Antiviral heterocyclic compounds

Abstract

Disclosed are compounds of Formula (I) or a pharmaceutically acceptable salt, ester or prodrug thereof, which inhibit human respiratory syncytial virus (HRSV) or human metapneumovirus (HMPV). The present invention further relates to pharmaceutical compositions comprising the above-mentioned compounds for administration to a subject suffering from HRSV or HMPV infection. The present invention also relates to methods of treating a subject for HRSV or HMPV infection by administering a pharmaceutical composition comprising a compound of the present invention.

Description

[0001] Related Applications

[0002] This application claims the benefits of U.S. Provisional Application No. 63 / 154,318, filed February 26, 2021; U.S. Provisional Application No. 63 / 168,705, filed March 31, 2021; U.S. Provisional Application No. 63 / 171,895, filed April 7, 2021; and U.S. Provisional Application No. 63 / 293,339, filed December 23, 2021. The entire teaching of these applications is incorporated herein by reference. Technical Field

[0003] This invention generally relates to compounds and pharmaceutical compositions used as inhibitors of respiratory syncytial virus (RSV) and human metapneumovirus (HMPV). Background Technology

[0004] Human respiratory syncytial virus (HRSV) is a negative-sense virus containing a non-segmented, single-stranded linear RNA genome. As a pneumovirus belonging to two serotypes of the paramyxovirus genus (Pneumoviridae), HRSV contains 10 genes encoding 11 proteins. The nucleocapsid protein (N), RNA polymerase protein (L), phosphoprotein (P), and transcription anti-termination factor (M2-1), together with the RNA genome, form the ribonucleoprotein (RNP) complex. Several small molecule compounds have been shown to target the RNP complex. In addition, the fusion protein (F), which is crucial for viral attachment to the host, has been extensively studied. High-resolution structures of the F protein interacting with inhibitors have been obtained, while structural studies of the N protein are in the early stages of development. A direct result of HRSV protein research is that the F, L, and N proteins have become a major focus of drug development efforts.

[0005] HRSV is a leading cause of acute lower respiratory tract infection (ALRI) in patients of all ages, hence the increasing efforts in HRSV drug development. Besides respiratory infections, high-risk patient groups for HRSV infection include the elderly, immunocompromised individuals, children under two years of age, and patients with chronic obstructive pulmonary disease (COPD) or chronic heart failure (CHF). Over a four-year period, HRSV was found to have caused 177,500 hospitalizations and 14,000 deaths in the elderly population in the United States. It is well known that almost all children will be infected with HRSV in the first three years of life, with HRSV infection being more severe in premature infants. In fact, HRSV is the most common cause of bronchiolitis and pneumonia in infants under one year of age in the United States. It is estimated that approximately 3.2 million children under five years of age worldwide are hospitalized due to HRSV, with 66,000 deaths. HRSV is associated with more infant deaths and hospitalizations in infants under one year of age than influenza.

[0006] HRSV infection can also affect healthy individuals, and reinfection with HRSV can even occur within a two-month timeframe. Symptoms in healthy individuals are similar to those of a cold; however, more severe cases may present with fever, wheezing, shortness of breath and difficulty breathing, as well as cyanosis.

[0007] Currently, treatment options for HRSV infection are quite limited, and there is no vaccine due to the unsuccessful attempts to date. Palizumab, a monoclonal antibody, is approved for prophylactic use, but its use is limited due to its high price. Palizumab is typically used only for high-risk infants, such as premature infants or those with heart / lung disease, but it is only 60% effective in reducing hospitalizations. Ribavirin is approved as an inhaled treatment option, but its efficacy is limited and there are associated safety concerns. Given the treatment options and the consistent seasonality of the HRSV epidemic, there is a need to develop new therapeutic agents for HRSV.

[0008] Several RSV fusion inhibitors have been disclosed in the following publications: WO2010 / 103306, WO 2012 / 068622, WO 2013 / 096681, WO 2014 / 060411, WO 2013 / 186995, WO 2013 / 186334, WO 2013 / 186332, WO 2012080451, WO 2012 / 080450, WO 2012 / 080449, WO 2012 / 080447, WO 2012 / 080446, WO 2015 / 110446, WO Examples of other N protein inhibitors for the treatment of HRSV have been disclosed in the following publications: WO 2004 / 026843, J.Med.Chem.2006,49,2311-2319, and J.Med.Chem.2007,50,1685-1692. Examples of L protein inhibitors of HRSV have been disclosed in the following publications: WO 2011 / 005842, WO 2005 / 042530, Antiviral Res. 2005, 65, 125-131, and Bioorg. Med. Chem. Lett. 2013, 23, 6789-6793. Examples of nucleoside / polymerase inhibitors have been disclosed in the following publications: WO 2011 / 005842, WO 2013 / 242525, WO 2014 / 031784, WO 2015 / 026792, WO 2016 / 0055791, WO 2016 / 138158, and J. Med. Chem. 2015, 58, 1862-1878.

[0009] Similarly, human metapneumovirus (HMPV), a negative-sense single-stranded RNA enveloped virus belonging to the genus Metapneumovirus of the family Pneumovirusidae, discovered by van Den Hoogen in 2001, is also a common cause of acute lower respiratory tract infections (ALTRI). Although usually mild, the virus can be serious and life-threatening in high-risk groups, such as children under 5 years of age, adults over 65 years of age, and adults with underlying medical conditions (such as chronic obstructive pulmonary disease (COPD), asthma, congestive heart failure, or diabetes). In healthy adults over 65 years of age, the annual incidence of HMPV infection is 1.2 / 1000, the incidence of disease (such as COPD) is 38%, and individuals are twice as likely to develop symptomatic illness and require medical care compared to healthy individuals. In immunocompromised individuals, HMPV-related respiratory infections account for 6% of all respiratory infections in lung transplant recipients and 3% of stem cell transplant-related lower respiratory tract infections. HMPV infection is also thought to be associated with acute transplant rejection.

[0010] Similar to HRSV, infection is thought to occur through glycoprotein (G) protein-protein interactions to attach to target cells, followed by fusion via F protein. The HMPV L protein sequence is homologous to the HRSV L protein.

[0011] HMPV infection is the second most common cause of lower respiratory tract infections in children (after HRSV), and it also poses a problem in the elderly. Four HMPV subtypes (A1, A2, B1, and B2) have been identified in clinical isolates. Reinfection can occur throughout childhood after the initial infection. Currently, there are no medications to treat HMPV infection.

[0012] Given the seasonality and predictability of HRSV and HMPV epidemics, the prevalence of HRSV in aged care facilities, and the severity of infection in high-risk infants, there is a clear need for robust and effective treatments for HRSV and HMPV. This invention discovers heterocyclic molecular compounds that are specific against HRSV-A / B and HMPV. This invention includes methods for preparing these molecules, methods for detecting RSV-GFP cells, methods for detecting HMPV-TN / 1501 / A1 cells, and small molecule compounds that have the potential to treat HRSV / HMPV infection. Summary of the Invention

[0013] This invention provides compounds represented by formula (I) and their pharmaceutically acceptable salts, esters, and prodrugs for the treatment or prevention of viral (especially HRSV or HMPV) infections:

[0014]

[0015] in:

[0016] A is selected from:

[0017] 1) The aryl group to be substituted is optional; and

[0018] 2) The substituted heteroaryl group is selected by choice;

[0019] R1 and R2 are each independently selected from:

[0020] 1) Hydrogen;

[0021] 2) Fluorine; and

[0022] 3) Optional substituted -C1-C6 alkyl groups;

[0023] Alternatively, R1 and R2 together with the carbon atoms they are attached to form optional substituted 3- to 6-membered rings;

[0024] Z is selected from:

[0025] 1) Hydrogen;

[0026] 2) Halogens; and

[0027] 3) Optional substituted -C1-C6 alkyl groups;

[0028] W is selected from:

[0029] 1) Hydrogen;

[0030] 2) The -C1-C6 alkoxy group is optionally substituted;

[0031] 3) Optional substituted -C1-C6 alkyl groups; and

[0032] 4) Optional substituted -C3-C6 cycloalkyl groups;

[0033] G is selected from:

[0034] 1)–C(O)OR 12 ;

[0035] 2)–C(O)NR 11 R 12 ;

[0036] 3) Optionally substituted -C1-C6 alkyl-CN;

[0037] 4) Optionally substituted -C1-C6 alkyl-C(O)NR 11 R 12 ;

[0038] 5) Optionally substituted -C1-C6 alkyl-C(O)NR 11 S(O)2R 12 ;

[0039] 6) Optionally substituted -C1-C6 alkyl-OC(O)NR 11 R 12 ;

[0040] 7) Optionally substituted -C1-C6 alkyl-NHR 13 ;

[0041] 8) Optionally substituted -C1-C6 alkyl-NHC(O)R 13 ;

[0042] n is 1, 2, or 3; preferably n is 1 or 2.

[0043] Y is O, S(O)2, or NR 14 ;

[0044] E is selected from:

[0045] 1) The aryl group to be substituted is chosen aryl;

[0046] 2) The substituted heteroaryl group is selected by choice;

[0047] 3) Selectively replace 3- to 8-membered heterocycles, and

[0048] 4) The substituted alkynyl group can be selected arbitrarily;

[0049] R3 is a hydroxyl group or fluorine;

[0050] R4 is selected from:

[0051] 1) Hydrogen;

[0052] 2) Optional substituted -C1-C6 alkyl groups;

[0053] 3) Optional substituted -C3-C8 cycloalkyl groups; and

[0054] 4) Any 3- to 8-membered heterocyclic rings may be substituted;

[0055] R 11 Selected independently each time it appears:

[0056] 1) Hydrogen;

[0057] 2) Optional substituted -C1-C8 alkyl groups;

[0058] 3) Optional substituted -C3-C8 cycloalkyl groups;

[0059] 4) Any 4- to 8-membered heterocycles may be substituted;

[0060] 5) The aryl group to be substituted is optional;

[0061] 6) The substituted aryl alkyl group may be selected;

[0062] 7) The substituted heteroaryl group is selected by choice; and

[0063] 8) Optional substituted heteroaryl alkyl groups;

[0064] R 12 Selected independently each time it appears:

[0065] 1) Hydrogen;

[0066] 2) Optional substituted -C1-C8 alkyl groups;

[0067] 3) Optional substituted -C3-C8 cycloalkyl groups;

[0068] 4) Any 4- to 8-membered heterocycles may be substituted;

[0069] 5) The aryl group to be substituted is optional;

[0070] 6) The substituted aryl alkyl group may be selected;

[0071] 7) The substituted heteroaryl group is selected by choice; and

[0072] 8) Optional substituted heteroaryl alkyl groups;

[0073] Or, R 11 and R 12 Together with the nitrogen atoms to which they are attached, they form 3- to 12-membered heterocycles, preferably, but not limited to, morpholino, piperidino, piperazine, pyrrolidinyl, and azetidine.

[0074] R 13 Selected independently each time it appears:

[0075] 1) Optional substituted -C1-C8 alkyl groups;

[0076] 2) Optional substituted -C3-C8 cycloalkyl groups;

[0077] 3) Any 4- to 8-membered heterocyclic rings may be substituted;

[0078] 4) The aryl group to be substituted is optional;

[0079] 5) The substituted aryl alkyl group may be selected;

[0080] 6) Optional substituted heteroaryl groups; and

[0081] 7) The substituted heteroaryl alkyl group is optionally used; and

[0082] R 14 Selected from:

[0083] 1) Hydrogen;

[0084] 2) Optional substituted -C1-C8 alkyl groups; and

[0085] 3) Optional substituted -C3-C8 cycloalkyl groups;

[0086] Each of the above preferred groups may be combined with one, any or all other preferred groups. Detailed Implementation

[0087] In one embodiment of the invention, a compound of formula (I) as described above, or a pharmaceutically acceptable salt thereof, is provided.

[0088] In some embodiments of the compound of formula (I), Y is O.

[0089] In some embodiments of the compound of formula (I), Y is 0 and n is 1 or 2.

[0090] In some embodiments of the compound of formula (I), R1 is hydrogen or F.

[0091] In some embodiments of the compound of formula (I), R2 is hydrogen or F.

[0092] In some embodiments of the compound of formula (I), Z is hydrogen, Cl or F.

[0093] In some embodiments of the compound of formula (I), R1 is hydrogen, R2 is hydrogen, and Z is hydrogen.

[0094] In some embodiments of the compound of formula (I), W is an optionally substituted methyl, optionally substituted ethyl, or optionally substituted cyclopropyl.

[0095] In some embodiments of the compound of formula (I), W is cyclopropyl, ethyl, -CH3, -CH2F, -CHF2 or -CF3.

[0096] In some embodiments of the compound of formula (I), R3 is -OH.

[0097] In some embodiments of the compound of formula (I), R4 is an optionally substituted methyl group or an optionally substituted cyclopropyl group.

[0098] In some embodiments of the compound of formula (I), R3 is OH and R4 is CF3 or cyclopropyl.

[0099] In some embodiments of the compound of formula (I), R1 is hydrogen, R2 is hydrogen, R3 is OH, and R4 is CF3.

[0100] In some embodiments of the compound of formula (I), G is –C(O)NR 11 R 12In some implementations, G is –C(O)NH2.

[0101] In some embodiments of the compound of formula (I), G is –CH2NHR 13 –CH2C(O)NR 11 R 12 –CH2NHC(O)R 13 –CH2OC(O)NR 11 R 12 –CH2CN or –CH2C(O)NR 11 S(O)2R 12 In some implementations, G is -CH2C(O)NH2.

[0102] In some embodiments of the compound of formula (I), A is selected from one of the following structures after removing a hydrogen atom:

[0103]

[0104] Each of these groups is optionally substituted.

[0105] In some embodiments of the compound of formula (I), A is selected from the following groups:

[0106]

[0107] Each of these groups is optionally substituted.

[0108] In some embodiments of the compound of formula (I), A is selected from the following groups:

[0109]

[0110] In some embodiments of the compound of formula (I), A is Where Ra represents hydrogen, halogen, -CN, -NO2, or -OR. 11 -NR 11 R 12 -NR 11 C(O)R 12 -NR 11 S(O)2R 12 -S(O)2R 12 -S(O)2NR 11 R 12 -NR 11 C(O)NR 11 R 12 -C(O)R 11 -C(O)OR 11 -C(O)NR 11 R12 Optionally substituted -C1-C6 alkyl, optionally substituted -C3-C8 cycloalkyl, optionally substituted 3- to 8-membered heterocyclic group, optionally substituted aryl or optionally substituted heteroaryl; Rb and Rb' are each independently selected from hydrogen, halogen, -OR 11 -NR 11 R 12 Optionally substituted -C1-C6 alkyl, optionally substituted -C3-C8 cycloalkyl, optionally substituted 3- to 8-membered heterocyclic groups, optionally substituted aryl and optionally substituted heteroaryl groups. Alternatively, Rb and Rb' together with the carbon atoms to which they are attached form 4- to 7-membered rings fused with the benzene ring.

[0111] In some embodiments of the compound of formula (I), E is an optionally substituted aryl group, preferably an optionally substituted phenyl group.

[0112] In some embodiments of the compound of formula (I), E is selected from one of the following structures after removing a hydrogen atom:

[0113]

[0114] Each of these groups is optionally substituted.

[0115] In some embodiments of the compound of formula (I), E is selected from the following groups:

[0116]

[0117] In some embodiments of the compound of formula (I), E is selected from the following groups:

[0118]

[0119] In one embodiment of the invention, the compound of formula (I) is represented by formula (Ia) or formula (Ib), or is a pharmaceutically acceptable salt, ester, or prodrug thereof:

[0120]

[0121] A, R1, R2, Z, W, G, n, Y, E, R3, and R4 are as defined above.

[0122] In a preferred embodiment, the compound of formula (I) has the stereochemical structure shown in formula (Ib).

[0123] In one embodiment of the invention, the compound of formula (I) is represented by formula (II), or is a pharmaceutically acceptable salt, ester, or prodrug thereof:

[0124]

[0125] A, R1, R2, W, G, n, E, R3, and R4 are defined as previously.

[0126] In one embodiment of the invention, the compound of formula (I) is represented by formula (III), or is a pharmaceutically acceptable salt, ester, or prodrug thereof:

[0127]

[0128] A, W, G, n, Y, E, R3, and R4 are defined as previously.

[0129] In one embodiment of the invention, the compound of formula (I) is represented by one of formulas (IV-1) to (IV-2), or by its pharmaceutically acceptable salt, ester, or prodrug:

[0130]

[0131] A, W, G, Y, E, R3, and R4 are defined as previously.

[0132] In one embodiment of the invention, the compound of formula (I) is represented by one of formulas (V-1) to (V-4), or by its pharmaceutically acceptable salt, ester, or prodrug:

[0133]

[0134] Among them, A, W, G, E, R 14 R3 and R4 are as defined above.

[0135] In one embodiment of the invention, the compound of formula (I) is represented by one of formulas (VI-1) to (VI-4), or by its pharmaceutically acceptable salt, ester, or prodrug:

[0136]

[0137] Among them, A, W, G, E, R 14 R3 and R4 are as defined above. Preferably, W is an optionally substituted methyl group; more preferably, W is -CH3 or -CF3.

[0138] In one embodiment of the invention, the compound of formula (I) is represented by one of formulas (VII-1) to (VII-12), or by its pharmaceutically acceptable salt, ester, or prodrug:

[0139]

[0140] Among them, A, W, E, R 11 R 12 R 13 R 14R3 and R4 are as defined above. Preferably, W is cyclopropyl, ethyl, -CH3, -CH2F, -CHF2 or -CF3; more preferably, W is -CH3 or -CF3.

[0141] In one embodiment of the invention, the compound of formula (I) is represented by one of formulas (VIII-1) to (VIII-12), or by its pharmaceutically acceptable salt, ester, or prodrug:

[0142]

[0143]

[0144] Among them, A, W, E, R 11 R 12 R 13 R 14 R3 and R4 are as defined above. Preferably, W is cyclopropyl, ethyl, -CH3, -CH2F, -CHF2 or -CF3; more preferably, W is -CH3 or -CF3.

[0145] In one embodiment of the invention, the compound of formula (I) is represented by one of formulas (IX-1) to (IX-4), or by its pharmaceutically acceptable salt, ester, or prodrug:

[0146]

[0147] Each R 21 Independently, the substituted methyl group, halogen, -CN, or -OR can be substituted. 11 or -NR 11 R 12 m is 0, 1, 2, 3, 4, or 5; A, W, G, R 11 R 12 R 14 R1, R2, and R3 are as defined above. Preferably, each R1... 21 Independently, it is a halogen or optionally substituted methyl group, and m is 1 or 2. More preferably, each R 21 It can be independently -F, -Cl, -CN, -CF3, -CH2F or -CHF2, and m is 1 or 2.

[0148] In one embodiment of the invention, the compound of formula (I) is represented by one of formulas (X-1) to (X-4), or by its pharmaceutically acceptable salt, ester, or prodrug:

[0149]

[0150] Where R 21 m, A, W, G, R 14R1, R2, and R3 are as defined above. Preferably, each R1... 21 The methyl group is halogen or optionally substituted, and m is 1 or 2.

[0151] In one embodiment of the invention, the compound of formula (I) is represented by one of formulas (XII-1) to (XII-12), or by its pharmaceutically acceptable salt, ester, or prodrug:

[0152]

[0153] Where m' is 0, 1, or 2. A, R3, R4, R 21 R 11 R 12 and R 13 As defined above. m' is preferably 2.

[0154] In one embodiment of the invention, the compound of formula (I) is represented by one of formulas (XIII-1) to (XIII-12), or by its pharmaceutically acceptable salt, ester, or prodrug:

[0155]

[0156] Where R 21 m' A R3 R4 R 11 R 12 and R 13 As defined above. m' is preferably 2.

[0157] In one embodiment of the invention, the compound of formula (I) is represented by one of formulas (XIV-1) to (XIV-6), or by a pharmaceutically acceptable salt, ester, or prodrug thereof:

[0158]

[0159] Among them, each R 22 It is a halogen on its own; -CN; -NO2; -OR 11 ;-NR 11 R 12 ;-NR 11 C(O)R 12 ;-NR 11 S(O)2R 12 ;-S(O)2R 12 ;-S(O)2NR 11 R 12 ;-NR 11 C(O)NR 11 R 12 ;-C(O)R 11 ;-C(O)OR 11 ;-C(O)NR11 R 12 ; optionally substituted -C1-C6 alkyl; optionally substituted -C3-C8 cycloalkyl; optionally substituted 3- to 8-membered heterocycle; optionally substituted aryl; or optionally substituted heteroaryl, and W, m', R3, R4, R 21 R 11 and R 12 As defined above. Preferably, R 21 It is a halogen, R3 is -OH, R4 is -CH3, -CF3, or cyclopropyl, and W is cyclopropyl, ethyl, -CH3, -CH2F, -CHF2, or -CF3. In some embodiments, two adjacent R... 22 The groups, together with the carbon atoms to which they are attached, form 4- to 12-membered carbon rings or heterocycles, which are fused with phenyl or quinolinyl groups.

[0160] In one embodiment of the invention, the compound of formula (I) is represented by one of formulas (XV-1) to (XV-6), or by its pharmaceutically acceptable salt, ester, or prodrug:

[0161]

[0162] Among them, W, m', R3, R4, R 21 R 22 R 11 and R 12 As defined above. Preferably, R 21 It is a halogen, R3 is -OH, R4 is -CH3, -CF3, or cyclopropyl, and W is cyclopropyl, ethyl, -CH3, -CH2F, -CHF2, or -CF3. In some embodiments, two adjacent R... 22 The groups, together with the carbon atoms to which they are attached, form 4- to 12-membered carbon rings or heterocycles, which are fused with phenyl or quinolinyl groups.

[0163] In one embodiment of the invention, the compound of formula (I) is represented by one of formulas (XVI-1) to (XVI-12), or by its pharmaceutically acceptable salt, ester, or prodrug:

[0164]

[0165] Among them, R 23 It is hydrogen, halogen, -CN, -NO2, -OR 11 -NR 11 R 12 -NR 11 C(O)R 12 -NR 11 S(O)2R 12-S(O)2R 12 -S(O)2NR 11 R 12 -NR 11 C(O)NR 11 R 12 -C(O)R 11 -C(O)OR 11 -C(O)NR 11 R 12 Optionally substituted -C1-C6 alkyl, optionally substituted -C3-C8 cycloalkyl, optionally substituted 3- to 8-membered heterocycle, optionally substituted aryl or optionally substituted heteroaryl; and W, R 21 R 22 、m'、R3、R4、R 11 and R 12 As defined above. Preferably, R 21 It is a halogen, R3 is -OH, R4 is -CH3, -CF3 or cyclopropyl, and W is cyclopropyl, ethyl, -CH3, -CH2F, -CHF2 or -CF3.

[0166] In one embodiment of the invention, the compound of formula (I) is represented by one of formulas (XVII-1) to (XVII-12), or by its pharmaceutically acceptable salt, ester, or prodrug:

[0167]

[0168] Among them, W and R 21 R 22 R 23 、m'、R3、R4、R 11 and R 12 As defined above. Preferably, R 21 It is a halogen, R3 is -OH, R4 is -CH3, -CF3 or cyclopropyl, and W is cyclopropyl, ethyl, -CH3, -CH2F, -CHF2 or -CF3.

[0169] In one embodiment of the invention, the compound of formula (I) is represented by one of formulas (XVIII-1) to (XVIII-14), or a pharmaceutically acceptable salt, ester, or prodrug thereof:

[0170]

[0171] Where R 14 R 21 and R 22 As defined above.

[0172] In one embodiment of the invention, the compound of formula (I) is represented by one of formulas (XIX-1) to (XIX-14), or by its pharmaceutically acceptable salt, ester, or prodrug:

[0173]

[0174] Where R 14 R 21 and R 22 As defined above.

[0175] In one embodiment of the invention, the compound of formula (I) is represented by one of formulas (XX-1) to (XX-20), or as a pharmaceutically acceptable salt, ester, or prodrug thereof:

[0176]

[0177]

[0178] Each R 31 Independently, it is either a substituted -C1-C3 alkyl group or a halogen; R 32 Independently, it is halogen, -OR 11 -NR 11 R 12 -NR 11 C(O)R 12 -C(O)NR 11 R 12 -C(O)R 11 Optionally substituted -C1-C6 alkyl or optionally substituted -C3-C8 cycloalkyl; and R 11 R 12 and R 14 As defined above. Preferably, R 31 It's halogen, R 32 It is a halogen, -NH2, optionally substituted methyl group, optionally substituted cyclopropyl group, R 14 It is the cyclic propyl group that is optionally substituted.

[0179] In one embodiment of the invention, the compound of formula (I) is represented by one of formulas (XXI-1) to (XXI-20), or by a pharmaceutically acceptable salt, ester, or prodrug thereof:

[0180]

[0181]

[0182] Where R 31 R 32 R 11 R 12 and R14 As defined above. Preferably, R 31 It's halogen, R 32 It is a halogen, -NH2, optionally substituted methyl group, optionally substituted cyclopropyl group, R 14 It is the cyclic propyl group that is optionally substituted.

[0183] In one embodiment of the invention, the compound of formula (I) is represented by one of formulas (XXII-1) to (XXII-14), or by a pharmaceutically acceptable salt, ester, or prodrug thereof:

[0184]

[0185] Where R 24 Is it hydrogen or R? 22 W, R4, R 14 R 21 and R 22 As defined above. Preferably, R4 is an optionally substituted -C3-C6 cycloalkyl group. More preferably, R4 is an optionally substituted cyclopropyl group or an optionally substituted cyclobutyl group.

[0186] In one embodiment of the invention, the compound of formula (I) is represented by one of formulas (XXIII-1) to (XXIII-14), or by a pharmaceutically acceptable salt, ester, or prodrug thereof:

[0187]

[0188] Among them, W, R4, R 14 R 21 R 22 and R 24 As defined above. Preferably, R4 is an optionally substituted -C3-C6 cycloalkyl group. More preferably, R4 is an optionally substituted cyclopropyl or optionally substituted cyclobutyl group, and W is an optionally substituted methyl group.

[0189] In one embodiment of the invention, the compound of formula (I) is represented by formula (XXIV), or is a pharmaceutically acceptable salt, ester, or prodrug thereof:

[0190]

[0191] A, E, R3, and R4 are defined as previously.

[0192] In one embodiment of the invention, the compound of formula (I) is represented by formula (XXIV), or by its pharmaceutically acceptable salt, ester or prodrug, wherein R3 is -OH; and R4 is optionally substituted methyl or optionally substituted -C3-C6 cycloalkyl.

[0193] A is selected from:

[0194]

[0195] E is selected from:

[0196]

[0197] Or E is selected from

[0198]

[0199] In one embodiment of the invention, the compound of formula (I) is represented by formula (XXV), or is a pharmaceutically acceptable salt, ester, or prodrug thereof:

[0200]

[0201] Where R 41 R 42 R 43 R 44 and R 45 Each of these is independently selected from hydrogen, halogen, optionally substituted methyl, optionally substituted methoxy, or -CN. R3, R4, R 14 and R 24 As defined above. Preferably, R3 is -OH; R4 is an optionally substituted methyl group or an optionally substituted -C3-C6 cycloalkyl group; R 14 It is an optional substituted -C3-C6 cycloalkyl group or an optional substituted methyl group; R 24 It can be either a methoxy group or a halogen that is substituted.

[0202] In one embodiment of the invention, a compound of formula (XXV) or a pharmaceutically acceptable salt, ester, or prodrug thereof, wherein R... 42 and R 43 Together with the carbon atoms they are attached to, they form fused 4- to 7-membered carbon rings or heterocycles.

[0203] In one embodiment of the invention, a compound of formula (XXV) or a pharmaceutically acceptable salt, ester, or prodrug thereof, wherein R... 41 and R 42 Together with the carbon atoms they are attached to, they form fused 4- to 7-membered carbon rings or heterocycles.

[0204] In one embodiment of the invention, the compound of formula (I) is represented by one of formulas (XXVI-1) to (XXVI-3), or by its pharmaceutically acceptable salt, ester, or prodrug:

[0205]

[0206] Among them, R3, R 14 R 24R 41 R 42 R 43 R 44 and R 45 As defined above.

[0207] In one embodiment of the invention, the compound of formula (I) is represented by formula (XXVII), or is a pharmaceutically acceptable salt, ester, or prodrug thereof:

[0208]

[0209] Where R 14 R 24 R 41 R 42 R 43 R 44 and R 45 As defined above.

[0210] In one embodiment of the invention, the compound of formula (I) is represented by formula (XXVIII), or is a pharmaceutically acceptable salt, ester, or prodrug thereof:

[0211]

[0212] Where R 14 R 24 R 41 R 42 R 43 R 44 and R 45 As defined above.

[0213] In one embodiment of the invention, a compound of formula (XXVII) or formula (XXVIII) or a pharmaceutically acceptable salt, ester, or prodrug thereof, wherein R 42 and R 43 Together with the carbon atoms they are attached to, they form fused 4- to 7-membered carbon rings or heterocycles.

[0214] In one embodiment of the invention, a compound of formula (XXVII) or formula (XXVIII) or a pharmaceutically acceptable salt, ester, or prodrug thereof, wherein R 43 and R 44 Together with the carbon atoms they are attached to, they form fused 4- to 7-membered carbon rings or heterocycles.

[0215] In one embodiment of the invention, the compound of formula (I) is represented by one of formulas (XXIV), (XXV), (XXVI-1) to (XXVI-3), (XXVII) or (XXVIII), or a pharmaceutically acceptable salt, ester or prodrug thereof, wherein R 14It is an optional substituted -C3-C6 cycloalkyl group or an optional substituted methyl group; R 24 It is either a substituted methoxy group or a halogen; R 41 R 42 R 43 R 44 and R 45 Each of them is independently selected from hydrogen, -F, -Cl, -CH3, -CF3, -OCH3, or -OCF3.

[0216] In one embodiment of the invention, the compound of formula (I) is represented by formula (XXIV), formula (XXV), formula (XXVI-1) to formula (XXVI-3), formula (XXVII) or formula (XXVIII), or a pharmaceutically acceptable salt, ester or prodrug thereof, wherein R 14 It is -CHF2, R 24 It is -F, -Cl, or -OCH3; Selected from:

[0217]

[0218] Selected from:

[0219]

[0220] In one embodiment of the invention, the compound of formula (I) is represented by formulas (XXIX-1) to (XXIX-3), or is a pharmaceutically acceptable salt, ester, or prodrug thereof:

[0221]

[0222] Where R 46 It is a substituted methyl group or an optionally substituted cyclopropyl group; R 47 It is hydrogen, Cl, F, or an optional substituted methoxy group, R 41 R 42 R 43 R 44 and R 45 As defined above. In certain embodiments of compounds of formula (XXIX-1), (XXIX-2), and (XXIX-3), R 46 It is cyclopropyl, 1-fluorocyclopropyl, or difluoromethyl.

[0223] In one embodiment of the invention, a compound of one of formulas (XXIX-1) to (XXIX-3) or a pharmaceutically acceptable salt, ester, or prodrug thereof, wherein R 42 and R 43 Together with the carbon atoms they are attached to, they form fused 4- to 7-membered carbon rings or heterocycles.

[0224] In one embodiment of the invention, a compound of one of formulas (XXIX-1) to (XXIX-3) or a pharmaceutically acceptable salt, ester, or prodrug thereof, wherein R 43 and R 44 Together with the carbon atoms they are attached to, they form fused 4- to 7-membered carbon rings or heterocycles.

[0225] It is understood that the description of this invention should be interpreted in accordance with the rules and principles of chemical bonding. In some cases, it may be necessary to remove a hydrogen atom to accommodate a substituent at any given position.

[0226] The design aims to define any substituent or variable (e.g., R1, R2, etc.) at a specific position in the molecule independently of its definition at other positions in the molecule.

[0227] It is also understood that the compounds of the present invention may contain one or more asymmetric carbon atoms and may exist in racemic, diastereomeric, and optically active forms. It is also understood that some compounds of the present invention may exist in different tautomer forms. All tautomers are considered within the scope of the present invention.

[0228] In some embodiments, the present invention provides a method for preventing or treating RSV activity and treating RSV infection in a subject in need. The method comprises administering a therapeutically effective amount of a compound of formula (I) to the subject.

[0229] The present invention also provides the use of the compound of formula (I) in the preparation of a medicament for the prevention or treatment of RSV.

[0230] Therefore, in one embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is combined with a steroidal anti-inflammatory compound such as budesonide or fluticasone. In a preferred embodiment, the steroid is administered in a low dose to minimize immunosuppressive effects. In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is combined with a nonsteroidal anti-inflammatory compound, a phosphodiesterase 4 inhibitor, a TNFα inhibitor, or NSAIDs. Nonsteroidal anti-inflammatory compounds include leukotriene antagonists such as montelukast (Merck) or Astra Zeneca, phosphodiesterase 4 inhibitors such as roflumilast (Altana), and TNFα inhibitors such as Enbrel (Amgen), Remicade (Centocor), Humira (Abbott), or CDP870 (Celltech). In another embodiment, the compound of formula (I) is combined with an interleukin-8 inhibitor or an interleukin-9 inhibitor. Therefore, the present invention also relates to products containing a compound of formula (I) or a pharmaceutically acceptable salt thereof and an anti-inflammatory compound for simultaneous, separate, or sequential treatment of RSV.

[0231] This invention also relates to combinations of compounds of formula (I) or pharmaceutically acceptable salts thereof with anti-influenza compounds, and the use of such combinations in the treatment of RSV and influenza co-infections. Therefore, this invention also relates to a product containing a compound of formula (I) or a pharmaceutically acceptable salt thereof and an anti-influenza compound for simultaneous, separate, or sequential treatment of RSV and influenza co-infections. The compounds of this invention can be administered in various dosage forms. Therefore, they can be administered orally, for example as tablets, lozenges, tablets, aqueous or oily suspensions, dispersible powders, or granules. The compounds of this invention can also be administered subcutaneously, intravenously, intramuscularly, intrasternally, transdermally, or parenterally via infusion techniques. The compounds can also be administered as suppositories.

[0232] In one embodiment, the compounds of the present invention are administered via intranasal or intrabronchial administration. The present invention also provides inhalers or nebulizers containing a drug comprising (a) a derivative of formula (I) as defined above or a pharmaceutically acceptable salt thereof, and (b) a pharmaceutically acceptable carrier or diluent.

[0233] The present invention also provides a pharmaceutical composition containing this benzodiazepine. Derivatives or their pharmaceutically acceptable salts, and pharmaceutically acceptable carriers or diluents.

[0234] The compounds of the present invention are typically formulated for administration with pharmaceutically acceptable carriers or diluents. For example, solid oral dosage forms may contain the active compound along with diluents, lubricants, binders, disintegrants, effervescent mixtures, dyes, sweeteners, wetting agents, and non-toxic and pharmacologically inactive substances commonly used in pharmaceutical formulations, wherein: diluents, such as lactose, glucose, sucrose, cellulose, corn starch, or potato starch; lubricants, such as silica, talc, stearic acid, magnesium stearate, or calcium stearate and / or polyethylene glycol; binders, such as starch, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, or polyvinylpyrrolidone; disintegrants, such as starch, alginate, alginate, or sodium starch glycolate; and wetting agents, such as lecithin, polysorbate, or dodecyl sulfate. Such pharmaceutical formulations can be prepared in known ways, for example by mixing, granulation, tableting, sugar coating, or film coating processes.

[0235] Orally administered liquid dispersions can be syrups, emulsions, and suspensions. Syrups may contain sucrose as a carrier, or sucrose with glycerol and / or mannitol and / or sorbitol.

[0236] Suspensions and emulsions may contain carriers such as natural gums, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. Suspensions or solutions for intramuscular injection may contain the active compound along with a pharmaceutically acceptable carrier, such as sterile water, olive oil, ethyl oleate, glycols such as propylene glycol, and, if necessary, an appropriate amount of lidocaine hydrochloride.

[0237] Solutions for injection or infusion may contain, for example, sterile water as a carrier, or preferably, they may be in the form of sterile, aqueous, isotonic saline solutions.

[0238] This invention also relates to novel compounds as defined above; or pharmaceutically acceptable salts thereof, for use in methods of treating human or animal bodies. This invention also relates to pharmaceutical compositions comprising novel compounds as defined above and pharmaceutically acceptable diluents or carriers. Preferably, the pharmaceutical composition comprises a pharmaceutically acceptable salt of the novel compound as defined above. Pharmaceutically acceptable salts are as defined above. The novel compounds of this invention are generally administered in the manner defined above, and said compounds are generally formulated for administration in the manner defined above.

[0239] Preferably, the pharmaceutical composition comprises an optically active isomer of the novel compound of the present invention. Thus, for example, preferred novel compounds of the present invention containing only one chiral center include substantially pure R enantiomers, substantially pure S enantiomers, and enantiomer mixtures containing excess R or excess S enantiomers. Particularly preferred are pharmaceutical compositions comprising substantially pure optically active isomers of the compound of the present invention. To avoid confusion, the novel compounds of the present invention may be used in solvate form if desired.

[0240] Another aspect of the invention is a method for preparing any of the compounds described herein using any of the synthetic methods described herein.

[0241] Definitions

[0242] The following lists the definitions of various terms used to describe the invention. These definitions apply to terms used throughout this specification and claims, unless otherwise specified, individually or as part of a larger group.

[0243] As used herein, the term "aryl" refers to a monocyclic, bicyclic, or polycyclic carbocyclic system containing at least one aromatic ring, including but not limited to phenyl, naphthyl, tetrahydronaphthyl, indenyl, and indenyl. Polycyclic aryl is a polycyclic ring system containing at least one aromatic ring. Polycyclic aryl can include fused rings, covalently linked rings, or combinations thereof.

[0244] As used herein, the term "heteroaryl" refers to a monocyclic, bicyclic, or polycyclic aromatic group having one or more ring atoms selected from S, O, and N; the remaining ring atoms are carbon, and any N or S contained within the ring may optionally be oxidized. Heteroaryls include, but are not limited to, pyridyl, pyrazinyl, pyrimidinyl, pyrroleyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, and quinoxalinyl. Polycyclic heteroaryls may comprise fused rings, covalently linked rings, or combinations thereof.

[0245] According to the present invention, the aromatic groups may be substituted or unsubstituted.

[0246] The term "bicyclic aryl" or "bicyclic heteroaryl" refers to a ring system consisting of two rings, at least one of which is aromatic; and the two rings may be fused or covalently linked.

[0247] As used in this article, the term "alkyl" refers to a saturated straight-chain or branched hydrocarbon group. "C1-C3 alkyl", "C1-C6 alkyl", "C1-C6 alkyl", "C1-C6 alkyl" are also used interchangeably. 10 "alkyl", "C2-C4 alkyl" or "C3-C6 alkyl" refers to an alkyl group containing one to three, one to six, one to ten, two to four, and three to six carbon atoms, respectively. Examples of C1-C8 alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, neopentyl, n-hexyl, heptyl, and octyl groups.

[0248] As used herein, the term "alkenyl" refers to a straight-chain or branched hydrocarbon group that has at least one carbon-carbon double bond by removing a single hydrogen atom. "C2-C" 10 "Alkenyl", "C2-C8 alkenyl", "C2-C4 alkenyl" or "C3-C6 alkenyl" refer to alkenyl groups containing two to ten, two to eight, two to four, or three to six carbon atoms, respectively. Alkenyl groups include, but are not limited to, vinyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, heptenyl, octenyl, etc.

[0249] As used herein, the term "alkynyl" refers to a straight-chain or branched hydrocarbon group that has at least one carbon-carbon triple bond by removing a single hydrogen atom. "C2-C" 10 "Alynyl", "C2-C8 alkynyl", "C2-C4 alkynyl" or "C3-C6 alkynyl" refer to alkynyl groups containing two to ten, two to eight, two to four or three to six carbon atoms, respectively. Representative alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 1-butynyl, heptynyl, octyynyl, etc.

[0250] As used herein, the term "cycloalkyl" refers to a monocyclic or polycyclic saturated carbide ring or a fused, bridged, or helical bicyclic or tricyclic group, wherein the carbon atom may optionally be substituted with an oxo or an exocyclic alkene, imino, or oxime double bond. Preferred cycloalkyl groups include C3-C6 groups. 12 Cycloalkyl, C3-C6 cycloalkyl, C3-C8 cycloalkyl, and C4-C7 cycloalkyl. C3-C 12 Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentyl, cyclooctyl, 4-methylene-cyclohexyl, bicyclo[2.2.1]heptyl, bicyclo[3.1.0]hexyl, spiro[2.5]octyl, 3-methylenebicyclo[3.2.1]octyl, spiro[4.4]nonyl, etc.

[0251] As used herein, the term "cycloalkenyl" refers to a bicyclic or tricyclic group having at least one carbon-carbon double bond, comprising a monocyclic or polycyclic carbocyclic or fused, bridged, or spirocyclic system, wherein the carbon atom may optionally be substituted with an oxo or an exocyclic alkene, imino, or oxime double bond. Preferred cycloalkenyl groups include C3-C... 12 Cycloalkenyl, C3-C8 cycloalkenyl, or C5-C7 cycloalkenyl. C3-C 12 Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, bicyclo[2.2.1]hept-2-enyl, bicyclo[3.1.0]hex-2-enyl, spiro[2.5]oct-4-enyl, spiro[4.4]non-1-enyl, bicyclo[4.2.1]non-3-en-9-yl, etc.

[0252] As used herein, the term "arylalkyl" refers to a functional group in which an alkylene chain is attached to an aryl group, such as -CH2CH2-phenyl. The term "substituted arylalkyl" refers to an arylalkyl functional group in which an aryl group is substituted. Similarly, the term "heteroarylalkyl" refers to a functional group in which an alkylene chain is attached to a heteroaryl group. The term "substituted heteroarylalkyl" refers to a heteroarylalkyl functional group in which a heteroaryl group is substituted.

[0253] As used herein, unless otherwise stated, the term "alkoxy" alone or in combination with other terms refers to an alkyl group having a specified number of carbon atoms bonded to the remainder of the molecule by an oxygen atom, such as, for example, methoxy, ethoxy, 1-propoxy, 2-propoxy (isopropoxy), and higher homologues and isomers. Preferred alkoxy groups are (C1-C3) alkoxy groups.

[0254] It should be understood that any alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, and cycloalkenyl moieties described herein may also be aliphatic or alicyclic groups.

[0255] An "aliphatic" group is a non-aromatic portion consisting of any combination of carbon atoms, hydrogen atoms, halogen atoms, oxygen, nitrogen, or other atoms, and optionally contains one or more unsaturated units, such as double and / or triple bonds. Examples of aliphatic groups are functional groups such as alkyl, alkenyl, alkynyl, O, OH, NH, NH2, C(O), S(O)2, C(O)O, C(O)NH, OC(O)O, OC(O)NH, OC(O)NH2, S(O)2NH, S(O)2NH2, NHC(O)NH2, NHC(O)C(O)NH, NHS(O)2NH, NHS(O)2NH2, C(O)NHS(O)2, C(O)NHS(O)2NH or C(O)NHS(O)2NH2, etc., groups including one or more functional groups, non-aromatic hydrocarbons (optionally substituted), and groups in which one or more carbons of the (optionally substituted) non-aromatic hydrocarbon are substituted by functional groups. The carbon atoms of the aliphatic group may optionally be substituted with oxygen. The aliphatic group may be straight-chain, branched, cyclic, or a combination thereof, and preferably contains about 1 to about 24 carbon atoms, more typically about 1 to about 12 carbon atoms. In addition to aliphatic hydrocarbon groups, as used herein, aliphatic groups explicitly include, for example, alkoxyalkyl, polyalkoxyalkyl, such as polyalkylene glycols, polyamines, and polyimides. The aliphatic group may optionally be substituted.

[0256] The term "carbocyclic" or "carbocyclic" refers to a saturated, partially unsaturated, or aromatic cyclic group in which each atom within the ring is carbon. Examples of carbocyclic groups include cycloalkyl, cycloalkenyl, and aryl groups.

[0257] The terms “heterocyclic” or “heterocyclic alkyl” are used interchangeably and refer to a bicyclic or tricyclic group that is not an aromatic ring or a fused, bridged or spirocyclic system, wherein (i) each ring system contains at least one heteroatom independently selected from oxygen, sulfur and nitrogen, (ii) each ring system may be saturated or unsaturated, (iii) the nitrogen heteroatom and sulfur heteroatom may optionally be oxidized, (iv) the nitrogen heteroatom may optionally be quaternized, (v) any of the above rings may be fused to an aromatic ring, and (vi) the remaining ring atom is a carbon atom that may optionally be oxidized or optionally substituted with an exocyclic alkene, imino or oxime double bond. Representative heterocyclic alkyl groups include, but are not limited to, 1,3-dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolinyl, imidazolinyl, imidazolinyl, piperidinyl, piperazinyl, oxazolinyl, isoxazolinyl, morpholinyl, thiazolinyl, isothiazolinyl, quinoxolinyl, pyridazinyl, 2-azabicyclo[2.2.1]heptyl, 8-azabicyclo[3.2.1]octyl, 5-azaspiro[2.5]octyl, 1-oxa-7-azaspiro[4.4]nonyl, 7-oxooxetane-4-yl, and tetrahydrofuranyl. These heterocyclic groups may be further substituted. Heteroaryl or heterocyclic groups may be C-linked or N-linked (if possible).

[0258] It should be understood that any alkyl, alkenyl, alkynyl, alicyclic, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclic, aliphatic moiety, etc., described herein, when used as a linker connecting two or more groups or substituents (which may be located on the same or different atoms), may also be divalent or polyvalent groups. Those skilled in the art can readily determine the valence of any such group from the context in which it appears.

[0259] The term "substituted" refers to substitution achieved by independently replacing one, two, three, or more hydrogen atoms with a substituent, including but not limited to -F, -Cl, -Br, -I, -OH, and -Cl-C. 12 Alkyl; -C2-C 12 alkenyl, -C2-C 12 alkynyl group, -C3-C 12 Cycloalkyl, protected hydroxyl, -NO2, -N3, -CN, -NH2, protected amino, oxo, thio, -NH-Cl-C 12 Alkyl, -NH-C2-C8 alkenyl, -NH-C2-C8 ynyl, -NH-C3-C 12 Cycloalkyl, -NH-aryl, -NH-heteroaryl, -NH-heterocycloalkyl, dialkylamino, diarylamino, diheteroarylamino, -O-Cl-C 12 Alkyl, -O-C2-C8 alkenyl, -O-C2-C8 ynyl, -O-C3-C 12 Cycloalkyl, -O-aryl, -O-heteroaryl, -O-heterocycloalkyl, -C(O)–C2-C8 alkenyl, -C(O)-C2-C8 ynyl, -C(O)-C3-C 12 Cycloalkyl, -C(O)-aryl, -C(O)-heteroaryl, -C(O)-heterocycloalkyl, -CONH2, -CONH-C1-C 12 Alkyl, -CONH-C2-C8 alkenyl, -CONH-C2-C8 ynyl, -CONH-C3-C 12 Cycloalkyl, -CONH-aryl, -CONH-heteroaryl, -CONH-heterocycloalkyl, -OCO2-C1-C 12 Alkyl, -OCO2-C2-C8 alkenyl, -OCO2-C2-C8 ynyl, -OCO2-C3-C 12 Cycloalkyl, -OCO2-aryl, -OCO2-heteroaryl, -OCO2-heterocycloalkyl, -CO2-C1-C 12 Alkyl, -CO2-C2-C8 alkenyl, -CO2-C2-C8 ynyl, CO2-C3-C 12Cycloalkyl, -CO2-aryl, CO2-heteroaryl, CO2-heterocycloalkyl, -OCONH2, -OCONH-C1-C 12 Alkyl, -OCONH-C2-C8 alkenyl, -OCONH-C2-C8 ynyl, -OCONH-C3-C 12 Cycloalkyl, -OCONH-aryl, -OCONH-heteroaryl, -OCONH-heterocycloalkyl, -NHC(O)H, -NHC(O)-C1-C 12 Alkyl, -NHC(O)-C2-C8 alkenyl, -NHC(O)-C2-C8 ynyl, -NHC(O)-C3-C 12 Cycloalkyl, -NHC(O)-aryl, -NHC(O)-heteroaryl, -NHC(O)-heterocycloalkyl, -NHCO2-C1-C 12 Alkyl, -NHCO2-C2-C8 alkenyl, -NHCO2-C2-C8 alkynyl, -NHCO2-C3-C 12 Cycloalkyl, -NHCO2-aryl, -NHCO2-heteroaryl, -NHCO2-heterocycloalkyl, -NHC(O)NH2, -NHC(O)NH-C1-C 12 Alkyl, -NHC(O)NH-C2-C8 alkenyl, -NHC(O)NH-C2-C8 ynyl, -NHC(O)NH-C3-C 12 Cycloalkyl, -NHC(O)NH-aryl, -NHC(O)NH-heteroaryl, -NHC(O)NH-heterocycloalkyl, -NHC(S)NH2, -NHC(S)NH-C1-C 12 Alkyl, -NHC(S)NH-C2-C8 alkenyl, -NHC(S)NH-C2-C8 ynyl, -NHC(S)NH-C3-C 12 Cycloalkyl, -NHC(S)NH-aryl, -NHC(S)NH-heteroaryl, -NHC(S)NH-heterocycloalkyl, -NHC(NH)NH2, -NHC(NH)NH-C1-C 12 Alkyl, -NHC(NH)NH-C2-C8 alkenyl, -NHC(NH)NH-C2-C8 alkynyl, -NHC(NH)NH-C3-C 12 Cycloalkyl, -NHC(NH)NH-aryl, -NHC(NH)NH-heteroaryl, -NHC(NH)NH-heterocycloalkyl, -NHC(NH)-C1-C 12 Alkyl, -NHC(NH)-C2-C8 alkenyl, -NHC(NH)-C2-C8 alkynyl, -NHC(NH)-C3-C 12Cycloalkyl, -NHC(NH)-aryl, -NHC(NH)-heteroaryl, -NHC(NH)-heterocycloalkyl, -C(NH)NH-C1-C 12 Alkyl, -C(NH)NH-C2-C8 alkenyl, -C(NH)NH-C2-C8 ynyl, -C(NH)NH-C3-C 12 Cycloalkyl, -C(NH)NH-aryl, -C(NH)NH-heteroaryl, -C(NH)NH-heterocycloalkyl, -S(O)-C1-C 12 Alkyl, -S(O)-C2-C8 alkenyl, -S(O)-C2-C8 ynyl, -S(O)-C3-C 12 Cycloalkyl, -S(O)-aryl, -S(O)-heteroaryl, -S(O)-heterocycloalkyl, -SO2NH2, -SO2NH-C1-C 12 Alkyl, -SO2NH-C2-C8 alkenyl, -SO2NH-C2-C8 ynyl, -SO2NH-C3-C 12 Cycloalkyl, -SO2NH-aryl, -SO2NH-heteroaryl, -SO2NH-heterocycloalkyl, -NHSO2-C1-C 12 Alkyl, -NHSO2-C2-C8 alkenyl, -NHSO2-C2-C8 ynyl, -NHSO2-C3-C 12 Cycloalkyl, -NHSO2-aryl, -NHSO2-heteroaryl, -NHSO2-heterocycloalkyl, -CH2NH2, -CH2SO2CH3, -aryl, -arylalkyl, -heteroaryl, -heteroarylalkyl, -heterocycloalkyl, -C3-C 12 Cycloalkyl, polyalkoxyalkyl, polyalkoxy, -methoxymethoxy, -methoxyethoxy, -SH, -S-Cl-C 12 Alkyl, -S-C2-C8 alkenyl, -S-C2-C8 ynyl, -S-C3-C 12Cycloalkyl, -S-aryl, -S-heteroaryl, -S-heterocycloalkyl, or methylthio-methyl. In some embodiments, the substituents are independently selected from: halogens, preferably Cl and F; C1-C4 alkyl, preferably methyl and ethyl; halo-C1-C4 alkyl, such as fluoromethyl, difluoromethyl, and trifluoromethyl; C2-C4 alkenyl; halo-C2-C4 alkenyl; C3-C6 cycloalkyl, such as cyclopropyl; C1-C4 alkoxy, such as methoxy and ethoxy; halo-C1-C4 alkoxy, such as fluoromethoxy, difluoromethoxy, and trifluoromethoxy; -CN; -OH; NH2; C1-C4 alkylamino; di(C1-C4 alkyl)amino; and NO2. It should be understood that aryl, heteroaryl, alkyl, etc., can be further substituted. In some cases, when possible, each substituent in the substituted moiety may be optionally substituted by one or more groups, each group being independently selected from C1-C4 alkyl groups; -CF3, -OCH3, -OCF3, -F, -Cl, -Br, -I, -OH, -NO2, -CN, and -NH2.

[0260] In some embodiments, the substituted alkyl, alkenyl, or alkoxy group is replaced by one or more halogen atoms (preferably fluorine or chlorine atoms). Such substituted alkyl groups include fluoromethyl, difluoromethyl, and trifluoromethyl groups. Such substituted alkoxy groups include fluoromethoxy, difluoromethoxy, and trifluoromethoxy groups.

[0261] As used herein, the term “halogenated” or “halogen” alone or as part of another substituent refers to a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.

[0262] As used herein, the term "optionally substituted" means that the mentioned group may be substituted or unsubstituted. In one embodiment, the mentioned group is optionally substituted with zero substituents, i.e., the mentioned group is unsubstituted. In another embodiment, the mentioned group is optionally substituted with one or more additional groups, which are individually and independently selected from the groups described herein.

[0263] The term "hydrogen" includes both hydrogen and deuterium. Additionally, the atom mentioned may include other isotopes of that atom, provided the resulting compound is pharmaceutically acceptable.

[0264] In some embodiments, the compound of each formula herein is defined as a compound comprising isotope labeling. An "isotope-labeled compound" is a compound in which at least one atomic position is enriched with a specific isotope of a specified element to a level significantly greater than the natural abundance of that isotope. For example, one or more hydrogen atom positions in a compound may be enriched with deuterium to a level significantly greater than the natural abundance of deuterium, for example, to at least 1%, preferably at least 20% or at least 50%. Such deuterated compounds may, for example, be metabolized more slowly than their non-deuterated analogues and thus exhibit a longer half-life when administered to a subject. These compounds can be synthesized using methods known in the art, for example, by using deuterated starting materials. Unless stated otherwise, isotope-labeled compounds are pharmaceutically acceptable.

[0265] As used herein, the term "hydroxyl-activating group" refers to an unstable chemical moiety known in the art that activates a hydroxyl group so that it leaves during synthesis, for example, in a substitution or elimination reaction. Examples of hydroxyl-activating groups include, but are not limited to, methanesulfonate, toluenesulfonate, trifluoromethanesulfonate, p-nitrobenzoate, phosphonate, etc.

[0266] As used herein, the term "activated hydroxyl group" refers to a hydroxyl group activated with a hydroxyl-activating group as defined above, including, for example, methanesulfonic acid group, toluenesulfonic acid group, trifluoromethanesulfonic acid group, p-nitrobenzoic acid group, and phosphonic acid group.

[0267] As used herein, the term "hydroxyl protecting group" refers to an unstable chemical moiety known in the art that protects hydroxyl groups from unwanted reactions during synthesis. The hydroxyl protecting group described herein can be selectively removed after the synthetic process. Hydroxyl protecting groups known in the art are generally described in THGreene and PGMWuts. Protective Groups in Organic Synthesis In the 3rd edition, John Wiley & Sons, New York (1999). Examples of hydroxyl protecting groups include benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, tert-butoxycarbonyl, isopropoxycarbonyl, diphenylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, allyloxycarbonyl, acetyl, formyl, chloroacetyl, trifluoroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, methyl, tert-butyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, allyl, benzyl, triphenyl-methyl (triphenylmethyl), methoxymethyl, methylthiomethyl, benzyloxymethyl, 2-(trimethylsilyl)-ethoxymethyl, methanesulfonyl, trimethylsilyl, triisopropylsilyl, etc.

[0268] As used herein, the term "protected hydroxyl group" refers to a hydroxyl group protected by a hydroxyl protecting group as defined above, including, for example, benzoyl, acetyl, trimethylsilyl, triethylsilyl, and methoxymethyl.

[0269] As used herein, the term "hydroxy prodrug group" refers to a precursor moiety known in the art that transiently alters the physicochemical properties of a parent drug and thus its biological properties by covering or masking a hydroxyl group. Following the synthetic process, the hydroxy prodrug group described herein must be able to revert to a hydroxyl group in vivo. Hydroxy prodrug groups known in the art are generally described in the works of Kenneth B. Sloan. Prodrugs, Topical and Ocular Drug Delivery , (Drugs and the Pharmaceutical Sciences; Volume 53), Marcel Dekker, Inc., New York (1992) and "Prodrugs of Alcohols and Phenols" by SSDhareshwar and VJStella, in Prodrugs Challenges and Rewards Part-2 (Biotechnology:PharmaceuticalAspects), edited by VJStella, et al, Springer and AAPSPress, 2007, pp. 31-99.

[0270] As used herein, the term "amino protecting group" refers to an unstable chemical moiety known in the art that protects an amino group from undesirable reactions during synthesis. Following the synthetic process, the amino protecting group described herein can be selectively removed. Amino protecting groups known in the art are generally described in THGreene and PGMWuts. Protective Groups in Organic Synthesis In the 3rd edition, John Wiley & Sons, New York (1999). Examples of amino protecting groups include, but are not limited to, methoxycarbonyl, tert-butoxycarbonyl, 9-fluorenyl-methoxycarbonyl, benzyloxycarbonyl, etc.

[0271] As used herein, the term "protected amino" refers to an amino group protected by an amino protecting group as defined above.

[0272] The term "leaving group" refers to a functional group or atom that can be replaced by another functional group or atom in a substitution reaction, such as a nucleophilic substitution reaction. For example, representative leaving groups include chloride, bromide, and iodide groups; sulfonate groups, such as methanesulfonate, toluenesulfonate, p-bromobenzenesulfonate, p-nitrobenzenesulfonate, etc.; and acyloxy groups, such as acetoxy, trifluoroacetoxy, etc.

[0273] As used herein, the term "aprotic solvent" refers to a solvent that is relatively inert to protons, i.e., does not act as a proton donor. Examples include, but are not limited to, hydrocarbons, heterocyclic compounds, and ethers; hydrocarbons such as hexane and toluene, for example, halogenated hydrocarbons such as dichloromethane, dichloroethane, chloroform, etc.; heterocyclic compounds such as tetrahydrofuran and N-methylpyrrolidone; and ethers such as diethyl ether and dimethoxymethyl ether. These compounds are well known to those skilled in the art, and it will be apparent to them that, for a particular compound and reaction conditions, a single solvent or a mixture thereof may be preferred, depending on factors such as, for example, the solubility of the reagent, the reactivity of the reagent, and the preferred temperature range. Further discussion of aprotic solvents can be found in organic chemistry textbooks or specialized monographs, for example: Organic Solvents Physical Properties and Methods of Purification ,4th ed.,edited by John A. Riddick etal.,Vol.II,in the Techniques of Chemistry Series John Wiley & Sons, NY, 1986.

[0274] As used herein, the term "proton-donating solvent" refers to a solvent that tends to donate protons, such as alcohols, including methanol, ethanol, propanol, isopropanol, butanol, tert-butanol, etc. These solvents are well known to those skilled in the art, and it will be apparent to them that, for a particular compound and reaction conditions, a single solvent or a mixture thereof may be preferred, depending on factors such as, for example, the solubility of the reagent, the reactivity of the reagent, and the preferred temperature range. Further discussion of proton-donating solvents can be found in organic chemistry textbooks or specialized monographs, for example: Organic Solvents Physical Properties and Methods of Purification ,4th ed.,edited by JohnA.Riddick et al.,Vol.II,in the Techniques of Chemistry Series John Wiley & Sons, NY, 1986.

[0275] The combinations of substituents and variables contemplated in this invention are only those that result in the formation of stable compounds. As used herein, the term "stable" means that a compound has sufficient stability to be manufactured and to maintain the integrity of the compound for a sufficiently long period of time for use for the purposes detailed herein (e.g., therapeutic or prophylactic administration to a subject).

[0276] The synthesized compounds can be isolated from the reaction mixture and further purified by methods such as column chromatography, high-performance liquid chromatography, or recrystallization. Those skilled in the art will understand that other methods for synthesizing compounds of the formulas herein will be readily apparent to those of ordinary skill in the art. Furthermore, various synthetic processes can be carried out in an alternating sequence or order to obtain the desired compounds. Synthetic chemical transformations and protecting group methods (protection and deprotection) used to synthesize the compounds described herein are known in the art, including, for example, those employed by R. Larock, Comprehensive Organic Transformations ,2 nd Ed.Wiley-VCH(1999);TWGreene and PGMWuts, Protective Groups in Organic Synthesis ,3rd Ed.,John Wiley and Sons(1999);L.Fieser andM.Fieser, Fieser and Fieser's Reagents for Organic Synthesis , John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis Those described in John Wiley and Sons (1995) and subsequent editions.

[0277] As used herein, the term "subject" refers to an animal. Preferably, the animal is a mammal. More preferably, the mammal is a human. Subjects also refer to animals such as dogs, cats, horses, cattle, pigs, guinea pigs, fish, birds, etc.

[0278] The compounds of the present invention can be modified to enhance selective biological properties by adding appropriate functional groups. These modifications are known in the art and may include those that increase biopermeability into a given biological system (e.g., blood, lymphatic system, central nervous system), increase oral bioavailability, increase solubility to allow administration by injection, alter metabolism, and change excretion rates.

[0279] The compounds described herein contain one or more asymmetric centers, thus producing enantiomers, diastereomers, and other stereoisomers, which can be defined according to absolute stereochemistry as (R)- or (S)-, or for amino acids as (D)- or (L)-. This invention is intended to include all such possible isomers, as well as their racemic and optically pure forms. Optical isomers can be prepared from their respective optically active precursors by the methods described above or by resolving racemic mixtures. Resolution can be carried out in the presence of a resolving agent by chromatography, by repeated crystallization, or by some combination of these techniques known to those skilled in the art. Further details regarding resolution can be found in Jacques, et al. Enantiomers Racemates, and Resolutions(John Wiley & Sons, 1981). When the compounds described herein contain an alkene double bond, other unsaturated bonds, or other geometrically asymmetric centers, it is intended, unless otherwise stated, that the compound includes E and Z geometric isomers or cis- and trans-isomers. Similarly, it is intended to include all tautomer forms. Tautomers can be cyclic or acyclic. Any carbon-carbon double bond configuration appearing herein is chosen for convenience only and is not intended to specify a particular configuration unless stated herein; therefore, any carbon-carbon double bond or carbon-heteroatom double bond described herein as trans can be cis, trans, or a mixture of both in any proportion.

[0280] Some compounds of the present invention can also exist in separable, different stable conformations. Torsional asymmetry, resulting from restricted rotation around asymmetric single bonds, such as due to steric hindrance or ring strain, allows for the separation of different conformational isomers. The present invention includes each conformational isomer of these compounds and mixtures thereof.

[0281] As used herein, the term "pharmaceutically acceptable salt" means that, within reasonable medical judgment, it is suitable for contact with tissues of humans and lower animals without undue toxicity, irritation, allergic reactions, etc., and in proportion to a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, SMBerge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66:1-19 (1977). These salts can be prepared in situ during the final isolation and purification of the compounds of the present invention, or by reacting a free base functional group with a suitable organic acid. Examples of pharmaceutically acceptable salts include, but are not limited to, non-toxic acid addition salts, which are salts formed by an amino group with an inorganic or organic acid, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, and such organic acid as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or salts formed by using other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentane-propionate, disaccharide, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucono-heptahydrate, glyceryl phosphate, gluconate, hemisulfate, heptahydrate, hexanoate, hydroiodate, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, neopentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium salts. Other pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium cations, quaternary ammonium cations, and amine cations, which are formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl groups having 1-6 carbon atoms, sulfonate, and arylsulfonate groups.

[0282] Pharmaceutically acceptable salts can also be prepared by deprotonating the parent compound with a suitable base, thereby forming the anionic conjugate base of the parent compound. In such salts, the counter ion is a cation. Suitable cations include ammonium cations and metal cations, such as alkali metal cations, including Li. + Na + K + and Cs + and alkaline earth metal cations, such as Mg 2+ and Ca2+ .

[0283] As used herein, the term "pharmaceutically acceptable ester" refers to esters that are hydrolyzed in vivo, including those that readily break down in the human body, leaving behind a parent compound or its salts. Suitable ester groups include, for example, those derived from pharmaceutically acceptable aliphatic carboxylic acids (particularly alkyl, olefinic, cycloalkyl, and alkanedioic acids), wherein each alkyl or alkenyl moiety advantageously has no more than six carbon atoms. Examples of specific esters include, but are not limited to, esters of C1-C6 alkyl acids, such as acetates, propionates, butyrates, and neopentylates.

[0284] In some embodiments, the present invention provides pharmaceutically acceptable prodrugs of the compounds disclosed herein. As used herein, the term "pharmaceuticalally acceptable prodrug" refers to those prodrugs of compounds formed by the methods of the present invention that, within reasonable medical judgment, are suitable for contact with tissues of humans and lower animals without producing undue toxicity, irritation, allergic reactions, etc., in proportion to a reasonable benefit / risk ratio, and are effective for their intended use, and, where possible, are zwitterionic forms of the compounds of the present invention. As used herein, "prodrug" refers to any compound that can be converted in vivo via metabolic pathways (e.g., via hydrolysis) to provide the structurally described compound of the present invention. Various forms of prodrugs are known in the art, for example in Bundgaard, (ed.), Design of Prodrugs, Elsevier (1985); Widder, et al. (ed.), Methods in Enzymology, Vol. 4, Academic Press (1985); Krogsgaard-Larsen, et al. (ed.), Design and Application of Prodrugs, Textbook of Drug Design and Development, Chapter 5, 113-191 (1991); Bundgaard, et al., Journal of Drug Deliver Reviews, 8:1-38 (1992); Bundgaard, J. of Pharmaceutical Sciences, 77:285 et seq. (1988); Higuchi and Stella (eds.), Prodrugs as Novel Drug Delivery Systems, American Chemical Society (1975); and Bernard Testa & Joachim Those discussed in Mayer, “Hydrolysis In Drug And Prodrug Metabolism: Chemistry, Biochemistry And Enzymology,” John Wiley and Sons, Ltd. (2002).

[0285] Other types of prodrugs are also included. For example, the free carboxyl group can be derived into an amide or alkyl ester. The free hydroxyl group can be derived using groups including, but not limited to, hemisuccinate, ethyl succinate, phosphate ester, dimethylaminoacetate, and phosphoryloxymethoxycarbonyl, as outlined in Advanced Drug Delivery Reviews, 1996, 19, 115. Also included are urethane prodrugs with hydroxyl and amino groups, as well as carbonate prodrugs, sulfonates, and sulfates with hydroxyl groups. Also included are hydroxyl-derived prodrugs to (acyloxy)methyl and (acyloxy)ethyl ethers, wherein the acyl group can be an alkyl ester, optionally substituted with groups including, but not limited to, ether, amine, and carboxylic acid functional groups, or wherein the acyl group is an amino acid ester as described above. Such prodrugs are described in J. Med. Chem. 1996, 39, 10. Free amines can also be derived into amides, sulfonamides, or phosphonamides. All these prodrug moieties can be incorporated into groups including, but not limited to, ether, amine, and carboxylic acid functional groups. In some embodiments, the compounds of the present invention may incorporate two or more groups that are metabolically removed in vivo to produce an active parent compound.

[0286] As used herein, the term "treatment" means relief, reduction, elimination, regulation, or improvement, that is, causing the remission of a disease state or symptom. Treatment may also include suppression, that is, preventing the development of an existing disease state or symptom, and relief or improvement, that is, causing the remission of an existing disease state or symptom, for example, when a disease state or symptom may have already been present.

[0287] As used herein, the term “prevention” means the complete or near-complete prevention of the occurrence of a disease state or condition in a patient or subject, especially when the patient or subject is susceptible to or at risk of contracting a disease state or condition.

[0288] Furthermore, the compounds of the present invention, such as salts of the compounds, may exist in hydrated or unhydrated (anhydrous) form or as solvates with other solvent molecules. Non-limiting examples of hydrates include monohydrates, dihydrates, etc. Non-limiting examples of solvates include ethanol solvates, acetone solvates, etc.

[0289] A solvate is a solvent addition form containing either stoichiometric or non-stoichiometric solvent molecules. Some compounds have a tendency to trap a fixed molar ratio of solvent molecules in their crystalline solid state, thus forming a solvate. If the solvent is water, the solvate formed is a hydrate; when the solvent is an alcohol, the solvate formed is an alcohol. Hydrates are formed by the combination of one or more water molecules with a substance in which water retains its molecular state, such as H₂O; this combination can form one or more hydrates.

[0290] As used herein, the term "analogue" refers to a compound that is structurally similar to another compound but differs slightly in composition (e.g., an atom is replaced by an atom of a different element, a specific functional group is present, or a functional group is replaced by another functional group). Therefore, an analogue is a compound that is functionally similar to or equivalent to the reference compound.

[0291] The combinations of substituents and variables contemplated in this invention are only those that result in the formation of stable compounds. As used herein, the term "stable" means that a compound has sufficient stability to be manufactured and to maintain the integrity of the compound for a sufficiently long period of time for use for the purposes detailed herein (e.g., therapeutic or prophylactic administration to a subject).

[0292] The synthesized compounds can be isolated from the reaction mixture and further purified by methods such as column chromatography, high-performance liquid chromatography, or recrystallization. Furthermore, the various synthetic steps can be performed in an alternating sequence or order to obtain the desired compounds. Additionally, the solvents, temperatures, reaction durations, etc., described herein are for illustrative purposes only, and variations in reaction conditions can produce the desired bridged macrocyclic products of this invention. Synthetic chemical transformations and protecting group methods (protection and deprotection) are used to synthesize the compounds described herein, including, for example, R. larock. Comprehensive Organic ,VCH Publishers(1989);TWGreene and PGMWuts, Transformations Protective ,2d.Ed.,John Wiley and Sons(1991);L.Fieser andM.Fieser, Groups in Organic Synthesis , John Wiley and Sons (1994); and L. Paquette, ed., Fieser and Fieser's Reagents for Organic Synthesis Those described in John Wiley and Sons (1995).

[0293] The compounds of the present invention can be modified by adding various functional groups via the synthetic methods described herein to enhance selective biological properties. These modifications include those that increase biopermeability into a given biological system (e.g., blood, lymphatic system, central nervous system), increase oral bioavailability, increase solubility to allow for administration by injection, alter metabolism, and change excretion rates.

[0294] Encyclopedia of Reagents for Organic Synthesis

[0295] The pharmaceutical compositions of the present invention comprise a therapeutically effective amount of the compound of the present invention formulated with one or more pharmaceutically acceptable carriers. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic, inert solid, semi-solid, or liquid filler, diluent, encapsulating material, or any type of formulation adjuvant. Some examples of materials that can be used as pharmaceutically acceptable carriers are: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth gum; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols, such as propylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol and phosphate buffer solutions; and other non-toxic, compatible lubricants, such as sodium lauryl sulfate and magnesium stearate. Colorants, releasing agents, coating agents, sweeteners, flavorings and aromas, preservatives, and antioxidants may also be present in the composition, according to the formulator's judgment. The pharmaceutical compositions of the present invention can be administered orally, rectally, parenterally, intracerebrospinally, intravaginally, intraperitoneally, topically (e.g., by powder, ointment or drops), sublingually, or as oral or nasal sprays to humans and other animals.

[0296] The pharmaceutical compositions of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, or orally, via implantable containers, preferably orally or by injection. The pharmaceutical compositions of the present invention may contain any conventionally non-toxic, pharmaceutically acceptable carrier, adjuvant, or excipient. In some cases, the pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases, or buffers to enhance the stability of the formulated compound or its delivery form. The term parenterally as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.

[0297] Oral liquid dosage forms include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compound, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oils (particularly cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitol, and mixtures thereof. Besides inert diluents, oral compositions may also include adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and aromatizers.

[0298] Injectable formulations, such as sterile aqueous or oily suspensions for injection, can be formulated using suitable dispersants or wetting agents and suspending agents according to known techniques. Sterile injectable formulations can also be sterile injectable solutions, suspensions, or emulsions in non-toxic, parenteral-acceptable diluents or solvents, such as solutions in 1,3-butanediol. Among acceptable carriers and solvents, water, Ringer's solution, USP, and isotonic sodium chloride solution can be used. Furthermore, sterile fixed oils are commonly used as solvents or suspension media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. Additionally, fatty acids such as oleic acid are used in injectable formulations.

[0299] Injectable formulations can be sterile, for example, filtered through a bacterial trap filter, or by incorporating a sterile solid composition in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable media prior to use.

[0300] To prolong the effect of a drug, it is often necessary to slow down its absorption from subcutaneous or intramuscular injection. This can be achieved by using liquid suspensions of poorly water-soluble crystalline or amorphous substances. The absorption rate of the drug depends on its dissolution rate, which in turn depends on the crystal size and crystal form. Alternatively, delayed absorption of parenteral dosage forms can be achieved by dissolving or suspending the drug in an oil carrier. Injectable reservoir forms are prepared by forming microcapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolic acid. The rate of drug release can be controlled depending on the drug-to-polymer ratio and the properties of the specific polymer used. Other examples of biodegradable polymers include poly(orthoester) and poly(anhydride). Reservoir-type injectable formulations can also be prepared by encapsulating the drug in tissue-compatible liposomes or microemulsions.

[0301] Compositions for rectal or vaginal administration are preferably suppositories, which can be prepared by mixing the compounds of the invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol, or suppository waxes, which are solid at ambient temperature but liquid at body temperature, thus melting and releasing the active compound in the rectal or vaginal cavity.

[0302] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, an active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or: a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and silica; b) binders, such as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; c) humectants, such as glycerin; d) disintegrants, such as agar, calcium carbonate, potato or cassava starch, alginate, certain silicates, and sodium carbonate; e) solution blockers, such as paraffin; f) absorption enhancers, such as quaternary ammonium compounds; g) wetting agents, such as cetyl alcohol and glyceryl monostearate; h) absorbents, such as kaolin and bentonite; and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also contain buffers.

[0303] Similar solid compositions can also be used as fillers in soft-filled and hard-filled gelatin capsules, which use excipients such as lactose or lactose and high molecular weight polyethylene glycol.

[0304] The active compound can also be formulated into microcapsule form with one or more of the excipients described above. Solid dosage forms such as tablets, lozenges, capsules, pills, and granules can be prepared using coatings and shells (such as enteric coatings, controlled-release coatings, and other coatings known in the field of pharmaceutical formulation). In such solid dosage forms, the active compound can be mixed with at least one inert diluent (such as sucrose, lactose, or starch). According to conventional practice, such dosage forms may also contain substances other than inert diluents, such as tableting lubricants and other tableting aids, such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets, and pills, the dosage form may also contain buffers. They may optionally contain light-blocking agents and may also be compositions in which they optionally release the active ingredient in a delayed manner, either alone or preferably in a portion of the intestine. Examples of encapsulation compositions that can be used include polymers and waxes.

[0305] Dosage forms for topical or transdermal application of the compounds of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalers, or patches. The active ingredient is mixed under aseptic conditions with a pharmaceutically acceptable carrier and any necessary preservatives or buffers. Ophthalmic preparations, ear drops, eye ointments, powders, and solutions are also considered to be within the scope of this invention.

[0306] In addition to the active compounds of the present invention, ointments, pastes, creams and gels may also contain excipients such as animal and vegetable fats, oils, waxes, paraffin waxes, starches, tragacanth gums, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc and zinc oxide or mixtures thereof.

[0307] In addition to the compounds of this invention, powders and aerosols may contain excipients such as lactose, talc, silica, aluminum hydroxide, calcium silicate, and polyamide powder, or mixtures thereof. Aerosols may also contain conventional propellants such as chlorofluorocarbons (CFCs).

[0308] The added advantage of transdermal patches is the controlled delivery of compounds into the body. This dosage form can be prepared by dissolving or dispersing the compound in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by providing a rate-controlled membrane or by dispersing the compound in a polymer matrix or gel.

[0309] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly known to a person skilled in the art. All publications, patents, published patent applications, and other references mentioned herein are incorporated herein by reference in their entirety.

[0310] Drug Compositions

[0311] The abbreviations used in the following description of the schemes and embodiments are:

[0312] ACN stands for acetonitrile;

[0313] AD-mix-β represents (9S)-(9”S)-9,9”-[1,4-phthalazinediylbis(oxy)]bis[10,11-dihydro-6'-methoxycinchonan];

[0314] Bn represents benzyl;

[0315] BOP stands for (benzotriazol-1-yloxy)tris(dimethylamino)phosphine hexafluorophosphate;

[0316] BzCl represents benzoyl chloride;

[0317] mCPBA represents m-chloroperbenzoic acid;

[0318] Cbz represents benzyloxycarbonyl;

[0319] CDI stands for carbonyl diimidazole;

[0320] DAST stands for diethylaminosulfur trifluoride;

[0321] DBU stands for 1,8-diazabicycloundec-7-ene;

[0322] DCE stands for dichloroethane;

[0323] DCM stands for dichloromethane;

[0324] Dess-Martin periodane represents 1,1,1-tris(acetoxy)-1,1-dihydro-1,2-benzoiodol-3-(1H)-one;

[0325] DIAD stands for diisopropyl azodicarbonate;

[0326] DIBAL-H represents diisobutylaluminum hydride;

[0327] DMAP stands for N,N-dimethylaminopyridine;

[0328] DME stands for 1,2-dimethoxyethane;

[0329] DMF stands for N,N-dimethylformamide;

[0330] DMSO stands for dimethyl sulfoxide;

[0331] DPPA stands for diphenylphosphohydride or diphenylphosphohydride;

[0332] dppf represents 1,1'-bis(diphenylphosphino)ferrocene;

[0333] EDCI or EDC represents 1-(3-diethylaminopropyl)-3-ethylcarbodiimide hydrochloride;

[0334] EA or EtOAc represents ethyl acetate;

[0335] Ghosez's reagent represents 1-chloro-N,N,2-trimethyl-1-propenylamine;

[0336] HATU represents O(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylureonium hexafluorophosphate;

[0337] HCl represents hydrochloric acid;

[0338] Hunig base represents diisopropylethylamine;

[0339] PyBOP represents (benzotriazol-1-yloxy)tripyrrolidinephosphine hexafluorophosphate;

[0340] LDA stands for lithium diisopropylamine;

[0341] Pd-C indicates palladium on carbon;

[0342] PE is petroleum ether;

[0343] Ph represents phenyl;

[0344] RT stands for reverse transcription;

[0345] RT-PCR stands for reverse transcription-polymerase chain reaction;

[0346] TBME stands for tert-butyl methyl ether;

[0347] TEA stands for triethylamine;

[0348] Tf2O represents trifluoromethanesulfonic anhydride;

[0349] TFA stands for trifluoroacetic acid;

[0350] THF stands for tetrahydrofuran;

[0351] (TMS)2NH represents hexamethyldisilazane;

[0352] TBS stands for tert-butyldimethylsilyl;

[0353] TBDPS stands for tert-butyldiphenylsilyl;

[0354] TMS stands for trimethylsilyl;

[0355] TPAP stands for tetrapropylammonium perruthenate;

[0356] TPP or PPh3 represents triphenylphosphine;

[0357] Ts or toluenesulfonyl group indicates p-CH3C6H4SO2-;

[0358] tBOC or Boc represents tert-butoxycarbonyl; and

[0359] Xantphos is represented by 4,5-bis-diphenylphosphono-9,9-dimethyl-9H-xanton.

[0360] Abbreviations

[0361] The compounds and methods of the present invention will be better understood in conjunction with the following synthetic schemes, which illustrate methods for preparing the compounds of the present invention. These synthetic schemes are intended to be illustrative only and do not limit the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art, and such changes and modifications can be made without departing from the spirit of the invention and the scope of the appended claims, including but not limited to those involving the chemical structures, substituents, derivatives, and / or methods of the invention.

[0362] Scheme 1 illustrates a method for preparing compounds of formula 11 from compounds 1 and 2, wherein n = 1, 2, or 3; P is a hydroxyl protecting group; Ar is E; E is as defined above. Alkylation of hydroxypyridine 1 with a hydroxy epoxide using Mitsunobu reaction conditions yields epoxide 4. Alternatively, the hydroxy epoxide is converted to 3 having a leaving group, such as, but not limited to, toluenesulfonyl and methanesulfonyl, and then alkylated in the presence of a base, such as, but not limited to, K₂CO₃ and Cs₂CO₃, to yield 4. Intramolecular ring-opening of the epoxide mediated by a base, such as, but not limited to, LDA, yields compound 5. Protecting the hydroxyl compound 5 with a suitable protecting group, such as, but not limited to, TBDPS and TBS, yields compound 6. Trifluoromethyl ketone 7 is obtained by iodine-magnesium exchange of compound 6 followed by esterification (e.g., but not limited to, ethyl 2,2,2-trifluoroacetate). Trifluoromethyl ketone 7 is cross-coupled with various metal conjugates 8 (but not limited to boric acid, borate esters, organotin reagents, organozinc reagents, organomagnesium reagents, organosilicon reagents, etc.), and catalyzed by suitable Pd, Ni, Cu, etc., to give compound 9. Nitromethane is added to compound 9 in the presence of a base (e.g., but not limited to K2CO3 and Cs2CO3) to give compound 10. The nitro group is reduced with a reducing agent (e.g., but not limited to zinc and acetic acid) to give the key intermediate 11. Scheme 1

[0363]

[0364] As seen in Scheme 2, where Ar1 is A; Ar is E; and R is R. 11 n is 1, 2, or 3; A, E, R 11 As defined above, key intermediate 11 is coupled with various carboxylic acids to yield amide 14. Amide 14 then reacts with various electrophilic agents to generate various ethers, esters, and carbamates of formula 15. Amide 14 is also oxidized to aldehyde 16, which is then subjected to reductive amination to give various amines 17. The -CH2OH hydroxyl group in amide 14 is activated and subsequently cyanidated to convert to cyanomethyl 18. In the presence of a catalyst (e.g., but not limited to Parkin catalyst), compound 14-1 is further converted to acetamide 19.

[0365] Option 2

[0366]

[0367] As seen in Scheme 3, where Ar1 is A; Ar is E; and R is R. 11 A, E, R 11 As defined above, aldehyde 16 is converted to a benzyl-protected amine via reductive amination. Hydrogenolysis yields the free amine 20, which is then replaced by various electrophilic reagents to obtain the N-substituted compound 21.

[0368] Option 3

[0369]

[0370] As seen in Scheme 4, Ar1 is A; Ar is E; R' is -C1-C6 alkyl, -C3-C6 cycloalkyl, aryl, or heteroaryl; n is 1, 2, or 3; A and E are as defined above. After aldehyde 16 is oxidized to acid 22, it is further converted to amide 23 and sulfonamide 24 using conventional methods (e.g., but not limited to HATU and DIPEA). This diversification is thus achieved to produce a variety of esters and amides.

[0371] Option 4

[0372]

[0373] Scheme 5 illustrates another method for preparing compounds of formula 11, where Ar is E; P is a hydroxyl protecting group; n is 1, 2, or 3; and E is as defined above. Ketone 9 is converted to compounds of formula 26 via olefination. Alternatively, 26 is obtained from: 1) 6 being cross-coupled with a metal coupling agent 6-1 under suitable catalysts such as Pd, Ni, or Cu to obtain compound 25, which can be, but is not limited to, boric acid, borate esters, organotin reagents, organozinc reagents, organomagnesium reagents, organosilicon reagents, etc.; 2) 25 being converted to compound 26 as described in Scheme 1 above. In the case of 26, compounds of formula 27 are prepared by dihydroxylation followed by the formation of an epoxide. Compound 27 is then subjected to epoxide ring-opening with an amine equivalent (e.g., but not limited to NH4OH and NH3) to obtain compounds of formula 11.

[0374] Option 5

[0375]

[0376] Scheme 6 illustrates another method for preparing compounds of formula 23, wherein Ar1 is A; Ar is E; R' is -C1-C6 alkyl, -C3-C6 cycloalkyl, aryl, or heteroaryl; n is 1, 2, or 3; A and E are as previously defined. Amine 11 is protected with a protecting group (e.g., but not limited to Boc and Cbz). After deprotection of the hydroxyl protecting group, it is subsequently oxidized to give acid 30. Compound 30 is coupled with various amines to give amide 31. The amine protecting group is deprotected, and then the following amide is formed to give compounds of formula 23.

[0377] Option 6

[0378]

[0379] Scheme 7 illustrates an alternative route for synthesizing the desired compound. The difference lies in that it begins with oxidation and amide coupling to generate amide 33 at the start of the synthesis. Ethylation and arylation are then performed sequentially to yield the bicoupled product 34. An asymmetric dihydroxylation reaction is then carried out, followed by activation and substitution, to give the amino alcohol precursor. Finally, amide coupling with the corresponding arylic acid yields the desired compound described in compound 36.

[0380] Option 7

[0381]

[0382] Synthesis Methods

[0383] The compounds and methods of the present invention will be better understood in conjunction with the following examples, which are for illustrative purposes only and not for limiting the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art, and such changes and modifications may be made without departing from the spirit of the invention and the scope of the appended claims, including but not limited to those related to the chemical structure, substituents, derivatives, formulations and / or methods of the invention.

[0384] Certain synthetic methods that can be used to prepare the compounds of the present invention are disclosed in U.S. Patent Application No. 16 / 930622, the contents of which are incorporated herein by reference in their entirety.

[0385] Example 1

[0386]

[0387] Example 1, Step a

[0388]

[0389] A solution of 3-bromo-4-hydroxybenzoate (16 g, 69.25 mmol), Cs₂CO₃ (68 g, 207.75 mmol), KI (46 g, 277.00 mmol), and bromocyclopropane (21 g, 173.12 mmol) in NMP (30 mL) was stirred in a Parr reactor at 180 °C for 16 h. The resulting solution was diluted with water and extracted with EtOAC. The combined organic compounds were dried and concentrated. The resulting solution was purified by reversed-phase C18 column chromatography (CH₃CN / H₂O) to give the desired product (3 g, 22%) as a yellow solid. ESI-MS m / z: 257.05 [M+H] + (The methyl ester product was also isolated and used).

[0390] Example 1, step b

[0391]

[0392] A solution of the compound from step a (250 mg, 0.98 mmol), Pd(dppf)Cl2 (142 mg, 0.19 mmol), 2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaneborane-2-yl)pyridine (425 mg, 1.94 mmol), H2O (0.1 mL), and Cs2CO3 (950 mg, 2.91 mmol) in dioxane (3 mL) was stirred at 90 °C under a N2 atmosphere for 2 hours. The resulting solution was purified by reversed-phase C18 column chromatography (MeOH / 0.1% FA in H2O) to give the desired product (180 mg, 68%) as a white solid. ESI-MS m / z: 270.15 [M+H] + .

[0393] Example 1, step c

[0394]

[0395] An amine (30 mg, 0.075 mmol) and an acid (19.18 mg, 0.075 mgmol) were added to a 2-dram vial equipped with a stir bar, and the material was dissolved in DMF (0.2 M). Hunig base (0.053 mL, 0.30 mmol) was added, and the vial was cooled to 0 °C. HATU (43 mg, 0.113 mmol) was added, and the reaction mixture was stirred for 10 min, warmed to room temperature, and monitored by LCMS (1 h). The reaction mixture was diluted with EtOAc and quenched with water. The aqueous solution was extracted and concentrated using EtOAc and DCM / MeOH with a phase separator. The material was purified by preparative HPLC (20–90%, MeCN / water, 25 min) to give the title compound (23.6 mg, 48%) as a white solid. ESI-MS m / z: 651.25 [M+H] + .

[0396] Example 2

[0397]

[0398] Example 2, step a

[0399]

[0400] Methyl 8-hydroxyquinoline-6-carboxylate (1.500 g, 7.38 mmol) and potassium carbonate (2.040 g, 14.76 mmol) were added to a 50 mL round-bottom flask equipped with a stir bar, and the solids were dissolved in DMF (0.5 M). Then, tert-butyl 2-bromoacetate (1.308 mL, 8.86 mmol) was added, and the reaction mixture was stirred at 40 °C and monitored by LC-MS (2 h). The reaction mixture was cooled to room temperature, diluted with EtOAc, and quenched with water. The aqueous solution was extracted with EtOAc, and the combined organic matter was dried, filtered, and concentrated. The residue was analyzed by automated column chromatography (silica gel, in hexane with 50% ethyl acetate, R...). f =0.27) Purification yielded a white solid (1.93 g, 82%). ESI-MS m / z: 262.0 [M+H] + .

[0401] Example 2, step b

[0402]

[0403] A stir bar was added to a 100 mL round-bottom flask containing the product from step a (1.93 g, 6.08 mmol), and the solid was dissolved in DCM (0.5 M). The flask was cooled to 0 °C, and TFA (4.69 mL, 60.8 mmol) was added. The reaction mixture was stirred for 10 min, warmed to room temperature, and monitored by LCMS (more than 5.0 equivalents of TFA were added after 3 hours, for a total of 5.5 hours). The mixture was quenched with water and diluted with DCM. The solid precipitated. It was further diluted with DCM and stirred vigorously for 10 min. The solid was collected by filtration, washed several times with DCM, and dried under high vacuum to give a light brown, fluffy solid (2.21 g, 97%). ESI-MS m / z: 262.0 [M+H] + .

[0404] Example 2, step c

[0405]

[0406] Add step b (125 mg, 0.333 mmol) to a 40 mL vial equipped with a stir bar. Dissolve the solid in DMF and cool to 0 °C. Add DIPEA (407 μl, 2.332 mmol), followed by 1-methylcyclopropane-1-amine hydrochloride (124 mg, 1.148 mmol). Then add PyBOP (260 mg, 0.500 mmol) in a fraction. Stir the reaction mixture for 10 min, warm to room temperature, and monitor by LCMS (1.5 h). Dilute the reaction mixture with EtOAc and quench with water. Extract the aqueous layer with EtOAc and a phase separator, and concentrate the combined organic compounds. Purify the residue by automated column chromatography (silica gel, 0-20% methanol in dichloromethane) to give the title compound (98 mg, 82%). ESI-MS m / z: 216.0 [M+H] + .

[0407] Example 2, step d

[0408]

[0409] General hydrolysis instructions: In some cases, the reactants are heated to 45°C to force the material into solution and accelerate hydrolysis. After hydrolysis, the products are separated by precipitation. If no precipitate is formed, the product is extracted or the aqueous solution is concentrated (the material is dried and the crude product is used). The main MS of all these compounds... + m / z represents CC fragmentation: ESI-MS m / z: 202.0 [M+H] + .

[0410] A stir bar was added to a 20 mL vial containing the product from step c of Example 2 (9 mg, 0.312 mmol). The compound was dissolved in MeOH, THF, and water (0.2 M, 2:1:1). Lithium hydroxide hydrate (62 mg, 1.56 mmol) was added, and the reaction mixture was stirred at room temperature and monitored by LCMS. The stir bar was removed, and the vial was cooled to 0 °C. The reaction mixture was acidified with 2 M HCl, and the pH was adjusted to approximately 4–5 (if the acidity was too strong, 1 M NaOH was used). The product was extracted three times with 10% MeOH / DCM using a phase separator and concentrated. The product was dried under high vacuum to give the title compound (45 mg, 50%). ESI-MS m / z: 202.0 [M+H] + .

[0411] Example 2, step e

[0412]

[0413] The following examples were prepared using the same procedures as in step c of Example 1, using the corresponding acid and amine hydrochloride (25 mg) conjugate from step d. The residues were purified by Gilson preparative HPLC (20-90%, MeCN / water, 25 min) to give the title compound (8 mg, 20%). ESI-MS m / z: 682.2.

[0414] Intermediate 1

[0415]

[0416] Intermediate 1 step a

[0417]

[0418] In a vial, 4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxane (566 mg, 3.67 mmol), methyl 2-bromobenzo[d]thiazolium-6-carboxylate (500 mg, 1.837 mmol), PdCl2(dppf)DCM (75 mg, 0.092 mmol), and Na2CO3 (302 mg, 2.85 mmol) were dissolved in dioxane (6.9 mL) and water (2.3 mL). The reaction mixture was heated to 100 °C for 3 h. The reaction mixture was cooled to room temperature and diluted with water. The aqueous layer was washed with EtOAc. The combined organic layers were dried over MgSO4 and concentrated. The crude reaction mixture was purified by silica gel chromatography, eluting with 0–50% EtOAc / hexane, to give the title compound (180 mg, 0.821 mmol, 45%).

[0419] Intermediate 1, steps b and c

[0420]

[0421] In a vial, the compound from step a (120 mg, 0.547 mmol) was dissolved in MeOH (5.47 mL). Pd / C (5.82 mg, 0.055 mmol) was added and the vial was purged with H2. The reaction mixture was stirred at room temperature for 1 hour under H2. The reaction mixture was purged with N2 and filtered through diatomaceous earth. The crude reaction mixture was concentrated and purified by silica gel chromatography, eluting with 0–50% EtOAc / hexane to give the title compound (80 mg, 0.362 mmol, 66%).

[0422] Ester hydrolysis was carried out in a manner similar to step d of Example 2. The title compound was separated by precipitation to give the desired product (50 mg, 0.241 mmol, 67%).

[0423] Intermediate 2

[0424]

[0425] In a vial, potassium cyclopropyltrifluoro-4-borane (326 mg, 2.205 mmol), methyl 2-bromobenzo[d]thiazolium-6-carboxylate (200 mg, 0.735 mmol), palladium tetraoxide (42.5 mg, 0.037 mmol), and potassium phosphate (468 mg, 2.205 mmol) were dissolved in toluene (2.76 mL) and water (0.919 mL). The reaction mixture was heated to 100 °C and stirred overnight. The reaction mixture was cooled to room temperature and water was added. The aqueous layer was washed with EtOAc. The combined organic layers were dried over MgSO4 and concentrated. The crude reaction mixture was purified by silica gel chromatography, eluting with 0–50% EtOAc / hexane, to give the title compound (80 mg, 0.343 mmol, 47%).

[0426] Ester hydrolysis was carried out in a manner similar to step d of Example 2. The title compound was separated by precipitation to give the desired product (50 mg, 0.228 mmol, 67%).

[0427] Intermediate 3

[0428]

[0429] Intermediate step 3a

[0430]

[0431] A solution of methyl 2-amino-4-methoxybenzo[d]thiazol-6-carboxylic acid (2 g), CuBr2 (3.7 g, 16.78 mmol), and t-BuNO2 (1.7 g, 16.77 mmol) in CH3CN was stirred at room temperature for 16 hours under a nitrogen atmosphere. The resulting solution was diluted with water, extracted with EtOAc, and the organic layer was dried and concentrated. The resulting solution was purified by silica gel column chromatography (EtOAc in hexane) to give the desired product (1.6 g, 63%) as an orange solid. ESI-MS m / z: 301.90 [M+H] + .

[0432] intermediate step 3b

[0433]

[0434] The title compound was synthesized using the compound from intermediate 3 (step a) in a manner similar to that of intermediate 1. Ester hydrolysis was performed in a manner similar to that of step d in Example 2. The title compound was separated by precipitation to give the desired product (30 mg, 79%).

[0435] Intermediate 4

[0436]

[0437] The title compound was synthesized using the compound from intermediate 3 (step a) in a manner similar to that of intermediate 2. Ester hydrolysis was performed in a manner similar to that of step d in Example 2. The title compound was separated by precipitation to give the desired product (57 mg, 75%).

[0438] Intermediate 5

[0439]

[0440] Intermediate step 5a

[0441]

[0442] In a vial, methyl 6-amino-5-bromonicotinic acid (500 mg, 2.164 mmol) was slurried in DCM (3.4 mL) and pyridine (2.0 mL). The solution was cooled to 0 °C. Cyclopropylformyl chloride (590 μl, 6.49 mmol) was added dropwise until the solution became homogeneous. The reaction mixture was stirred for 2 hours. The reaction was terminated upon the addition of MeOH, and then concentrated. MeCN was added and the reaction mixture was evaporated to remove pyridine.

[0443] Dissolve in 1:1 THF / MeOH (5 mL) and cool to 0 °C. Slowly add NaOMe to MeOH (620 μL, 2.7 mmol) and stir for 30 min. Add AcOH (250 μL) to solidify the reaction mixture. Crumble the solid and add water. Stir the solution for another 10 min, then filter and wash the solid with water to give the title compound (546 mg, 1.825 mmol, 84%).

[0444] Intermediate step 5b

[0445]

[0446] In a vial, the compound from step a (400 mg, 1.337 mmol) was suspended in THF (5.35 mL), and Lawson's reagent (595 mg, 1.471 mmol) was added. The reaction mixture was heated to 65 °C overnight. The reaction mixture was cooled to room temperature and allowed to stand before concentration. The crude reaction mixture was purified by silica gel chromatography, eluting with 0–50% EtOAc / hexane to give the title compound (400 mg, 1.27 mmol, 95%).

[0447] Intermediate step 5, steps c and d

[0448]

[0449] In a vial, the compound from step b (434 mg, 1.377 mmol) was dissolved in DMSO (3.44 mL). Sodium hydride (60.6 mg, 1.515 mmol) was added, and the mixture was stirred for 5 minutes. The vial was then sealed and heated to 70 °C for 5 hours. After cooling, the reaction mixture was diluted with water and extracted with EtOAc. The aqueous phase was washed with EtOAc. The combined organic phases were washed with brine, dried over MgSO4, filtered, and concentrated. The crude reaction mixture was purified by silica gel chromatography, eluting with 0–50% EtOAc / hexane to give the title compound (130 mg, 0.555 mmol, 40%).

[0450] Ester hydrolysis was carried out in a manner similar to step d of Example 2. The title compound was separated by precipitation to give the desired product (50 mg, 0.232 mmol, 77%).

[0451] Intermediate 6

[0452]

[0453] Intermediate step 6a

[0454]

[0455] To a solution of 5-bromo-1H-indazole (500 mg, 2.54 mmol) in EtOAc (5 mL), 2,2-difluoro-2-(fluorosulfonyl)acetic acid (542 mg, 3.05 mmol) and K₂CO₃ (701 mg, 5.08 mmol) were added. The resulting mixture was stirred at room temperature for 2 hours and then evaporated. The residue was purified by combiflash chromatography, eluting with 0–30% EtOAc / hexane to give the desired product (380 mg, 60.6%) as a white foam. ESI-MS m / z: 248.9 [M+H] + .

[0456] Intermediate step 6, steps b and c

[0457]

[0458] To a DMF / EtOH (6 mL, 2:1) solution, add the compound from step a (240 mg, 0.866 mmol), Pd(OAc)₂ (23.34 mg, 0.104 mmol), 1,3-bis(diphenylphosphine)propane (86 mg, 0.208 mmol), and triethylamine (362 μl, 2.60 mmol). After degassing, perform CO₂ balloon experiments. The reaction mixture was heated at 80 °C for 12 hours, cooled to room temperature, and diluted with EtOAc (150 mL). The solution was washed with brine, dried, and purified by flash chromatography, eluting with 0–50% EtOAc / hexane to give the desired product (130 mg, 55.5%) as a white solid. ESI-MS m / z: 271.2 [M+H] + .

[0459] LiOH (112 mg, 4.68 mmol) was added to a solution of the compound from step b (120 mg, 0.468 mmol) in THF / water (5:1, 6 mL). The resulting mixture was stirred at 45 °C for 20 hours, and then most of the THF was evaporated. The remaining mixture was neutralized to pH approximately 3 by adding 1 M hydrochloric acid aqueous solution. The precipitated solid was filtered, washed with water, and dried to obtain the desired product (105 mg, 93.0%) as a white solid. ESI-MS m / z: 243.21 [M+H] + .

[0460] Intermediate step 7, steps a and b

[0461]

[0462] To a solution of ethyl 7-chloro-2-(difluoromethyl)-2H-indazole-5-carboxylate (200 mg, 0.728 mmol) in 1,4-dioxane / water (2.1 mL, 20:1), cyclopropylboronic acid (125 mg, 1.456 mmol), XPhos Pd G2 (28.6 mg, 0.036 mmol), and K3PO4 (773 mg, 3.64 mmol) were added. After degassing, the mixture was heated at 80 °C for 2 hours until the reaction was complete. The reaction mixture was diluted with EtOAc (100 mL), washed with brine, dried, and purified by flash chromatography, eluting with 0–30% EtOAc / hexane to obtain the desired product (93 mg, 45.6%) as a brown solid. ESI-MS m / z: 281.10 [M+H] + .

[0463] LiOH (77 mg, 3.21 mmol) was added to a solution of the compound from step a (90 mg, 0.321 mmol) in THF / water (5:1, 6 mL). The resulting mixture was stirred at 45 °C for 20 hours, and then most of the THF was evaporated. The remaining mixture was neutralized to pH approximately 3 by adding 1 M hydrochloric acid aqueous solution. The precipitated solid was filtered, washed with water, and dried to obtain the desired product (60 mg, 74.1%) as a brown solid. ESI-MS m / z: 253.20 [M+H] + .

[0464] Intermediate 8

[0465]

[0466] intermediate step 8a

[0467]

[0468] 3-Bromo-1,1-trifluoroprop-2-one (752 mg, 3.94 mmol) was added to a solution of 5-bromo-3-methoxypyridin-2-amine (400 mg, 1.970 mmol) in EtOH (6 mL). The resulting mixture was heated overnight at 80 °C. After evaporation of the solvent, the residue was purified by flash chromatography, eluting with 0–40% EtOAc / hexane, to give the title compound (300 mg, 51.6%) as a colorless oil. ESI-MS m / z: 296.20 [M+H] + .

[0469] intermediate 8 steps b and c

[0470]

[0471] The title compound was prepared according to the six-step intermediate procedure. The crude material was purified by flash chromatography, eluting with 0-50% EtOAc / hexane to give the desired product (200 mg, 68.2%) as a white solid. ESI-MS m / z: 289.17 [M+H] + .

[0472] LiOH (166 mg, 6.94 mmol) was added to a solution of the compound from step b (200 mg, 0.694 mmol) in THF / water (5:1, 6 mL). The resulting mixture was stirred at room temperature for 4 hours, and then most of the THF was evaporated. The remaining mixture was neutralized to pH approximately 3 by adding 1 M hydrochloric acid aqueous solution. The precipitated solid was filtered, washed with water, and dried to obtain the desired product (120 mg, 665%) as a white solid. ESI-MS m / z: 261.20 [M+H] + .

[0473] Intermediate 9

[0474]

[0475] Intermediate step 9a

[0476]

[0477] 2-Bromo-1-cyclopropylethane-1-one (1.99 g, 11.82 mmol) was added to a solution of 5-bromo-3-methoxypyridin-2-amine (2.0 g, 8.75 mmol) in EtOH (30 mL). The resulting mixture was heated at 80 °C for three days. After evaporation of the solvent, the residue was purified by flash chromatography, eluting with 0–100% EtOAc / hexane followed by 0–5% MeOH / DCM, to give the title compound (1.88 g, 85%) as a brown foam. ESI-MS m / z: 268.20 [M+H] + .

[0478] Example 671 Steps b and c

[0479]

[0480] BBr3 (3.74 mL, 3.74 mmol) was slowly added to a solution of the compound from step a (500 mg, 1.872 mol) in DCM (10 mL). The resulting mixture was stirred at room temperature for 20 hours, then quenched with water (2 mL). After dilution with DCM (50 mL), the organic layer was separated, dried, and evaporated. The residue was purified by Gilson preparative HPLC (20–90%, MeCN / water, 25 min) to give the desired product (40 mg, 8.44%) as a brown foam. ESI-MS m / z: 254.10 [M+H] + .

[0481] To a solution of the compound from step b (40 mg, 0.158 mmol) in DMF (2 mL), K₂CO₃ (43.7 mg, 0.316 mmol) and 2,2-difluoroethyl-4-methylbenzenesulfonate (187 mg, 0.79 mmol) were added. The resulting mixture was heated in a sealed container at 60 °C for 20 hours. After evaporation of the solvent, the residue was purified by flash chromatography, eluting with 0–30% EtOAc / hexane to give the desired product (35 mg, 85%) as a pale yellow foam. ESI-MS m / z: 318.99 [M+H] + .

[0482] intermediate 9 steps d and e

[0483]

[0484] Add the compound from step c (35 mg, 0.11 mmol), Pd(OAc)₂ (2.97 mg, 0.013 mmol), 1,3-bis(diphenylphosphine)propane (10.92 mg, 0.026 mmol), and triethylamine (46 μl, 0.33 mmol) to a DMF / EtOH (6 mL, 2:1) solution. After degassing, perform CO₂ balloon experiments. Heat the reaction mixture at 80 °C for 20 hours, cool to room temperature, and dilute with EtOAc (50 mL). Wash the solution with brine, dry, and purify by flash chromatography, eluting with 0–50% EtOAc / hexane to give the desired product (17 mg, 49.6%) as a white solid. ESI-MS m / z: 311.10 [M+H] + .

[0485] 2NNaOH (0.2 mL) was added to a solution of the compound from step d (17 mg, 0.055 mmol) in THF / water (5:1, 1 mL). The resulting mixture was stirred at room temperature for 40 hours, and then most of the THF was evaporated. The remaining mixture was neutralized to pH approximately 3 by adding 1 M hydrochloric acid aqueous solution. After extraction of the mixture with 10% MeOH / DCM (50 mL × 3), the organic layers were combined, dried, and evaporated to give the desired product (7 mg, 45.3%) as a white solid. ESI-MS m / z: 283.07 [M+H] + .

[0486] Intermediate 10

[0487]

[0488] Intermediate 10 steps a

[0489]

[0490] BH3-THF (325 mL, 325.10 mmol) was added dropwise to a solution of 2-amino-5-bromo-3-methoxybenzoic acid (4.00 g, 16.25 mmol) in THF (60 mL) under ice / water bath conditions, and the reaction mixture was stirred overnight at 50 °C. The mixture was cooled to 0 °C, quenched with MeOH, and concentrated. The residue was diluted with aqueous Na2CO3 solution and extracted with EA. The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to give a yellow oily crude product (4.4 g). ESI-MS m / z: 231.95 [M+H] + .

[0491] Intermediate 10, steps b and c

[0492]

[0493] The compound from step a (4.40 g, 18.96 mmol) and MnO2 (8.24 g, 94.80 mmol) were mixed in DCM (50 mL) and stirred overnight at room temperature. The resulting mixture was filtered, and the filter cake was washed with DCM. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EA (10:1) to give the desired product (2.9 g, 66%) as a red solid. ESI-MS m / z: 229.95 [M+H] + .

[0494] At 0°C, NaNO2 (1.09 g, 15.80 mmol) in water (5 mL) was added to a solution of the compound from step b (2.90 g, 12.60 mmol) in HCl (6 M) (30 mL). After 30 minutes, the ice bath was removed, and the reaction mixture was stirred at room temperature for 30 minutes. The solid was removed by filtration, the mother liquor was cooled to 0°C, and treated with NaN3 (0.82 g, 12.60 mmol) in water (5 mL). The ice bath was removed, and stirring was continued for 30 minutes. The resulting solid (3.8 g) was collected by filtration.

[0495] Intermediate 10 steps d

[0496]

[0497] The compound from step c (3.80 g, 14.84 mmol) and aminocyclopropane (1.27 g, 22.26 mmol) were treated with a molecular sieve in toluene (50 mL). The reaction mixture was stirred at room temperature for 2 hours, then heated under reflux overnight. After cooling to room temperature, the mixture was filtered through diatomaceous earth and the solution was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EA (1:1) to give the desired product (2.4 g, 60%) as a brown oil. ESI-MS m / z: 267.05 [M+H] + .

[0498] Intermediate step 10, steps e and f

[0499]

[0500] In a pressure vessel, DMF (10 mL), TEA (2.5 mL), Pd(OAc)₂ (0.40 g, 1.78 mmol), and dppp (1.48 g, 3.59 mmol) were added to a solution of the compound from step d (2.40 g, 8.98 mmol) in EtOH (10 mL). The mixture was pressurized to 15 atm with carbon monoxide overnight at 100 °C. The reaction mixture was cooled to room temperature and concentrated under vacuum. The residue was purified by silica gel column chromatography, eluting with PE / EA (2:1) to give the desired product (2.2 g, 94%) as a yellow oil. ESI-MS m / z: 261.10 [M+H] + .

[0501] The compound from step e (2.10 g, 8.07 mmol) and MeOH (30 mL) were added to a 100 mL round-bottom flask at room temperature. A solution of LiOH (1.93 g, 80.59 mmol) in H₂O (10 mL) was added, and the mixture was stirred at room temperature for 2 hours. The resulting mixture was concentrated under reduced pressure. The mixture was acidified to pH 5 with HCl (2 M aqueous solution). The product was collected by filtration and washed with water (1.28 g). ESI-MS m / z: 233.05 [M+H] + .

[0502] Intermediate 11, steps a, b, and c

[0503]

[0504] A solution of methyl 1H-indazole-5-carboxylate (3 g, 17.03 mmol) and F-TEDA-BF4 (12 g, 34.06 mmol) in DMF was stirred at 80 °C for 16 hours under a nitrogen atmosphere. The resulting solution was purified by reversed-phase C18 column chromatography (CH3CN / H2O) to give the desired product (1.5 g, 45%) as a yellow solid. ESI-MS m / z: 194.95 [M+H] + .

[0505] A solution of the compound from step a (1.5 g, 7.72 mmol), CH3I (2.2 g, 15.45 mmol), and Cs2CO3 (7.6 g, 23.17 mmol) in DMF was stirred at room temperature for 4 hours. The resulting solution was diluted with water, extracted with EA, and the organic layer was dried and concentrated. The resulting solution was purified by reversed-phase C18 column chromatography (CH3CN / H2O) to give the desired product (600 mg, 37%) as a yellow solid. ESI-MS m / z: 209.00 [M+H] + .

[0506] A solution of the compound from step b (600 mg, 2.88 mmol) and LiOH (690 mg, 28.82 mmol) in THF / H₂O = 10:1 (11 mL) was stirred at room temperature for 16 hours. The resulting mixture was concentrated under vacuum. The resulting solution was purified by reversed-phase C18 column chromatography (CH₃CN / H₂O) to give the desired product (211.9 mg, 38%) as a pale yellow solid. ESI-MS m / z: 195.05 [M+H] + .

[0507] Intermediate 12

[0508]

[0509] Intermediate 12 Step a

[0510]

[0511] At -5°C, 0.77 g (11.19 mmol) of NaNO₂ in H₂O (2 mL) was added dropwise to a mixture of 4-amino-3-fluoro-5-methylbenzylnitrile (1.60 g, 10.65 mmol) in HCl (8 M) (15 mL). The reaction mixture was stirred for 30 min, and 2-methyl-2-propanethiol (0.96 g, 10.64 mmol) was added to EtOH (5 mL) at 0°C and stirred for 1 h. The reaction was quenched with water / ice and extracted with EA. The combined organic layers were concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EA (20:1) to give the desired product (2.7 g, 99%) as an orange oil. ESI-MS m / z: 252.00 [M+H] + .

[0512] Intermediate 12 step b

[0513]

[0514] The mixture of the compound from step a (2.70 g, 10.74 mmol) and t-BuOK (9.64 g, 85.91 mmol) in DMSO (25 mL) was stirred at room temperature for 30 minutes. The resulting mixture was diluted with water and extracted with EA. The combined organic layers were concentrated under reduced pressure. The residue was purified by reversed-phase flash chromatography to give the desired product (940 mg, 54%) as a yellow solid.

[0515] Intermediate 12 steps c and d

[0516]

[0517] The compound from step b (900 mg, 5.58 mmol), ethane iodide (1.05 g, 6.7 mmol), and K₂CO₃ (1.54 g, 11.17 mmol) were stirred overnight in DMF (8 mL) at room temperature. The resulting mixture was poured into water and extracted with EA. The combined organic layers were concentrated under reduced pressure. The residue was purified by reversed-phase flash chromatography to give the desired product (510 mg, 48%) as an off-white solid.

[0518] At room temperature, the compound from step c (510 mg, 2.69 mmol), KOH (2.27 g, 40.46 mmol), EtOH (21 mL), and H₂O (7 mL) were added to a 100 mL round-bottom flask. The resulting mixture was stirred overnight at 80 °C. The mixture was concentrated under reduced pressure. The residue was acidified to pH 5 with HCl (2 M aqueous solution). The product was collected by filtration and washed with water to give the title compound (545 mg). ESI-MS m / z: 209.15 [M+H] + .

[0519] Intermediate 13, steps a and b

[0520]

[0521] A mixture of the compound from intermediate 12b (900 mg, 5.58 mmol), K₂CO₃ (1.54 g, 11.14 mmol), and iodoethane (1.05 g, 6.70 mmol) in DMF (8 mL) was stirred overnight at room temperature. The resulting mixture was poured into water and extracted with EA. The combined organic layers were concentrated under reduced pressure. The residue was purified by reversed-phase flash chromatography to give the desired product (280 mg, 26%) as an off-white solid.

[0522] At room temperature, the compound from step a (280 mg, 1.48 mmol), KOH (1.25 g, 22.28 mmol), EtOH (21 mL), and H₂O (7 mL) were added to a 100 mL round-bottom flask. The resulting mixture was stirred overnight at 80 °C. The mixture was concentrated under reduced pressure. The residue was acidified to pH 5 with HCl (2 M aqueous solution). The product was collected by filtration and washed with water to give the title compound (263 mg). ESI-MS m / z: 209.15 [M+H] + .

[0523] Intermediate 14

[0524]

[0525] Intermediate 14 step a

[0526]

[0527] 4-Bromo-2-chloro-6-methylpyridine (4.5 g, 22 mmol) and THF (100 mL) were added to a 250 mL three-necked round-bottom flask. The flask was cooled to -60 °C, and LDA (4.43 mL, 33 mmol) was added dropwise while stirring for 30 min. Then N-methoxy-N-methylcyclopropaneformamide (4.2 g, 33 mmol) was added. The resulting mixture was stirred at -60 °C under a nitrogen atmosphere for 1 h. The reaction was monitored by TLC. The reaction was quenched with saturated NH4Cl (aqueous solution), and the aqueous layer was extracted with EtOAc. The resulting mixture was concentrated under reduced pressure. The residue was purified by reversed-phase flash chromatography under the following conditions: column, C18 silica gel; mobile phase, MeCN in water, gradient of 10% to 50% over 25 min; detector, UV 254 nm, to give the desired compound (2.8 g, 47%) as an off-white solid. ESI-MS m / z: 273.95 [M+H] + .

[0528] Intermediate 14 step b

[0529]

[0530] At room temperature, the compound from step a (2.8 g, 10 mmol), NH₂OH, HCl (3.5 g, 51 mmol), NaOH (2 g, 51 mmol), and MeOH (30 mL) were added to a 100 mL round-bottom flask. The resulting mixture was stirred overnight at 60 °C under a nitrogen atmosphere. The reaction was monitored by LCMS. The aqueous layer was extracted with EtOAc. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EtOAc (1:1) to give the desired compound (2.2 g, 75%) as a white solid. ESI-MS m / z: 288.95 [M+H] + .

[0531] Intermediate 14 steps c and d

[0532]

[0533] At room temperature, the compound from step b (2.2 g, 8 mmol), TFAA (1.8 g, 8 mmol), TEA (3.8 g, 38 mmol), and DME (15 mL) were added to a 100 mL round-bottom flask. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 2 hours. The reaction was monitored by LCMS. The desired product could be detected by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was purified by reversed-phase flash chromatography under the following conditions: column, C18 silica gel; mobile phase, MeCN in water, gradient of 10% to 50% over 25 minutes; detector, UV 254 nm, yielding the desired compound (1.8 g, 87%) as an off-white solid. ESI-MS m / z: 270.95 [M+H] + .

[0534] The compound from step c (1.8 g, 7 mmol), ferrous chloride (84 mg, 0.7 mmol), and DME (10 mL) were added to a 40 mL vial at room temperature. The resulting mixture was stirred at 80 °C under a nitrogen atmosphere for 2 hours. The reaction was monitored by TLC. The aqueous layer was extracted with EtOAc. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EtOAc (1:1) to give the desired compound (1.2 g, 67%) as a white solid. ESI-MS m / z: 270.95 [M+H] + .

[0535] intermediate 14 steps e

[0536]

[0537] The compound from step d (1.2 g, 4.4 mmol), MeONa (716 mg, 13 mmol), and THF (10 mL) were added to a 40 mL vial at room temperature. The resulting mixture was stirred for 2 hours at room temperature under a nitrogen atmosphere. The reaction was monitored by LCMS. The desired product could be detected by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was purified by reversed-phase flash chromatography under the following conditions: column, C18 silica gel; mobile phase, MeCN in water, gradient from 10% to 50% over 25 minutes; detector, UV 254 nm, yielding the desired compound (800 mg, 68%) as an off-white solid. ESI-MS m / z: 267.00 [M+H] + .

[0538] Intermediate step 14, steps f and g

[0539]

[0540] At room temperature, the compound from step e (400 mg, 1.5 mmol), Pd(AcO)₂ (101 mg, 0.45 mmol), DPPP (371 mg, 0.9 mmol), EtOH (4 mL), Et₃N (1 mL), and DMF (4 mL) were added to a 30 mL pressure vessel reactor. The resulting mixture was stirred overnight at 100 °C under a CO atmosphere and at 10 atm. The reaction was monitored by TLC. The resulting mixture was filtered, and the filter cake was washed with EtOH. The filtrate was concentrated under reduced pressure. The residue was purified by reversed-phase flash chromatography under the following conditions: column, C18 silica gel; mobile phase, MeCN in water, gradient from 10% to 70% over 25 minutes; detector, UV 254 nm, yielding the desired compound (360 mg, 92%) as a white solid. ESI-MS m / z: 261.10 [M+H] + .

[0541] At room temperature, the compound from step f (360 mg, 1.4 mmol), LiOH (166 mg, 7 mmol), MeOH (5 mL), and H₂O (1 mL) were added to a 50 mL round-bottom flask. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 2 hours. The reaction was monitored by LCMS. The mixture was acidified to pH 6 with HCl (aqueous solution). The resulting mixture was concentrated under reduced pressure. The precipitated solid was collected by filtration and washed with water (3 × 3 mL) to give the desired compound (280 mg, 87%) as an off-white solid. ESI-MS m / z: 233.00 [M+H] + .

[0542] Intermediate 15

[0543]

[0544] Intermediate 15 Step a

[0545]

[0546] A solution of 5-bromo-3-methoxypyridine-2-amine (5 g, 24.63 mmol) and 2-bromo-1-cyclopropyl ethyl ketone (8.03 g, 49.26 mmol) in EtOH (20 mL) was stirred overnight at 80 °C. The residue was purified by reversed-phase flash chromatography to give the desired product (4.4 g, 67%) as a yellow solid. ESI-MS m / z: 267.00 [M+H] + .

[0547] Intermediate 15, steps b and c

[0548]

[0549] A solution of the compound from step a (4.4 g, 16.47 mmol), Pd(OAc)₂ (740 mg, 3.29 mmol), and dppp (2.7 g, 6.59 mmol) in DMF (12 mL), EtOH (12 mL), and TEA (3 mL) was stirred overnight at 100 °C under a CO atmosphere. The residue was purified by silica gel column chromatography, eluting with PE / EA, to give the desired product (3 g, 70%) as a yellow solid. ESI-MS m / z: 261.00 [M+H] + .

[0550] The solution of the compound from step b (3 g, 11.53 mmol) and LiOH (2.76 g, 115.25 mmol) in EtOH (10 mL) and H₂O (10 mL) was stirred at room temperature for 2 hours. The residue product was purified by reversed-phase flash chromatography to give 1.3 g of the desired product as a yellow solid. ESI-MS m / z: 233.00 [M+H] + .

[0551] Intermediate 16

[0552]

[0553] Intermediate 16 steps a

[0554]

[0555] A solution of 5-bromo-3-methoxypyridine-2-amine (4 g, 19.7 mmol) and methyl 3-bromo-2-oxopropionate (7 g, 39.4 mmol) in EtOH (10 mL) was stirred at 80 °C for 16 hours. The residue was purified by reversed-phase flash chromatography to give the desired product (4.6 g, 82%) as a yellow solid. ESI-MS m / z: 285.00 [M+H] + .

[0556] Intermediate step 16, steps b and c

[0557]

[0558] The compound from step a (3 g, 10.5 mmol) and a solution of DIBAL-H (30 mL) in THF (30 mL) were stirred at 0 °C for 1 hour. The residue was purified by silica gel column chromatography, eluting with DCM / MeOH (10 / 1) to give the desired product (1.6 g, 59%) as a yellow solid. ESI-MS m / z: 257.00 [M+H] + .

[0559] The solution of the compound from step b (1 g, 3.9 mmol) and MnO2 (3.4 g, 38.9 mmol) in DCM (20 mL) was stirred overnight at room temperature. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. 600 mg of the crude product was used directly in the next step. ESI-MS m / z: 255.00 [M+H] + .

[0560] Intermediate 16 steps d

[0561]

[0562] A solution of the compound from step c (600 mg, 2.4 mmol) and DAST (1 mL) in DCM (10 mL) was stirred at 0 °C for 2 hours. The aqueous layer was extracted with EA. The residue was purified by silica gel column chromatography, eluting with PE / EA, to give the desired product (400 mg, 61%) as a yellow solid. ESI-MS m / z: 277.00 [M+H] + .

[0563] Intermediate 16 steps e

[0564]

[0565] A solution of the compound from step d (400 mg, 1.4 mmol) and Pd(dppf)Cl2 (211 mg, 0.29 mmol) in DMF (10 mL), H2O (10 mL), and TEA (2 mL) was stirred at 100 °C and CO (15 atm) for 8 hours. The residue product was purified by reversed-phase flash chromatography to give the desired product (188 mg, 54%) as a yellow solid. ESI-MS m / z: 243.00 [M+H] + .

[0566] Intermediate 17

[0567]

[0568] Intermediate 17 Step a

[0569]

[0570] Add BINAP (687 mg, 1.10 mmol) and Pd(OAc)₂ (124 mg, 0.55 mmol) to a toluene solution of methyl 4-amino-3-methoxyphenylcarboxylate (500 mg, 2.76 mmol) and 2-bromocyclopent-1-ene-1-carboxaldehyde (1.45 g) under stirring. Heat the reaction mixture to 90 °C for 3 hours under a nitrogen atmosphere. Extract the resulting mixture with EtOAc (3 x 0 mL). Wash the combined organic layers with EtOAc (3 x 10 mL) and dry with anhydrous Na₂SO₄. Filter and concentrate the filtrate under reduced pressure. Purify the residue by silica gel column chromatography, eluting with PE / EtOAc (1:1) to give the desired product (600 mg, 79%) as a yellow oil. ESI-MS m / z: 276.00 [M+H] + .

[0571] Intermediate 17 steps b and c

[0572]

[0573] The solution of the compound from step a (500 mg) and In(TfO)₂ (3 g) in xylene (5 mL) was stirred at 130 °C under a N₂ atmosphere for 12 hours. The resulting mixture was concentrated under reduced pressure and used directly in the next step. ESI-MS m / z: 258.00 [M+H] + .

[0574] The solution of the compound from step c (1 g, 3.89 mmol) and LiOH (93 mg, 3.89 mmol) in H₂O (10 mL) and MeOH (10 mL) was stirred at room temperature for 12 hours. The crude product was purified by reversed-phase flash chromatography to give the desired product (184.7 mg) as a white solid. ESI-MS m / z: 244.00 [M+H] + .

[0575] Intermediate 18

[0576]

[0577] Intermediate 18, steps a and b

[0578]

[0579] A solution of (E)-2-bromocrotonaldehyde (10 g crude) and methyl 4-amino-3-methoxybenzoate (34 g, 134.24 mmol) in AcOH:HCl (2:3, 50 mL) was stirred at 100 °C for 1 hour under a nitrogen atmosphere. The resulting solution was concentrated to give the desired product (crude product), a yellow solid. ESI-MS m / z: 295.90 [M+H] + .

[0580] A solution of the compound from step a, ethyl iodine (21 g, 135.08 mmol), and Cs₂CO₃ (66 g, 202.62 mmol) in DMF was stirred overnight at room temperature. The resulting solution was diluted with water, extracted with EA (×3), the organic layer was dried, and concentrated. The resulting solution was purified by silica gel column chromatography (PE:EA) to give the desired product (1 g) as a yellow solid. ESI-MS m / z: 323.90 [M+H] + .

[0581] intermediate 18 steps c and d

[0582]

[0583] A solution of the compound from step b (1 g, 3.08 mmol), tricyclohexylphosphine (0.9 g, 3.08 mol), cyclopropylboronic acid (0.8 g, 9.25 mmol), Pd(OAc)₂ (140 mg, 0.62 mmol), and K₃PO₄ (2 g, 9.25 mmol) in toluene and H₂O was stirred overnight at 110 °C under a nitrogen atmosphere. The resulting mixture was concentrated under vacuum. The resulting solution was purified by silica gel column chromatography (PE:EA) to give the desired product as a yellow solid. ESI-MS m / z: 286.05 [M+H] + .

[0584] The compound from step c and a solution of LiOH (441 mg, 18.43 mmol) in MeOH (10 mL) and H₂O (10 mL) were stirred at room temperature for 1 hour. The resulting mixture was concentrated under vacuum. The resulting solution was purified by reversed-phase C18 column chromatography (CH₃CN / H₂O) to give the desired product (428 mg) as a yellow solid. ESI-MS m / z: 258.10 [M+H] + .

[0585] Intermediate 19, steps a and b

[0586]

[0587] A solution of intermediate 18b (200 mg, 0.62 mmol), L-proline (142 mg, 1.23 mmol), trichloromethylamine (117 mg, 1.23 mmol), and Cu₂O (176 mg, 1.23 mol) in EtOH was stirred at 80 °C for 16 hours. The resulting solution was diluted with water, extracted with EA (×3), and the organic layer was dried and concentrated. The resulting solution was purified by silica gel column chromatography (PE:EA) to give the desired product (70 mg, 41%) as a yellow solid. ESI-MS m / z: 279.95 [M+H] + .

[0588] A solution of the compound from step a (70 mg, 0.25 mmol) and LiOH (60 mg, 2.50 mmol) in THF (10 mL) and H₂O (1 mL) was stirred at room temperature for 1 hour. The resulting mixture was concentrated under vacuum. The resulting solution was purified by reversed-phase C18 column chromatography (MeCN / H₂O) to give the desired product (50 mg, 79%) as a yellow solid. ESI-MS m / z: 251.95 [M+H] + .

[0589] Intermediate 20, steps a and b

[0590]

[0591] A solution of intermediate 18b (200 mg, 0.62 mmol), methyl 2,2-difluoro-2-sulfoacetate (237 mg, 1.23 mmol), KF (72 mg, 1.23 mmol), and CuI (235 mg, 1.23 mol) in NMP was stirred at 120 °C for 4 hours. The resulting solution was diluted with water, extracted with EA (×3), the organic layer was dried, and concentrated. The resulting solution was purified by silica gel column chromatography (PE:EA) to give the desired product (140 mg, 72%) as a yellow solid. ESI-MS m / z: 313.95 [M+H] + .

[0592] A solution of the compound from step a (140 mg, 0.45 mmol) and LiOH (107 mg, 4.47 mmol) in MeOH (10 mL) and H₂O (1 mL) was stirred at room temperature for 1 hour. The resulting mixture was concentrated under vacuum. The resulting solution was purified by reversed-phase C18 column chromatography (CH₃CN / H₂O) to give the desired product (70 mg, 55%) as a white solid. ESI-MS m / z: 285.90 [M+H] + .

[0593] Intermediate 21

[0594]

[0595] Intermediate 21 step a

[0596]

[0597] At 5°C, NaNO2 (0.7 g in 5 mL of water) was added dropwise to a stirred solution of methyl 4-amino-3-iodobenzoate (2.7 g, 10 mmol) in HCl (6 mL) for 1 hour. Piperidine (1 mL) was then added dropwise to the mixture at 5°C. The resulting mixture was stirred at room temperature for another 1 hour. The mixture was extracted with EA, the combined organic layers were washed with water, and dried over anhydrous Na2SO4. The residue was purified by silica gel column chromatography (EtOAc in hexane) to give the desired product (2.7 g) as a yellow solid. ESI-MS m / z: 374.00 [M+H] + .

[0598] intermediate 21 step b

[0599]

[0600] Magnesium bromo(prop-1-yn-1-yl)bromo(4.3 g, 29.94 mmol) was added to a dry, N2-washed 50 mL Schlenk tube equipped with a magnetic stirrer and a diaphragm. The solution was cooled to -30 °C, and ZnBr2 (5.08 g, 22.56 mmol) was added dropwise to the reaction mixture. The reaction mixture was heated to room temperature for 30 min. The compound from step a (2 g, 5.36 mmol) was added, followed by (PPh3)4 (309 mg, 0.27 mmol). The reaction mixture was stirred at room temperature for 2 h and quenched with a saturated aqueous NH4Cl solution. The aqueous solution was extracted with EtOAc, dried, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (EtOAc in hexane) to give the desired product (2 g, 97%) as a yellow solid. ESI-MS m / z: 286.00 [M+H] + .

[0601] intermediate 21 step c

[0602]

[0603] A solution of the compound from step b (1.5 g, 5.26 mmol) and HBr in water (850 mg, 10.51 mmol) in acetone (10 mL) was stirred at room temperature for 2 hours. The resulting mixture was extracted with EtOAc, and the combined organic layers were washed with water and dried over anhydrous Na₂SO₄. The residue was purified by silica gel column chromatography (EtOAc in hexane) to give the desired product (900 mg, 61%) as a yellow solid. ESI-MS m / z: 281.00 [M+H] + .

[0604] intermediate 21 step d

[0605]

[0606] The solution of the compound from step c (900 mg, 3.2 mmol) and Pd / C (681 mg, 6.40 mmol) in MeOH (20 mL) was stirred for 2 hours at room temperature under a H2 atmosphere. The resulting mixture was filtered, and the solution was concentrated for direct use in the next step. ESI-MS m / z: 205.00 [M+H] + .

[0607] intermediate 21, steps e and f

[0608]

[0609] The compound from step d and a solution of MnO2 (1.5 g, 17.67 mmol) in THF (20 mL) were stirred at room temperature for 2 hours. The crude product was purified by reversed-phase flash chromatography to give the desired product (253 mg, 51%) as a yellow solid. ESI-MS m / z: 203.00 [M+H] + .

[0610] In a vial, the compound from step e (100 mg, 0.495 mmol) and lithium hydroxide (118 mg, 4.95 mmol) were dissolved in THF (2.2 mL), MeOH (2.2 mL), and water (0.55 mL). The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was diluted with water and the pH was adjusted to 3–4 with 1 M hydrochloric acid aqueous solution. The aqueous layer was washed with DCM and 9:1 DCM / MeOH. The combined organic layers were dried over MgSO4 and concentrated under reduced pressure to give the title compound (45 mg, 48%). ESI-MS m / z: 188.68 [M+H] + .

[0611] Intermediate 21a

[0612]

[0613] intermediate 21a step a

[0614]

[0615] The title compound was prepared using methyl 4-amino-3-iodo-5-methoxybenzoate (4 g, 13.03 g) in a manner similar to step a of intermediate 21. (3.5 g) ESI-MS m / z: 404.00 [M+H] + .

[0616] Intermediate 21a Step b

[0617]

[0618] The following examples were prepared using cyclopropylacetylene in a manner similar to step b of intermediate 21. The title compound was purified by silica gel chromatography to give a brown oily title compound (3.6 g). ESI-MS m / z: 342.00 [M+H] + .

[0619] intermediate 21a step c

[0620]

[0621] The following examples were prepared using a procedure similar to step c of intermediate 21, yielding the title compound (600 mg). ESI-MS m / z: 336.00 [M+H] + .

[0622] intermediate 21a step d

[0623]

[0624] The following examples were prepared using a procedure similar to step d of intermediate 21, yielding the title compound (500 mg). ESI-MS m / z: 261.00 [M+H] + .

[0625] intermediate 21a, steps e and f

[0626]

[0627] The following examples were prepared using procedures similar to steps e and f in intermediate 21, yielding the title compound (200 mg, 60%). ESI-MS m / z: 245.00 [M+H] + .

[0628] Intermediate 22

[0629]

[0630] Intermediate 22 step a

[0631]

[0632] At room temperature, a solution of 2-amino-5-bromo-3-methoxybenzaldehyde (2.2 g, 9.56 mmol), piperazine (2.5 g, 28.69 mmol), and methyl acetoacetate (2.2 g, 19.13 mmol) in MeOH was stirred for 1 hour. The resulting solution was diluted with water, extracted with EA (×3), and the organic layer was dried and concentrated. The resulting solution was purified by silica gel column chromatography (PE:EA) to give the desired product (2.5 g, 84%) as a yellow solid. ESI-MS m / z: 309.85 [M+H] + .

[0633] Intermediate step 22, steps b, c, and d

[0634]

[0635] A solution of the compound from step a (1 g, 28.69 mmol) in THF was treated with DIBAL-H (20 mL) for 1 hour at room temperature and under a nitrogen atmosphere. The resulting solution was diluted with water, extracted with EA (×3), the organic layer was dried, and concentrated. The resulting solution was purified by silica gel column chromatography (PE:EA) to give the desired product (1 g crude product) as a yellow solid. ESI-MS m / z: 281.90 [M+H] + .

[0636] A solution of the compound from step b (1 g crude), Pd(OAc)₂ (159 mg, 0.71 mmol), DPPP (585 mg, 1.42 mmol), and TEA (2.00 mL) in EtOH and DMF was stirred at 100 °C under a CO atmosphere (15 atm) for 16 hours. The resulting mixture was concentrated under vacuum. The resulting solution was purified by silica gel column chromatography (PE:EA) to give the desired product (600 mg crude) as a yellow solid. ESI-MS m / z: 276.10 [M+H] + .

[0637] A solution of the compound from step c (600 mg crude) and LiOH (522 mg, 21.79 mmol) in MeOH / H₂O = 10:1 (11 mL) was stirred at room temperature for 1 hour. The resulting mixture was concentrated under vacuum. The resulting solution was purified by reversed-phase C18 column chromatography (MeCN / H₂O) to give the desired product (504.7 mg) as a pale yellow solid. ESI-MS m / z: 247.95 [M+H]+ .

[0638] Intermediate 23

[0639]

[0640] intermediate 23 step a

[0641]

[0642] A solution of methyl 4-amino-3-hydroxybenzoate (5 g, 29.91 mmol) and NCS (4.8 g, 35.89 mmol) in CAN (20 mL) was stirred at 80 °C for 5 hours. The residue was purified by silica gel column chromatography, eluting with PE / EA (10%) to give the desired product (1.2 g) as a yellow solid. ESI-MS m / z: 202.00 [M+H] + .

[0643] Intermediate 23 steps b and c

[0644]

[0645] A solution of the compound from step a (1.2 g, 5.95 mmol) and BrCN (3.2 g, 29.74 mmol) in MeOH (42 mL) and H₂O (18 mL) was stirred at 50 °C for 48 hours. The resulting mixture was filtered, and the filter cake was washed with EA and water to give the desired product (700 mg, 52%). ESI-MS m / z: 227.00 [M+H] + .

[0646] A solution of the compound from step b (500 mg, 2.21 mmol) and LiOH (264 mg, 11.03 mmol) in THF (10 mL) and H₂O (10 mL) was stirred at room temperature for 1 hour. The mixture was acidified to pH 7 with HCl (1 M). The crude product was recrystallized from solution to give the desired product (231 mg, 49%) as a brown solid. ESI-MS m / z: 213.00 [M+H] + .

[0647] Intermediate 24

[0648]

[0649] Intermediate 24 steps a

[0650]

[0651] A mixture of 5-bromo-3-chlorobenzene-1,2-diamine (2.5 g, 11.29 mmol), cyclopropanecarboxylic acid (1.26 g, 14.64 mmol), HATU (4.29 g, 11.29 mmol), DIEA (2.9 g, 22.58 mmol), and DMF (50 mL) was stirred overnight at room temperature. The reaction was monitored by LCMS. The resulting mixture was poured into water and extracted with EOAc. The combined organic layers were washed with brine and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to give the desired product (6.7 g crude product) as a brown oil. ESI-MS m / z: 289.00 [M+H] + .

[0652] Intermediate step 24, steps b and c

[0653]

[0654] A solution of the compound from step a (6.7 g, 23.14 mmol) and acetic acid (60 mL) was stirred overnight at 100 °C. The resulting mixture was concentrated under vacuum. The residue was purified by reversed-phase flash chromatography under the following conditions: column, C18 silica gel; mobile phase, MeCN in water, gradient from 0% to 100% over 30 min; detector, UV 254 nm, to give the desired product (2 g, 32%) as a yellow solid. ESI-MS m / z: 271.00 [M+H] + .

[0655] NaH (0.35 g, 14.73 mmol) was added dropwise to a stirred solution of the compound from step b (2 g, 7.37 mmol) in 20 mL of DMF under a nitrogen atmosphere at 0 °C. The resulting mixture was stirred under a nitrogen atmosphere at the same temperature for 30 min. SEM-Cl (2.46 g, 14.76 mmol) was added dropwise and stirred at room temperature for 2 h. The reaction was quenched with water / ice at room temperature. The resulting mixture was extracted with EtOAc. The combined organic layers were washed with brine and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to give the desired product (2.6 g, 88%) as a yellow oil. ESI-MS m / z: 401.00 [M+H] + .

[0656] Intermediate step d and step e

[0657]

[0658] In a pressure vessel, DPPP (1 g, 2.59 mmol) and Pd(OAc)₂ (0.29 g, 1.29 mmol) were added to a solution of the compound from step c (2.6 g, 6.47 mmol) in DMF (10 mL), H₂O (10 mL), and TEA (2.5 mL). The mixture was pressurized to 15 atm with carbon monoxide and stirred at 100 °C for 2 days. The reaction mixture was cooled to room temperature and filtered to remove insoluble solids. The resulting mixture was concentrated under reduced pressure. The residue was purified by reversed-phase flash chromatography under the following conditions: column, C18 silica gel; mobile phase, MeCN in water (0.05% FA), gradient from 10% to 80% over 25 minutes; detector, UV 254 nm, to give crude desired product (1.6 g, 67%) as a yellow solid. ESI-MS m / z: 367.10 [M+H] + .

[0659] The solution of the compound from step d (1.6 g, 4.4 mmol) and HCl (6 M, 5 mL) in EtOH (6 mL) and H₂O (6 mL) was stirred at 80 °C for 16 hours. The crude product was purified by reversed-phase flash chromatography to give the desired product (300 mg, 29%) as a white solid. ESI-MS m / z: 237.00 [M+H] + .

[0660] Intermediate 25 steps a and b

[0661]

[0662] A solution of lithium chloride (0.5 M, in THF) (10.00 mL, 5.00 mmol) and methyl d3-iodide (1.0 M, in diethyl ether) (5.00 mL, 5.00 mol) was treated with zinc chloride (1.9 M, in 2-MeTHF) (1.316 mL, 2.500 mmol) (1.316 mL, 2.500 mmol). A solution of IPr PEPPSI (0.017 g, 0.025 mmol) and methyl 7-bromo-1H-indazole-5-carboxylic acid (0.128 g, 0.500 mmol) dissolved in 1.5 mL NMP was added dropwise, and the resulting reaction mixture was stirred overnight at room temperature. After completion, the reaction mixture was poured into a 5% aqueous citric acid solution and extracted with EtOAc. The organic extract was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The title compound (84 mg, 0.435 mmol, 87% yield) was purified by flash column chromatography on silica gel as an off-white solid. ESI-MS m / z: 193.7 [M+H] + .

[0663] The solution of step a (0.083 g, 0.430 mmol) in THF (5 mL) was treated with potassium trimethylsilanol (0.276 g, 2.148 mmol) and stirred at room temperature. After completion, the reaction was quenched with MeOH and silica gel, concentrated, and the resulting free-flowing mixture was directly purified by flash column chromatography on silica gel to give the title compound (0.075 g, 97% yield) as a white solid. ESI-MS m / z: 179.6 [M+H] + .

[0664] Intermediate 26

[0665]

[0666] Intermediate 26 steps a

[0667]

[0668] Selenium dioxide (183 mg, 1.654 mmol) and 2-methylbenzo[d]thiazol-6-carboxylic acid (213 mg, 1.102 mmol) were dissolved in 1,4-dioxane (2.205 mL) at room temperature. The reaction mixture was heated at 90 °C for 16 hours. The mixture was diluted with ethyl acetate and passed through a short silica gel stopper. The stopper was washed with ethyl acetate, and the resulting solution was concentrated and used as the crude product. ESI-MS m / z: 207.61 [M+H] + .

[0669] intermediate 26 step b

[0670]

[0671] The product from step a (37 mg, 0.179 mmol) was dissolved in DCM (1 mL), and DAST (83 μl, 0.625 mmol) was added at 0 °C. The reaction mixture was stirred at 0 °C for 2 hours. The reaction mixture was diluted with ethyl acetate and poured into 1 N HCl solution. The aqueous layer was extracted three times with ethyl acetate. The combined organic layers were dried and purified by flash chromatography to give the title compound (15 mg, 36.6% yield). ESI-MS m / z: 230.00 [M+H] + .

[0672] Intermediate 27 steps a and b

[0673]

[0674] A solution of lithium chloride (0.5 M, in THF) (6.17 mL, 3.08 mmol) and methyl d3-iodide magnesium (1.0 M, in diethyl ether) (3.08 mL, 3.08 mmol) was treated with zinc chloride (1.9 M, in 2-MeTHF) (0.812 mL, 1.542 mmol) (0.812 mL, 1.542 mmol). A solution of IPr PEPPSI (10.51 mg, 0.015 mmol) and ethyl 3-bromo-8-methoxy-2-methylquinoline-6-carboxylate (0.1 g, 0.308 mmol) dissolved in 1.5 mL NMP was added dropwise, and the resulting reaction mixture was stirred overnight at room temperature. After completion, the reaction mixture was poured into a 5% aqueous citric acid solution and extracted with EtOAc. The organic extract was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by flash chromatography gave the desired product (50 mg, 61.8% yield) as a yellow solid. ESI-MS m / z: 263.02 [M+H] + .

[0675] Ester hydrolysis was performed according to step d of Example 2 to obtain the desired compound.

[0676] Example 3

[0677]

[0678] Example 3, step a

[0679]

[0680] To a solution of (R)-3-cyclopropoxy-4-(2-hydroxypropoxy)benzoic acid (100 mg, 0.396 mmol) in THF (1.982 mL), 4-methylmorpholine (47.9 μl, 0.436 mmol) and tert-butylchlorodimethylsilane (65.7 mg, 0.436 mol) were added. After stirring the reaction mixture for 30 min, 1H-tetrazole (4405 μl, 1.982 mmol) and dibenzyldiisopropylphosphatidine (400 μl, 1.189 mmol) were added. The reaction mixture was stirred at room temperature for 5 h. The reaction mixture was cooled to 0 °C, and then hydrogen peroxide (121 μl, 1.189 mmol) was added. The reaction mixture was slowly heated to room temperature over 30 min. Then, saturated sodium sulfite was added. After vigorous stirring for 30 min, the mixture was partitioned between ethyl acetate and saturated sodium sulfite. The pH of the combined aqueous layers was adjusted to 3 with 1 N HCl, and the resulting suspension was extracted with ethyl acetate. The combined organic layers were dried over MgSO4. The solvent was removed, and the residue was purified by flash chromatography to give the title compound (183 mg, 90% yield).

[0681] Steps b and c of Example 3

[0682]

[0683] According to step c of Example 1, amide coupling was performed to obtain the desired compound.

[0684] In a 40 mL vial, dissolve step b (193 mg, 0.216 mmol) in ethyl acetate (2.159 mL). Purge the vial three times with nitrogen. Add palladium carbide (22.98 mg, 0.022 mmol) to one portion. Purge the vial with a hydrogen balloon, and stir the reaction mixture at 25 °C for 2 hours. Remove the hydrogen and purge with nitrogen. Filter the solution through diatomaceous earth, concentrate the residue, and purge by HPLC. ESI-MS m / z: 714.13 [M+H] + .

[0685] Example 4

[0686]

[0687] Example 4, step a

[0688]

[0689] The following examples were prepared according to step c of Example 1, using the acid coupling compound 2-formyl-8-methoxy-3-methylquinoline-6-carboxylic acid (55 mg).

[0690] Example 4, step b

[0691]

[0692] The product from step a (36 mg, 0.057 mmol) was dissolved in EtOH, and sodium borohydride (4.35 mg, 0.115 mmol) was added at 0 °C. The reaction mixture was stirred at room temperature for 30 min. The reaction mixture was quenched by careful addition of saturated NH4Cl. The aqueous layer was extracted with ethyl acetate, the combined organic layers were dried, and purified by HPLC to give the title compound (14.5 mg, 40.1% yield). ESI-MS m / z: 629.27 [M+H] + .

[0693] Example 5

[0694]

[0695] The product from step a of Example 4 (38 mg, 0.061 mmol) was then dissolved in tBuOH and water. 2-Methyl-2-butene (129 μl, 1.213 mmol) and sodium dihydrogen phosphate (72.8 mg, 0.606 mmol) were added at room temperature. Sodium chlorite (68.6 mg, 0.606 mmol) was then added. The reaction mixture was stirred at room temperature for 30 minutes. The reaction was quenched by adding 15% Na₂S₂O₃ solution, and the aqueous layer was extracted with ethyl acetate. The combined organic layers were dried and purified by HPLC to give the title compound (14 mg, 0.022 mmol, 35.9% yield). ESI-MS m / z: 643.15 [M+H] + .

[0696] Intermediate 28 steps a and b

[0697]

[0698] In a 100 mL round-bottom flask equipped with a stir bar, methyl 4-amino-3-methoxybenzoate (2.86 g, 15.79 mmol) and (E)-2-ethylbut-2-enal (5.468 g, 18.94 mmol) were added, followed by AcOH (23.68 mL) and HCl (15.79 mL). The mixture was then heated to 100 °C for 2 hours. The mixture was then cooled to room temperature and partially concentrated at 60 °C under vacuum to give a viscous black oil. The oil was treated with 40 mL of EtOH and 250 μL of sulfuric acid, followed by heating to 80 °C for 2 hours. The mixture was cooled to room temperature, neutralized by adding 2 mL of NEt3, and concentrated. The resulting residue was purified by automated silica gel column chromatography (0 to 45% ethyl acetate in hexane) and dried under high vacuum to give a colorless oily title compound (49.8 mg, 1%). ESI-MS m / z: 274.2 [M+H] + .

[0699] Ester hydrolysis was carried out in a manner similar to step d in Example 2. The crude product was a white solid and was continued without further purification. ESI-MS m / z: 246.0 [M+H] + .

[0700] Intermediate 29

[0701]

[0702] 2-Methyl-1H-indole-5-carboxylic acid (125 mg, 0.714 mmol) was added to a 4 mL vial, followed by DMF (1.427 mL). The mixture was cooled to 0 °C, and then NaH (86 mg, 2.141 mmol) was slowly added. The mixture was stirred for 15 minutes, and then methyl iodine (134 μL, 2.141 mmol) was added. After 2 hours, the mixture was neutralized by adding 1 M HCl (aqueous solution), and then diluted with 2 mL of dichloromethane. The layers were separated, and the aqueous layer was washed with 5 × 1 mL of dichloromethane. The combined organic matter was washed with water, dried over Na₂SO₄, filtered and concentrated, and then purified by automated silica gel column chromatography and dried under high vacuum to give the title compound (65 mg, 38%) as a white solid. ESI-MS m / z: 189.7 [M+H] + .

[0703] Intermediate 30

[0704]

[0705] Intermediate 30 step a

[0706]

[0707] 3-Methyl-1H-pyrazole-5-amine (1.00 g, 10.3 mmol), AcOH (20.6 mL), and diethyl butyryl-2-yndioate (1.65 mL, 10.3 mmol) were placed in 20 mL vials. The mixture was stirred at 23 °C for 48 hours, then added to hexane:EtOAc (1:1, 100 mL) and stirred for 1 hour. The solid was collected by filtration to give the title compound (1.78 g, 78%) as a yellow solid. ESI-MS m / z: 221.9 [M+H] + .

[0708] Intermediate 30, steps b and c

[0709]

[0710] Add step a (250 mg, 1.13 mmol), cesium carbonate (736 mg, 2.26 mmol), and DMF (2.3 mL) to a 20 mL vial, followed by MeI (212 μl, 3.39 mmol). Stir the mixture for 16 hours. Then dilute the mixture with 5 mL of water and 5 mL of dichloromethane. Separate the layers and wash the aqueous layer with 5 × 5 mL of dichloromethane. Wash the combined organic matter with 2 × 1 mL of water, then with 1 mL of brine, dry with Na₂SO₄, concentrate, and purify by automated silica gel chromatography to give the title compound (205 mg, 77%) as a yellow oil. ESI-MS m / z: 235.9 [M+H] + .

[0711] Ester hydrolysis was carried out in a manner similar to step d in Example 2. After acidification, the product was collected by filtration as a yellow solid, and the process was continued without further purification. ESI-MS m / z: 207.7 [M+H] + .

[0712] Intermediate 31

[0713]

[0714] 6-Methylnicotinic acid (333 mg, 2.43 mmol) and 2-bromo-1-cyclopropylethane-1-one (396 mg, 2.43 mmol) were added to a 20 mL vial, followed by the addition of acetonitrile (12.0 mL) and heating to 70 °C for 16 h. Triethylamine (1.35 mL, 9.71 mmol) was then added, and the mixture was stirred for another 3 h. The mixture was then diluted with 5 mL of water and 5 mL of EtOAc. The layers were separated, and the aqueous layer was washed with 4 × 5 mL of EtOAc. The combined organic compounds were washed with 2 mL of water brine, dried over Na₂SO₄, filtered, concentrated, and purified by automated silica gel chromatography to give the title compound (78 mg, 16%). ESI-MS m / z: 201.8 [M+H] + .

[0715] Intermediate 32, steps a and b

[0716]

[0717] Add 6-bromo-2-cyclopropylimidazo[1,2-b]pyridazine (128 mg, 0.538 mmol), palladium(II) acetate (12 mg, 0.054 mmol), and 1,3-bis(diphenylphosphine)propane (44.3 mg, 0.108 mmol) to an 8 mL vial, and purge the headspace of the vial with N2 for 30 min. Degas a mixture of DMF (1.434 mL), ethanol (0.717 mL), and triethylamine (225 μl, 1.613 mmol) by N2 injection for 30 min, and then add it to the solid mixture. Place the vial under a CO2 balloon and heat the mixture to 80 °C for 16 h. Add additional palladium(II) acetate (12 mg, 0.054 mmol), and heat the mixture to 100 °C for 4 h. Raise the reaction vessel and degas by N2 injection, then concentrate the mixture and purify it by automated silica gel chromatography to give the title compound (35 mg, 28%) as a yellow oil. ESI-MS m / z: 232.0 [M+H] + .

[0718] Ester hydrolysis was carried out in a manner similar to step d of Example 2. The product could not be extracted from the aqueous layer. The aqueous layer was concentrated to give a mixture of the title compound and lithium chloride. ESI-MS m / z: 204.1 [M+H] + .

[0719] Intermediate 33

[0720]

[0721] Intermediate 33 step a

[0722]

[0723] 1H-pyrrole-1-amine (1 mL, 13.15 mmol) and diethyl 2-(ethoxymethylene)malonate (3.11 mL, 15.39 mmol) were placed in a 20 mL vial and heated to 125 °C for 1 hour. The mixture was diluted with a mixture of biphenyl (1 g) and diphenyl ether (3 mL) and then heated to 200 °C for 2 hours. The mixture was cooled to room temperature and then purified by automated silica gel chromatography (0 to 10% ethyl acetate in cyclohexane) to give the title compound (1.43 g, 53%) as a yellow solid. ESI-MS m / z: 207.1 [M+H] + .

[0724] intermediate 33 step b

[0725]

[0726] The compound from step a (300 mg, 1.46 mmol), CCl4 (1.5 mL), diatomaceous earth (150 mg), and triphenylphosphine (1.13 g, 4.36 mmol) were placed in an 8 mL vial. The mixture was heated to 75 °C for 16 hours. The reaction mixture was diluted with EtOAc and filtered through diatomaceous earth. The filtrate was concentrated and purified by automated silica gel chromatography to give the desired ester (243 mg, 74%) and the hydrolysis product (19 mg, 7%) as a fluorescent yellow solid, which proceeded directly to the next step. ESI-MS m / z: 197.0 / 199.0 [M+H] + .

[0727] Intermediate 34

[0728]

[0729] The ester (131 mg, 0.583 mmol) and ammonium formate (184 mg, 2.92 mmol) from intermediate 33b were placed in 8 mL vials. A mixture of dioxane (2.92 mL) and ethanol (2.92 mL) was degassed by jetting with N2 for 30 min and then added to the mixture. Palladium on carbon (62.1 mg, 0.583 mmol, 10 wt%) was added, and the mixture was stirred for 1 h, then filtered through diatomaceous earth. The filtrate was concentrated and purified by automated silica gel chromatography to give a fluorescent yellow solid product (77 mg, 70%). ESI-MS m / z: 191.1 [M+H] + .

[0730] Ester hydrolysis was carried out in a manner similar to step d of Example 2. After acidification, the title compound was collected by filtration as a fluorescent yellow solid (19 mg, 32%). ESI-MS m / z: 163.0 [M+H] + .

[0731] Intermediate 35 steps a and b

[0732]

[0733] Methyl 4-amino-3-bromobenzoate (345 mg, 1500 mmol), Pd(PPh3)2Cl2 (211 mg, 0.300 mmol), and copper iodide (I) (28.6 mg, 0.150 mmol) were added to an 8 mL vial. The reaction vessel was evacuated and backfilled three times with N2. Toluene (2 mL) and water (1 mL) were degassed by spraying with N2 for 15 min, and the solid was then added to the vial. Ethynylcyclopropane (508 μL, 6.00 mmol) and triethylamine (627 μL, 4.50 mmol) were added, and the mixture was stirred at 70 °C for 16 h. The mixture was then diluted with water and EtOAc, the phases were separated, the organic layer was concentrated, and purified by automated silica gel chromatography (0 to 10% ethyl acetate in cyclohexane). The title compound (298 mg, 92%) was given as a colorless oil. ESI-MS m / z: 216.1 [M+H] + .

[0734] Dimethyl sulfoxide (1 mL) and KOtBu (155 mg, 1.38 mmol) were added to an 8 mL vial containing the compound from step a (99 mg, 0.46 mmol), and the mixture was heated to 100 °C for 30 min. The mixture was cooled to room temperature, diluted with water and EtOAc, and then acidified to pH 3 with 1 M HCl (aqueous solution). The layers were separated, and the aqueous layer was washed with 4 × 3 mL of EtOAc. The combined organic layers were then washed twice with water, followed by brine, dried over Na2SO4, and purified by automated silica gel chromatography (0–50% (1% AcOH in EtOAc) in cyclohexane) to give the title compound (45 mg, 49%) as a white solid. ESI-MS m / z: 202.1 [M+H] + .

[0735] Intermediate 36

[0736]

[0737] Intermediate 36 steps a

[0738]

[0739] Add 3.80 g (10.27 mmol) and 100 mL of acetone (from step b of Example 1) to a 100 mL round-bottom flask. Cool the solution to 0 °C, then add Jones' reagent (1.9–2.2 M, 10 mL) dropwise (under internal temperature monitoring). Heat the reaction to room temperature and monitor by LCMS (3 hours). Cool the reaction to 0 °C and use… iThe mixture was quenched with PrOH and stirred for 15 minutes. The reaction was diluted with EtOAc and water. The aqueous solution was extracted, the combined organic matter was dried, and the mixture was concentrated under reduced pressure to give a crude product (3.95 g, 99%) as a yellow solid. ESI-MS m / z: 383.80 [M+H] + .

[0740] Intermediate step 36b

[0741]

[0742] To a 100 mL round-bottom flask, add the compound from step a (3.95 g, 10.28 mmol), NH4Cl (1.10 g, 20.57 mmol), and the solid dissolved in DMF (20 mL). Add Hunig base (5.27 mL, 30.84 mmol), cool the reaction to 0 °C, and add HATU (7.82 g, 20.56 mmol). Heat the reaction to room temperature and monitor by LCMS (1 h). Dilute the reaction mixture with EtOAc and water. Extract the aqueous solution, dry the combined organic matter, and concentrate. Purify the substance by automated column chromatography (silica gel, 0-100% EtOAc in hexane) to give the title compound (3 g, 76%). ESI-MS m / z: 382.95 [M+H] + .

[0743] Intermediate step 36c

[0744]

[0745] To a 100 mL round-bottom flask, add the compound from step b (3.00 g, 7.83 mmol), 3,3,3-trifluoropropyl-1-en-2-ylboronic acid (2.19 g, 15.65 mmol), Pd(dppf)Cl2 (1.15 g, 1.56 mmol), and the material dissolved in dioxane (40 mL) and H2O (5 mL). Then add K2CO3 (3.25 g, 23.50 mmol), and stir the resulting mixture at 90 °C for 1 hour under a nitrogen atmosphere. Cool the mixture to room temperature, pour it into water, extract with EtOAc, and concentrate the combined organic matter under reduced pressure. Purify the residue by column chromatography (silica gel, 0-100% EtOAc in hexane) to give the desired product (2.3 g, 83%) as a brown oil. ESI-MS m / z: 350.90 [M+H] + .

[0746] Intermediate 36 steps d

[0747]

[0748] To a stirred solution of step c (5.00 g, 14.24 mmol) and 3-chloro-4-fluorophenylboronic acid (3.72 g, 21.33 mmol) in THF (80 mL), Na₂CO₃ (3.32 g, 31.33 mmol), H₂O (20 mL), and Pd(PPh₃)₂Cl₂ (1.00 g, 1.42 mmol) were added. The resulting mixture was stirred at 70 °C under a nitrogen atmosphere for 1 h. The reaction was monitored by TLC and LCMS. The resulting mixture was extracted with EtOAc, the combined organic layers were washed with brine, dried, and concentrated under reduced pressure. The residue was purified by automated column chromatography (silica gel, 0-75% EtOAc in hexane) to give the title compound (5.8 g, 99%) as a yellow solid. ESI-MS m / z: 401.05 [M+H] + .

[0749] Intermediate step 36 e

[0750]

[0751] AD-mix-β (33.82 g, 43.41 mmol) and methanesulfonamide (1.38 g, 14.47 mmol) were added to a 500 mL round-bottom flask equipped with a stir bar. The solid was dissolved in tBuOH (60 mL) and H₂O (100 mL). The flask was cooled to 0 °C, and the compound from step d (5.80 g, 14.47 mmol) was slowly added as a tBuOH solution (40 mL). The reaction mixture was allowed to warm to room temperature naturally and stirred for 16 hours. The reaction was quenched with sodium sulfite (0.25 g / g AD-mix), and diluted with water and EtOAc. The layers were separated, and the aqueous layer was extracted with EtOAc. The combined organic matter was washed with brine, dried over Na₂SO₄, filtered, concentrated, and purified by automated column chromatography (silica gel, 0-100% EtOAc in hexane) to give the title compound (5.48 g, 87%) as a white solid. ESI-MS m / z: 435. [M+H] + .

[0752] Intermediate step 36 f

[0753]

[0754] The compound from step e (4.70 g, 10.81 mmol) and DCM (80 mL) were added to a 250 mL round-bottom flask at room temperature. The solution was cooled to 0 °C, and then DMAP (264 mg, 2.16 mmol), TEA (3.28 g, 32.43 mmol), and TsCl (2.47 g, 12.97 mmol) were added sequentially. The resulting mixture was stirred at 0 °C for 1 hour. The mixture was acidified to pH 4 with 2 M HCl, and the aqueous solution was extracted with DCM. The combined organic layers were dried over anhydrous Na₂SO₄ and concentrated under reduced pressure to give a crude product (6.2 g, 97%) as a pale yellow solid. ESI-MS m / z: 589.15 [M+H] + .

[0755] Intermediate 36 steps g

[0756]

[0757] NH3 in MeOH (35 mL) was added to a 100 mL round-bottom flask at room temperature, and the compound from step g (6.20 g, 10.52 mmol) was slowly added. The resulting mixture was stirred at room temperature and monitored by LCMS (5 h). The mixture was dissolved in EtOAc, washed with saturated sodium bicarbonate × 3, brine, dried, and concentrated to give the title compound (2.93 g, 64%). ESI-MS m / z: 434.05 [M+H] + .

[0758] Intermediate 37

[0759]

[0760] Intermediate 37 step a

[0761]

[0762] N-fluoro-N-(phenylsulfonyl)benzenesulfonamide (963 mg, 3.05 mmol) and methyl 7-bromo-5-iodide-2,3-dihydrofluoro[2,3-c]pyridine-3-carboxylic acid (586 mg, 1.526 mmol) were added to a 20 mL vial equipped with a stir bar, and the solids were dissolved in THF (0.25 M). The vial was cooled to -78 °C, and LDA (916 μl, 1.831 mmol) was slowly added. The reaction mixture was stirred at -78 °C for 1 hour. After 1 hour at -78 °C, the reaction mixture gelled. The reaction mixture was heated to room temperature and stirred, and monitored by LCMS. The reaction mixture was diluted with EtOAc and quenched with water and saturated ammonium chloride. The mixture was extracted once with EtOAc and once with DCM / MeOH using a phase separator. The mixture was then concentrated. The substance was purified by automated column chromatography (0-100% EtOAc / cHex) to give the title compound (483 mg, 71%). ESI-MS m / z: 401.86 / 403.86 [M+H] + .

[0763] intermediate 37 step b

[0764]

[0765] Add a stir bar to a 20 mL vial containing step a (483 mg, 1.081 mmol) and dissolve the substance in THF / MeOH and water (1:1:1, 0.25 M). Add lithium hydroxide hydrate (113 mg, 2.70 mmol), stir the reaction mixture at room temperature, and monitor the reaction by LCMS.

[0766] The reactants were diluted with EtOAc and acidified to pH 2 with 2M HCl. Extraction with EtOAc and DCM / MeOH was performed using a phase separator. The substance was then ground with DCM / hexane to give an orange solid. (455 mg, 92%) ESI-MS m / z: 387.82 / 387.82 [M+H] + .

[0767] intermediate 37 step c

[0768]

[0769] The above compound was prepared in a manner similar to intermediate 36b. The crude compound was purified by automated column chromatography (silica gel, 0-100% EtOAc / cHex) to give the title compound (243 mg, 43%) as an orange oil. ESI-MS m / z: 386.33 [M+H] + .

[0770] Intermediate 38

[0771]

[0772] The above-described examples, as a mixture of diastereomers, were prepared in a similar sequence to intermediate 36 to obtain the desired amino alcohol (42 mg). ESI-MS m / z: 404.09 [M+H] + .

[0773] The compounds in Table 1 were prepared using a method similar to step c (PyBOP or HATU) of Example 1. Most compounds were purified by Gilson preparative HPLC, and some were purified by automated column chromatography (silica gel). Many arylic acid conjugates were prepared according to intermediates 1-38 or similar procedures. In some cases, the arylic acid conjugates were commercially available or prepared by the methods previously described.

[0774] Table 1

[0775]

[0776]

[0777]

[0778]

[0779]

[0780]

[0781]

[0782]

[0783]

[0784]

[0785]

[0786]

[0787]

[0788]

[0789]

[0790]

[0791]

[0792]

[0793]

[0794]

[0795] Intermediate 39

[0796]

[0797] intermediate 39 step a

[0798]

[0799] At 0 °C, 4-bromo-2-chloro-6-methylpyridine (3 g, 15 mmol), methyl cyclopropanecarboxylate (22 mL, 22 mmol), and THF (50 mL) were added to a 250 mL three-necked round-bottom flask. LiHMDS (3.7 g, 22 mmol) was added dropwise to the mixture. The resulting mixture was stirred at 0 °C for another 2 hours. The reaction was monitored by TLC. The reaction was quenched by the addition of saturated NH4Cl (aqueous solution). The aqueous layer was extracted with EtOAc. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / Et (7:1) to give the desired compound (3.3 g, 83%) as a pale yellow solid. ESI-MS m / z: 273.95 [M+H] + .

[0800] intermediate 39 step b

[0801]

[0802] At room temperature, the compound from step a (3.3 g, 12 mmol), NaOAc (5 g, 60 mmol), hydroxylamine hydrochloride (4.2 g, 60 mmol), and MeOH (20 mL) were added to a 40 mL vial. The resulting mixture was stirred overnight at 60 °C under a nitrogen atmosphere. The reaction was monitored by LC-MS. The aqueous layer was extracted with EtOAc. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / Et (5:1) to give the desired compound (2.3 g, 66%) as a white solid. ESI-MS m / z: 288.95 [M+H] + .

[0803] intermediate 39 steps c and d

[0804]

[0805] At 0 °C, the compound from step b (1.1 g, 3.8 mmol) and DME (10 mL) were added to a 20 mL vial. TFAA (0.9 g, 4 mmol) was added dropwise to the mixture. The resulting mixture was stirred at room temperature for another 3 hours. The reaction was monitored by TLC. The residue was purified in water with MeCN by reversed-phase flash chromatography to give the desired compound (600 mg, 58%) as a white solid. ESI-MS m / z: 270.95 [M+H] + .

[0806] At room temperature, the compound from step c (600 mg, 2.2 mmol), ferrous chloride (56 mg, 0.44 mmol), and DME (10 mL) were added to a 20 mL vial. The resulting mixture was stirred at 80 °C under a nitrogen atmosphere for 2 h. The reaction was monitored by TLC. The aqueous layer was extracted with EtOAc. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / Et (5:1) to give the desired compound (400 mg, 67%) as a white solid. ESI-MS m / z: 270.95 [M+H] + .

[0807] intermediate 39 steps e

[0808]

[0809] At 0 °C, the compound from step d (400 mg, 1.5 mmol), cyclopropanol (257 mg, 4.4 mmol), NaH (71 mg, 3 mmol), and DMF (10 mL) were added to a 50 mL round-bottom flask. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 2 hours. The reaction was quenched by adding saturated NH4Cl (aqueous solution). The aqueous layer was extracted with EtOAc and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / Et (5:1) to give the desired compound (360 mg, 83%) as a white solid. ESI-MS m / z: 293.00 [M+H] + .

[0810] intermediate 39 steps f

[0811]

[0812] In a pressure vessel, Pd(OAc)₂ (83 mg, 0.4 mmol), DPPP (304 mg, 0.74 mmol), and TEA (1 mL) were added to a solution of the compound from step e (360 mg, 1.2 mmol) in EtOH (4 mL) and DMF (4 mL). The mixture was pressurized to 10 atm with carbon monoxide overnight at 100 °C. The reaction mixture was cooled to room temperature and filtered to remove insoluble solids. The residue was purified by reversed-phase flash chromatography to give the desired compound (300 mg, 85%) as a white solid. ESI-MS m / z: 287.10 [M+H] + .

[0813] intermediate 39 steps g

[0814]

[0815] At room temperature, the compound from step f (298 mg, 1 mmol), LiOH (125 mg, 5.2 mmol), MeOH (5 mL), and H₂O (1 mL) were added to a 50 mL round-bottom flask. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 2 hours. The reaction was monitored by LC-MS. The mixture was acidified to pH 6 with HCl (aqueous solution). The resulting mixture was concentrated under reduced pressure. The residue was purified by reversed-phase flash chromatography to give the desired compound (245 mg, 91%) as an off-white solid. ESI-MS m / z: 259.10 [M+H] + .

[0816] Intermediate 40

[0817]

[0818] Intermediate 40, steps a and b

[0819]

[0820] A mixture of methyl 4-amino-3-methoxybenzoate (50.00 g, 275.95 mmol) and acrolein (30.94 g, 551.87 mmol) in HCl (100 mL) and AcOH (100 mL) was stirred at 100 °C under a nitrogen atmosphere for 1 h. The resulting mixture was concentrated under vacuum and used directly in the next step. ESI-MS m / z: 204.10 [M+H] + .

[0821] A mixture of the compound from step a, ethyl iodine (76.76 g, 492.16 mmol), and Cs₂CO₃ (240.52 g, 738.20 mmol) in DMF was stirred overnight at room temperature. The resulting mixture was filtered, and the filter cake was washed with ethyl acetate (3 × 300 mL). The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with hexane / EA (1:1) to give a crude product (25 g) as a yellow solid. ESI-MS m / z: 232.05 [M+H] + .

[0822] intermediate 40 steps c

[0823]

[0824] A mixture of ethyl 8-methoxyquinoline-6-carboxylate (14.00 g, 60.54 mmol) and NIS (54.48 g, 242.16 mmol) in AcOH (300 mL) was stirred at 100 °C for 16 hours under nitrogen atmosphere. The aqueous layer was extracted with EA. The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography and reversed-phase flash chromatography to give the desired product (5.5 g, 14%) as a yellow solid. ESI-MS m / z: 358.00 [M+H] + .

[0825] intermediate 40 steps d

[0826]

[0827] A mixture of the compound from step c (4.30 g, 12.04 mmol) and (1,10-phenanthroline)(trifluoromethyl)copper(I) (5.65 g, 18.06 mmol) in DMF (70 mL) was stirred at 110 °C for 4 hours under nitrogen atmosphere. The mixture was cooled to room temperature. The resulting mixture was poured into water and extracted with EA. The combined organic layers were washed with brine and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / Et (4:1) to give the desired product (2.6 g, 72%) as a pale yellow solid. ESI-MS m / z: 300.15 [M+H] + .

[0828] intermediate 40 steps e

[0829]

[0830] The compound from step d (2.60 g, 8.69 mmol) and LiOH (2.08 g, 86.85 mmol) were mixed in MeOH (30 mL) and H₂O (10 mL) and stirred at room temperature for 2 hours. The mixture was acidified to pH 4 with HCl (2 M aqueous solution). The precipitated solid was collected by filtration and washed with water to give the desired product (2.29 g, 97%) as a white solid. ESI-MS m / z: 271.95 [M+H] + .

[0831] Intermediate 41

[0832]

[0833] The following compounds were prepared in a manner similar to intermediate 39. The residue was purified by reversed-phase flash chromatography to give the desired product (40 mg, 89%) as a pale yellow solid. ESI-MS m / z: 247.10 [M+H] + .

[0834] Intermediate 42

[0835]

[0836] The following compounds were prepared in a manner similar to intermediate 10. The crude residue was purified by reversed-phase flash chromatography to give the desired product (130 mg, 72%) as a white solid. ESI-MS m / z: 247.10 [M+H] + .

[0837] Intermediate 43

[0838]

[0839] Intermediate 43, steps a and b

[0840]

[0841] 400 mg of methyl 3-methoxyquinoline-6-carboxylate (1.841 mmol) was added to a 40 mL vial equipped with a stir bar and dissolved in DCM. mCPBA (883 mg, 3.68 mmol) was added, and the reaction mixture was stirred overnight for 20 hours. The reaction mixture was diluted with DCM and quenched with sodium thiosulfate. DCM / MeOH extraction was performed using a phase separator, and the organic matter was concentrated. The residue was purified by automated column chromatography (silica gel, 0-20% methanol in DCM) to give the desired compound (361 mg, 84%). ESI-MS m / z: 234.08 [M+H] + .

[0842] Add the reaction mixture from step a (143 mg, 0.613 mmol) to a 50 mL round-bottom flask equipped with a stir bar, and dissolve the solid in DCM. Add POCl3 (114 μl, 1.226 mmol), and heat the flask to 45 °C for 2 hours. Dilute the reaction mixture with DCM and quench with water and saturated sodium bicarbonate. Extract with DCM and DCM / MeOH using a phase separator, and concentrate the organic matter. Purify the residue by automated column chromatography (silica gel, 0-90% EtOAc in hexane) to give the desired compound (102 mg, 66%). ESI-MS m / z: 252.04 [M+H] + .

[0843] intermediate 43 step c

[0844]

[0845] Add Pd(PPh3)4 (4.59 mg, 3.97 μmol), step b (40 mg, 0.159 mmol), potassium carbonate (65.9 mg, 0.477 mmol), and methylboric acid (47.6 mg, 0.795 mmol) to a 20 mL vial equipped with a stir bar. Purge the vial with N2 for 10 min. Add 1,4-dioxane (1.060 mL) and heat the reaction mixture to 100 °C for 18 h. Dilute the reaction mixture with EtOAc and quench the reaction with water. Extract with EtOAc and DCM / MeOH using a phase separator and concentrate the organic matter. Purify the residue by automated column chromatography (silica gel, 0-80% EtOAc in hexane) to obtain the desired compound (36 mg, 98%). ESI-MS m / z: 232.10 [M+H] + .

[0846] Intermediate 43 step d

[0847]

[0848] Add step c (36 mg, 0.156 mmol) to a 20 mL vial equipped with a stir bar and dissolve the substance in THF:MeOH:water (1:1:1, 0.2 M). Add lithium hydroxide hydrate (32.7 mg, 0.778 mmol) and heat the reaction mixture to 45 °C, monitoring the reaction by LCMS. Cool the vial to 0 °C and acidify to pH 4 with 2 M HCl. No precipitate formed; concentrate the organic matter under vacuum. A solid precipitate formed. Cool to 0 °C for further precipitation. Collect the solid by vacuum filtration and dry under high vacuum to give the desired product (18 mg, 53%) as a white solid. ESI-MS m / z: 217.82 [M+H] + .

[0849] Example 233

[0850]

[0851] (S)-7-(4-fluorophenyl)-3-(hydroxymethyl)-5-((S)-1,1,1-trifluoro-2-hydroxy-3-(8-methoxy-2,3-dimethylquinoline-6-carboxamido)propyl-2-yl)-2,3-dihydrofluoro[2,3-c]pyridine-3-carboxamide hydrochloride (20 mg, 0.032 mmol) was dissolved in DCM (1 ml), and DAST (1 equivalent, 4.2 μl, 0.032 mmol) was added at 0 °C. The reaction mixture was stirred at 0 °C for 2 hours. The reaction mixture was quenched with MeOH, the solution was concentrated, and purified by HPLC to give (S)-3-(chloromethyl)-7-(4-fluorophenyl)-5-((S)-1,1,1-trifluoro-2-hydroxy-3-(8-methoxy-2,3-dimethylquinoline-6-carboxamido)propyl-2-yl)-2,3-dihydrofluoro[2,3-c]pyridine-3-carboxamide (3.68 mg, 5.69 μmol, yield 17.88%). ESI-MS m / z: 647.074 [M+H] + .

[0852] Intermediate 44

[0853]

[0854] The above-described examples, as a mixture of diastereomers, were prepared in a similar sequence to intermediate 36 to obtain the desired amino alcohol (320 mg). ESI-MS m / z: 426.04 [M+H] + .

[0855] Intermediate 45

[0856]

[0857] Intermediate 45 step a

[0858]

[0859] To a 250 mL round-bottom flask, add methyl 7-bromo-5-iodo-2,3-dihydrofluoro[2,3-c]pyridine-3-carboxylic acid (586 mg, 1.526 mmol), tetrahydrofuran (85 mL), and ((chloromethoxy)methyl)benzene (6.94 mL, 50.6 mmol). Cool the mixture to -78 °C and add lithium diisopropylamide (2 M solution in tetrahydrofuran / heptane / ethylbenzene, 9.27 mL, 18.54 mmol) dropwise over 15 minutes. Stir the mixture at -78 °C for 1 hour, then heat it to 23 °C for 30 minutes. Dilute the reaction mixture with EtOAc (100 mL) and water (50 mL). Separate the layers and extract the aqueous layer with EtOAc (3 × 50 mL). Dry the combined organic matter with Na₂SO₄, filter, and concentrate. The crude compound was purified by automated column chromatography (silica gel, 0-10% EtOAc in hexane) to give the title compound (4.69 g, 55%). ESI-MS m / z: 503.55 / 505.47 [M+H] + .

[0860] intermediate 45 step b

[0861]

[0862] Add the compound from step a (4.69 g, 9.30 mmol) and chloroform (47 mL) to a 100 mL round-bottom flask. Cool the mixture to 0 °C and add methanesulfonic acid. Heat the mixture to room temperature with stirring for 2 hours, monitoring by LCMS. Dilute the reaction mixture with EtOAc and water. Extract the aqueous solution, wash the combined organic matter with NaHCO3, dry with Na2SO4, filter and concentrate. Purify the substance by automated column chromatography (silica gel, 0-20% EtOAc in hexane) to give the title compound (2.25 g, 58%). ESI-MS m / z: 413.46 / 415.42 [M+H] + .

[0863] intermediate 45 step c

[0864]

[0865] Add the compound from step b (2.12 g, 5.12 mmol) to a 50 mL round-bottom flask, followed by dichloromethane (21 mL), and then 2,6-dimethylpyridine (890 μL, 7.68 mmol). Cool the mixture to -78 °C, and then add trifluoroformic anhydride (1 M solution in dichloromethane, 5.63 mL, 5.63 mmol) dropwise over 10 minutes. Stir the mixture at -78 °C for 30 minutes and monitor by LCMS. Heat the mixture to 0 °C and then dilute with water and dichloromethane. Separate the layers, wash the aqueous layer with dichloromethane (3 × 25 mL), wash the combined organic matter with NaHCO3 (saturated aqueous solution) (2 × 10 mL), and then concentrate. Dissolve the resulting oil in tetrahydrofuran (2 mL) and cool to 0 °C. Tetrabutylammonium fluoride (1M solution in tetrahydrofuran, 5.37 mL, 5.37 mmol) was added to the mixture, and the mixture was heated to room temperature for 19 hours. The reaction mixture was diluted with EtOAc and water. The aqueous solution was extracted with EtOAc (3 × 20 mL), and the combined organic compounds were dried over Na₂SO₄ and concentrated. The substance was purified by automated column chromatography (silica gel, 0–10% EtOAc in hexane) to give the title compound (2.00 g, 94%). ESI-MS m / z: 415.47 / 417.40 [M+H] + .

[0866] intermediate 45 steps d

[0867]

[0868] Ammonia (7M solution in MeOH, 20.6 mL, 144 mmol) was added to a 100 mL round-bottom flask containing the compound from step c (2.00 g, 4.81 mmol). The mixture was heated to 45 °C for 1.5 h, monitored by LCMS. The mixture was cooled to room temperature, concentrated, and the yellow solid was transferred to the next step without further purification (1.93 g, >99%). ESI-MS m / z: 400.46 / 402.41 [M+H] + .

[0869] Intermediate 45

[0870]

[0871] The above compound, as a mixture of diastereomers, was prepared in a similar sequence to intermediate 36 to give the desired amino alcohol (1.056 g, 85%). ESI-MS m / z: 417.97 [M+H] + .

[0872] Intermediate 46

[0873]

[0874] Intermediate 46 Step a

[0875]

[0876] Methyl 7-bromo-5-iodo-2,3-dihydrofluoro[2,3-c]pyridine-3-carboxylic acid (2.044 g, 5.32 mmol) was added to a 250 mL round-bottom flask equipped with a stir bar and dissolved in THF (21.29 mL). The flask was cooled to -78 °C, and (bromodifluoromethyl)trimethylsilane (1.656 mL, 10.65 mmol) was added, followed by dropwise addition of lithium diisopropylamide (2.93 mL, 5.86 mmol). The reaction mixture was stirred at -78 °C for 1 hour and then warmed to room temperature. After 1.5 hours, the reaction mixture was diluted with EtOAc and quenched with saturated ammonium chloride. The aqueous solution was extracted with EtOAc, and the organic matter was dried, filtered, and concentrated. The crude substance was purified by automated column chromatography (silica gel, 0-30% EtOAc in hexane, large column volume) to give the desired product (295.6 mg, 13%). ESI-MS m / z:433.87 / 435.86[M+H]+.

[0877] Intermediate step 46b

[0878]

[0879] A stir bar was added to a 40 mL vial containing step a (393.9 mg, 0.908 mmol), followed by the addition of ammonia (3890 μl, 27.2 mmol). The reaction was heated to 45 °C and monitored by LCMS (completed in 1.5 h). The stir bar was removed, and the reaction mixture was concentrated. The mixture was milled with DCM to give a light brown solid as the desired product (360 mg, 96%). ESI-MS m / z: 418.87 / 420.87 [M+H]+.

[0880] Intermediate 46

[0881]

[0882] The following compounds were prepared in a manner similar to intermediate 36 to obtain the desired amino alcohol (105 mg, 92%) as a mixture of diastereomers. ESI-MS m / z: 436.11 [M+H]+.

[0883] Table 2 below contains examples prepared using a method similar to step c (PyBOP or HATU) of Example 1. Amine conjugates were prepared as mixtures of diastereomers according to intermediates 44-46 or by similar procedures. Most compounds were purified by Gilson preparative HPLC. If described as a single diastereomer, the diastereomer was separated by Gilson preparative HPLC. Aryl acid conjugates were prepared according to intermediates 1-46 or by similar procedures with slight modifications, and also according to the procedure described in U.S. Patent Application No. 16 / 930622.

[0884] Table 2

[0885]

[0886]

[0887]

[0888]

[0889]

[0890]

[0891]

[0892] Table 3 below contains examples prepared using methods similar to step c (PyBOP or HATU) of Example 1. Most compounds were purified by Gilson preparative HPLC, and some were purified by automated column chromatography (silica gel). Aryl acid conjugates were prepared according to intermediates 1-43 or by similar procedures with slight modifications, and also according to the procedures described in U.S. Patent Application No. 16 / 930622.

[0893] Table 3

[0894]

[0895]

[0896]

[0897]

[0898] Table 4 below contains examples prepared using methods similar to step c (PyBOP or HATU) of Example 1. Most compounds were purified by Gilson preparative HPLC, and some were purified by automated column chromatography (silica gel). Aromatic acid conjugates are commercially available.

[0899] Table 4

[0900]

[0901]

[0902]

[0903]

[0904]

[0905]

[0906]

[0907]

[0908]

[0909]

[0910]

[0911]

[0912]

[0913]

[0914] Intermediate 47

[0915]

[0916] Intermediate 47 step a

[0917]

[0918] A mixture of (R)-7-bromo-5-iodo-3-methyl-2,3-dihydrofluoro[2,3-c]pyridine-3-carboxamide (3.5 g, 9.14 mmol), pinacol cyclopropyl vinylboronate (2.13 g, 10.96 mmol), Pd(dppf)Cl2 (1.34 g, 1.83 mmol), and K2CO3 (3.16 g, 22.84 mmol) in dioxane (18 mL) and H2O (2 mL) was stirred at 90 °C under a nitrogen atmosphere for 2 h. The reaction was monitored by LCMS. The resulting mixture was filtered, and the filter cake was washed with EA. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with 50% ethyl acetate in hexane) to give the desired product (2.2 g, 74%) as a brown oil. ESI-MS m / z: 322.90 [M+H] + .

[0919] Intermediate 47 step b

[0920]

[0921] A mixture of the compound from step a (1.5 g, 4.64 mmol), 4-fluorophenylboronic acid (0.97 g, 6.96 mmol), Pd(PPh3)2Cl2 (0.33 g, 0.46 mmol), and Na2CO3 (1.23 g, 11.60 mmol) in THF (10 mL) and H2O (2.5 mL) was stirred at 70 °C under a nitrogen atmosphere for 1 h. The reaction was monitored by TLC. The resulting mixture was filtered, and the filter cake was washed with EA. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with 33% ethyl acetate in hexane) to give the desired product (1.2 g, 76%) as a yellow oil. ESI-MS m / z: 339.05 [M+H] + .

[0922] intermediate 47 step c

[0923]

[0924] The solution of the compound from step b (1.2 g, 3.54 mmol) in t-BuOH (40 mL) and H₂O (40 mL) was cooled to 0 °C. Methanesulfonamide (0.34 g, 3.54 mmol) and AD-mix-β (8.29 g, 10.64 mmol) were added, and the reaction was stirred overnight at room temperature, monitored by LCMS. The resulting mixture was poured into water and extracted with ethyl acetate. The combined organic layers were concentrated under reduced pressure. The residue was purified by reversed-phase flash chromatography (MeCN in water, 0.1% FA) to give the desired product (1.1 g, 83%) as a white solid. ESI-MS m / z: 373.25 [M+H] + .

[0925] Intermediate 47 step d

[0926]

[0927] The solution of the compound from step c (1.7 g, 4.56 mmol) in DCM (70 mL) was cooled to 0 °C. TsCl (1.31 g, 6.84 mmol), TEA (1.39 g, 13.69 mmol), and DMAP (0.22 g, 1.82 mmol) were added, and the reaction mixture was stirred at room temperature for 3 hours. The reaction was monitored by LC-MS. The mixture was acidified to pH 4 with HCl (2 M aqueous solution). The resulting mixture was extracted with CH2Cl2. The combined organic layers were concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with 66% ethyl acetate in hexane) to give the desired product (1.6 g, 66%) as a yellow solid. ESI-MS m / z: 527.30 [M+H] + .

[0928] intermediate 47 step e

[0929]

[0930] At room temperature, NH3 (68 mL, 156 eq, 7 N, in MeOH) was added to a 250 mL round-bottom flask containing step d (1.6 g, 3.04 mmol). The resulting mixture was stirred at room temperature for 20 hours and monitored by LC-MS. The solvent was removed, and the crude mixture was dissolved in EtOAc. The organic matter was washed three times with saturated sodium bicarbonate and concentrated. The crude matter was prepared with DCM to give the desired product (590.9 mg, 52%) as an off-white solid. ESI-MS m / z: 372.15 [M+H] + .

[0931] Intermediate 48

[0932]

[0933] Intermediate 48 Step a

[0934]

[0935] In a vial, the compound from intermediate 36b (6.5 g, 17 mmol) and 1-fluoro-N-methoxy-N-methylcyclopropane-1-carboxamide (3 g, 20.39 mmol) were dissolved in THF (121 mL) and cooled to 0 °C. Isopropylmagnesium chloride (16.99 mL, 34.0 mmol) was slowly added, and the reaction mixture was stirred at 0 °C. After stirring for 3 hours, water was added at 0 °C, and the reaction mixture was warmed to room temperature. The aqueous layer was washed with EtOAc, and the combined organic layers were dried over MgSO4 and concentrated. The crude reaction mixture was purified by silica gel column chromatography, eluting with 0–50% EtOAc / hexane to give the title compound (2.8 g, 48%) as a foaming white solid. ESI-MS m / z: 343.17 [M+H] + .

[0936] intermediate 48 step b

[0937]

[0938] In a vial, the compound from step a (1.4 g, 4.08 mmol), (4-fluorophenyl)boronic acid (0.685 g, 4.90 mmol), PdCl2(dppf)DCM (0.200 g, 0.245 mmol), and K2CO3 (1.692 g, 12.24 mmol) were dissolved in 1,4-dioxane (16.32 mL) and water (4.08 mL). The reaction mixture was sprayed with N2 and sealed. The reaction mixture was heated to 90 °C. After 4 hours, the reaction mixture was cooled to room temperature and water was added. The aqueous layer was washed with EtOAc. The combined organic layers were washed with water and brine, then dried over MgSO4 and concentrated. The crude reaction mixture was purified by silica gel column chromatography, eluting with 0–70% EtOAc / hexane, to give the title compound (1.12 g, 77%). ESI-MS m / z: 359.06 [M+H] + .

[0939] intermediate 48 steps c

[0940]

[0941] Methyltriphenylphosphonium bromide (3.00 g, 8.41 mmol) and THF (18.7 mL) were added to a vial and then cooled to 0 °C in an ice bath. Potassium tert-butoxide (0.906 g, 8.07 mmol) was slowly added as a solution in THF (2 mL), and the yellow suspension was stirred at 0 °C for 30 min. The compound from step b (1.205 g, 3.36 mmol) was added as a solution in THF (15 mL). The reaction mixture was stirred at 0 °C for 2 h, then heated to room temperature and stirred for another 2 h. The reaction was quenched by adding MeOH (5 mL) followed by water (20 mL). The solution was diluted with EtOAc and the layers were separated. The aqueous layer was washed with EtOAc, the combined organic layers were washed with brine, dried over MgSO4, and concentrated. The crude reaction mixture was purified by silica gel column chromatography, eluting with 0–80% EtOAc / hexane, to give the title compound (741 mg, 61.8%). ESI-MS m / z: 357.39 [M+H] + .

[0942] intermediate 48 steps d

[0943]

[0944] The above compound was prepared in a manner similar to that of intermediate 36, step e. The reaction mixture was purified by silica gel column chromatography, eluting with 0–80% EtOAc / Hex, to give the title compound (480 mg, 59%). ESI-MS m / z: 391.33 [M+H] + .

[0945] intermediate 48 steps e

[0946]

[0947] The above compound was prepared in a manner similar to step f of intermediate 36. After water treatment, the title compound (585 mg, 87%) was ready for use without further purification. ESI-MS m / z: 545.38 [M+H] + .

[0948] intermediate 48 steps f

[0949]

[0950] The above compound was prepared in a manner similar to that of intermediate 36 step g. After water treatment, the title compound (381 mg, 91%) was ready for use without further purification. ESI-MS m / z: 390.35 [M+H] + .

[0951] Intermediate 49

[0952]

[0953] The above compound was prepared in a manner similar to intermediate 47 to give the desired amino alcohol (473 mg, 64%). ESI-MS m / z: 434.40 [M+H] + .

[0954] Intermediate 50

[0955]

[0956] The above compound was prepared in a manner similar to intermediate 36 to give the desired amino alcohol (493 mg, 92%). ESI-MS m / z: 452.25 [M+H] + .

[0957] Intermediate 51

[0958]

[0959] The above compound was prepared in a manner similar to intermediate 47 to give the desired amino alcohol (246 mg, 83%). ESI-MS m / z: 424.31 [M+H] + .

[0960] Intermediate 52

[0961]

[0962] The above compound was prepared in a manner similar to intermediate 47 to give the desired amino alcohol (147 mg, 95%). ESI-MS m / z: 422.36 [M+H] + .

[0963] Intermediate 53

[0964]

[0965] Intermediate 53 step a

[0966]

[0967] 2-Chloro-5-fluoropyridin-3-ol (10 g, 68 mmol), K₂CO₃ (19 g, 136 mmol), I₂ (19 g, 75 mmol), and H₂O (200 mL) were added to a 500 mL round-bottom flask at room temperature. The resulting mixture was stirred overnight at room temperature under a nitrogen atmosphere and monitored by LC-MS. The reaction was quenched at room temperature by the addition of saturated sodium thiosulfate (aqueous solution) and extracted with EtOAc. The residue was purified by silica gel column chromatography (eluting with 9% ethyl acetate in hexane) to give the desired compound (16.7 g, 90%) as a white solid. ESI-MS m / z: 273.90 [M+H] + .

[0968] intermediate 53 step b

[0969]

[0970] At room temperature, the compound from step a (12.4 g, 45 mmol), (2-methylethyleneoxy-2-yl)methyl-4-methylbenzenesulfonate, K₂CO₃ (13 g, 91 mmol), KI (9 g, 54 mmol), and DMF (50 mL) were added to a 250 mL vial. The resulting mixture was stirred overnight at 50 °C under a nitrogen atmosphere. The reaction was monitored by LCMS. The reaction was quenched with water, and the aqueous layer was extracted with EtOAc. The resulting mixture was concentrated under reduced pressure. The residue was purified by reversed-phase flash chromatography to give the desired compound (9.6 g, 63%) as a white solid. ESI-MS m / z: 343.80 [M+H] + .

[0971] intermediate 53 step c

[0972]

[0973] At 0 °C, the compound from step b (1.5 g, 4 mmol), THF (15 mL), and LDA (2.4 mL, 5 mmol) were added to a 50 mL three-necked round-bottom flask. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 1 hour. The reaction was monitored by TLC. At 0 °C, the reaction was quenched by the addition of saturated NH4Cl (aqueous solution). The aqueous layer was extracted with EtOAc. The residue was purified by reversed-phase flash chromatography to give the desired compound (430 mg, 29%) as a white solid. ESI-MS m / z: 343.80 [M+H] + .

[0974] intermediate 53 step d

[0975]

[0976] At 0 °C, the compound from step c (400 mg, 1 mmol), acetone (10 mL), and Jones' reagent (1 mL) were added to a 100 mL round-bottom flask. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 3 hours. The reaction was monitored by LCMS. The aqueous layer was extracted with EtOAc and concentrated under reduced pressure. The crude product was used directly in the next step without further purification. ESI-MS m / z: 357.85 [M+H] + .

[0977] intermediate 53 step e

[0978]

[0979] At room temperature, the crude compound from step d, CDI (726 mg, 4 mmol), and THF (5 mL) were added to a 50 mL round-bottom flask. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 2 hours. The mixture was then added dropwise to NH3·H2O (50 mL) and stirred for 1 hour. The reaction was monitored by TLC. The aqueous layer was extracted with EtOAc, and the residue was purified by reversed-phase flash chromatography to give the desired compound (218.8 mg, 69%) as a white solid. ESI-MS m / z: 356.90 [M+H] + .

[0980] Intermediate 54

[0981]

[0982] The following compounds were prepared in a sequence similar to steps a and b of intermediate 48. ESI-MS m / z: 336.95 [M+H] + .

[0983] Intermediate 55

[0984]

[0985] The above compounds were prepared in a manner similar to intermediate 36. After water treatment, the title compound (45 mg, 63%) was separated as a white solid. ESI-MS m / z: 386.40 [M+H] + .

[0986] Intermediate 56

[0987]

[0988] The following compounds were prepared in a sequence similar to steps a and b of intermediate 48. ESI-MS m / z: 351.05 [M+H] + .

[0989] Intermediate 57

[0990]

[0991] The above compounds were prepared using the corresponding arylboronic acids in a manner similar to intermediate 36. The crude substance was purified by automated column chromatography (silica gel, 10% MeOH in DCM) to give the desired product (1.80 g, 57%) as an off-white solid. ESI-MS m / z: 462.05 [M+H] + .

[0992] Intermediate 58

[0993]

[0994] The above compounds were prepared using the corresponding arylboronic acids in a manner similar to intermediate 36. The crude substance was purified by preparative TLC (silica gel, 10% MeOH in DCM, containing NH3) to give the desired product (508 mg, 74%) as a white solid. ESI-MS m / z: 468.05 [M+H] + .

[0995] Intermediate 59

[0996]

[0997] The above compounds were prepared using the corresponding arylboronic acids in a manner similar to intermediate 36. The crude substance was purified by preparative TLC (silica gel, 6% MeOH in DCM, containing NH3) to give the desired product (450 mg, 72%) as a white solid. ESI-MS m / z: 434.10 [M+H] + .

[0998] Intermediate 60

[0999]

[1000] The above compounds were prepared using the corresponding arylboronic acids in a manner similar to intermediate 36. The crude substance was purified by preparative TLC (silica gel, 6% MeOH in DCM, containing NH3) to give the desired product (468.9 mg, 92%) as a white solid. ESI-MS m / z: 433.95 [M+H] + .

[1001] Intermediate 61

[1002]

[1003] The above compounds were prepared using the corresponding arylboronic acids in a manner similar to intermediate 36. The crude substance was purified by preparative TLC (silica gel, 6% MeOH in DCM, containing NH3) to give the desired product as a white solid. ESI-MS m / z: 434.10 [M+H] + .

[1004] Intermediate 62

[1005]

[1006] The above compounds were prepared using the corresponding arylboronic acids in a manner similar to intermediate 36. The crude substance was purified by preparative TLC (silica gel, 6% MeOH in DCM, containing NH3) to give the desired product as a white solid. ESI-MS m / z: 452.00 [M+H] + .

[1007] Intermediate 63

[1008]

[1009] The above compounds were prepared from the corresponding arylboronic acid pinacol esters in a manner similar to intermediate 36. The arylboronic acid pinacol esters were prepared from the corresponding bromides via a palladium-catalyzed borylation reaction. The crude product was purified by preparative TLC (silica gel, 6% MeOH in DCM, containing NH3) to give the desired product (355 mg, 67%) as a white solid. ESI-MS m / z: 452.10 [M+H] + .

[1010] Intermediate 64

[1011]

[1012] The above compounds were prepared using the corresponding arylboronic acids in a manner similar to intermediate 36. The crude substance was purified by preparative TLC (silica gel, 6% MeOH in DCM, containing NH3) to give the desired product (570.3 mg, 87%) as a white solid. ESI-MS m / z: 452.10 [M+H] + .

[1013] Intermediate 65

[1014]

[1015] At room temperature, 3-bromo-7-fluoro-1-benzothiophene (1.29 g, 5.60 mmol), pinacol diboronate (1.42 g, 5.59 mmol), KOAc (1.65 g, 16.81 mmol), Pd(dppf)Cl2.CH2Cl2 (365 mg, 0.45 mmol), and dioxane (18 mL) were added to a 40 mL sealed tube. The resulting mixture was stirred at 110 °C under a nitrogen atmosphere for 1 hour. The reaction was monitored by LCMS. The mixture was then cooled to room temperature. (R)-7-bromo-3-methyl-5-(3,3,3-trifluoroprop-1-en-2-yl)-2,3-dihydrofuran[2,3-c]pyridine-3-carboxamide (787 mg, 2.24 mmol), K₂CO₃ (0.93 g, 6.72 mmol), Pd(dppf)Cl₂.CH₂Cl₂ (365 mg, 0.45 mmol), and H₂O (2 mL) were added, and the mixture was stirred at 80 °C for 2 hours. The resulting mixture was filtered, and the filter cake was washed with EA. The filtrate was concentrated under reduced pressure. The residue was purified by reversed-phase flash chromatography to give the desired product (460 mg, 48%) as a brown oil. ESI-MS m / z: 422.95 [M+H] + .

[1016] Intermediate 66

[1017]

[1018] The above compounds were prepared in a manner similar to that of intermediates 36 and 65. The crude material was purified by preparative TLC (silica gel, 8% MeOH in DCM, containing NH3) to give the desired product (251.3 mg, 73%) as a white solid. ESI-MS m / z: 456.00 [M+H] + .

[1019] Intermediate 67

[1020]

[1021] The above compounds were prepared using the corresponding arylboronic acids in a manner similar to intermediate 47. The crude substance was purified by preparative TLC (silica gel, 10% MeOH in DCM, containing NH3) to give the desired product (291.1 mg, 61%) as a white solid. ESI-MS m / z: 440.00 [M+H] + .

[1022] Intermediate 68

[1023]

[1024] The above compounds were prepared using the corresponding arylboronic acids in a manner similar to that of intermediates 47 and 65. The crude substances were purified by preparative TLC (silica gel, 6% MeOH in DCM, containing NH3) to give the desired product (114.2 mg, 49%) as a white solid. ESI-MS m / z: 428.05 [M+H] + .

[1025] Intermediate 69

[1026]

[1027] The above compounds were prepared using the corresponding arylboronic acids in a manner similar to intermediate 47. The crude substance was purified by preparative TLC (silica gel, MeOH in DCM, containing NH3) to give the desired product (220 mg, 64%) as a white solid. ESI-MS m / z: 406.10 [M+H] + .

[1028] Intermediate 70

[1029]

[1030] The above compounds were prepared using the corresponding arylboronic acids in a manner similar to intermediate 47. The crude substance was purified by preparative TLC (silica gel, MeOH in DCM, containing NH3) to give the desired product (267.8 mg, 64%) as a white solid. ESI-MS m / z: 406.10 [M+H] + .

[1031] Intermediate 71

[1032]

[1033] The above compounds were prepared using the corresponding arylboronic acids in a manner similar to intermediate 47. The crude substance was purified by preparative TLC (silica gel, MeOH in DCM, containing NH3) to give the desired product (262.1 mg, 53%) as a white solid. ESI-MS m / z: 406.15 [M+H] + .

[1034] Intermediate 72

[1035]

[1036] The above compounds were prepared using the corresponding arylboronic acids in a manner similar to intermediate 47. The crude substance was purified by preparative TLC (silica gel, 6% MeOH in DCM, containing NH3) to give the desired product (250 mg, 68%) as a white solid. ESI-MS m / z: 424.00 [M+H] + .

[1037] Intermediate 73 steps a and b

[1038]

[1039] Methyl 3-amino-4-fluorobenzoate (50 mg, 0.296 mmol), isocyanocyclopropane (24.56 mg, 0.296 mmol), and EtOH (2 mL) were added to a vial. The resulting mixture was stirred at room temperature for 20 hours. After solvent removal, the residue was purified by automated column chromatography (4 g silica gel column, eluted with 0-50% EtOAc / hexane) to give the desired product (30 mg, 40.2%) as a pale yellow foam. ESI-MS m / z: 253.10 [M+H] + .

[1040] Add step a (90 mg, 0.357 mmol) and 2N NaOH (1 mL) to a vial containing 2 mL of THF / H₂O (9:1) solvent. After stirring overnight at room temperature, neutralize the mixture to pH approximately 3 by adding 1N HCl. The solid precipitate out. Filter and dry in an oven to obtain the desired product (85 mg, 100%). ESI-MS m / z: 239.20 [M+H] + .

[1041] Intermediate 74

[1042]

[1043] Ethyl 6-hydroxyimidazo[1,2-a]pyridine-2-carboxylate (60 mg, 0.291 mmol), 2-bromopropane (165 μl, 1.164 mmol), and DMF (0.582 mL) were added to a 2 mL MW vial. Cesium carbonate (284 mg, 0.873 mmol) was then added. The vial was capped and heated in a microwave reactor at 140 °C for 2 hours. The reaction mixture was diluted with ethyl acetate and water. The organic layer was washed with brine, dried, and concentrated, and used for the next step without further purification. ESI-MS m / z: 249.12 [M+H] + .

[1044] Intermediate 75

[1045]

[1046] Ethyl 3-bromo-2-oxopropionate (68.5 μl, 0.546 mmol) and 5-(trifluoromethoxy)pyridine-2-amine (81 mg, 0.455 mmol) were dissolved in DME (1.137 mL) and MeOH (1.137 mL). The reaction mixture was heated at 80 °C for 18 hours. The solvent was removed, and the residue was purified by flash chromatography to give the desired product (78 mg, 62.6%). ESI-MS m / z: 275.057 [M+H] + .

[1047] Intermediate 76

[1048]

[1049] Methyl 2-(1-hydroxycyclopropyl)-7-methoxy-2H-indazole-5-carboxylic acid (55 mg, 0.210 mmol) was dissolved in dichloromethane (1.049 mL) at 0 °C. Thionyl chloride (61.2 μl, 0.839 mmol) was added, and the mixture was stirred at 50 °C for 18 hours. The reaction mixture was directly purified by flash chromatography to give the desired product (34 mg, 57.8%). ESI-MS m / z: 281.061 [M+H] + .

[1050] Intermediate 77

[1051]

[1052] Intermediate 77 step a

[1053]

[1054] At room temperature, 5-bromo-1-methoxy-2-methyl-3-nitrobenzene (1 g, 4 mmol), NBS (0.8 g, 4.5 mmol), AIBN (0.13 g, 0.8 mmol), and CCl4 (20 mL) were added to a 100 mL round-bottom flask. The resulting mixture was stirred overnight at 80 °C under a nitrogen atmosphere and monitored by LCMS. The mixture was extracted with EtOAc by quenching the reaction with water. The combined organic matter was dried over sodium sulfate, filtered, and concentrated. The crude product was used directly in the next step without further purification. ESI-MS m / z: 325.90 [M+H] + .

[1055] Intermediate 77 step b

[1056]

[1057] At room temperature, the compound from step a (1.33 g, 4 mmol), aminocyclopropane (2.34 g, 41 mmol), K₂CO₃ (1.13 g, 8 mmol), and DMF (10 mL) were placed in a 100 mL round-bottom flask. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 3 hours. The reaction was monitored by LC-MS. The aqueous layer was extracted with EtOAc. The residue was purified by silica gel column chromatography (eluting with 16% ethyl acetate in hexane) to give the desired compound (1.07 g, 87%) as a white solid. ESI-MS m / z: 301.00 [M+H] + .

[1058] Intermediate step 77c

[1059]

[1060] At 0 °C, the compound from step b (500 mg, 1.7 mmol) and THF (6 mL) were added to a 100 mL three-necked round-bottom flask. NaOH (664 mg, 17 mmol) in H₂O (6 mL) was added over 2 minutes, followed by the addition of zinc (326 mg, 5 mmol) in six portions over 3 hours, with stirring for another 1 hour. The reaction was monitored by LCMS. The mixture was acidified to pH 3 with HCl (aqueous solution). The aqueous layer was extracted with CH₂Cl₂ and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure and washed with hexane to give the desired compound (310 mg, 70%) as a white solid. ESI-MS m / z: 267.00 [M+H] + .

[1061] intermediate 77 step d

[1062]

[1063] At room temperature, the compound from step c (240 mg, 0.9 mmol), dppp (222 mg, 0.5 mmol), Pd(OAc)₂ (60 mg, 0.3 mmol), DMF (4 mL), EtOH (4 mL), and TEA (1 mL) were added to a 30 mL pressure vessel reactor. The resulting mixture was stirred overnight at 100 °C under a CO atmosphere and at 15 atm. The reaction was monitored by LCMS. The resulting mixture was filtered, and the filter cake was washed with EA. The filtrate was concentrated under reduced pressure. The residue was purified by reversed-phase silica gel chromatography (MeCN / H₂O) to give the desired compound (210 mg, 90%) as a white solid. ESI-MS m / z: 261.10 [M+H] + .

[1064] intermediate 77 step e

[1065]

[1066] At room temperature, the compound from step d (470 mg, 1.8 mmol), LiOH (216 mg, 9 mmol), MeOH (10 mL), and H₂O (3 mL) were added to a 50 mL round-bottom flask. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 1 hour. The reaction was monitored by LCMS. The mixture was acidified to pH 6 with HCl (aqueous solution). The resulting mixture was washed with water and evaporated to give the desired compound (317.8 mg, 76%) as a yellow solid. ESI-MS m / z: 233.10 [M+H] + .

[1067] Intermediate 78

[1068]

[1069] Intermediate 78 Step a

[1070]

[1071] At room temperature, methyl 3-[(cyclopropylamino)methyl]-5-methoxy-4-nitrobenzoate (3 g, 11 mmol), KOH (6 g, 107 mmol), MeOH (20 mL), and H₂O (2 mL) were added to a 50 mL round-bottom flask. The resulting mixture was stirred at 80 °C under a nitrogen atmosphere for 72 hours. The reaction was monitored by TLC. The mixture was acidified to pH 6 with HCl (aqueous solution). The residue was purified by reversed-phase silica gel chromatography (MeCN / H₂O) to give the desired compound (580 mg, 21%) as a white solid. ESI-MS m / z: 263.10 [M+H] + .

[1072] intermediate 78 step b

[1073]

[1074] At room temperature, 1.28 g (5 mmol) of the compound from step a and 6 mL of AcOH were added to a 40 mL vial. The resulting mixture was stirred at 120 °C under a nitrogen atmosphere for 3 days. The reaction was monitored by LC-MS. The resulting mixture was concentrated under reduced pressure. The residue was purified by reversed-phase silica gel chromatography (MeCN / H2O) to give the desired compound (0.96 g, 79%) as a brown solid. ESI-MS m / z: 249.05 [M+H] + .

[1075] Intermediate 79

[1076]

[1077] Intermediate 79 step a

[1078]

[1079] A solution of 5-bromo-7-methoxy-1H-indazole (3.00 g, 13.21 mmol) in MeCN (50 mL) was treated overnight at room temperature with 1-(benzenesulfonyl)cyclopropane-1-ol (3.14 g, 15.85 mmol) and TEA (1.60 g, 15.8 mmol). The reaction was monitored by LCMS. The resulting mixture was extracted with EtOAc and concentrated. The residue was purified by silica gel column chromatography (eluting with 50% ethyl acetate in hexane) to give the desired compound (2.40 g, 64%) as a yellow solid. ESI-MS m / z: 283.00 [M+H] + .

[1080] intermediate 79 step b

[1081]

[1082] A solution of the compound from step a (2.43 g, 8.58 mmol) in DCM (20 mL) was treated with BAST (5.69 g, 25.74 mmol) for 1.5 h at 0 °C to room temperature. The reaction was monitored by LCMS and quenched with water. The aqueous layer was extracted with EtOAc and concentrated. The residue was purified by silica gel column chromatography (eluting with 50% ethyl acetate in hexane) to give the desired compound (1.2 g, 49%) as a yellow solid. ESI-MS m / z: 285.00 [M+H] + .

[1083] Intermediate step 79, steps c and d

[1084]

[1085] The following compound was prepared following the same procedures as steps d and e of intermediate 77, yielding the desired product (312.6 mg, 58%) as a white solid. ESI-MS m / z: 250.95 [M+H] + .

[1086] Intermediate 80

[1087]

[1088] The following compound was prepared using the same procedure as intermediate 79, yielding the desired product (170.3 mg, 76%) as a white solid. ESI-MS m / z: 276.95 [M+H]+ .

[1089] Intermediate 81

[1090]

[1091] Intermediate 81, steps a and b

[1092]

[1093] A mixture of 5-bromo-1-fluoro-3-methyl-2-nitrobenzene (5 g, 21.37 mmol), cyclopropanol (2.5 g, 42.73 mmol), and Cs₂CO₃ (21 g, 64 mmol) in DMF (20 mL) was stirred at 50 °C under a nitrogen atmosphere for 2 hours. The resulting mixture was extracted with EtOAc (3 × 200 mL). The combined organic layers were washed with water and concentrated under reduced pressure to give the crude product without further purification.

[1094] A solution / mixture of the compound from step a (5 g, 18.38 mmol), Fe (10.3 g, 183.76 mmol), and NH4Cl (9.8 g, 183.75 mmol) in EtOH (40 mL) and H2O (20 mL) was stirred at 80 °C for 2 h. The residue was purified by silica gel column chromatography (ethyl acetate in hexane) to give the desired product (3.6 g, 81%) as a yellow solid. ESI-MS m / z: 242.00 [M+H] + .

[1095] intermediate 81 steps c and d

[1096]

[1097] At room temperature, 1.5 g (6.2 mmol) of the compound from step b was added to 9.8 mL of a 50% aqueous solution of fluoroboric acid and stirred for 5 minutes. The mixture was cooled in an ice bath for 10 minutes, and an aqueous solution of NaNO2 (900 mg, 13.04 mmol) in 1.7 mL of H2O was added to the mixture. The reaction mixture was stirred at 10 °C for 30 minutes, during which time the product precipitated. The cooled reaction mixture was filtered through a Buchner funnel, the solid product was washed with small amounts of H2O, MeOH, and Et2O, and dried under high vacuum to give a crude product as a brown solid. ESI-MS m / z: 253.00 [M+H] + .

[1098] In a dry flask, 18-crown-6 (0.05 g, 0.2 mmol) and potassium acetate (2.9 g, 29.4 mmol) were dried under high vacuum for 1 hour. CHCl3 (70 mL) was added and the mixture was stirred at room temperature for 10 minutes. Then, fractions from step c were added to the mixture under N2 atmosphere. The reaction mixture was stirred at room temperature for 3 hours, filtered, and the residue was washed with CHCl3. The filtrate was washed with water (3 × 40 mL), the organic layer was dried over Na2SO4, filtered, and concentrated under vacuum to give the crude product. The crude product was purified by flash column chromatography (hexane / EtOAc) to give the desired product (1.8 g, 82%) as a yellow solid. ESI-MS m / z: 253.00 [M+H] + .

[1099] intermediate 81 steps e, f and g

[1100]

[1101] The following intermediate was prepared in a manner similar to intermediate 6-step ac to obtain the desired product (330 mg, 87%) as a yellow solid. ESI-MS m / z: 269.10 [M+H] + .

[1102] Intermediate 82

[1103]

[1104] intermediate 82 steps ad

[1105]

[1106] The following intermediate was prepared using cyclopropane-methanol in a manner similar to step ad of intermediate 81, yielding the desired product (1.85 g, 66%) as a pale yellow solid. ESI-MS m / z: 266.90 [M+H] + .

[1107] intermediate 82 steps eh

[1108]

[1109] The following intermediate was prepared in a manner similar to step ad of intermediate 79 to obtain the desired product (298 mg, 60%) as a white solid. ESI-MS m / z: 291.10 [M+H] + .

[1110] Intermediate 83

[1111]

[1112] The following intermediate was prepared in a manner similar to intermediate 6 to obtain the desired product (260 mg, 69%) as a white solid. ESI-MS m / z: 247.00 [M+H] + .

[1113] Intermediate 84

[1114]

[1115] The following intermediate was prepared in a manner similar to intermediate 6 to provide the desired product. ESI-MS m / z: 231.00 [M+H] + .

[1116] Intermediate 85

[1117]

[1118] intermediate 85 step a

[1119]

[1120] A solution / mixture of methyl 6-chloro-5-methoxypyridine-3-carboxylic acid ester (1 g, 4.96 mmol), ethynylcyclopropane (0.66 g, 9.92 mmol), Pd(PPh3)4 (1.15 g, 0.99 mmol), CuI (0.47 g, 2.48 mmol), and Et3N (1.51 g, 14.88 mmol) in 1,4-dioxane (30 mL) was stirred overnight at 90 °C under a N2 atmosphere. The residue was purified by silica gel column chromatography (eluting with 50% ethyl acetate in hexane) to give the desired product (1 g, 87%) as a yellow solid. ESI-MS m / z: 232.00 [M+H] + .

[1121] intermediate 85 steps bd

[1122]

[1123] Under ice cooling, a solution of amino-2,4,6-trimethylbenzenesulfonate (1.21 g, 5.62 mmol) in CH₂Cl₂ (100 mL) was added to a solution of the compound from step a (1 g, 4.32 mmol) in CH₂Cl₂ (50 mL), and the reaction mixture was stirred for another hour. Et₂O (12 mL) was added to the reaction mixture to precipitate crystals. The filtrate was filtered off and then dried under reduced pressure to give a crude product as a pale yellow solid. ESI-MS m / z: 247.00 [M+H] + .

[1124] The compound from step b (500 mg, 2.02 mmol) and K₂CO₃ (559 mg, 4.04 mmol) were stirred in MeOH (50 mL) for 2 hours at room temperature under a N₂ atmosphere. The residue was purified by silica gel column chromatography (eluting with ethyl acetate in hexane) to give the desired product (220 mg, 44%) as a yellow solid. ESI-MS m / z: 247.00 [M+H] + .

[1125] A solution of the compound from step c (220 mg, 0.89 mmol) and LiOH (214 mg, 8.93 mmol) in MeOH (10 mL) and H₂O (10 mL) was stirred at room temperature for 2 hours. The mixture was acidified with HCl to pH 5-6. The resulting mixture was filtered, and the filter cake was collected to give the desired product (160 mg, 77%) as a white solid. ESI-MS m / z: 233.00 [M+H] + .

[1126] Intermediate 86

[1127]

[1128] intermediate 86 step a

[1129]

[1130] A mixture of 3-methoxy-6-bromopyridin-2-ylamine (1.0 g, 3.6 mmol) and chloroacetaldehyde (1.2 mL, 50 wt% in water) in EtOH (12 mL) was refluxed and stirred for 1 hour, then concentrated under reduced pressure. The residue was partitioned between saturated solutions of sodium bicarbonate and EtOAc. The organic layer was separated and washed with brine, dried over Na₂SO₄, filtered, and concentrated. The residue was purified by flash chromatography (Si-PPC, MeOH / Et₂O, gradient 0:100–1:99) to give the desired product (960 mg, 88%) as a beige solid. ESI-MS m / z: 227.00 [M+H] + .

[1131] intermediate 86 steps b and c

[1132]

[1133] The following examples were prepared in a manner similar to intermediate 77 to obtain the desired product (340 mg, 49%) as a yellow solid. ESI-MS m / z: 192.95 [M+H] + .

[1134] Intermediate 87

[1135]

[1136] Intermediate 87 steps a and b

[1137]

[1138] Pd(OAc)₂ (0.42 g, 1.87 mmol) and Xantphos (1.08 g, 1.87 mmol) were added to a solution of 2,6-dibromo-4-methoxypyridine (5 g, 18.73 mmol), tert-butyl carbamate (2.19 g, 18.73 mmol), and Cs₂CO₃ (12.21 g, 37.46 mmol) in 1,4-dioxane (60 mL). The reaction was heated to 100 °C for 16 h under a nitrogen atmosphere. After cooling to room temperature, water was added and the mixture was extracted with EA (50 mL × 3). The combined organic layers were washed with water and dried over anhydrous Na₂SO₄. The filtrate was concentrated under reduced pressure after filtration. The residue was purified by silica gel column chromatography, eluting with PE / EA, to give the desired product (2 g, 35%) as a yellow solid. ESI-MS m / z: 303.00 [M+H] + .

[1139] The solution of the compound from step a (1.5 g, 2.4 mmol) and TFA (8 mL) in DCM (20 mL) was stirred at room temperature for 2 hours. The residue product was purified by reversed-phase flash chromatography under the following conditions (water (0.05% FA) / MeOH) to give the desired product (520 mg) as a white solid. ESI-MS m / z: 203.00 [M+H] + .

[1140] intermediate 87 steps ce

[1141]

[1142] The following examples were prepared in a manner similar to intermediate 86 to obtain the desired product (300 mg, 86%) as a yellow solid. ESI-MS m / z: 192.95 [M+H] + .

[1143] Intermediate 88

[1144]

[1145] intermediate 88 step a

[1146]

[1147] A solution of 6-bromo-3-methoxypyridine-2-amine (1 g, 4.93 mmol) and 2-bromo-1-cyclopropyl ethyl ketone (963 mg, 5.91 mmol) in EtOH (10 mL) was stirred overnight at 80 °C. The reaction was quenched with water, and the resulting mixture was extracted with EA (3 × 200 mL). The combined organic layers were washed with NaHCO3 (3 × 200 mL) and dried over anhydrous NaSO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EA, to give the desired product (360 mg, 27%) as a yellow solid. ESI-MS m / z: 267.00 [M+H] + .

[1148] intermediate 88 steps b and c

[1149]

[1150] The following examples were prepared in a manner similar to intermediate 86 to obtain the desired product (110 mg, 62%) as a yellow solid. ESI-MS m / z: 233.00 [M+H]+.

[1151] Intermediate 89

[1152]

[1153] The following examples were prepared in a manner similar to intermediate 86 to provide the desired product. ESI-MS m / z: 206.95 [M+H]+.

[1154] Intermediate 90

[1155]

[1156] intermediate 90 step a

[1157]

[1158] A solution / mixture of 3-cyclopropoxy-2-nitropyridine (1.8 g, 1 mmol), Fe (5.58 g, 100 mmol), and NH4Cl (5.34 g, 100 mol) in EtOH (60 mL) and H2O (30 mL) was stirred at 80 °C for 2 hours. The residue was purified by silica gel column chromatography, eluting with PE / EA to give the desired product (1.4 g, 93%) as a yellow solid. ESI-MS m / z: 151.00 [M+H]+.

[1159] intermediate 90 step b

[1160]

[1161] The solution / mixture of the compound from step a (1.4 g, 9.32 mmol) and Br2 (2.98 g, 18.64 mmol) in AcOH (30 mL) was stirred overnight at room temperature. The resulting mixture was quenched with saturated sodium thiosulfate and extracted with EA. The combined organic layers were washed with NaHCO3 solution (3 × 200 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to give the desired product (1.4 g, 66%) as a yellow solid. ESI-MS m / z: 229.00 [M+H]+.

[1162] intermediate 90 steps ce

[1163]

[1164] The following examples were prepared in a manner similar to intermediate 86 to provide the desired product. ESI-MS m / z: 219.00 [M+H]+.

[1165] Intermediate 91

[1166]

[1167] Intermediate 91, steps a and b

[1168]

[1169] A solution of 3-hydroxy-4-aminobenzoic acid (10 g, 65.30 mmol) and methacrolein (9.15 g, 130.60 mmol) in HCl (40 mL) and AcOH (60 mL) was stirred at 100 °C under a nitrogen atmosphere for 1 hour. The combined layers were concentrated under reduced pressure to give the desired product (5.6 g, 42%) as a brown solid. ESI-MS m / z: 204.00 [M+H]+.

[1170] The solution / mixture of the compound from step a (5.6 g, 27.56 mmol) and H₂SO₄ (5 mL) in MeOH (50 mL) was refluxed and stirred at 80 °C for 1 hour. The mixture was neutralized to pH 7 with NaOH solution. The resulting mixture was extracted with EA (3 × 300 mL). The combined organic layers were concentrated under reduced pressure to give the desired product (2.5 g, 42%) as a yellow solid. ESI-MS m / z: 218.00 [M+H] + .

[1171] intermediate 91 steps c and d

[1172]

[1173] A solution of the compound from step b (500 mg, 2.30 mmol), 2-bromo-1,1-difluoroethane (667 mg, 4.6 mmol), and K₂CO₃ (954 mg, 6.91 mmol) in DMF (20 mL) was stirred overnight at 80 °C. The residue was purified by silica gel column chromatography, eluting with PE / EA to give the desired product (500 mg, 62%) as a yellow solid. ESI-MS m / z: 282.00 [M+H] + .

[1174] A solution of the compound from step c (500 mg, 1.78 mmol) and LiOH (426 mg, 17.78 mmol) in MeOH (20 mL) and H₂O (20 mL) was stirred at room temperature for 30 minutes. The mixture was acidified with HCl to pH 7. The crude product was recrystallized from water to give the desired product (270 mg, 57%) as a yellow solid. ESI-MS m / z: 267.95 [M+H] + .

[1175] Intermediate 92, steps a and b

[1176]

[1177] A solution of methyl 8-hydroxy-3-methylquinoline-6-carboxylate (3 g, 13.81 mmol), sodium 2-chloro-2,2-difluoroacetate (3.16 g, 20.72 mmol), and Cs₂CO₃ (9 g, 27.61 mmol) in DMF (30 mL) was stirred at 80 °C under a nitrogen atmosphere for 4 hours. The resulting mixture was extracted with EA (3 × 200 mL). The combined organic layers were washed with water (3 × 200 mL) and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EA, to give the desired product (2 g, 54%) as a yellow solid. ESI-MS m / z: 268.00 [M+H] + .

[1178] The solution of the compound from step a (3 g, 11.23 mmol), LiOH (2.7 g, 112.26 mmol), and MeOH (30 mL) in H₂O (30 mL) was stirred at room temperature for 4 hours. The mixture / residue was acidified to pH 7 with HCl, filtered, and the filter cake was washed with water. The residue was purified by reversed-phase flash chromatography under the following conditions (silica gel, MeCN in water, 10% to 50% gradient over 10 minutes) to give the desired product (800 mg, 28%) as a yellow solid. ESI-MS m / z: 254.20 [M+H] + .

[1179] Intermediate 93, steps a and b

[1180]

[1181] A mixture of methyl 4-amino-3-chlorobenzoate (1.5 g, 8.08 mmol) and (2Z)-2-chlorobut-2-enal (1.27 g, 12.12 mmol) in HCl (10 mL) and AcOH (15 mL) was stirred at 100 °C for 20 min. The reaction was monitored by LC-MS. The mixture was cooled to room temperature and concentrated under vacuum. The residue was dissolved in MeOH (20 mL) and H₂SO₄ (2 mL) and stirred at 80 °C for 1 h. The resulting mixture was poured into water and extracted with EA. The combined organic layers were concentrated under reduced pressure. The residue was purified by reversed-phase flash chromatography to give the desired product (110 mg, 5%) as a yellow solid. ESI-MS m / z: 269.90 [M+H] + .

[1182] At room temperature, the compound from step a (110 mg, 0.40 mmol), THF (4 mL), MeOH (1 mL), LiOH (97 mg, 4.07 mmol), and H₂O (1 mL) were added to a 100 mL round-bottom flask. The resulting mixture was stirred at room temperature for 3 hours. The mixture was acidified to pH 5 with HCl (1 M aqueous solution). The product precipitated. The precipitate was collected by filtration and washed with water to give the desired product (65 mg). ESI-MS m / z: 256.00 [M+H] + .

[1183] Intermediate 94

[1184]

[1185] Intermediate 94 step a

[1186]

[1187] A solution of methyl 3-bromo-5-methoxy-4-nitrobenzoate (3.60 g, 12.43 mmol) in toluene (20 mL) was treated with (1-ethoxyvinyl)tin tributyl ester (5.39 g, 14.91 mmol) and Pd(dppf)Cl2 (1.82 g, 2.49 mmol) and stirred at 110 °C under nitrogen atmosphere for 2 h. The reaction was monitored by LCMS. The resulting mixture was filtered, and the filter cake was washed with EA. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with 25% ethyl acetate in hexane) to give a yellow oily compound (2.69 g, 77%). ESI-MS m / z: 282.20 [M+H] + .

[1188] intermediate 94 step b

[1189]

[1190] A solution of the compound from step a (2.69 g, 9.56 mmol) in DCM (30 mL) was treated overnight at room temperature with HCl (3 mL). The reaction was monitored by LC-MS. The aqueous layer was extracted with CH2Cl2. The residue was purified by silica gel column chromatography (eluting with 50% ethyl acetate in hexane) to give the compound as a white solid (1.82 g, 75%). ESI-MS m / z: 253.85 [M+H] + .

[1191] intermediate 94 steps c and d

[1192]

[1193] The compound from step b (1.82 g, 7.18 mmol) was added to a 250 mL round-bottom flask, and a solution of NaOAc (21.83 g, 266.09 mmol) in MeOH (90 mL) and THF (90 mL) was treated with SnCl2 (18.19 g, 94.92 mmol), and stirred at room temperature under a nitrogen atmosphere for 16 hours. The reaction was monitored by LC-MS. The resulting mixture was filtered, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EA (7:3), to give the compound (851 mg, 54%) as a white solid. ESI-MS m / z: 222.10 [M+H] + .

[1194] The compound from step c (840 mg, 3.80 mmol) and THF (40 mL) were added to a 250 mL round-bottom flask at room temperature. A solution of LiOH (909 mg, 37.97 mmol) in MeOH (10 mL) and H₂O (10 mL) was added, and the mixture was stirred at room temperature for 5 hours. The reaction was monitored by LCMS. The mixture was acidified to pH 4 with HCl, and the product was precipitated. The precipitated solid was collected by filtration and washed with H₂O to give the compound (646 mg, 81%) as an orange solid. ESI-MS m / z: 207.95 [M+H] + .

[1195] Intermediate 95

[1196]

[1197] The following compound was prepared following a similar procedure to that used for intermediate 94, yielding the desired product (741 mg, 88%) as an orange solid. ESI-MS m / z: 178.10 [M+H] + .

[1198] Table 5 below contains examples prepared using methods similar to step c (PyBOP or HATU) of Example 1. Most compounds were purified by preparative HPLC (ACN / H2O, 20-90%, 25 min), and some were purified by automated column chromatography (silica gel). Aryl acid conjugates were prepared according to intermediates 1-95 or by similar procedures with slight modifications, and also according to the procedures described in U.S. Patent Application No. 16 / 930622.

[1199] Table 5

[1200]

[1201]

[1202]

[1203]

[1204]

[1205]

[1206]

[1207]

[1208]

[1209]

[1210]

[1211]

[1212]

[1213]

[1214]

[1215]

[1216]

[1217]

[1218]

[1219]

[1220]

[1221]

[1222]

[1223]

[1224]

[1225]

[1226]

[1227]

[1228]

[1229]

[1230]

[1231]

[1232]

[1233]

[1234] Intermediate 96

[1235]

[1236] Intermediate 96 Step a

[1237]

[1238] 5-Bromoindigo (1 g, 4 mmol) and DCM (5 mL) were added to a 100 mL round-bottom flask at room temperature. DAST (1.43 g, 9 mmol) was added dropwise. The resulting mixture was stirred at 0 °C under a nitrogen atmosphere for 1 hour. The reaction was monitored by LC-MS. The reaction was quenched with water, and the aqueous layer was extracted with EA. The residue was purified by silica gel column chromatography (eluting with 25% ethyl acetate in hexane) to give the desired compound (900 mg, 82%) as a pale yellow solid.

[1239] intermediate 96 step b

[1240]

[1241] A solution of the compound from step a (300 mg, 1.2 mmol), pinacol diboronate (461 mg, 1.8 mmol), Pd(dppf)Cl₂CH₂Cl₂ (197 mg, 0.24 mmol), and KOAc (356 mg, 4 mmol) in a mixture of dioxane (5 mL) was stirred at 90 °C for 2 hours under a nitrogen atmosphere. The reaction was monitored by TLC. The aqueous layer was extracted with EtOAc. The resulting mixture was concentrated under reduced pressure. The residue was purified by reversed-phase flash chromatography to give the desired compound (297 mg, 83%) as a yellow solid.

[1242] Intermediate 97

[1243]

[1244] The following compound is prepared in a manner similar to step b of intermediate 96 described above to provide the desired product, which is used directly in the next step.

[1245] Intermediate 98

[1246]

[1247] In a 50 mL round-bottom flask, under a nitrogen atmosphere and at -78 °C, n-BuLi (2.8 mL, 5.6 mmol) was added dropwise to a solution of 1-bromo-2-chloro-3,4-difluorobenzene (850 mg, 3.7 mmol) in THF (30 mL). The reaction mixture was stirred at -78 °C for 5 minutes. Then, B(OMe)3 (583 mg, 5.6 mmol) was added dropwise, and the mixture was stirred at room temperature for another 30 minutes. The mixture was acidified to pH 5 with HCl (3 M aqueous solution). The reaction was quenched with saturated NH4Cl, and the mixture was then extracted with EtOAc. The combined organic extracts were washed with brine, dried over anhydrous Na2SO4, and concentrated under vacuum to give the desired crude compound (790 mg, 109%) as a yellow oil. The crude product was used directly in the next step without further purification.

[1248] Example 890

[1249]

[1250] Example 890, Step a

[1251]

[1252] THF (24.16 mL), acetone (24.16 mL), and water (24.16 mL) were added to a 250 mL round-bottom flask containing intermediate 36c (5.09 g, 14.50 mmol). The flask was cooled to 0 °C, and NMO (4.25 g, 36.2 mmol) was added, followed by potassium cinnamate dihydrate (0.230 g, 0.623 mmol). The reaction mixture was stirred for 10 min, heated to room temperature, and stirred overnight for 20 h. Sodium sulfite was added, and the mixture was diluted with water and stirred for 20 min. The mixture was diluted with EtOAc, and the aqueous layer was extracted. The combined organic compounds were dried over sodium sulfate, filtered, and concentrated. The crude mixture was purified by automated column chromatography (silica gel, 0-100% ethyl acetate in hexane) to give the desired product (4.12 g, 74%) as a white solid and a mixture of diastereomers. ESI-MS m / z: 385.21 [M+H] + .

[1253] Example 890, step b

[1254]

[1255] Add DCM (36 mL, 0.3 M) to a 250 mL round-bottom flask containing step a (4.118 g, 10.69 mmol). Add DMAP (0.065 g, 0.535 mmol), followed by TEA (4.47 mL, 32.1 mmol). Cool the flask to 0 °C and add TsCl (2.242 g, 11.76 mmol). Stir for 10 min, heat to room temperature, and monitor by LCMS (1 h). Concentrate the reaction mixture and purify the crude residue by automated column chromatography (silica gel, 0-100% ethyl acetate in hexane) to give the desired product (3.92 g, 99%) as a white solid, a mixture of diastereomers, and a mixture of epoxide (major) and toluenesulfonate. ESI-MS m / z: 367.19 [M+H] + .

[1256] Example 890 Step c

[1257]

[1258] Ammonia (239 mL, 1670 mmol, 7 N, in MeOH) was added to a 500 mL round-bottom flask containing step b (3.92 g, 10.70 mmol) at 0 °C. The reaction mixture was stirred for 10 min, heated to room temperature, and monitored by LCMS (3.5 h). The stirring was then removed and the reaction mixture was concentrated. The crude compound was dissolved in EtOAc and washed three times with saturated sodium bicarbonate. The organic compound was concentrated and the crude compound was prepared with DCM / hexane to give a white solid (2.41 g, 59%) as a mixture of diastereomers. ESI-MS m / z: 384.21 [M+H] + .

[1259] Example 890, step d

[1260]

[1261] Add step c (1.000 g, 2.60 mmol) and 2-cyclopropyl-7-methoxy-2H-indazole-5-carboxylic acid (0.605 g, 2.60 mmol) to a 100 mL round-bottom flask equipped with a stir bar. Dissolve the solid in N,N-dimethylformamide (13.02 mL, 0.2 M) and add DIPEA (0.909 mL, 5.21 mmol). Cool the vial to 0 °C and add PyBOP (1.626 g, 3.12 mmol). Stir the reaction mixture for 10 min, heat to room temperature, and monitor by LCMS (1 h). Quench the reaction with saturated ammonium chloride and dilute with EtOAc. Extract the aqueous solution with EtOAc, dry the combined organic matter with sodium sulfate, filter, and concentrate. Purify the crude mixture by automated column chromatography (silica gel, 0-100% ethyl acetate in hexane) to give the desired product as an oil. The oil was dissolved in EtOAc, washed with water and brine to remove DMF, and concentrated to give a product as a white solid (597 mg, 38%), which is a mixture of diastereomers. ESI-MS m / z: 598.12 [M+H] + .

[1262] Example 891

[1263]

[1264] PdCl2 (dppf) (6.11 mg, 8.36 μmol), potassium carbonate (26.0 mg, 0.188 mmol), (4-cyclopropylphenyl)boronic acid (16.24 mg, 0.100 mmol), and Example 890 (50 mg, 0.084 mmol) were added to a 2-dram vial equipped with a stir bar and diaphragm cap. The substances were dissolved in 1,4-dioxane (0.3 mL, 4:1, 0.2 M) and water (0.084 mL) and purged with nitrogen. The reaction was heated to 90 °C and monitored by LCMS (2 h). The reactants were diluted with EtOAc, filtered through a silica gel septum, the septum was washed with EtOAc, and the organic matter was concentrated. The crude substance was purified, and diastereomers were separated by preparative HPLC (ACN / H2O, 20–90%, 25 min) to give the desired product (3 mg, 6%) as a white solid. ESI-MS m / z: 636.26 [M+H] + .

[1265] Example 892

[1266]

[1267] The following examples show that the sample was purified from Example 891 to a white solid (3 mg, 6%) by preparative HPLC (ACN / H₂O, 20-90%, 25 min). ESI-MS m / z: 636.26 [M+H] + .

[1268] Table 6 below contains examples prepared using methods similar to those in Example 890. If further conversion is desired, more palladium and boric acid may be added. Most compounds were purified by preparative HPLC (ACN / H₂O, 20-90%, 25 min), and diastereomers were separated. If reported as a mixture of diastereomers, they may not be separable by HPLC.

[1269] Table 6

[1270]

[1271]

[1272]

[1273]

[1274]

[1275]

[1276]

[1277]

[1278]

[1279] Table 7 below contains examples prepared using methods similar to step c (PyBOP or HATU) of Example 1. Most compounds were purified by preparative HPLC (ACN / H2O, 20-90%, 25 min). If described as a single diastereomer, they were separated during preparative HPLC purification. Amine conjugates were prepared in a manner similar to intermediates 45 and 46. Aryl acid conjugates were prepared according to intermediates 1-95 or by similar procedures with slight modifications, and also according to the procedure described in U.S. Patent Application No. 16 / 930622.

[1280] Table 7

[1281]

[1282]

[1283]

[1284]

[1285]

[1286]

[1287] Intermediate 99

[1288]

[1289] Intermediate 99 steps a and b

[1290]

[1291] 5-(benzyloxy)-2-(hydroxymethyl)-1H-pyridin-4-one (5 g, 21.62 mmol), Trt-Cl (6.63 g, 23.78 mmol), and DMAP (2.91 g, 23.78 mmol) were suspended in DMF (50 mL), and the mixture was stirred overnight at 95 °C. The reaction was monitored by LCMS. After cooling to room temperature, the mixture was treated with cold water and stirred for 30 min. The precipitated solid was collected by filtration, washed with water and MeOH, and dried to give the desired product (8.8 g, 86%) as a light gray solid. ESI-MS m / z: 474.30 [M+H] + .

[1292] Under a nitrogen atmosphere, Pd / C (2.2 g, 20.67 mmol) was added to a solution of the compound from step a (4.4 g, 9.29 mmol) in THF (120 mL), H₂O (12 mL), and 2 M NaOH aqueous solution (6 mL). The resulting mixture was stirred at room temperature under a hydrogen atmosphere for 2 hours. The reaction was monitored by TLC. The resulting mixture was filtered, and the filter cake was washed with acetonitrile. The filtrate was concentrated under reduced pressure. DMAP (2.27 g, 18.58 mmol) and thiophosgene (1.58 g, 13.75 mmol) were added to the suspension of the residue in DCM (100 mL). The reaction was monitored by TLC after stirring at room temperature for 1 hour. The reaction was quenched with water and extracted with CH₂Cl₂. The combined organic layers were washed with brine and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with P / EA (10:1) to give the desired product (3.3 g, 83%) as a yellow solid. ESI-MS m / z: 448.20 [M+Na] + .

[1293] intermediate 99 steps c and d

[1294]

[1295] While maintaining a temperature below -60°C, HF pyridine (33 mL) and DBDMH (3.77 g, 13.18 mmol) were added to a solution of the compound from step b (3.3 g, 7.75 mmol) in DCM (100 mL). The reaction mixture was heated to 0°C over 20 minutes. After stirring at this temperature for 2 hours, the reaction was quenched with 2 M NaOH aqueous solution and extracted with CH2Cl2. The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EA (1:1) to give the desired product (740 mg, 50%) as a yellow oil. ESI-MS m / z: 190.05 [M+H] + .

[1296] intermediate 99 steps e

[1297]

[1298] To a solution of the compound from step d (550 mg, 2.94 mmol) in acetone (20 mL) and H₂O (10 mL), 2-methyl-2-butene (2.06 g, 29.40 mmol), NaH₂PO₄ (529 mg, 4.41 mmol), and NaClO₂ (532 mg, 5.88 mmol) were added. The resulting mixture was stirred at room temperature for 2 hours. The mixture was treated with NaHSO₃ (1.07 g, 10.28 mmol) and concentrated under reduced pressure to remove acetone. The mixture was treated with brine (10 mL) and extracted twice with EA / THF (1:1). The combined organic layers were dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reversed-phase flash chromatography to give the desired product (151.8 mg, 25%) as a white solid. ESI-MS m / z: 203.90 [M+H] + .

[1299] Intermediate 100

[1300]

[1301] Intermediate 100, steps a and b

[1302]

[1303] Under a nitrogen atmosphere and at room temperature, a mixture of 5-bromo-7-methoxy-1H-indazole (500 mg, 2.2 mmol), Cs₂CO₃ (2.15 g, 6.6 mmol), and CBr₂F₂ (5 mL) in ACN (25 mL) was stirred overnight. The residue was purified by reversed-phase flash chromatography to give the desired product (200 mg, 26%) as a yellow oil. ESI-MS m / z: 355.00 [M+H] + .

[1304] A solution of the compound from step a (400 mg, 1.12 mmol) and AgBF4 (656 mg, 3.37 mmol) in DCM (20 mL) was stirred for 2 hours at room temperature. The resulting mixture was washed with 3 × 100 mL of NaHCO3. The resulting mixture was extracted with EA (3 × 100 mL). The combined organic layers were washed with water (3 × 100 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EA, to give the desired product (300 mg, 90%) as a yellow solid. ESI-MS m / z: 295.00 [M+H] + .

[1305] intermediate 100 steps c and d

[1306]

[1307] The following compound was prepared following the same procedures as in steps d and e of intermediate 77, yielding the desired product (200 mg, 72%) as a white solid. ESI-MS m / z: 275.00 [M+H] + .

[1308] Intermediate 101

[1309]

[1310] Intermediate 101 steps a, b and c

[1311]

[1312] A mixture of 2-amino-5-bromo-3-methoxybenzaldehyde (9.6 g, 41.73 mmol) and urea (37.59 g, 625.92 mmol) was stirred for 2 hours at 180 °C. The mixture was cooled to room temperature and poured into ice water. The precipitated solid was collected by filtration, washed with water, and dried under vacuum to give a crude product (11 g) as a gray solid. ESI-MS m / z: 254.85 [M+H] + .

[1313] A solution of the compound from step a (11 g, 43.12 mmol) in phosphorus oxychloride (100 mL) was stirred for 5 hours at 110 °C. The mixture was cooled to room temperature and concentrated under reduced pressure. The residue was dissolved in EA and poured into ice water with vigorous stirring. The resulting mixture was extracted with EA. The combined organic layers were washed with brine and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EA (2:1) to give the desired product (2.4 g, 20%) as a pale yellow solid. ESI-MS m / z: 272.95 [M+H] + .

[1314] The mixture of the compound from step b (2.4 g, 8.77 mmol) and NaOMe (0.47 g, 8.77 mmol) in MeOH (30 mL) was stirred at room temperature for 1 hour and then refluxed for 1 hour. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was stirred with water. The precipitated solid was collected by filtration, washed with water, and dried under vacuum to give the desired product (2.2 g, 93%) as a pale yellow solid. ESI-MS m / z: 268.85 [M+H] + .

[1315] intermediate 101 steps d and e

[1316]

[1317] The following compound was prepared following the same procedures as in steps d and e of intermediate 77, yielding the desired product (658.6 mg, 77%) as a white solid. ESI-MS m / z: 235.00 [M+H] + .

[1318] Intermediate 102

[1319]

[1320] Intermediate 102, steps a and b

[1321]

[1322] Isobutylaldehyde (3.87 g, 55.19 mmol) was added to a solution of methyl 4-amino-3-methoxybenzoate (4 g, 22.07 mmol) in HCl (15 mL). The resulting mixture was stirred at 100 °C for 5 hours. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The crude product was used directly in the next step without further purification. This yielded 8-methoxy-3-methylquinoline-6-carboxylic acid (4 g, 83%) as a brown crude solid. ESI-MS m / z: 218.05 [M+H] + .

[1323] SOCl2 (8.76 g, 73.65 mmol) was added to a solution of the compound from step a (4 g, 18.41 mmol) in MeOH (120 mL). The resulting mixture was stirred at 80 °C for 40 min. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was dissolved in water (40 mL). The mixture was alkalized to pH 7 with 1 M NaHCO3 aqueous solution, extracted, and evaporated. The residue was purified by silica gel column chromatography, eluting with PE / EA (1:2), to give the compound (1.8 g, 42%) as a brown solid. ESI-MS m / z: 232.00 [M+H] + .

[1324] intermediate 102 steps c and d

[1325]

[1326] At 0 °C, m-CPBA (4.03 g, 23.35 mmol) was added fractionally to a solution of the compound from step b (1.8 g, 7.78 mmol) in DCM (20 mL). The resulting mixture was stirred overnight at room temperature. The reaction was monitored by LCMS. The resulting mixture was washed with 1 M NaOH. The aqueous layer was alkalized to pH 2 with concentrated HCl. The resulting mixture was filtered, and the filter cake was washed with water. The filtrate was concentrated under reduced pressure. The crude product was used directly in the next step without further purification. ESI-MS m / z: 233.95 [M+H] + .

[1327] A solution of the compound from step c (5 g crude) and POCl3 (42.73 g, 278.707 mmol, 13 equivalents) was stirred for 1 hour at 95 °C. The reaction mixture was slowly added to water (20 mL), and the reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was purified by reversed-phase flash chromatography to give the title compound (100 mg, 1.85%) as a white solid. ESI-MS m / z: 251.95 [M+H] + .

[1328] Intermediate 103

[1329]

[1330] This intermediate was prepared in a similar manner to intermediate 102 (80 mg, 70%). ESI-MS m / z: 277.95 [M+H] + .

[1331] Intermediate 104

[1332]

[1333] The following compound (64 mg, 74%) was prepared in a manner similar to that used for intermediate 102 described above. ESI-MS m / z: 318.10 [M+H] + .

[1334] Intermediate 105

[1335]

[1336] The above compound (32 mg, 46%) was prepared in a manner similar to that used for intermediate 102 described above. ESI-MS m / z: 268.15 [M+H] + .

[1337] Intermediate 106

[1338]

[1339] Intermediate 106 steps a, b and c

[1340]

[1341] At room temperature, 2,5-dichloro-3-fluoropyridine (10 g, 60.25 mmol), (4-methoxyphenyl)methanol (9.16 g, 66.27 mmol), Cs₂CO₃ (39.26 g, 121 mmol), and DMF (40 mL) were added to a 250 mL round-bottom flask. The resulting mixture was stirred at 80 °C for 1 hour. The reaction was monitored by LCMS. The aqueous layer was extracted with EtOAc. The resulting mixture was washed with brine. The mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography to give the desired compound (14 g, 81%) as a white solid. ESI-MS m / z: 284.25 [M+H] + .

[1342] At room temperature, add the compound from step a (5 g, 18 mmol) and DCM (24 mL) to a 100 mL round-bottom flask. Add CF3COOH (6 g, 52.62 mmol) dropwise to the mixture. Stir the resulting mixture overnight at room temperature. Monitor the reaction by LCMS. Extract the aqueous layer with CH2Cl2. Concentrate the resulting mixture under vacuum; the crude product is used directly in the next step without further purification. ESI-MS m / z: 165.10 [M+H] + .

[1343] At room temperature, the compound from step c (6.2 g, 37.81 mmol), I₂ (10.56 g, 41.59 mmol), K₂CO₃ (10.45 g, 75.61 mmol), and H₂O (40 mL) were added to a 100 mL round-bottom flask. The resulting mixture was stirred at room temperature for 2 hours. The reaction was monitored by LC-MS. The reaction was quenched with saturated sodium thiosulfate (aqueous solution) at 0 °C. The mixture was acidified to pH 6 with concentrated HCl. The aqueous layer was extracted with EtOAc. The residue was purified by silica gel column chromatography to give the desired compound as a white solid. ESI-MS m / z: 290.15 [M+H] + .

[1344] intermediate 106 steps d and e

[1345]

[1346] At room temperature, the compound from step c (3.9 g, 13.45 mmol), (2-methylethyleneoxy-2-yl)methyl4-methylbenzenesulfonate (3.91 g, 16.14 mmol), KI (2.2 g, 13.45 mmol), K₂CO₃ (3.7 g, 26.91 mmol), and DMF (20 mL) were added to a 250 mL round-bottom flask. The resulting mixture was stirred overnight at 50 °C. The reaction was monitored by LC-MS. The reaction was quenched at 0 °C with saturated NH₄Cl (aqueous solution). The aqueous layer was extracted with EtOAc. The residue was purified by reversed-phase flash chromatography to give the desired compound (3.0 g, 65%) as a yellow oil. ESI-MS m / z: 359.95 [M+H] + .

[1347] At 0 °C, the compound from step d (1.85 g, 5.13 mmol) and LDA (2 M in THF) (3.9 mL) were added to a 50 mL three-necked round-bottom flask. The mixture was stirred for 1 hour at room temperature under a nitrogen atmosphere. The reaction was monitored by LCMS. The desired product was detectable by LCMS. The reaction was quenched with saturated NH4Cl (aqueous solution) and extracted with EA. The resulting mixture was concentrated under reduced pressure. The residue was purified by reversed-phase flash chromatography to give the desired compound as a yellow oil. ESI-MS m / z: 360.15 [M+H] + .

[1348] intermediate 106 steps f and g

[1349]

[1350] Under a nitrogen atmosphere and at 0 °C, the compound from step e (6.6 g, 18.33 mmol), acetone (20 mL), and Jones' reagent (8 mL, 40.39 mmol) were added in portions to a 250 mL round-bottom flask. The resulting mixture was stirred at room temperature and under a nitrogen atmosphere for 3 hours. The reaction was monitored by LCMS. The aqueous layer was extracted with EtOAc. The resulting liquid was dried under vacuum. The crude product was used directly in the next step without further purification. ESI-MS m / z: 374.20 [M+H] + .

[1351] At room temperature, the compound from step f (3.5 g, 9.35 mmol), CDI, and THF (50 mL) were added to a 250 mL round-bottom flask. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 2 hours. The mixture was then added dropwise to NH3H2O ​​(400 mL) and stirred for 2 hours. The reaction was monitored by LCMS. The aqueous layer was extracted with EtOAc. The residue was purified by reversed-phase flash chromatography to give the desired compound (1 g, 28.65%) as a white solid. ESI-MS m / z: 372.90 [M+H] + .

[1352] Intermediate 107

[1353]

[1354] The above compound (95 mg, 91%) was prepared using the corresponding arylboronic acid in a manner similar to intermediate 47. ESI-MS m / z: 470.16 [M+H] + .

[1355] Intermediate 108

[1356]

[1357] The above compound (476 mg, 86%) was prepared using the corresponding arylboronic acid in a manner similar to intermediate 47. ESI-MS m / z: 452.13 [M+H] + .

[1358] Intermediate 109

[1359]

[1360] The above compound (382 mg, 68%) was prepared using the corresponding arylboronic acid in a manner similar to intermediate 47. ESI-MS m / z: 470 / 10 [M+H] + .

[1361] Intermediate 110

[1362]

[1363] The above compounds were prepared using the corresponding arylboronic acids in a manner similar to intermediate 47. The crude product was purified by preparative TLC (silica gel, MeOH in DCM, containing NH3) to give the desired product (305 mg, 59%) as a white solid. ESI-MS m / z: 434.15 [M+H] + .

[1364] Intermediate 111

[1365]

[1366] The above compounds were prepared using the corresponding arylboronic acids in a manner similar to intermediate 47. The crude product was purified by preparative TLC (silica gel, MeOH in DCM, containing NH3) to give the desired product (170 mg, 72%) as a white solid. ESI-MS m / z: 406.16 [M+H] + .

[1367] Intermediate 112

[1368]

[1369] The above compounds were prepared using the corresponding arylboronic acids in a manner similar to intermediate 47. The crude substance was concentrated to dryness, redissolved in EtOAc, and washed with 5 × 1 mL of NaHCO3 (saturated aqueous solution). The organic layer was dried with Na2SO4, filtered, and concentrated to give the desired product (1.311 g, 80%) as a white powder. ESI-MS m / z: 436.23 [M+H] + .

[1370] Intermediate 113

[1371]

[1372] The above compounds were prepared using the corresponding arylboronic acids in a manner similar to intermediate 47. The crude substance was concentrated to dryness, redissolved in EtOAc, and washed with 5 × 1 mL of NaHCO3 (saturated aqueous solution). The organic layer was dried with Na2SO4, filtered, and concentrated to give the desired product (589.7 mg, 82%) as a yellow powder. ESI-MS m / z: 408.29 [M+H] + .

[1373] Table 8 below contains examples prepared using methods similar to step c (PyBOP or HATU) of Example 1. Most compounds were purified by preparative HPLC (ACN / H2O, 20-90%, 25 min), and some by automated column chromatography (silica gel). Aromatic acid and amine conjugates were prepared according to intermediates 1-113, or by similar methods with slight modifications, and also according to the method described in U.S. Patent Application No. 16 / 930,622.

[1374] Table 8

[1375]

[1376]

[1377]

[1378]

[1379]

[1380]

[1381]

[1382]

[1383]

[1384]

[1385]

[1386]

[1387]

[1388] Intermediate 114

[1389]

[1390] To a dried vial fitted with a stir bar, add 6-bromo-2,2,3,3-tetrafluoro-2,3-dihydrobenzofuran (46 mg, 0.17 mmol) and 1,4-dioxane (0.85 mL). Then add Pd(dppf)Cl2 (13 mg, 0.018 mmol), pinacol diboronate (65 mg, 0.26 mmol), and potassium acetate (33 mg, 0.34 mmol), and spray the reaction mixture with N2 for 5 minutes. The vial is then resealed and heated to 90 °C. After 3 hours, the reaction mixture is cooled to room temperature, filtered through a silica gel pad, and washed with EtOAc. The filtrate is concentrated to give crude borate ester X as a dark brown oil (50 mg, assuming 100% yield), which is used without further purification.

[1391] Intermediate 115

[1392]

[1393] The following intermediate was prepared in a manner similar to that of Example 890 to obtain the desired product, a pale yellow solid (1.63 g, 62%), which was a mixture of diastereomers. ESI-MS m / z: 572.28 [M+H] + .

[1394] Table 9 below contains examples prepared using Example 890 and Intermediate 115 in a manner similar to that of Example 891. If further conversion is desired, add more palladium and boric acid. Purify most compounds by separating diastereomers using preparative HPLC (ACN / H2O, 20-90%, 25 min). If reported as a mixture of diastereomers, they may not be separable by HPLC.

[1395] Table 9

[1396]

[1397]

[1398]

[1399]

[1400]

[1401] Example 1268

[1402]

[1403] Example 1228 (15 mg, 0.024 mmol) was dissolved in EtOH (0.5 mL) and MeOH (0.5 mL), and palladium on carbon (10 wt%, 0.8 mg, 0.00075 mmol) was added. The air in the headspace of the reaction vessel was replaced with H2 from the balloon, and the mixture was stirred under H2 for 24 hours. The reaction mixture was then filtered through a diatomaceous earth saddle, and the filtrate was concentrated to give the product (14 mg, 93%). ESI-MS m / z: 638.29 [M+H] + .

[1404] Example 1269

[1405]

[1406] In a vial, Example 890 (30 mg, 0.05 mmol) was dissolved in DMF (1 mL). Morpholine (44 mg, 0.5 mmol) and K₂CO₃ (69 mg, 0.5 mmol) were added, the vial was sealed, and the mixture was heated to 100 °C for 24 hours. The reaction was cooled to room temperature and diluted with H₂O (1 mL). The aqueous layer was washed with EtOAc, and the combined organic layers were dried over MgSO₄ and concentrated under reduced pressure. The crude product was purified by preparative HPLC (ACN / H₂O, 20–90%, 25 min) to give the title compound (5 mg, 16%). ESI-MS m / z: 605.27 [M+H] + .

[1407] Example 1270

[1408]

[1409] The following examples were prepared in a manner similar to Example 1269. ESI-MS m / z: 639.20 [M+H] + .

[1410] Example 1271

[1411]

[1412] The following examples were prepared in a manner similar to Example 1269. ESI-MS m / z: 639.20 [M+H] + .

[1413] Table 10 below contains examples prepared using Example 890 and Intermediate 115, following methods similar to step c (PyBOP or HATU) of Example 1 and Example 891. Most compounds were purified by preparative HPLC (ACN / H2O, 20-90%, 25 min), with some purified by automated column chromatography (silica gel). Aromatic acid and amine conjugates were prepared according to Intermediates 1-113, or by similar methods with slight modifications, and also according to the method described in U.S. Patent Application No. 16 / 930,622.

[1414] Table 10

[1415]

[1416]

[1417]

[1418]

[1419]

[1420]

[1421] The following examples in Table 11 were prepared using methods similar to those described above:

[1422]

[1423]

[1424]

[1425]

[1426]

[1427]

[1428]

[1429]

[1430]

[1431]

[1432]

[1433]

[1434]

[1435]

[1436] Examples Assays

[1437] Methods for RSV-A determination

[1438] Hep-2 cells (originally derived from tumors grown in irradiated corticalized weaned rats injected with epidermoid carcinoma tissue from a 56-year-old male laryngeal fossa, but later found to be indistinguishable from HeLa cells by PCR DNA analysis) were used to culture genotype A, "growing" RSV. RSV was inoculated into flasks, and the virus stock was collected once the cytopathic effect (CPE) exceeded 90%. The virus stock in 25% sucrose medium was flash-frozen with liquid nitrogen to improve viral stability. The virus stock titer was 50% of the tissue culture infectious dose (TCID). 50 For quantification, 8000 cells were used per well, and the virus was diluted 3-fold in a 96-well plate and cultured for 4 days. The viral stock titer can also be quantified using the plaque-forming unit assay, the specific method of which can be found elsewhere.

[1439] After extensive parameter testing, the final determination was performed as follows: Hep-2 cells were seeded at a volume of 50 μL (8000 cells per well) into the inner 60 wells of a 96-well plate using growth medium (DMEM without phenol red, 1% L-Glut, 1% Penn / Strep, 1% non-essential amino acids, 10% heat-inactivated FBS). Two-fold serial dilutions of the control and test compounds were added to each well, for a total volume of 25 μL. Then, the viral stock solution was added to each well at a multiple of infection (MOI) of 0.1, at a volume of 25 μL, for a total volume of 100 μL per well. MOI was calculated using PFU / mL, or TCID if unavailable. 50Calculations were performed. Each 96-well plate contained 6 control columns (negative control, maximum CPE) containing cells and virus but no compound, 6 columns (positive control, minimum CPE) containing cells but no compound or virus, and 6 columns (background / reagent control) containing no cells, virus, or compound. An additional 25 μL of growth medium containing the same volume of sucrose as the wells receiving the viral stock solution was added to the control wells containing cells but no virus to maintain consistency in medium and volume conditions. 125 μL of moat media (DMEM, 1% Penn / Strep) was packed into the outer wells of the plate to act as a heat and evaporation moat around the test wells. After a 5-day incubation period, the plate was read using ATPlite (50 μL per well), quantifying the amount of ATP present in each well (a measure of cell health). The assay plate was read using an Envision photometer. These data were used to calculate the EC50 of each compound. 50 (Table 12). EC 50 The ranges are as follows: A < 0.2 μM; B > 0.2 μM.

[1440] Table 12 Summary of RSV-A activity

[1441]

[1442]

[1443]

[1444]

[1445]

[1446]

[1447]

[1448]

[1449]

[1450]

[1451]

[1452]

[1453]

[1454]

[1455] Methods for HMPV antiviral assay

[1456] Method A:

[1457] The antiviral activity of HMPV was evaluated using a recombinant form of HMPV CAN97-83, which was modified to contain an enhanced green fluorescent protein (eGFP) coding sequence (MPV-GFP1, ViraTree) at the 3' end of the viral genome. The day before analysis, Vero E6 cells (ATCC#CCL-7) were seeded in 96-well plates at a density of 12,000 cells / 100 μL / well. On the day of selection, cell culture medium was aspirated from the wells, and cells were washed twice with serum-free Eagle Modified Essential Medium (EMEM, ATCC#) (Invitrogen) (SF-EMEM) containing 1% penicillin-streptomycin. Cell washing was performed by dispensing 100 μL of SF-EMEM to each well and immediately aspirating the washing medium from the well. Following the second washing step, serum-free OptiMEM (Invitrogen, Cat No.) (SF-OptiMEM) containing 0.5 μg / mL TPCK-trypsin (VENDOR) and 1% penicillin-streptomycin was added to the cells at 50 μL / well. The compounds were added to 96-well plates using a JANUS automated liquid handling system (VENDOR). Before transferring to assay plates (25 μL / well), the compounds were first diluted 1:50 to intermediate 96-well plates containing SF-OptiMEM. Each test compound was tested in replicate wells, starting at a final concentration of 8 μM or 2 μM, using stepwise 1 / 2-stage dilutions for a total of 8 spots. Viral infection was performed by preparing a working stock of MPV-GFP1 with a multiplicity of infection (MOI) of 0.05 / 25 μL and aliquoting 25 μL of viral inoculum into the compound and positive control wells. SF-OptiMEM (25 μL / well) was added to the appropriate wells as a virus-free negative control for assay. The final DMSO concentration in all wells was 0.5%. The plate was incubated at 32°C and 5% CO2 for 5 days.

[1458] After 5 days of incubation, eGFP fluorescence intensity was measured at (x) nM wavelength using a Spectramax i3X plate reader (VENDOR). The percentage of viral inhibition was calculated using the following equation:

[1459] y = [100 - (X)] Q / X P )]x100

[1460] Among them, X Q The fluorescence intensity, X, was measured in wells containing compound-treated cells infected with recombinant MPV-GFP1. PThe average fluorescence intensity was measured in wells containing untreated cells infected with recombinant virus. EC was then calculated via nonlinear regression using a four-parameter curve logistic equation. 50 Value. The curve fitting model used is XLFit Dose ResponseOne Site Model 200:

[1461] y=(A+(B / (1+((x / C)^D))))

[1462] Where A is the minimum y value, B is the maximum y value, and C is logEC. 50 The value, D, is the slope factor. These data are used to calculate the EC value for each compound. 50 (Table 13). EC 50 The ranges are as follows: A < 0.5 μm; B > 0.5 μm.

[1463] Table 13 Summary of HMPV activity

[1464]

[1465]

[1466]

[1467] Method B:

[1468] The in vitro antiviral activity of HMPV was evaluated using clinical isolates TN / 1501 / A1 cells and LLC-MK2 cells (ATCC#CCL-7), an immortalized renal epithelial cell line derived from macaques (Macaca mulatta).

[1469] The compound was resuspended in dimethyl sulfoxide (DMSO) at 10 mM and added to a 384-well source plate. The compound was diluted and transferred to a 384-well analytical plate using an Echo-650 automated liquid handling system (Beckman Coulter, Indiana). The test compound was evaluated in duplicate at a maximum concentration of 10 μM, followed by 3-fold serial dilutions to obtain a total of 10 concentration points. The plate also included DMSO control wells, which were either infected or uninfected, serving as positive and negative controls.

[1470] TN / 1501 / A1 virus infection was performed using LLC-MK2 cell suspensions. Cells were washed twice with PBS and removed from the cell culture flasks with 0.25% trypsin-EDTA (Thermo Fisher Scientific, MA). Trypsin-EDTA was inactivated by resuspending in 2% fetal bovine serum (FBS) and OptiMEM (Thermo Fisher Scientific, MA) containing 1% penicillin-streptomycin. Cells were pelleted by centrifugation at 800 rpm for 5 min, the supernatant was removed, and the cells were resuspended in PBS supplemented with 100 μg / mL CaCl2. This step was performed twice. Cells were then resuspended in serum-free (SF)-OptiMEM containing 4 μg / mL TPCK-trypsin (Sigma Aldrich, MO), 1% penicillin-streptomycin (Thermo Fisher Scientific, MA), and 100 μg / mL CaCl2. Cells were counted and seeded at a density of 5000 cells / well, 12.5 μL / well.

[1471] Viral infection was performed with 12.5 μL of 0.014 multiples of infection (MOI) per well. 12.5 μL / well of SF-OptiMEM was added to the appropriate wells as a virus-free negative control for this assay. The final concentration of TPCK-trypsin was 2 μg / mL. Plates were incubated at 37°C with 5% CO2 for 6 days.

[1472] After 6 days of culture, 12.5 μL of ATP-Lite (PerkinElmer, MA) was added to each well, and the raw luminescence value was measured using an Envision 2104 (PerkinElmer, MA). The average raw luminescence value of cells and positive control wells containing only virus was subtracted from all test conditions, and the percentage of healthy cells was determined by dividing these values ​​by the average of negative control wells containing only cells. EC was then calculated using a four-parameter curve logistic equation via nonlinear regression. 50 The curve fitting model used is XLFit Dose Response One Site Model 200.

[1473] y = (A + (B / (1 + ((x / C)^D)))), where A is the minimum y value, B is the maximum y value, and C is log E(x / C). 50 The value, D, is the slope factor. These data are used to calculate the EC value of each compound. 50 (Table 14). EC 50 The ranges are as follows: A < 0.5 μM; B > 0.5 μM.

[1474] Table 14 summarizes the activity of WT1501 at 4h MPV.

[1475]

[1476]

[1477]

[1478]

[1479]

[1480]

[1481]

[1482]

[1483] Although the invention has been specifically shown and described with reference to preferred embodiments thereof, those skilled in the art will understand that various changes in form and detail may be made therein without departing from the scope of the invention as covered by the appended claims.

Claims

1. A compound selected from the following compounds or pharmaceutically acceptable salts thereof:

2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is 。 3. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is 。 4. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is 。 5. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is 。 6. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is 。 7. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is 。 8. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is 。 9. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is 。 10. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is 。 11. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is 。 12. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is 。 13. A compound or a pharmaceutically acceptable salt thereof, said compound being 。 14. A pharmaceutical composition comprising a compound according to any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

15. Use of the compound of any one of claims 1 to 13 in the preparation of a medicament for treating or preventing RSV infection in a subject of need.

16. Use of the compound of any one of claims 1 to 13 in the preparation of a medicament for treating or preventing HMPV infection in a subject of need.

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