WO2006094003A1

WO2006094003A1 - Novel cathepsin c inhibitors and their use

Info

Publication number: WO2006094003A1
Application number: PCT/US2006/007162
Authority: WO
Inventors: David T. Fosbenner; Ronggang Liu; Michael L. Moore
Original assignee: Glaxo Group Limited
Priority date: 2005-03-02
Filing date: 2006-03-01
Publication date: 2006-09-08
Also published as: EP1858509A1; EP1858509A4; JP2008531706A

Abstract

The invention is directed to novel cathepsin C inhibitors and their use in the treatment of diseases mediated by the cathepsin C enzyme. Specifically, the invention is directed to compounds according to Formula (I): wherein X and n are defined below, and to pharmaceutically-acceptable salts thereof. The compounds of the invention are cathepsin C inhibitors and can be used in the treatment of diseases mediated by the cathepsin C enzyme, such as COPD. Accordingly, the invention is further directed to pharmaceutical compositions comprising a compound of the invention. The invention is still further directed to methods of inhibiting cathepsin C and treatment of conditions associated therewith using a compound of the invention or a pharmaceutical composition comprising a compound of the invention.

Description

NOVEL CATHEPSIN C INHIBITORS AND THEIR USE

FIELD OF THE INVENTION

The invention is directed to certain novel cathepsin C inhibitors and their use in the treatment of diseases mediated by the cathepsin C enzyme.

BACKGROUND OF THE INVENTION

Cathepsins are a family of enzymes included in the papain superfamily of cysteine proteases. Cathepsins B, C, F₁ H, K, L, S, V, and X have been described in the scientific literature. Cathepsin C is also known in the literature as Dipeptidyl Peptidase I or "DPPI."

A number of recently published studies have begun to describe the role cathepsin

C plays in certain inflammatory processes. See E.g. Adkison et al., The Journal of

Clinical Investigation 109:363-371 (2002); Tran et al., Archives of Biochemistry and

Biophysics 403:160-170 (2002); Thiele et al., The Journal of Immunology 158: 5200-5210 (1997); Bidere et al., The Journal of Biological Chemistry 277: 32339-32347 (2002);

Mabee et al., The Journal of Immunology 160: 5880-5885; McGuire et al., The Journal of

Biological Chemistry, 268: 2458-2467; and Paris et al., FEBS Letters 369: 326-330

(1995). From these studies, it appears that cathepsin C is co-expressed with certain serine proteases, which are released from inflammatory cells recruited to cites of inflammation, and acts as a physiological activator of these proteases. Once activated, these proteases are capable of degrading various extracellular matrix components, which can lead to tissue damage and chronic inflammation.

For example, Chronic Obstructive Pulmonary Disease ("COPD") is a chronic inflammatory disease where cathepsin C appears to play a role. The American Thoracic Society defines COPD as "a disease characterized by the presence of airflow obstruction due to chronic bronchitis or emphysema; the airflow obstruction is generally progressive, may be accompanied by airway hyperreactivity, and may be partially reversible." American Journal of Respiratory and Critical Care Medicine 152: S77-S120 (1995). Chronic bronchitis is generally characterized by a chronic productive cough, whereas emphysema is generally characterized by permanent enlargement of the airspaces distal to the terminal bronchioles and airway wall destruction. Chronic bronchitis and emphysema usually occur together in COPD patients.

Cigarette smoking is a significant risk factor for developing COPD. Exposure to cigarette smoke and other noxious particles and gases may result in chornic inflammation of the lung. In response to such exposure, inflammatory cells such as CD8+ Tcells, macrophages, and neutrophils are recruited to the area. These recruited inflammatory cells release proteases, which are believed to play a major role in the disease etiology by degrading airway walls. Proteases believed to be involved in this process include the serine proteases neutrophil elastase ("NE"), chymase ("CY"), cathepsin G, proteinase 3 and granzymes A and B. Cathepsin C appears to be involved in activating these enzymes.

Rheumatoid arthritis ("RA") is another chronic inflammatory disease where cathepsin C appears to play a role. Neutrophils are recruited to the site of joint inflammation and release cathepsin G, NE, and proteinase 3, which are believd to be responsible for cartilage destruction associated with RA. Cathepsin C appears to be involved in activating these enzymes.

Other conditions where cathepsin C may play a role include osteoarthritis, asthma, and Multiple Sclerosis. See E.g. Matsui K. Yuyama N. Akaiwa M. Yoshida NL. Maeda M. Sugita Y. Izuhara K., Identification of an alternative splicing variant of cathepsin C/dipeptidyl-peptidase I. Gene. 293(1 -2): 1-7, 2002 Jun 26; Wolters PJ. Laig-Webster M. Caughey GH., Dipeptidyl peptidase I cleaves matrix-associated proteins and is expressed mainly by mast cells in normal dog airways, American Journal of Respiratory Cell & Molecular Biology. 22(2): 183-90, 2000.

One approach to treating these conditions is to inhibit the activity of the serine proteases involved in the inflammatory process, especially NE activity. See E.g.. Ohbayashi, "Neutrophil elastase inhibitors as treatment for COPD", Expert Opin. Investig. Drugs 11 (7): 965-980 (2002); Shapiro, "Neutrophil Elastase: Path Clearer, Pathogen Killer, or Just Pathologic?", Am. J. Respir. Cell MoI. Biol. 26: 266-268 (2002). In light of the role cathepsin C plays in activating certain serine proteases, especially NE, it is desirable to prepare compounds that inhibit its activity, which thereby inhibit serine protease activity. Thus, there is a need to identify compounds that inhibit cathepsin C, which can be used in the treatment of a variety of conditions mediated by cathepsin C.

SUMMARY OF THE INVENTION

The invention is directed to novel cathepsin C inhibitors and their use in the treatment of diseases mediated by the cathepsin C enzyme. Specifically, the invention is directed to compounds according to Formula I:

CN wherein X and n are defined below, and to pharmaceutical ly-acceptable salts thereof.

The compounds of the invention are cathepsin C inhibitors and can be used in the treatment of diseases mediated by the cathepsin C enzyme, such as COPD. Accordingly, the invention is further directed to pharmaceutical compositions comprising a compound of the invention. The invention is still further directed to methods of inhibiting cathepsin C and treatment of conditions associated therewith using a compound of the invention or a pharmaceutical composition comprising a compound of the invention.

DETAILED DESCRIPTION OF THE INVENTION In describing the invention, chemical elements are identified in accordance with the Periodic Table of the Elements. Abbreviations and symbols utilized herein are in accordance with the common usage of such abbreviations and symbols by those skilled in the chemical and biological arts.

Terms and Definitions

"Alkyl" refers to a monovalent saturated hydrocarbon chain having the specified number of member atoms. For example, C1 -C8 alkyl refers to an alkyl group having from 1 to 8 member atoms. Alkyl groups may be optionally substituted with one or more substituents as defined herein. Alkyl groups may be straight or branched. Representative branched alkyl groups have one, two, or three branches. Alkyl includes methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl, and t-butyl), pentyl (n-pentyl, isopentyl, and neopentyl), and hexyl.

"Alkylene" refers to a divalent saturated hydrocarbon chain having the specified number of member atoms. For example, C1-C4 alkylene refers to an alkylene group having from 1 to 4 member atoms. Alkylene groups may be optionally substituted with one or more substituents as defined herein. Alkylene groups may be straight or branched. Representative branched alkylene groups have one, two, or three branches. Alkylene includes methylene, ethylene, propylene (n-propylene and isopropylene), and butylene (n-butylene, isobutylene, and t-butylene). "Alkenylene" refers to a divalent unsaturated hydrocarbon chain having the specified number of member atoms and having one carbon-carbon double bond within the chain. For example, C2-C4 alkenylene refers to an alkenylene group having from 2 to 4 member atoms. Alkenylene groups may be optionally substituted with one or more substituents as defined herein. Alkenylene groups may be straight or branched. Representative branched alkenylene groups have one, two, or three branches. Alkenylene includes ethylenylene, propenylene, and butenylene.

"Aryl" refers to a monovalent aromatic hydrocarbon ring. Aryl groups are monocyclic ring systems or bicyclic ring systems. Monocyclic aryl ring refers to phenyl. Bicyclic aryl ring refers to napthyl, biphenyl, and to rings wherein phenyl is fused to a cycloalkyl or cycloalkenyl ring having 5, 6, or 7 member atoms. Aryl groups may be optionally substituted with one or more substituents as defined herein.

"Cycloalkyl" refers to a monovalent saturated or unsaturated hydrocarbon ring having the specified number of member atoms. For example, C3-C6 cycloalkyl refers to a cycloalkyl group having from 3 to 6 member atoms. Cycloalkyl groups are not aromatic. Cycloalkyl groups are monocyclic ring systems. Cycloalkyl groups may be optionally substituted with one or more substituents as defined herein. Cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

"Enantiomerically enriched" refers to products whose enantiomeric excess is greater than zero. For example, enantiomerically enriched refers to products whose enantiomeric excess is greater than 50% ee, greater than 75% ee, and greater than 90% ee.

"Enantiomeric excess" or "ee" is the excess of one enantiomer over the other expressed as a percentage. As a result, since both enantiomers are present in equal amounts in a racemic mixture, the enantiomeric excess is zero (0% ee). However, if one enantiomer was enriched such that it constitutes 95% of the product, then the enantiomeric excess would be 90% ee (the amount of the enriched enantiomer, 95%, minus the amount of the other enantiomer, 5%).

"Enantiomerically pure" refers to products whose enantiomeric excess is 99% ee or greater.

"Half-life" ( or "half-lives") refers to the time required for half of a quantity of a substance to be converted to another chemically distinct specie in vitro or in vivo.

"Halo" refers to the halogen radical fluoro, chloro, bromo, or iodo.

"Haloalkyl" refers to an alkyl group that is substituted with one or more halo substituents. Haloalkyl includes trifrouromethyl.

"Heteroaryl" refers to a monovalent aromatic ring containing from 1 to 4 heteroatoms as member atoms in the ring. Heteroaryl groups containing more than one heteroatom may contain different heteroatoms. Heteroaryl groups may be optionally suDstiTuted with one or more substitυents as defined herein. Heteroaryl groups are monocyclic ring systems or are fused, spiro, or bridged bicyclic ring systems. Monocyclic heteroaryl rings have from 5 to 7 member atoms. Bicyclic heteroaryl rings have from 7 to 11 member atoms. Bicyclic heteroaryl rings include those rings wherein phenyl and a monocyclic heterocycloalkyl ring are attached forming a fused, spiro, or bridged bicyclic ring system, and those rings wherein a monocyclic heteroaryl ring and a monocyclic cycloalkyl, cycloalkenyl, heterocycloalkyl, or heteroaryl ring are attached forming a fused, spiro, or bridged bicyclic ring system. Heteroaryl includes pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, furanyl, furazanyl, thienyl, triazolyl, pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, tetrazinyl, tetrazolyl, indolyl, isoindolyl, indolizinyl, indazolyl, purinyl, quinolinyl, isoquinolinyl, quinoxalinyl, quinazolinyl, pteridinyl, cinnolinyl, benzimidazolyl, benzopyranyl, benzoxazolyl, benzisoxazolyl, benzofuranyl, isobenzofuranyl, benzothiazolyl, benzisothiazolyl, benzothienyl, furopyridinyl, and napthyridinyl. "Heteroatom" refers to a nitrogen, sulphur, or oxygen atom.

"Member atoms" refers to the atom or atoms that form a chain or ring. Where more than one member atom is present in a chain and within a ring, each member atom is covalently bound to an adjacent member atom in the chain or ring. Atoms that make up a substituent group on a chain or ring are not member atoms in the chain or ring. "Optionally substituted" indicates that a group, such as alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl, heterocycioalkyl, or heteroaryl, may be unsubstituted or substituted with one or more substituents as defined herein. "Substituted" in reference to a group indicates that a hydrogen atom attached to a member atom within a group is replaced. It should be understood that the term "substituted" includes the implicit provision that such substitution be in accordance with the permitted valence of the substituted atom and the substituent and that the substitution results in a stable compound (i.e. one that does not spontaneously undergo transformation such as by rearrangement, cyclization, or elimination). In certain embodiments, a single atom may be substituted with more than one substituent as long as such substitution is in accordance with the permitted valence of the atom. Suitable substituents are defined herein for each substituted or optionally substituted group.

"Pharmaceutically acceptable" refers to those compounds, materials, compositions, and dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

Compounds

The invention is directed to compounds according to Formula I:

wherein: n is 1 , 2, or 3;

X is -C(O)RI , -C(O)ORI , -C(O)R2, -C(O)OR2, -C(O)YR2, or -C(O)OYR2; R1 is C1 -C8 alkyl or C3-C6 cycloalkyl;

R2 is aryl or heteroaryl; wherein said aryl is optionally substituted with one or more substituents independently selected from the group consisting of: halo, C1-C3 haloalkyl, -CN, -NO₂,

Ra, -ORa, -C(O)ORa, -C(O)Ra, -NHC(O)Ra, heteroaryl and heterocycloalkyl, where said heteroaryl and heterocycloalkyl are optionally substituted with one or more substituents independently selected from the group consisting of: halo, C1 -C3 haloalkyl, Ra, -ORa, -

C(O)ORa, -C(O)Ra, and -NHC(O)Ra; and wherein said heteroaryl is optionally substituted with one or more substituents independently selected from the group consisting of: halo, C1-C3 haloalkyl, -CN, -NO₂, Ra, -ORa, -C(O)ORa, -C(O)Ra, and - NHC(O)Ra;

Y is C1-C4 alkylene, C2-C4 alkenylene, or -(CH₂)_I-O-(CH₂W;

I is 1 , 2, or 3; m is 0, 1 , or 2;

I + m is 1 , 2, 3, or 4; and Ra is C1-C3 alkyl; provided that when n is 1 and X is -C(O)ORI , R1 is a group other than f-butyl.

The meaning of any functional group or substituent thereon at any one occurrence in Formula I, or any subformula thereof, is independent of its meaning, or any other functional group's or substituent's meaning, at any other occurrence, unless stated otherwise. The compounds according to Formula I may contain one or more asymmetric center (also referred to as a chiral center) and may, therefore, exist as individual enantiomers, diastereomers, or other stereoisomeric forms, or as mixtures thereof. Chiral centers, such as chiral carbon atoms, may also be present in a substituent such as an alkyl group. Where the stereochemistry of a chiral center present in Formula I, or in any chemical structure illustrated herein, is not specified the structure is intended to encompass any stereoisomer and all mixtures thereof. Thus, compounds according to Formula I containing one or more chiral center may be used as racemic mixtures, enantiomerically enriched mixtures, or as enantiomerically pure individual stereoisomers. Individual stereoisomers of a compound according to Formula I which contain one or more asymmetric center may be resolved by methods known to those skilled in the art. For example, such resolution may be carried out (1) by formation of diastereoisomeric salts, complexes or other derivatives; (2) by selective reaction with a stereoisomer- specific reagent, for example by enzamatic oxidation or reduction; or (3) by gas-liquid or liquid chromatography in a chiral enviornment, for example, on a chiral support such as silica with a bound chiral ligand or in the presence of a chiral solvent. The skilled artisan will appreciate that where the desired stereoisomer is converted into another chemical entity by one of the separation procedures described above, a further step is required to liberate the desired form. Alternatively, specific stereoisomers may be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one enantiomer to the other by asymmetric transformation.

The compounds according to Formula I may also contain double bonds or other centers of geometric asymmetry. Where the stereochemistry of a center of geometric asymmetry present in Formula I, or in any chemical structure illustrated herein, is not specified, the structure is intended to encompass the trans (E) geometric isomer, the cis (Z) geometric isomer, and all mixtures thereof. Likewise, all tautomeric forms are also included in Formula I whether such tautomers exist in equilibrium or predominately in one form.

The skilled artisan will appreciate that pharmaceutically-acceptable salts of the compounds according to Formula I may be prepared. Indeed, in certain embodiments of the invention, pharmaceutically-acceptable salts of the compounds according to Formula I may be preferred over the respective free base or free acid because such salts impart greater stability or solubility to the molecule thereby facilitating formulation into a dosage form. Accordingly, the invention is further directed to the use of pharmaceutically- acceptable salts of the compounds according to Formula I. /Λ5 uaeα πerein, tne term "pharmaceutically-acceptable salts" refers to salts that retain the desired biological activity of the subject compound and exhibit minimal undesired toxicological effects. These pharmaceutically-acceptable salts may be prepared in situ during the final isolation and purification of the compound, or by separately reacting the purified compound in its free acid or free base form with a suitable base or acid, respectively.

In certain embodiments, compounds according to Formula I may contain an acidic functional group. Suitable pharmaceutically-acceptable salts include salts of such acidic functional groups. Representative salts include pharmaceutically-acceptable metal salts such as sodium, potassium, lithium, calcium, magnesium, aluminum, and zinc salts; carbonates and bicarbonates of a pharmaceutically-acceptable metal cation such as sodium, potassium, lithium, calcium, magnesium, aluminum, and zinc; pharmaceutically- acceptable organic primary, secondary, and tertiary amines including aliphatic amines, aromatic amines, aliphatic diamines, and hydroxy alkylamines such as methylamine, ethylamine, 2-hydroxyethylamine, diethylamine, triethylamine, ethylenediamine, ethanolamine, diethanolamine, and cyclohexylamine.

In certain embodiments, compounds according to Formula I may contain a basic functional group and are therefore capable of forming pharmaceutically-acceptable acid addition salts by treatment with a suitable acid. Suitable acids include pharmaceutically- acceptable inorganic acids amd pharmaceutically-acceptable organic acids. Representative pharmaceutically-acceptable acid addition salts include hydrochloride, hydrobromide, nitrate, methylnitrate, sulfate, bisulfate, sulfamate, phosphate,_, acetate, hydroxyacetate, phenylacetate, propionate, butyrate, isobutyrate, valerate, maleate, hydroxymaleate, acrylate, fumarate, malate, tartrate, citrate, salicylate, p- aminosalicyclate, glycollate, lactate, heptanoate, phthalate, oxalate, succinate, benzoate, o-acetoxybenzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, mandelate, tannate, formate, stearate, ascorbate, palmitate, oleate, pyruvate, pamoate, malonate, laurate, glutarate, glutamate, estolate, methanesulfonate (mesylate), ethanesulfonate (esylate), 2-hydroxyethanesulfonate, benzenesulfonate (besylate), p-aminobenzenesulfonate, p-toluenesulfonate (tosylate), and napthalene-2- sulfonate.

As used herein, the term "compounds of the invention" means both the compounds according to Formula I and the pharmaceutically-acceptable salts thereof. The term "a compound of the invention" also appears herein and refers to both a compound according to Formula I and its pharmaceutically-acceptable salts. The compounds of the invention may exist in solid or liquid form. In the solid state, the compounds of the invention may exist in crystalline or noncrystalline form, or as a mixture thereof. For compounds of the invention that are in crystalline form, the skilled artisan will appreciate that pharmaceutically-acceptable solvates may be formed wherein solvent molecules are incorporated into the crystalline lattice during crystallization. Solvates may involve nonaqueous solvents such as ethanol, isopropanol, DMSO, acetic acid, ethanolamine, and ethyl acetate, or they may involve water as the solvent that is incorporated into the crystalline lattice. Solvates wherein water is the solvent that is incorporated into the crystalline lattice are typically referred to as "hydrates." Hydrates include stoichiometric hydrates as well as compositions containing vaiable amounts of water. The invention includes all such solvates.

The skilled artisan will further appreciate that certain compounds of the invention that exist in crystalline form, including the various solvates thereof, may exhibit polymorphism (i.e. the capacity to occur in different crystalline structures). These different crystalline forms are typically known as "polymorphs." The invention includes all such polymorphs. Polymorphs have the same chemical composition but differ in packing, geometrical arangement, and other descriptive properties of the crystalline solid state. Polymorphs, therefore, may have different physical properties such as shape, density, hardness, deformability, stability, and dissolution properties. Polymorphs typically exhibit different melting points, IR spectra, and X-ray powder diffraction patterns, which may be used for identification. The skilled artisan will appreciate that different polymorphs may be produced, for example, by changing or adjusting the reaction conditions or reagents, used in making the compound. For example, changes in temperature, pressure, or solvent may result in polymophs. In addition, one polymorph may spontaneously convert to another polymorph under certain conditions.

Representative Embodiments

As defined above, X is -C(O)RI , -C(O)ORI , -C(O)R2, -C(O)OR2, -C(O)YR2, or - C(O)OYR2. In one embodiment, X is -C(O)R2, -C(O)OR2, -C(O)YR2, or -C(O)OYR2. In another embodiment, X is -C(O)R2. In yet another embodiment, X is -C(O)OR2. In yet another embodiment, X is -C(O)YR2. In yet another embodiment, X is -C(O)OYR2.

As defined above, R2 is aryl or heteroaryl optionally substituted as defined above.

In one embodiment, X is -C(O)R2, -C(O)OR2, -C(O)YR2, or -C(O)OYR2 and R2 is aryl optionally substituted as defined above. In another embodiment, R2 is phenyl optionally substituted as defined above. In yet another embodiment, R2 is napthyl optionally substituted as defined above. As defined above, Y is C1 -C4 alkylene, C2-C4 alkenylene, or -(CH₂)_rO-(CH₂)_m-. In one embodiment, Y is C1-C4 alkylene, or -(CH₂)r0-(CH₂)_m-- ^ln another embodiment, Y is methylene.

Compound Preparation

The compounds according to Formula I are prepared using conventional organic syntheses. A suitable synthetic route is depicted below in the following general reaction scheme.

The skilled artisan will appreciate that if a substituent described herein is not compatible with the synthetic methods described herein, the substituent may be protected with a suitable protecting group that is stable to the reaction conditions. The protecting group may be removed at a suitable point in the reaction sequence to provide a desired intermediate or target compound. Suitable protecting groups and methods for protecting and de-protecting different substituents using such suitable protecting groups are well known to those skilled in the art; examples of which may be found in T. Greene and P. Wuts, Protecting Groups in Chemical Synthesis (3rd ed.), John Wiley & Sons, NY (1999). In some instances, a substituent may be specifically selected to be reactive under the reaction conditions used. Under these circumstances, the reaction conditions convert the selected substituent into another substituent that is either useful as an intermediate compound or is a desired substituent in a target compound.

Scheme 1

n=1-3 n=1-3

Commercially available bis-Boc-hydrazine is condensed with 1 ,3-dibromopropane in the presence of sodium hydride to form the bis-Boc-pyrazolidine. The corresponding hexahydropyridazine and hexahydroazepine are prepared in an analogous manner from 1 ,4-dibromobutane and 1 ,5-dibromopentane, respectively. The Boc groups are removed with HCi/dioxane and the cyano group is introduced using cyanogen bromide. This intermediate cyanopyrazolidine is derivatized in situ without isolation. Carbamates are prepared by treatment with commercially available chloroformates. Chloroformates not commercially available are prepared by treatment of commercially available alchohols with phosgene and are used directly without purification. Amides are prepared from commercially available acids using diisopropylcarbodiimide, or from commercially available acid chlorides. Final products are purified by silica gel chromatography.

Biological Assays

The compounds according to Formula I are cathepsin C inhibitors, which indirectly inhibit the activity of serine proteases that are activated by cathepsin C, such as NE. The compounds according to Formual I, therefore, are useful in the treatment of COPD and other conditions involving cathepsin C and/or such serine proteases. The biological activity of the compounds according to Formula I can be determined using any suitable assay for determining the activity of a candidate compound as a cathepsin C inhibitor or for determining the ability of a candidate compound to prevent the cathepsin C mediated activation of certain serine proteases, as well as suitable tissue and in vivo models. Examples 7, 9-21 , and 24-50 were tested for activity as cathepsin C inhibitors. All tested compounds were found to be cathepsin C inhibitors.

A. Neutrophil Isolation:

Reagents: Histopaque-1077 (Sigma H-8889)

Dulbecco's Phosphate Buffered Saline (DPBS) without CaCl2 and without MgCl2 6% Dextran T500 (Pharmacia 17-0320-02 or 17-0320-01):

Dissolve 6Og in 1 -Liter of DPBS without Ca++ and Zg++, aliquot into 50 ml tubes, store at -20⁰C. Do Not Filter.

Method:

[blood and reagents at room temperature]

• Pipette 20 ml Histopaque into each 50 ml tube

• Layer 25-30 ml of blood on top of Histopaque - gently • Spin at 1600 rpm (40Og) for 30 minutes (brake off)

• Aspirate and discard the top 2 layers

• Add DPBS without Ca++ and Mg++ to obtain a final volume of 35 ml

• Add 12 ml of 6% Dextran

• Mix by inversion and allow to sit and sediment for 45 minutes • Aspirate and discard the top layer and carefully pipette the supernatant into another 50 ml tube add DPBS without Ca++ and Mg++ to 50 ml (avoid aspirating the red blood cells)

• Centrifuge at 1000 rpm (brake on)

• Pour (or aspirate leaving only risidual supernatant) off and discard the supernatant saving the pellet

• Mix the pellet with the residual supernatant by tapping the base of the tube

• Add 20 ml of H2O to the cells, mix and allow to sit for 40 seconds

• Add 2 ml of 10X DPBS without Ca++ and Mg++

• Fill the tube with 1X DPBS without Ca++ and Mg++ and allow to sit for 10 minutes - all the red cells should be lysed at this point • Spin at 1000 rpm for 5 minutes (brake on)

• Aspirate or pour off supernatant, tap to resuspend ceils in residual supernatant

• Resuspend at desired concentration and in desired media (we're using RPMI- 1640 without phenol red and with 0.1%BSA)

B. Human Neutrophil Elastase Release Assay: Reagents:

Incubation media: 0.1 % BSA in RPMI-1640 without phenol red

BSA: Sigma A-6003 PMA (phorboM 2-myristate acetate): Sigma P-1585

> prepare stock solutions 1.6mM in DMSO, aliquot, store at -20⁰C

> use final concentration of 2OnM (this will yield a less than maximal activation response)

Substrate: N-methoxy succinyl-Ala-Ala-Pro-Val-p-nitroanilide

Sigma M-4765 NE Assay Reagent: 2 mM substrate, 0.1 M Tris HCI (pH 8), 0.5M NaCI

Prepare NE assay reagent at 2X final concentration:- For one 96-well plate

Dissolve 28.3 mg of substrate in 200 ul DMSO Add 3.4 ml H₂O, mix till substrate goes back in solution Add 2.4 ml 1 M Tris HCI, pH 8 Add 6.0 ml 2M NaCI Method:

• Isolate human neutrophils according to protocol. Resuspend the final cell pellet at 2x10⁶ cells / ml in incubation media (0.1 % BSA in RPMI-1640 without phenol red).

Note: depending on response the concentration of cells can be increased (0.2x1 (f cells - 4x1 (f cells per well)

Incubation:

[prepare PMA and drugs at 4X desired final concentration in incubation media]

• Add 50 ul of 8OnM PMA (final concentration = 2OnM) to the appropriate wells of a 96-well (round bottom) plate Include controls w/o PMA to demonstrate that upon addition of PMA the cells are activated • Add 50 ul of drugs to appropriate wells - prepare drugs at 4X desired final concentration in incubation media

Mix

• Add 100 ul of neutrophil suspension • Mix (shake gently on microplate shaker for 5 minutes)

• Incubate 2 hours at 37⁰C in 5% CO₂ in air

• Pellet at 2000-RPM for 10 minutes

• Transfer 100 ul aliquots of supernatant to a clear flat-bottom 96-well plate

NE Assay: in a clear flat-bottom 96-well plate

• 100 ul supernatant (see above)

• 100 ul 2X NE assay reagent

• mix on microplate shaker for 5 minutes keep covered during color development

• allow color to develop (we routinely use 30-60min but you can go longer if needed - people routinely allow to go overnight)

• Read absorbance at 405 nm

Human neutrophil elastase can be added as standard or reference for the assay.

Human Sputum Elastase [Elastin Products Co / Catalog #SE563]

Lot #14994 = 87.5units/0.1 mg per vial dissolve in 175 ul of solution containing 50%glycerol and 50%

0.02MnaOAc, pH5.0 (store at -2O⁰C)

Add ~50m units for positive assay reference

Add 2.5 - 50m units for a standard curve

See also: Keiko Fujie, Yasuhiko Shinguh, Noriaki Inamura, Ritsuko Yasumitsu, Masanori Okamoto, Masakuni Okuhara, Release of neutrophil elastase and its role in tissue injury in acute inflamation: effect of the elastase inhibitor, FR134043, Eur. J. Pharm. 374: 117- 125, 1999 and Kazuhiko Yoshimura, Sawako Nakagawa, Sekiya Koyama, Toshio Kobayashi, and Tatsuji Homma, Roles of neutrophil elastase and superoxide anion in leukotriene LTB^-induced lung injury in rabbit J. Appl. Physiol. 76(1 ): 91 -96, 1994. C. Recombinant Cathepsin C in vitro assay:

The activity of recombinant human cathepsin C is measured by the cleavage of a fluorogenic substrate, (H-Gly-Arg)2-rhodamine110. Briefly, 40 pM cathepsin C and 0.01 mM (H-Gly-Arg)2-rhodamine110 are incubated in a buffer consisting of 50 mM sodium acetate, 30 mM sodium chloride, 1 mM CHAPS, 1 mM dithiothreitol, 1 mM EDTA, pH 5.5 at 25 ⁰C for one hour. The reaction is stopped by the addition of 1/10 volume of 10 mM 2-Aldrithiol. The reaction product is measured on a fluorescence reader set at an excitation wavelength of 485 nm and emission wavelength of 535 nm.

Methods of Use

The compounds of the invention inhibit the cathepsin C enzyme and can be useful in the treatment of conditions wherein the underlying pathology is (at least in part) attributable to cathepsin C involvement or in conditions wherein cathepsin C inhibition offers some clinical benefit even though the underlying pathology is not (even in part) attributable to cathepsin C involvement. Examples of such conditions include COPD, rheumatoid arthritis, osteoarthritis, asthma, and Multiple Sclerosis. Accordingly, in another aspect the invention is directed to methods of treating such conditions.

The methods of treatment of the invention comprise administering a safe and effective amount of a compound of the invention to a patient in need thereof. As used herein, "treatment" in reference to a condition means: (1) the amelioration or prevention of the condition being treated or one or more of the biological manifestations of the condition being treated, (2) the interference with (a) one or more points in the biological cascade that leads to or is responsible for the condition being treated or (b) one or more of the biological manifestations of the condition being treated, or (3) the alleviation of one or more of the symptoms or effects associated with the condition being treated.

As indicated above, "treatment" of a condition includes prevention of the condition. The skilled artisan will appreciate that "prevention" is not an absolute term. In medicine, "prevention" is understood to refer to the prophylactic administration of a drug to substantially diminish the likelihood or severity of a condition or biological manifestation thereof, or to delay the onset of such condition or biological manifestation thereof.

As used herein, "safe and effective amount" in reference to a compound of the invention or other pharmaceutically-active agent means an amount of the compound sufficient to significantly induce a positive modification in the condition to be treated but low enough to avoid serious side effects (at a reasonable benefit/risk ratio) within the scope of sound medical judgment. A safe and effective amount of a compound of the invention will vary with the particular compound chosen (e.g. consider the potency, efficacy, and half-life of the compound); the route of administration chosen; the condition being treated; the severity of the condition being treated; the age, size, weight, and physical condition of the patient being treated; the medical history of the patient to be treated; the duration of the treatment; the nature of concurrent therapy; the desired therapeutic effect; and like factors, but can nevertheless be routinely determined by the skilled artisan.

As used herein, "patient" refers to a human or other animal.

The compound of the invention may be administered by any suitable route of administration, including both systemic administration and topical administration. Systemic administration includes oral administration, parenteral administration, transdermal administration, rectal administration, and administration by inhalation. Parenteral administration refers to routes of administration other than enteral, transdermal, or by inhalation, and is typically by injection or infusion. Parenteral administration includes intravenous, intramuscular, and subcutaneous injection or infusion. Inhalation refers to administration into the patient's lungs whether inhaled through the mouth or through the nasal passages. Topical administration includes application to the skin as well as intraocular, otic, intravaginal, and intranasal administration. The compound of the invention may be administered once or according to a dosing regimen wherein a number of doses are administered at varying intervals of time for a given period of time. For example, doses may be administered one, two, three, or four times per day. Doses may be administered until the desired therapeutic effect is achieved or indefinitely to maintain the desired therapeutic effect. Suitable dosing regimens for a compound of the invention depend on the pharmacokinetic properties of that compound, such as absorption, distribution, and half-life, which can be determined by the skilled artisan. In addition, suitable dosing regimens, including the amount administered and the duration such regimens are administered, for a compound of the invention depend on the condition being treated, the severity of the condition being treated, the age and physical condition of the patient being treated, the medical history of the patient to be treated, the nature of concurrent therapy, the particular route of administration chosen, the desired therapeutic effect, and like factors within the knowledge and expertise of the skilled artisan. It will be further understood by such skilled artisans that suitable dosing regimens may require adjustment given an individual patient's response to the dosing regimen or over time as individual patient needs change. Typical daily dosages range from 10 mg to 1000 mg. Additionally, the compounds of the invention may be administered as prodrugs. As used herein, a "prodrug" of a compound of the invention is a functional derivative of the compound which, upon administration to a patient, eventually liberates the compound of the invention in vivo. Administration of a compound of the invention as a prodrug may enable the skilled artisan to do one or more of the following: (a) modify the onset of the compound in vivo; (b) modify the duration of action of the compound in vivo; (C) modify the transportation or distribution of the compound in vivo; (d) modify the solubility of the compound in vivo; and (e) overcome or overcome a side effect or other difficulty encountered with the compound. Typical functional derivatives used to prepare prodrugs include modifications of the compound that are chemically or enzymatically cleaved in vivo. Such modifications, which include the preparation of phosphates, amides, esters, thioesters, carbonates, and carbamates, are well known to those skilled in the art.

Compositions The compound of the invention will normally, but not necessarily, be formulated into a pharmaceutical composition prior to administration to a patient. Accordingly, in another aspect the invention is directed to pharmaceutical compositions comprising a compound of the invention and a pharmaceutically-acceptable excipient.

The pharmaceutical compositions of the invention may be prepared and packaged in bulk form wherein a safe and effective amount of a compound of the invention can be extracted and then given to the patient such as with powders, syrups, and solutions for injection. Alternatively, the pharmaceutical compositions of the invention may be prepared and packaged in unit dosage form wherein each physically discrete unit contains a safe and effective amount of a compound of the invention. When prepared in unit dosage form, the pharmaceutical compositions of the invention typically contain from 1 mg to 1000 mg.

The pharmaceutical compositions of the invention typically contain one compound of the invention. However, in certain embodiments, the pharmaceutical compositions of the invention contain more than one compound of the invention. For example, in certain embodiments the pharmaceutical compositions of the invention contain two compounds of the invention. In addition, the pharmaceutical compositions of the invention may optionally further comprise one or more additional pharmaceutically active compounds. Conversely, the pharmaceutical compositions of the invention typically contain more than one pharmaceutically-acceptable excipient. However, in certain embodiments, the pharmaceutical compositions of the invention contain one pharmaceutically-acceptable excipient. As used herein, "pharmaceutically-acceptable excipient" means a pharmaceutically acceptable material, composition or vehicle involved in giving form or consistency to the pharmaceutical composition. Each excipient must be compatible with the other ingredients of the pharmaceutical composition when commingled such that interactions which would substantially reduce the efficacy of the compound of the invention when administered to a patient and interactions which would result in pharmaceutical compositions that are not pharmaceutically acceptable are avoided. In addition, each excipient must of course be of sufficiently high purity to render it pharmaceutically-acceptable. The compound of the invention and the pharmaceutically-acceptable excepient or excepients will typically be formulated into a dosage form adapted for administration to the patient by the desired route of administration. For example, dosage forms include those adapted for (1) oral administration such as tablets, capsules, caplets, pills, troches, powders, syrups, elixers, suspensions, solutions, emulsions, sachets, and cachets; (2) parenteral administration such as sterile solutions, suspensions, and powders for reconstitution; (3) transdermal administration such as transdermal patches; (4) rectal administration such as suppositories; (5) inhalation such as aerosols and solutions; and (6) topical administration such as creams, ointments, lotions, solutions, pastes, sprays, foams, and gels. Suitable pharmaceutically-acceptable excipients will vary depending upon the particular dosage form chosen. In addition, suitable pharmaceutically-acceptable excipients may be chosen for a particular function that they may serve in the composition. For example, certain pharmaceutically-acceptable excipients may be chosen for their ability to facilitate the production of uniform dosage forms. Certain pharmaceutically- acceptable excipients may be chosen for their ability to facilitate the production of stable dosage forms. Certain pharmaceutically-acceptable excipients may be chosen for their ability to facilitate the carrying or transporting the compound or compounds of the invention once administered to the patient from one organ, or portion of the body, to another organ, or portion of the body. Certain pharmaceutically-acceptable excipients may be chosen for their ability to enhance patient compliance.

Suitable pharmaceutically-acceptable excipients include the following types of excipients: Diluents, fillers, binders, disinteg rants, lubricants, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweetners, flavoring agents, flavor masking agents, coloring agents, anticaking agents, hemectants, chelating agents, plasticizers, viscosity increasing agents, antioxidants, preservatives, stabilizers, surfactants, and buffering agents. The skilled artisan will appreciate that certain pharmaceutically-acceptable excipients may serve more than one function and may serve alternative functions depending on how much of the excipient is present in the formulation and what other ingredients are present in the formulation.

Skilled artisans possess the knowledge and skill in the art to enable them to select suitable pharmaceutically-acceptable excipients in appropriate amounts for use in the invention. In addition, there are a number of resources that are available to the skilled artisan which describe pharmaceutically-acceptable excipients and may be useful in selecting suitable pharmaceutically-acceptable excipients. Examples include Remington's Pharmaceutical Sciences (Mack Publishing Company), The Handbook of Pharmaceutical Additives (Gower Publishing Limited), and The Handbook of Pharmaceutical Excipients (the American Pharmaceutical Association and the Pharmaceutical Press).

The pharmaceutical compositions of the invention are prepared using techniques and methods known to those skilled in the art. Some of the methods commonly used in the art are described in Remington's Pharmaceutical Sciences (Mack Publishing Company).

In one aspect, the invention is directed to a solid oral dosage form such as a tablet or capsule comprising a safe and effective amount of a compound of the invention and a diluent or filler. Suitable diluents and fillers include lactose, sucrose, dextrose, mannitol, sorbitol, starch (e.g. corn starch, potato starch, and pre-gelatinized starch), cellulose and its derivatives (e.g. microcrystalline cellulose), calcium sulfate, and dibasic calcium phosphate. The oral solid dosage form may further comprise a binder. Suitable binders include starch (e.g. corn starch, potato starch, and pre-geiatinized starch), gelatin, acacia, sodium alginate, alginic acid, tragacanth, guar gum, povidone, and cellulose and its derivatives (e.g. microcrystalline cellulose). The oral solid dosage form may further comprise a disintegrant. Suitable disintegrants include crospovidone, sodium starch glycolate, croscarmelose, alginic acid, and sodium carboxymethyl cellulose. The oral solid dosage form may further comprise a lubricant. Suitable lubricants include stearic acid, magnesuim stearate, calcium stearate, and talc.

In another aspect, the invention is directed to a dosage form adapted for administration to a patient by inhalation. For example, the compound of the invention may be inhaled into the lungs as a dry powder, an aerosol, a suspension, or a solution.

Dry powder compositions for delivery to the lung by inhalation typically comprise a compound of the invention as a finely divided powder together with one or more pharmaceutically-acceptable excipients as finely divided powders. Pharmaceutically- acceptable excipients particularly suited for use in dry powders are known to those skilled in the art and include lactose, starch, mannitol, and mono-, di-, and polysaccharides.

The dry powder may be administered to the patient via a reservoir dry powder inhaler (RDPI) having a reservoir suitable for storing multiple (un-metered doses) of medicament in dry powder form. RDPIs typically include a means for metering each medicament dose from the reservoir to a delivery position. For example, the metering means may comprise a metering cup, which is movable from a first position where the cup may be filled with medicament from the reservoir to a second position where the metered medicament dose is made available to the patient for inhalation. Alternatively, the dry powder may be presented in capsules (e.g. gelatin or plastic), cartridges, or blister packs for use in a multi-dose dry powder inhaler (MDPI). MDPIs are inhalers wherein the medicament is comprised within a multi-dose pack containing (or otherwise carrying) multiple defined doses (or parts thereof) of medicament. When the dry powder is presented as a blister pack, it comprises multiple blisters for containment of the medicament in dry powder form. The blisters are typically arranged in regular fashion for ease of release of the medicament therefrom. For example, the blisters may be arranged in a generally circular fashion on a disc-form blister pack, or the blisters may be elongate in form, for example comprising a strip or a tape. Each capsule, cartridge, or blister may, for example, contain between 20μg~10mg of the compound of the invention. Aerosols may be formed by suspending or dissolving a compound of the invention in a liquified propellant. Suitable propellants include halocarbons, hydrocarbons, and other liquified gases. Representative propellants include: trichlorofluoromethane (propellant 11), dichlorofluoromethane (propellant 12), dichlorotetrafluoroethane (propellant 114), tetrafluoroethane (HFA-134a), 1 ,1-difluoroethane (HFA-152a), difluoromethane (HFA-32), pentafluoroethane (HFA-12), heptafluoropropane (HFA-227a), perfluoropropane, perfluorobutane, perfluoropentane, butane, isobutane, and pentane. Aerosols comprising a compound of the invention will typically be administered to a patient via a metered dose inhaler (MDI). Such devices are known to those skilled in the art. The aerosol may contain additional pharmaceutically-acceptable excipients typically used with MDIs such as surfactants, lubricants, cosolvents and other excipients to improve the physical stability of the formulation, to improve valve performance, to improve solubility, or to improve taste.

Suspensions and solutions comprising a compound of the invention may also be administered to a patient via a nebulizer. The solvent or suspension agent utilized for nebulization may be any pharmaceutically-acceptable liquid such as water, aqueous saline, alcohols or glycols, e.g., ethanol, isopropylaicohol, glycerol, propylene glycol, polyethylene glycol, etc. or mixtures thereof. Saline solutions utilize salts which display little or no pharmacological activity after administration. Both organic salts, such as alkali metal or ammonium halogen salts, e.g., sodium chloride, potassium chloride or organic salts, such as potassium, sodium and ammonium salts or organic acids, e.g., ascorbic acid, citric acid, acetic acid, tartaric acid, etc. may be used for this purpose.

Other pharmaceutically-acceptable excipients may be added to the suspension or solution. The compound of the invention may be stabilized by the addition of an inorganic acid, e.g., hydrochloric acid, nitric acid, sulphuric acid and/or phosphoric acid; an organic acid, e.g., ascorbic acid, citric acid, acetic acid, and tartaric acid, etc., a complexing agent such as EDTA or citric acid and salts thereof; or an antioxidant such as antioxidant such as vitamin E or ascorbic acid. These may be used alone or together to stabilize the compound of the invention. Preservatives may be added such as benzalkonium chloride or benzoic acid and salts thereof. Surfactant may be added particularly to improve the physical stability of suspensions. These include lecithin, disodium dioctylsulphosuccinate, oleic acid and sorbitan esters.

Examples

The following examples illustrate the invention. These examples are not intended to limit the scope of the present invention, but rather to provide guidance to the skilled artisan to prepare and use the compounds, compositions, and methods of the present invention. While particular embodiments of the present invention are described, the skilled artisan will appreciate that various changes and modifications can be made without departing from the spirit and scope of the invention.

Preparation Examples

Example 1

Pyrazolidine dihvdrochloride (1) Sodium hydride (18.08 g, 60% w/w, 0.452 mol) was suspended in 215 mL anhydrous DMF and the mixture was stirred at room temperature under argon for 2.5 hours. The suspension was then cooled with a cold water bath and a solution of bis-Boc hydrazine (50.0 g, 0.215 mol) in 360 ml DMF was added dropwise over 55 minutes. The reaction mixture was stirred an additional 1.5 hours and then a solution of 1 ,3-dibromopropane (21.9 mL, 0.215 mol) in 70 mL DMF was added dropwise. The reaction mixture was allowedd to come to room temperature and was stirred for 24 hours. The reaction mixture was then cooled in an ice/water bath and quenched by the careful addition of 215 mL of water. The reaction mixture was poured into 2150 mL of water and extracted with ethyl acetate (1 x 750 mL, 2 x 650 mL). The combined extracts were dried over sodium sulfate and evaporated to dryness to give 88.79 g of the crude bis-Boc pyrazolidine as a colorless oil. This oil was purified by dissolving in 10% diethyl ether in hexane (85 mL) and applying to a BioTage 75L silica column and eluting sequentially with 4 L of 10% diethyl ether in hexane, 4 L of 25% diethyl ether in hexane, and 7.1 L of 30% diethyl ether in hexane. The appropriate fractions were combined and evaporated to give the purified bis-Boc pyrazolidine (47.0 g, 80.2%). The protected cyclic hydrazine was then dissolved in 4 N HCI/dioxane (800 mL). The flask was stoppered, cooled in an ice bath, and then allowedd to warm to room temperature overnight with stirring. After 24 hours, the reaction mixture was filtered under argon and the solid obtained was washed with cold dioxane (200 mL). The solid was dried in a vacuum oven at 35 ⁰C for 36 hours, giving the titled compound as a white solid (24.13 g , 96.4%), ESMS 72.8 (M+H⁺).

Example 2

Hexahydropyridazine dihvdrochloride (2)

Using the procedure of Example 1 and substituting 1 ,4-dibromobutane (25.7 mL, 0.215 mol) gave the bis-Boc hexahydropyridazine as a white solid (66.75 g) which did not require purification. Removal of the Boc groups and workup as in Example 1 gave the titled compound as a slightly pink solid (32.59 g, 95.3%), ESMS 87.2 (M+H⁺).

Example 3

Hexahydro-1 H-1 ,2-diazepine dihvdrochloride (3) Using the procedure of Example 1 and substituting 1 ,5-dibromopentane (29.4 mL, 0.215 mol) gave the crude bis-Boc cyclic hydrazine as an oil (80.81 g). This was purified as in Example 1 , eluting with 8.3 L 10% diethyl ether in hexane followed by 4.0 L of 15% diethyl ether in hexane, followed by 6.3 L of 20% diethyl ether in hexane. Evaporation of the appropriate fractions gave the purified bis-Boc cyclic hydrazine (52.12 g, 80.7%). Removal of the Boc groups and workup as in Example 1 gave the titled compound as a slightly pink solid (28.93 g, 96.3%), ESMS 101.0 (M+H⁺). Example 4

1 -Cyanopyrazolidine (4)

To a suspension of 1 (100 mg, 0.69 mmoi) in 5 ml of dichloromethane at room temperature was added diisopropylethylamine (721 uL, 4.14 mmol) and the mixture was stirred at room temperature until a homogeneous solution was obtained. Cyanogen bromide (80.4 mg, 0.76 mmol) was the added in one portion and the reaction mixture was stirred for two hours at room temperature. This reaction mixture was used directly without workup or purification.

Example 5 1 -Cyanohexahydropyridazine (5)

Prepared according to Example 4, substituting hexahydropyridazine dihyrdochloride (100 mg, 0.63 mmol) and using 657 uL of diisopropylethylamine (3.77 mmol) and 100.1 mg cyanogen bromide (0.94 mmol). This reaction mixture was used directly without workup or purification.

Example 6

1 -Cyanohexahvdro-1 H- 1 ,2-diazepine (6)

Prepared according to Example 4, substituting hexahydro-1 H-1 ,2-diazepine dihydrochloride ( 100mg, 0.58 mmo!) and using 606 uL diisopropylethylamine (3.48 umol) and 174 uL of 5 M cyanogen bromide in acetonitrile (0.87 mmol). This reaction mixture was used directly without workup or purification.

wumμounα examples

Example 7

Benzyl 2-cvanopyrazolidine-1-carboxylate (7)

4 was prepared by the method of Example 4 starting with 1 (50 mg, 0.34 mmol) and diisopropylethylamine (296 uL, 1.7 mmol) followed by cyanogen bromide (54 mg, 0.51 mmol). To this was added benzyl chloroformate (73 uL, 0.51 mmol) in one portion, and the reaction mixture was stirred at room temperature overnight. After removal of solvent by rotary evaporation, the title compound was purified by reverse phase HPLC (10% acetonitrile/water to 90% acetonitrile/water over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 7 (8.1 mg, 10%), ESMS 232.2 (M+H⁺).

Example 8

Benzyl 2-cvanotetrahvdropyridazine-1-carboxylate (8)

5 was prepared according to Example 5, starting with 2 (50 mg, 0.31 mmol) and diisopropylethylamine (270 uL, 1.55 mmol), followed by 93 uL of 5 M cyanogen bromide in acetonitrile (0.47 mmol). To this was added benzyl choloroformate (66 uL, 0.47 mmol) in one portion, and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by reverse phase HPLC (10% acetonitrile/water to 90% acetonitrile/water over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 8 (7.7 mg, 10%), ESMS 246.6 (M+H⁺). Example 9

Benzyl 2-cyanotetrahydro-1 H- 1 ,2-diazepine-1 -carboxylate (9)

6 was prepared according to Example 6, starting with 3 (50 mg, 0.29 mmol) and diisopropylethylamine (270 uL, 1.55 mmol), followed by 88 uL of 5 M cyanogen bromide in acetonitrile (0.44 mmol). To this was added benzyl choloroformate (62 uL, 0.44 mmol) in one portion, and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by reverse phase HPLC (10% acetonitrile/water to 90% acetonitrile/water over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 9 (11.5 mg, 15.2%), ESMS 260.2 (M+H⁺).

Example 10

4-Methoxyphenyl 2-cvanopyrazolidine-1-carboxylate (10)

4 was prepared according to Example 4. To this was added 4-methoxyphenyl chloroformate (104.4 mg, 0.69 mmol) in one portion and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by reverse phase HPLC (10% acetonitrile/water to 90% acetonitrile/water over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 10 (3.4 mg, 2.0%), ESMS 248.2 (M+H⁺). Example 11

2'-Nitrophenyl 2-cyanopyrazolidine-i-carboxylate (11)

4 was prepared according to Example 4. To this was added 2-nitrophenyl chloroformate (139.1 mg, 0.69 mmol) in one portion and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by reverse phase HPLC (10% acetonitrile/water to 90% acetonitrile/water over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 11 (5.3 mg, 2.9%), ESMS 263.2 (M+H⁺).

Example 12

4'-Methylphenyl 2-cvanopyrazolidine-1 -carboxylate (12)

4 was prepared according to Example 4. To this was added p-tolyl chloroformate (117.7 mg, 0.69 mmol) in one portion and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by reverse phase HPLC (10% acetonitrile/water to 90% acetonitrile/water over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 12 (4.9 mg, 3.1%), ESMS 232.2 (M+H⁺).

Example 13

2'-Methoxyphenyl 2-cvanopyrazolidine-1-carboxylate (13)

4 was prepared according to Example 4. To this was added 2-methoxyphenyl chloroformate (104.4 mg, 0.69 mmol) in one portion and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by silica gel chromatography (CombiFlash, 20% ethyl acetate in hexanes to 100% ethyl acetate over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 13 (72.8 mg, 42.6%), ESMS 248.6 (M+H⁺).

Example 14 2'-Chlorophenyl 2-cvanopyrazolidine-1-carboxylate (14)

4 was prepared according to Example 4. To this was added 2-chlorophenyl chloroformate (131.8 mg, 0.69 mmol) in one portion and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by silica gel chromatography (CombiFlash, 20% ethyl acetate in hexanes to 100% ethyl acetate over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 13 (51.4 mg, 29.6%), ESMS 252.2 (M+H⁺).

Example 15

2'-Methoxyphenyl 2-cyanohexahyclropyriclazine-1 -carboxylate (15)

5 was prepared according to Example 5. To this was added 2-methoxyphenyl chloroformate (117.6 mg, 0.63 mmol) in one portion and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by reverse phase HPLC (10% acetonitriie/water to 90% acetonitrile/water over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 15 (3.7 mg, 2.2%), ESMS 262.2 (M+H⁺).

Example 16

2^f-Chlorophenyl 2-cyanohexahvdropyridazine-1 -carboxylate (16)

5 was prepared according to Example 5. To this was added 2-chlorophenyl chloroformate (120.3 mg, 0.63 mmol) in one portion and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by reverse phase HPLC (10% acetonitrile/water to 90% acetonitrile/water over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 16 (5.6 mg, 3.3%), ESMS 266.2 (M+H⁺).

Example 17

2'-Nitrophenyl 2-cvanohexahvdropyridazine-1 -carboxylate (17)

5 was prepared according to Example 5. To this was added 2-nitrophenyl chloroformate (127.0 mg, 0.63 mmol) in one portion and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by reverse phase HPLC (10% acetonitrile/water to 90% acetonitrile/water over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 17 (5.5 mg, 3.2%), ESMS 277.2 (M+H⁺).

Example 18

2'-Chlorobenzyl 2-cvanohexahvdropyridazine-1-carboxylate (18)

5 was prepared according to Example 5. To this was added 2-chlorobenzyl chloroformate (129.2 mg, 0.63 mmol) in one portion and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by reverse phase HPLC (10% acetonitrile/water to 90% acetonitrile/water over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 18 (10.1 mg, 5.7%), ESMS 280.2 (M+H⁺).

Example 19

2'-Naphthyl 2-cvanohexahvdropyridazine-1 -carboxylate (19)

5 was prepared according to Example 5. To this was added 2-naphthyl chloroformate (130.2 mg, 0.63 mmol) in one portion and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by reverse phase HPLC (10% acetonitrile/water to 90% acetonitrile/water over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 19 (7.4 mg, 4.2%), ESMS 282.2 (M+H⁺).

Example 20

1 '-Naphthyl 2-cvanohexahvdropyridazine-1 -carboxylate (20)

5 was prepared according to Example 5. To this was added 1-naphthyl chloroformate (130.2 mg, 0.63 mmol) in one portion and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by reverse phase HPLC (10% acetonitrile/water to 90% acetonitrile/water over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 20 (6.9 mg, 3.9%), ESMS 282.2 (M+H⁺).

Example 21

4'-r(Mθthyloxv)carbonvπphenvl 2-cvanohexahvdropvridazine-1 -carboxvlate (21)

5 was prepared according to Example 5. To this was added 4-methoxycarbonylphenyl chloroformate (135.2 mg, 0.63 mmol) in one portion and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by reverse phase HPLC (10% acetonitrile/water to 90% acetonitrile/water over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 21 (0.2 mg, >1 %), ESMS 290.2 (M+H⁺).

Example 22

2'-Nitrophenyl 2-cvanohexahvdro-1 HA ,2-diazepine-1 -carboxylate (22)

6 was prepared according to Example 6, using 174 uL of 5 N cyanogen bromide in acetonitrile (0.87 mmol). To this was added 2-nitrophenyl chloroformate (116.9 mg, 0.58 mmol) in one portion and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by reverse phase HPLC (10% acetonitrile/water to 90% acetonitrile/water over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 22 (7.4 mg, 4.4%), ESMS 291.2 (M+H⁺). Example 23

2^f-r(Phenylmethyl)oxy1ethyl 2-cyanohexahydro-1 H-1 ,2-diazepine-1 -carboxylate (23)

6 was prepared according to Example 6, using 174 uL of 5 N cyanogen bromide in acetonitrile (0.87 mmol). To this was added 2-(benzyloxy)ethyl chloroformate (124.4 mg, 0.58 mmol) in one portion and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by reverse phase HPLC (10% acetonitrile/water to 90% acetonitrile/water over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 23 (3.7 mg, 2.1%), ESMS 304.2 (M+H⁺).

Example 24 Phenyl 2-cyanopyrazolidine-1 -carboxylate (24)

4 was prepared according to Example 4. To this was added phenyl chloroformate (86.6 mg, 0.69 mmol) in one portion and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by silica gel chromatography (CombiFlash, 20% ethyl acetate in hexanes to 100% ethyl acetate over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 24 (77.8 mg, 51.9%), ESMS 218.4 (M+H⁺). Example 25

1 '-Naphthyl 2-cvanopyrazolidine-1 -carboxylate (25)

4 was prepared according to Example 4. To this was added 1 -naphthyl chloroformate (142.5 mg, 0.69 mmol) in one portion and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by silica gel chromatography (CombiFlash, 20% ethyl acetate in hexanes to 100% ethyl acetate over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 25 (74.0 mg, 40.1 %), ESMS 268.4 (M+H⁺).

Example 26

2'-Naphthyl 2-cvanopyrazolidine-1 -carboxylate (26)

4 was prepared according to Example 4. To this was added 2-naphthyl chloroformate (142.5 mg, 0.69 mmol) in one portion and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by silica gel chromatography (CombiFlash, 20% ethyl acetate in hexanes to 100% ethyl acetate over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 26 (86.1 mg, 46.6%), ESMS 268.4 (M+H⁺).

Example 27

4'-Chlorophenyl 2-cyanopyrazolidine-1-carboxylate (27)

4 was prepared according to Example 4. To this was added 4-chlorophenyl chloroformate (131.8 mg, 0.69 mmol) in one portion and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by silica gel chromatography (CombiFlash, 20% ethyl acetate in hexanes to 100% ethyl acetate over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 27 (55.0 mg, 31.6%), ESMS 252.4 (M+H⁺).

Example 28

4'-Methylphenyl 2-cvanohexahvdropyridazine-1 -carboxylate (28)

5 was prepared accorting to Example 5. To this was added p-tolyl chloroformate (87.7 uL, 0.63 mmol) in one portion and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by silica gel chromatography (CombiFlash, 20% ethyl acetate in hexanes to 100% ethyl acetate over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 28 (66.7 mg, 43.1%), ESMS 246.6 (M+H⁺).

Example 29

4'-r(Methyloxy)carbonvnphenyl 2-cyanopyrazolidine-1 -carboxylate (29)

4 was prepared according to Example 4. To this was added 4-(methoxycarbonyl)phenyl chloroformate (148.1 mg, 0.69 mmol) in one portion and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by silica gel chromatography (CombiFlash, 20% ethyl acetate in hexanes to 100% ethyl acetate over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 29 (85.9 mg, 45.2%), ESMS 276.4 (M+H⁺).

Example 30 4'-(4-Acetylpiperazino)phenyl 2-cvanopyrazolidine-1 -carboxylate (30)

4-(4-Acetylpiperazino)phenyl chloroformate was prepared by dissolving 1 -acetyl-4-(4- hydroxyphenyl)piperazine (152.0 mg, 0.69 mmol) was in 3 ml_ dichloromethane followed by diisopropylethylamine (145 uL, 0.83 mmol). 20% Phosgene in toluene (473 uL, 0.83 mmol) was added dropwise and the reaction mixture was allowed to stir at room temperature for 2 hours.

4 was prepared according to Example 4. To this was added the 4-(4- acetylpiperazino)phenyl chloroformate in one portion and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by silica gel chromatography (CombiFlash, 20% ethyl acetate in hexanes to 100% ethyl acetate over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 30 (6.4 mg, 2.7%), ESMS 344.4 (M+H⁺).

Example 31 2-Naphthalenylmethyl 2-cvanopyrazolidine-1-carboxylate (31)

2-Naphthalenylmethyl chloroformate was prepared by dissolving 2-naphthalenemethanol (119.0 mg, 0.69 mmol) was in 3 ml_ dichloromethane followed by diisopropylethylamine (145 uL, 0.83 mmol). 20% Phosgene in toluene (473 uL, 0.83 mmol) was added dropwise and the reaction mixture was allowed to stir at room temperature for 2 hours. 4 was prepared according to Example 4. To this was added the 2-naphthalenylmethyl chloroformate in one portion and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by silica gel chromatography (CombiFlash, 20% ethyl acetate in hexanes to 100% ethyl acetate over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 31 (47.9 mg, 24.6%), ESMS 282.4 (M+H⁺).

Example 32

(3,4-Dichlorophenyl)methyl 2-cyanopyrazolidine-1 -carboxylate (32)

(3,4-Dichlorophenyl)methyl chloroformate was prepared by dissolving 3,4-dichlorobenzyl alcohol (122.0 mg, 0.69 mmol) was in 3 mL dichloromethane followed by diisopropylethylamine (145 uL, 0.83 mmol). 20% Phosgene in toluene (473 uL, 0.83 mmol) was added dropwise and the reaction mixture was allowed to stir at room temperature for 2 hours. 4 was prepared according to Example 4. To this was added the 3,4- (dichlorophenyl)methyl chloroformate in one portion and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compounα was purified by silica gel chromatography (CombiFlash, 20% ethyl acetate in hexanes to 100% ethyl acetate over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 32 (57.9 mg, 28.0%), ESMS 300.4 (M+H⁺).

Example 33

1 ,3-Benzodioxol-5-yl 2-cvanopyrazolidine-1-carboxylate (33)

1 ,3-Benzodioxol-5-yl chloroformate was prepared by dissolving sesamol (94.5 mg, 0.69 mmol) was in 3 mL dichloromethane followed by diisopropylethylamine (145 uL, 0.83 mmol). 20% Phosgene in toluene (473 uL, 0.83 mmol) was added dropwise and the reaction mixture was allowed to stir at room temperature for 2 hours. 4 was prepared according to Example 4. To this was added the 1 ,3-benzodioxol-5-yl chloroformate in one portion and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by silica gel chromatography (CombiFlash, 20% ethyl acetate in hexanes to 100% ethyl acetate over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 33 (86.3 mg, 47.9%), ESMS 262.2 (M+H⁺).

Example 34

(6'-Cyano)-2-naphthalenyl 2-cyanopyrazolidine-i -carboxylate (34)

(6'-Cyano)-2-naphthalenyl chloroformate was prepared by dissolving 6-cyano-2-naphthol (116.7 mg, 0.69 mmol) was in 3 mL dichloromethane followed by diisopropylethylamine (145 uL, 0.83 mmol). 20% Phosgene in toluene (473 uL, 0.83 mmol) was added dropwise and the reaction mixture was allowed to stir at room temperature for 2 hours. 4 was prepared according to Example 4. To this was added the (6'-cyano)-2-naphthalenyl chloroformate in one portion and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by silica gel chromatography (CombiFlash, 20% ethyl acetate in hexanes to 100% ethyl acetate over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 34 (52.3 mg, 25.9%), ESMS 293.4 (M+H⁺).

Example 35

4'-Trifluoromethylphenyl 2-cvanopyrazolidine-1 -carboxylate (35)

4'-Trifluoromethylphenyl chloroformate was prepared by dissolving 4-trifluoromethylphenol (111.9 mg, 0.69 mmol) was in 3 mL dichloromethane followed by diisopropylethylamine (145 uL, 0.83 mmol). 20% Phosgene in toluene (473 uL, 0.83 mmol) was added dropwise and the reaction mixture was allowed to stir at room temperature for 2 hours.

4 was prepared according to Example 4. To this was added the 4'-trifluoromethylphenyl chloroformate in one portion and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by silica gel chromatography (CombiFlash, 20% ethyl acetate in hexanes to 100% ethyl acetate over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 35 (48.1 mg, 24.4%), ESMS 286.2 (M+H⁺). Example 36

4'-Acetylaminophenyl 2-cvanopyrazolidine-1-carboxylate (36)

4'-Acetylaminophenyl chloroformate was prepared by dissolving 4-acetylamidophenol (104.3 mg, 0.69 mmol) was in 3 ml_ dichloromethane followed by diisopropylethylamine (145 uL, 0.83 mmol). 20% Phosgene in toluene (473 uL, 0.83 mmol) was added dropwise and the reaction mixture was allowed to stir at room temperature for 2 hours.

4 was prepared according to Example 4. To this was added the 4'-acetylaminophenyl chloroformate in one portion and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by silica gel chromatography (CombiFlash, 20% ethyl acetate in hexanes to 100% ethyl acetate over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 36 (9.9 mg, 5.2%), ESMS 275.4 (M+H⁺).

Example 37

2,3-Dichlorophenyl 2-cvanopyrazolidine-1-carboxylate (37)

2,3-Dichlorophenyl chloroformate was prepared by dissolving 2,3-dichlorophenol (112.5 mg, 0.69 mmol) was in 3 ml_ dichloromethane followed by diisopropylethylamine (145 uL, 0.83 mmol). 20% Phosgene in toluene (473 uL, 0.83 mmol) was added dropwise and the reaction mixture was allowed to stir at room temperature for 2 hours. 4 was prepared according to Example 4. To this was added the 4'-acetylaminophenyl chloroformate in one portion and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by silica gel chromatography (CombiFlash, 20% ethyl acetate in hexanes to 100% ethyl acetate over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 37 (90.1 mg, 45.6%), ESMS 286.2 (M+H⁺).

Example 38 4'-(1-lmidazolyl)phenyl 2-cvanopyrazolidine-1 -carboxylate (38)

4'-(1-lmidazolyl)phenyl chloroformate was prepared by dissolving 4'-(1-imidazolyl)phenol (110.5 mg, 0.69 mmol) was in 3 ml. dichloromethane followed by diisopropylethylamine

(145 uL, 0.83 mmol). 20% Phosgene in toluene (473 uL, 0.83 mmol) was added dropwise and the reaction mixture was allowed to stir at room temperature for 2 hours.

4 was prepared according to Example 4. To this was added the 4'-acetylaminophenyl chloroformate in one portion and the reaction mixture was stirred overnight at room temperature. An additional aliquot of 20% phosgene in toluene (473 uL, 0.83 mmol) was added and the reaction mixture stirred at room temperature for an additional 24 hours.

After removal of solvent by rotary evaporation, the title compound was purified by silica gel chromatography (CombiFlash, 20% ethyl acetate in hexanes to 100% ethyl acetate over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 38 (13.0 mg, 6.7%), ESMS 284.4 (M+H⁺).

Example 39

3',4'-Dimethvlphenylmethyl 2-cvanopvrazolidine-1 -carboxylate (39)

3',4'-Dimethylphenylmethyl chloroformate was prepared by dissolving 3,4-dimethylbenzyl alcohol (94.0 mg, 0.69 mmol) was in 3 ml_ dichloromethane followed by diisopropylethylamine (145 uL, 0.83 mmol). 20% Phosgene in toluene (473 uL, 0.83 mmol) was added dropwise and the reaction mixture was allowed to stir at room temperature for 2 hours.

4 was prepared according to Example 4. To this was added the 4'-acetylaminophenyl chloroformate in one portion and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by silica gel chromatography (CombiFlash, 20% ethyl acetate in hexanes to 100% ethyl acetate over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 39 (57.0 mg, 31.9%), ESMS 260.4 (M+H⁺).

Example 40

6-Quinolinyl 2-cvanopyrazolidine-1-carboxylate (40)

6-Quinolinly chloroformate was prepared by dissolving 6-hydroxyquinoline (100.2 mg,

0.69 mmol) was in 2 ml_ dichloromethane/1 mL N-methylpyrrolidine followed by diisopropylethylamine (145 uL, 0.83 mmol). 20% Phosgene in toluene (473 uL, 0.83 mmol) was added dropwise and the reaction mixture was allowed to stir at room temperature for 2 hours.

4 was prepared according to Example 4. To this was added the 4'-acetylaminophenyl chloroformate in one portion and the reaction mixture was stirred overnight at room temperature. An additional aliquot of 20% phosgene in toluene (473 uL, 0.83 mmol) was added and the reaction mixture stirred at room temperature for an additional 24 hours. ATter removal of solvent by rotary evaporation, the title compound was purified by silica gel chromatography (CombiFlash, 20% ethyl acetate in hexanes to 100% ethyl acetate over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 40 (37.6 mg, 20.3%), ESMS 269.4 (M+H⁺).

Example 41

7-lsoquinolinyl 2-cvanopyrazolidine-1-carboxylate (41)

7-lsoquinolinly chloroformate was prepared by dissolving 7-hydroxyisoquinoline (100.2 mg, 0.69 mmol) was in 2 mL dichloromethane/1 ml_ N-methylpyrrolidine followed by diisopropylethylamine (145 uL, 0.83 mmol). 20% Phosgene in toluene (473 uL, 0.83 mmol) was added dropwise and the reaction mixture was allowed to stir at room temperature for 2 hours.

4 was prepared according to Example 4. To this was added the 4'-acetylaminophenyl chloroformate in one portion and the reaction mixture was stirred overnight at room temperature. An additional aliquot of 20% phosgene in toluene (473 uL, 0.83 mmol) was added and the reaction mixture stirred at room temperature for an additional 24 hours. After removal of solvent by rotary evaporation, the title compound was purified by silica gel chromatography (CombiFlash, 20% ethyl acetate in hexanes to 100% ethyl acetate over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 41 (4.2 mg, 2.3%), ESMS 269.4 (M+H⁺).

Example 42

5-Quinolinyl 2-cvanopyrazolidine-1-carboxylate (42)

5-Quinolinly chloroformate was prepared by dissolving 5-hydroxyquinoline (100.2 mg, 0.69 mmol) was in 2 mL dichloromethane/1 mL N-methylpyrrolidine followed by diisopropylethylamine (145 uL, 0.83 mmol). 20% Phosgene in toluene (473 uL, 0.83 mmol) was added dropwise and the reaction mixture was allowed to stir at room temperature for 2 hours.

4 was prepared according to Example 4. To this was added the 5-quinolinly chloroformate in one portion and the reaction mixture was stirred overnight at room temperature. An additional aliquot of 20% phosgene in toluene (473 uL, 0.83 mmol) was added and the reaction mixture stirred at room temperature for an additional 24 hours. After removal of solvent by rotary evaporation, the title compound was purified by silica gel chromatography (CombiFlash, 20% ethyl acetate in hexanes to 100% ethyl acetate over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 42 (15.8 mg, 8.5%), ESMS 269.4 (M+H⁺).

Example 43

2-f4'-(Methylphenv0acetvπ-1 -pyrazolidinecarbonitrile (43)

4 was prepared according to Example 4, using 200 mg pyrazolidine dihydrochloride (1.38 mmol) and 1.4 ml_ diisopropylethylamine (8.27 mmol), followed by 414 uL 5 M cyanogen bromide in acetonitrile (2.07 mmol). To this was added p-tolueneacetic acid (311.0 mg, 2.07 mmol) followed by diisopropyl carbodiimide (324 uL, 2.07 mmol) in one portion, and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by silica gel chromatography (CombiFlash, 20% ethyl acetate in hexanes to 100% ethyl acetate over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 43 (31.4 mg, 9.9%), ESMS 230.6 (M+H⁺).

Example 44 2-r(2£)-3-(2-Naphthalenyl)-2-propenovn-1-pyrazolidinecarbonitrile (44)

4 was prepared according to Example 4. To this was added 3-(2-naphthalene)acrylic acid (136.8 mg, 0.69 mmol) followed by diisopropyl carbodiimide (129.6 uL, 0.69 mmol) in one portion, and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by silica gel chromatography (CombiFlash, 20% ethyl acetate in hexanes to 100% ethyl acetate over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 44 (52.3 mg, 52.2%), ESMS 278.4 (M+H⁺). Example 45

2-(2-Naphthalenyl)acetyl-1 -pyrazolidinecarbonitrile (45)

4 was prepared according to Example 4. To this was added 2-naphthalene acetic acid (128.5 mg, 0.69 mmol) followed by diisopropyl carbodiimide (129.6 uL, 0.69 mmol) in one portion, and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by silica gel chromatography (GombiFlash, 20% ethyl acetate in hexanes to 100% ethyl acetate over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 45 (107.8 mg, 58.9%), ESMS 266.2 (M+H⁺).

Example 46 2-(1 -Naphthalenyl)acetyl-1 -pyrazolidinecarbonitrile (46)

4 was prepared according to Example 4. To this was added 1 -naphthalene acetic acid (128.5 mg, 0.69 mmol) followed by diisopropyl carbodiimide (129.6 uL, 0.69 mmol) in one portion, and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by silica gel chromatography (CombiFlash, 20% ethyl acetate in hexanes to 100% ethyl acetate over

10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 46 (94.5 mg, 51.6%), ESMS 266.2 (M+H⁺). Example 47

2-(3-Phenylpropanoyl)-1 -pyrazolidinecarbonitrile (47)

4 was prepared according to Example 4. To this was added phenylpropionic acid (103.6 mg, 0.69 mmol) followed by diisopropyl carbodiimide (129.6 uL, 0.69 mmol) in one portion, and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by silica gel chromatography (CombiFlash, 20% ethyl acetate in hexanes to 100% ethyl acetate over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 47 (94.5 mg, 59.7%), ESMS 230.4 (M+H⁺).

Example 48 2-(4'-Fluorophenylacetyl)-1 -pyrazolidinecarbonitrile (48)

4 was prepared according to Example 4. To this was added 4-fluorophenylacetyl chloride (94.6 uL, 0.69 mmol) in one portion, and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by silica gel chromatography (CombiFlash, 20% ethyl acetate in hexanes to 100% ethyl acetate over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 48 (90.1 mg, 45.6%), ESMS 234.4 (M+H⁺).

Example 49

2-(4'-MethvlphenvQcarbonvl-1 -pyrazolidinecarbonitrile (49)

4 was prepared according to Example 4. To this was added p-toluyl chloride (91.2 uL, 0.69 mmol) in one portion, and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by silica gel chromatography (CombiFlash, 20% ethyl acetate in hexanes to 100% ethyl acetate over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 49 (50.5 mg, 34.0%), ESMS 216.4 (M+H⁺).

Example 50

2-(1 -Naphthalenyl)carbonyl-1 -pyrazolidinecarbonitrile (50)

4 was prepared according to Example 4. To this was added 1 -naphthoyl chloride (103.9 uL, 0.69 mmol) in one portion, and the reaction mixture was stirred overnight at room temperature. After removal of solvent by rotary evaporation, the title compound was purified by silica gel chromatography (CombiFlash, 20% ethyl acetate in hexanes to 100% ethyl acetate over 10 minutes.) The appropriate fractions were collected, combined and evaporated to dryness to yield 50 (84.5 mg, 48.7%), ESMS 252.4 (M+H⁺).

Claims

wnat is claimed is:

1. A compound according to Formula I:

Formula I

wherein: n is 1 , 2, or 3;

X is -C(O)RI , -C(O)ORI , -C(O)R2, -C(O)OR2, -C(O)YR2, or -C(O)OYR2; R1 is C1 -C8 alkyl or C3-C6 cycloalkyl; R2 is aryl or heteroaryl; wherein said aryl is optionally substituted with one or more substituents independently selected from the group consisting of: halo, C1-C3 haloalkyl, -CN, -NO₂, Ra, -ORa, -C(O)ORa, -C(O)Ra, -NHC(O)Ra, heteroaryl and heterocycloalkyl, where said heteroaryl and heterocycloalkyl are optionally substituted with one or more substituents independently selected from the group consisting of: halo, C1 -C3 haloalkyl, Ra, -ORa, - C(O)ORa, -C(O)Ra, and -NHC(O)Ra; and wherein said heteroaryl is optionally substituted with one or more substituents independently selected from the group consisting of: halo, C1-C3 haloalkyl, -CN, -NO₂, Ra, -ORa, -C(O)ORa, -C(O)Ra, and - NHC(O)Ra; Y is C1 -CA alkylene, C2-C4 alkenylene, or -(CH₂)_rO-(CH₂)_m-;

I is 1 , 2, or 3; m is 0, 1 , or 2; I + m is 1 , 2, 3, or 4; Ra is C1 -C3 alkyl; provided that when n is 1 and X is -C(O)ORI , R1 is a group other than f-butyl; or a pharmaceutically-acceptable salt thereof.

2. A compound or salt according to Claim 1 wherein X is -C(O)RI or -C(O)ORI .

3. A compound or salt according to Claim 1 wherein X is -C(O)RI , -C(O)ORI , - C(0)R2, -C(0)0R2, -C(0)YR2, or -C(0)OYR2.

4. A compound or salt according to Claim 3 wherein R2 is aryl.

5. A compound or salt according to Claim 4 wherein R2 is phenyl.

6. A compound or salt according to Claim 4 wherein R2 is napthyl.

7. A compound or salt according to Claim 3 wherein Y is C1-C4 alkylene or -(CH₂)r O-(CH₂)_m-.

8. A compound or salt according to Claim 3 wherein Y is C1 -C4 alkylene.

9. A compound or salt according to Claim 8 wherein Y is methylene or ethylene.

10. A compound or salt according to Claim 8 wherein Y is methylene.

11 . A compound or salt according to Claim 8 wherein R2 is phenyl.

12. A compound or salt according to Claim 8 wherein R2 is napthyl.

13. A compound or salt according to Claim 8 wherein n is 1.

14. A compound or salt according to Claim 8 wherein n is 2.

15. A compound or salt according to Claim 8 wherein n is 3.

16. A compound or salt according to Claim 8 wherein Ra is methyl.

17. A pharmaceutical composition comprising a compound or salt according to claim 1 and one or more pharmaceutically-acceptable excipient.

18. A method for treating COPD comprising administering a safe and effective amount of a compound or salt according to Claim 1 to a patient in need thereof.