of lithium bis- trimethylsilylamide in THF (1.0M, 5 mL, 5 mmol) added dropwise. The reaction mixture was stirred at 0 °C for 1 hour, before being warmed to ambient temperature and stirred for 20 hours. The reaction mixture was quenched with saturated aqueous ammonium chloride solution (5 mL) and water (5 mL), ethyl acetate (5 mL) was added and the mixture stirred for 10 minutes. The separated aqueous layer was extracted with ethyl acetate (3 x 10 mL), the combined organic phases passed through a phase separator and concentrated in vacuo to afford the title compound (152 mg, 95%), as a light brown solid.1H NMR (300 MHz, DMSO) δ 8.63 (s, 1H), 8.12 (t, J = 6.3 Hz, 1H), 7.19 (s, 2H), 5.11 (s, 1H), 3.49 (td, J = 13.6, 6.2 Hz, 2H), 2.70 - 2.58 (m, 4H), 1.46 (t, J = 19.1 Hz, 3H), 1.04 (s, 3H). LC-MS (ESI+) Method 1 RT 0.30 minutes m / z = 302.2 (M+H).INTERMEDIATE 8: Benzyl N- -[3-[1-(2,2-difluoropropylcarbamoyl)-3-hydroxy-3- methyl-cyclobutyl]imidazo[1,2-b] triazin-6-yl]-[4- (trifluoromethyl)cyclohexyl]methyl] carbamateA mixture of 1-(3-amino-1,2,4-triazin-5-yl)-N-(2,2-difluoropropyl)-3-hydroxy-3-methyl- cyclobutanecarboxamide (prepared according to the procedure of Intermediate 7) (2.46 g, 8.16 mmol), benzyl N-[(1S)-3-bromo-2-oxo-1-[4-(trifluoromethyl)cyclohexyl]propyl] carbamate (prepared according to the procedure of Intermediate 16) (4.6 g, 11 mmol) and activated 4Å molecular sieves (4.5g) was suspended in 1-butanol (50 mL) and 2,6-lutidine (2.1 mL, 18 mmol) added with stirring. The mixture was heated to 65oC for 18 hours. The reaction mixture was cooled to room temperature, filtered through Celite® and concentrated in vacuo. The residue was dissolved in ethylacetate (300 mL) and the organic phase washed with water (2 x 300mL), brine (300mL), separated and dried over anhydrous sodium sulphate. The mixture was filtered and concentrated in vacuo to provide a crude residue that was purified by column chromatography on silica eluting with iso-hexanes and ethylacetate to provide the title compound as a yellow / orange solid (3.95g, 73%).1H NMR (400 MHz, DMSO) δ 8.59 (s, 1H), 8.23 (t, J = 6.3 Hz, 1H), 8.13 (s, 1H), 7.79 (d, J = 9.3 Hz, 1H), 7.45 – 7.12 (m, 5H), 5.23 (s, 1H), 5.03 (d, J = 2.5 Hz, 2H), 4.67 (dd, J = 9.2, 7.1 Hz, 1H), 3.50 (td, J = 13.7, 6.2 Hz, 2H), 2.80 (q, J = 12.4 Hz, 4H), 2.17 (m 1H), 1.86 (m 4H), 1.62 (d, J = 12.3 Hz, 1H), 1.50 (t, J = 19.1 Hz, 3H), 1.27 – 1.11 (m, 4H), 1.08 (s, 3H). LCMS: Method 1 RT 1.46 minutes m / z 639 (M+H)+INTERMEDIATE 9: 1-[6-[(S)-amino-[4- (trifluoromethyl)cyclohexyl]methyl]imidazo[1,2-b][1,2,4]triazin-3-yl]-N-(2,2- difluoropropyl)-3-hydroxy-3-methyl-cyclobutanecarboxamideTo a solution of benzyl N-[(S)-[3-[1-(2,2-difluoropropylcarbamoyl)-3-hydroxy-3-methyl- cyclobutyl]imidazo[1,2-b][1,2,4]triazin-6-yl]-[4-(trifluoromethyl)cyclohexyl]methyl] carbamate (prepared according to the procedure of Intermediate 8) (3.05g 4.78 mmol) in 2- propanol (70 mL) under an atmosphere of nitrogen was added ammonium formate (6g, 94 mmol). Under a stream of nitrogen was added a slurry of 10% palladium on carbon, type 48750% water (1g) in 2-propanol (5 mL) to the reaction mixture. The reaction was allowed to stir at ambient temperature for 18 hours. The reaction mixture was diluted with DCM (200 mL) and filtered through Celite® eluting with DCM. The organic phase was washed with saturated aqueous sodium bicarbonate solution (200 mL) and brine (2 x 200mL). The organic phase was dried over anhydrous sodium sulphate, filtered and concentrated in vacuo. The resultant crude residue was purified by column chromatography on silica eluting with a gradient of 0 to 30% methanol in DCM to provide the title compound (believed to be in the desired syn configuration) as a pale yellow solid (1.67g, 69%).1H NMR (400 MHz, DMSO) δ 8.57 (s, 1H), 8.25 (t, J = 6.3 Hz, 1H), 8.15 (s, 1H), 5.23 (s, 1H), 3.80 (d, J = 5.5 Hz, 1H), 3.50 (td, J = 13.7, 6.3 Hz, 2H), 2.90 – 2.71 (m, 4H), 2.15 (d, J = 10.3 Hz, 1H), 2.03 – 1.78 (m, 5H), 1.78 – 1.58 (m, 2H), 1.49 (t, J = 19.1 Hz, 3H), 1.29 – 1.11 (m, 4H), 1.07 (s, 3H).LCMS: Method 1 RT 1.16 minutes m / z 505 (M+H)+INTERMEDIATE 10: (NE)-2-Methyl- 4-(trifluoromethyl)cyclohexyl] sulfinamideTo a solution of trans-4-(trifluoromethyl)cyclohexanecarboxaldehyde (16.1 g, 74.2 mmol) in DCM (500 mL) was added (S)-(-)-2-methyl-2-propanesulfinamide (8.70 g, 70.0 mmol), followed by titanium(IV) ethoxide (52 mL, 223.2 mmol). The mixture was heated under reflux for 2 h, then cooled to r.t. and treated with water (50 mL). After vigorous stirring, the fine suspension was filtered through Celite®. The filtrate was passed through a hydrophobic frit and concentrated in vacuo to afford the title compound (18.8 g, 89%) as an off-white solid. [α]25D +145° (c 1.00, DCM). δH (300 MHz, CDCl3) 8.00 (d, J 4.3 Hz, 1H), 2.57-2.40 (m, 1H), 2.17-1.96 (m, 5H), 1.54-1.28 (m, 4H), 1.21 (s, 9H). 19F {1H} NMR (282 MHz, CDCl3) δ -73.84 (s, 3F). INTERMEDIATE 11: N-{(S)-Cyano[trans-4-(trifluoromethyl)cyclohexyl]methyl}-2- methylpropane-2-sulfinamideTo a solution of (NE)-2-Methyl-N-{[trans-4-(trifluoromethyl)cyclohexyl]methylene} propane-2-sulfinamide (prepared according to the procedure according to Intermediate 10) (18.8 g, 66.3 mmol) in DCM (180 mL) at 25°C was added cesium fluoride (1.00 g, 6.58 mmol), followed by trimethylsilyl cyanide (18.0 mL, 134 mmol). The mixture was stirred for 17 h at 25°C. Saturated aqueous sodium bicarbonate solution (150 mL) was added, and the mixture was stirred for 1 h. The biphasic mixture was separated and the organic layers were concentrated in vacuo. The resulting pale yellow oil was purified by flash columnchromatography (0-100% EtOAc / isohexane), then recrystallised from EtOAc:isohexane (1:2; 90 mL), to afford the title compound (single diastereoisomer) (7.20 g, 35%) as a white solid. δH (400 MHz, CDCl3) 4.05 (dd, J 8.0, 6.4 Hz, 1H), 3.66 (d, J 8.0 Hz, 1H), 2.15-1.99 (m, 5H), 1.91-1.74 (m, 1H), 1.45-1.32 (m, 2H), 1.28 (s, 9H), 1.26-1.11 (m, 2H). 19F {1H} NMR (282 MHz, CDCl3) δ -73.80 (s, 3F). INTERMEDIATE 12: (2S)-2-Amino-2-[trans-4-(trifluoromethyl)cyclohexyl]acetonitrile hydrochlorideTo a solution of N-{(S)-cyano[trans-4-(trifluoromethyl)cyclohexyl]methyl}-2- methylpropane-2-sulfinamide (prepared according to the procedure of Intermediate 11) (7.00 g, 22.6 mmol) in MeOH (17 mL) was added HCl in 1,4-dioxane (4N, 12 mL), resulting in a white precipitate. After 4 h, the mixture was concentrated under reduced pressure. To the resulting oily slurry was added isohexane (100 mL). After vigorous stirring, the resultant white solid was filtered out, to afford the title compound (5.20 g, 95%). δH (300 MHz, DMSO-d6) 9.15 (s, 3H), 4.57 (d, J 5.5 Hz, 1H), 2.39-2.20 (m, 1H), 2.11-1.79 (m, 5H), 1.40-1.01 (m, 4H). INTERMEDIATE 13: (2S)-2-Amino-2- 4-(trifluoromethyl)cyclohexyl]acetic acidhydrochlorideAcetic acid (35 mL) and HCl in H2O (33%, 50 mL) were added to (2S)-2-amino-2-[trans- 4-(trifluoromethyl)cyclohexyl]acetonitrile hydrochloride (prepared according to theprocedure of Intermediate 12) (5.20 g, 21.0 mmol). The mixture was heated under reflux for 4 h, then cooled with stirring to 20°C. After 18 h, the white precipitate was filtered off, and dried under a flow of air, to afford the title compound (4.40 g, 74%) as a white solid. [α]25D +22.1° (c 1.0, MeOH). δH (300 MHz, DMSO-d6) 13.81 (s, 1H), 8.46 (s, 3H), 3.75 (d, J 4.3 Hz, 1H), 2.34-2.05 (m, 1H), 2.03-1.66 (m, 5H), 1.47-1.04 (m, 4H).19F {1H} NMR (282-MHz, DMSO-d6) δ -72.33 (s, 3F). INTERMEDIATE 14: -2-(Benzyloxycarbonylamino)-2-[4-(trifluoromethyl) acetic acidTo a suspension of (2S)-2-Amino-2-[trans-4-(trifluoromethyl)cyclohexyl]acetic acid hydrochloride (prepared according to the procedure of Intermediate 13) (5.00 g, 19.1 mmol) and triethylamine (10 mL, 71.7 mmol) in DCM (100 mL) at 0°C was added N- (benzyloxycarbonyloxy)succinimide (4.47 g, 17.6 mmol) in three portions. The reaction mixture was allowed to warm to r.t. overnight. The reaction mixture was diluted with DCM (10 mL), washed with 5% hydrochloric acid (2 x 15 mL) and water (15 mL), then dried (Na2SO4) and concentrated in vacuo, to give the title compound (6.34 g, 92%) as a white solid. δH (300 MHz, DMSO-d6) 12.65 (s, 1H), 7.54 (d, J 8.4 Hz, 1H), 7.40-7.28 (m, 5H), 5.04 (s, 2H), 3.89 (dd, J 8.4, 5.9 Hz, 1H), 2.26-2.05 (m, 1H), 1.96-1.80 (m, 2H), 1.80-1.59 (m, 3H), 1.32-1.07 (m, 4H). INTERMEDIATE 15: Benzyl N-{(1S)-3-[dimethyl(oxo)-λ6-sulfanylidene]-2-oxo-1-[4- (trifluoromethyl)-cyclohexyl]propyl}carbamateTrimethylsulfoxonium iodide (70.00 g, 0.318 mol) was dissolved in THF (350 mL) and 1M potassium tert-butoxide in THF (300 mL, 0.300 mol) was added. The resulting mixture was stirred at 70°C (external temperature) under nitrogen for 2 h, then cooled to -5°C in an ice / salt bath under nitrogen, to provide an ylide. Meanwhile, in a separate flask, (2S)-2- (benzyloxycarbonylamino)-2-[4-(trifluoromethyl)cyclohexyl]acetic acid (prepared according to the procedure of Intermediate 14) (37.00 g, 0.103 mol) was dissolved in THF (350 mL) and HATU (49.00 g, 0.129 mol) was added, followed by DIPEA (24 mL, 0.137 mol). The reaction mixture was stirred under nitrogen for 150 minutes, then added dropwise over ~50 minutes to the cooled solution of the ylide, maintaining internal temperature below 1°C. The reaction mixture was stirred at -4°C under nitrogen for 5 minutes, then quenched at -4°C by addition of water (700 mL) and saturated aqueous NaHCO3 solution (700 mL). The resulting suspension was extracted with TBME (3 L). The organic layer was washed with brine (500 mL) and concentrated to dryness under vacuum. The residue was suspended in TBME (500 mL) and water (50 mL), then heated to 50°C and cooled to r.t., then filtered, then washed with TBME (100 mL) and heptane (100 mL). An additional three crops were obtained as more solid precipitated from solution in the filtrate each time. The four crops of solid were combined, then water (400 mL) was added. The mixture was sonicated to break up big lumps, then cooled to 0°C, filtered and washed with water (150 mL). the residue was dried in a vacuum oven overnight to afford the title compound (36.15 g, 81%) as a colourless solid. δH (400 MHz, DMSO-d6) 7.42- 7.27 (m, 5H), 7.06 (d, J 9.2 Hz, 1H), 5.01 (s, 2H), 4.87 (s, 1H), 3.67 (dd, J 9.1, 6.6 Hz, 1H), 3.43 (s, 6H), 2.20-2.03 (m, 1H), 1.92-1.78 (m, 2H), 1.76-1.54 (m, 3H), 1.26-0.99 (m, 4H). LCMS (Method 6): [M+H]+434.2, RT 2.70 minutes.INTERMEDIATE 16: Benzyl N-{(1S)-3-bromo-2-oxo-1-[4- (trifluoromethyl)cyclohexyl]propyl}carbamateBenzyl N-{(1S)-3-[dimethyl(oxo)-λ6-sulfanylidene]-2-oxo-1-[4-(trifluoromethyl)- cyclohexyl]propyl}carbamate (prepared according to the procedure of Intermediate 15) (28.08 g, 64.8 mmol) was dissolved in THF (300 mL) and cooled to 0°C (external temperature) under nitrogen. LiBr (5.69 g, 64.8 mmol) was added, and the mixture was stirred until it fully dissolved (~2 minutes). Methanesulfonic acid (4.2 mL, 64.7 mmol) was added and the mixture was stirred at 0°C under nitrogen for 5 minutes, then allowed to warm to r.t. and stirred for 30 minutes under nitrogen. The resulting suspension was warmed to 58°C (internal temperature; heating block at 65°C) over approximately 30 minutes, and stirred under nitrogen at this temperature for 1 h. The reaction mixture was allowed to cool to r.t. and quenched by the addition of saturated aqueous NaHCO3solution (450 mL). The resulting mixture was diluted with water (200 mL) and extracted with EtOAc (600 mL). The organic layer was washed with saturated aqueous NaBr solution (400 mL), then dried (Na2SO4) and concentrated to dryness under vacuum. The residue was purified by FCC (750 g Biotage KP-Sil cartridge, wet loaded in 150 mL DCM, eluting with 10-20% EtOAc in heptane), and the product fractions were concentrated to dryness under vacuum, to afford the title compound (19.80 g, 68%) as a colourless solid. A mixed fraction was isolated and concentrated to dryness under vacuum, then re-purified by FCC (100 g Biotage Sfar Duo cartridge, wet loaded in 20 mL DCM, eluting with 10-20% EtOAc in heptane), and the product fractions were concentrated to dryness under vacuum, to afford a second crop of the title compound (4.90 g, 17%) as a colourless solid. δH (500 MHz, CDCl3) 7.44-7.29 (m, 5H), 5.31 (d, J 8.7 Hz, 1H), 5.11 (s, 2H), 4.66 (dd, J 8.7, 4.6 Hz, 1H),4.03 (q, J 13.3 Hz, 2H), 2.03-1.92 (m, 3H), 1.92-1.83 (m, 2H), 1.63 (d, J 13.0 Hz, 1H), 1.42-1.18 (m, 3H), 1.06 (qd, J 13.0, 3.2 Hz, 1H). LCMS (Method 4): [M+H]+436.0 / 438.0, RT 3.27 minutes. IV. EXAMPLES EXAMPLE 1: N-[(S)-[3-[1-(2,2-difluoropropylcarbamoyl)-3-hydroxy-3-methyl- cyclobutyl]imidazo[1,2-b][1,2,4]triazin-6-yl]-[4-(trifluoromethyl)cyclohexyl]methyl]-4- methyl-1,2,5-oxadiazole-3-carboxamide (desired syn stereoisomer):A mixture of 1-[6-[(S)-amino-[4-(trifluoromethyl)cyclohexyl]methyl]imidazo[1,2- b][1,2,4]triazin-3-yl]-N-(2,2-difluoropropyl)-3-hydroxy-3-methyl- cyclobutanecarboxamide (believed to be syn stereoisomer, prepared according to the procedure of Intermediate 9) (250mg, 0.50 mmol) and 4-methyl-1,2,5-oxadiazole-3- carboxylic acid (82 mg, 0.64 mmol) in acetonitrile (5mL) was cooled to 0oC (ice bath) and EDCI.HCl (150 mg, 0.77 mmol) added. The mixture was allowed to warm to room temperature and stirred for 18 hours. The reaction mixture was diluted with DCM (30 mL) and washed with 0.5M HCl (30 mL), sat. aq. NaHCO3solution (30mL) and brine (30 mL). The organics were dried over anhydrous sodium sulphate, filtered and concentrated in vacuo to provide a crude residue which was purified by column chromatography on silica eluting in a gradient from 0 to 100% EtOAc in iso-hexanes to provide the title compound as a pale yellow solid (240mg, 79%).1H NMR (400 MHz, DMSO) δ 9.45 (d, J = 8.9 Hz, 1H), 8.61 (s, 1H), 8.29 (s, 1H), 8.24 (t, J = 6.3 Hz, 1H), 5.24 (s, 1H), 5.11 (t, J = 8.6 Hz, 1H), 3.50 (td, J = 13.8, 6.2 Hz, 2H), 2.80 (q, J = 12.7 Hz, 4H), 2.48 (s, 3H), 2.26 (t, J = 17.4 Hz, 1H), 2.02 (m, 2H), 1.97 – 1.89 (m, 1H), 1.85 (d, J = 11.5 Hz, 1H), 1.66 (d, J = 11.4 Hz,1H), 1.50 (t, J = 19.1 Hz, 3H), 1.32 – 1.17 (m, 4H), 1.08 (s, 3H). LCMS: Method 2 RT 1.96 minutes; m / z 615 (M+H)+V. BIOLOGICAL ASSAYS V.I: Inhibition of IL-17A induced IL-6 release from primary Human Dermal Fibroblast Cells The purpose of this assay is to test the neutralising ability to IL-17 proteins, in a human primary cell system. Stimulation of normal human dermal fibroblasts (HDF) with IL-17 alone produces only a very weak signal but in combination with certain other cytokines, such as TNFα, a synergistic effect can be seen in the production of inflammatory cytokines, i.e. IL-6. HDFs were stimulated with IL-17A (50 pM) in combination with TNF-α (25 pM). The resultant IL-6 response was then measured using a homogenous time-resolved FRET kit from Cisbio. The kit utilises two monoclonal antibodies, one labelled with Eu-Cryptate (Donor) and the second with d2 or XL665 (Acceptor). The intensity of the signal is proportional to the concentration of IL-6 present in the sample (Ratio is calculated by 665 / 620 x 104). The ability of a compound to inhibit IL-17 induced IL-6 release from human dermal fibroblasts is measured in this assay. HDF cells (Sigma #106-05n) were cultured in complete media (DMEM + 10% FCS + 2 mM L-glutamine) and maintained in a tissue culture flask using standard techniques. Cells were harvested from the tissue culture flask on the morning of the assay using TrypLE (Invitrogen #12605036). The TrypLE was neutralised using complete medium (45 mL) and the cells were centrifuged at 300 x g for 3 minutes. The cells were re-suspended in complete media (5 mL) counted and adjusted to a concentration of 3.125 x 104cells / mL before being added to the 384 well assay plate (Corning #3701) at 40 μL per well. The cells were left for a minimum of three hours, at 37°C / 5% CO2, to adhere to the plate. The compound of formula (I) was serially diluted in DMSO before receiving an aqueous dilution into a 384 well dilution plate (Greiner #781281), where 5 μL from the titration plate was transferred to 45 μL of complete media and mixed to give a solution containing 10% DMSO.Mixtures of TNFα and IL-17 cytokine were prepared in complete media to final concentrations of TNFα 25 pM / IL-17A 50 pM, then 30 μL of the solution was added to a 384 well reagent plate (Greiner #781281). 10 μL from the aqueous dilution plate was transferred to the reagent plate containing 30 μL of the diluted cytokines, to give a 2.5% DMSO solution. The compounds were incubated with the cytokine mixtures for 5 h at 37°C. After the incubation, 10 μL was transferred to the assay plate, to give a 0.5% DMSO solution, then incubated for 18-20 h at 37°C / 5% CO2. From the Cisbio IL-6 FRET kit (Cisbio #62IL6PEB) europium cryptate and Alexa 665 were diluted in reconstitution buffer and mixed 1:1, as per kit insert. To a white low volume 384 well plate (Greiner #784075) were added FRET reagents (10 μL), then supernatant (10 μL) was transferred from the assay plate to Greiner reagent plate. The mixture was incubated at room temperature for 3 h with gentle shaking (<400 rpm) before being read on a Synergy Neo 2 plate reader (Excitation: 330 nm; Emission: 615 / 645 nm). When tested in the primary HDF cell assay as described above, the compound of formula (I), believed to be (IA’) (desired syn stereoisomer), was found to exhibit a pIC50 value of about 8.2. V.II: Primary Th17-Human Dermal Fibroblast Co-culture cell assay Peripheral blood monocytes were isolated from healthy donors, CD4+ T cells enriched and then CD4+CCR6+ T cells (Th17 cells) were sorted using a BD FACS Aria. Human dermal fibroblasts (HDF) were allowed to adhere and grow in a 384-well assay plate for 24 hours at 37oC. The following day, the compound of formula (I) was pre-diluted or anti-IL17 antibody or DMSO vehicle controls and T cell stimulants anti-CD3 / CD28 beads were added to the HDF 384-well assay plate. Finally, the CD4+CCR6+ cells (Th17 cells) were added to the assay plate and incubation continued at 37oC for 17-18 hours. An aliquot of the cell supernatant was then diluted into a homogenous time-resolved fluorescence (HTRF from Cisbio) buffer reagent plus substrate to determine the concentration of IL6 cytokine produced. The HTRF ratios were expressed as % inhibition relative to a minimum response in the presence of DMSO control and a maximum response in the presence of anti-IL17A antibody, and plotted as a function of log-transformed compound concentrations. A four-parameter fitting model was used to determine IC50 and IC90 values as a measure of compound potency. The compound of formula (I) was tested in a minimum of 6 independent human blood donors and the composite data expressed as a geomean. When tested in the primary Th-17-HDF co-culture cell assay as described above, the compound of formula (I), believed to be (IA’) (desired syn stereoisomer), was found to exhibit a pIC50value of about 7.2. V.III Kinetic measurements of affinity by Surface Plasmon Resonance (SPR) The SPR assay was performed on Biacore T200, S200 and 8K (Cytiva, Sweden) consisting of immobilizing human IL17AA on the surface of CM5 chip followed by titration of compound in the single cycle kinetics format. Human IL17AA was diluted in 10mM sodium acetate and immobilized on a CM5 Series S Sensor Chip via amine coupling chemistry at 25oC to a level of ~1200 response units (RU). The immobilisation was run in HBS P buffer (10mM HEPES pH 7.4, 0.15M NaCl, 0.005 % Surfactant P20, (Cytiva, Sweden) at a flow rate of 10μL / min. A reference surface was prepared by activating and deactivating the appropriate flow cell. Binding assays were performed after equilibration at 37oC in HBS-P buffer with 5% DMSO at a flow rate of 50μL / min. The assay was performed in the single cycle kinetics mode with compound titrated at 5 concentrations in the ascending order to 30μM over the immobilized human IL17AA and reference flow cell, followed by 17200s dissociation. After dissociation a second injection of the same IL17 NCE top concentration was added to confirm binding. Background subtraction binding curves for compound were analyzed using the Biacore Evaluation Software, Version (Cytiva, Sweden), using the 1:1 binding model fitted. When tested in the SPR assay as described above, the compound of formula (I), believed to be (IA’) (desired syn stereoisomer), was found to exhibit an advantageous SPR on rate.

Claims

Claims:

1. A compound of formula (I)or a salt and / or solvate thereof.

2. A compound of formula (I) as claimed in claim 1, which is a compound of formula (IA’), namely N-[(S)-[3-[1-(2,2-difluoropropylcarbamoyl)-3-hydroxy-3-methyl- cyclobutyl]imidazo[1,2-b][1,2,4]triazin-6-yl]-[4-(trifluoromethyl)cyclohexyl]methyl]-4- methyl-1,2,5-oxadiazole-3-carboxamide (syn isomer):or a salt and / or solvate thereof.

3. The compound of formula (I) or a salt and / or solvate thereof as claimed in claim 1 or claim 2, wherein the salt and / or solvate is a pharmaceutically acceptable salt and / or solvate.

4. The compound of formula (I) or a pharmaceutically acceptable salt and / or solvate thereof as claimed in claim 3, for use in therapy.

5. The compound of formula (I) or a pharmaceutically acceptable salt and / or solvate thereof as claimed in claim 3, for use in the treatment and / or prevention of disorders for which the administration of a modulator of IL-17 function is indicated.

6. The compound of formula (I) or a pharmaceutically acceptable salt and / or solvate thereof as claimed in claim 3, for use in the treatment and / or prevention of an inflammatory or autoimmune disorder.

7. A pharmaceutical composition comprising the compound of formula (I) or a pharmaceutically acceptable salt and / or solvate thereof as claimed in claim 3, in association with a pharmaceutically acceptable carrier.

8. The use of the compound of formula (I) or a pharmaceutically acceptable salt and / or solvate thereof as claimed in claim 3, for the manufacture of a medicament for the treatment and / or prevention of disorders for which the administration of a modulator of IL- 17 function is indicated.

9. The use of the compound of formula (I) or a pharmaceutically acceptable salt and / or solvate thereof as claimed in claim 3, for the manufacture of a medicament for the treatment and / or prevention of an inflammatory or autoimmune disorder.

10. A method for the treatment and / or prevention of disorders for which the administration of a modulator of IL-17 function is indicated, which comprises administering to a patient in need of such treatment an effective amount of the compound of formula (I), or a pharmaceutically acceptable salt and / or solvate thereof as claimed in claim 3.

11. A method for the treatment and / or prevention of an inflammatory or autoimmune disorder, which comprises administering to a patient in need of such treatmentan effective amount of the compound of formula (I), or a pharmaceutically acceptable salt and / or solvate thereof as claimed in claim 3.or a salt and / or solvate thereof.

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