ADVANCED PARTNERSHIP PLATFORM FOR EARLY DRUG DISCOVERY FRAMEWORK OF PRODUCTS AND SERVICES lifechemicals.com

Fully Integrated Resources For Drug Discovery

HTS Compounds Contract research services • HTS compound collection • Custom Synthesis • Pre-plated diversity sets • Computational Chemistry • Fragment Libraries • GLP Primary Drug Trials – Key Preclinicals • Targeted & Focused Libraries

Advanced Building Blocks Fine Chemicals in Multigram Scale

• Off-the-shelf Building Blocks • IR-Dyes • Tangible Building Blocks • APIs • Cutting-edge Design of Building Blocks • Fine Reagents for Organic Synthesis • Custom Synthesis of Building Blocks • Scale-up Synthesis (up to 100kg) and Intermediates • Process Optimization

2 OUR FIELDS OF EXPERTISE lifechemicals.com

 Strong position in organic synthesis  Scale-up from grams to kilograms and medicinal chemistry  Recognized scientific portfolio  Successful experience in custom  Cutting-edge techniques and approaches synthesis projects of high complexity, including multistep procedures  Primary drug trial services  Co-design with customers  Qualified and experienced staff  Synthesis of reference compounds  Customer-tailored terms and conditions  Synthesis of impurities  IP protection  Broadest range of compounds and  Timely and efficient data transfer chemical transformations

3 ORDER HANDLING lifechemicals.com

Life Chemicals guarantees high quality service and flexibility

Inquiry Order Synthesis QC Delivery Payment

Inquiry: quotation within 0-3 business days Order: the process is launched immediately after the order Synthesis (for non-stock compounds): normally 2-6 weeks, progress reports are provided upon request Quality control: high quality standards (purity of 90 – 95 % confirmed with NMR and/or LCMS) Delivery: 3-7 days by FedEx to any destination (customer’s FedEx account can be used) Payment: by cheque, credit card or wire transfer to the account in Germany or in Canada, payment terms are NET 30 days.

For small stock orders you can go directly to our shop at https://shop.lifechemicals.com/

4 QUALITY ASSURANCE AND ORDER PROCESSING lifechemicals.com

• Structure of compounds is validated by analytical data, 400 MHz NMR and/or LCMS analysis. Analytical data is readily available upon the customer’s request

• ISIS .db or .sdf files are enclosed with every shipment

• Sample weights: 1 mg - 0.5 g with 0.1 mg weighing precision, mg, micromole amounts

• Compounds forms: - dry powders - frozen DMSO solutions

• Formatting: - Standard 4mL, 15mm*45mm, amber borosilicate glass vials with rubber-lined plastic screw caps - 96 well PP-masterblocks, Greiner bio-one ref: 780215, U-bottom 1.0ml - 96 well plates, Matrix cat.# 4247, racked round-bottom tubes, 1,4 ml

• Each plate/box of vials is barcoded and labeled to meet the customer’s requirements

5 LIFE CHEMICALS GEOGRAPHY lifechemicals.com

Production space: Total number of employees: 2,500 m2 Over 110, including 61 chemists (1 Prof, 11 PhDs, 35 MSc, 14 BSc)

6 SCREENING COMPOUNDS lifechemicals.com

Our Products  460,000 compounds in stock  2,900 original scaffolds  Fsp³ enriched Library: 38,000 compounds  900,000 tangible compounds  Pre-plated diversity sets (non-overlapping): 5,000, 10,000, 15,000 and 20,000 compounds, with all these sets enabling to obtain a “blanket” collection of 50,000 unique structures

Quality and Handling standards:  Over 90 % purity, confirmed by LCMS and/or 400MHz NMR  Prompt delivery in any container as powder or DMSO solution  Bar-coding and labeling according to customers’ requirements

7 NEW PATENT FREE SCAFFOLDS lifechemicals.com

• Novelty checked in - e-molecules - SciFinder

• Average values: - MW 184 - ClogP –0.14

• Enough room for lead-oriented library design, even if rigorous MW/ClogP limitations are applied

Inquiry Quotation Validation Optimization Decoration

8 lifechemicals.com HTS LIBRARY DESIGN Compound Collection Genesis

9 HTS LIBRARY DESIGN lifechemicals.com Main principles and scientific background

• The concept of lead-oriented synthesis: limitation on MW, cLogP, heteroatom count, RotB and Fsp3; structural filters Churcher et al (GSK) Angew. Chem. Int. Ed. 2012, 51, 1114–1122

• Using drug/natural product-based privileged structural motifs (BioCores etc.) Kombarov et al Mol. Divers. 2010, 14, 193–200

• Controlled selection of building blocks for scaffold decoration Goldberg et al (AstraZeneca) Drug Discov. Today 2015, 20, 11–17

• Weighted approach to diversity analysis Langdon et al (UK Institute of Cancer Research) In: Scaffold Hopping in Medicinal Chemistry, Wiley, 2013, pp. 39–60

• Even population of the variation points in the scaffolds 20x20 library is optimal; 10x40 – good compromise; 2x200 – awkward

10 HTS LIBRARY DESIGN lifechemicals.com Design of scaffold library

1. Scaffold selection is a crucial step in design of HTS library.

2. Around 200 scaffolds are selected yearly.

3. Chemoinformatics definition of scaffold is used to obtain scaffold is a combination of core ring systems, linkers combining them, and attachment points. Therefore, different side chains are assigned to the same scaffold.

4. The resulting scaffold contains one strong and 1–2 weak diversity points.

5. The resulting scaffold contains 2–4 (preferably 2–3) rings and 1–2 ring systems

6. Diversity points should be present in every ring system, preferably in every ring. For bi- and polycyclic scaffolds with one ring system, diversity points must be present in at least two different rings, preferably distant ones.

11 HTS LIBRARY DESIGN lifechemicals.com Design of scaffold library, continued

7. The scaffold should represent an attractive chemotype: • No bad groups, reactive groups, PAINS etc. • No isolated benzene rings • No more than 1 benzene ring (even fused) • No chain amides/sulfamides/ureas/sulfides etc. • HAC < 20 (preferably ≤ 15) • Rotatable bonds ≤ 4 (preferably ≤ 3) • Heteroatom count: 1–6 (preferably 2–5) • At least 1 aliphatic and 1 aromatic ring • Approved by MedChem visual expertize 8. The scaffold does not belong to the existing Life Chemicals Stock HTS Collection

9. The scaffold should not be represented in E-molecules (≤ 50 cpds, ≤ 100 cpds for especially attractive chemotypes) 10. The scaffold should show <85% similarity to templates from LC database

12 HTS LIBRARY DESIGN lifechemicals.com Design of scaffolds selected

Examples of scaffolds:

13 HTS LIBRARY DESIGN lifechemicals.com Design of scaffold library, continued

Scaffold-based building blocks

1. At least 4 variations are for a weak diversity point of a scaffold, preferably 5–6 and up to 10 variations can be included.

2. Desirable Substituents: • H, Me, i-Pr, CF3, CF3CH2, c-Pr, c-Bu, i-Bu, t-Bu, OH, OMe, CH2OH, SO2Me, F(arylation agents are avoided), C(O)NH2, THP, Py and other hetaryls, fused alicyclic rings (for 2 adjacent week DPs), etc • n-Alkyl groups are avoided • limited use of benzene derivatives is allowed (e.g. fluorine-substituted)

14 HTS LIBRARY DESIGN lifechemicals.com Design of scaffold library, continued

Examples of Scaffold Building Blocks:

15 HTS LIBRARY DESIGN lifechemicals.com Reagent Database

• A database of ~1000 appropriate reagents is involved.

• The following types of reactions and reagents are used: 50% - arylation / alkylation / epoxides / aldehydes; 20% - acylation / sulfonylation / urea synthesis; 30% - amines.

• The following rules are applied: Ro2 compliant; <5% containing isolated benzene; 1–2 rings (except the short list of 10–20 acyclic); no bad groups / PAINS / reactive (after modification), etc.

16 HTS LIBRARY DESIGN lifechemicals.com Filtering by PhysChem / Structure

1. All available filters for bad, PAINS, reactive compounds, etc. are applied 2. Lead-likeness PhysChem parameters: • MW 200…450 • ClogP –1…4 • HDonors ≤ 5 • HAcceptors ≤ 8 • Rotatable Bonds ≤ 6 • Molecular complexity by Wyeth > 20 • TPSA < 140 • LogS > –6 • No too saturated without H-donor 3. Less than 2 benzenes, ≤ 2 S, ≤ 2 Cl, Br, ≤ 5 F; no chains longer than 6 bonds; at least 2 heteroatoms. 4. Additional structural filters depending on a particular set.

17 HTS LIBRARY DESIGN lifechemicals.com Final Steps

5. Filtering by Novelty

1. 98% Tanimoto dissimilarity to competitors (E-molecules). 2. 95% Tanimoto dissimilarity to Life Chemicals HTS collection.

6. Chemotype control

3. Checking for Fsp3 distribution (ideally Fsp3 = 0.6–0.7). 4. Checking for amide/sulfonamide/urea % (<30%).

7. Final diversity selection

5. 30-50 compounds per building block (the rest is removed by Tanimoto). 6. Check for scaffold population (≤ 30 compounds per scaffold)

18 HTS LIBRARY DESIGN lifechemicals.com Examples of Final Compounds:

19 SCREENING LIBRARIES BACKGROUND lifechemicals.com

20 PRE-PLATED DIVERSITY SETS lifechemicals.com

• Sets of 5000, 10000, 15000, 20000 and 50000 compounds – non-overlapping selections with high diversity and attractive prices

• Format: assay-ready 384-well plates formatted as 50 µL of 10 mM DMSO-solutions, 320 compounds per plate

• Custom made diversity sets upon request (dry powders in 96-well plates, DMSO-solutions in 96-, 384- well plates)

Diversity Average values Set MW HbD HbAc ClogP RotB Fsp3 Tanimoto PSA

50000 360.70 1.50 4.00 2.62 5.00 0.31 0.82 117.80

20000 378.07 1.53 4.12 2.85 5.54 0.30 0.82 115.92

15000 368.21 1.50 4.13 2.62 5.10 0.31 0.73 118.26

10000 359.16 1.47 4.10 2.54 5.02 0.31 0.66 119.69

5000 338.97 1.47 3.97 2.49 4.59 0.30 0.59 119.97

21 FSP3-ENRICHED COMPOUNDS LIBRARY lifechemicals.com

The Library containing 38,400 Fsp3-enriched compounds was designed, considering an increased importance of molecular complexity and presence of chiral centers in organic compounds, that rises their potential as good drug candidates and, thus, efficiently covers biologically relevant chemical space.

Average Parameter Range Values Molecular Weight 175 to 450 334

Fsp3 ≥ 0.45 0.56

ClogP < 4 1.7

TPSA < 140Å2 80 Å2

H-acceptors ≤ 8 3.9

H-donors ≤ 4 1.4

Rotatable Bonds ≤ 8 4.9

Number of Carbonaromatics ≤ 1 0.51

22 FRAGMENT LIBRARIES lifechemicals.com

The Fragment-based approach is now well established as an efficient tool in modern drug discovery. This method has proven to be more versatile and beneficial over conventional HTS-driven paradigms. Life Chemicals Fragment Collection with variable parameters comprises more than 68,000 screening compounds, containing over 38,000 stock and 30,000 tangible structures. In response to modern FBDD trends Life Chemicals has designed a number of unique Fragment Libraries:

• General Fragment Library • Ultimate Fragment Library • Fluorine Fragment Library • Fluorine Fragment Cocktails • Bromine Fragment Library • Low MW Fragment Library • Fsp3-enriched Fragment Library • 3D Fragment Library • PPI Fragment Library • Covalent Fragments Library • Soluble Fragments with Experimental Data • Natural Product-like Fragment Library • Innovative Design of Compound Library for FBDD

23 GENERAL FRAGMENT LIBRARY lifechemicals.com

• 38,000 stock available fragments with MW<300 and ClogP<3 • Advanced subset of 9,400 Fragments satisfying expanded Ro3 criteria that are perfectly suitable for FBDD purposes

Advanced Subset

Parameter Range Average values 7.43·10 MW 100 … 300 Å2 ClogP –2 … 3 8 TPSA < 100 Å2 7 HAcceptors ≤ 4 6 HDonors ≤ 3 5 2 Rotatable Bonds ≤ 3 2.58·10 3.35 4 Da LogS ≥ –3.5 3 Rings count 1 – 4 1.37 1.29 2 –NO2 group and Br count ≤ 1 S, Cl count ≤ 2 1 –CN group count ≤ 1 0 Benzene count ≤ 1 MW Acc Donor clogP TPSA

PAINS and compounds with undesired functionalities are removed.

24 GENERAL FRAGMENT LIBRARY lifechemicals.com

Examples of structures:

Advanced Subset:

25 ULTIMATE FRAGMENT LIBRARY lifechemicals.com

• 3,600 fragments with excellent ADME profile for lead generation. • Ultimately rigorous selection parameters are applied

Parameter Range Average values MW 150 – 300 6 cLogP –2 – 3 4.76·10Å2 TPSA ≤ 80 Å2 5 H-acceptors ≤ 3 4 H-donors ≤ 3 2 Rotatable Bonds ≤ 3 3 2.28·10 Da clogSw > –3 2.25 1.59 Halogens (except F) ≤ 1 2 1.20 S atoms ≤ 1 1 Ring count 1 – 3 Fused rings ≤ 2 0 Benzene count ≤ 1 MW Acc Donor clogP TPSA

PAINS and compounds with undesired functionalities are removed.

26 ULTIMATE FRAGMENT LIBRARY lifechemicals.com

Examples of structures:

27 FLUORINE FRAGMENT LIBRARY lifechemicals.com

• 19F NMR-based fragment screening can be used as a very efficient tool for rapid and sensitive detection of fragment hits. • 3,300 fluorine-containing fragments including Advanced Subset of 2,000 compounds are available in stock.

Fluorine Fragment Library – Advanced subset

Parameter Range ClogP ≤ 3 Molecular weight < 300

CF3 group or F count 1 Br count ≤ 1 HbAc 1 – 3 HbD ≤ 3 Rotatable bonds ≤ 5 Cyano and Sulfonyl group ≤ 1

NO2 group ≤ 1 S and Cl count ≤ 2 Number of rings 1 – 3 Number of acid groups ≤ 1 Number of base groups ≤ 2

Metal containing and reactive compounds, undesired chemotypes, “Fragment space” coverage of Life Chemicals Fluorinated salts and PAINS are discarded. fragments (in black) is similar to the coverage of all commercially available fluorinated fragments (in red).

28 FLUORINE FRAGMENT LIBRARY lifechemicals.com

Examples of structures:

29 FLUORINE FRAGMENT COCKTAILS lifechemicals.com

• The efficiency of fragment screening can be increased substantially by pooling compounds in pools of up to 15 fragments. Identification of the bound fragments by NMR, X-ray or ligand-observed LC/MS experiments then becomes a case of determining the best fragment-fits. • Life Chemicals prepared an exclusive set of fluorine-containing fragment cocktails based on 1,000 fragments selected from Fluorine-based Fragment Library.

• Each fragment pool contains 10 fragments.

• Fragments are carefully selected to ensure most different 19F chemical shifts within each pool. • 19F NMR spectra are available for every fragment

30 FLUORINE FRAGMENT COCKTAILS lifechemicals.com

Carefully collected 100+ cocktails comprise drug-like fluorine fragments satisfying the following parameters: • Mostly only 1 peak in 19F NMR spectrum • Purity 95%+ • DMSO soluble at 200 mM

Structure Filters applied: PhysChem Parameters:

• no reactive functional groups • 130 < MW < 300 • Lilly MedChem rules • -1.7 < ClogP < 3.0 • PAINS • H-bond Donors ≤ 3 • Ring count 1 – 3 • H-bond Acceptors ≤ 4 • TPSA ≤ 110 Å2

31 FLUORINE FRAGMENT COCKTAILS lifechemicals.com

Example of 19F NMR spectrum of the fragment cocktail with signals associated with each compound

32 BROMINE FRAGMENT LIBRARY lifechemicals.com

• Bromine atom can be located uniquely due to anomalous scattering that enables recognizing the structure of fragment-target complex

• 1,300 bromine-containing fragment to be used in crystallographic FBDD

• Compounds with MW ≤ 350 and ClogP ≤ 3.5 are selected. All the fragments contain only one Br atom. Reactive compounds, undesired chemotypes, PAINS are removed.

• An advanced subset of more than 800 compounds is also available selected by the following criteria: ClogP < 3.5 Molecular weight < 350 Da H-bond acceptors = 0−4 H-bond donors = 0−3 Rotatable bonds <= 4 Number of rings = 1−3

33 BROMINE FRAGMENT LIBRARY lifechemicals.com

Examples of structures:

34 LOW MW FRAGMENT LIBRARY lifechemicals.com

• The library contains leads with lower molecular weight and higher hydrophilicity. • Also included are over 3,000 high quality stock available Fragments selected by means of strict control of molecular weight (100-225 Da) and other essential physico-chemical parameters (cLogP, HDonors, HAcceptors, TPSA, RotB) • Finally, the Library is passed through a number of structural filters (MedChem, PAINS). • An advanced low molecular weight fragment set of nearly 1,500 compounds was obtained by using even more rigorous physicochemical and structural cut-offs.

Average value Filtering Parameter range Low MW Fragment Advanced Low MW Library Set Molecular weight 100…225 177 186 ClogP –3…3 0.7 0.9 H-acceptors 0…6 2.5 2.1 H-donors 0…3 1.3 1.3 TPSA, Å2 0…100 55 47 Rotatable bonds 0…2 1.3 1.3 Ring count 1…3 1.8 2.0

35 LOW MW FRAGMENT LIBRARY lifechemicals.com

Examples of structures:

36 FSP3-ENRICHED FRAGMENT LIBRARY lifechemicals.com

• Fsp3-enriched Fragment Library comprises more than 11,500 compounds with inherent high selectivity towards pharmacological targets.

• Physicochemical parameters of advanced Fsp3-enriched fragments (about 3,200 compounds) are summarized in the table below:

3 Parameter Range Average Value Fsp distribution

Molecular Weight ≤ 300 224 Fsp3 > 0.45 0.65 cLogP < 3 0.96 TPSA < 90 Å2 52 Å2 H-acceptors ≤ 3 2.2 H-donors ≤ 3 1.3 Rotatable bonds ≤ 3 2,2

Number of benzene rings ≤ 1 0.5

37 FSP3-ENRICHED FRAGMENT LIBRARY lifechemicals.com

Examples of structures:

38 3D FRAGMENT LIBRARY lifechemicals.com

• 1,500 carefully designed drug-like 3D fragments • Special selection by physicochemical properties and principal moments of inertia (PMI) to improve 3D-shape functionality and diversity of the molecules • Diversity filtering using linear fingerprints (Tanimoto coefficient 88%)

Parameter Range

Molecular Weight 100-300 Fsp3 > 0.47 TPSA < 100 A2 H-acceptors ≤ 4 H-donors ≤ 3 Rotatable Bonds ≤ 3 Chiral centers ≥ 1 Functionalization points ideally 2

Ring count 1 - 4

CN, NO2, Br ≤ 1 Life Chemical 3D fragments library covers various shapes: rod-like, S, Cl ≤ 2 disk-like and spherical with sufficient diversity.

PAINS and compounds with undesired functionalities are removed.

39 3D FRAGMENT LIBRARY lifechemicals.com

Examples of structures:

40 PPI FRAGMENT LIBRARY lifechemicals.com • About 2,500 in-stock fragments potentially capable of PPI inhibition • Selected using advanced set of descriptors and specially designed ranking mechanism1 • Hydrophobic and more spatial structures (sp3-enriched) to enhance selectivity2 • Excellent coverage of 3D shape space with good diversity3

Parameter Range Average value

Fsp3 ≥ 0.4 0.51

ClogP 2.5–4.5 3.3

TPSA < 75 Å2 58.9 Å2

Molecular weight 150–450 345

H-acceptors ≤ 5 2.9

H-donors ≤ 3 1.1

Rotatable bonds 0–8 4.6

Hydrophobic cores 1–6 2.6 PMI plot showing distribution of Life Chemicals PPI Ring count >0 3.1 fragments in 3D shape space

1 Reynès C., Host H., Camproux A.C. et al. PLoS Comput. Biol., 2010, 6(3):e1000695 2 Fry D.C. Protein-protein interactions as targets for small molecule drug discovery. Biopolymers, 2006, 84, 535–52. 3 Carr R.A.E. et al. Fragment-based lead discovery: leads by design. Drug Discov. Today, 2005, 10, 987–92.

41 PPI FRAGMENT LIBRARY lifechemicals.com

Examples of structures:

42 COVALENT FRAGMENT LIBRARY lifechemicals.com

 1,300 carefully selected Ro3 compliant covalent binders  Compounds with specific structural fragments (functional groups) that are known to form covalent bonds with amino acid residues in binding sites of target proteins: Lys, Cys, Ser, Asp, Glu, His and Tyr:

. Acetals . Other Michael acceptors . Acrylamides . Quinones . Acrylonitriles . Sulfonate esters . -Dicarbonyl compounds . Sulfonyl halides . Aliphatic thiols . Terminal acetylenes . Aromatic thiols . Thioureas and thiones . Epoxides . Vinyl sulfones and vinyl sulfonamides . Ketals . Other types . Maleimides and related compounds

Molecules with highly reactive electrophilic and nucleophilic groups, compounds with non drug-like cores were removed using selected unwanted cores filters and selected PAINS filters.

43 COVALENT FRAGMENT LIBRARY lifechemicals.com

Examples of structures:

44 SOLUBLE FRAGMENTS WITH EXPERIMENTAL DATA lifechemicals.com

We have around 12,500 carefully selected fragments with experimentally measured solubility at 200 mM in DMSO and 1 mM and 5 mM in pH 7.5 phosphate buffer solution (turbidimetry method).

• Average MW = 243 Da, average cLogP = 1.3

• Guaranteed solubility of all compounds in DMSO at very high concentration (200 mM)

• More than 75 % of fragments are also soluble in PBS buffer at 1mM and 60 % are soluble at 5mM

45 SOLUBLE FRAGMENTS WITH EXPERIMENTAL DATA lifechemicals.com

Examples of structures:

46 NATURAL PRODUCT-LIKE FRAGMENT LIBRARY lifechemicals.com

• A new fragment library recently added to Life Chemicals collection • Designed using Scaffold Tree analysis1 of Universal Natural Product Database2 (comprising more than 220,000 Natural Products) as the starting point • More than 3,200 fragments obtained from NP-derived or NP-like scaffolds

1 Schuffenhauer, A.; Ertl, P.; Roggo, S.; Wetzel, S.; Koch, M. A.; Waldmann H. J Chem Inf Model. 2007, 47, 47–58. 2 Gu, J.; Gui, Y.; Chen, L.; Yuan, G.; Lu, H.-Z.; Xu, X. PLoS ONE, 2013, 8, e62839.

47 INNOVATIVE DESIGN OF COMPOUND LIBRARY FOR FBDD lifechemicals.com

• Specially designed and synthesized for FBDD

• High novelty of chemical structures Diversity analysis VS eMolecules and most common vendors databases

• Diversification of the Library includes: Linear fingerprint, pharmacophore and 3D analysis

• High quality of molecules: rigorous physicochemical parameters, PAINS and in-house developed structure filters

• Rapid SAR generation potential Quick selection and synthesis of molecules for follow-up testing analogues

48 DEDICATED DESIGN OF COMPOUND LIBRARY FOR FBDD lifechemicals.com

1st Step. Virtual coupling

1,139 Building Blocks carefully selected novel Fsp3-enriched molecules + 1,246 Decorating reagents pharmacologically privileged and selected by Phys-Chem parameters structures

463,000 compounds

49 DEDICATED DESIGN OF COMPOUND LIBRARY FOR FBDD lifechemicals.com

2nd Step.

Phys-Chem parameters Structural filtering parameters

“Rule of three”-like restrictions: PAINS & in-house developed filters for • 150 < MW < 300 undesired and reactive functionalities • –2 < ClogP < 3 At least one ring in structure; no more than • HDon ≤ 3 two fused aromatic rings • HAcc ≤ 4 No more than one S, Cl, Br; no more than • RotB ≤ 3 three F Min one additional atom N, O apart from Other requirements the main functional group • TPSA ≤ 70 Å2 No N-arylsulfonamides, no tetrazoles, no • clogSw > –3 more than 5% of compounds with isolated • Heavy atoms (HAC) ≤ 20 benzene ring

50 DEDICATED DESIGN OF COMPOUND LIBRARY FOR FBDD lifechemicals.com

3rd Step. Novelty verification

98 % Tanimoto dissimilarity to eMolecules database 98 % Tanimoto dissimilarity to Major Competitors Fragment Databases 90 % Tanimoto dissimilarity to Life Chemicals Stock Collection

62,000 compounds

51 DEDICATED DESIGN OF COMPOUND LIBRARY FOR FBDD lifechemicals.com

4th Step. Complex diversity approach

Tanimoto 3D Shape dissimilary analysis selection 87%

Pharmacophore Complexity diversity optimization analysis

21,200 compounds

52 DEDICATED DESIGN OF COMPOUND LIBRARY FOR FBDD lifechemicals.com

5th Step. Final set formation

Chemotype control Possibility of evolution (ideally 2 points) Synthetic considerations Scaffold population analysis

Advanced Set of Tangible Fragments 5,202 compounds

53 DEDICATED DESIGN OF COMPOUND LIBRARY FOR FBDD lifechemicals.com Physicochemical properties analysis:

25 mean Fsp3= 0.58 20

cpds 15 Maybridge Ro3 set: 10 mean Fsp3 = 0.30

% the of % 5 0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Fsp3

mean HAC = 16.5 15

10

5 % of % cpds the

0 10 11 12 13 14 15 16 17 18 19 20 HAC

54 DEDICATED DESIGN OF COMPOUND LIBRARY FOR FBDD lifechemicals.com Physicochemical properties analysis:

30 mean MW = 232 50 mean HAcc=2.9 25 40 20 30 15 20 10

10 % cpds the of % % cpds the of % 5 0 0 150 200 250 300 0 1 2 3 4 MW HAcc

80 30 mean HDon= 0.3 mean TPSA =39 Å2 25 60 20 40 15 10

20 % cpds the of % % cpds the of % 5 0 0 0 1 2 3 30 40 50 60 70 HDon TPSA

55 DEDICATED DESIGN OF COMPOUND LIBRARY FOR FBDD lifechemicals.com Physicochemical properties analysis:

60 mean LogP = 1.1 25 mean logP Sw = –1.5 50 20 40 15 30 20 10

10 5 % cpds the of % 0 cpds the of % 0 -2 -1 0 1 2 3 -3 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 LogP logP Sw

50 mean RotB = 2.0 70 mean RingCount = 2.5 60 40 50 30 40 20 30 20

10 % cpds the of % % cpds the of % 10 0 0 0 1 2 3 0 1 2 3 4 5 RotB Ring Count

56 DEDICATED DESIGN OF COMPOUND LIBRARY FOR FBDD lifechemicals.com

Examples of building blocks:

57 DEDICATED DESIGN OF COMPOUND LIBRARY FOR FBDD lifechemicals.com

Examples of reagents:

58 DEDICATED DESIGN OF COMPOUND LIBRARY FOR FBDD lifechemicals.com

Examples of fragments:

59 DEDICATED DESIGN OF COMPOUND LIBRARY FOR FBDD lifechemicals.com

Examples of already synthesized fragments: More than 1000 compounds with stock weight of over 100 mg

60 DEDICATED DESIGN OF COMPOUND LIBRARY FOR FBDD lifechemicals.com

References

1. Boyd S. M., de Kloe G. E. Fragment library design: efficiently hunting drugs in chemical space. Drug Discovery Today: Technologies 2010, 7, 173–180. 2. Collins I., Jones A. M. Diversity-oriented synthetic strategies applied to cancer chemical biology and drug discovery. Molecules, 2014, 19, 17221–17255. 3. Langdon S. R., Brown N., Blagg J. Scaffold diversity of exemplified medicinal chemistry space. J. Chem. Inf. Model. 2011, 51, 2174–2185. 4. Joseph-McCarthy D., Campbell A. J., Kern G., Moustakas D. Fragment-based lead discovery and design. J. Chem. Inf. Model., 2014, 54, 693−704. 5. Koutsoukas A., Paricharak S., Galloway W. R. J. D. et. al. How diverse are diversity assessment methods? A comparative analysis and benchmarking of molecular descriptor space. J. Chem. Inf. Model. 2014, 54, 230−242. 6. Morley A. D., Pugliese A., Birchall K. et al. Fragment-based hit identification: thinking in 3D. Drug Discovery Today, 2013, 18, 23–24, 1221–1227. 7. Boyd S. M., Turnbull A. P. , Walse B. Fragment library design considerations. Comput. Mol. Sci., 2012, 2, 868–885. 8. Schulz M. N., Landström J., Bright K., Hubbard R. E. Design of a fragment library that maximally represents available chemical space. J. Comput. Aided Mol. Des., 2011, 25, 611–620.

61 lifechemicals.com TARGETED AND FOCUSED LIBRARIES

• Receptor-based Libraries: Based on flexible docking to a target structure(-s) obtained with X- ray analysis or homology modeling

• Ligand-based Libraries: Based on known compounds’ activity, structure-activity relationships, pharmacophore features and structural 2D and 3D similarity

• Cheminformatics-based Libraries: Based on combination of physicochemical parameters and descriptors, ADMET characteristics and presence of certain chemical groups and features

• Broad Focus Libraries: Designed with either ligand-, receptor- or cheminformatics-based approach but focused towards general pharmacological activity rather than particular drug target or drug target family

62 TARGETED AND FOCUSED LIBRARIES lifechemicals.com

Receptor-based & Ligand-based Libraries

63 KINASES TARGETED LIBRARIES lifechemicals.com

The design of Life Chemicals GPCRs Targeted Library implied application of several computational methods, such as homology modeling, reference set selection, molecular dynamics, molecular docking and in silico version of high-throughput screening (HTS).

Kinase Targeted Docking Library ~ 13,500 compounds

• CDK2 (cyclin dependent kinase 2) • GSK3 (glycogen synthase kinase 3) • PKB (protein kinase B) • SRC kinases (2 protein structures) • EGFR (epidermal growth factor receptor)

Sharp-aimed Kinase Targeted Libraries ~23,876 compounds

64 SHARP-AIMED KINASE TARGETED LIBRARIES lifechemicals.com ~23,876 compounds

• Aurora A Kinase Focused Library (600 compounds) • Aurora B Kinase Focused Library (1,000 compounds) • Cyclin-dependent Kinase 2 (CDK2) Focused Library (400 compounds) • P38 Mitogen-Activated Protein (MAP) Kinase Focused Library (1,200 compounds) • Vascular Endothelial Growth Factor Receptor Kinase 2 (VGFR-2) Focused Library (1,000 compounds) Aurora A kinase • Human Fibroblast Growth Factor Receptor 1 Tyrosine Kinase (FGFR1K) Focused Library (700 compounds) • c-Met Kinase (Hepatocyte Growth Factor Receptor (HGFR)) Focused Library (1,100 compounds) • 3-Phosphoinositide-dependent Kinase 1 (PDK-1) Focused Library (800 compounds) • cAMP Dependent Protein Kinase (PKA) Focused Library (750 compounds) CDK2 kinase • Phosphatidylinositol 3-kinase (PI3K) Focused Library (950 compounds) • Human Janus Kinase (JAK) Focused Library (600 compounds) • Rho-associated Protein Kinase (ROCK) Focused Library (1,500 compounds) • Extracellular-signal-regulated Kinase 5 (ERK5) Focused Library (1,000 compounds)

c-Met kinase

65 KINASE FOCUSED LIBRARY lifechemicals.com Tanimoto Similarity Search

• The list of 20,000 compounds that were active in biological assays (more than 87 different kinase targets) was used as a Reference Set (ChEMBL database) • Collection was searched for compounds similar to the Reference Set using MDL public keys and the Tanimoto similarity cut-off of 85 % • About 30,000 potential compounds were selected to Kinase Focused Library

• 3-phosphoinositide dependent protein kinase-1 • Bcr/Abl fusion protein; Tyrosine-protein kinase ABL • CaM kinase II delta • Casein kinase • c-Jun N-terminal kinase • Cyclin-dependent kinase • Death-associated protein kinase • Dual specificity mitogen-activated protein kinase 1 • Ephrin type-B receptor 4 • Epidermal growth factor receptor • Glycogen synthase kinase • MAP kinase • Tyrosine-protein kinase ABL; Bcr/Abl fusion protein • ets.

66 KINASE FOCUSED LIBRARY lifechemicals.com Markush Similarity Search

• Similarity search based on 964 Markush structures of known kinase inhibitors

• Tanimoto similarity search based on 216 structures of reference compounds

• Library consists of 2,090 inhibitors & activators

• Focused on the key kinases in cancer and inflammation: ABL, AKT, AMPK, Aur, CDK, CHK1, CK2, cKIT, ERK, FLT3, GSK3, HER, JAKs, LCK, MEK1, mTOR, p38a, PAK, PDK1, PI3K, PIM, PKM2, PLK1, ROCK, Src, Syk, Tyk2, VEGFR

67 GPCRs TARGETED LIBRARY lifechemicals.com The design of Life Chemicals GPCRs Targeted Library required application of several computational methods, such as homology modeling, reference set selection, molecular dynamics, molecular docking and in silico version of high-throughput screening (HTS).

Individual receptors: Sphingosine 1-phosphate, Muscarinic acethylcholine, Serotonine, Dopamine, GABA.

Receptor families: 8,500 compounds were identified Adenosine, Chemokine, Opioid, Histamine

68 NUCLEAR RECEPTORS TARGETED LIBRARY lifechemicals.com

Over 5,100 compounds were selected with protein-ligand docking method.

69 ION CHANNELS FOCUSED LIBRARY lifechemicals.com Pharmacophore search

The pharmacophore search was performed on the basis of the reference set of compounds derived from ChEMBL (2,750 compounds) and BindingDB (3,157 compounds) with the use of following techniques:

1. Hierarchical clustering of the reference set (which provided a subset distribution by 10-atom core similarity) revealed 150 top-level groups of compounds. Almost 50 of them were chosen as the weightiest branches. 2. Molecular properties of the reference set (MW, TPSA, PSA) were calculated to obtain parameters for stock collection filtering. 3. A substructure search in the filtered stock collection of compounds revealed about 25 scaffolds (represented by about 1,800 compounds) similar to the clustering result. 4. Search process was improved by modeling a series of GALAHAD (SYBYL-X) pharmacophores for each set of reference compounds.

Subsequent flexible 3D alignment of compounds based on pharmacophore features, such as HDA, ACC, Hydrophobicity, steric features, resulted in the library of 5,900 compounds available in stock.

70 ION CHANNELS FOCUSED LIBRARY lifechemicals.com Similarity Search

 The reference set of known ion channel blockers was taken from ChEMBL database.  Fingerprint-based similarity search was performed against following 28 ion channel targets:

• Amiloride-sensitive cation channel 3 • Transient receptor potential cation channel subfamily A member 1 • Apoptosis regulator Bcl-2 • Transient receptor potential cation channel subfamily M member 8 • Calcium-activated potassium channel subunit alpha-1 • Transient receptor potential cation channel subfamily V member 4 • Glutamate [NMDA] receptor subunit epsilon 2 • Vanilloid receptor 1 (TRPV1) • Glutamate receptor ionotropic kainate 1 • Voltage-gated L-type calcium channel alpha-1S subunit • HERG • Voltage-gated N-type calcium channel alpha-1B subunit • MCOLN3 protein • Amyloid beta A4 precursor protein-binding family A member 1 • Neuronal acetylcholine receptor protein alpha-7 subunit • Voltage-gated T-type calcium channel alpha-1G subunit • P2X purinoceptor 7 • Voltage-gated T-type calcium channel alpha-1H subunit • Serotonin (5-HT) receptor • Voltage-gated T-type calcium channel alpha-1I subunit • Serotonin 3 (5-HT3) receptor • Voltage-gated potassium channel subunit Kv1.3 • Serotonin 3a (5-HT3a) receptor • Voltage-gated potassium channel subunit Kv1.5 • Short transient receptor potential channel 3 • Voltage-gated potassium channel subunit Kv7.2 • Sodium channel protein type IX alpha subunit • Voltage-gated potassium channel subunit Kv7.4

 Application of Tanimoto similarity threshold of 85 % resulted in a library of 5,400 compounds.

71 PPI PDZ DOMAINS TARGETED LIBRARIES lifechemicals.com

PDZ proteins are the keys to cell life, their therapeutic potential includes neurology, cancer and immunology drug discovery. Three libraries of small molecules targeting PDZ domains of Magi1 (d1) and PSD-95 (d2) proteins were designed using Life Chemicals’ proprietary stock collection.

First PDZ domain of Magi1(d1)

Second PDZ domain of PSD-95 (2 screening models)

72 PPI PDZ DOMAINS TARGETED LIBRARIES lifechemicals.com First PDZ Domain of Magi1(d1)

The Unity query model based on 2I04 PDB entry was created as the superposition of the features from ligand human papillomavirus (HPV E6) residues and substrate binding groove of PDZ domain. Therefore, the final Unity model contained peculiarities both from PDZ binding site residues and HPV E6 peptide pharmacophore.

Screening model includes: • 9 H-bond Donor features • 6 H-bond Acceptor features • 2 hydrophobic features • 1 Surface-Volume constraint

2,075 compounds were identified as hits. Donor sites (green), acceptor sites (violet) and Hydrophobic feature (brown) according to the Unity color scheme

73 PPI PDZ DOMAINS TARGETED LIBRARIES lifechemicals.com Second PDZ Domain of PSD-95

Two docking models of binding, considering site of PSD95 PDZ domain 2 were created based on the protein structure recorded in 1QLC and 2KA9 PDB entries.

The query features were identified from the key residues of the PDZ domain responsible for ligand binding considering some differences in the binding groove conformations of PDZ domain observed in 2KA9 and 1QLC models.

2KA9 docking model: 1,000 hits were identified Example of a hit compound F3225-8235 binding conformation. QFIT = 65.03. The compound forms two hydrogen bonds with 1QLC docking model: Ile114 and Asn120 of the PSD95 PDZ2 substrate binding site. 1,075 hits were found

74 PPI INHIBITORS lifechemicals.com Similarity Search Libraries

PPI Focused Library 2D Similarity Search to Timbal DB 1,400 compounds were extracted from Life Chemicals stock HTS collection by 2D fingerprint similarity search towards Timbal DB with Tanimoto 85% threshold.

PPI Focused Library 2D Similarity Search to Binding DB and Pubmed DB A set of 10,000 reference compounds with activity in 28 PPI assays towards 7 PPI targets was collected using Binding and Pubmed Databases:

• toll-like receptor 4 • Hepatitis C virus core protein • Tyrosine-protein kinase TYRO11 • runt-related transcription factor 1 isoform AML1c • core-binding factor beta subunit isoform 1 • mitogen-activated protein kinase 2 (MAP2) • mitogen-activated protein kinase 3 (MAP3)

Similarity search through Life Chemicals Stock Collection with Tanimoto 90 % cutoff led to the Library comprising about 22,000 potential PPI inhibitors. http://www-cryst.bioc.cam.ac.uk/databases/timbal http://www.bindingdb.org http://www.ncbi.nlm.nih.gov/pccompound

75 PPI PEPTIDOMIMETIC LIBRARY lifechemicals.com

More than 5,000 α-helix and β-turn mimetics were selected with ligand-based approach, including various similarity analysis techniques, such as structural similarity search against known peptidomimetic inhibitors and scaffolds, 3D shape screening, pharmacophore screening and complex substructure search:

• 2D similarity analysis has been done against known peptidomimetic inhibitors and scaffolds with Tanimono index ≥ 0.8

• 3D shape screening has been done with Pharmacophore Types volume scoring against more than 1 million conformers generated from Life Chemicals Stock Compound Collection. The screening was based on 3D structures of α-helices and β-turns (type I and II) obtained with the use of donor\acceptor quantum mechanics calculations.

• Pharmacophore screening included pharmacophore model construction based on 3D structures of β-turns (type I and II) obtained with quantum mechanics calculations. The model contained specific exclusion volume constraint and donor\acceptor atom positions in order to allow hydrogen bond formation in β-turn mimetics.

• Complex substructure search involved search queries constructed on the base of α-helix and β-turn peptidomimetic scaffolds known from literature, such as various bicyclic, spiro, macrocyclic, pyrrolidine, terphenyl, oligobenzamide, anthracene and other scaffolds.

76 PPI PEPTIDOMIMETIC LIBRARY lifechemicals.com

Compound aligned against pharmacophore sites. Example of pharmacophore search based on β-turn structure.

1. Peptidomimetics in Organic and Medicinal Chemistry. Andrea Trabocchi, Antonio Guarna. John Wiley & Sons, Apr 7, 2014. 2. Maryanna E. Lanning and Steven Fletcher, Multi-Facial, Non-Peptidic α-Helix Mimetics, Biology 2015, 4, 540-555; 3. Andrew J. Wilson, Helix mimetics: Recent developments, Progress in Biophysics and Molecular Biology (2015) 4. Madura K. P. Jayatunga, Sam Thompson, Andrew D. Hamilton, a-Helix mimetics: Outwards and upwards, Bioorganic & Medicinal Chemistry Letters 24 (2014) 717–724. 5. Maryanna Lanning & Steven Fletcher, Recapitulating the a-helix: nonpeptidic, low-molecular-weight ligands for the modulation of helix- mediated protein–protein interactions, Future Med. Chem. (2013) 5(18), 2157–2174. 6. Landon R. Whitby, Yoshio Ando, Vincent Setola, Peter K. Vogt, Bryan L. Roth, and Dale L. Boger, Design, Synthesis, and Validation of a β-Turn Mimetic Library Targeting Protein-Protein and Peptide-Receptor Interactions. J. Am. Chem. Soc. – 2011 – Vol. 133, p. 10184– 10194. 7. Jari J. Koivisto, Esa T. T. Kumpulainen and Ari M. P. Koskine, Conformational ensembles of flexible b-turn mimetics in DMSO-d6. Org. Biomol. Chem. – 2010 – Vol. 8, p. 2103–2116. 8. Ralph F. Hirschmann et al., The β-D-Glucose Scaffold as a β-Turn Mimetic. Acc Chem Res. – 2009 – Vol. 42(10), p. 1511–1520.

77 PPI INHIBITORS MACHINE LEARNING lifechemicals.com Method (MLM) Library

Machine learning method (decision tree, DT) was used as background for the construction of Life Chemicals PPI Inhibitors Enriched Library.

Accumulation of compounds with best matching to our parameters with PCA

2,600 potential PPI inhibitors were found

78 EPIGENETIC DOCKING LIBRARIES lifechemicals.com

The Library contains 7,019 compounds selected by virtual screening against the following targets:

DNA Methyltransferase: • DNMT1 • DNMT3A • DNMT3B • Lysine-Specific Histone Demethylase 1 Histone Deacetylase: • HDAC I • HDAC II Histone Acetyltransferase: • P300 Histone-Lysine N-Methyltransferase: • DOTL1 • Histone H3 Lysine 4 Specific Methyltransferase (KMT4) Protein Arginine Methyltransferase: • PRMT1 • PRMT3 • PRMT4/CARM1 • PRMT5

79 EPIGENETIC DOCKING LIBRARIES lifechemicals.com SIRT Targeted Library

Sirtuins (SIRT) is a class of proteins that possess either mono-ADP-ribosyltransferase or deacylase activity, including the activity that is classified as epigenetic factor.

• Two docking models were developed to find potential sirtuin inhibitors by virtual screening:

1. Model based on SIRT 1, 2, 5 and 6 (due to similarity of their binding site, 2 hydrogen bonds) 2. Model based on the structure of SIRT 3 (3 hydrogen bonds)

About 667 compounds were identified as potential hits.

80 EPIGENETIC LIBRARY lifechemicals.com Similarity Search

• The Library contains 660 compounds carefully selected with 2D Fingerprint Similarity search (SYBYL-X software). • Reference sets of compounds were obtained from ChEMBL database. The upper IC50 value threshold for all reference compounds was 1.1μM against each target.

81 COVALENT INHIBITORS LIBRARY lifechemicals.com

6,700 Ro5 compliant compounds were extracted by characteristic structural fragments (functional groups) that were described to form covalent bonds with residues in binding sites of target proteins: Lys, Cys, Ser, Asp, Glu, Tyr.

Substructure fragments given below were used for selection of in-stock compounds:

β-lactams Thioles Alkyl halides Rodanides Epoxides, aziridines Thioureas and thioketones Michael acceptors: o-quinones α,β-unsaturated ketones, -nitriles, -esters; p-quinones maleimide-like compounds; Ketales activated vinyl derivatives Acetales Cyanoacrylamides Disulfides Sulfonate esters Terminal acetylenes Sulfonyl fluorides Sulfoalkenes

82 COVALENT INHIBITORS LIBRARY lifechemicals.com

Cysteine Focused

The Cys focused compound set of 1,400 potential covalent inhibitors was created on the basis of specific structural moieties that could react reversibly or irreversibly with active site cysteine residues.

The following structural fragments were used for selection of possible covalent inhibitors focused on Cys residue from Life Chemicals’ collection of Ro5 compliant compounds:

α,β-unsaturated ketones α-chloroacetamides Phenyl sulfonate esters Vinyl sulfonamides Acrylamides Acrylonitriles Aminomethyl methyl acrylates Methyl vinyl sulfones Epoxides Activated acetylenes Sulfonyl fluorides

83 COVALENT INHIBITORS LIBRARY lifechemicals.com

Serine Focused

Serine Focused Library containing 1,800 covalent inhibitors was designed on the basis of combination of specific structural fragments (functional groups) that were reported to form covalent bonds with serine residue in binding sites of proteins and drug-like filters (“Lipinsky’s Rule of Five”, “Veber rules”, reactive groups and selected PAINS filters).

Compounds with the following structural fragments were included into the library:

Carbamates Boronic acids Activated aliphatic nitriles Aryl ureas (no isolated benzene ring) (retro Michael acceptors) Trifluoroacetamides Epoxides α-hydroxy ketones β-propiolactones α-ketoalkyl oxazoles γ-butyrolactones N-carbamoyl azoles β-lactams 1,2-diazetidones

84 PROTEASE FOCUSED LIBRARY lifechemicals.com

The library of 1,400 compounds was generated with similarity search against a set of compounds displaying activity towards corresponding classes of protease targets:

• Serine proteases • Cysteine proteases • Aspartate proteases • Glutamic acid proteases • Metalloproteases • Threonine proteases

85 Protease LASSO Screening Libraries lifechemicals.com

SimBioSys and LifeChemicals teamed up to provide focused libraries for selected targets on the LifeChemicals' stock catalog. Using the LASSO ligand-similarity search tool The Life Chemicals Database was screened for the following libraries:

• HIV Protease (4,300 compounds) • HIV Reverse Transcriptase (3,100 compounds) • Factor Xa (FXa) (1,700 compounds) • Thrombin (2,400 compounds) • Chymotrypsin (100 compounds) • Trypsin-like Protease (900 compounds)

86 CYSTEINE PROTEASE FOCUSED LIBRARY lifechemicals.com

To design Cysteine Protease Targeted Library a similarity search was used as the most universal method.

A reference database of 9,618 biologically active compounds from assays related to Cysteine Proteases was compiled using the data available from patents and literature publications.

Life Chemicals collection was searched for compounds similar to compounds from the reference database using MDL public keys and Tanimoto similarity cut-off of 90 %.

As a result, 3,200 compounds were extracted to Life Chemicals Cysteine Protease Targeted Library.

87 CASPASE FOCUSED LIBRARY lifechemicals.com

To identify potential inhibitors for a number of caspases a TopomerSearch method, ligand- based fast virtual screening tool (SYBYLX), was used. As a template we used potent caspase inhibitors that were co-crystalized with corresponding proteins. The query features were created based on crystallographic conformations of the ligands. Compounds with unwanted structures were filtered out using the PAINS filters.

The following targets and corresponding pdb entries were used for the Library preparation: • CASP1 (1RWX pdb) • CASP2 (1PYO pdb) • CASP3 (2CNO and 2CNL pdbs) • CASP6 (3OD5 pdb) • CASP7 (3EDR and 2QL9 pdbs) • CASP8 (1QTN and 3KJN pdbs) • CASP9 (1NW9 pdb) • Paracaspase (3V4O pdb)

Almost 2,800 potential hits were identified.

88 PHOSPHATASE FOCUSED LIBRARY lifechemicals.com

 38 phosphatase targets were considered to create the reference set for similarity search:

• Receptor-type tyrosine-protein phosphatase S • Receptor-type tyrosine-protein phosphatase beta • Dual specificity phosphatase 22 • Receptor-type tyrosine-protein phosphatase F (LAR) • Serine/threonine-protein phosphatase • Protein-tyrosine phosphatase G1 • Tyrosine-protein phosphatase yopH • Protein-tyrosine phosphatase 1C • Protein-tyrosine phosphatase 1E • Dual specificity protein phosphatase 3 • Tyrosine-protein phosphatase non-receptor type 3 • Dual specificity phosphatase Cdc25B • Dual specificity protein phosphatase 15 • Dual specificity phosphatase Cdc25A • Dual specificity protein phosphatase 23 • Protein-tyrosine phosphatase 2C • Receptor-type tyrosine-protein phosphatase mu • Low molecular weight phosphotyrosine protein phosphatase • Protein-tyrosine phosphatase 4A3 • Receptor-type tyrosine-protein phosphatase alpha • Dual specificity protein phosphatase 10 • Dual specificity phosphatase Cdc25C • Tyrosine-protein phosphatase non-receptor type 14 • Protein-tyrosine phosphatase 1 • HPr kinase/phosphatase • Secreted effector protein • Protein phosphatase 2A regulatory subunit B • Protein-tyrosine phosphatase LC-PTP • Serine-threonine protein phosphatase 2A regulatory • Receptor-type tyrosine-protein phosphatase gamma subunit • Serine/threonine protein phosphatase PP1-alpha catalytic subunit • Protein phosphatase 2C beta • Serine/threonine protein phosphatase PP1-gamma catalytic subunit • Protein-tyrosine phosphatase 1B • Probable low molecular weight protein-tyrosine-phosphatase • Hematopoietic cell protein-tyrosine phosphatase 70Z-PEP • Phosphotyrosine-protein phosphatase PTPB • T-cell protein-tyrosine phosphatase

 4,500 compounds with activity value IC50<10000nM were extracted into the reference set.  Tanimoto similarity cut-off of 85 % using 2D fingerprints (Unity module of Sybyl-X) was applied for search in The Life Chemicals Stock Compounds Collection.  As a result, over 1,100 potential hits were included into our Phosphate Focused Library.

89 CYSTEINE PROTEASE FOCUSED LIBRARY lifechemicals.com

 To design Cysteine Protease Targeted Library a similarity search was used as the most universal method.

 A reference database of 9,618 biologically active compounds from assays related to Cysteine Proteases was compiled using the data available from patents and literature publications.

 The Life Chemicals collection was searched for compounds similar to compounds from the reference database using MDL public keys and Tanimoto similarity cut-off of 90 %.

 As a result, 3,200 compounds were extracted to The Life Chemicals Cysteine Protease Targeted Library.

90 AQUAPORINS TARGETED LIBRARY lifechemicals.com

A library of 2,000 compounds targeted at aquaporins 1, 4 and 5 was designed using our in-house developed procedure of docking models preparation and virtual screening:

1. Search for a test set of compounds. 2. Ligprep tool was used for generation of 3D coordinates of compounds with different ionization state. 3. Ft-MAP server was used for site finding. 4. Flexible Glide Docking was applied for preparation of maps. 5. Minimization of docked ligand poses and nearest protein environment was carried out in SybylX. 6. Development of docking models by marking atoms that have to form donor/acceptor interactions with ligand atoms, hydrophobic cores.

91 DEUBIQUITINASE (DUB) TARGETED/FOCUSED LIBRARIES lifechemicals.com

Both structure-based and ligand-based approaches were implemented to identify compounds displaying activity towards DUB targets: first pair of representatives (USP1/USP2) of the main ubiquitin-specific protease superfamily and UBA5 of E1 class.

DUB Targeted Library Docking of entire The Life Chemicals Stock Database in the active site of E1 activating protein. To improve docking results a set of constraints was defined. The resulting library includes 417 stock available compounds.

DUB Focused Library Pharmacophore modeling for search of DUB-specific compounds was used. 1,239 prospective small- molecule inhibitors of ubiquitin-specific proteases (USP1/USP2) were identified.

92 PHOSPHODIESTERASE 10 (PDE10) TARGETED LIBRARY lifechemicals.com

Different computational approaches were combined to develop a unique method for in silico search of potential PDE10 inhibitors.

• A general model 3D pharmacophore model was created based on the structure of three most potent PDE10 inhibitors. The model was validated using the reference set of 150 active models definition and 10 non-active compounds (ChEMBLdb) • TopomerSearch was used for pharmacophore search of active moleculesdefinition. • Consequently, a structure of binding site of PDE10 including water molecules was modeled. • The protein structure displayed highest stability during calculations was used for Docking.

427 small molecules were found as potential PDE10 inhibitors from Life Chemicals Stock Compounds Collection.

93 ATPASE FOCUSED LIBRARIES lifechemicals.com

Life Chemicals offers its ATPase Targeted Libraries prepared with 2D fingerprint similarity search (Tanimoto 85 % cut-off) against 2 reference databases using MDL public keys:

ATPase Targeted Library I A subset of 4,500 carefully selected off-the-shelf compounds. Similarity search against the reference set extracted from ChEMBL database was applied. The following targets were considered:

• ATPase family AAA domain-containing protein 5 • Potassium-transporting ATPase • Vacuolar H(+)-ATPase

ATPase Targeted Library II A list of 3,400 biologically active compounds was made by similarity. Similarity search against reference data on 127 ATPase related assays obtained from Patents, literature publications and other sources.

94 HELICASE FOCUSED LIBRARY lifechemicals.com

• The reference database of 13,850 biologically active compounds from ChEMBL was prepared. The following targets were considered:

• ATP-dependent DNA helicase Q1 • Werner syndrome ATP-dependent helicase

• The Life Chemicals Stock Compound Collection was searched for compounds similar to the structures from the reference database using MDL public keys and Tanimoto similarity cut-off of 85 %. • 1,000 potential hits were extracted to The Helicase Focused Library

95 POLYMERASE FOCUSED LIBRARIES lifechemicals.com

• Polymerase Focused Library I 1,200 advanced compounds were extracted from the stock by similarity search against the reference set of 20,000 active compounds from ChEMBL database displaying activity towards the following targets:

• DNA polymerase beta • DNA polymerase iota • DNA polymerase eta • Poly [ADP-ribose] polymerase-1 • Polymerase Focused Library II A list of 4,567 biologically active compounds (reference compound set) from 15 polymerase assays representing the following polymerases was compiled using the data available from patents and literature publications:

• RNA polymerase beta subunit (EC 2.7.7.6) • Ribonuclease HI (RNase HI) (Ribonuclease H) (RNase H) • DNA Polymerase III Holoenzyme • Measles Virus RNA-Dependent RNA Polymerase • Reverse Transcriptases (HIV-1, HIV-2, West Nile Virus NS2bNS3) Similarity analysis of The Life Chemicals Stock Compounds Collection VS the reference compound set using MDL public keys and Tanimoto similarity cut-off 90% identified 17,500 compounds.

96 RNA FOCUSED LIBRARY lifechemicals.com

The Life Chemicals RNA-focused Library comprises over 3,500 compounds with predicted RNA-binding activity. The library was designed with two ligand-based approaches:

1. 2D Similarity Search A set of about 750 known RNA-binding molecules has been collected from literature. It was narrowed down by binding activity via discarding compounds with low dissociation constant (Kd > 10μM). Further, 2D similarity search was performed against The Life Chemicals Stock Compound Collection with Tanimoto coefficient > 0.65 and Fragment Based Fingerprints. PAINS compounds, compounds with "bad" and reactive groups were discarded.

2. Bayesian Modeling The same training set of known RNA-binding molecules was used to build a Bayesian model (Bayesian categorization methodology) to distinguish “good” molecules from the compounds with known activity. Both molecular fingerprints (circular FCFP6 fingerprints) and molecular properties (MW, No. of HBA, No. of HBD, logP, PSA, No. of rotatable bonds, No. of rings) were involved in the construction of Bayesian model. The model was applied to Life Chemical’s stock collection in order to predict RNA-binding compounds.

97 CYP INHIBITORS LIBRARY lifechemicals.com

A set of 1,900 compounds was found to be 85 % similar with the ChMBL derived reference set of 19,000 compounds possessing activity towards a family of Cytochrome 450 enzymes:

• Cytochrome P450 1A2 • Cytochrome P450 11B1 • Cytochrome P450 19A1 • Cytochrome P450 3A4 • Cytochrome P450 2D6 • Cytochrome P450 1A1 • Cytochrome P450 2A6 • Cytochrome P450 2C9 • Cytochrome P450 2C19 • Cytochrome P450 3A4/3A5

98 GLUTAMATE RECEPTORS FOCUSED LIBRARY lifechemicals.com

 The Life Chemicals Stock Database was searched for compounds similar to active compounds from the ChMBL database reference set using 80 % similarity cut-off (Tanimoto) on MDL public keys fingerprints.  The approach increases chances for good biological activity by selecting compounds similar enough to the known active compoundsbut at the same time rather different from those to be outside.  As a result, over 1,100 Glutamate Receptors Focused compounds were selected.

 Targets used for the Library generation: • Metabotropic glutamate receptor 1 • Metabotropic glutamate receptor 5 • Glutamate receptor ionotropic kainate 1 • Metabotropic glutamate receptor 2 • Metabotropic glutamate receptor 4

99 TARGETED AND FOCUSED LIBRARIES lifechemicals.com

Cheminformatics-based Libraries & Broad Focus Libraries

100 NATURAL PRODUCT-LIKE COMPOUNDS lifechemicals.com

Life Chemicals presents a collection of Natural Product-like compounds, containing 2 subsets generated through by the following approaches:

Similarity Search Based Library Commercial databases were used as a reference: Timetek, Specnet, Analyticon, Selleck. 900 compounds in stock.

Chemoinformatics & Substructure search based Library Superposition of chemical descriptors calculation and natural-likeness scoring revealed 2,000 compounds in stock.

O O OH O O O OH N O O N O HO O O N N

101 CNS LIBRARY lifechemicals.com

Parameter Range • Careful literature analysis was performed to identify MW 150 – 400 parameters describing the compounds capable of ClogP 1.3 – 3.0 permeation of blood-brain barrier (BBB). PSA ≤ 65 Å2 HbD ≤ 3 HbAc ≤ 6 • 6,400 small organic molecules from the Life Chemicals RotB ≤ 6 database that meet stated parameters were selected. Rings 1 – 5 Total H-bonding < 8 Carboxylic acid group ≤ 1 • PAINS and toxicophore filters were applied to the library. S atoms ≤ 2 Cl atoms ≤ 2 Basic Nitrogen ≤ 2 No quaternary nitrogens

References: 1. Pardridge W. M. Blood–brain barrier delivery. Drug Discovery Today. 2007, 12 (1/2); 54–61. 2. Pajouhesh H., Lenz G. R. Medicinal chemical properties of successful central nervous system drugs. NeuroRx., 2005, 2; 541–553. 3. Vlieghe1 P., Khrestchatisky M. Medicinal chemistry based approaches and nanotechnology-based systems to improve CNS drug targeting and delivery. Medicinal Research Reviews, 2012, 00; 1–60.

102 VETERINARY LIBRARY lifechemicals.com

The Life Chemicals Veterinary Focused Library appeared due to our interest in biology and veterinary medicine.

• 205 FDA approved veterinary drugs taken from DrugBank (https://www.drugbank.ca) were used as the reference set for 2D similarity search. • Similarity search was done with Fragment Based Fingerprints and Tanimoto index >7.5 against The Life Chemicals Stock Compound Collection • The resulting Life Chemicals Veterinary Focused Library of 241 compounds includes full structural analogs or structurally similar compounds to those from the reference set of FDA approved veterinary drugs. • For each compound, the Similarity information information (similarity value and corresponding reference compound from DrugBank) is indicated for each compound.

103 AGROCHEMICAL LIBRARIES lifechemicals.com

The Library was created using The Life Chemicals Stock Compound Collection to pick up compounds with appropriate physicochemical properties and structural features.  Physicochemical restrictions applied: 250 < MW < 500 0,3 < ClogP < 4.5 HbD ≤ 2 HbA < 10  Similarity search (Tanimoto 80 % cut-off) VS the referent set of known agrochemical compounds (www.pesticideinfo.org) was performed.  In addition, a substructure search was applied. Substructural cores of each class of agrochemical compounds were selected from the literature and e-databases.  4 subsets of potential agrochemical agents were collected: Insecticides (1,900 compounds) Fungicides (1,100 compounds) Herbicides (7,900 compounds) Microbiocides (2,000 compounds)

104 BIOACTIVE COMPOUNDS LIBRARY lifechemicals.com

The Life Chemicals Library of Bioactive Compounds contains over 8,700 small molecules that have been tested in functional, binding and other biological assays and have confirmed bioactivity interest in vitro and/or in vivo against various biological targets of pharmaceutical interest. The Library has been designed on the basis of ChMBL database version 21 which contains: • 1,929,473 compound records • 1,212,831 assays • 1,592,191 compounds • 11,019 targets • 13,968,617 activities

The Library of Bioactive Compounds comprises compound records that meet the following criteria:

• Inhibition > 30% • XC50 < 10µM • Ki < 10µM • Residual Activity < 50% • EC50 < 10µM • AC50 < 10 µM • CC50 < 10µM • IZ > 10mm -1 • Kd < 10µM • MIC < 10 µg.mL • Potency < 10µM • MIC < 10µM

Each compound is supplied with a link to its Compound Report Card and Bioactivity summary.

105 ANTICANCER LIBRARY lifechemicals.com

 A reference set of 7,000 compounds with confirmed activity towards cancer-related targets (>10,000nM) was extracted from ChMBL and Binding databases.

 The Life Chemicals Stock Database was subjected to similarity search against reference compounds using the 80 % similarity cut-off (Tanimoto).

 As a result, over 700 potential anticancer agents were selected in the result.

106 ANTIBACTERIAL LIBRARY lifechemicals.com

• 2D Similarity search through The Life Chemicals Stock Database was carried out against the reference set of active molecules (ChEMBLdb and Bindingdb) that were reported to target more than 60 pathogenic bacteria and their resistant strains.

• Compounds with similarity index more than 80 % were included in the library.

• The final set of selected compounds was processed with reactive groups filtering and PhysChem restriction (e.g. 150 ≤ MW ≤ 650, -2 ≤ ClogP ≤ 6, at least 1 ring).

• The resulting Antibacterial Library included 5,500 small molecules.

• All the data on selected entities are available: similarity index to the reference structure, reference molecule ID, target (organism, bacteria) name, protein target, activity data (IC50, EC50), gram-information.

107 ANTIVIRAL LIBRARY lifechemicals.com Similarity Search

The Library was extracted from The Life Chemicals Stock Database with 2D fingerprint similarity search (Tanimoto similarity cut-off of 90 %) against the reference set of 6,937 biologically active compounds from 33 therapeutically relevant viral assays representing the following species of viruses:

Human herpesvirus 4 type 2 Influenza B viruses: Human immunodeficiency viruses 1, 2 B-Lee-40 Avian sarcoma virus B-Memphis-3-93 Influenza A viruses: B-Nashville-6-89 Puerto Rico-8-34(H1N1) Strain b-victoria-3-85 A-Puerto Rico-8-34-Mount Measles virus Sinai(H1N1) Rous sarcoma virus (strain Schmidt-Ruppin A) A-Shangdong-9-1993(H3N2) West Nile virus A-Tokyo-3-1967(H2N2) Bluetongue virus A-tern-Australia-G70C-1975(H11N9) Dengue virus A-Singapore-1-1957(H2N2)

18,400 compounds were found to be potential antiviral agents.

108 ANTIVIRAL LIBRARY lifechemicals.com Combined Ligand-based and Structure-based Approach

Life Chemicals prepared a set of mini libraries, containing targets potentially active against several most interesting and widely spread representative targets:

SARS coronavirus 3C-like proteinase Dengue virus type 2 NS3 protein Human herpesvirus 5 DNA polymerase Hepatitis C virus NS3 protease Human herpesvirus 6 DNA polymerase Hepatitis C virus NS5B RNA-dependent RNA polymerase Human herpesvirus 5 capsid protein Human rhinovirus A protease P40 Human immunodeficiency virus type 1 reverse transcriptase Human herpesvirus 1 DNA polymerase Human immunodeficiency virus type 1 integrase • A combination of ligand-based and structure-based approaches provided a higher degree of accuracy and cross-validation step. • To identify some features of binding mechanisms RCSB database for protein crystal structures and Chembl DB for reference compounds were used. • All most active compounds (IC50<1–1.5uM) were clustered and top of each group were docked into crystal structure to obtain bioactive conformation. • The aligned structures were used for pharmacophore modeling. Both Glide docking and UNITY pharmacophore search methods were applied to select the most promising compounds. • About 3,600 potential antiviral agents were identified within The Life Chemicals Stock Collection.

109 ANTITUBERCULOSIS LIBRARY lifechemicals.com

The library was designed using receptor-based approach in search for potential drug candidates possessing activity towards InhA enzyme - specific protein of M.tuberculosis that is responsible for cell wall synthesis of the bacteria but is absent in mammals.

A set of 3,700 potential antituberculosis agents Ligand F2269-0132 forms a strong hydrogen bond capable of binding with InhA protein was obtained with Tyr158 and protein coenzyme NAD+. Strong hydrophobic interaction observed between fused aromatic rings and large hydrophobic pocket of InhA.

110 ANTIPROTOZOAL LIBRARY lifechemicals.com

The Library comprises compounds resulting from similarity search against reference sets of compounds displaying activity towards the most dangerous protozoa.

• The reference sets were taken from the most reliable inhibitor database derived from ChEMBL. • According to the activity data and number of compounds in the reference subsets the Tanimoto similarity index was set up in a range from 0.8 to 1 • The list of targeted protozoa families and species:

Trypanosoma brucei Giardia intestinalis Trypanosoma cruzi Toxoplasma gondii Cryptosporidium parvum Leishmania mexicana Leishmania major

Over 4,500 potentially active stock available compounds were included in the Antiprotozoal Library.

111 LOW-TO-MEDIUM THROUGHPUT WHOLE-ORGANISM SCREENING lifechemicals.com ASSAYS AGAINST SEVEN NTDS

• A diverse library of 10,000 compounds selected out of the set of 50,000 compounds was purchased from Life Chemicals Inc. • This set was screened against 7 (malaria, leishmaniasis, human African trypanosomiasis [HAT], Chagas’ disease, schistosomiasis, onchocerciasis and lymphatic filariasis) NTDs • The screen of 10,000 compounds revealed 744 structures to be active against at least one pathogen • Cytotoxicity data were used to aid selection and prioritization of compounds to proceed to the next stage of the lead discovery process

Examples of compounds active against NTDs:

N O S H N O HN N S N H N O N N N O H S O H C 3 N S

O CH2 NH O

CH3 O O H N F H3C S H S N N N S O H3C N O O O CH2 O

Neglected Tropical Diseases (2011), V.5., Iss.12.

112 ANTI-HEPATITIS C VIRUS LIBRARY lifechemicals.com

The hepatitis C virus belongs to the genus Hepacivirus, a member of the family Flaviviridae, and it is known as one of the most dangerous diseases with very expensive treatment. Three proteins of capside were used as a target for our search for potential agents against HCV.

• A reference database of 3,486 biologically active compounds with confirmed potency in 22 HCV assays was compiled using the data available from patents and literature publications. These compounds show activities in the following 3 targets of the virus: NS3 Core protein LYPLA1

• Life Chemicals collection was searched for compounds similar to compounds from the reference database using MDL public keys and the Tanimoto similarity cut-off of 90 %.

As a result, a selection of 16,100 compounds was found and included in The Life Chemicals Anti- HCV Library.

113 ANTIINFLAMMATORY LIBRARY lifechemicals.com

Taking into account information on protein interaction signaling pathways and the availability of protein structural data (RCSB protein data bank) along with active inhibitors (ChMBL DB), Life Chemicals prepared its proprietary antiinflammatory library. The following targets involved in activation of other modulators or increase of defense mechanisms have been considered:

Pi3KC SK1 PTP1B SPL1 LTA4 SYK JAK3 RORγ COX2 LRRK2 iNOS

1,400 compounds were selected in total.

114 CUSTOM SYNTHESIS lifechemicals.com

OUR CUSTOM SYNTHESIS SERVICES

Synthetic Chemistry Medicinal Chemistry • Discovery chemistry • Design and synthesis of drug-like compounds • Synthesis of building blocks and scaffolds • Synthesis of peptidomimetics • Synthesis of reference compounds and • contaminants Hit-to-lead projects • Synthesis of metabolites • Lead optimization • Synthesis of new chemicals or analogs for • Characterization of physical and chemical properties hit-to-lead development • Parallel synthesis of compound libraries Process Optimization (10-1000 members) • Route scouting • Reaction and process optimization • Catalyst screening • Material supply (GMP and non-GMP)

115 CUSTOM SYNTHESIS PROJECTS lifechemicals.com

General Procedure: • For each project a research team is assigned for each project • Both FTE and FFS business models can be offered • Regular reporting as agreed with the customer • Regular communication by means of telephone and Skype conferences • Confidential Disclosure Agreements • “Need to know” principle • Hard copies data locked and data on server protected • IP rights can be transferred to the customer

116 lifechemicals.com CUSTOM SYNTHESIS PROJECTS

117 CATALYTIC HYDROGENATION lifechemicals.com

• A dedicated laboratory facility (“key” laboratory) • Know-how in use and optional employment of Ni, Pd, Pt, Rh catalysts • Selective reduction of functional groups • Selective hydrogenation of aromatic and heteroaromatic systems

H N N N O N N N N H NH2 NH2

NH F 2 CO2H F N Up to 100 g F from 1 run O N H

N N N N

N N H H O

118 SULPHUR TETRAFLUORIDE IN HF lifechemicals.com

Synthesis of fluorinated amines

CF3

HO2C NH2 HCl *HCl

SF4 / HF 69% 2 O CO H O CF N 2 92% N 3 H H BH3*Me2S 1 82% CF N 3 H 3

CH3NO2 HCl Br Br NO2 NO DMSO HOAc 2

EtO C K CO 87% 2 2 3 EtO C HO C 54% 2 2 4 5 6

SF NO2 4 Zn/HCl NH2 *HCl 91% 67% F3C F3C 7 8*HCl

119 CHEMISTRY WITH GASEOUS DIAZOCOMPOUNDS lifechemicals.com

Safety of the process involving the use of highly explosive and toxic CH2H2 is achieved by: • The use of a procedure in which diazomethane is generated in low concentration in octane solution (generator) and then is blown out by argon to the reactors; therefore,

dangerous concentration of CH2N2 is not possible • Neutralization of unreacted diazomethane by acetic acid (trap) • Strict monitoring of the reaction in order to avoid accumulation of diazomethane in the reactors

Trifluoromethyl diazomethane: • The reagent is synthesized in free state and reacts as a gas • Know-how in use and optional employment of Cu, Rh catalysts

120 FLAMMABLE REDUCING AGENTS lifechemicals.com

Reagent Source Scale LAH Rockwood Lithium (sieved, tablets and solution) Up to 200g in 1 run DIBAL Aldrich (Sure-Pac™, 1kg reagent grade) Up to 100g in 1 run

AlH3 in-house generation Up to 60g in 1 run Vitride® Aldrich (Sure/Seal™, 65% solution in toluene) Up to 100g in 1 run L-Selectride® Aldrich (Sure-Pac™, 1M in THF) Up to 1.8L of solution in 1 run Super-Hydride® Aldrich (Kilo-Lab™, 1M in THF) Up to 2L of solution in 1 run BH Me S, BH THF, BH py, BH Me N, “BH ” 3 2 3 3 3 3 Up to 50g of “BH ”equivalent in 1 run 3 in-house generation 3

LiAlH4 + AlCl3

ethylene- O AlH O glycol, H+ 3 O O OH 91% 92% 3 1 2 100 g of 2 was introduced into reaction in 1 run

121 FLAMMABLE STRONG BASES lifechemicals.com

Reagent Source Scale

LiHMDS Aldrich (Kilo-Lab™, 1M in THF of hexane), in-house preparation in pure form Up to 2L of solution in 1 run n-BuLi Rockwood Lithium (2.5M solution in hexane) Up to 2L of solution in 1 run t-BuLi Rockwood Lithium (1.9M in hexane) Up to 2L of solution in 1 run s-BuLi Aldrich (1.4M in cyclohexane) Up to 2L of solution in 1 run MeLi Rockwood Lithium (3M in DME) Up to 2L of solution in 1 run LDA In-house generation in solutions or in-house synthesis in pure form Up to 30g of pure reagent in 1 run LTMP In-house generation in solutions or in-house synthesis in pure form Up to 30g of pure reagent in 1 run

Optimization of reaction conditions by varying base, solvent, concentration, temperature, etc.

122 MG, AL, ZN ORGANODERIVATIVES lifechemicals.com

Reagent Source Scale MeMgCl Rockwood Lithium (3M solution in THF) Up to 2L of solution in 1 run EtMgCl Rockwood Lithium (2.8M solution in THF) Up to 2L of solution in 1 run c-HexMgCl Rockwood Lithium (1.3M solution in THF) Up to 2L of solution in 1 run

CH2=CH−CH2MgBr Aldrich (0.5M solution in THF) Up to 1L of solution in 1 run PhMgCl Aldrich, in-house generation Up to 2L of solution in 1 run t-BuMgCl Rockwood Lithium (1.7M solution in THF) Up to 2L of solution in 1 run

CH2=CHMgBr Aldrich (Kilo-Lab™, 1M solution in THF) Up to 2L of solution in 1 run i-PrMgCl Aldrich (2M solution in THF) Up to 1L of solution in 1 run

AlMe3 Aldrich (2M solution in toluene) Up to 1L of solution in 1 run

EtAlCl2 Aldrich (Sure/Seal™, 1M solution in toluene or in heptane) Up to 200ml of solution in 1 run

Me2AlCl Aldrich (Sure/Pac™) Up to 50g in 1 run

Et3Al Aldrich (Sure/Pac™) Up to 1L of solution in 1 run

Et2Zn Rockwood Lithium (0.1M solution in hexane) Up to 1L of solution in 1 run

Kulinkovich-de Meijere reaction in multi-gram scale

120g of 3 was synthesized in 1 run

123 TOXIC ORGANOTIN DERIVATIVES lifechemicals.com

Reagent Source Scale

Bu3SnCl Avokado Up to 500g in 1 run Tributyl(1-ethoxyvinyl)tin Aldrich, in-house synthesis Up to 20g in 1 run

Bu3SnCH=CH2 Advanced Tec Up to 100g in 1 run

Bu3SnC≡CH Rintec, in-house synthesis Up to 20g in 1 run

Bu3SnH Aldrich Up to 100g in 1 run

124 PHOTOCHEMISTRY lifechemicals.com

The photoreactor designed to carry out medium-scale synthesis (10 – 50 g) It is important to use the highly purified argon for the inert atmosphere (trap)

PHOTOREACTOR

O OH O O O O Br SeO 2 MeOH NBS Br Ph O CCl N 2 N H2SO4 N 4 o h N 24 120 C 86% 20 28 70% 92%

AgNO3 74%

H2O-MeOH H H O N O N N O O N N H O H2, PtO2 2 4

CF3COOH MeOH H CH OH 95% N 3 N TRAP H 97% N 4 8 16

125 MICROVAWE-ASSISTED CHEMISTRY lifechemicals.com

CO2H R2 R1 = Alk R2 = Alk, Ar, Het N R1 R3 = H O

O MeCN, MW O KAl(SO4)26H2O O

R1 R3 N in situ R2 generated O R4 O R O MeCN, MW OH R1 = Alk Biotage® Initiator R4 R2 = Alk, Ar, Het Microwave Synthesizer with Accessories O R3 = H for Automated Sample Processing R2 N R4 = EWG R1

126 COMPUTATIONAL CHEMISTRY SERVICES lifechemicals.com

• Computer-aided rational design of small organic molecules against any biological target • Drug-likeness and lead-likeness prediction, diversity calculations, 2D/3D similarity and substructure search, compound filtering by any physicochemical properties, compound clustering • Target profiling: given a molecule, perform an in silico target profiling against more than 2,800 protein targets • Scaffold hopping and fragment linking • ADME/Tox prediction • Design of custom target-focused libraries • Receptor-based virtual screening (molecular docking) • Ligand-based virtual screening (QSAR, shape similarity, pharmacophore search) • Molecular dynamics simulation of macromolecules including their complexes with natural or synthetic ligands • Protein structure modeling, de novo or by homology

127 COMPUTATIONAL CHEMISTRY SERVICES lifechemicals.com Software

Molecular modeling/docking/virtual screening • Schrödinger • GOLD • MOE • SYBYL • DOCK • ICM

Cheminformatics • E-dragon • Datawarrior • Canvas

Homology Modeling • Swiss-Model server, Moldeller - homology modeling web servers • Orchestrar, Fugue from SYBYLX • Prime from Scrödinger • BLAST

Molecular Dynamics, Geometry Optimization • GROMACS MD simulation and protein structure optimization

128 KEY PRECLINICALS lifechemicals.com

The Key Preclinicals laboratory for primary drug trials provides an advanced highly-efficient and solid framework of services to companies and academic institutions involved in pharmaceutical and biotech research. Available among our products is a variety of in vitro and in vivo tests, including:

SPECIFIC DRUG SAFETY PHARMACOLOGICAL ACTIVITY DRUG IN VITRO • General toxicology BIOAVAILABILITY AND IN VIVO • Safety pharmacology • Antihypertensive STUDIES ADMET TESTS (ICH S7A and S7B) • Anti-inflammatory • Anti-ischaemic • Antiarrhythmic

Today Key Preclinicals can expertly carry out short term oral toxicity studies and short term intramuscular toxicity studies in compliance with the Principles of GLP and according to the Directive 2004/9/EC and Directive 2004/10/EC, that is equivalent to the OECD Principles of Good Laboratory Practice ENV/MC/Chem (98)17. The Laboratory also meets requirements of GSTU ISO 9001: 2009 (ISO 9001: 2008, IDT). There are about 60 people on the staff of Key Preclinicals, with 34 of them directly involved in preclinical drug testing.

129 KEY PRECLINICALS lifechemicals.com

In vitro and in vivo ADMET tests

It is generally known that the early stage of drug discovery involves ADMET screening to investigate such characteristics as solubility, permeability, microcomal stability and CYP inhibition that enable identifying metabolically stable and potent substances for further analysis. At Key Preclinicals, we offer the following spectrum of in vitro and in vivo ADMET tests:

In Vitro Metabolism: In Vivo DMPK: Drug Interaction Permeability and transporters: - Metabolic stability - Animal Pharmacokinetics - CYP induction/inhibition - Stability in plasma and - Caco-2 permeability buffer - Uptake and influx transporters

Physicopchemistry and binding: In Vitro Toxicology :

- Solubility - Cytotoxicity screening - Lipophilicity (logD/P) - Genotoxicity screening (Ames test, chromatin aberration) - Plasma protein binding - Cadriotoxicoty screening (hERG testing, Q-T - Red blood cells binding interval measurement in perfused isolated - Tissue binding Langendorff heart, drug’s anti- or proarrhythmic activity using maximal follow frequency (MFF) - Microsome/hepatocyte binding method which refers to the sequence of consecutive contractions of the papillary muscle following each electrical stimulus)

130 KEY PRECLINICALS lifechemicals.com

Additionally, a range of microbiological studies can also be provided:

• screening of potentially active antibacterial and antifungal drugs

• investigation of susceptibility/resistance of microorganisms to antibiotics and antifungal drugs

• studies of antibiofilm activity of drugs on different surfaces

• evaluation of potential drug activity on animal model of sepsis, cutaneous surgical and burn wounds, subcutaneous abscesses and conjunctivitis

131 ADVANCED BUILDING BLOCKS lifechemicals.com FOR LIFE AND MATERIALS SCIENCES

• There are about 50,000 structures, based on rare chemotypes in our collection. Most of the compounds are not commonly available: more than 50 % of the database have < 5 suppliers in ACD

• There are more than 11,000 building blocks in stock.

• High quality standards: over 95 % purity confirmed with NMR and/or LCMS

• Physicochemical properties favorable for lead-oriented synthesis

• Building blocks of interest for many areas of natural sciences: pharmaceutical, agrochemical, materials sciences.

• You can see availability and prices immediately at https://shop.lifechemicals.com/

132 ADVANCED BUILDING BLOCKS FOR LIFE AND MATERIALS SCIENCES lifechemicals.com

Diverse functionalities

Total: 62,000 Primary amines: 23.4 % Secondary amines: 18.7 % Carboxylic acids: 10.0 % Organofluorine cpds: 9.3 % Alkylation agents: 7.3 % Ketones: 5.0 %

Aldehydes: 1.8 % Sulfonyl chlorides: 1.4 %

133 ADVANCED BUILDING BLOCKS FOR LIFE AND MATERIALS SCIENCES lifechemicals.com

Lead oriented

11.9 1.17 30 30

20 20

10 10

% % ofthe cpds % ofthe cpds

0 0 5 6 7 8 9 10 11 12 13 14 15 16 17 18 -4 -3 -2 -1 0 1 2 3 4 5 HAc ClogP

2.2 16 0.52 30 14 12 10 20 8 6 10

4

% % ofthe cpds % % ofthe cpds 2 0 0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 1 2 3 4 5 6 7 8 Fsp3 RotB

134 ADVANCED BUILDING BLOCKS FOR LIFE AND MATERIALS SCIENCES lifechemicals.com

Primary amines:

135 ADVANCED BUILDING BLOCKS FOR LIFE AND MATERIALS SCIENCES lifechemicals.com

Secondary amines:

136 ADVANCED BUILDING BLOCKS FOR LIFE AND MATERIALS SCIENCES lifechemicals.com

Carboxylic acids:

137 ADVANCED BUILDING BLOCKS FOR LIFE AND MATERIALS SCIENCES lifechemicals.com

Alkylation agents:

138 ADVANCED BUILDING BLOCKS FOR LIFE AND MATERIALS SCIENCES lifechemicals.com

Sulfonyl chlorides:

139 ADVANCED BUILDING BLOCKS FOR LIFE AND MATERIALS SCIENCES lifechemicals.com

Organofluorine compounds:

140 ADVANCED BUILDING BLOCKS FOR LIFE AND MATERIALS SCIENCES lifechemicals.com

Special reagent classes:

Alkyl hydrazines Epoxides Trifluoroborates

141 ADVANCED BUILDING BLOCKS FOR LIFE AND MATERIALS SCIENCES lifechemicals.com

Special reagent classes:

Bicyclic amino acids Bicyclic diamines

142 ADVANCED BUILDING BLOCKS FOR LIFE AND MATERIALS SCIENCES lifechemicals.com

Examples of stock available building blocks:

143 FINE REAGENTS IN MULTIGRAM SCALE lifechemicals.com

Life Chemicals offers

• Multi-kg synthesis of rare reagents for organic synthesis (25L, 50L and 75 L reactors are available)

• Scale-up of procedures from mg/g to kg quantities

• Compounds for materials sciences: dyes, extragents, calixarenes, monomers, ligands, etc.

• Proprietary catalogue of functional dyes and fine reagents in multigram scale

• GMP synthesis in terms of subcontracting agreements

144 LIFE CHEMICALS FACILITIES lifechemicals.com Some Facts on our Resources: • Over 20 years’ experience in parallel synthesis, library design and medicinal chemistry • Up to 50 synthetic chemists can be involved • Purification techniques: • Up to 1000m2 production facilities are available - TLC, NP LC (CombiFlash) - RP HPLC (Shimadzu) Renovated building – 2000m2 space

145 SYNTHESIS FACILITIES lifechemicals.com

Chemistry labs are supplied with high-end equipment for organic synthesis:

• Biotage Microwave station • Paar autoclaves • Photochemistry lab • Specialized facility for handling highly toxic reagents

146 PREPARATIVE CHROMATOGRAPHY LAB lifechemicals.com

• CombiFlash Rf (SiO2 phase) devices • Shimadzu HPLC machines (direct phase and reverse phase columns): semi-preparative scale 0.5g/run • Preparative Thin Layer Chromatography

147 ANALYTICAL LAB lifechemicals.com Certified by ISO/IEC 17025:2005

NMR lab Capacity: 200 1H NMR spectra daily • Varian Gemini 2000 400 MHz with an Oxford Instruments superconducting magnet • Varian VXR-300 300 MHz • Varian Gemini 200 MHz Ready for 1H, 13C, 19F, 29Si, 31P measurements and advanced 2D NMR techniques (COSY, NOESY, HMQC, HSQC, INADEQUATE)

LCMS lab Capacity: 200 samples per day • Agilent 1100 • Agilent 1200 • over 10 columns of various types

GCMS • Agilent 7820A GC/MSD

148 ANALYTICAL LAB lifechemicals.com Certified by ISO/IEC 17025:2005 • Water determination by Karl Fischer Titration • Qualitative and quantitative determination of functional groups in organic compounds • Qualitative and quantitative determination of salt form of organic compounds • QC control of solvents and inorganic compounds • Measuring Optical Activity (POLAX – 2L, ATAGO polarimeter) • Melting Point verification OptiMelt MPA100

Titroline KF SCHOTT Instruments

149 X-RAY DIFFRACTOMETER lifechemicals.com

Bruker SMART APEX II 2 Enraf-Nonius CAD-4 four-circle diffractometers

150 WHY LIFE CHEMICALS? lifechemicals.com

The Company has strong and recognized position in organic synthesis and medicinal chemistry.

• Novel chemistry and highest quality • Our product catalogue includes over 460,000 screening compounds and 12,000 in-house synthesized building blocks • Hit resupply • Experienced project management • Experience in creating of discovery products (targeted libraries, FBDD) • Developed platform (analytic labs, logistics, etc.) • Access to literature (Reaxys, SciFinder, DiscoveryGate) • Chemical Informatics support • Our chemists can tackle tasks of highest complexity • We work as a fully integrated platform to offer best products and services.

151 lifechemicals.com

CASE STUDIES

152 CASE STUDIES (IN-HOUSE) lifechemicals.com

CO H CO H F3C 2 2 H N COOH H 2 COOH NBoc NBoc H F3C H N H

NH 2 NHBoc NH2 NH2

H2N N NH2 H2N NHBoc Ph NH2

N N N NH2 NH NH F C CF H 3 N 3 H CF F3C CF3 3 CO2H

153 CASE STUDIES (IN-HOUSE) lifechemicals.com

O HO O O O HO N O N N N O O O O OH N O N O H N O S N N P O N N N Fe NH O N P O N H NH Ph O

154 CASE STUDIES (IN-HOUSE) lifechemicals.com

HO O H N NH O N NHFmoc H N 2 N N F O F N N NH F H H BocHN

N N NBoc N O N N N O N N O

+ O N NH N H H 2 H H H N N O H H O + N + O N N S H O O H BocHN O 2 2 O O

155 CASE STUDIES 2 lifechemicals.com

Advanced building blocks synthesis project Customer – US Big Pharma

Starting point: • 500 novel building blocks were designed as per customer’s requirements • A set of 100 advanced building blocks was selected by the customer

Team task (3.5 months): • Development/validation of synthetic schemes for the 100 most challenging building blocks • Synthesis of these building blocks weighing 25 g, with their purity being over 95 %

Result: • 86 of 100 building blocks were delivered

156 CASE STUDIES 3 lifechemicals.com

CCE Project Customer – UK BigPharma

Starting point: • A library to comply with rigorous criteria for lead-likeness, diversity and novelty was needed for compound collection enhancement

Team task (8 months): • Design of the library (non-exclusive basis) • Synthesis of key intermediates • Synthesis of the library (10 umol, > 90 % by LCMS)

Results: • 3,500-member library based on 15 templates was synthesized

157 CASE STUDIES 4 lifechemicals.com

The synthetic scheme was designed and optimized for multi-gram scale-up:

Cl

F3C O OMe HO 16 steps N O N N N N O H N O

NH2 H N 31 steps N N Ph O N N F S O NH O N N N N N HO OH O

O 15 steps N N N H F N N O HN O Cl O S Ph O OH O Cl H NH2 OH N 12 steps 16 steps H

158 FP 7 PROJECT lifechemicals.com

Combined Highly Active Anti-Retroviral Microbicides

EC Contribution: 12,000,000 Euros Duration: 60 month Starting date: 01.01.2010

Spoluka/Life Chemicals objective: to develop new CCR5 inhibitors

159