CONFORMATIONALLY-CONSTRAINED INHIBITORS OF 3C OR 3C-LIKE PROTEASES
20240307419 ยท 2024-09-19
Inventors
- Kyeong-Ok Chang (Manhattan, KS)
- Yunjeong Kim (Manhattan, KS, US)
- William C. Groutas (Wichita, KS)
- Stanley Perlman (Iowa City, IA, US)
Cpc classification
C07D207/263
CHEMISTRY; METALLURGY
A61K31/5377
HUMAN NECESSITIES
C07F9/65844
CHEMISTRY; METALLURGY
A61K31/438
HUMAN NECESSITIES
A61K31/4025
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61K31/675
HUMAN NECESSITIES
C07D207/267
CHEMISTRY; METALLURGY
C07D403/12
CHEMISTRY; METALLURGY
International classification
A61K31/675
HUMAN NECESSITIES
C07D207/267
CHEMISTRY; METALLURGY
A61K31/4025
HUMAN NECESSITIES
C07F9/6584
CHEMISTRY; METALLURGY
C07D403/12
CHEMISTRY; METALLURGY
Abstract
Compounds and treatment methods with compounds exhibiting antiviral activity and/or inhibition of viral replication against viruses, particularly those belonging to the picornavirus-like supercluster, including coronavirus.
Claims
1. A viral inhibitor compound comprising formula I, or a prodrug, deuterated form, or pharmaceutically-acceptable salt thereof: ##STR00088## wherein, n is 0-6; each R.sub.0 is a branched or unbranched alkyl, cycloalkyl, aryl, arylalkyl, alkenyl, alkynyl, natural or unnatural amino acid side chain, or a combination thereof, each X is a polycyclic cycloalkane, bridged polycycle, nitrogen-, oxygen-, phosphorous-, or sulfur-containing heterocycle or polycycle, azetidine, or spirocycle; and each Z is selected from the group consisting of C.sub.1-C.sub.6 hydroxyalkyl, aldehydes, alpha-ketoamides, bisulfite salts, and bisulfite adducts of alpha-ketoamides and alpha-ketoheterocycles.
2. The compound of claim 1, wherein X is a polycyclic cycloalkane, bridged polycycle, nitrogen-containing heterocycle or polycycle, azetidine, or spirocycle.
3. The compound of claim 2, wherein the nitrogen-containing heterocycle comprises at least one ring nitrogen atom and 0-3 additional ring heteroatoms selected from nitrogen, oxygen, phosphorous, and sulfur.
4. The compound of claim 1 or claim 2, wherein X is selected from the group consisting of ##STR00089## ##STR00090## ##STR00091## and deuterated forms thereof, prodrugs thereof, or pharmaceutically-acceptable salts thereof.
5. A compound of Table 2, Table 6, Table 7, Table 8, Table 9, Table 10, Table 11, or Table 12, or a pharmaceutically acceptable salt thereof.
6. The compound of claim 5, wherein the compound comprises ##STR00092## or the deuterated forms thereof, prodrugs thereof, or pharmaceutically-acceptable salts thereof.
7. The compound of any one of claims 1-6, wherein said compound inhibits viral replication of coronavirus as well as against other viruses that belong to the picornavirus-like supercluster, including caliciviruses and picornaviruses.
8. The compound of any one of claims 1-7, wherein said compound inhibits 3C-like protease activity of said virus.
9. The compound of any one of claims 1-8, wherein said compound has broad spectrum activity effective against multiple viruses, including Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2.
10. A method of treating or preventing viral infection from one or more coronaviruses, caliciviruses, or picornaviruses in a subject, said method comprising administering to said subject a therapeutically-effective amount of a first viral inhibitor compound according to any one of claims 1-9.
11. The method of claim 10, wherein said compound is dispersed in a pharmaceutically-acceptable carrier.
12. The method of claim 10 or 11, further comprising providing a unit dosage form of said compound dispersed in said pharmaceutically-acceptable carrier prior to said administering.
13. The method of any one of claims 10 to 12, further comprising administering a second viral inhibitor compound to said subject.
14. The method of claim 13, wherein said second compound is a viral inhibitor compound according to any one of claims 1 to 8, said first compound being different from said second compound.
15. The method of claim 13 or 14, wherein said first and second compounds are co-administered.
16. The method of any one of claims 13 to 15, both of said compounds being dispersed or dissolved in a pharmaceutically-acceptable carrier.
17. The method of any one of claims 10 to 16, wherein said subject is suffering from a viral infection from coronaviruses, caliciviruses, or picornaviruses prior to said administering.
18. The method of any one of claims 10 to 16, wherein said subject is free from a viral infection from coronaviruses, caliciviruses, or picornaviruses prior to said administering.
19. The method of any one of claims 10 to 18, wherein said compound is administered intramuscularly, subcutaneously, intradermally, intranasally, intravenously, orally, or via a transdermal patch.
20. The method of any one of claims 10 to 19, wherein said subject is a human.
21. The method of any one of claims 10 to 19, wherein said subject is a non-human animal.
22. A broad spectrum antiviral composition comprising a first viral inhibitor compound according to any one of claims 1 to 9 dispersed in a pharmaceutically-acceptable carrier.
23. The composition of claim 22, wherein said carrier is selected from the group consisting of sterile isotonic aqueous buffer, normal saline, phosphate buffered saline, DMSO, sterile water, oil-in-water emulsion, water-in-oil emulsion, and mixtures thereof.
24. The composition of claim 22 or 23, said composition comprising from about 5% to about 95% by weight of said viral inhibitor compound, based upon the total weight of said composition taken as 100% by weight.
25. The composition of any one of claims 22 to 24, further comprising a second compound, both of said compounds being dispersed in said pharmaceutically-acceptable carrier.
26. The composition of claim 25, wherein said second compound is a viral inhibitor compound according to any one of claims 1 to 9, said first compound being different from said second compound.
27. The composition of any one of claims 22 to 26, further comprising adjuvants, other active agents, preservatives, buffering agents, salts, and mixtures thereof.
28. A kit comprising: a viral inhibitor compound according to any one of claims 1 to 9; and instructions for administering said compound to a subject in need thereof.
29. The kit of claim 28, wherein said compound is provided in unit dosage form.
30. The kit of claim 28 or 29, wherein said compound is provided in a first container, said kit further comprising a carrier in a second container; and instructions for preparing said antiviral compound for administration to said subject.
31. A method of preventing or inhibiting replication of a virus in a cell, said method comprising contacting said cell with a viral inhibitor compound according to any one of claims 1 to 9, wherein said virus is a coronavirus, calicivirus, or picronavirus.
32. The method of claim 31, wherein said virus is selected from the group consisting of MERS-CoV, SARS-CoV, and SARS-CoV-2.
33. Use of a viral inhibitor compound according to any one of claims 1 to 9 to prepare a therapeutic or prophylactic medicament for the treatment or prevention of a viral infection from coronaviruses, caliciviruses, or picronaviruses in a subject.
34. A method of synthesizing a viral inhibitor compound that inhibits 3C-like protease activity of coronavirus as well as against other viruses that belong to the picornavirus-like supercluster, including caliciviruses and picornaviruses, said method comprising: activating a polycyclic cycloalkane, bridged polycycle, nitrogen-, oxygen-, phosphorous-, or sulfur-containing heterocycle or polycycle, azetidine, or spirocycle compound with a reactive primary or secondary alcohol group with N,N-disuccinimidyl carbonate (DSC) to yield a mixed carbonate; coupling said mixed carbonate with an amino alcohol precursor of formula I to yield an intermediate alcohol compound; and oxidizing said intermediate alcohol with Dess-Martin periodinane (DMP) to generate the corresponding aldehyde of said intermediate alcohol, wherein said aldehyde is then optionally converted to a ketoamide, bisulfite salt, bisulfite adduct of an alpha-ketoamide, or bisulfite adduct of an alpha-ketoheterocycle.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
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DETAILED DESCRIPTION
[0044] A series of novel protease inhibitors has been synthesized and shown to possess broad-spectrum activity against multiple coronaviruses including MERS-CoV, SARS-CoV, and SARS-CoV-2, as well as against other viruses that belong to the picornavirus-like supercluster, including caliciviruses and picornaviruses, in enzymatic and cell-based assays. The efficacy of the compounds in an animal model of MERS-CoV infection is also demonstrated. Members of this series of compounds are highly effective as antiviral therapeutics targeting a specific virus or, more importantly, they are broad-spectrum antivirals targeting multiple viruses. The wide applicability of the latter constitutes a significant advance in antiviral research and public health.
[0045] Embodiments described herein include antiviral compounds having broad-spectrum (multivalent) activity against coronaviruses as well as against other viruses that belong to the picornavirus-like supercluster, including caliciviruses and picornaviruses. The compounds are small-molecule based antivirals that effectively target and inhibit viral 3CL protease activity across multiple virus species, strains, and subtypes, thereby preventing formation of the mature virus and inhibiting virus replication in the host cell. In some embodiments, the compounds are prodrugs that are converted into active compounds that target and inhibit viral 3CL protease activity.
[0046] In some embodiments, antiviral compounds comprising (consisting essentially or even consisting of) formula (I), the series derivatives B, D, E, and F, described herein, or the pharmaceutically-acceptable salts thereof, are provided. In such compounds, the design element (X group) is selected from the group consisting of polycyclic cyclohexane derivatives, preferably bridged polycycles (bi- and tri-), as well as nitrogen, oxygen, phosphorous, or sulfur-containing heterocycles and polycycles, particularly 5-member heterocycles, such as piperidine derivatives, pyrrolidine derivatives, pyrrolidinones, fluorinated pyrrolidine, phospholane derivatives, as well as 4-member azetidines, and 4-, 5-, and 6-member spirocyclic compounds.
[0047] The recognition element in the inhibitor compounds encompasses the R.sub.0 sidechain (preferably leucine) and glutamine surrogate fragment which drive substrate specificity to the viral protease, as well as the peptidyl design element (X group), which is configured to control spatial orientation of the compound for enhanced binding as well as other pharmacokinetic features of the inhibitor compounds. The design element X can be derived from one of following precursor compounds having a reactive primary or secondary alcohol group for conjugation with the glutamine surrogate and side chain fragment for synthesis of the inhibitors. In an improved reaction scheme, as illustrated in
TABLE-US-00001 TABLE 1 X Design Moieties-Alcohol Inputs Conformationally constrained cyclohexane derivatives (Series B)
[0048] Further embodiments contemplated herein for X design elements include conformationally-constrained piperidine bicycles:
##STR00033##
where Y is (CR.sub.iR.sub.j).sub.n, where n can be 0 (meaning Y is not present and the oxygen is directly bonded to carbon of the ring) or 1-2 (meaning Y?CR.sub.iR.sub.j where R.sub.i and R.sub.j can both be H or D, or both alkyl (e.g., methyl), or one H or D and the other alkyl (methyl)), and R is COOR, SO.sub.2R, CONHR, alkyl, arylalkyl, substituted or unsubstituted phenyl, or CN. An exemplary synthesis of these compounds is illustrated below.
##STR00034##
[0049] The initial step starts with reaction of the starting material scaffold with the desired derivative R group (e.g., as carboxylic acid, RSO.sub.2Cl, phenyl, substituted phenyl, etc.), followed by mixing with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI) to form the acid, and then treated with diisopropylethylamine (DIEA), using a traditional approach as described in U.S. Pat. No. 11,013,779, followed by reduction to form the alcohol, which can then be conjugated to the glutamine surrogate/side chain fragment using the new synthesis approach illustrated in
[0050] Additional substituents for any of the foregoing X design elements may be defined further below with respect to specific compounds. Each of the foregoing structures is subject to the proviso that at least one group is an OH group for participation in the reaction described above. For example, at least one position in the structure contains an OH group, and when that position contains the primary or secondary alcohol for reaction, the other position(s) in the compound can be derivatized. As a nonlimiting example, an N-Boc protecting group can be removed during the synthesis process, to give a secondary amine, which can be derivatized by introducing an alkyl, arylalkyl, substituted aryl, C?O)alkyl, C?O)Oalkyl, SO.sub.2alkyl, and the like, such that the other positions in the structure contain a substituent comprising an OH group (e.g., CHOH, C(OH)alkyl, (CH.sub.2)2OH, CH.sub.2CD.sub.2OH, and the like. Similarly, typically when there is a nitrogen in the scaffold (e.g., piperidines or constrained piperidines, pyrrolidines or constrained pyrrolidines, etc.), it is preferably that the R group on the nitrogen will selected from H, alkyl, aryl, arylalkyl, substituted aryl, CN, (C?O)R, (C?O)OR, or SO.sub.2R, CH.sub.3SO.sub.2, CONHR, PhCH.sub.2CO, n-C.sub.6H.sub.11CO as well as a Cbz (COOC(benzyl) or Boc protecting group (COOC(CH.sub.3).sub.3), where R is an alkyl or arylalkyl. As used herein, the term alkyl refers to straight chained and branched saturated hydrocarbon groups containing one to thirty carbon atoms, for example, one to twenty carbon atoms, or one to ten carbon atoms, preferably one to six carbon atoms. The term C.sub.n means the alkyl group has n carbon atoms. For example, C.sub.4 alkyl refers to an alkyl group that has 4 carbon atoms. C.sub.1-C.sub.6 alkyl refers to an alkyl group having a number of carbon atoms encompassing the entire range (e.g., 1 to 6 carbon atoms), as well as all subgroups (e.g., 1-6, 2-7, 1-5, 3-6, 1, 2, 3, 4, 5, and 6 carbon atoms). Nonlimiting examples of alkyl groups include, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl (2-methylpropyl), and t-butyl. Unless otherwise indicated, an alkyl group can be an unsubstituted alkyl group or a substituted alkyl group.
[0051] In some embodiments, particularly preferred X moieties include the following structures below connected to the backbone via the dashed line.
##STR00035## ##STR00036## ##STR00037##
[0052] As noted elsewhere, it is contemplated that in any of the foregoing structures, the X moiety may be connected directly to the oxygen in the inhibitor backbone, or via a branched or unbranched alkyl linkage.
[0053] In one or more embodiments, using the foregoing backbone and alcohol input structures, exemplary aldehyde or bisulfite adduct inhibitors would thus comprise a structure such as:
##STR00038##
[0054] The present disclosure encompasses deuterated forms of the foregoing compounds, for example where hydrogen groups within the metabolically active sites of the X design element cyclic rings and/or in the adjacent carbon groups (e.g., methylene linkage) can be substituted with deuterium to generate deuterated variants of the compounds. It will be appreciated that pharmaceutically-acceptable salts of any of the compounds described here as well as their prodrug forms are also contemplated herein.
[0055] The term pharmaceutically-acceptable salt refers to an acid or base salt of a compound of the disclosure, which salt possesses the desired antiviral activity and is neither biologically nor otherwise undesirable. The present disclosure also includes prodrugs (ester, amide, carbamate, carbonate, ether, imine, phosphate, etc. derivatives) of the disclosed compounds. For example, the warhead, Z moiety, can be modified to generate prodrug forms of the compounds, which are described in detail in U.S. Pat. No. 11,033,600, incorporated by reference herein in its entirety.
[0056] Prophylactic and/or therapeutic compositions with specific or broad-spectrum antiviral activities are also disclosed. Combinations of one or more of the foregoing compounds are also contemplated. The compositions comprise an antiviral compound described herein dispersed in a pharmaceutically-acceptable carrier. The term carrier is used herein to refer to diluents, excipients, vehicles, and the like, in which the antiviral may be dispersed for administration. Suitable carriers will be pharmaceutically acceptable. As used herein, the term pharmaceutically acceptable means not biologically or otherwise undesirable, in that it can be administered to a subject without excessive toxicity, irritation, or allergic response, and does not cause unacceptable biological effects or interact in a deleterious manner with any of the other components of the composition in which it is contained. A pharmaceutically-acceptable carrier would be selected to minimize any degradation of the compound or other agents and to minimize any adverse side effects in the subject. Pharmaceutically-acceptable ingredients include those acceptable for veterinary use as well as human pharmaceutical use, and will depend on the route of administration. For example, compositions suitable for administration via injection are typically solutions in sterile isotonic aqueous buffer. Exemplary carriers include aqueous solutions such as normal (n.) saline (?0.9% NaCl), phosphate buffered saline (PBS), sterile water/distilled autoclaved water (DAW), various oil-in-water or water-in-oil emulsions, as well as dimethyl sulfoxide (DMSO) or other acceptable vehicles, and the like.
[0057] The composition can comprise a therapeutically effective amount of the compound dispersed in the carrier. As used herein, a therapeutically effective amount refers to the amount that will elicit the biological or medical response of a tissue, system, or subject that is being sought by a researcher or clinician, and in particular elicit some desired therapeutic or prophylactic effect as against the viral infection by slowing and/or inhibiting 3CL protease activity and/or viral replication. One of skill in the art recognizes that an amount may be considered therapeutically effective even if the condition is not totally eradicated or prevented, but it or its symptoms and/or effects are improved or alleviated partially in the subject. In some embodiments, the composition will comprise from about 5% to about 95% by weight of an antiviral compound described herein, and preferably from about 30% to about 90% by weight of the antiviral compound, based upon the total weight of the composition taken as 100% by weight. In some embodiments, combinations of more than one type of the described antiviral compounds can be included in the composition, in which case the total levels of all such compounds will preferably fall within the ranges described above.
[0058] Other ingredients may be included in the composition, such as adjuvants, other active agents, preservatives, buffering agents, salts, other pharmaceutically-acceptable ingredients. The term adjuvant is used herein to refer to substances that have immunopotentiating effects and are added to or co-formulated in a therapeutic composition in order to enhance, elicit, and/or modulate the innate, humoral, and/or cell-mediated immune response against the active ingredients. Other active agents that could be included in the composition include other antiviral compounds (e.g., cathepsins) or any immunogenic active components (e.g., antigens) such as those that resemble a disease-causing microorganism or infectious agent, and/or are made from weakened or killed forms of the same, its toxins, subunits, particles, and/or one of its surface proteins, such that it provokes an immune response to that microorganism or infectious agent. In addition to live, modified, or attenuated vaccine components, active agents using synthetic peptides, carbohydrates, or antigens can also be used.
[0059] Compositions according to the embodiments disclosed herein are useful in inhibiting protease activity. More specifically, the compositions can be used to inhibit viral infection or viral replication, such as by treating and/or preventing viral infection from a variety of causes, including caliciviruses (noroviruses), picornaviruses, and/or coronaviruses in a subject. Viruses in the picornavirus-like supercluster include important human and animal pathogens. For example, caliciviruses include noroviruses (Norwalk virus [NV]), feline calicivirus, MD145, murine norovirus [MNV], vesicular exanthema of swine virus, and rabbit hemorrhagic disease virus. Picornaviruses include enteroviruses (such as enterovirus 71), poliovirus, coxsackievirus, foot-and-mouth disease virus (FMDV), hepatitis A virus (HAV), porcine teschovirus, and rhinovirus (cause of common cold). Coronaviruses include human coronavirus (cause of common cold such as 229E strain), transmissible gastroenteritis virus (TGEV), murine hepatitis virus (MHV), bovine coronavirus (BCV), feline infectious peritonitis virus (FIPV), severe acute respiratory syndrome coronavirus (SARS-Co), SARS-CoV2 (causative agent of COVID-19), and Middle East respiratory syndrome coronavirus (MERS-CoV).
[0060] Compositions according to the embodiments disclosed herein are useful in treating and/or preventing viral infection from coronaviruses as well as against other viruses that belong to the picornavirus-like supercluster, including caliciviruses and picornaviruses in a subject. Thus, embodiments described herein have broad-spectrum therapeutic and/or prophylactic uses. The terms therapeutic or treat, as used herein, refer to processes that are intended to produce a beneficial change in an existing condition (e.g., viral infection, disease, disorder) of a subject, such as by reducing the severity of the clinical symptoms and/or effects of the infection, and/or reducing the duration of the infection/symptoms/effects. The terms prophylactic or prevent, as used herein, refer to processes that are intended to inhibit or ameliorate the effects of a future viral infection or disease to which a subject may be exposed (but is not currently infected with). In some cases, the composition may prevent the development of observable morbidity from viral infection (i.e., near 100% prevention). In other cases, the composition may only partially prevent and/or lessen the extent of morbidity due to the viral infection (i.e., reduce the severity of the symptoms and/or effects of the infection, and/or reduce the duration of the infection/symptoms/effects, or increase the rate of recovery from the condition). In either case, the compounds are still considered to prevent the target infection or disease.
[0061] In use, a therapeutically-effective amount of an antiviral compound is administered to a subject. In some embodiments, a composition comprising a therapeutically-effective amount of an antiviral compound is administered to a subject. Regardless, the compound or pharmaceutically acceptable salt thereof will preferably be administered to the subject in an amount sufficient to provide antiviral compound levels (independent of salt, if any) of from about 0.1 mg to about 1,000 mg of compound per kg of body weight of the subject, preferably from about 1 mg/kg to about 100 mg/kg of body weight of the subject, and more preferably from about 10 mg/kg to about 50 mg/kg of body weight of the subject. Thus, it will be appreciated that in the case of compound salts, for example, the formulation may be administered in amounts greater than the above ranges to provide sufficient levels of the active compound.
[0062] In some embodiments, the subject is afflicted with or suffering from a condition (e.g., infection, disease, or disorder) before the compounds are administered, wherein methods described herein are useful for treating the condition and/or ameliorating the effects of the condition. Preferably, the antiviral compound is administered as soon as possible after infection, preferably within about 7 days from onset of observable symptoms, more preferably within about 5 days from onset of observable symptoms, even more preferably within 3 days from onset of observable symptoms. It will be appreciated that the sooner the compound(s) is administered, the increased chance of successfully reducing effects of the viral infection. In other embodiments, the subject is free of a given condition before administering the compound, wherein the methods described herein are useful for preventing the occurrence or incidence of the condition and/or preventing the effects of the condition, as described above.
[0063] The disclosed embodiments are suitable for various routes of administration, depending upon the particular carrier and other ingredients used. For example, the prophylactic and/or therapeutic compounds or compositions can be injected intramuscularly, subcutaneously, intradermally, or intravenously. They can also be administered via mucosa such as intranasally or orally. The compounds or compositions can also be administered through the skin via a transdermal patch.
[0064] In some embodiments, the compound or compositions can be provided in unit dosage form in a suitable container. The term unit dosage form refers to a physically discrete unit suitable as a unitary dosage for human or animal use. Each unit dosage form may contain a predetermined amount of a compound disclosed herein (and/or other active agents) in the carrier calculated to produce a desired effect. In other embodiments, the compound can be provided separate from the carrier (e.g., in its own vial, ampule, sachet, or other suitable container) for on-site mixing before administration to a subject. A kit comprising the antiviral compound(s) is also disclosed herein. The kit further comprises instructions for administering the compound to a subject. The antiviral compound(s) can be provided as part of a dosage unit, already dispersed in a pharmaceutically-acceptable carrier, or it can be provided separately from the carrier. The kit can further comprise instructions for preparing the antiviral compounds for administration to a subject, including for example, instructions for dispersing the compounds in a suitable carrier.
[0065] It will be appreciated that therapeutic and prophylactic methods described herein are applicable to humans as well as any suitable animal, including, without limitation, dogs, cats, and other pets or captive animals (e.g., zoo animals, research subjects), as well as, rodents, primates, horses, cattle, pigs, etc. The methods can be also applied for clinical research and/or study. Additional advantages of the various embodiments of the disclosure will be apparent to those skilled in the art upon review of the disclosure herein and the working examples below. It will be appreciated that the various embodiments described herein are not necessarily mutually exclusive unless otherwise indicated herein. For example, a feature described or depicted in one embodiment may also be included in other embodiments, but is not necessarily included. Thus, the present disclosure encompasses a variety of combinations and/or integrations of the specific embodiments described and claimed herein.
[0066] As used herein, the phrase and/or, when used in a list of two or more items, means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed. For example, if a composition is described as containing or excluding components A, B, and/or C, the composition can contain or exclude A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.
[0067] The present description also uses numerical ranges to quantify certain parameters relating to various embodiments of the disclosure. It should be understood that when numerical ranges are provided, such ranges are to be construed as providing literal support for claim limitations that only recite the lower value of the range as well as claim limitations that only recite the upper value of the range. For example, a disclosed numerical range of about 10 to about 100 provides literal support for a claim reciting greater than about 10 (with no upper bounds) and a claim reciting less than about 100 (with no lower bounds).
EXAMPLES
[0068] The following examples set forth methods in accordance with the disclosure. It is to be understood, however, that these examples are provided by way of illustration and nothing therein should be taken as a limitation upon the overall scope of the disclosure. Except where noted, precursor, intermediate, and final compounds described in the synthesis reactions below are independently numbered in each Example. Structures are indicated in the Tables below for avoidance of doubt.
Introduction
[0069] Coronaviruses are enveloped, positive-sense, single-stranded RNA viruses that belong to the family Coronaviridae. Among human coronaviruses, several strains (229E, NL63, OC43, and KHU1) are the cause of mild upper respiratory infections; however, a few coronaviruses have emerged from animals that cause severe respiratory disease, including Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Of particular concern is SARS-CoV-2, the highly pathogenic causative agent of COVID-19 which is associated with a high mortality rate and is a significant threat to public health worldwide. The problem is further compounded by the current lack of effective vaccines or small molecule therapeutics for the treatment of SARS-CoV-2 infections, underscoring the urgent and dire need for the development of prophylactic and therapeutic countermeasures to combat infections by pathogenic coronaviruses.
[0070] The SARS-CoV-2 genome is large (?30 kb) and similar to the genomes of SARS-CoV and MERS-CoV (?80% and ?50% sequence identity, respectively). It contains two open reading frames (ORF1a and ORF1b) and encodes multiple structural and nonstructural proteins. Translation of the genomic mRNA of ORF1a yields a polyprotein (pp1a), while a second polyprotein (pp1b) is the product of a ribosomal frameshift that joins ORF1a together with ORF1b. The two polyproteins are processed by a 3C-like protease (3CLpro, also referred to as Main protease, M.sup.rpo) (11 cleavage sites) and a papain-like cysteine protease (PLpro), resulting in 16 mature nonstructural proteins which are involved in the replication-transcription complex. The two proteases are essential for viral replication, making them attractive targets for therapeutic intervention.
[0071] SARS-CoV-2 3CLpro is a homodimer with a catalytic Cys-His dyad (Cys.sup.145-His.sup.41) and an extended binding cleft. Substrate specificity profiling studies have shown that the protease displays a strong preference for a YZ-Leu-Gln-X sequence, where X is a small amino acid, Y is a hydrophobic amino acid, and Z is solvent-exposed and fairly diverse (V/T/K), corresponding to the subsites S.sub.4-S.sub.3S.sub.2S.sub.1-S.sub.1. Cleavage is at the P.sub.1-P.sub.1 scissile bond. The 3D structure of SARS-CoV-2 3CLpro is similar to that of SARS-CoV 3CLpro, however, the S.sub.2 subsite of SARS-CoV-2 3CLpro displays considerable plasticity and can accommodate natural and unnatural amino acids with smaller side chains. High-resolution crystal structures with bound inhibitors have been determined, enabling the use of structure-guided approaches in the design of inhibitors. In continuing our foray in this area, we report herein the results of preliminary studies related to the inhibition of SARS-CoV-2 protease by a series of inhibitors (I) that incorporate in their structure a conformationally-constrained cyclohexane moiety envisaged to exploit new chemical space and to optimally engage in favorable binding interactions with the active site of the protease.
[0072] Inhibitor Design Rationale. The design of inhibitor (I) (Scheme) included the use of a P.sub.1 glutamine surrogate residue and a P.sub.2 Leu residue as recognition elements congruent with the substrate specificity of the protease, as well as an aldehyde warhead or a latent aldehyde bisulfite adduct. The design of inhibitor (I) was further abetted by insights gained from examining the available X-ray crystal structures of the protease with inhibitors.
##STR00039##
Example 1
[0073] In this Example, a series of non-deuterated and deuterated dipeptidyl aldehyde and masked aldehyde inhibitors (2 (a-o)) and bisulfite adducts thereof (3 (a-o)) that incorporate in their structure a conformationally-constrained cyclohexane moiety was synthesized and found to potently inhibit SARS-CoV-2 3CL protease in biochemical and cell-based assays. Several of the inhibitors were also found to be nanomolar inhibitors of MERS-CoV 3CL protease. The corresponding latent aldehyde bisulfite adducts were found to be equipotent to the precursor aldehydes. High-resolution cocrystal structures confirmed the mechanism of action and illuminated the structural determinants involved in binding. The spatial disposition of the compounds disclosed herein provides an effective means of accessing new chemical space and optimizing pharmacological activity. The cellular permeability of the identified inhibitors and lack of cytotoxicity warrants their advancement as potential therapeutics for COVID-19.
Results and Discussion
Inhibitor Design Rationale
[0074] The design of the inhibitors (
Chemistry
[0075] The synthesis of inhibitors 2(a-o) and 3(a-o) was readily accomplished by activating the precursor primary or secondary alcohol inputs (Table 2) with N,N-disuccinimidyl carbonate (DSC) and coupling the mixed carbonate with the readily-accessible Leu-Gln surrogate amino alcohol to yield alcohol product 1 which was oxidized with Dess-Martin periodinane (DMP) to generate the corresponding aldehyde 2 (
TABLE-US-00002 TABLE 2 Series B Alcohol Inputs for derivatives of inhibitors 2 (aldehyde) and 3 (bisulfite adducts)
Biochemical Studies
[0076] The inhibitory activity of the aldehydes (compounds 2 (a-o)) and bisulfite adducts (compounds 3 (a-o)) against SARS-CoV-2 3CL protease and their activity in a cell-based system, were determined as described in the experimental section. The IC.sub.50 values (50% inhibitory concentration in the enzyme assay); EC.sub.50 values (50% effective concentration in cell culture) for two representative inhibitors (2a/3a), and the CC.sub.50 values (50% cytotoxic concentration in cell-based assays) in Huh-7, CRFK, or CCL1 cells are summarized in Table 3 and they are the average of at least two determinations. The inhibitory activity of compounds 2a/3a, 2f/3f, and 2k/3k against IVERS-CoV 3CL protease was also determined as described previously and the IC.sub.50 values are listed in Table 4.
TABLE-US-00003 TABLE 3 IC.sub.50 and CC.sub.50 values of SARS-CoV-2 3CLpro inhibitors 2-3(a-o) Compound IC.sub.50 (?M) CC.sub.50 (?M) 2a 0.18 ? 0.03.sup.a >100 3a 0.17 ? 0.02.sup.a >100 2b 0.29 ? 0.05 >100 3b 0.29 ? 0.01 >100 2c 0.31 ? 0.06 >100 3c 0.26 ? 0.05 >100 2d 0.28 ? 0.08 >100 3d 0.30 ? 0.03 >100 2e 0.26 ? 0.05 >100 3e 0.28 ? 0.03 >100 2f 0.14 ? 0.02 >100 3f 0.10 ? 0.01 >100 2g 1.90 ? 0.14 >100 3g 1.71 ? 0.16 >100 2h 0.39 ? 0.02 >100 3h 0.37 ? 0.03 >100 2i 0.27 ? 0.02 21 ? 1 3i 0.30 ? 0.02 21 ? 3 2j 0.31 ? 0.03 20 ? 3 3j 0.31 ? 0.06 23 ? 1 2k 0.18 ? 0.04 >100 3k 0.15 ? 0.04 >100 2l 0.29 ? 0.04 >100 3l 0.27 ? 0.04 >100 2m 0.27 ? 0.02 >100 3m 0.25 ? 0.04 >100 2n 0.82 ? 0.19 >100 3n 1.03 ? 0.24 >100 2o 0.74 ? 0.26 >100 3o 0.78 ? 0.18 >100 .sup.aThe EC.sub.50 values for inhibitors 2a and 3a against SARS-CoV-2 in Vero E6 cells were 0.035 ? 0.001 and 0.032 ? 0.001 ?M, respectively.
TABLE-US-00004 TABLE 4 IC.sub.50 values of MERS-CoV 3CLpro inhibitors 2a/3a, 2f/3f and 2k/3k. Compound IC.sub.50 (?M) 2a 0.052 ? 0.001 3a 0.049 ? 0.002 2f 0.063 ? 0.003 3f 0.058 ? 0.002 2k 0.055 ? 0.002 3k 0.053 ? 0.003
[0077] It is clearly evident from the results shown in Table 3 that the synthesized compounds display high potency in biochemical assays, with most IC.sub.50 values in the sub-micromolar range. Furthermore, the inhibitors were found to be devoid of cytotoxicity and the Safety Index (SI), defined as the CC.sub.50/IC.sub.50 ratio, ranged between ?78 to 1110. The potency of deuterated variants 2b/3b decreased ?1.6-fold (aldehydes) and ?1.7-fold (bisulfite adducts) as compared to the respective non-deuterated compounds 2a/3a and remained essentially the same in the case of non-deuterated 2n/3n and deuterated 2o/3o inhibitors, respectively. A change in geometry from a cyclohexene (2e/3e) to a cyclohexane (2f/3f) resulted in a 2 to 3-fold increase in potency. The ?5-fold decrease in potency of compounds 2n/3n compared to 2k/3k presumably reflects the inimical effect on potency of the 3? hydroxyl group. Importantly, the EC.sub.50 values of two representative inhibitors (2a/3a) against SARS-CoV-2 in Vero E6 cells were found to be ?4.6-fold lower (EC.sub.50 0.035 and 0.032 ?M, respectively) than the corresponding IC.sub.50 values, and the selectivity indices of compounds 2a/3a were very high (2857 and 3125, respectively). The significance of these findings was further augmented by the notable inhibition of MERS-CoV 3CL protease by a select number of inhibitors (Table 3, compounds 2a/3a, 2f/3f and 2k/3k), demonstrating the broad spectrum of antiviral activity displayed by this series of compounds.
[0078] Emergence of viral resistance to antiviral drugs is a major concern. We previously reported that GC376 has a high barrier to resistance to feline infectious peritonitis virus (FIPV) in cell culture and naturally infected animals with long term treatment. We also examined several compounds similar to the series in this report for emergence of viral resistance by serial passaging FIPV in the presence of each compound in cell culture. The EC.sub.50 values of the compounds did not increase at up to 10 passage number, and the 3CLpro of viruses passaged with each compound has the same sequence as mock-passaged viruses. These results suggest that this series of compounds have a high barrier to resistance.
X-Ray Crystallography Studies
[0079] In order to elucidate the mechanism of action of the inhibitors as well as identify the structural determinants associated with the binding of inhibitors to the active site of SARS-CoV-2 3CL protease, high-resolution cocrystal structures were determined for inhibitors 2a and its deuterated analog 3b, 2f, 2k, 3c and its deuterated analog 3d and 3e. The structure of SARS-CoV-2 3CLpro in complex with compound 2a contained prominent difference electron density consistent with the inhibitor covalently bound to Cys 145 in each subunit (
[0080] Likewise, the structure of 3c shows similar binding mode properties as observed for 2a (
[0081] Similarly, inhibitors 2f, 2k and 3e in complex with SARS-CoV-2 were found to adopt similar binding modes in the active site of the protease as shown in
Conclusions
[0082] Given the major clinical importance associated with the SARS-CoV-2 pandemic and the current paucity of effective countermeasures, the results of the studies described herein can serve as a launching pad for conducting further pre-clinical studies. Most of the compounds exhibited high potency in biochemical assays and, for two of the compounds tested, in cellular assays. Furthermore, members of this series were also found to potently inhibit MERS-CoV 3CL protease, suggesting that the compounds can be developed into broad-spectrum antivirals. Since there are no known human proteases that have a primary substrate specificity P.sub.1 residue that is Gln, these inhibitors could also display high selectivity and diminished off-target effects. Furthermore, the utilization of an aldehyde warhead, or a latent aldehyde functionality that can rapidly generate the aldehyde in vivo, in the design of transition state inhibitors is advantageous for several reasons, including rapid engagement with the target leading to the reversible formation of a covalent adduct. The high reactivity of aldehydes is generally viewed as a toxicity alert, however, the safety indices for most of the compounds reported herein were found to be high. Indeed, a number of pharmaceuticals that incorporate in their structure an aldehyde functionality are currently in clinical use and, furthermore, toxicity arising from the presence of the aldehyde is context-specific, as is presumably the case here. Finally, the present study also sought to exploit the kinetic isotope effect associated with the H/D bioisosteric replacement in order to dampen oxidative metabolism at the CH.sub.2O metabolic soft spot in the inhibitors, as well as to reduce toxicity. Thus, the availability of equipotent deuterated analogs that display improved pharmacokinetics (PK) characteristics enhances further the significance of the results reported herein. Evaluation of a select number of inhibitors in a mouse model of SARS-CoV-2 infection is in progress and the results will be reported in due course. In conclusion, a series of potent transition state inhibitors of SARS-CoV-2 3CL protease that incorporate in their structures a conformationally-constrained cyclohexyl moiety is reported.
Experimental Section
General
[0083] Reagents and dry solvents were purchased from various chemical suppliers (Sigma-Aldrich, Acros Organics, Chem-Impex, TCI America, Oakwood chemical, APExBIO, Cambridge Isotopes, Alpha Aesar, Fisher and Advanced Chemblocks) and were used as obtained. Silica gel (230-450 mesh) used for flash chromatography was purchased from Sorbent Technologies (Atlanta, GA). Thin layer chromatography was performed using Analtech silica gel plates. Visualization was accomplished using UV light and/or iodine. NMR spectra were recorded in CDCl.sub.3 or DMSO-d.sub.6 using Varian XL-400 spectrometer. Melting points were recorded on a Mel-Temp apparatus and are uncorrected. High resolution mass spectrometry (HRMS) was performed at the Wichita State University Mass Spectrometry lab using Orbitrap Velos Pro mass spectrometer (ThermoFisher, Waltham, MA) equipped with an electrospray ion source. The purity of all final compounds was >95% as evidenced by NMR analysis.
Synthesis of Compounds
[0084] Preparation of compounds 1(a-o). General procedure. As illustrated in
[0085] To a solution of Leu-Gln surrogate amino alcohol (1.0 eq) in dry methylene chloride (10 mL/g of amino alcohol) was added TEA (1.5 eq) and the reaction mixture was stirred for 20 min at room temperature (solution 1). In a separate flask, the mixed carbonate was dissolved in dry methylene chloride (10 mL/g of carbonate) (solution 2). Solution 1 was added to solution 2 and the reaction mixture was stirred 3 h at room temperature. Methylene chloride was added to the organic phase (40 mL/g of carbonate) and then washed with saturated aqueous NaHCO.sub.3(2?20 mL/g alcohol), followed by brine (20 mL/g alcohol). The organic phase was dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The resultant crude product was purified by flash chromatography (hexane/ethyl acetate) to yield the dipeptidyl alcohol 1 for each respective precursor, as a white solid.
[0086] ((1R,5S)-Bicyclo[3.3.1]nonan-3-yl)methyl ((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (1a). Yield (3 5%); .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.75 (d, J=7.2 Hz, 1H), 6.24 (s, 1H), 5.31 (d, J=9.1 Hz, 1H), 4.29-4.10 (m, 1H), 4.10-3.93 (m, 1H), 3.89 (d, J=6.3 Hz, 1H), 3.71-3.54 (m, 2H), 3.48 (d, J=2.0 Hz, 1H), 3.39-3.27 (m, 2H), 2.55-2.25 (m, 3H), 2.03 (d, J=11.9 Hz, 3H), 1.96-1.77 (m, 4H), 1.77-1.57 (m, 4H), 1.57-1.44 (m, 1H), 1.37 (d, J=9.5 Hz, 4H), 1.27-1.15 (m, 1H), 1.09 (dd, J=12.8, 2.6 Hz, 1H), 0.95 (d, J=6.3 Hz, 6H), 0.91-0.80 (m, 2H).
[0087] ((1R,3S,5S)-Bicyclo[3.3.1]nonan-3-yl)methyl-d2 ((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (1b). Yield (36%); .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.77 (d, J=7.1 Hz, 1H), 6.25 (s, 1H), 5.30 (s, 1H), 4.27-3.93 (m, 2H), 3.69-3.55 (m, 2H), 3.39-3.32 (m, 2H), 2.53-2.35 (m, 2H), 2.09-1.96 (m, 4H), 1.96-1.77 (m, 3H), 1.77-1.58 (m, 5H), 1.58-1.47 (m, 1H), 1.45-1.30 (m, 5H), 1.09 (d, J=12.8 Hz, 1H), 0.95 (d, J=6.4 Hz, 6H), 0.88 (d, J=13.1 Hz, 2H).
[0088] ((1S,5R)-Bicyclo[3.3.1]non-6-en-3-yl)methyl((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (1c). Yield (3 5%). .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.74 (s, 1H), 5.98 (s, 1H), 5.85 (t, J=8.0 Hz, 1H), 5.58 (t, J=9.7 Hz, 1H), 5.19 (s, 1H), 4.21-4.16 (m, 1H), 4.07 (d, J=2.5 Hz, 2H), 4.02-3.95 (m, 1H), 3.68-3.56 (m, 2H), 3.38-3.32 (m, 2H), 2.50-2.36 (m, 2H), 2.36-2.31 (m, 1H), 2.31-2.25 (m, 2H), 2.17-2.09 (m, 1H), 2.03-1.95 (m, 1H), 1.95-1.87 (m, 1H), 1.87-1.81 (m, 2H), 1.81-1.73 (m, 2H), 1.73-1.59 (m, 3H), 1.56-1.46 (m, 2H), 1.46-1.35 (m, 2H), 0.95 (d, J=6.5 Hz, 6H).
[0089] ((1S,3S,5R)-Bicyclo[3.3.1]non-6-en-3-yl)methyl-d2((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (1d). Yield (36%). .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.72 (s, 1H), 6.04 (s, 1H), 5.85 (t, J=8.1 Hz, 1H), 5.62-5.55 (m, 1H), 5.20 (s, 1H), 4.23-4.15 (m, 1H), 4.02-3.93 (m, 1H), 3.68-3.54 (m, 2H), 3.39-3.33 (m, 2H), 2.49-2.31 (m, 3H), 2.30-2.26 (m, 1H), 2.16-2.09 (m, 1H), 2.03-1.95 (m, 2H), 1.93-1.78 (m, 4H), 1.78-1.59 (m, 4H), 1.59-1.46 (m, 2H), 1.46-1.35 (m, 2H), 0.95 (d, J=6.5 Hz, 6H).
[0090] Bicyclo[2.2.1]hept-5-en-2-ylmethyl ((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (1e). Yield (44%). .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.74 (s, 1H), 6.38 (s, 1H), 6.14 (dd, J=5.7, 3.0 Hz, 1H), 5.93 (dd, J=5.8, 2.9 Hz, 1H), 5.41-5.32 (m, 1H), 4.27-4.19 (m, 1H), 4.19-4.07 (m, 1H), 4.06-3.89 (m, 1H), 3.88-3.80 (m, 1H), 3.69-3.53 (m, 3H), 3.35 (dd, J=10.6, 4.2 Hz, 2H), 2.87-2.77 (m, 2H), 2.52-2.34 (m, 2H), 2.10-1.87 (m, 1H), 1.87-1.77 (m, 1H), 1.77-1.57 (m, 3H), 1.57-1.47 (m, 1H), 1.47-1.40 (m, 1H), 1.37-1.18 (m, 1H), 1.18-1.10 (m, 1H), 0.96 (d, J=6.5 Hz, 6H), 0.53 (ddd, J=11.7, 4.5, 2.6 Hz, 1H).
[0091] Bicyclo[2.2.1]heptan-2-ylmethyl ((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (1f). Yield (44%). .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.73 (s, 1H), 6.30 (s, 1H), 5.30 (s, 1H), 4.30-4.15 (m, 1H), 4.15-4.04 (m, 1H), 4.04-3.95 (m, 1H), 3.95-3.85 (m, 1H), 3.78 (d, J=8.0 Hz, 1H), 3.71-3.52 (m, 2H), 3.39-3.32 (m, 2H), 2.48-2.36 (m, 2H), 2.24-2.17 (m, 1H), 2.17-1.96 (m, 1H), 1.97-1.75 (m, 1H), 1.75-1.56 (m, 3H), 1.56-1.41 (m, 3H), 1.41-1.21 (m, 3H), 1.21-0.99 (m, 3H), 0.95 (d, J=6.4 Hz, 6H), 0.66 (dd, J=12.6, 5.1 Hz, 1H).
[0092] tert-Butyl (1R,3s,5S)-3-((((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)oxy)-8-azabicyclo[3.2.1]octane-8-carboxylate (1g). Yield (45%). .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.85 (s, 1H), 6.29 (s, 1H), 5.31 (d, J=9.3 Hz, 1H), 4.94 (s, 1H), 4.29-4.02 (m, 3H), 4.01-3.97 (m, 1H), 3.64-3.59 (m, 2H), 3.39-3.31 (m, 2H), 2.44-2.39 (m, 2H), 2.20-1.89 (m, 8H), 1.84 (dd, J=11.3, 9.0 Hz, 1H), 1.76 (d, J=15.2 Hz, 2H), 1.73-1.60 (m, 2H), 1.60-1.48 (m, 1H), 1.46 (s, 9H), 0.99-0.91 (m, 6H).
[0093] Benzyl (1R,3s,5S)-3-((((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)oxy)-8-azabicyclo[3.2.1]octane-8-carboxylate (1h). Yield (49%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 7.60-7.56 (m, 2H), 7.47-7.10 (m, 6H), 5.11 (d, J 10.53 Hz, 2H), 5.02-4.88 (m, 1H), 4.83-4.61 (m, 1H), 4.30-4.15 (m, 2H), 4.05 (d, J=7.11 Hz, 1H), 3.97-3.88 (m, 2H), 3.79-3.71 (m, 1H), 3.65-3.56 (m, 2H), 2.79-2.71 (m, 1H), 2.30-1.35 (m, 5H), 1.35-0.90 (m, 9H), 0.87 (td, J=9.48, 8.02, 8.02 Hz, 6H).
[0094] (4-Pentylbicyclo[2.2.2]octan-1-yl)methyl ((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (1i). Yield (3 3%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 7.63-7.45 (m, 1H), 7.09-6.97 (m, 2H), 6.94-4.71 (m, 2H), 4.69-4.57 (m, 5H), 2.90 (s, 1H), 2.78-2.70 (m, 1H), 1.95-1.87 (m, 5H), 1.32 (d, J=7.74 Hz, 6H), 1.23-1.15 (m, 19H), 0.86 (dd, J=13.86, 6.91 Hz, 6H).
[0095] (4-Pentylbicyclo[2.2.2]octan-1-yl)methyl-d2 ((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (1j). Yield (20%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 7.62-7.47 (m, 1H), 7.10-6.97 (m, 2H), 6.97-4.75 (m, 2H), 4.69-4.59 (m, 3H), 2.92 (s, 1H), 2.78-2.76 (m, 1H), 1.95-1.87 (m, 5H), 1.39 (d, J=7.74 Hz, 6H), 1.23-1.17 (m, 19H), 0.86 (dd, J=13.86, 6.91 Hz, 6H).
[0096] ((3S,5S,7S)-Adamantan-1-yl)methyl ((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (1k). Yield (7 5%). .sup.1HNMR (400 MHz, CDCl.sub.3) ? 7.75 (d, J=7.1 Hz, 1H), 6.15 (s, 1H), 5.27 (d, J=8.1 Hz, 1H), 4.23-4.18 (m, 1H), 4.03-3.96 (m, 1H), 3.74-3.57 (m, 4H), 3.39-3.31 (m, 2H), 2.49-2.34 (m, 2H), 1.99-1.95 (m, 4H), 1.88-1.79 (m, 1H), 1.76-1.60 (m, 9H), 1.58-1.46 (m, 7H), 0.96 (dd, J=6.4, 2.4 Hz, 6H).
[0097] ((3S,5S,7S)-Adamantan-1-yl)methyl-d2 ((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (1i). Yield (26%). .sup.1H NMR (400 MHz, cdcl.sub.3) ? 7.73 (d, J=7.3 Hz, 1H), 6.19 (s, 1H), 5.26 (d, J=8.1 Hz, 1H), 4.22-4.18 (m, 1H), 4.03-3.96 (m, 1H), 3.67-3.54 (m, 2H), 3.39-3.31 (m, 2H), 2.49-2.33 (m, 2H), 2.06-2.02 (m, 1H), 1.99-1.95 (m, 3H), 1.88-1.79 (m, 2H), 1.78-1.60 (m, 9H), 1.57-1.48 (m, 6H), 0.96 (dd, J=6.4, 2.1 Hz, 6H).
[0098] 2-((3S,5S,7S)-Adamantan-1-yl)ethyl ((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (1m). Yield (65%). .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.73 (d, J=7.0 Hz, 1H), 6.24 (s, 1H), 5.28 (d, J=8.3 Hz, 1H), 4.25-4.20 (m, 1H), 4.17-4.07 (m, 2H), 4.04-3.96 (m, 1H), 3.70-3.44 (m, 2H), 3.39-3.31 (m, 2H), 2.52-2.34 (m, 2H), 2.09-1.97 (m, 1H), 1.97-1.91 (m, 3H), 1.90-1.76 (m, 1H), 1.74-1.57 (m, 9H), 1.55-1.45 (m, 7H), 1.40 (t, J=7.5 Hz, 2H), 0.95 (d, J=6.4 Hz, 6H).
[0099] ((1R,3R,5R,7S)-3-Hydroxyadamantan-1-yl)methyl ((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (In). Yield (8%). 1H NMR (400 MHz, CDCl.sub.3) ? 7.69 (d, J=7.3 Hz, 1H), 6.34 (s, 1H), 5.45 (d, J=8.0 Hz, 1H), 4.20-4.16 (m, 1H), 4.05-3.87 (m, 1H), 3.74-3.54 (m, 4H), 3.37-3.28 (m, 2H), 2.45-2.40 (m, 2H), 2.21 (s, 2H), 2.11-1.94 (m, 1H), 1.88-1.79 (m, 1H), 1.79-1.59 (m, 8H), 1.59-1.54 (m, 2H), 1.54-1.47 (m, 3H), 1.47-1.32 (m, 3H), 0.99-0.88 (m, 6H).
[0100] ((1r,3R,5R,7S)-3-Hydroxyadamantan-1-yl)methyl-d2 ((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (1o). Yield (7%). .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.70 (d, J=7.1 Hz, 1H), 6.26 (s, 1H), 5.41 (d, J=7.9 Hz, 1H), 4.23-4.14 (m, 1H), 4.03-3.96 (m, 1H), 3.68-3.54 (m, 2H), 3.39-3.32 (m, 2H), 2.43 (s, 2H), 2.27-2.19 (m, 2H), 2.11-1.91 (m, 1H), 1.88-1.80 (m, 1H), 1.77-1.60 (m, 8H), 1.60-1.54 (m, 1H), 1.54-1.47 (m, 4H), 1.43-1.38 (m, 3H), 0.99-0.88 (m, 6H).
[0101] Preparation of compounds 2(a-o). General procedure. To a solution of dipeptidyl alcohol 1 (1 eq) in anhydrous dichloromethane (300 mL/g dipeptidyl alcohol) kept at 0-5? C. under a N.sub.2 atmosphere was added DMP reagent (3.0 eq) and the reaction mixture was stirred for 3 h at 15-20? C. The organic phase was washed with 10% aq Na.sub.2S.sub.2O.sub.3 (2?100 mL/g dipeptidyl alcohol), followed by saturated aqueous NaHCO.sub.3(2?100 mL/g dipeptidyl alcohol), distilled water (2?100 mL/g dipeptidyl alcohol), and brine (100 mL/g dipeptidyl alcohol). The organic phase was dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The resulting crude product was purified by flash chromatography (hexane/ethyl acetate) to yield aldehyde 2 as a white solid.
[0102] ((1R,3s,5S)-Bicyclo[3.3.1]nonan-3-yl)methyl ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)carbamate (2a). Yield (86%). .sup.1H NMR (400 MHz, CDCl.sub.3) ? 9.50 (s, 1H), 8.31 (s, 1H), 5.21 (d, J=8.7 Hz, 1H), 4.38-4.28 (m, 2H), 3.96-3.87 (m, 2H), 3.40-3.31 (m, 2H), 2.54-2.36 (m, 2H), 2.08-1.99 (m, 3H), 1.99-1.81 (m, 5H), 1.76-1.63 (m, 6H), 1.60-1.49 (m, 1H), 1.46-1.29 (m, 4H), 1.15-1.05 (m, 1H), 0.97 (d, J=6.3 Hz, 6H), 0.88 (d, J=13.4 Hz, 2H). HRMS m/z: [M+H].sup.+ Calculated for C.sub.24H.sub.40N.sub.3O.sub.5: 450.2968, Found: 450.2958, m/z: [M+Na].sup.+ Calculated for C.sub.24H.sub.39N.sub.3NaO.sub.5: 472.2788, Found: 472.2776.
[0103] ((1R,3s,5S)-Bicyclo[3.3.1]nonan-3-yl)methyl-d2 ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)carbamate (2b). Yield (85%). .sup.1H NMR (400 MHz, CDCl.sub.3) ? 9.50 (s, 1H), 8.30 (s, 1H), 6.16 (s, 1H), 5.24 (d, J=8.6 Hz, 1H), 4.38-4.29 (m, 2H), 3.41-3.30 (m, 2H), 2.52-2.34 (m, 2H), 2.12-1.99 (m, 3H), 1.98-1.78 (m, 4H), 1.78-1.62 (m, 4H), 1.61-1.51 (m, 1H), 1.41-1.30 (m, 6H), 1.13-1.06 (m, 1H), 0.97 (d, J=6.3 Hz, 6H), 0.96-0.83 (m, 2H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.24H.sub.37D.sub.2N.sub.3NaO.sub.5: 474.2913, Found: 474.2897.
[0104] ((1S,5R)-Bicyclo[3.3.1]non-6-en-3-yl)methyl ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)carbamate (2c). Yield (5 3%). .sup.1HNMR (400 MHz, CDCl.sub.3) ? 9.49 (s, 1H), 8.27 (s, 1H), 6.04 (s, 1H), 5.86 (t, J=7.1 Hz, 1H), 5.63-5.55 (m, 1H), 5.18 (d, J=8.5 Hz, 1H), 4.38-4.27 (m, 2H), 4.10-4.03 (m, 1H), 4.03-3.94 (m, 1H), 3.41-3.31 (m, 2H), 2.52-2.37 (m, 1H), 2.28 (s, 2H), 2.18-2.08 (m, 1H), 2.01-1.92 (m, 3H), 1.92-1.83 (m, 3H), 1.83-1.71 (m, 1H), 1.71-1.66 (m, 4H), 1.62-1.49 (m, 3H), 1.49-1.36 (m, 1H), 0.97 (d, J=6.4 Hz, 6H). HRMS m/z: [M+H].sup.+ Calculated for C.sub.24H.sub.38N.sub.3O.sub.5: 448.2811, Found: 448.2810, m/z: [M+Na].sup.+ Calculated for C.sub.24H.sub.37N.sub.3NaO.sub.5: 470.2631, Found: 470.2628.
[0105] ((1S,3S,5R)-Bicyclo[3.3.1]non-6-en-3-yl)methyl-d2 ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)carbamate (2d). Yield (88%). .sup.1H NMR (400 MHz, CDCl.sub.3) ? 9.49 (s, 1H), 8.28 (d, J=6.1 Hz, 1H), 6.33 (s, 1H), 5.85 (t, J=8.0 Hz, 1H), 5.58 (d, J=9.9 Hz, 1H), 5.24 (d, J=8.7 Hz, 1H), 4.38-4.29 (m, 2H), 3.43-3.30 (m, 2H), 2.54-2.32 (m, 2H), 2.31-2.26 (m, 1H), 2.20-2.08 (m, 1H), 2.08-1.62 (m, 9H), 1.62-1.46 (m, 4H), 1.45-1.38 (m, 2H), 0.97 (d, J=6.1 Hz, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.24H.sub.35D.sub.2N.sub.3NaO.sub.5: 472.2757, Found: 472.2743.
[0106] Bicyclo[2.2.1]hept-5-en-2-ylmethyl ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)carbamate (2e). Yield (84%). .sup.1H NMR (400 MHz, CDCl.sub.3) ? 9.50 (s, 1H), 8.30 (d, J=6.7 Hz, 1H), 6.39 (s, 1H), 6.14 (dd, J=5.8, 3.1 Hz, 1H), 5.93 (dd, J=5.7, 2.9 Hz, 1H), 5.27 (s, 1H), 4.38-4.31 (m, 2H), 3.89-3.80 (m, 1H), 3.69-3.60 (m, 1H), 3.43-3.30 (m, 2H), 2.89-2.78 (m, 1H), 2.56-2.31 (m, 3H), 2.11-1.96 (m, 1H), 1.96-1.90 (m, 1H), 1.90-1.78 (m, 2H), 1.78-1.63 (m, 2H), 1.63-1.49 (m, 1H), 1.48-1.33 (m, 1H), 1.29-1.18 (m, 1H), 1.19-1.10 (m, 1H), 0.97 (d, J=6.3 Hz, 6H), 0.58-0.50 (m, 1H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.22H.sub.33N.sub.3NaO.sub.5: 442.2318, Found: 442.2310.
[0107] Bicyclo[2.2.1]heptan-2-ylmethyl ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)carbamate (2f). Yield (88%). .sup.1H NMR (400 MHz, CDCl.sub.3) ? 9.50 (s, 1H), 8.29 (s, 1H), 6.34 (s, 1H), 5.26 (s, 1H), 4.39-4.29 (m, 2H), 4.14-4.03 (m, 1H), 3.97-3.86 (m, 1H), 3.84-3.72 (m, 1H), 3.43-3.30 (m, 2H), 2.54-2.34 (m, 2H), 2.29-2.15 (m, 2H), 2.16-2.08 (m, 1H), 2.08-1.77 (m, 1H), 1.77-1.63 (m, 3H), 1.59-1.43 (m, 2H), 1.41-1.23 (m, 4H), 1.22-1.02 (m, 2H), 0.97 (d, J=6.3 Hz, 6H), 0.67 (ddd, J=12.5, 5.4, 2.3 Hz, 1H). IRMS m/z: [M+Na].sup.+ Calculated for C.sub.22H.sub.35N.sub.3NaO.sub.5: 444.2475, Found: 444.2467.
[0108] tert-Butyl (1R,3s,5S)-3-((((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)carbamoyl)oxy)-8-azabicyclo[3.2.1]octane-8-carboxylate (2g). Yield (90%). .sup.1H NMR (400 MHz, CDCl.sub.3) ? 9.49 (s, 1H), 8.43 (d, J=5.6 Hz, 1H), 6.07 (s, 1H), 5.20 (d, J=8.6 Hz, 1H), 4.96 (s, 1H), 4.35-4.28 (m, 2H), 4.25-4.09 (m, 2H), 3.41-3.33 (m, 2H), 2.53-2.36 (m, 2H), 2.23-1.94 (m, 8H), 1.94-1.82 (m, 1H), 1.81-1.64 (m, 4H), 1.61-1.52 (m, 1H), 1.46 (s, 9H), 0.98 (d, J=5.0 Hz, 6H). HRMS m/z: [M+H].sup.+ Calculated for C.sub.26H.sub.43N.sub.4O.sub.7: 523.3131, Found: 523.3116. HRMS m/z: [M+Na].sup.+ Calculated for C.sub.26H.sub.42N.sub.4NaO.sub.7: 545.2951, Found: 545.2938.
[0109] Benzyl (1R,3s,5S)-3-((((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)carbamoyl)oxy)-8-azabicyclo[3.2.1]octane-8-carboxylate (2h). Yield (66%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 9.39 (s, 1H), 8.55-8.35 (m, 1H), 8.11-8.02 (m, 1H), 7.64 (s, 1H), 7.45-7.19 (m, 5H), 5.75 (s, 1H), 5.09 (d, J=10.47 Hz, 2H), 4.83-4.71 (m, 2H), 4.21 (s, 2H), 3.73-3.57 (m, 2H), 3.24-3.00 (m, 2H), 2.34-1.79 (m, 3H), 1.79-1.34 (m, 2H), 1.28-1.12 (m, 9H), 0.98-0.77 (m, 6H). HRMS m/z: [M+H].sup.+ Calculated for C.sub.29H.sub.41N.sub.4O.sub.7: 557.2970, Found: 557.2962. HRMS m/z: [M+Na].sup.+ Calculated for C.sub.29H.sub.40N.sub.4NaO.sub.7: 579.2789, Found: 579.2773.
[0110] (4-Pentylbicyclo[2.2.2]octan-1-yl)methyl ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)carbamate (2i). Yield (59%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 9.49 (s, 1H), 7.66-7.58 (m, 1H), 7.54-7.44 (m, 2H), 5.72-5.64 (m, 2H), 3.71-3.53 (m, 5H), 3.21-3.13 (m, 1H), 2.93-2.85 (m, 1H), 2.77-2.69 (m, 1H), 2.30-2.22 (m, 1H), 1.91 (s, 1H), 1.63-1.55 (m, 5H), 1.42-0.96 (m, 20H), 0.87 (td, J=19.27, 6.96, 6.96 Hz, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.28H.sub.47N.sub.3NaO.sub.5: 528.3414, Found: 528.3391.
[0111] (4-Pentylbicyclo[2.2.2]octan-1-yl)methyl-d2 ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)carbamate (2j). Yield (49%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 9.49 (s, 1H), 7.66-7.58 (m, 1H), 7.54-7.44 (m, 2H), 5.72-5.64 (m, 2H), 3.71-3.63 (m, 3H), 3.31 (s, 1H), 2.93-2.85 (m, 1H), 2.85-2.69 (m, 1H), 2.30-2.22 (m, 1H), 1.91 (s, 1H), 1.42-0.96 (m, 25H), 0.87 (td, J=19.21, 6.96, 6.96 Hz, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.28H.sub.45D.sub.2N.sub.3NaO.sub.5: 530.3539, Found: 530.3536.
[0112] ((3S,5S,7S)-Adamantan-1-yl)methyl((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)carbamate (2k). Yield (90%). .sup.1H NMR (400 MHz, CDCl.sub.3) ? 9.50 (s, 1H), 8.31 (d, J=5.8 Hz, 1H), 6.36 (s, 1H), 5.27 (s, 1H), 4.39-4.29 (m, 2H), 3.66 (s, 2H), 3.41-3.30 (m, 2H), 2.55-2.34 (m, 2H), 2.08-1.92 (m, 3H), 1.92-1.78 (m, 2H), 1.78-1.60 (m, 9H), 1.52 (d, J=2.9 Hz, 7H), 1.00-0.94 (m, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.25H.sub.39N.sub.3NaO.sub.5: 484.2788, Found: 484.2780.
[0113] ((3S,5S,7S)-Adamantan-1-yl)methyl-d2 ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)carbamate (2). Yield (8 8%). .sup.1H NMR (400 MHz, cdcl.sub.3) ? 9.49 (s, 1H), 8.32 (d, J=5.9 Hz, 1H), 6.13 (d, J=8.1 Hz, 1H), 5.23 (s, 1H), 4.68-4.58 (m, 1H), 4.38-4.25 (m, 1H), 3.41-3.27 (m, 2H), 2.52-2.34 (m, 2H), 2.06-2.01 (m, 2H), 2.00-1.89 (m, 3H), 1.88-1.78 (m, 4H), 1.78-1.55 (m, 9H), 1.52 (d, J=2.9 Hz, 3H), 1.00-0.91 (m, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.25H.sub.37D.sub.2N.sub.3NaO.sub.5: 486.2913, Found: 486.2910.
[0114] 2-((3S,5S,7S)-Adamantan-1-yl)ethyl ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)carbamate (2m). Yield (83%). .sup.1H NMR (400 MHz, CDCl.sub.3) ? 9.50 (s, 1H), 8.30 (d, J=5.9 Hz, 1H), 6.09 (d, J=11.7 Hz, 1H), 5.19 (d, J=8.7 Hz, 1H), 4.38-4.28 (m, 2H), 4.26-4.05 (m, 2H), 3.41-3.33 (m, 2H), 2.54-2.35 (m, 2H), 2.01-1.91 (m, 4H), 1.91-1.79 (m, 1H), 1.74-1.58 (m, 9H), 1.51 (d, J=2.9 Hz, 6H), 1.41 (t, J=7.4 Hz, 2H), 1.28-1.23 (m, 1H), 0.97 (d, J=6.4 Hz, 6H). HRMS m/z: [M+H].sup.+ Calculated for C.sub.26H.sub.42N.sub.3O.sub.5: 476.3124, Found: 476.3124. HRMS m/z: [M+Na].sup.+ Calculated for C.sub.26H.sub.41N.sub.3NaO.sub.5: 498.2944, Found: 498.2938.
[0115] ((1r,3R,5R,7S)-3-Hydroxyadamantan-1-yl)methyl ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)carbamate (2n). Yield (47%). .sup.1H NMR (400 MHz, CDCl.sub.3) ? 9.49 (s, 1H), 8.28 (d, J=23.9 Hz, 1H), 6.19 (s, 1H), 5.19 (s, 1H), 4.34-4.03 (m, 2H), 4.02-3.61 (m, 2H), 2.89-2.58 (m, 2H), 2.58-2.28 (m, 2H), 2.28-2.13 (m, 3H), 2.09-1.76 (m, 2H), 1.78-1.60 (m, 6H), 1.57-1.31 (m, 9H), 1.01-0.91 (m, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.25H.sub.39N.sub.3NaO.sub.6: 500.2737, Found: 500.2739.
[0116] ((1r,3R,5R,7S)-3-Hydroxyadamantan-1-yl)methyl-d2 ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)carbamate (2o). Yield (44%). .sup.1H NMR (400 MHz, CDCl.sub.3) ? 9.48 (s, 1H), 8.28 (d, J=6.5 Hz, 1H), 6.39 (s, 1H), 5.12 (s, 1H), 4.40-4.09 (m, 2H), 2.89-2.59 (m, 2H), 2.56-2.30 (m, 2H), 2.30-2.20 (m, 3H), 2.18-1.77 (m, 2H), 1.77-1.61 (m, 6H), 1.59-1.39 (m, 9H), 1.01-0.89 (m, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.25H.sub.37D.sub.2N.sub.3NaO.sub.6: 502.2862, Found: 502.2860.
[0117] Preparation of compounds 3(a-o). General procedure. To a solution of dipeptidyl aldehyde 2 (1 eq) in ethyl acetate (10 mL/g of dipeptidyl aldehyde) was added absolute ethanol (5 mL/g of dipeptidyl aldehyde) with stirring, followed by a solution of sodium bisulfite (1 eq) in water (1 mL/g of dipeptidyl aldehyde). The reaction mixture was stirred for 3 h at 50? C. The reaction mixture was allowed to cool to room temperature and then vacuum filtered. The solid was thoroughly washed with absolute ethanol and the filtrate was dried over anhydrous sodium sulfate, filtered, and concentrated to yield a white solid. The white solid was stirred with dry ethyl ether (3?10 mL/g of dipeptidyl aldehyde), followed by careful removal of the solvent using a pipette and dried using a vacuum pump for 2 h to yield dipeptidyl bisulfite adduct 3 as a white solid.
[0118] Sodium (2S)-2-((2S)-2-(((((1R,5S)-bicyclo[3.3.1]nonan-3-yl)methoxy)carbonyl)amino)-4-methylpentanamido)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propane-1-sulfonate (3a). Yield (66%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 7.61 (d, J=9.0 Hz, 1H), 7.57-7.44 (m, 1H), 7.28-7.08 (m, 1H), 5.62 (d, J=6.1 Hz, 1H), 5.47 (d, J=5.9 Hz, 1H), 4.12-3.91 (m, 2H), 3.91-3.73 (m, 2H), 3.16-3.00 (m, 2H), 2.24-2.07 (m, 2H), 2.06-1.95 (m, 2H), 1.83 (dt, J=17.2, 7.2 Hz, 4H), 1.74-1.51 (m, 5H), 1.51-1.38 (m, 2H), 1.35-1.15 (m, 6H), 1.14-1.00 (m, 2H), 0.99-0.81 (m, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.24H.sub.40N.sub.3Na.sub.2O.sub.8S: 576.2332, Found: 576.2329, m/z: [M].sup.? Calculated for C.sub.24H.sub.40N.sub.3O.sub.8S: 530.2536, Found: 530.2529.
[0119] Sodium (2S)-2-((S)-2-(((((1R,3s,5S)-bicyclo[3.3.1]nonan-3-yl)methoxy-d2)carbonyl)amino)-4-methylpentanamido)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propane-1-sulfonate (3b). Yield (75%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 7.63-7.43 (m, 2H), 7.20 (dd, J=20.3, 8.2 Hz, 1H), 5.52-5.33 (m, 1H), 4.06-3.78 (m, 2H), 3.20-2.99 (m, 2H), 2.33-2.04 (m, 3H), 2.04-1.93 (m, 2H), 1.94-1.74 (m, 2H), 1.74-1.50 (m, 3H), 1.50-1.38 (m, 2H), 1.38-1.19 (m, 5H), 1.11-0.98 (m, 2H), 0.97-0.80 (m, 10H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.24H.sub.38D.sub.2N.sub.3Na.sub.2O.sub.8S: 578.2457, Found: 578.2432.
[0120] Sodium (2S)-2-((2S)-2-(((((1S,5R)-bicyclo[3.3.1]non-6-en-3-yl)methoxy)carbonyl)amino)-4-methylpentanamido)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propane-1-sulfonate (3c). Yield (66%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 7.52 (d, J=9.5 Hz, 1H), 7.47 (d, J=4.5 Hz, 1H), 7.18 (ddd, J=19.5, 8.5, 2.7 Hz, 1H), 5.89-5.79 (m, 1H), 5.58-5.52 (m, 1H), 3.97 (h, J=8.1 Hz, 1H), 3.90-3.70 (m, 1H), 3.27-3.00 (m, 2H), 2.35-2.19 (m, 3H), 2.19-2.02 (m, 3H), 2.02-1.68 (m, 6H), 1.68-1.49 (m, 4H), 1.49-1.25 (m, 6H), 1.13-1.04 (m, 1H), 0.91-0.79 (m, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.24H.sub.38N.sub.3Na2O.sub.8S: 574.2175, Found: 574.2163, m/z: [M].sup.? Calculated for C.sub.24H.sub.38N.sub.3O.sub.8S: 528.2379, Found: 528.2367.
[0121] Sodium (2S)-2-((S)-2-(((((1S,3S,5R)-bicyclo[3.3.1]non-6-en-3-yl)methoxy-d2)carbonyl) amino)-4-methylpentanamido)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propane-1-sulfonate (3d). Yield (53%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 7.62-7.39 (m, 2H), 7.18 (dd, J=24.9, 8.5 Hz, 1H), 5.89-5.80 (m, 1H), 5.59-5.52 (m, 1H), 5.43-5.26 (m, 1H), 4.06-3.67 (m, 2H), 3.22-2.98 (m, 2H), 2.37-2.03 (m, 5H), 2.03-1.64 (m, 5H), 1.64-1.52 (m, 3H), 1.52-1.29 (m, 5H), 1.13-1.02 (m, 1H), 0.89-0.80 (m, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.24H.sub.36D.sub.2N.sub.3Na.sub.2O.sub.8S: 576.2301, Found: 576.2275.
[0122] Sodium (2S)-2-((2S)-2-(((bicyclo[2.2.1]hept-5-en-2-ylmethoxy)carbonyl)amino)-4-methylpentanamido)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propane-1-sulfonate (3e). Yield (56%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 7.68-7.37 (m, 2H), 7.32-7.17 (m, 1H), 6.20-6.02 (m, 1H), 6.02-5.86 (m, 1H), 5.59-5.30 (m, 1H), 4.11-3.79 (m, 2H), 3.79-3.41 (m, 2H), 3.21-2.98 (m, 2H), 2.87-2.66 (m, 2H), 2.39-2.04 (m, 3H), 2.04-1.85 (m, 1H), 1.85-1.70 (m, 1H), 1.70-1.51 (m, 2H), 1.50-1.36 (m, 2H), 1.28 (dd, J=39.4, 7.9 Hz, 2H), 1.18-1.00 (m, 1H), 0.93-0.79 (m, 6H), 0.47 (d, J=11.6 Hz, 1H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.22H.sub.34N.sub.3Na.sub.2O.sub.8S: 546.1862, Found: 546.1842.
[0123] Sodium (2S)-2-((2S)-2-(((bicyclo[2.2.1]heptan-2-ylmethoxy)carbonyl)amino)-4-methyl pentanamido)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propane-1-sulfonate (3f). Yield (66%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 7.61-7.39 (m, 2H), 7.32-7.08 (m, 1H), 5.41 (dd, J=55.1, 5.9 Hz, 1H), 4.07-3.55 (m, 2H), 3.18-2.99 (m, 2H), 2.28-2.04 (m, 6H), 2.04-1.81 (m, 1H), 1.81-1.53 (m, 3H), 1.53-1.36 (m, 5H), 1.36-1.18 (m, 3H), 1.18-0.91 (m, 2H), 0.90-0.75 (m, 6H), 0.70-0.59 (m, 1H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.22H.sub.36N.sub.3Na.sub.2O.sub.8S: 548.2019, Found: 548.1999.
[0124] Sodium (2S)-2-((S)-2-(((((1R,3s,5S)-8-(tert-butoxycarbonyl)-8-azabicyclo[3.2.1]octan-3-yl)oxy)carbonyl)amino)-4-methylpentanamido)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propane-1-sulfonate (3g). Yield (52%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 7.64-7.51 (m, 1H), 7.46 (d, J=3.8 Hz, 1H), 7.30 (dd, J=19.2, 8.3 Hz, 1H), 5.55-5.31 (m, 1H), 4.76 (s, 1H), 4.02-3.78 (m, 4H), 3.22-2.98 (m, 2H), 2.27-1.68 (m, 9H), 1.66-1.57 (m, 4H), 1.51-1.43 (m, 2H), 1.40 (s, 9H), 1.13-1.02 (m, 1H), 0.91-0.81 (m, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.26H.sub.43N.sub.4Na.sub.2O.sub.10S: 649.2496, Found: 649.2500.
[0125] Sodium (2S)-2-((S)-2-(((((1R,3s,5S)-8-((benzyloxy)carbonyl)-8-azabicyclo[3.2.1]octan-3-yl)oxy)carbonyl)amino)-4-methylpentanamido)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propane-1-sulfonate (3h). Yield (53%). 1H NMR (400 MHz, DMSO-d.sub.6) ? 7.37 (t, J=4.74, 4.74 Hz, 8H), 5.09 (d, J=11.06 Hz, 2H), 4.29-4.13 (m, 5H), 3.96-3.82 (m, 1H), 3.79-3.69 (m, 1H), 3.17-3.09 (m, 1H), 3.06-2.98 (m, 1H), 2.05-1.81 (m, 5H), 1.74-1.63 (m, 3H), 1.58-1.49 (m, 4H), 1.47-1.33 (m, 2H), 1.08 (dd, J=13.72, 6.78 Hz, 1H), 0.84 (ddd, J=10.35, 8.11, 4.59 Hz, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.29H.sub.41N.sub.4Na.sub.2O.sub.10S: 683.2339, Found: 683.2317.
[0126] Sodium (2S)-1-hydroxy-2-((S)-4-methyl-2-((((4-pentylbicyclo[2.2.2]octan-1-yl)methoxy) carbonyl)amino)pentanamido)-3-((S)-2-oxopyrrolidin-3-yl)propane-1-sulfonate (3i). Yield (57%). 1H NMR (400 MHz, DMSO-d.sub.6) ? 7.51-7.40 (m, 3H), 4.00-3.53 (m, 5H), 3.49-3.40 (m, 1H), 3.16-2.98 (m, 4H), 1.94-1.85 (m, 3H), 1.65-0.96 (m, 25H), 0.85 (dd, J=12.73, 5.76 Hz, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.28H.sub.48N.sub.3Na.sub.2O.sub.8S: 632.2958, Found: 632.2932. HRMS m/z: [M].sup.? Calculated for C.sub.28H.sub.48N.sub.3O.sub.8S: 586.3168, Found: 586.3163.
[0127] Sodium (2S)-1-hydroxy-2-((S)-4-methyl-2-((((4-pentylbicyclo[2.2.2]octan-1-yl)methoxy-d2)carbonyl)amino)pentanamido)-3-((S)-2-oxopyrrolidin-3-yl)propane-1-sulfonate (3j). Yield (50%). 1H NMR (400 MHz, DMSO-d.sub.6) ? 7.51-7.40 (m, 3H), 4.00-3.53 (m, 3H), 3.40-3.26 (m, 1H), 3.16-2.90 (m, 3H), 2.56-2.44 (m, 1H), 1.95-1.85 (m, 3H), 1.65-0.96 (m, 25H), 0.85 (dd, J=12.73, 5.76 Hz, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.25H.sub.46D.sub.2N.sub.3Na.sub.2O.sub.8S: 634.3083, Found: 634.3071. HRMS m/z: [M].sup.? Calculated for C.sub.28H.sub.46D.sub.2N.sub.3O.sub.8S: 588.3287, Found: 588.3399.
[0128] Sodium (2S)-2-((S)-2-(((((3S,5S,7S)-adamantan-1-yl)methoxy)carbonyl)amino)-4-methylpentanamido)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propane-1-sulfonate (3k). Yield (52%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 7.66-7.41 (m, 2H), 7.25-7.10 (m, 1H), 5.40 (dd, J=47.1, 6.0 Hz, 1H), 4.10-3.70 (m, 2H), 3.69-3.47 (m, 2H), 3.18-3.00 (m, 2H), 2.24-2.03 (m, 2H), 2.03-1.83 (m, 4H), 1.83-1.55 (m, 8H), 1.55-1.33 (m, 6H), 1.13-1.02 (m, 1H), 0.93-0.80 (m, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.25H.sub.40N.sub.3Na.sub.2O.sub.8S: 588.2332, Found: 588.2310.
[0129] Sodium (2S)-2-((S)-2-(((((3S,5S,7S)-adamantan-1-yl)methoxy-d2)carbonyl)amino)-4-methylpentanamido)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propane-1-sulfonate (3l). Yield (39%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 7.63-7.50 (m, 1H), 7.47-7.36 (m, 1H), 7.38-7.28 (m, 1H), 5.41 (dd, J=36.5, 6.0 Hz, 1H), 4.27-4.19 (m, 1H), 3.99-3.94 (m, 1H), 3.49-3.34 (m, 1H), 3.21-3.01 (m, 2H), 2.25-1.99 (m, 2H), 1.99-1.90 (m, 3H), 1.89-1.73 (m, 3H), 1.72-1.52 (m, 9H), 1.48 (s, 5H), 0.90-0.76 (m, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.25H.sub.38D.sub.2N.sub.3Na.sub.2O.sub.8S: 590.2457, Found: 590.2447.
[0130] Sodium (2S)-2-((S)-2-(((2-((3S,5S,7S)-adamantan-1-yl)ethoxy)carbonyl)amino)-4-methyl pentanamido)-1-hydroxy-3-((S)-2-oxopyrrolidin-3-yl)propane-1-sulfonate (3m). Yield (60%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 7.62-7.49 (m, 1H), 7.49-7.43 (m, 1H), 7.24-7.07 (m, 1H), 5.40 (dd, J=48.6, 6.1 Hz, 1H), 4.05-3.81 (m, 4H), 3.20-3.00 (m, 2H), 2.26-2.06 (m, 2H), 2.01-1.87 (m, 3H), 1.84-1.72 (m, 1H), 1.70-1.54 (m, 10H), 1.49 (d, J=3.0 Hz, 6H), 1.45-1.38 (m, 1H), 1.38-1.28 (m, 2H), 0.89-0.80 (m, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.26H.sub.42N.sub.3Na.sub.2O.sub.8S: 602.2488, Found: 602.2480.
[0131] Sodium (2S)-1-hydroxy-2-((S)-2-(((((1r,3R,5R,7S)-3-hydroxyadamantan-1-yl)methoxy) carbonyl)amino)-4-methylpentanamido)-3-((S)-2-oxopyrrolidin-3-yl)propane-1-sulfonate (3n). Yield (69%). .sup.1H NMR (400 MHz, acetone) 6 7.08-7.03 (m, 1H), 7.00-6.95 (m, 1H), 6.45-6.41 (m, 1H), 4.38-3.89 (m, 2H), 3.89-3.53 (m, 2H), 2.96-2.53 (m, 1H), 2.53-2.25 (m, 2H), 2.16 (s, 2H), 1.99-1.70 (m, 4H), 1.70-1.62 (m, 5H), 1.62-1.53 (m, 4H), 1.53-1.35 (m, 5H), 1.34-1.16 (m, 1H), 0.99-0.85 (m, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.25H.sub.40N.sub.3Na.sub.2O.sub.9S: 604.2282, Found: 604.2273.
[0132] Sodium (2S)-1-hydroxy-2-((S)-2-(((((1r,3R,5R,7S)-3-hydroxyadamantan-1-yl)methoxy-d2)carbonyl)amino)-4-methylpentanamido)-3-((S)-2-oxopyrrolidin-3-yl)propane-1-sulfonate (3o). Yield (73%). .sup.1H NMR (400 MHz, acetone) 6 7.10-7.05 (m, 1H), 6.91-6.87 (m, 1H), 6.43-6.38 (m, 1H), 4.33-4.06 (m, 2H), 3.33-3.19 (m, 2H), 2.50-2.24 (m, 1H), 2.22-2.10 (m, 3H), 2.03-1.71 (m, 2H), 1.70-1.61 (m, 5H), 1.61-1.52 (m, 5H), 1.52-1.38 (m, 6H), 1.01-0.85 (m, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.25H.sub.38D.sub.2N.sub.3Na.sub.2O.sub.9S: 606.2406, Found: 606.2409.
Biochemical Studies
[0133] Enzyme assays and inhibition studies. Cloning and expression of the 3CL protease of SARS-CoV-2 and FRET enzyme assays. The codon-optimized cDNA of full length of 3CLpro of SARS-CoV-2 (GenBank number MN908947.3) fused with sequences encoding 6 histidine at the N-terminal was synthesized by Integrated DNA (Coralville, IA). The synthesized gene was subcloned into the pET-28a(+) vector. The expression and purification of SARS-CoV-2 3CLpro were conducted following a standard procedure. Briefly, a stock solution of an inhibitor was prepared in DMSO and diluted in assay buffer comprised of 20 mM HEPES buffer, pH 8, containing NaCl (200 mM), EDTA (0.4 mM), glycerol (60%), and 6 mM dithiothreitol (DTT). The SARS-CoV-2 protease was mixed with serial dilutions of inhibitor or with DMSO in 25 ?L of assay buffer and incubated at 37? C. for 1 h, followed by the addition of 25 ?L of assay buffer containing substrate (FAM-SAVLQ/SG-QXL?520, AnaSpec, Fremont, CA). The substrate was derived from the cleavage sites on the viral polyproteins of SARS-CoV. Fluorescence readings were obtained using an excitation wavelength of 480 nm and an emission wavelength of 520 nm on a fluorescence microplate reader (FLx800; Biotec, Winoosk, VT) 1 h following the addition of substrate. Relative fluorescence units (RFU) were determined by subtracting background values (substrate-containing well without protease) from the raw fluorescence values using established procedures. The dose-dependent FRET inhibition curves were fitted with a variable slope by using GraphPad Prism software (GraphPad, La Jolla, CA) in order to determine the IC.sub.50 values of the compounds. The expression and purification of the 3CLpro of MERS-CoV, as well as the FRET enzyme assays were performed using an established procedure.
[0134] Cell-based assay for antiviral activity. Representative compounds 2a and 3a were investigated for their antiviral activity against the replication of SARS-CoV-2. Briefly, confluent Vero E6 cells were inoculated with SARS-CoV-2 at 50-100 plaque forming units/well, and medium containing various concentrations of each compound and agar was applied to the cells. After 48-72 hr, plaques in each well were counted. The 50% effective concentration (EC.sub.50) values were determined by GraphPad Prism software using a variable slope (GraphPad, La Jolla, CA).
[0135] Nonspecific cytotoxic effects/In vitro cytotoxicity. Confluent cells grown in 96-well plates were incubated with various concentrations (1 to 100 ?M) of each compound for 72 h. Cell cytotoxicity was measured by a CytoTox 96 nonradioactive cytotoxicity assay kit (Promega, Madison, WI), and the CC.sub.50 values were calculated using a variable slope by GraphPad Prism software. The in vitro Safety Index was calculated by dividing the CC.sub.50 by the IC.sub.50.
X-Ray Crystallographic Studies
[0136] Crystallization and Data Collection. Purified SARS-2 3CL protease (SARS-2 3CLpro) in 100 mM NaCl, 20 mM Tris buffer, pH 8.0, was concentrated to 9.6 mg/mL (0.28 mM) for crystallization screening. All crystallization experiments were set up using an NT8 drop-setting robot (Formulatrix Inc.) and UVXPO MRC (Molecular Dimensions) sitting drop vapor diffusion plates at 18? C. 100 nL of protein and 100 nL crystallization solution were dispensed and equilibrated against 50 ?L of the latter. Stock solutions of the inhibitors (100 mM) were prepared in DMSO and the complexes were obtained by mixing 1 ?L of the ligand (2 mM) with 49 ?L (0.28 mM) of SARS-2 3CLpro and incubating on ice for 1 h. Crystals were obtained in 1-2 days from the following conditions. 2a and 3b: Berkeley screen (Rigaku Reagents) condition C5 (20% (w/v) PEG 4000, 100 mM Tris pH 8.0), 2f: Index HT screen (Hampton Research) condition H6 (20% (w/v) PEG 3350, 200 mM sodium formate), 2k: Proplex HT screen (Molecular Dimensions) condition D7 (15% (w/v) PEG 6000, 100 mM sodium citrate pH 5.5), 3c and 3d: the Berkeley screen (Rigaku Reagents) condition D9 (20% (w/v) PEG 3350, 100 mM Bis-Tris pH 6.5, 100 mM ammonium phosphate dibasic, 5% (v/v) 2-propanol) and 3e: Index HT screen (Hampton Research) condition C.sub.5 (15% (w/v) PEG 3350, 100 mM succinic acid pH 7.0). Samples were transferred to cryoprotectant solutions, prior to plunging in liquid nitrogen, composed of 80% crystallization solution and 20% (v/v) PEG 200 except for 3c and 3d for which 20% (v/v) ethylene glycol was used as the cryoprotectant. X-ray diffraction data were collected at the Advanced Photon Source IMCA-CAT beamline 17-ID except for the data for the complex with 3c which were collected at the National Synchrotron Light Source II (NSLS-II) AMX beamline 17-ID-1.
[0137] Structure Solution and Refinement. Intensities were integrated using XDS (X-ray detector software) via Autoproc and the Laue class analysis and data scaling were performed with Aimless. Structure solution was conducted by molecular replacement with Phaser using a previously determined structure of SARS-2 3CLpro (PDB 6XM1K) as the search model. Structure refinement and manual model building were conducted with Phenix and Coot, respectively. Disordered side chains were truncated to the point for which electron density could be observed. Structure validation was conducted with Molprobity and structure analysis/figure preparation were carried out using the CCP4 mg package. Crystallographic data are provided in Table 5. Coordinates and structure factors for the following SARS2 3CLpro complexes with inhibitors were deposited to the Worldwide Protein Databank (wwPDB) with the accession codes: 7LKR (2a), 7LKS (2f), 7LKT (2k), 7LKU (3b), 7LKV (3c), 7LKW (3d) and 7LKX (3e).
TABLE-US-00005 TABLE 5 Crystallographic data for SARS-CoV-2 3CLpro inhibitor complexes. Inhibitor PDB Code 2a 2f 2k 3b 3c 3d 3e (7LKR) (7LKS) (7LKT) (7LKU) (7LKV) (7LKW) (7LKX) Data Collection Unit-cell a = 55.47 a = 55.19 a = 55.10 a = 54.91 a = 55.59 a = 54.91 a = 55.30 parameters b = 98.62 b = 98.76 b = 98.86 b = 98.39 b = 98.88 b = 98.39 b = 98.75 (?, ?) c = 58.94 c = 58.85 c = 59.42 c = 58.19 c = 58.96 c = 58.19 c = 59.09 ? = 108.2.sub. ? = 107.6.sub. ? = 107.9.sub. ? = 107.4.sub. ? = 108.3.sub. ? = 107.4.sub. ? = 108.0.sub. Space group P2.sub.1 P2.sub.1 P2.sub.1 P2.sub.1 P2.sub.1 P2.sub.1 P2.sub.1 Resolution (?).sup.1 48.69-1.65 49.38-1.70 49.08-1.50 49.19-1.65 46.56-1.55 49.19-1.65 48.83-1.60 (1.68-1.65) (1.73-1.70) (1.53-1.50) (1.68-1.65) (1.58-1.55) (1.68-1.65) (1.63-1.60) Wavelength (?) 1.0000 1.0000 1.0000 1.0000 0.9201 1.0000 1.0000 Temperature (K) 100 100 100 100 100 100 100 Observed 248,528 429,846 336,406 246,165 299,258 246,165 277,540 reflections Unique 72,217 65,020 96,612 70,811 86,929 70,811 77,631 reflections <I/?(I)>.sup.1 8.6 (2.4) 11.4 (1.7) 10.5 (1.8) 9.8 (1.9) 8.9 (1.8) 9.8 (1.9) 9.1 (1.8) Completeness 99.9 (98.4) 98.4 (100) 99.9 (99.9) 99.9 (100) 99.4 (99.3) 99.9 (100) 97.9 (96.4) (%).sup.1 Multiplicity.sup.1 3.4 (2.7) 6.6 (7.0) 3.5 (3.2) 3.5 (3.5) 3.4 (3.6) 3.5 (3.5) 3.6 (3.7) R.sub.merge (%).sup.1, 2 9.3 (46.0) 7.9 (141.5) 6.3 (67.5) 5.4 (55.3) 7.2 (69.9) 5.4 (55.3) 7.1 (81.4) R.sub.meas (%).sup.1, 4 11.0 (57.0) 8.6 (153.0) 7.4 (81.5) 6.4 (65.3) 8.5 (82.2) 6.4 (65.3) 8.4 (95.3) R.sub.pim (%).sup.1, 4 5.8 (33.1) 3.3 (57.6) 3.9 (45.0) 3.4 (34.3) 4.5 (42.9) 3.4 (34.3) 4.4 (49.2) CC.sub.1/2.sup.1, 5 0.989 (0.819) 0.999 (0.801) 0.997 (0.573) 0.998 (0.883) 0.996 (0.718) 0.998 (0.883) 0.996 (0.696) Refinement Resolution (?).sup.1 37.01-1.65 36.00-1.70 37.21-1.50 31.66-1.65 46.56-1.55 45.72-1.70 35.99-1.60 Reflections 68,631/3,538 61,732/3,127 91,730/4,828 67,198/3,470 82,504/4,340 61,623/3,225 73934/3,644 (working/test).sup.1 R.sub.factor/R.sub.free 18.0/21.9 18.4/22.1 17.8/20.5 17.8/21.1 17.8/20.8 18.6/22.7 18.4/21.2 (%).sup.1, 3 # of atoms 4,467/128/ 4,492/120/ 4,558/132/ 4,524/128/ 4,495/128/ 4,448/128/ 4,460/120/ (Protein/ 369 282 420 323 442 224 329 Ligand/Water) Model Quality R.m.s deviations Bond lengths (?) 0.009 0.010 0.010 0.008 0.009 0.010 0.007 Bond angles (?) 0.959 1.068 1.070 0.932 1.012 1.051 0.901 Mean B-factor (?.sup.2) All Atoms 27.0 33.4 26.0 31.6 27.5 36.0 30.7 Protein 26.3 33.0 25.2 31.0 26.5 35.5 29.8 Ligand 31.5 35.3 23.3 35.2 29.6 41.6 34.9 Water 53.2 39.1 34.6 39.3 37.0 40.6 37.3 Coordinate error 0.20 0.21 0.17 0.21 0.21 0.19 0.21 (maximum likelihood) (?) Ramachandran Plot Most favored 98.1 97.5 97.6 98.7 97.8 97.6 98.1 (%) Additionally 1.9 2.5 2.4 1.3 2.2 2.2 1.9 allowed (%) .sup.1Values in parenthesis are for the highest resolution shell. .sup.2R.sub.merge = ?.sub.hkl?.sub.i |I.sub.i(hkl) ? <I(hkl)>|/?.sub.hkl?.sub.i I.sub.i(hkl), where I.sub.i(hkl) is the intensity measured for the ith reflection and <I(hkl)> is the average intensity of all reflections with indices hkl. .sup.3R.sub.factor = ?.sub.hkl ||F.sub.obs (hkl) | ? |F.sub.calc (hkl) ||/?.sub.hkl |F.sub.obs (hkl)|; R.sub.free is calculated in an identical manner using 5% of randomly selected reflections that were not included in the refinement. .sup.4R.sub.meas = redundancy-independent (multiplicity-weighted) R.sub.merge. R.sub.pim = precision-indicating (multiplicity-weighted) R.sub.merge. .sup.5CC.sub.1/2 is the correlation coefficient of the mean intensities between two random half-sets of data.
Example 2
[0138] Several compounds were synthetized based upon the Series F compounds using the backbone I and screened against SARS-CoV-2 and MVERS-CoV as described in Example 1 above.
##STR00049##
Synthesis of F1 Series
[0139] The synthesis of the compounds shown follows previously published procedures (Heinrich et al J Med Chem 62 (2019) 11119-11134) to make the appropriate precursor carboxylic acids followed by treatment with carbonyl diimidazole and sodium borohydride to furnish the corresponding alcohols which were then used to make the inhibitors. Briefly, a solution of cyclopropyl Meldrum's acid (1.3 eq) in acetonitrile:DMF (10:3 mixture) was reacted with an appropriate amine (RNH2, 1 eq) at 60? C. for 12 h. The solvents were removed in vacuo and the residue was dissolved in 10% aqueous sodium hydroxide and extracted with ethyl acetate. The layers were separated, and the aqueous layer was then acidified and extracted with ethyl acetate to yield the acid, which was dried thoroughly under vacuum. A solution of the acid (1 eq) in THE was treated with carbonyl diimidazole (1.5 eq) and stirred for 30 minutes. An aqueous solution of sodium borohydride (2.5 eq) was added dropwise and the mixture was stirred overnight. The solution was acidified, the solvent was removed in vacuo and the residue was extracted with ethyl acetate to yield the desired alcohol (91% yield). The reaction sequence shown in Scheme 1 was then used to generate the inhibitors.
##STR00050##
[0140] R.sub.0 can be any natural or unnatural amino acid side chain (preferably leucine/isobutyl), and R can be any aliphatic or aromatic amine (substituted or unsubstituted), or a heterocyclic amine.
TABLE-US-00006 TABLE 6 Alcohol input (F1)
Synthesis of F2 Series
[0141] A mixture of dimethyl itaconate (15 mmol), amine (15 mmol) and methanol (1.5 mL) was kept at RT overnight. The reaction mixture was then refluxed for 2 h and the solvent removed. Water (30 mL) was added to the residue and the mixture was extracted with ethyl acetate (3?30 mL). The combined organic extracts were dried over anhydrous sodium sulfate and the drying agent was filtered off. The filtrate was concentrated and the crude product was purified using flash chromatography (silica gel/ethyl acetate/hexane). Lithium borohydride reduction yielded the corresponding alcohols which were then used to make the inhibitors using Scheme 1.
##STR00053##
[0142] R.sub.0 can be any natural or unnatural amino acid side chain (preferably leucine/isobutyl), and R can be any aliphatic or aromatic amine (substituted or unsubstituted), or a heterocyclic amine.
TABLE-US-00007 TABLE 7 Alcohol input (F2)
Synthesis of F3 Series
[0143] Briefly, (L)Boc-glutamic acid dimethyl ester (1 eq) was treated with 4M HCl in dioxane (10 eq) and stirred for 3 h. Removal of the solvent yielded the product, which was dissolved in methanol and treated with triethylamine (4 eq). After stirring for 30 minutes, the mixture was cooled to 0? C., followed by the addition of benzaldehyde (1.1 eq) and sodium borohydride (2 eq) and stirring continued at 0? C. for 2 h. A mixture of 20% HCl and ether (1:1 mixture) was added and the organic layer was removed in vacuo. The aqueous layer was neutralized with solid sodium carbonate and extracted with ether. The isolated amine was dissolved in acetonitrile and then refluxed for 4 h to yield the desired cyclized product. The ester was then treated with lithium borohydride (3 eq) to yield the alcohol which was used to generate the inhibitors as shown in Scheme 1.
##STR00057##
TABLE-US-00008 TABLE 8 Alcohol input (F-3)
Series F4 (1,3,2-Oxazaphospholidin-3-One) Synthesis
[0144] The general procedures for generating the P ring (first 3 steps) are reported in Thomson C M et al J Org Chem 55 (1990) 111-116. Briefly, (S) serine methyl ester hydrochloride (0.065 mmol) was dissolved in anhydrous methanol and cooled to 0? C. Triethylamine (0.065 mmol) was added and the reaction mixture was stirred for 10 minutes. Benzaldehyde (0.065 mmol) was added and the reaction mixture was stirred for 2 h, at which time sodium borohydride (0.13 mmol) was added portionwise over 30 minutes. The solution was partitioned between 20% HCl (50 mL) and ether (50 mL). The organic phase was extracted twice with 20-mL portions of 20% HCl and the combined aqueous layers were washed with ether (20 mL). The combined aqueous later was carefully neutralized with solid sodium carbonate and extracted with diethyl ether (3?20 mL). The combined ether extracts were washed with brine (30 mL) and dried over anhydrous sodium sulfate. Evaporation of the solvent yielded (S)-N-benzyl serine methyl ester (70% yield) which was used in the next step without further purification. A solution of (S)-benzyl serine methyl ester (4.78 mmol) in dry toluene (20 mL) was cooled to 0? C. and then treated with triethylamine (10 mmol) followed by phosphorous oxychloride (4.78 mmol) and stirred for 3 h. The reaction mixture was diluted with dry THE (50 mL) and the solution was filtered through a pad of Celite. Evaporation of the solvent yielded a syn- and anti-mixture of diastereomers (if desired, these can be separated by flash chromatography). Alternatively, a solution of the mixture in toluene (15 mL/g of starting material) was treated with an alcohol (R40H) (150 mol %) followed by the addition of triethylamine (100 mol %) with stirring. The reaction was monitored by TLC. The reaction mixture was partitioned between ethyl ether and saturated sodium carbonate. The organic phase was washed sequentially with aqueous sodium carbonate and brine. The ether layer was dried over anhydrous sodium sulfate, and the crude mixture of syn- and anti-methyl esters was purified by flash chromatography. Reduction of the esters with lithium borohydride yielded a mixture of the corresponding alcohols which was then used to synthesize the inhibitors (Scheme 1).
[0145] These compounds can exist in two distinct conformations (syn and anti), where the R.sub.4 group is oriented in space in two different ways and therefore each series can access different regions of space in S.sub.4 subsite. The two series can be isolated separately, although in the examples thus far, we generated them as a mixture of the syn and anti series (hence these were screened as mixtures).
##STR00061##
[0146] If the starting material used is threonine methyl ester hydrochloride, then the structure will have a methyl group which will be the R.sub.5 group (R.sub.5=methyl)
##STR00062##
TABLE-US-00009 TABLE 9 Alcohol Input (F4)
Series F5 Synthesis
[0147]
TABLE-US-00010 TABLE 10 Alcohol Inputs (F5)
Example 3
Series D (Azetidines) and E (Spirocycles)
[0148] We report herein the results of preliminary studies related to the structure-guided design of potent and permeable inhibitors of SARS-CoV-2 3CLpro that incorporate in their structure a spirocyclic component as a design element to optimally exploit new chemical space in the active site of the protease. Finally, for comparative purposes, a series of azetidine-derived inhibitors were also synthesized and evaluated in biochemical and cell-based assays.
##STR00068##
Results and Discussion
[0149] Inhibitor design rationale. There are an array of advantages accrued from the judicious use of spirocycles in drug design, including improved physicochemical and PK characteristics, structural novelty, reduced conformational flexibility, and the capture of favorable binding interactions by probing and exploiting poorly-explored regions of chemical space. Importantly, the structural motifs embodied in spirocycles make possible the rigorous control of the spatial disposition of exit vectors; consequently, it was envisaged that the attachment of a suitably-decorated spirocycle capable of engaging in favorable binding interactions with the S.sub.4 subsite region of SARS-CoV-2 3CLpro to a recognition element that is congruent with the known substrate specificity of the enzyme (in the case of SARS-CoV-2 3CLpro, a Leu-Gln surrogate fragment), would yield a molecule with high inhibitory prowess. The validity of the approach and the design of the inhibitors was further facilitated by the use of high resolution cocrystal structures. Lastly, we sought to harness the benefits accrued through deuteration, particularly the potential improvement of pharmacokinetics and physicochemical properties, consequently, a select number of deuterated inhibitors are also made. For the general spirocyclic and azetidine alcohol inputs for these Series see Table 1.
[0150] Chemistry. The inhibitors were synthesized by attaching an azetidine-based spirocyclic alcohol to a Leu-Gln surrogate fragment incorporating an aldehyde warhead or latent aldehyde bisulfite adduct. The spirocyclic and azetidine-based precursor alcohols were either commercially available or readily synthesized using commercially available ketone or carboxylic acid precursors.
[0151] The appropriate spirocyclic and azetidine alcohol inputs (Tables 11 and 12) were treated with N, N-disuccinimidyl carbonate (DSC), followed by coupling of the resulting mixed carbonate to amino alcohol A. Dess-Martin periodinane oxidation of dipeptidyl alcohol a generated the desired aldehydes b which were subsequently transformed into the corresponding aldehyde bisulfite adducts c (see Scheme 1).
[0152] Biochemical studies. The inhibitory activity of the compounds toward SARS-CoV-2 3CL protease in biochemical and cell-based assays, as well as the cytotoxicity of the compounds, are determined and the results are listed in Tables 11 and 12. For comparative purposes, the interaction of a select number of compounds with MERS-CoV-2 3CL protease is also investigated. Selected compounds were tested in a cell-based assay against SARS-CoV-2 as described in the experimental section. The IC.sub.50 values, EC.sub.50 values for a select number of inhibitors, and the CC.sub.50 values in CRFK cells are summarized in Tables 11 and 12 and they are the average of at least two determinations
[0153] In this study, we used another BSL2 cell-based replicon assay in 293T cells, mimicking the natural cycle of SARS-CoV-2 replication. As a control, we used GC376 and the EC.sub.50 was calculated at 0.037?0.01 ?M in the assay. The EC.sub.50 is comparable to the value (0.02 ?M in 293T cells) previously reported with the same system. Four compounds were selected for the determination of EC.sub.50s, and inhibition curves by each compound were consistent with a dose-dependent mode and R.sup.2>0.9 (
TABLE-US-00011 TABLE 11 IC.sub.50 values of spirocyclic inhibitors 1-11b/c against SARS-CoV-2 3CL.sup.pro and MERS-CoV 3CL.sup.pro, and CC.sub.50 values.
TABLE-US-00012 TABLE 12 IC.sub.50 values of azetidine inhibitors 12-17b/c against SARS-CoV-2 3CL.sup.pro and MERS-CoV 3CL.sup.pro, and CC.sub.50 values. IC.sub.50 (?M) Compound SARS-CoV-2 MERS- Code Structure Z 3CL.sup.pro CoV 3CL.sup.pro CC.sub.50 (?M) 12b 12c
X-Ray Crystallographic Studies
[0154] In order to gain insight and understanding into the binding of the spirocyclic inhibitors to the active site of the protease, as well as identify the structural determinants associated with binding, high-resolution cocrystal structures of SARS-CoV-2 3CL.sup.pro and MERS-CoV 3CL.sup.pro were obtained in complex with spirocyclic and azetidine-derived inhibitors. For all structures described below, the electron density was consistent both the R and S enantiomers at the stereocenter formed by covalent attachment of the S? atom of Cys 145 or Cys 148 in SARS-CoV-2 3CL.sup.pro and MVERS-CoV 3CL.sup.pro, respectively. Therefore, the alternate conformations were modeled as each enantiomer with 0.5 occupancy.
[0155] Azetidine-derived inhibitor bound structures. In the case of the azetidine inhibitor 14c, the active site contained prominent difference electron density consistent with the inhibitor covalently bound to Cys 148 and Cys 145 in each subunit (
[0156] 2-Azaspiro [3.3]-derived inhibitor bound structures. Similar to the azetidine inhibitors above, difference electron density consistent with inhibitors 2c, 3c and 4c bound in the SARS-CoV-2 3CLpro active site covalently to Cys 145 (
[0157] 6-Azaspiro [3.5]-derived inhibitor bound structures. Interestingly, the spirocyclic inhibitors that contained the larger 6-membered nitrogen heterocycle did not display the same degree of disorder observed for 2c, 3c and 4c, which contain the 4-membered rings. This was revealed by the structure determination of 7c, 8c, 9c, 10c and 11c in complex with SARS-CoV-2 3CL.sup.Pro in which the electron density was well-defined for the majority of these inhibitors (
[0158] Notably, the methyl sulfonyl group of 10c is in proximity to Pro 168 but too far to form an interaction (3.4 ?). The interaction between Pro 168 and 9c results in the movement (?2.6 ?) of a nearby loop that includes Leu 167, Pro 168 and Thr 169 relative to the other structures, such as 10c (
[0159] Similarly, the structures of MERS-CoV 3CL.sup.pro with 8c, 9c and 10c yielded well-defined electron density overall (
[0160] Structure-Activity Relationships. A representative series of spirocyclic inhibitors derived from 2-azaspiro[3.3]-, 2-azaspiro[3.4]-, 6-azaspiro[3.4]-, and 6-azaspiro[3.5]-spirocycles displaying different exit vectors were synthesized and evaluated in biochemical and cell-based assays. It is evident from the results shown in Table 11 that the synthesized compounds generally display high inhibitory activity toward SARS-CoV-2 3CL.sup.pro and MERS-CoV 3CL.sup.pro, with the IC.sub.50 values of most of the inhibitors in the submicromolar range. Furthermore, the compounds are devoid of cytotoxic effects. The IC.sub.50 values of spirocycles 7b and 3b were found to be >9-fold and nearly 13-fold lower than that of compound 1b, respectively, suggesting that directional and recognition effects associated with the nature of the spirocycle and X group, respectively, are important in enhancing potency. The importance of exit vectors is also evident in comparing the relative potency of aldehyde inhibitors 1b, 5b and 6b which are derived from different spirocycles. The potency of compounds 8b, 9b, 10b and 11b was high and remained invariant to the nature of the R group. Several of the inhibitors were found to be broadly active against both SARS-CoV-2 3CLpro and MERS-CoV 3CL.sup.pro, suggesting a high likelihood for identifying a broad-spectrum pre-clinical candidate. The EC.sub.50 values of the aldehyde and corresponding bisulfite adduct pairs tested were comparable, and one pair was in the nM range (Table 11, compounds 7b/7c) The Safety Index (SI), defined as CC.sub.50/EC.sub.50, for the compounds was very high (?1250). The results shown in Table 11 are congruent with the crystallographic studies (vide supra) and validate the use of spirocyclic inhibitors in exploring and exploiting new chemical space in the S.sub.4 region of SARS-CoV-2 3CL.sup.pro.
[0161] In the azetidine series, biochemical evaluation of the synthesized azetidine inhibitors revealed that the compounds were fairly potent against both SARS-CoV 3CLP.sup.pro and MERS-CoV 3CLpro (Table 12). The IC.sub.50 values of compounds 14b/14c having an extra methylene group were >6-fold better than those of the 12b/12c pair. Furthermore, in the series of compounds 14b, 15b, 16b and 17b, potency was found to be sensitive to the nature of the group attached to the azetidine nitrogen, with compound 14b being 12-fold more potent than 17b and with an EC.sub.50 value of 0.38 ?M.
CONCLUSIONS
[0162] There is currently a need for the development of direct-acting antivirals to complement the use of vaccines and biologics for the treatment of COVID-19. In this study we have sought to exploit the directional and stereochemical control afforded by spirocycles to optimize potency. The results indicate that the incorporation of spirocyclic elements embellished with appropriate recognition moieties, combined with structural information gained from cocrystal structures, into the design of process, has resulted in the identification of highly effective broad-spectrum inhibitors of SARS-CoV-2 3CLpro and MERS-CoV 3CL.sup.pro, with EC.sub.50 values and Safety Indices in the 0.08-0.43 ?M and >2000 range, respectively. The structural determinants associated with binding and the mechanism of action involving participation of the catalytic dyad Cys145 and His41 and the formation of a tetrahedral adduct, were elucidated using X-ray crystallography. These studies provide a solid foundation for conducting further preclinical studies.
EXPERIMENTAL SECTION
General
[0163] Reagents and dry solvents were purchased from various chemical suppliers (Advanced ChemBlocks, Sigma-Aldrich, Acros Organics, Chem-Impex, TCI America, Oakwood chemical, APExBIO, SynQuest, Fisher and Bachem) and were used as obtained. The synthesized compounds were purified using flash chromatography and silica gel (230-450 mesh) (Sorbent Technologies, Atlanta, GA). Normal phase chromatography was performed on a Teledyne ISCO CombiFlash system using RediSep normal phase silica cartridges (35-70 ?m particle size range). Thin layer chromatography was performed using Analtech silica gel plates. Visualization was accomplished using UV light and/or iodine. .sup.1H NMR spectra were recorded in CDCl.sub.3 or DMSO-d.sub.6 using a Varian XL-400 spectrometer. Chemical shifts and coupling constants are reported in parts per million and hertz, respectively. The following abbreviations are used to describe splitting patterns: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. The purity of the final compounds was found to be ?90%, as determined by absolute qNMR analysis using a Bruker AV III 500 NMR spectrometer equipped with a CPDUL CRYOprobe and CASE autosampler (the University of Kansas Nuclear Magnetic Resonance Laboratory). Dimethyl sulfone TraceCERT? was used as the internal calibrant. High resolution mass spectrometry (HRMS) was performed at the Wichita State University Mass Spectrometry lab using Orbitrap Velos Pro mass spectrometer (ThermoFisher, Waltham, MA) equipped with an electrospray ion source.
Synthesis of Compounds
[0164] Preparation of compounds 1-17a. General procedure. To a solution of alcohol (1 eq) (Table 11) in anhydrous acetonitrile (10 mL/g alcohol) was added N,N-disuccinimidyl carbonate (1.2 eq) and TEA (3.0 eq) and the reaction mixture was stirred for 4 h at room temperature. The solvent was removed in vacuo and the residue was dissolved in ethyl acetate (40 mL/g alcohol). The organic phase was washed with saturated aqueous NaHCO.sub.3(2?20 mL/g alcohol), followed by brine (20 mL/g alcohol). The organic layers were combined and dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuo to yield the mixed carbonate which was used in the next step without further purification.
[0165] To a solution of Leu-Gln surrogate amino alcohol A (1.0 eq) in dry methylene chloride (10 mL/g of amino alcohol) was added TEA (1.5 eq) and the reaction mixture was stirred for 20 min at room temperature (solution 1). In a separate flask, the mixed carbonate was dissolved in dry methylene chloride (10 mL/g of carbonate) (solution 2). Solution 1 was added to solution 2 and the reaction mixture was stirred 3 h at room temperature. Methylene chloride was added to the organic phase (40 mL/g of carbonate) and then washed with saturated aqueous NaHCO.sub.3(2?20 mL/g alcohol), followed by brine (20 mL/g alcohol). The organic phase was dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The resultant crude product was purified by flash chromatography (hexane/ethyl acetate) to yield dipeptidyl alcohol a as a white solid.
[0166] Preparation of compounds 1-17b. General procedure. To a solution of dipeptidyl alcohol a (1 eq) in anhydrous dichloromethane (300 mL/g dipeptidyl alcohol) kept at 0-5? C. under a N.sub.2 atmosphere was added Dess-Martin periodinane reagent (3.0 eq) and the reaction mixture was stirred for 3 h at 15-20? C. The organic phase was washed with 10% aq Na.sub.2S.sub.2O.sub.3 (2?100 mL/g dipeptidyl alcohol), followed by saturated aqueous NaHCO.sub.3(2?100 mL/g dipeptidyl alcohol), distilled water (2?100 mL/g dipeptidyl alcohol), and brine (100 mL/g dipeptidyl alcohol). The organic phase was dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The resulting crude product was purified by flash chromatography (hexane/ethyl acetate) to yield aldehyde b as a white solid.
[0167] Tert-butyl 6-((((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((R)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)carbamoyl)oxy)-2-azaspiro[3.3]heptane-2-carboxylate (1b). .sup.1H NMR (500 MHz, DMSO-d6) ? 9.38 (d, J=6.9 Hz, 1H), 8.44 (d, J=7.6 Hz, 1H), 7.53 (s, 1H), 7.33 (d, J=8.1 Hz, 1H), 4.74-4.60 (m, 1H), 4.08-3.89 (m, 2H), 3.81 (d, J=26.2 Hz, 4H), 3.19-3.04 (m, 2H), 2.30-2.02 (m, 7H), 1.98-1.74 (m, 2H), 1.71-1.38 (m, 3H), 1.36 (s, 9H), 0.86 (ddd, J=14.0, 10.5, 6.4 Hz, 6H). Yield (74%). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.25H.sub.40N.sub.4NaO.sub.7 531.2795; Found 531.2776.
[0168] 2-Isobutyryl-2-azaspiro[3.3]heptan-6-yl ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((R)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)carbamate (2b). Yield (24%). .sup.1H NMR (400 MHz, cdcl.sub.3) ? 9.58 (s, 1H), 6.69 (s, 1H), 5.88 (s, 1H), 5.68 (s, 1H), 5.23-4.79 (m, 2H), 4.38-4.09 (m, 2H), 4.02-3.89 (m, 2H), 3.78-3.66 (m, 2H), 3.63-3.54 (m, 2H), 3.51-3.24 (m, 4H), 2.69-2.19 (m, 2H), 2.19-1.98 (m, 1H), 1.98-1.37 (m, 5H), 1.19-1.10 (m, 6H), 1.03-0.79 (m, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.24H.sub.38N.sub.4NaO.sub.6 501.2689; Found 501.2672.
[0169] 2-(2-Phenylacetyl)-2-azaspiro[3.3]heptan-6-yl ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((R)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)carbamate (3b). Yield (69%). .sup.1H NMR (400 MHz, cdcl.sub.3) ? 9.46 (s, 1H), 8.95 (d, J=5.1 Hz, 1H), 7.40-7.22 (m, 5H), 6.61 (s, 1H), 5.87 (s, 1H), 5.17 (d, J=8.5 Hz, 1H), 4.94-4.86 (m, 1H), 4.35-4.25 (m, 1H), 4.25-4.17 (m, 1H), 3.63-3.53 (m, 2H), 3.53-3.39 (m, 4H), 3.37-3.29 (m, 2H), 2.51-2.35 (m, 2H), 2.33-2.10 (m, 2H), 2.09-1.96 (m, 1H), 1.94-1.62 (m, 5H), 1.57-1.44 (m, 1H), 1.01-0.89 (m, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.28H.sub.38N.sub.4NaO.sub.6 549.2689; Found 549.2675.
[0170] 2-(Methylsulfonyl)-2-azaspiro[3.3]heptan-6-yl ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((R)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)carbamate (4b). Yield (16%). .sup.1H NMR (400 MHz, cdcl.sub.3) ? 9.45 (s, 1H), 8.12 (s, 1H), 6.67 (s, 1H), 6.24 (s, 1H), 5.03-4.79 (m, 1H), 4.23 (t, J=11.4 Hz, 1H), 4.00-3.87 (m, 1H), 3.71-3.54 (m, 4H), 3.44-3.16 (m, 6H), 2.99 (s, 3H), 2.52-2.28 (m, 2H), 2.26-1.71 (m, 3H), 1.68-1.45 (m, 3H), 1.05-0.78 (m, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.21H.sub.34N.sub.4NaO.sub.7S 509.2046; Found 509.1988.
[0171] Tert-butyl 2-((((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((R)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)carbamoyl)oxy)-6-azaspiro[3.4]octane-6-carboxylate (5b). Yield (88%). 1H NMR (400 MHz, cdcl.sub.3) ? 9.49 (s, 1H), 8.34 (s, 1H), 6.06 (s, 1H), 5.28-5.17 (m, 1H), 5.02-4.89 (m, 1H), 4.38-4.12 (m, 2H), 3.47-3.19 (m, 6H), 2.55-2.29 (m, 4H), 2.19-1.80 (m, 7H), 1.80-1.61 (m, 2H), 1.61-1.49 (m, 1H), 1.45 (s, 9H), 1.01-0.89 (m, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.26H.sub.42N.sub.4NaO.sub.7 545.2951; Found 545.2931.
[0172] Tert-butyl 6-((((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((R)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)carbamoyl)oxy)-2-azaspiro[3.4]octane-2-carboxylate (6b). Yield (67%). .sup.1H NMR (400 MHz, DMSO-d6) ? 9.40 (d, J=2.0 Hz, 1H), 7.71-7.44 (m, 2H), 7.16 (dt, J=51.0, 7.3 Hz, 1H), 5.01-4.83 (m, 1H), 4.65 (t, J=5.6 Hz, 1H), 4.19 (td, J=7.7, 4.0 Hz, 1H), 4.07-3.84 (m, 1H), 3.87-3.49 (m, 5H), 3.39-3.03 (m, 3H), 2.75-2.70 (m, 1H), 2.32-1.94 (m, 3H), 1.94-1.69 (m, 4H), 1.69-1.57 (m, 3H), 1.37 (s, 9H), 0.95-0.81 (m, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.26H.sub.42N.sub.4NaO.sub.7 545.2951; Found 545.2928.
[0173] Tert-butyl 2-((((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((R)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)carbamoyl)oxy)-7-azaspiro[3.5]nonane-7-carboxylate (7b). Yield (67%). .sup.1H NMR (500 MHz, DMSO-d6) ? 7.63 (s, 1H), 7.51 (d, J=6.9 Hz, 1H), 7.22-7.16 (m, 1H), 4.86-4.78 (m, 1H), 4.07-3.91 (m, 2H), 3.28-3.03 (m, 6H), 2.30-2.02 (m, 4H), 1.89-1.77 (m, 2H), 1.75-1.50 (m, 4H), 1.49-1.40 (m, 7H), 1.40 (s, 9H), 0.93-0.80 (m, 6H). HRMS m/z: [M+H].sup.+ Calculated for C.sub.27H.sub.45N.sub.4O.sub.7 537.3288; Found 537.3257.
[0174] 7-Isobutyryl-7-azaspiro[3.5]nonan-2-yl ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((R)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)carbamate (8b). Yield (55%). .sup.1H NMR (400 MHz, cdcl.sub.3) ? 9.49 (s, 1H), 8.33 (s, 1H), 6.21-6.13 (m, 1H), 5.29-5.22 (m, 1H), 5.01-4.93 (m, 1H), 4.32 (s, 2H), 3.61-3.45 (m, 2H), 3.44-3.28 (m, 4H), 2.82-2.69 (m, 1H), 2.54-2.27 (m, 5H), 2.12-1.93 (m, 2H), 1.92-1.81 (m, 3H), 1.79-1.63 (m, 1H), 1.56 (s, 5H), 1.10 (d, J=6.7 Hz, 6H), 0.97 (d, J=6.2 Hz, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.26H.sub.42N.sub.4NaO.sub.6 529.3002; Found 529.2985.
[0175] 7-(2-Phenylacetyl)-7-azaspiro[3.5]nonan-2-yl ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((R)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)carbamate (9b). Yield (87%). .sup.1H NMR (400 MHz, cdcl.sub.3) ? 9.48 (s, 1H), 8.32 (s, 1H), 7.35-7.15 (m, 5H), 6.19 (d, J=13.8 Hz, 1H), 5.28-5.21 (m, 1H), 5.00-4.86 (m, 1H), 4.37-4.10 (m, 2H), 3.72 (s, 2H), 3.59-3.44 (m, 2H), 3.42-3.23 (m, 4H), 2.52-2.34 (m, 2H), 2.34-2.18 (m, 2H), 2.12-1.90 (m, 1H), 1.90-1.76 (m, 3H), 1.74-1.60 (m, 1H), 1.58-1.43 (m, 4H), 1.41-1.32 (m, 3H), 0.96 (d, J=6.1 Hz, 6H). HRMS m/z: [M+H].sup.+ Calculated for C.sub.30H.sub.43N.sub.4O.sub.6 555.3182; Found 555.3156.
[0176] 7-(Methylsulfonyl)-7-azaspiro[3.5]nonan-2-yl ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((R)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)carbamate (10b). Yield (62%). .sup.1H NMR (400 MHz, cdcl.sub.3) ? 9.49 (s, 1H), 8.33 (d, J=5.8 Hz, 1H), 6.10 (s, 1H), 5.25 (d, J=8.6 Hz, 1H), 5.03-4.89 (m, 1H), 4.31 (s, 2H), 3.44-3.29 (m, 2H), 3.21-3.00 (m, 4H), 2.76 (s, 3H), 2.55-2.20 (m, 4H), 2.09-1.80 (m, 4H), 1.69 (td, J=12.3, 7.5 Hz, 7H), 1.54 (t, J=8.8 Hz, 1H), 0.97 (d, J=6.2 Hz, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.23H.sub.38N.sub.4NaO.sub.7S 537.2359; Found 537.2341. 7-Cyano-7-azaspiro[3.5]nonan-2-yl ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((R)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)carbamate (Jib). Yield (53%). .sup.1H NMR (400 MHz, cdcl.sub.3) ? 9.49 (s, 1H), 8.36 (d, J=5.7 Hz, 1H), 5.95 (s, 1H), 5.21 (d, J=8.3 Hz, 1H), 5.04-4.89 (m, 1H), 4.38-4.25 (m, 2H), 3.45-3.30 (m, 2H), 3.19-3.08 (m, 4H), 2.56-2.22 (m, 4H), 2.01-1.81 (m, 4H), 1.77-1.62 (m, 7H), 1.61-1.48 (m, 1H), 0.97 (d, J=5.8 Hz, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.23H.sub.35N.sub.5NaO.sub.5 484.2536; Found 484.2522.
[0177] Tert-butyl 3-((((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((R)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)carbamoyl)oxy)azetidine-1-carboxylate (12b). Yield (74%). .sup.1H NMR (500 MHz, DMSO-d6) ? 7.78 (s, 1H), 7.68-7.61 (m, 1H), 7.54-7.47 (m, 1H), 5.01-4.90 (m, 1H), 4.19-4.05 (m, 2H), 4.05-3.61 (m, 4H), 3.26-3.04 (m, 2H), 2.27-2.02 (m, 3H), 1.86-1.71 (m, 2H), 1.70-1.39 (m, 4H), 1.38-1.34 (m, 9H), 0.92-0.79 (m, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.22H.sub.36N.sub.4NaO.sub.7 491.2482; Found 491.2461.
[0178] Tert-butyl 3-methyl-3-((((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((R)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)carbamoyl)oxy)azetidine-1-carboxylate (13b). Yield (76%). .sup.1H NMR (400 MHz, DMSO-d6) ? 9.40 (d, J=4.9 Hz, 1H), 8.45 (d, J=7.8 Hz, 1H), 7.63 (s, 1H), 7.50 (d, J=7.7 Hz, 1H), 4.22 (ddd, J=11.6, 7.7, 3.9 Hz, 1H), 4.08-3.93 (m, 1H), 3.88 (d, J=9.3 Hz, 2H), 3.78 (d, J=9.4 Hz, 2H), 3.23-3.02 (m, 2H), 2.34-2.07 (m, 2H), 1.96-1.84 (m, 1H), 1.63 (ddt, J=16.1, 11.8, 6.3 Hz, 3H), 1.55 (s, 3H), 1.46 (qd, J=8.4, 3.9 Hz, 2H), 1.37 (s, 9H), 0.93-0.82 (m, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.23H.sub.38N.sub.4NaO.sub.7 505.2638; Found 505.2621.
[0179] Tert-butyl 3-(((((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((R)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)carbamoyl)oxy)methyl)azetidine-1-carboxylate (14b). Yield (90%). .sup.1H NMR (400 MHz, DMSO-d6) ? 9.40 (s, 1H), 8.45 (d, J=7.5 Hz, 1H), 7.63 (s, 1H), 7.40 (d, J=7.9 Hz, 1H), 4.21-3.99 (m, 3H), 3.92-3.82 (m, 2H), 3.62-3.52 (m, 2H), 3.21-3.05 (m, 2H), 2.83-2.72 (m, 2H), 2.34-2.06 (m, 2H), 1.95-1.83 (m, 2H), 1.70-1.56 (m, 3H), 1.52-1.42 (m, 1H), 1.37 (s, 9H), 0.92-0.83 (m, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.23H.sub.38N.sub.4NaO.sub.7 505.2638; Found 505.2609.
[0180] (1-(2-Phenylacetyl)azetidin-3-yl)methyl ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((R)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)carbamate (15b). Yield (63%). .sup.1H NMR (400 MHz, cdcl.sub.3) ? 9.46 (s, 1H), 8.73-8.66 (m, 1H), 7.40-7.16 (m, 5H), 6.38 (d, J=32.7 Hz, 1H), 6.14 (d, J=22.3 Hz, 1H), 5.32 (d, J=16.0 Hz, 1H), 4.35-3.89 (m, 4H), 3.81-3.65 (m, 2H), 3.60-3.43 (m, 2H), 3.40-3.13 (m, 4H), 2.57-2.19 (m, 2H), 2.06 (s, 1H), 1.98-1.77 (m, 2H), 1.75-1.61 (m, 2H), 1.57-1.45 (m, 1H), 1.03-0.82 (m, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.26H.sub.36N.sub.4NaO.sub.6 523.2533; Found 523.2518.
[0181] (1-(Bicyclo[2.2.1]heptane-2-carbonyl)azetidin-3-yl)methyl ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((R)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)carbamate (16b). Yield (75%). .sup.1H NMR (400 MHz, cdcl.sub.3) ? 9.47 (s, 1H), 8.85 (s, 1H), 6.28 (d, J=42.6 Hz, 1H), 6.10 (d, J=32.7 Hz, 1H), 5.32-5.28 (m, 1H), 4.45-3.98 (m, 5H), 3.94-3.70 (m, 1H), 3.69-3.52 (m, 2H), 3.51-3.16 (m, 3H), 2.69-2.56 (m, 1H), 2.56-2.21 (m, 5H), 1.96-1.67 (m, 6H), 1.66-1.46 (m, 2H), 1.43-1.23 (m, 3H), 1.18 (q, J=8.3 Hz, 1H), 0.97 (d, J=5.6 Hz, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.26H.sub.40N.sub.4NaO.sub.6 527.2846; Found 527.2837.
[0182] (1-(Methylsulfonyl)azetidin-3-yl)methyl ((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((R)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)carbamate (17b). Yield (14%). .sup.1H NMR (400 MHz, cdcl.sub.3) ? 9.48 (s, 1H), 8.28 (d, J=7.5 Hz, 1H), 6.56 (s, 1H), 5.54 (s, 1H), 4.46-3.91 (m, 2H), 3.90-3.73 (m, 2H), 3.70-3.10 (m, 4H), 3.02-2.71 (m, 2H), 2.57-2.16 (m, 3H), 2.16-1.78 (m, 1H), 1.75-1.48 (m, 3H), 1.46-1.36 (m, 2H), 1.26 (s, 3H), 1.08-0.78 (m, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.19H.sub.32N.sub.4NaO.sub.7S 483.1890; Found 483.1832.
[0183] Preparation of compounds 1-17c. General procedure. To a solution of dipeptidyl aldehyde b (1 eq) in ethyl acetate (10 mL/g of dipeptidyl aldehyde) was added absolute ethanol (5 mL/g of dipeptidyl aldehyde) with stirring, followed by a solution of sodium bisulfite (1 eq) in water (1 mL/g of dipeptidyl aldehyde). The reaction mixture was stirred for 3 h at 50? C. The reaction mixture was allowed to cool to room temperature and then vacuum filtered. The solid was thoroughly washed with absolute ethanol and the filtrate was dried over anhydrous sodium sulfate, filtered, and concentrated to yield a white solid. The white solid was stirred with dry ethyl ether (3?10 mL/g of dipeptidyl aldehyde), followed by careful removal of the solvent using a pipette and dried using a vacuum pump for 2 h to yield dipeptidyl bisulfite adduct c as a white solid.
[0184] Sodium (2S)-2-((S)-2-((((2-(tert-butoxycarbonyl)-2-azaspiro[3.3]heptan-6-yl)oxy) carbonyl)amino)-4-methylpentanamido)-1-hydroxy-3-((R)-2-oxopyrrolidin-3-yl)propane-1-sulfonate (1c). Yield (56%). .sup.1H NMR (400 MHz, DMSO-d6) ? 7.52 (d, J=9.3 Hz, 1H), 7.44 (s, 1H), 7.18 (d, J=8.2 Hz, 1H), 5.71 (d, J=5.9 Hz, 1H), 4.74-4.59 (m, 2H), 4.08-3.58 (m, 5H), 3.23-2.99 (m, 2H), 2.29-1.94 (m, 4H), 1.91-1.71 (m, 1H), 1.69-1.38 (m, 7H), 1.35 (s, 9H), 0.91-0.79 (m, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.25H.sub.41N.sub.4Na.sub.2O.sub.10S 635.2339; Found 635.2379.
[0185] Sodium (2S)-1-hydroxy-2-((S)-2-((((2-isobutyryl-2-azaspiro[3.3]heptan-6-yl)oxy) carbonyl)amino)-4-methylpentanamido)-3-((R)-2-oxopyrrolidin-3-yl)propane-1-sulfonate (2c). Yield (69%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 7.85 (s, 1H), 7.64 (s, 1H), 7.19 (s, 1H), 5.80-5.66 (m, 1H), 4.90-4.60 (m, 2H), 4.28-3.81 (m, 2H), 3.81-3.59 (m, 2H), 3.25-2.98 (m, 4H), 2.47-2.34 (m, 1H), 2.34-1.75 (m, 7H), 1.75-1.29 (m, 4H), 1.01 (d, J=6.8 Hz, 6H), 0.95-0.77 (m, 6H). HRMS m/z: [M+H].sup.+ Calculated for C.sub.24H.sub.40N.sub.4NaO.sub.9S 583.2413; Found 583.2675.
[0186] Sodium (2S)-1-hydroxy-2-((S)-4-methyl-2-((((2-(2-phenylacetyl)-2-azaspiro[3.3]heptan-6-yl)oxy)carbonyl)amino)pentanamido)-3-((R)-2-oxopyrrolidin-3-yl)propane-1-sulfonate (3c). Yield (97%). .sup.1H NMR (400 MHz, DMSO-d6) ? 8.41-8.32 (m, 1H), 8.21 (dd, J=13.7, 7.3 Hz, 1H), 7.47 (d, J=3.9 Hz, 1H), 7.35-7.14 (m, 5H), 5.55 (dd, J=188.2, 6.3 Hz, 1H), 4.86-4.70 (m, 1H), 4.07-3.87 (m, 2H), 3.86-3.54 (m, 2H), 3.49-3.42 (m, 2H), 3.42-3.31 (m, 4H), 3.29-2.99 (m, 2H), 2.32-1.85 (m, 6H), 1.70-1.49 (m, 2H), 1.49-1.39 (m, 2H), 0.93-0.79 (m, 6H). HRMS m/z: [M+H].sup.+ Calculated for C.sub.28H.sub.40N.sub.4NaO.sub.9S 631.2853; Found 631.2413.
[0187] Sodium (2S)-1-hydroxy-2-((S)-4-methyl-2-((((2-(methylsulfonyl)-2-azaspiro[3.3]heptan-6-yl)oxy)carbonyl)amino)pentanamido)-3-((R)-2-oxopyrrolidin-3-yl)propane-1-sulfonate (4c). Yield (90%). .sup.1H NMR (400 MHz, DMSO-d6) ? 7.63 (s, 1H), 7.37 (d, J=8.0 Hz, 1H), 7.19-7.15 (m, 1H), 5.73-5.67 (m, 1H), 5.01-4.78 (m, 2H), 4.78-4.59 (m, 1H), 4.09-3.67 (m, 6H), 3.23-2.98 (m, 4H), 2.91 (s, 3H), 2.38-2.06 (m, 4H), 2.06-1.76 (m, 2H), 1.73-1.53 (m, 1H), 1.53-1.33 (m, 1H), 0.98-0.78 (m, 6H). HRMS m/z: [M+H].sup.+ Calculated for C.sub.21H.sub.36N.sub.4NaO.sub.10S.sub.2 591.1770; Found 591.1647.
[0188] Sodium (2S)-2-((S)-2-((((6-(tert-butoxycarbonyl)-6-azaspiro[3.4]octan-2-yl)oxy)carbonyl)amino)-4-methylpentanamido)-1-hydroxy-3-((R)-2-oxopyrrolidin-3-yl)propane-1-sulfonate (5c). Yield (22%). .sup.1H NMR (400 MHz, DMSO-d6) ? 7.63 (s, 1H), 7.53 (s, 1H), 7.24-7.20 (m, 1H), 5.73-5.68 (m, 1H), 4.86-4.77 (m, 1H), 4.07-3.77 (m, 2H), 3.67-3.38 (m, 4H), 3.28-2.95 (m, 6H), 2.37-2.20 (m, 2H), 2.20-2.05 (m, 1H), 2.05-1.88 (m, 2H), 1.88-1.74 (m, 3H), 1.74-1.45 (m, 2H), 1.39 (s, 9H), 0.92-0.81 (m, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.26H.sub.43N.sub.4Na.sub.2O.sub.10S 649.2496; Found 649.2458.
[0189] Sodium (2S)-2-((2S)-2-((((2-(tert-butoxycarbonyl)-2-azaspiro[3.4]octan-6-yl)oxy)carbonyl)amino)-4-methylpentanamido)-1-hydroxy-3-((R)-2-oxopyrrolidin-3-yl)propane-1-sulfonate (6c). Yield (7%). .sup.1H NMR (400 MHz, DMSO-d6) ? 7.63-7.38 (m, 2H), 7.30-7.03 (m, 1H), 5.30 (dt, J=54.1, 5.9 Hz, 1H), 5.00-4.81 (m, 1H), 4.66 (t, J=5.6 Hz, 1H), 4.02-3.86 (m, 2H), 3.83-3.48 (m, 4H), 3.37-2.97 (m, 3H), 2.29-1.96 (m, 3H), 1.96-1.67 (m, 5H), 1.67-1.48 (m, 4H), 1.37 (s, 9H), 0.94-0.77 (m, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.26H.sub.43N.sub.4Na.sub.2O.sub.10S 649.2496; Found 649.2454.
[0190] Sodium (2S)-2-((S)-2-((((7-(tert-butoxycarbonyl)-7-azaspiro[3.5]nonan-2-yl)oxy) carbonyl)amino)-4-methylpentanamido)-1-hydroxy-3-((R)-2-oxopyrrolidin-3-yl)propane-1-sulfonate (7c). Yield (87%). .sup.1H NMR (400 MHz, DMSO-d6) ? 7.57-7.50 (m, 1H), 7.45 (s, 1H), 7.28 (dd, J=35.4, 8.4 Hz, 1H), 5.33 (dd, J=56.7, 6.1 Hz, 1H), 4.88-4.77 (m, 2H), 4.42-4.10 (m, 1H), 4.07-3.76 (m, 4H), 3.27-3.00 (m, 6H), 2.36-1.85 (m, 4H), 1.85-1.66 (m, 1H), 1.65-1.50 (m, 1H), 1.43 (d, J=14.3 Hz, 6H), 1.38 (s, 9H), 0.89-0.79 (m, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.27H.sub.45N.sub.4Na.sub.2O.sub.10S 663.2652; Found 663.2690.
[0191] Sodium (2S)-1-hydroxy-2-((S)-2-((((7-isobutyryl-7-azaspiro[3.5]nonan-2-yl)oxy) carbonyl)amino)-4-methylpentanamido)-3-((R)-2-oxopyrrolidin-3-yl)propane-1-sulfonate (8c). Yield (80%). .sup.1H NMR (400 MHz, DMSO-d6) ? 7.63 (s, 1H), 7.46 (s, 1H), 7.36-7.28 (m, 1H), 5.42 (dd, J=64.4, 6.1 Hz, 1H), 4.87-4.81 (m, 1H), 4.52-4.12 (m, 2H), 4.09-3.80 (m, 2H), 3.22-2.97 (m, 4H), 2.88-2.79 (m, 2H), 2.37-2.18 (m, 3H), 2.18-1.96 (m, 1H), 1.96-1.68 (m, 3H), 1.68-1.32 (m, 8H), 1.00-0.94 (m, 6H), 0.92-0.80 (m, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.26H.sub.43N.sub.4Na.sub.2O.sub.9S 633.2546; Found 633.2526.
[0192] Sodium (2S)-1-hydroxy-2-((S)-4-methyl-2-((((7-(2-phenylacetyl)-7-azaspiro[3.5]nonan-2-yl)oxy)carbonyl)amino)pentanamido)-3-((R)-2-oxopyrrolidin-3-yl)propane-1-sulfonate (9c). Yield (68%). .sup.1H NMR (400 MHz, DMSO-d6) ? 7.60-7.49 (m, 2H), 7.45 (s, 1H), 7.35-7.11 (m, 5H), 5.38 (dd, J=60.0, 6.1 Hz, 1H), 4.86-4.73 (m, 2H), 4.44-4.12 (m, 1H), 4.06-3.77 (m, 4H), 3.71-3.61 (m, 4H), 3.22-2.99 (m, 2H), 2.35-2.03 (m, 4H), 2.03-1.79 (m, 1H), 1.78-1.65 (m, 1H), 1.63-1.49 (m, 1H), 1.48-1.27 (m, 7H), 0.91-0.79 (m, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.30H.sub.43N.sub.4Na.sub.2O.sub.9S 681.2546; Found 681.2522.
[0193] Sodium (2S)-1-hydroxy-2-((S)-4-methyl-2-((((7-(methylsulfonyl)-7-azaspiro[3.5]nonan-2-yl)oxy)carbonyl)amino)pentanamido)-3-((R)-2-oxopyrrolidin-3-yl)propane-1-sulfonate (10c). Yield (71%). .sup.1H NMR (400 MHz, DMSO-d6) ? 7.62 (d, J=9.3 Hz, 1H), 7.45 (s, 1H), 7.38-7.31 (m, 1H), 5.41 (dd, J=73.2, 6.1 Hz, 1H), 4.88-4.76 (m, 1H), 4.28-3.76 (m, 2H), 3.21-2.91 (m, 6H), 2.83 (s, 3H), 2.35-1.98 (m, 3H), 1.96-1.68 (m, 4H), 1.67-1.50 (m, 6H), 1.49-1.32 (m, 2H), 1.14-1.01 (m, 1H), 0.91-0.78 (m, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.23H.sub.39N.sub.4Na.sub.2O.sub.10S.sub.2 641.1903; Found 641.1874.
[0194] Sodium (2S)-2-((S)-2-((((7-cyano-7-azaspiro[3.5]nonan-2-yl)oxy)carbonyl)amino)-4-methylpentanamido)-1-hydroxy-3-((R)-2-oxopyrrolidin-3-yl)propane-1-sulfonate (11c). Yield (74%). .sup.1H NMR (400 MHz, DMSO-d6) ? 8.43 (d, J=7.6 Hz, 1H), 7.63 (s, 1H), 7.30 (d, J=8.0 Hz, 1H), 4.86-4.76 (m, 1H), 4.26-4.08 (m, 1H), 4.06-3.80 (m, 1H), 3.40-3.24 (m, 2H), 3.22-3.00 (m, 4H), 2.36-2.02 (m, 4H), 1.95-1.63 (m, 2H), 1.62-1.49 (m, 7H), 1.49-1.30 (m, 2H), 1.15-1.02 (m, 2H), 0.96-0.76 (m, 6H). HRMS m/z: [M+H].sup.+ Calculated for C.sub.23H.sub.37N.sub.5NaO.sub.8S 566.2260; Found 566.2238.
[0195] Sodium (2S)-2-((S)-2-((((1-(tert-butoxycarbonyl)azetidin-3-yl)oxy)carbonyl)amino)-4-methylpentanamido)-1-hydroxy-3-((R)-2-oxopyrrolidin-3-yl)propane-1-sulfonate (12c). Yield (64%). .sup.1H NMR (400 MHz, DMSO-d6) ? 7.66 (d, J=11.1 Hz, 2H), 7.58-7.42 (m, 1H), 5.01-4.90 (m, 2H), 4.71-4.64 (m, 1H), 4.23-3.84 (m, 3H), 3.84-3.51 (m, 2H), 3.19-3.04 (m, 2H), 2.34-2.01 (m, 2H), 2.00-1.73 (m, 1H), 1.71-1.43 (m, 5H), 1.38 (s, 9H), 0.92-0.81 (m, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.22H.sub.37N.sub.4Na.sub.2O.sub.10S 595.2026; Found 595.1995.
[0196] Sodium (2S)-2-((S)-2-((((1-(tert-butoxycarbonyl)-3-methylazetidin-3-yl)oxy)carbonyl) amino)-4-methylpentanamido)-1-hydroxy-3-((R)-2-oxopyrrolidin-3-yl)propane-1-sulfonate (13c). Yield (33%). .sup.1H NMR (400 MHz, DMSO-d6) ? 7.64 (d, J=10.0 Hz, 1H), 7.58-7.35 (m, 2H), 4.29-4.10 (m, 1H), 4.08-3.86 (m, 3H), 3.77-3.69 (m, 3H), 3.18-2.98 (m, 2H), 2.37-2.04 (m, 2H), 2.02-1.77 (m, 1H), 1.77-1.50 (m, 6H), 1.48-1.34 (m, 11H), 0.93-0.80 (m, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.23H.sub.39N.sub.4Na.sub.2O.sub.10S 609.2183; Found 609.2160.
[0197] Sodium (2S)-2-((S)-2-((((1-(tert-butoxycarbonyl)azetidin-3-yl)methoxy)carbonyl)amino)-4-methylpentanamido)-1-hydroxy-3-((R)-2-oxopyrrolidin-3-yl)propane-1-sulfonate (14c). Yield (57%). .sup.1H NMR (400 MHz, DMSO-d6) ? 7.59 (dd, J=9.2, 5.5 Hz, 1H), 7.43 (s, 1H), 7.36-7.23 (m, 1H), 5.34 (dd, J=69.8, 6.1 Hz, 1H), 4.14-4.01 (m, 2H), 4.01-3.76 (m, 3H), 3.62-3.47 (m, 2H), 3.20-2.98 (m, 3H), 2.87-2.67 (m, 1H), 2.24-2.06 (m, 3H), 2.04-1.80 (m, 1H), 1.72-1.48 (m, 3H), 1.46-1.39 (m, 1H), 1.37 (s, 9H), 0.92-0.80 (m, 6H). HRMS m/z: [M+Na].sup.+ Calculated for C.sub.23H.sub.39N.sub.4Na.sub.2O.sub.10S 609.2183; Found 609.2205.
[0198] Sodium (2S)-1-hydroxy-2-((S)-4-methyl-2-((((1-(2-phenylacetyl)azetidin-3-yl)methoxy) carbonyl)amino)pentanamido)-3-((R)-2-oxopyrrolidin-3-yl)propane-1-sulfonate (15c). Yield (92%). .sup.1H NMR (400 MHz, DMSO-d6) ? 8.16 (s, 1H), 7.64 (s, 1H), 7.52-7.44 (m, 1H), 7.34-7.14 (m, 5H), 4.22 (d, J=6.5 Hz, 2H), 4.14-3.79 (m, 4H), 3.72-3.54 (m, 2H), 3.50-3.38 (m, 2H), 3.23-3.00 (m, 4H), 2.38-1.95 (m, 3H), 1.93-1.72 (m, 1H), 1.72-1.53 (m, 2H), 1.53-1.30 (m, 2H), 0.92-0.80 (m, 6H). HRMS m/z: [M+H].sup.+ Calculated for C.sub.26H.sub.38N.sub.4NaO.sub.9S 605.2257; Found 605.2698.
[0199] Sodium (2S)-2-((2S)-2-((((1-(bicyclo[2.2.1]heptane-2-carbonyl)azetidin-3-yl)methoxy) carbonyl)amino)-4-methylpentanamido)-1-hydroxy-3-((R)-2-oxopyrrolidin-3-yl)propane-1-sulfonate (16c). Yield (71%). .sup.1H NMR (400 MHz, DMSO-d6) ? 7.78 (s, 1H), 7.64 (s, 1H), 7.38 (s, 1H), 4.18 (s, 1H), 4.13-3.79 (m, 3H), 3.72-3.54 (m, 2H), 3.26-2.96 (m, 4H), 2.70-2.55 (m, 1H), 2.36-2.02 (m, 4H), 2.00-1.76 (m, 1H), 1.75-1.32 (m, 9H), 1.29-1.19 (m, 4H), 1.19-1.00 (m, 2H), 0.93-0.77 (m, 6H). HRMS m/z: [M+H].sup.+ Calculated for C.sub.26H.sub.42N.sub.4NaO.sub.9S 609.2570; Found 609.3013.
[0200] Sodium (2S)-1-hydroxy-2-((S)-4-methyl-2-((((1-(methylsulfonyl)azetidin-3-yl)methoxy) carbonyl)amino)pentanamido)-3-((R)-2-oxopyrrolidin-3-yl)propane-1-sulfonate (17c). Yield (88%). .sup.1H NMR (400 MHz, DMSO-d6) ? 7.64 (s, 1H), 7.16 (s, 1H), 6.96 (s, 1H), 4.67 (s, 2H), 4.29-3.83 (m, 5H), 3.81-3.52 (m, 3H), 3.24-2.96 (m, 2H), 2.36-2.02 (m, 2H), 1.95-1.73 (m, 1H), 1.59 (s, 4H), 1.37 (s, 3H), 1.24 (s, 1H), 0.97-0.77 (m, 6H). HRMS m/z: [M+H].sup.+ Calculated for C.sub.19H.sub.34N.sub.4NaO.sub.10S.sub.2 565.1614; Found 565.1878.
Biochemical Studies
[0201] Enzyme assays and inhibition studies. Cloning and expression of the 3CL protease of SARS-CoV-2 and FRET enzyme assays. The codon-optimized cDNA of full length of 3CLpro of SARS-CoV-2 (GenBank number MN908947.3) fused with sequences encoding 6 histidine at the N-terminal was synthesized by Integrated DNA (Coralville, IA). The synthesized gene was subcloned into the pET-28a(+) vector. The expression and purification of SARS-CoV-2 3CLpro were conducted following a standard procedure. Briefly, a stock solution of an inhibitor was prepared in DMSO and diluted in assay buffer comprised of 20 mM HEPES buffer, pH 8, containing NaCl (200 mM), EDTA (0.4 mM), glycerol (60%), and 6 mM dithiothreitol (DTT). The SARS-CoV-2 protease was mixed with serial dilutions of inhibitors 1-17b/c or with DMSO in 25 ?L of assay buffer and incubated at 37? C. for 1 h, followed by the addition of 25 ?L of assay buffer containing substrate (FAM-SAVLQ/SG-QXL?520, AnaSpec, Fremont, CA). The substrate was derived from the cleavage sites on the viral polyproteins of SARS-CoV. Fluorescence readings were obtained using an excitation wavelength of 480 nm and an emission wavelength of 520 nm on a fluorescence microplate reader (FLx800; Biotec, Winoosk, VT) 1 h following the addition of substrate. Relative fluorescence units (RFU) were determined by subtracting background values (substrate-containing well without protease) from the raw fluorescence values. The dose-dependent FRET inhibition curves were fitted with a variable slope by using GraphPad Prism software (GraphPad, La Jolla, CA) in order to determine the IC.sub.50 values of the compounds.
[0202] Antiviral Assays/Cell-based inhibition assays. To assess antiviral effects of selected compounds (dissolved in DMSO) in cell culture, the SARS-CoV-2 replicon system with pSMART-T7-scv2-replicon (pSMART? BAC V2.0 Vector Containing the SARS-CoV-2, Wuhan-Hu-1 Non-Infectious Replicon) was used. The synthetic SARS-CoV-2 replicon RNA was prepared from the pSMART-T7-scv2-replicon, and the Neon Electroporation system (ThermoFisher, Chicago, IL) was used for the RNA electroporation to 293T cells. After the electroporation, cells were incubated with DMSO (0.1%) or each compound at 2, 0.5, 0.1 and 0.02 uM for 30 hr, and luciferase activities were measured for antiviral effects. The dose-dependent inhibition curve for each compound was prepared and the 50% effective concentration (EC.sub.50) values were determined by GraphPad Prism software using a variable slope (GraphPad, La Jolla, CA).
[0203] Nonspecific cytotoxic effects/Measurement of in vitro cytotoxicity. Confluent cells grown in 96-well plates were incubated with various concentrations (1 to 100 ?M) of each compound for 72 h. Cell cytotoxicity was measured by a CytoTox 96 nonradioactive cytotoxicity assay kit (Promega, Madison, WI), and the CC.sub.50 values were calculated using a variable slope by GraphPad Prism software. The in vitro Safety Index was calculated by dividing the CC.sub.50 by the EC.sub.50.
X-Ray Crystallographic Studies
[0204] Crystallization and Data Collection. Purified MERS-CoV 3CL.sup.pro and SARS-CoV-2 3CL.sup.pro in 100 mM NaCl, 20 mM Tris pH 8.0 were concentrated to 10 mg/mL (0.3 mM) for crystallization screening. Stock solutions of the inhibitors were prepared in DMSO at 100 mM and the complexes with the 3CL proteases were prepared by adding 2 mM of each compound and incubating the complexes on ice for 1 hour. All crystallization experiments were setup using an NT8 drop-setting robot (Formulatrix Inc.) and UVXPO MRC (Molecular Dimensions) sitting drop vapor diffusion plates at 18? C. 100 nL of protein and 100 nL crystallization solution were dispensed and equilibrated against 50 uL of the latter. Crystals of the MERS-CoV 3CL.sup.pro complexes were obtained from the following conditions. Index HT screen (Hampton Research) 9c: condition E7 (30% (w/v) PEG 550 MME, 100 mM Hepes pH 7.5, 50 mM magnesium chloride), 8c: condition F7 (20% (w/v) PEG 3350, 100 mM Bis-Tris pH 6.5, 200 mM ammonium sulfate) and 10c: condition F5 (17% (w/v) PEG 10000, 100 mM Bis-Tris pH 5.5, 100 mM ammonium acetate). Proplex HT screen (Molecular Dimensions) 14c: condition E2 (25% (w/v) PEG 3350, 100 mM Hepes pH 7.5, 200 mM magnesium chloride). Crystals of the SARS-CoV-2 3CL.sup.pro complexes were obtained from the following conditions. PACT screen (Molecular Dimensions) 2c: condition C2 (25% (w/v) PEG 1500, 100 mM PCTP pH 5.0), 3c: condition C1 (25% (w/v) PEG 1500, 100 mM PCTP pH 4.0), 11c: condition E1 (20% (w/v) PEG 3350, 20 mM sodium/potassium phosphate) and 10c: condition D4 (25% (w/v) PEG 1500, 100 MMT pH 7.0), Index HT screen (Hampton Research) 4c: condition F5 (17% (w/v) PEG 10000, 100 mM Bis-Tris pH 5.5, 100 mM ammonium acetate), 8c: condition F10 (25% (w/v) PEG 3350, 100 mM Bis-Tris pH 5.5, 200 mM NaCl), 14c: condition F11 (25% (w/v) PEG 3350, 100 mM Bis-Tris pH 6.5, 200 mM sodium chloride), 9c: condition G4 (20% (w/v) PEG 3350, 100 mM Hepes pH 7.5, 200 mM lithium sulfate) and Berkeley screen (Rigaku Reagents) 7c: condition B6 (20% (w/v) PEG 3350, 200 mM sodium fluoride). Cryoprotectants containing 80% crystallant and 20% (v/v) PEG 200 were layered onto the drop, samples were harvested and stored in liquid nitrogen. For MERS-CoV 3CL.sup.pro in complex with 9c, the crystallization solution served as the cryoprotectant. X-ray diffraction data were collected at the Advanced Photon Source beamline 17-ID (IMCA-CAT) and National Synchrotron Light Source-II, beamline 19-ID (NYX).
[0205] Structure Solution and Refinement. Intensities were integrated using XDS via Autoproc and the Laue class analysis and data scaling were performed with Aimless. Structure solution was conducted by molecular replacement with Phaser using a previously determined inhibitor bound structures of MERS-CoV (SWKK) and SARS-CoV-2 3CL.sup.pro (PDB 6XM1K) as the search models. Structure refinement and manual model building were conducted with Phenix and Coot, respectively. Disordered side chains were truncated to the point for which electron density could be observed. Structure validation was conducted with Molprobity and figures were prepared using the CCP4MG package. Crystallographic data are provided in Tables 13 and 14 below.
TABLE-US-00013 TABLE 13 Crystallographic data for MERS 3CLpro. Compound Code 8c 9c 10c 15c PDB Code 7T3Y 7T3Z 7T40 7T41 Data Collection Unit-cell parameters a = 75.23 a = 49.66 a = 101.61 a = 101.34 (?, ?) b = 91.33 b = 106.13, b = 58.06 b = 57.87 c = 99.17 c = 58.70 c = 49.90 c = 49.73 ? = 108.9 ? = 112.7 ? = 112.1 Space group P2.sub.12.sub.12.sub.1 P2.sub.1 C2 C2 Resolution (?).sup.1 45.66-1.90 46.99-1.95 46.87-1.70 46.96-2.10 (1.94-1.90) (2.00-1.95) (1.73-1.70) (2.16-2.10) Wavelength (?) 1.0000 1.0000 1.0000 0.9795 Temperature (K) 100 100 100 100 Observed reflections 369,090 268,646 100,588 50,969 Unique reflections 54,463 40,703 29,415 15,305 <I/?(I)>.sup.1 14.8 (1.4) 10.2 (2.4) 10.5 (1.7) 7.9 (2.0) Completeness (%).sup.1 99.9 (99.6) 97.7 (97.7) 99.5 (99.7) 97.6 (99.3) Multiplicity.sup.1 6.8 (7.0) 6.6 (6.8) 3.4 (3.1) 3.3 (3.4) R.sub.merge (%) .sup.1, 2 6.0 (175.0) 11.3 (80.9) 6.5 (67.3) 12.2 (72.0) R.sub.meas (%) .sup.1, 4 6.5 (188.7) 12.3 (87.4) 7.7 (81.9) 14.5 (85.5) R.sub.pim (%) .sup.1, 4 2.5 (70.2) 4.7 (32.8) 4.1 (46.1) 7.8 (45.5) CC.sub.1/2 .sup.1, 5 0.999 (0.723) 0.997 (0.776) 0.998 (0.749) 0.993 (0.599) Refinement Resolution (?) .sup.1 45.66-1.90 46.99-1.95 46.87-1.70 37.55-2.10 Reflections 51,603/2,736 38,631/2,039 27,953/1,448 14,602/698 (working/test).sup.1 R.sub.factor/R.sub.free (%).sup.1, 3 20.5/25.8 15.7/20.6 15.6/19.5 16.5/23.7 No. of atoms 4,484/144/189 4,581/120/342 2,276/70/232 2,233/68/78 (Protein/Ligand/Water) Model Quality R.m.s deviations Bond lengths (?) 0.012 0.011 0.011 0.011 Bond angles (?) 1.121 1.071 0.995 1.115 Average B-factor (?.sup.2) All Atoms 51.2 30.5 25.2 33.6 Protein 51.1 30.0 24.4 33.3 Ligand 58.9 32.7 22.2 40.1 Water 48.7 35.9 33.2 34.6 Coordinate 0.29 0.21 0.18 0.26 error(maximum likelihood) (?) Ramachandran Plot Most favored (%) 98.2 98.5 99.3 97.7 Additionally allowed 1.8 1.5 0.7 1.7 (%) Values in parenthesis are for the highest resolution shell. R.sub.merge = ?.sub.hkl?.sub.i |I.sub.i(hkl) ? <I(hkl)>|/?.sub.hkl?.sub.i I.sub.i(hkl), where I.sub.i(hkl) is the intensity measured for the ith reflection and <I(hkl)> is the average intensity of all reflections with indices hkl. R.sub.factor = ?.sub.hkl ||F.sub.obs (hkl) | ? |F.sub.calc (hkl) ||/?.sub.hkl |F.sub.obs (hkl)|; R.sub.Free is calculated in an identical manner using 5% of randomly selected reflections that were not included in the refinement. R.sub.meas = redundancy-independent (multiplicity-weighted) R.sub.merge. R.sub.pim = precision?indicating (multiplicity-weighted) R.sub.merge. CC.sub.1/2 is the correlation coefficient of the mean intensities between two random half-sets of data.
TABLE-US-00014 TABLE 14 Crystallographic data for SARS-CoV-2 3CLpro. Compound Code 2c 3c 4c 7c PDB Code 7T42 7T43 7T44 7T45 Data Collection Unit-cell parameters a = 55.10 a = 55.29 a = 55.55 a = 55.30 (?, ?) b = 98.57 b = 98.84 b = 98.75 b = 98.72 c = 58.69 c = 58.86 c = 59.32 c = 58.90 ? = 107.7 ? = 107.9 ? = 108.3 ? = 107.9 Space group P2.sub.1 P2.sub.1 P2.sub.1 P2.sub.1 Resolution (?).sup.1 49.28-1.60 48.74-1.70 49.38-1.45 49.36-1.65 (1.63-1.60) (1.73-1.70) (1.47-1.45) (1.68-1.65) Wavelength (?) 0.9795 0.9795 0.9795 1.0000 Temperature (K) 100 100 100 100 Observed reflections 532,226 213,050 364,972 246,835 Unique reflections 78,397 64,486 105,771 72,006 <I/?(I)>.sup.1 9.7 (1.7) 9.9 (2.0) 10.0 (1.9) 11.1 (1.4) Completeness (%).sup.1 99.7 (99.6) 97.6 (98.7) 98.7 (96.4) 99.6 (97.8) Multiplicity.sup.1 6.8 (6.9) 3.3 (3.3) 3.5 (3.1) 3.4 (2.7) R.sub.merge (%) .sup.1, 2 8.8 (111.0) 4.9 (53.5) 6.5 (58.6) 5.3 (69.6) R.sub.meas (%) .sup.1, 4 9.5 (119.8) 5.8 (63.8) 7.7 (70.5) 6.2 (87.1) R.sub.pim (%) .sup.1, 4 3.6 (44.8) 3.2 (34.4) 4.1 (38.8) 3.3 (51.6) CC.sub.1/2 .sup.1, 5 0.998 (0.796) 0.999 (0.831) 0.995 (0.746) 0.998 (0.592) Refinement Resolution (?) .sup.1 36.97-1.60 48.74-1.70 37.12-1.45 36.02-1.65 Reflections 74,621/3,681 61,337/3,104 100,574/5,153 68,413/3,534 (working/test).sup.1 R.sub.factor/R.sub.free (%).sup.1, 3 18.0/21.2 17.7/21.3 19.5/22.6 18.5/22.1 No. of atoms 4,563/122/257 4,585/117/217 4,598/116/358 4,472/64/334 (Protein/Ligand/Water) Model Quality R.m.s deviations Bond lengths (?) 0.008 0.007 0.004 0.011 Bond angles (?) 0.939 0.878 0.727 1.050 Average B-factor (?.sup.2) All Atoms 29.8 30.6 24.9 32.8 Protein 29.5 30.4 24.5 32.2 Ligand 28.8 30.4 25.1 37.4 Water 34.9 34.6 31.0 37.6 Coordinate 0.18 0.17 0.17 0.22 error(maximum likelihood) (?) Ramachandran Plot Most favored (%) 98.2 98.5 98.5 96.5 Additionally allowed 1.8 1.5 1.5 3.5 (%) Compound Code 9c 10c 11c 15c PDB Code 7T48 7T49 7T4A 7T4B Data Collection Unit-cell parameters a = 113.66 a = 55.31 a = 55.56 a = 55.42 (?, ?) b = 52.59 b = 98.33 b = 98.81 b = 99.03 c = 46.31 c = 58.79 c = 58.81 c = 59.11 ? = 102.9 b = 108.1 ? = 107.7 ? = 108.2 Space group C2 P2.sub.1 P2.sub.1 P2.sub.1 Resolution (?).sup.1 47.51-1.90 49.16-1.75 48.73-1.80 48.85-1.60 (1.94-1.90) (1.78-1.75) (1.84-1.80) (1.63-1.60) Wavelength (?) 1.0000 1.0000 1.0000 0.9795 Temperature (K) 100 100 100 100 Observed reflections 70,653 201,886 192,466 269,373 Unique reflections 20,929 59,451 55,643 79,076 <I/?(I)>.sup.1 8.4 (2.0) 10.1 (1.8) 12.7 (1.7) 11.5 (1.7) Completeness (%).sup.1 99.0 (96.9) 98.9 (99.3) 99.4 (99.9) 99.2 (99.9) Multiplicity.sup.1 3.4 (3.3) 3.4 (3.4) 3.5 (3.3) 3.4 (3.5) R.sub.merge (%) .sup.1, 2 9.6 (88.1) 5.9 (66.9) 5.0 (75.2) 5.8 (82.0) R.sub.meas (%) .sup.1, 4 11.4 (106.2) 7.0 (79.2) 5.9 (87.5) 6.9 (97.1) R.sub.pim (%) .sup.1, 4 6.1 (58.3) 3.7 (42.2) 3.1 (49.0) 3.7 (51.4) CC.sub.1/2 .sup.1, 5 0.993 (0.682) 0.998 (0.808) 0.999 (0.677) 0.996 (0.650) Refinement Resolution (?) .sup.1 39.60-1.90 33.44-1.75 31.89-1.80 48.85-1.60 Reflections 19,843/1,011 56,581/2,787 52,872/2,725 75,315/3,715 (working/test).sup.1 R.sub.factor/R.sub.free (%).sup.1, 3 18.2/24.2 19.0/24.1 17.4/22.5 18.5/21.6 No. of atoms 2,261/80/150 4,475/122/248 4,396/118/306 4,499/122/369 (Protein/Ligand/Water) Model Quality R.m.s deviations Bond lengths (?) 0.012 0.010 0.011 0.009 Bond angles (?) 1.121 1.068 1.153 1.049 Average B-factor (?.sup.2) All Atoms 27.2 37.8 36.4 28.6 Protein 26.6 37.5 35.9 27.9 Ligand 32.5 41.6 40.1 31.6 Water 32.9 41.5 41.1 35.4 Coordinate 0.25 0.20 0.21 0.21 error(maximum likelihood) (?) Ramachandran Plot Most favored (%) 96.6 97.3 97.8 98.0 Additionally allowed 3.0 2.5 2.2 2.0 (%) Values in parenthesis are for the highest resolution shell. R.sub.merge = ?.sub.hkl?.sub.i |I.sub.i(hkl) ? <I(hkl)>|/?.sub.hkl?.sub.i I.sub.i(hkl), where I.sub.i(hkl) is the intensity measured for the ith reflection and <I(hkl)> is the average intensity of all reflections with indices hkl. R.sub.factor = E.sub.hkl ||F.sub.obs (hkl) | ? |F.sub.calc (hkl) ||/E.sub.hkl |F.sub.obs (hkl)|; R.sub.Free is calculated in an identical manner using 5% of randomly selected reflections that were not included in the refinement. R.sub.meas = redundancy-independent (multiplicity-weighted) R.sub.merge. R.sub.pim = precision-indicating (multiplicity-weighted) R.sub.merge. CC.sub.1/2 is the correlation coefficient of the mean intensities between two random half-sets of data.
Accession Codes
[0206] Coordinates and structure factors for complexes with the following with inhibitors were deposited to the Worldwide Protein Databank (wwPDB) with the accession codes: MERS-CoV 3CLpro complexes: 8c (7T3Y), 9c (7T3Z), 10c (7T40), 14c (7T41) and SARS-CoV-2 3CL.sup.pro complexes: 2c (7T42), 3c (7T43), 4c (7T44), 7c (7T45), 8c (7T46), 9c (7T48), 10c (7T49), 11c (7T4A), 14c (7T4B). Atomic coordinates are available upon publication.
Example 4
Additional Compounds
[0207] Additional compounds have been synthesized and screened against SARS-CoV-2 and MERS-CoV 3C-like protease. Briefly, to a solution of benzyl chloromethyl ether (31.6 mmol; 2 eq) in anhydrous DMF (40 mL) kept at ?40? C. was added dropwise a 2M solution of sodium cyclopentadienide in THE (16 mmol; 1 eq) with vigorous stirring for 20 minutes. The reaction mixture was poured into a cold (4? C.) mixture of pentane (120 mL) and water (60 mL). After extraction with cold pentane (2?60 mL) the combined pentane extracts were washed with icy water, dried over anhydrous sodium sulfate and filtered. The pentane was removed by distillation under reduced pressure at 0? C. to yield a cold yellow oily residue of 5-benzyloxymethyl cyclopentadiene which was used in the next step. Using essentially the same procedure as that described in Katsuaki M et al, EPA 0249953A1, Dec. 23, 1987), the acrylic acid ester (2.4 g; 13 mmol) was dissolved in a mixture of DCM/petroleum ether (24 mL of a 7/1 v/v mixture) and the solution was cooled to ?15? C. A 1M solution of titanium tetrachloride in petroleum ether (6.5 mL) was added dropwise and stirred for 30 minutes at 15? C. The reaction mixture was cooled to ?60? C. and 5-benzyloxymethyl cyclopentadiene (31.9 mmol) was added and the mixture was stirred at ?60? C. for 10 h. The temperature was raised to 0? C. and then sodium carbonate decahydrate (13.4 g) was added and the mixture was stirred at room temperature for 0.5 h. The precipitate was filtered off and the filtrate was concentrated. The crude residue was purified by flash chromatography (silica gel/hexane/ethyl ether). The ester was hydrolyzed to the acid by stirring with lithium hydroxide in aqueous THE and the acid was treated with carbonyl diimidazole followed by NaBH.sub.4 or NaBD.sub.4 as described for other synthesis protocols to yield the nondeuterated and deuterated alcohols which were elaborated further to yield the corresponding aldehydes ADR-VI-01 and ADR-VI-02 which were screened against SARS-CoV-2 and MERS-CoV as described in the Examples above. The IC.sub.50 values are given in the table below.
##STR00087##
TABLE-US-00015 IC.sub.50 uM SARS-CoV-2 MERS-CoV 3CLpro 3CLpro ADR-VI-01 (R.sub.i = R.sub.j = H) 0.22 0.10 ADR-VI-02 (R.sub.i = R.sub.j = D) 0.45 0.23