NL2033609B1 - DNA Gyrase Inhibitors - Google Patents

DNA Gyrase Inhibitors Download PDF

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NL2033609B1
NL2033609B1 NL2033609A NL2033609A NL2033609B1 NL 2033609 B1 NL2033609 B1 NL 2033609B1 NL 2033609 A NL2033609 A NL 2033609A NL 2033609 A NL2033609 A NL 2033609A NL 2033609 B1 NL2033609 B1 NL 2033609B1
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compound
mmol
mhz
nmr
alkyl
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NL2033609A
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J H B N Van Den Berg Richard
Kotsogianni Ioli
M Punt Jeroen
A A Van Boeckel Constant
Liu Bing
P Van Wezel Gilles
T Bakker Alexander
Ghilarov Dmitry
Van Der Stelt Mario
Piermarini Diana
I Martin Nathaniel
Avalos Mariana
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Innes John Centre
Univ Leiden
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Priority to NL2033609A priority Critical patent/NL2033609B1/en
Priority to PCT/NL2023/050618 priority patent/WO2024112201A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
    • A61K31/4725—Non-condensed isoquinolines, e.g. papaverine containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D215/36—Sulfur atoms
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links

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Abstract

Provided are compounds useful fortreating bacterial infections, e.g. Gram-negative bacterial infection. The compounds may be of formula I as defined herein: R4 R1 R2 O | RBa 0%ll/N\)\ ( % R7 S N H X1 \ R5 || X2 / \X3 ( R6 )n . 5 The compounds may be capable of binding a horseshoe-like hydrophobic pocket on the DNA-binding surface GyrA subunit of DNA gyrase. Also provided are formulations comprising such compounds, as well uses of such compounds or formulations, e.g. forthe treatment of bacterial infection, and/or as a DNA gyrase inhibitor.

Description

P345823NL 1
DNA Gyrase Inhibitors
[0001] This invention relates to compounds useful for treating bacterial infections. The compounds may be capable of binding a horseshoe-like hydrophobic pocket on the DNA-binding surface GyrA subunit of
DNA gyrase. Also provided are formulations comprising such compounds, as well uses of such compounds or formulations, e.g. for the treatment of bacterial infection, and/or as a DNA gyrase inhibitor.
BACKGROUND
[0002] The global threat posed by antimicrobial resistance (AMR) and the associated death toll continues to rise every year. In 2019, an estimated 1.27 million deaths worldwide were directly attributable to AMR, 20% of which were linked to resistant Escherichia coli.’ Multidrug-resistant (MDR) Gram-negative pathogens, such as E. coli and Klebsiella pneumoniae strains displaying resistance to third generation cephalosporins, carbapenems, and fluoroquinolones, are now widespread? and pose a daunting challenge to healthcare systems. At the same time, resistance to antibiotics of last resort® has also been detected in several countries. The emergence of mobilized colistin resistance-1 (mcr-1) in 20154 and tigecycline resistance (tetX3-tetX5) genes in 20195 threatens to render Gram-negative MDR infections untreatable. Clearly, new antibiotics with a novel mode-of-action (MoA) are urgently needed to keep pace with drug-resistant Gram-negative bacteria.
[0003] Target-based screening is an effective approach to identify novel small molecules as hits in a drug discovery program, but it is less successful in the search for novel Gram-negative-specific antibiotics.87
Lack of target validation and the additional outer membrane (OM) of Gram-negative bacteria constitute major bottlenecks for target-based drug discovery. Rational modification of existing drugs?® and phenotypic screening have instead emerged as promising strategies to overcome these challenges.10.1í A major advantage of phenotypic screening is the opportunity to discover compounds with an unprecedented MoA, and historically the mining of libraries of natural product extracts presented a fruitful strategy to search for new target-drug combinations.12-13 Today, however, the risk of re-discovery along with the challenges associated with the structure elucidation and chemical synthesis of complex natural products has led to a slowdown in antibiotic discovery from such sources. As an alternative, phenotypic screening using libraries of small synthetic compounds ensures both synthetic accessibility and structural novelty.
[0004] For decades, the bacterial DNA gyrase has proven to be an excellent target for antibiotics with a number of clinically used anti-infectives operating by inhibiting its activity.?® Given that DNA gyrase lacks a direct human homolog, has multiple target sites, and is essential to bacterial DNA function, inhibitors have the potential to selectively target bacterial cells vs host cells. Notably, the DNA negative supercoiling activity of DNA gyrase is a multi-step process that can be interrupted at multiple stages through pharmacological intervention. Key examples are the classical fluoroquinolone antibiotics®! and the more recent NBTIs22 that intercalate in DNA, the aminocoumarins® that compete with ATP, and SD8% which prevents DNA binding with the gyrase complex. Despite successful clinical application, resistance to fluoroquinolones is increasingly abundant35:38 and the mammalian toxicity of fluoroquinolones?7, NBTIs38 and aminocoumarins present serious clinical concerns. In this light there is a clear need and opportunity for the development of improved next-generation DNA gyrase inhibitors.
P345823NL 2
[0005] An aim of certain embodiments of the present invention is to provide compounds with antibiotic activity against clinically relevant bacteria, such as Gram-negative bacteria, e.g. E. coli and K. pneumoniae.
[0006] An aim of certain embodiments of the present invention is to provide compounds that inhibit bacterial DNA gyrase.
[0007] An aim of certain embodiments of the present invention is to provide compounds that have activity against bacterial strains that have developed a resistance to prior art compounds, e.g. fluoroquinolones.
BRIEF SUMMARY OF THE DISCLOSURE
[0008] According to a first aspect of the invention there is provided a compound of formula I:
R+ R' R? 0 3a . ed
H
A A RS x2 ANE x (Ry (0) wherein: a single one of Xt, X2 and X3 is N and the remaining two of Xt, X2 and X3 are CRY;
R! is selected from H, halo, C+4-C4 alkyl and C1-C4 haloalkyl;
R2 is selected from H, halo, C+4-C4 alkyl and C1-C4 haloalkyl; or Rt and R2 together form a 1, 2 or 3 membered alkylene chain, optionally substituted, where chemically possible, with one, two or three groups independently selected from halo, C+-C4 alkyl, C+-Ca haloalkyl, CN; each R® and R* is independently selected from halo, C1-C4 alkyl, C1-C4 haloalkyl, CN, OR? and
NRPRP, wherein Rais selected from H, C1-C4 alkyl and C+-C4 haloalkyl and each RP is independently selected from H and C1-C4 alkyl;
R+ is selected from H, C+-C4 alkyl, C1-C4 haloalkyl,
RS is selected from phenyl and a 5- or 8-membered heteroaryl group, optionally substituted with 1 or 2 R* groups; each R® is independently selected from halo, C+-C4 alkyl, C1-C4 haloalkyl, CN, -0-C+-C4 alkyl and -O-C1-C4 haloalkyl; each R7 is independently selected from H, halo, C1-C4 alkyl, C+-C4 haloalkyl, CN, -O-C1-Ca alkyl and -O-C+1-C4 haloalkyl, nis 0,1, 2or3; and pis0,1,20r3; or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof.
[0009] In an embodiment, the compound of formula | is a compound of formula II:
P345823NL 3
R* R' R2 of (R33),
Ì k (R®) q
CO
NLs EX oh, wherein R1, R2, R% R3 R+ and p are as defined above for compounds of formula I; and q is 0, 1 or 2; X* is N, CH, or CR%; X5 is N, CH, or CR"; R1 is selected from halo, C-C4 alkyl, C4-C4 haloalkyl and CN; and R" is selected from halo, C1-C4 alkyl, C1-C4 haloalkyl and CN, with the proviso that when X is N, X5 5 isCHor CR", and when X5 is N, X* is CH or CR, or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof.
[0010] In an embodiment, the compound of formula | is a compound of formula Ill:
R4 R1 R2 o 7 wee Sei
Ne” N
H
CO ’
NA an, wherein Rt, R2, R22, R+, R5 and p are as defined above for compounds of formula |, or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof.
[0011] In an embodiment, the compound of formula | is a compound of formula IV:
R+ R' R2
O 3a
R7 on, í SC )p 3b
NN = pa )q x AA | AX x (Rn ow, wherein X', X2, X3, R1, R2, R%, R3b, R+, RS, R7, n and p are as defined above for compounds of formula I; and q is 0, 1 or 2; X4is N, CH, or CR; X5 is N, CH, or CR"; R" is selected from halo, C1-C4 alkyl, C1-C4 haloalkyl and CN; and R" is selected from halo, C+-C4 alkyl, C1-C4 haloalkyl and CN, with the proviso that when X* is N, X5 is CH or CR", and when X5 is N, X4 is CH or CR1°, or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof.
[0012] In an embodiment, the compound of formula | is a compound of formula V:
P345823NL 4 o FR (R°2) r7 OSA ° )m x IAN R® RO RS fl 2 id . (R°)n (v), wherein X', X2, X3, R% R+, RS RS, R7, n and p are as defined above for compounds of formula I; and each R3 and R? is independently selected from H, halo, C+-C4 alkyl, C+-C4 haloalkyl and CN; and m is 0, 1 or 2, or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof.
[0013] In an embodiment, the compound of formula | is a compound of formula VI: of 3a
ON (R™)p )m 3b (R“*)
AN RS R9 a
NLs | X53 xX¥ wn, wherein R?, R3b, R+ and p are as defined above for compounds of formula I; and q is 0, 1 or 2; X+ is N,
CH, or CR'%; X5 is N, CH, or CR1t; R19 is selected from halo, C1-C4 alkyl, C1-C4 haloalkyl and CN; R'is selected from halo, C1-C4 alkyl, C1-C4 haloalkyl and CN, with the proviso that when X is N, X5 is CH or
CR, and when XS is N, X4 is CH or CR12; each R® and RS is independently selected from H, halo, C+4-C4 alkyl, C+-C4 haloalkyl and CN; and mis 0, 1 or 2, or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof.
[0014] In an embodiment, the compound of formula (1) is a compound of formula VII: of RK
R32
ON (Rp
S
Im
R® 9
Xx R R5
N
A (VIN, wherein R22, R+, R5 and p are as defined above for compounds of formula |; and each R® and R? is independently selected from H, halo, C+-Ca alkyl, C+-C4 haloalkyl and CN; and mis 0, 1 or 2, ora pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof.
[0015] In an embodiment, the compound of formula | is a compound of formula VIII:
P345823NL oF KR
Im 3b ee OR Ra fl xd” 6 | xXx (R°)n (VID, wherein X', X2, X3, R32, R3 R4 RS, R7, n and p are as defined above for compounds of formula |; q is 0, 1 or 2; X+ is N, CH, or CR; X5 is N, CH, or CR; R10 is selected from halo, C+-Ca4 alkyl, C4+-C4 haloalkyl and
CN; and R" is selected from halo, C+-C4 alkyl, C+-C4 haloalkyl and CN, with the proviso that when X is N, 5 X5is CH or CRY, and when X5 is N, X4 is CH or CR'?; each R8 and R? is independently selected from H, halo, C+-C4 alkyl, C4-C4 haloalkyl and CN; and m is 0, 1 or 2, or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof.
[0016] In an embodiment, the compound of formula | is a compound of formula IX: o FR
R7 Og A
Im
De R® R® R5
XA . (Rn (1X), wherein Xt, X2, X3, R33, R+, R5, RS, R7, n and p are as defined above for compounds of formula |; and each R® and R? is independently selected from H, halo, C+-Ca alkyl, C+-C4 haloalkyl and CN; and m is 0, 1 or 2, or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof.
[0017] In an embodiment, the compound of formula | is a compound of formula X:
TX
- 6 Vs (R®), [ID we xt, wherein R32, R3b, R+ and p are as defined above for compounds of formula |; q is 0, 1 or 2; X*is N, CH, or
CR; XS js N, CH, or CR"; Rf? is selected from halo, C1-C4 alkyl, C4-C4 haloalkyl and CN; and R'! is selected from halo, C1-Ca alkyl, C1-C4 haloalkyl and CN, with the proviso that when X* is N, X5 is CH or
CRY, and when X5 is N, X* is CH or CR'?; each R® and RS is independently selected from H, halo, C+-C4 alkyl, C1-C4 haloalkyl and CN; and m is 0, 1 or 2, or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof.
[0018] In an embodiment, the compound of formula | is a compound of formula XI:
P345823NL 6 oN (R°2)
Ond N —_ p dm 8 9 x R R RS
N
A XD, wherein R33, R+, R5 and p are as defined above for compounds of formula I; each R® and R® is independently selected from H, halo, C+-Ca alkyl, C1-C4 haloalkyl and CN; and mis 0, 1 or 2, or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof.
[0019] In an embodiment, the compound of formula | is a compound of formula XII: oF AK 3a
ON (Rp
ROS
Im 3b (R*) ll
X2 =z _X5
NE RS x4 (R°)n (XIN, wherein X1, X2, X3, R32, RP, R+ RS, R7, n and p are as defined above for compounds of formula I; q is 0, 1 or 2; X% is N, CH, or CR19; X5 is N, CH, or CR"; R1? is selected from halo, C1-C4 alkyl, C+-C4 haloalkyl and
CN; and R!! is selected from halo, C1-C4 alkyl, C+-C4 haloalkyl and CN, with the proviso that when X* is N,
X%is CH or CR", and when X5 is N, X4 is CH or CR; each R® and R¢ is independently selected from H, halo, C4-C4 alkyl, C+-C4 haloalkyl and CN; and mis C, 1 or 2, or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof.
[0020] In an embodiment, the compound of formula | is a compound of formula XIII: o FR (R°2) rr OSV ° )m
NU R® R® RS fl
X2 =z
DE R6 (R°)n (KID), wherein Xt, X2, X3, R22, R+, R5, RS, R7, n and p are as defined above for compounds of formula |; and each R3 and RS is independently selected from H, halo, C+-C4 alkyl, C+-C4 haloalkyl and CN; and m is 0, 1 or 2, or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof.
[0021] In an embodiment, the compound of formula | is a compound of formula XIV:
P345823NL 7 of 3a
ON (R°p )m 3b (R%)
AN RS RO a
NLs | 2X5
XP (xiv, wherein R32, R22, R+ and p are as defined above for compounds of formula |; q is 0, 1 or 2; X+ is N, CH, or
CR; X5 is N, CH, or CR!t; R10 is selected from halo, C1-Cas alkyl, C+-C4 haloalkyl and CN; and R11 is selected from halo, C+-C4 alkyl, C1-C4 haloalkyl and CN, with the proviso that when X4 is N, X5 is CH or
CRY, and when X5 is N, X4 is CH or CR; each R3 and RS? is independently selected from H, halo, C+-C4 alkyl, C1-C4 haloalkyl and CN; and m ís 0, 1 or 2, or a pharmaceutically acceptable salt, sterecisomer, solvate, or prodrug thereof.
[0022] In an embodiment, the compound of formula | is a compound of formula XVI of RK
R32
Ox JN ( )p
S
Im
R® 9
Dy R R5
N
A xv), wherein R% R+, R5 and p are as defined above for compounds of formula I; each R® and RS is independently selected from H, halo, C1-C4 alkyl, C1-Ca4 haloalkyl and CN; and mis 0, 1 or 2, or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof.
[0023] In an embodiment, the compound of formula | is a compound of formula XVI: of UK (R%)
R7 OLN P
Im 3b (R*) ll
X2 = _X5 “x3 RS x4 (R°)n (XVI), wherein X1, X2, X3, R% R3 R+ RS, R7, n and p are as defined above for compounds of formula |; q is 0, 1 or 2; X*is N, CH, or CR10; X5 is N, CH, or CR"; R'0 is selected from halo, C1-C4 alkyl, C+-C4 haloalkyl and
CN; and Rt! is selected from halo, C1-Cs alkyl, C1-C4 haloalkyl and CN, with the proviso that when X is N,
X5 is CH or CR", and when X® is N, X* is CH or CR'®; each R3 and RS is independently selected from H, halo, C1-C4 alkyl, C+-C4 haloalkyl and CN; and m is 0, 1 or 2, or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof.
[0024] The following embodiments apply to compounds of any of formulae (I)-(XVI). These embodiments are independent and interchangeable. Any one embodiment may be combined with any other embodiment, where chemically allowed. In other words, any of the features described in the following
P345823NL 8 embodiments may (where chemically allowable) be combined with the features described in one or more other embodiments. In particular, where a compound is exemplified or illustrated in this specification, any two or more of the embodiments listed below, expressed at any level of generality, which encompass that compound may be combined to provide a further embodiment which forms part of the present disclosure.
[0025] It may be that X' is N. It may be that X3 is N. Preferably, X2 is N. It may be that a single one of
X1, X2 and XG is N and the remaining two of X1, X2 and X3 are CH. It may be that X2 is N and X! and X2 are both CH.
[0026] Each R7 may be independently selected from H, halo, C+-C2 alkyl, C1-C2 haloalkyl, CN, -O-C+-C2 alkyl and -O-C1-C2 haloalkyl. Each R7 may be independently selected from H, halo, CHa, C1 haloalkyl,
CN, -OCH: and -O-C: haloalkyl. Each R7 may be independently selected from halo or H. Each R7 may be independently selected from halo. Preferably, each R7 is H. It may be that X2 is N and X* and X? are independently selected from CH and CR?2, wherein each R?2 is independently selected from F and CF:.
[0027] It may be that nis O or 1. Preferably, n is 0.
[0028] Each R® may be independently selected from halo, C4-C2 alkyl, C1-C2 haloalkyl, CN, -O-C1-C: alkyl and -O-C1-C: haloalkyl. Each R® may be independently selected from H, halo, CHs, C+ haloalkyl, CN, -
OCH: and -O-C+ haloalkyl. It may be that each R® is independently selected from halo or H, e.g. each R® may be independently selected from halo. It may be that nis 1 and RS is halo, e.g. F.
[0029] It may be that R! is selected from H, halo, C+4-C2 alkyl and C+-Cz haloalkyl. R' may be selected from H, halo, CHs and C1 haloalkyl. It may be that Rt is selected from H and halo. Rt may be H. It may be that R2 is selected from H, halo, C+1-C2 alkyl and C+-C2 haloalkyl. R2 may be selected from H, halo,
CH: and C+ haloalkyl. It may be that R2 is selected from H and halo. R2 may be H. R* and R2 may be H.
[06030] Alternatively, Rt and R2 together may form a 1, 2 or 3 membered alkylene chain, optionally substituted, where chemically possible, with one, two or three groups independently selected from halo,
C1-C4 alkyl, C1-C4 haloalkyl and CN. R' and R2 together may form a 1, 2 or 3 membered alkylene chain, optionally substituted, where chemically possible, with one, two or three groups independently selected from halo, C1-C2 alkyl, C+-C2 haloalkyl and CN. Rt and R2 together may form a 1, 2 or 3 membered alkylene chain, optionally substituted, where chemically possible, with one, two or three groups independently selected from halo, CHs, CF: and CN. It may be that R1 and R2 together form an unsubstituted 1, 2 or 3 membered alkylene chain.
[0031] R! and R2 together may form a 2 or 3 membered alkylene chain, optionally substituted with one, two or three groups independently selected from halo, C+-C4 alkyl, C+-C4 haloalkyl and CN. R' and R2 together may form a 2 or 3 membered alkylene chain, optionally substituted with one, two or three groups independently selected from halo, C+4-C2 alkyl, C1-C2 haloalkyl and CN. Rt and R? together may form a 2 or 3 membered alkylene chain, optionally substituted with one, two or three groups independently selected from halo, CHs, CFs and CN. It may be that Rt and R2 together form an unsubstituted 2 or 3 membered alkylene chain.
[0032] R' and R2 together may form a 2 membered alkylene chain, optionally substituted, with one, two or three groups independently selected from halo, C4-C4 alkyl, C1-C4 haloalkyl and CN. Rt and R2 together may form a 2 membered alkylene chain, optionally substituted, with one, two or three groups
P345823NL 9 independently selected from halo, C+-C2 alkyl, C+-C2 haloalkyl and CN. Rt and R? together may form a 2 membered alkylene chain, optionally substituted, with one, two or three groups independently selected from halo, CHs, CF: and CN. Preferably, R' and R? together may form an unsubstituted 2 membered alkylene chain.
[0033] It may be thatpis 0, 1 or 2. Itmay be that p is Oor 1. p may be 1. p may be 0.
[0034] It may be that each R32 is independently selected from halo, C4-C2 alkyl, C1-C2 haloalkyl, CN, ORa and NRPRP, wherein Rais selected from H, C+-C2 alkyl and C+-C: haloalkyl and each RP is independently selected from H and C+-C2 alkyl.
[0035] It may be that each R32 is independently selected from halo, CHs, CF3, OH, CN and NH2. Each
R% may be independently selected from halo, e.g. F. It may be that p is 1 and R32is halo, e.g. F.
[0036] When p is 2, it may be that an R32 is substituted at a carbon atom adjacent to the carbon atom to which RS is connected. When p is 1 it may be that R32 is substituted at the carbon atom adjacent to the carbon atom to which R5 is connected.
[0037] R+ may be selected from H, C1-C2 alkyl, C+-Cz haloalkyl. R+ may be selected from H, CH: and
CFs. Preferably, R4 is H.
[0038] R5 may be phenyl. R5 may be unsubstituted phenyl. R5 may be phenyl substituted with 1 or 2 R% groups. R5 may be phenyl substituted with 1 R3P group.
[0039] R5 may be a 5- or 6-membered heteroaryl group. R5 may be an unsubstituted 5- or 6-membered heteroaryl group. R5 may be a 5- or 6-membered heteroaryl group, optionally substituted with 1 or 2 R2 groups. R5 may be a 5- or 6-membered heteroaryl group, optionally substituted with 1 R32 group.
[0040] R5 may be a 6-membered heteroaryl group, e.g. pyridinyl. R5 may be an unsubstituted 6- membered heteroaryl group, e.g. pyridinyl. R5 may be a 6-membered heteroaryl group, e.g. pyridinyl, optionally substituted with 1 or 2 R% groups. R5 may be a 6-membered heteroaryl group, e.g. pyridinyl, optionally substituted with 1 R® group.
[0041] R5 may be a 5-membered heteroaryl group. R5 may be an unsubstituted 5-membered heteroaryl group. R5 may be a 5-membered heteroaryl group, optionally substituted with 1 or 2 R groups. R5 may be a 5-membered heteroaryl group, optionally substituted with 1 R3® group.
[0042] Exemplary five-membered heteroaryl groups include furanyl, thiophenyl, pyrrolyl, oxazolyl, triazolyl and thiazolyl. It may be that RS is furanyl or thiophenyl. It may be that RS is thiophenyl.
[0043] It may be that each R3? is independently selected from halo, C1-C2 alkyl, C+-C2 haloalkyl, CN, OR: and NRPRP, wherein R2is selected from H, C+-C2 alkyl and C+-C2 haloalkyl and each RP is independently selected from H and C+-C: alkyl. It may be that each R*® is independently selected from halo, CHs, CFs,
OH, CN and NH:. Each R32 may be independently selected from halo, CHs and CN.
C
S
[0044] R> may be: ==/ , wherein / indicates the point of attachment to the rest of the compound.
P345823NL 10 (R%),
C7
[0045] R5 may be: x4 , wherein: X4 is N, CH, or CR; X5 is N, CH, or CR"; Rf? is selected from halo, C+-Ca4 alkyl, C1-C4 haloalkyl and CN; R' is selected from halo, C1-C4 alkyl, C+-C4 haloalkyl and
CN; qis 0,1 or 2; and / indicates the point of attachment to the rest of the compound, with the proviso that when X4 is N, X% is CH or CR", and when X5 is N, X? is CH or CR.
[00486] In these embodiments, X+ may be N. It may be that X* is N and X5 is CH. It may be that X*is N and X5 is CR". X3 may be N. It may be that X5 is N and X4 is CH. It may be that X5 is CH or CR12. X5 may be CH. It may be that X* is CH or CR10. X*may be is CH. It may be that X* and X5 are each CH. It may be that X* is CH and X5 is CR"
[0047] R'® may be selected from halo, CHs, CF3 and CN. R'® may be CN. R" may be selected from halo, CHa, and CF: and CN. R' may be halo, e.g. F.
[0048] It may be that q is 0. It may be that q is 1. It may be that q is 1 and R*® is selected from halo,
CHs, CF3 and CN. It may be that q is 1 and R® is Me.
[0049] It may be that each R3 and RS is independently selected from H, halo, C1-C2 alkyl, C+-C2 haloalkyl and CN. For example, each R3 and R® may be independently selected from H, halo, CHs, C1 haloalkyl and CN. It may be that R% is H. It may be that each R® is H. It may be that mis 1 or 2. Preferably, m is 1. It may be that mis 1 and R® and R® are both H.
[0050] The compound of formula | may be a compound selected from: odin OF Ney © odin
H
NF Na No © N.~# pa
OH OH OH
Os Ny ® Os. Ny Os.N ny
H H H DD
Ng ® EN NE A CN
OH OH OH
Osy Os Noy Ole N ny
On 70 2 oC oC QO
NS „N NLS NLs of Nn F on F of N~, F 0 : TO
CO piss 0,00 © 4 PP NLs Na F Nus F
F F
4 & Ì aN me N 2 H 0 H 9 H (7
O=S-NH N Oz5-NH N O=5-NH N 7 F » 5
Ns Ns Na
P345823NL 11 yr À 4 0” om Á 7 A or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof.
[0051] The compound of formula (I) may be:
A eg cd 0 , or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof.
DNA gyrase binding
[0052] The present inventors have identified DNA gyrase as the target responsible for the observed antimicrobial activity of the compounds described herein. DNA gyrase is a well-validated essential bacterial topoisomerase that is targeted by ciprofloxacin (CIP), a fluoroquinolone for which widespread resistance has been observed in the clinic. importantly, the compounds described herein (such as compound 102) did not show any cross-resistance with CIP or vice versa.
[0053] The present inventors carried out extensive structural biology studies using cryo-electron microscopy and identified an unprecedented allosteric binding mode of 102 at a site distinct from CIP.
This novel binding site (also referred to herein as “a binding pocket”) is a horseshoe-like hydrophobic pocket on the DNA-binding surface GyrA subunit of DNA gyrase.
[0054] According to a second aspect of the invention there is provided a compound capable of binding a horseshoe-like hydrophobic pocket on the DNA-binding surface GyrA subunit of DNA gyrase. The compound may be a compound according to the first aspect. The binding pocket is described in more detail hereinbelow.
[0055] The inventors’ identification of the isoquinoline sulfonamides as allosteric DNA gyrase inhibitors paves the way for the development of a novel class of antibiotics targeting bacteria, including those that have become resistant to fluoroquinolones. Due to the amino acids defining the binding pocket being broadly conserved across many species of bacteria, the inventors believe that the compounds described herein may have antibiotic activity against Gram-negative and/or Gram-positive bacteria. Suitably, the compounds described herein may have antibiotic activity against Gram-negative bacteria (for example
Escherichia coli, as described in more detail in the Examples section of the present disclosure).
[0056] Suitably, the binding pocket may be defined by residues 92, 97, 98, 169, 172, 266, 267, 268 and 269 of SEQ ID NO: 1 (amino acid sequence of DNA gyrase subunit A of Escherichia coli), or corresponding amino acid residues in homologs thereof. Suitably, residues 92, 97, 98, 169, 172, 266, 267, 268 and 269 of SEQ ID NO: 1 are respectively, M92, S97, L98, N169, S172, Y266, Q267, V268 and
N269 of SEQ ID NO: 1, or conservative modifications thereof. What constitutes a conservative modification will be known to a person skilled in the art. However, examples of conservative modifications are provided herein below.
P345823NL 12
[0057] In the context of the present disclosure the reference polypeptide may be the amino acid sequence according to SEQ ID NO: 1. SEQ ID NO: 2, 3,4,5,6,7,8,9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 and 26 provide non-limiting examples of homologs from other bacterial species.
[0058] The term “corresponding amino acid” refers to an amino acid which is present within a corresponding region and which is the counterpart of a given amino acid of SEQ ID NO: 1 in a sequence alignment (for example as shown in Figures 1 to 26). Merely by way of example, as shown in Figure 1, the amino acid of SEQ ID NO: 2 that corresponds tc amino acid residue M92 of SEQ ID NO: 1 is M96.
The amino acid of SEQ ID NO: 2 that corresponds to the amino acid residue S97 of SEQ ID NO: 1 is
S100. The amino acid of SEQ ID NO: 2 that corresponds to the amino acid residue L98 of SEQ ID NO: 1 is Q101. The amino acid of SEQ ID NO: 2 that corresponds to the amino acid residue N169 of SEQ ID
NO: 1 is N172. The amino acid of SEQ ID NO: 2 that corresponds to the amino acid residue S172 of SEQ
ID NO: 1is S175. The amino acid of SEQ ID NO: 2 that corresponds to the amino acid residue Y266 of
SEQ ID NO: 1 is Y268. The amino acid of SEQ ID NO: 2 that corresponds to the amino acid residue
Q267 of SEQ ID NO: 1 is Q269.
[6059] The amino acid of SEQ ID NO: 2 that corresponds to the amino acid residue V268 of SEQ ID NO: 1 is V270.The amino acid of SEQ ID NO: 2 that corresponds to the amino acid residue N269 of SEQ ID
NO: 1 is N271. It will be appreciated that the corresponding amino acid does not have to be the same amino acid as in the reference polypeptide. Suitably, the corresponding amino acid may be a similar amino acid. Similar amino acid residues are grouped by chemical characteristics of the side chains into families. Said families are described below for “conservative amino acid substitutions”.
[0060] Methods for obtaining sequence alignments in order to determine corresponding amino acids are well known in the art. Merely by way of example, sequence alignment may be obtained by using bioinformatics tools such as EMBOSS Needle (pair wise alignment; available at www.ebi.ac.uk). When the same position in the sequences to be compared is occupied by the same amino acid residue, then the respective molecules are identical at that very position. Accordingly, the “percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100%.
For instance, if 6 out of 10 sequence positions are identical, then the identity is 60%. The percent identity between two protein sequences can, e.g., be determined using the Needleman and Wunsch algorithm (NEEDLEMAN, S. B. and Wunsch, C. D. A general method applicable to the search for similarities in the amino acid sequence of two proteins. Journal of Molecular Biology 1970, vol. 48, p. 443-453) which has been incorporated into EMBOSS Needle. The % identity is typically determined over the entire length of the query sequence on which the analysis is performed. Two molecules having the same primary amino acid sequence are identical irrespective of any chemical and/or biological modification. For example, two antibodies having the same primary amino acid sequence but different glycosylation patterns are identical by this definition. Similar protein sequences are those which, when aligned, share similar amino acid residues and most often, but not mandatorily, identical amino acid residues at the same positions of the sequences to be compared.
[0061] It will be appreciated that whilst the inventors have identified that the novel binding pocket is defined by residues M92, S97, L98, N169, 8172, Y266, Q267, V268 and N269 of SEQ ID NO: 1 (amino
P345823NL 13 acid sequence of DNA gyrase subunit A of Escherichia col), or corresponding amino acid residues in homologs thereof, the binding pocket may also be defined by amino acids that are conservative modifications of these amino acids.
[0062] The compounds described herein may inhibit DNA gyrase by binding a horseshoe-like hydrophobic pocket on the DNA-binding surface GyrA subunit of DNA gyrase. Suitably, the compounds described herein may bind one or more amino acid residues selected from the group consisting 92, 97, 98, 169, 172, 266, 267, 268 and 269 of SEQ ID NO: 1 (amino acid sequence of DNA gyrase subunit A of
Escherichia coli), or corresponding amino acid residues in homologs thereof.
[0063] Suitably, the compounds described herein may bind one or more amino acid residues selected from the group consisting of M92, S97, L98, N189, S172, Y266, Q267, V268 and N269 of SEQ ID NO:1, or corresponding amino acid residues in homologs thereof, or conservative modifications thereof.
[0064] Suitably, the compounds may bind one amino acid residue selected from the group consisting of
M92, 897, L98, N169, S172, Y266, Q267, V268 and N269 of SEQ ID NO:1, or corresponding amino acid residues in homologs thereof, or conservative modifications thereof. Suitably, the compounds may bind two amino acid residues selected from the group consisting of M92, 897, L98, N169, S172, Y266, Q267,
V268 and N269 of SEQ ID NO:1, or corresponding amino acid residues in homologs thereof, or conservative modifications thereof. Suitably, the compounds may bind three amino acid residues selected from the group consisting of M92, S97, L98, N169, S172, Y266, Q267, V268 and N269 of SEQ ID NO:1, or corresponding amino acid residues in homologs thereof, or conservative modifications thereof.
Suitably, the compounds may bind four amino acid residues selected from the group consisting of M92,
S97, L98, N169, S172, Y266, Q267, V268 and N269 of SEQ ID NO:1, or corresponding amino acid residues in homologs thereof, or conservative modifications thereof. Suitably, the compounds may bind five amino acid residues selected from the group consisting of M92, S97, L98, N169, S172, Y266, Q267,
V268 and N269 of SEQ ID NO:1, or corresponding amino acid residues in homologs thereof, or conservative modifications thereof. Suitably, the compounds may bind six amino acid residues selected from the group consisting of M92, S97, L98, N169, S172, Y266, Q267, V268 and N269 of SEQ ID NO:1, or corresponding amino acid residues in homologs thereof, or conservative modifications thereof.
Suitably, the compounds may bind seven amino acid residues selected from the group consisting of M82,
S97, L98, N1869, S172, Y268, Q267, V268 and N269 of SEQ ID NO:1, or corresponding amine acid residues in homologs thereof, or conservative modifications thereof. Suitably, the compounds may bind eight amino acid residues selected from the group consisting of M92, S87, L98, N169, S172, Y268, Q267,
V268 and N269 of SEQ ID NO:1, or corresponding amino acid residues in homologs thereof, or conservative modifications thereof. Suitably, the compounds may bind all nine amino acid residues selected from the group consisting of M82, 897, L98, N169, S172, Y266, Q267, V268 and N269 of SEQ
ID NO:1, or corresponding amino acid residues in homologs thereof, or conservative modifications thereof.
[0065] Suitably, the compounds may bind to amino acid residue S97 or S172 of SEQ ID NO: 1, ora corresponding amino acid residue in a homolog thereof, or a conservative modifications thereof. Suitably, the compounds may bind to amino acid residue S87 and S172 of SEQ ID NO: 1, or a corresponding amino acid residue in a homolog thereof, or a conservative modifications thereof. Suitably, the binding to
P345823NL 14 residue S87 and/or S172 of SEQ ID NO: 1, or a corresponding amino acid residue in a homolog thereof, or a conservative modification thereof, is by hydrogen bonding.
[0066] Binding of a compound of the invention to the novel binding pocket can be confirmed using suitable structural biology techniques known in art, such as cryogenic electron microscopy (Cryo EM).
This can be combined with functional assay to confirm inhibitory activity. A suitable functional assay to confirm inhibitory activity is a DNA gyrase supercoiling assay as disclosed herein. The structural biology technique and/or the functional assay is preferably performed using an enzyme comprising a polypeptide of SEQ ID NO: 1 or a homolog (such as SEQ ID NO: 2, 3,4,5,6,7,8,9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26).
[0067] A compound of the invention (such as a compound of the first aspect or second aspect) may have an ICs of less than about 10 pM in a DNA gyrase supercoiling assay. For example, the compound may have an ICs of less than about 1 pM in a DNA gyrase supercoiling assay. The compound may have an
ICso of less than about 500 nM in a DNA gyrase supercoiling assay. For example, the compound may have an ICs of less than about 400 nM in a DNA gyrase supercoiling assay. The compound may have an IC: of less than about 300 nM (e.g. less than about 200 nM) in a DNA gyrase supercoiling assay.
Preferably, the compound may have an ICs of less than about 100 nM (e.g. less than about 80 nm) in a
DNA gyrase supercoiling assay.
Formulations
[0068] According to a third aspect of the invention there is provided a pharmaceutical formulation or composition including a compound of the invention, optionally in admixture with at least one pharmaceutically acceptable adjuvant, diluent or carrier.
[0069] The formulation or composition may be a parenteral formulation or an oral formulation. The formulation may be a parenteral formulation, for example a formulation for intravenous injection. The formulation may be an oral formulation.
[0070] Compounds, formulations or compositions of the invention may be administered orally, topically, intravenously, subcutaneously, buccally, rectally, dermally, nasally, tracheally, bronchially, by any other parenteral route, as an oral or nasal spray or via inhalation. The compounds may be administered in the form of pharmaceutical preparations comprising the compound either as a free compound or, for example, a pharmaceutically acceptable non-toxic organic or inorganic acid or base addition salt, in a pharmaceutically acceptable dosage form. Depending upon the disorder and patient to be treated and the route of administration, the compositions may be administered at varying doses.
[0071] Typically, therefore, the pharmaceutical compounds of the invention may be administered parenterally (“parenterally” as used herein, refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion) or orally to a host to obtain an antibacterial effect. For example, the pharmaceutical compounds of the invention may be administered by intravenous injection or infusion. In the case of larger animals, such as humans, the compounds may be administered alone or as compositions in combination with pharmaceutically acceptable diluents, excipients or carriers.
P345823NL 15
[0072] Actual dosage levels of active ingredients in the pharmaceutical formulations and pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular patient, compositions and mode of administration. The selected dosage level will depend upon the activity of the particular compound, the route of administration, the severity of the condition being treated and the condition and prior medical history of the patient being treated. However, it is within the skill of the art to start doses of the compound at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. Suitable doses are generally in the range of from 0.01 — 100 mg/kg/day, for example in the range of 0.1 to 50 mg/kg/day.
[0073] Pharmaceutical formulations or compositions of this invention for parenteral (e.g. intravenous) injection may comprise pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. Formulations or compositions for parenteral injection may represent preferred formulations or compositions of the invention.
[0074] These compositions may also contain adjuvants such as preservative, wetting agents, emulsifying agents and dispersing agents. Inhibition of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol or phenol sorbic acid.
It may also be desirable to include isotonic agents, such as sugars or sodium chloride, for example.
Prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents (for example, aluminium monostearate and gelatine) which delay absorption.
[0075] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
In such solid dosage forms, the active compound is typically mixed with at least one inert, pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate and/or one or more: a) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol and silicic acid; b) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; c) humectants, such as glycerol; d) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; e) solution retarding agents, such as paraffin; f) absorption accelerators, such as quaternary ammonium compounds; g) wetting agents, such as cetyl alcohol and glycerol monostearate; h) absorbents, such as kaolin and bentonite clay and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycol, for example.
P345823NL 16
[0076] Oral formulations may contain a dissolution aid. Examples of dissolution aids include nonionic surface active agents, such as sucrose fatty acid esters, glycerol fatty acid esters, sorbitan fatty acid esters (e.g. sorbitan tricleate), polyethylene glycol, polyoxyethylene hydrogenated castor oil, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene alkyl ethers, methoxypolyoxyethylene alkyl ethers, polyoxyethylene alkylphenyl ethers, polyethylene glycol fatty acid esters, polyoxyethylene alkylamines, polyoxyethylene alkyl thioethers, polyoxyethylene polyoxypropylene copolymers, polyoxyethylene glycerol fatty acid esters, pentaerythritol fatty acid esters, propylene glycol monofatty acid esters, polyoxyethylene propylene glycol monofatty acid esters, polyoxyethylene sorbitol fatty acid esters, fatty acid alkylolamides, and alkyamine oxides; bile acid and salts thereof (e.g. chenodeoxycholic acid, cholic acid, deoxycholic acid, dehydrocholic acid and salts thereof, and glycine or taurine conjugate thereof); ionic surface active agents, such as sodium laurylsulfate, fatty acid soaps, alkylsufonates, alkylphosphates, ether phosphates, fatty acid salts of basic amino acids; triethanolamine soap, and alkyl quaternary ammonium salts; and amphoteric surface active agents, such as betaines and aminocarboxylic acid salts.
[0077] The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and may also be of a composition such that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, and/or in delayed fashion. Examples of embedding compositions include polymeric substances and waxes.
[0078] The active compounds may also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.
[0079] The active compounds may be in finely divided form, for example it may be micronized.
[0080] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohal, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, clive, castor and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan and mixtures thereof. Besides inert diluents, the oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavouring and perfuming agents. Suspensions, in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminium metahydroxide, bentonite, agar-agar, and traganacanth and mixtures thereof.
[0081] Compositions for rectal or vaginal administration may be in the form of suppositories which can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
P345823NL 17
[0082] Dosage forms for topical administration of a compound of this invention include powders, sprays, creams, foams, gels, ointments and inhalants. The active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives, buffers or propellants which may be required. Ophthalmic formulations, eye ointments, powders and solutions are also contemplated as being within the scope of this invention.
[0083] Liquid (e.g. aqueous) formulations and compositions, whether intended for parenteral or oral use, may comprise additional compound(s) to help prevent precipitation of the active compound. Compounds of the invention are glycopeptide derivatives. Precipitation of such compounds in aqueous solution may be avoided or minimised by including a monosaccharide in the solution. For example, aqueous formulations or compositions may comprise glucose. In particular, a parenteral (e.g. intravenous injection) formulation or composition may comprise a compound of the invention, water for injection and glucose.
[0084] Insofar as they do not interfere with the activity of the compounds, the formulations or compositions according to the present subject matter may contain other active agents intended, in particular, for use in treating a bacterial infection.
[0085] The formulations according to the present subject matter may also contain inactive components.
Suitable inactive components are well known in the art and are described in standard textbooks, such as
Goodman and Gillman’s: The Pharmacological Bases of Therapeutics, 13Ed., Brunton et al., Eds.
McGraw-Hill Education (2017), and Remington's Pharmaceutical Sciences, 17!» Ed., Mack Publishing
Co., Easton, Pa. (1990), both of which are incorporated by reference herein in their entirety.
[0086] The formulations may be used in combination with an additional pharmaceutical dosage form to enhance their effectiveness in treating any of the disorders described herein. In this regard, the present formulations may be administered as part of a regimen additionally including any other pharmaceutical and/or pharmaceutical dosage form known in the art as effective for the treatment of any of these disorders.
Uses
[0087] According to a fourth aspect of the invention there is provided a compound or formulation of the invention for use as a medicament.
[0088] According to a fifth aspect of the invention there is provided a compound or formulation of the invention for use in the treatment of a bacterial infection.
[0089] It may be that the bacterial infection is a Gram-negative bacterial infection. lllustrative Gram- negative bacterial infections include infections caused by bacteria of a genus selected from
Campylobacter, Vibrio, Escherichia, Haemophilus, Klebsiella, Enterobacter, Salmonella, Shigella,
Legionella, Yersinia, Pseudomonas, or Acinetobacter.
[0090] It may be that the Gram-negative bacterial infection is an infection caused by bacteria selected from Campylobacter jejuni, Vibrio cholerae, Escherichia coli, Haemophilus influenzae, Klebsiella pneumoniae, Enferobacter cloacae, Salmonella enterica, Shigella dysenteriae, Legionella pneumophila,
Yersinia pestis, Pseudomonas aeruginosa, or Acinetobacter baumannii. It may be that the Gram- negative bacterial infection is an infection caused by bacteria selected from E. coli, K. pneumoniae,
P345823NL 18
Klebsiella, Acinetobacter, P. aeruginosa, Salmonella, Helicobaceter. It may be that the Gram-negative bacterial infection is an infection caused by E. coli or K. pneumoniae.
[0091] The Gram-negative bacterial infection may be an infection caused by drug-resistant Gram- negative bacteria, e.g. ciprofloxacin-resistant Gram-negative bacteria.
[0092] It may be that the bacterial infection is a Gram-positive bacterial infection. Illustrative Gram- positive bacterial infections include infections caused by bacteria of a genus selected from Bacillus,
Corynebacterium, Enterococcus, Erysipelothrix, Listeria, Streptococcus, Staphylococcus, Clostridioides or
Mycobacterium.
[0093] It may be that the Gram-positive bacterial infection is an infection caused by bacteria selected from Bacillus anthracis, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium,
Erysipelothrix rhusiopathiae, Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Clostridioides difficile, Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium avium, or Mycobacterium abscessus.
[0094] Exemplary bacterial (e.g. Gram-negative) infections that may be treated by compounds of the invention include skin and structure infections, lower respiratory tract infections, bacteremia, sepsis, septicemia, infective endocarditis, peritonitis (e.g. associated with continuous ambulatory peritoneal dialysis), enterocolitis (e.g. staphyloccocal), mastitis, Clostridium difficile infection-associated diarrhoea and colitis. The infection that may be treated may be selected from skin and structure infections and bacteremia. Exemplary skin and structure infections include cellulitis/erysipelas, major cutaneous abscesses, and wound infections. Exemplary lower respiratory tract infections include pneumonia, community-acquired pneumonia (CAP), nosocomial pneumonia, and pleural empyema.
Methods of Treatment
[0095] According to a sixth aspect of the invention there is provided provides a method of treating a bacterial infection in a patient, comprising administering to the patient an effective amount of a compound or formulation of the invention.
[0096] The bacterial infection may be as described above in relation to the fifth aspect of the invention.
[0097] According to a seventh aspect of the invention there is provided the use of a compound or formulation of the invention as a DNA gyrase inhibitor; optionally wherein the use in in vitro.
BRIEF DESCRIPTION OF THE DRAWINGS
[0098] Embodiments of the invention will now be described, by way of example only, with reference to the accompanying figures, in which:
Figure 1 shows sequence alignment of Escherichia coli (strain K12) sp|POAES4|GYRA_ECOLI (SEQ ID NO: 1) with Bacillus anthracis ADAOF7R8R3|ACAOF7R8R3_BACAN DNA gyrase subunit A (SEQ ID NO: 2). Amino acids defining the binding pocket are shown in bold and underlined.
Figure 2 shows sequence alignment of Escherichia coli (strain K12) sp|POAES4|GYRA_ECOLI (SEQ ID NO: 1) with Corynebacterium diphtheriae ACABJ4WQKS|ADABJ4WQKSE(SEQ ID NO: 3). Amino acids defining the binding pocket are shown in bold and underlined.
P345823NL 19
Figure 3 shows sequence alignment of Escherichia coli (strain K12) sp|POAES4|GYRA_ECOLI (SEQ ID NO: 1) with Enterococcus faecalis ACA2Z6BNK2|A0A2Z6BNK2_ENTFL(SEQ ID NO: 4). Amino acids defining the binding pocket are shown in bold and underlined.
Figure 4 shows sequence alignment of Escherichia coli (strain K12) sp|POAES4|GYRA_ECOLI (SEQ ID NO: 1) with Enterococcus faecium AOA8B2RLX8|A0A8B2ZRLX6_ENTFC{SEQ ID NO: 5). Amino acids defining the binding pocket are shown in bold and underlined
Figure 5 shows sequence alignment of Escherichia coli (strain K12) sp|POAES4|GYRA_ECOLI (SEQ ID NO: 1) with Erysipelothrix rhusiopathiae ETFX39|E7FX39_ERYRH (SEQ ID NO: 6). Amino acids defining the binding pocket are shown in bold and underlined.
Figure 6 shows sequence alignment of Escherichia coli (strain K12) sp|POAES4|GYRA_ECOLI (SEQ ID NO: 1) with Listeria monocytogenes AOASLS8YRA1|A0A5L8YRA1_LISMN(SEQ ID NO: 7). Amino acids defining the binding pocket are shown in bold and underlined.
Figure 7 shows sequence alignment of Escherichia coli (strain K12) sp|POAES4|GYRA_ECOLI (SEQ ID NO: 1) with Streptococcus pneumoniae serotype 4 (strain ATCC BAA-334 /
TIGR4sp|P72524|GYRA_STRPN(SEQ ID NO: 8). Amino acids defining the binding pocket are shown in bold and underlined.
Figure 8 shows sequence alignment of Escherichia coli (strain K12) sp|POAES4|GYRA_ECOLI (SEQ ID NO: 1) with Streptococcus pyogenes serotype M3 (strain SSI- 1)sp|PODGO3|GYRA_STRPQ(SEQ ID NO: 9). Amino acids defining the binding pocket are shown in bold and underlined.
Figure 9 shows sequence alignment of Escherichia coli (strain K12) sp|POAES4|GYRA_ECOLI (SEQ ID NO: 1) with Staphylococcus aureus (strain NCTC 8325 / PS 47)sp|Q2G2Q0|GYRA_STAAS (SEQ ID NO: 10). Amino acids defining the binding pocket are shown in bold and underlined.
Figure 10 shows sequence alignment of Escherichia coli (strain K12) sp|POAES4|GYRA_ECOLI (SEQ ID NO: 1) with Clostridioides difficile
AOA3S9XYK7|[A0A3S9XYK7_ CLODI(SEQ ID NO: 11). Amino acids defining the binding pocket are shown in bold and underlined.
Figure 11 shows sequence alignment of Escherichia coli (strain K12)
Sp|POAES4|GYRA_ECOLI (SEQ ID NO: 1) with Mycobacterium tuberculosis (strain ATCC 25618 /
H37Rv)sp|PSWG47|GYRA_MYCTU (SEQ ID NO: 12). Amino acids defining the binding pocket are shown in bold and underlined.
Figure 12 shows sequence alignment of Escherichia coli (strain K12)
Sp|POAES4|GYRA_ECOLI (SEQ ID NO: 1) with Mycobacterium leprae (strain TN) sp|Q57532|GYRA_MYCLE DNA (SEQ ID NO: 13). Amino acids defining the binding pocket are shown in bold and underlined.
Figure 13 shows sequence alignment of Escherichia coli (strain K12)
Sp|POAES4|GYRA_ECOLI (SEQ ID NO: 1) with Mycobacterium avium subsp. avium tr]ACAGBOBAVO|ACAGBOBAVO_MYCAYV (SEQ ID NO: 14). Amino acids defining the binding pocket are shown in bold and underlined.
P345823NL 20
Figure 14 shows sequence alignment of Escherichia coli (strain K12) sp|PCAES4|GYRA_ECOLI (SEQ ID NO: 1) with Mycobacteroides abscessus
SV=1trfACA8B3CXT4|A0A8B3CXT4_SMYCO (SEQ ID NO: 15). Amino acids defining the binding pocket are shown in bold and underlined.
Figure 15 shows sequence alignment of Escherichia coli (strain K12)
Sp|POAES4|GYRA_ECOLI (SEQ ID NO: 1) with Campylobacter jejuni subsp. jejuni serotype O:2 (strain
ATCC 700819 / NCTC 11168)sp|Q03470|GYRA_CAMJE(SEQ ID NO: 16). Amino acids defining the binding pocket are shown in bold and underlined.
Figure 16 shows sequence alignment of Escherichia coli (strain K12) sp|POAES4|GYRA_ECOLI (SEQ ID NO: 1) with Vibrio cholerae trJAOASC2B1C1]|ACASC2B1C1_VIBCL(SEQ ID NO: 17). Amino acids defining the binding pocket are shown in bold and underlined.
Figure 17 shows sequence alignment of Escherichia coli (strain K12) sp|POAES4|GYRA_ECOLI (SEQ ID NO: 1) with Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd) sp|P43700|GYRA_HAEIN(SEQ ID NO: 18). Amino acids defining the binding pocket are shown in bold and underlined.
Figure 18 shows sequence alignment of Escherichia coli (strain K12)
Sp|POAES4|GYRA_ECOLI (SEQ ID NO: 1) with Klebsiella pneumoniae subsp. pneumoniae trJACGA7IODT23|AOA7IODT23_KLEPN (SEQ ID NO: 19). Amino acids defining the binding pocket are shown in bold and underlined.
Figure 19 shows sequence alignment of Escherichia coli (strain K12)
Sp|POAES4|GYRA_ECOLI (SEQ ID NO: 1) with Enterobacter cloacae trJAGA411GIW2Z|AOA411GIW2_ENTCL(SEQ ID NO: 20). Amino acids defining the binding pocket are shown in bold and underlined.
Figure 20 shows sequence alignment of Escherichia coli (strain K12) sp|POAES4|GYRA_ECOLI (SEQ ID NO: 1) with Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720)sp|P37411|GYRA_SALTY (SEQ ID NO: 21). Amino acids defining the binding pocket are shown in bold and underlined.
Figure 21 shows sequence alignment of Escherichia coli (strain K12) sp|POAES4|GYRA_ECOLI (SEQ ID NO: 1) with Shigella dysenteriae trJACGA2X2IF02|ACAZ2X2IF02_SHIDY (SEQ ID NO: 22). Amino acids defining the binding pocket are shown in bold and underlined.
Figure 22 shows sequence alignment of Escherichia coli (strain K12)
Sp|POAES4|GYRA_ECOLI (SEQ ID NO: 1) with Legionella pneumophila subsp. pneumophila tr]ACASABUAY2|ACA3ABUAY2_LEGPN (SEQ ID NO: 23). Amino acids defining the binding pocket are shown in bold and underlined.
Figure 23 shows sequence alignment of Escherichia coli (strain K12)
Sp|POAES4|GYRA_ECOLI (SEQ ID NO: 1) with Yersinia pestis subsp. pestis bv. Medievalis
P345823NL 21 tTAOASPSRXDS|ACA5P8RXD6_ YERPE(SEQ ID NO: 24). Amino acids defining the binding pocket are shown in bold and underlined.
Figure 24 shows sequence alignment of Escherichia coli (strain K12)
Sp|POAES4|GYRA_ECOLI (SEQ ID NO: 1) with Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) sp|P48372|GYRA_PSEAE (SEQ
ID NO: 25). Amino acids defining the binding pocket are shown in bold and underlined.
Figure 25 shows sequence alignment of Escherichia coli (strain K12) sp|POAES4|GYRA_ECOLI (SEQ ID NO: 1) with Acinetobacter baumannii trJAOA335FJS7|ACA335FJS7_ACIBA (SEQ ID NO: 26). Amino acids defining the binding pocket are shown in bold and underlined.
Figure 26: shows the hemolytic activity of certain compounds of the invention after 20 h incubation with sheep blood cells.
Figure 27: Time-kill kinetics of compound 102 (LEI-800) and CIP against E. coli W3110. Points represent mean values from n=2 biologically independent experiments, LOQ = limit of quantification.
Figure 28: shows Microscopy images of E. coli BW25113 from the BCP experiment.
Conditions are, from top to bottom: DMSO, ampicillin (AMP), rifampicin (RIF), tetracycline (TET), ciprofloxacin (CIP), 99, 102 (LEI-800). From left to right the channels indicate: DIC, DAPI, FM4-84, and
DAPI/FM4-64 combined. The bacteria treated with 99 displayed a phenotype that is consistent with the
DMSO control, while incubation with 102 (LEI-800) led to a phenotype resembling the CIP and TET morphological profile. Scale bars represent 10 um.
Figure 29: shows (a), Individual graphs of six exemplary features extracted from BCP microscopy after image analysis. Each dot represents the measured value for an individual bacterium. (b),
PCA plot of all the morphological features extracted from three independent BCP experiments.
Figure 30: shows (a) Agar containing 60 (5x MIC) was inoculated with 107 CFU of E. coli. After one day, nine viable colonies were isolated and used for MIC testing and whole-genome sequencing (WGS). (b) Susceptibility assays confirm spontanecus resistance to 60 and WGS identified mutations located in the DNA gyrase, predominantly in subunit A. Similar mutations are similarly coloured. Cross- resistance is observed for conformationally restricted compounds 101 and 102. (¢) Topology of the mutations in the core of the DNA gyrase heterotetramer outline a possible binding site of isoquinoline sulfonamides. (d) All mutations are found in the cleavage-reunion domain of DNA gyrase. Domain organization of DNA gyrase, GyrB (coral) and GyrA (beige) subunits.
Figure 31: shows Whole-genome sequencing results.
Figure 32: shows (a)Schematic representation of negative supercoiling. In a supercoiling assay, relaxed (R) DNA is transformed into supercoiled (SC) DNA by DNA gyrase. (b) Three compounds tested for supercoiling inhibition. CIP (green) is used as positive control, 99 (blue) is used as antimicrobially inactive compound, and 102 (LEI-800) (red) is used as lead compound. (c) Dose-response curves of DNA gyrase supercoiling inhibition, based on n = 3 gels. Error bars and dotted lines represent the standard deviation at each concentration and the 95% CI respectively. (d) Representative agarose gels for E. coli DNA gyrase supercoiling inhibition by the three compounds. (e) Table showing
P345823NL 22 supercoiling inhibition alongside antimicrobial activity in WT E. coli and in genetic and pharmacological
LPS disruption backgrounds. Genetic disruption is accomplished by deletion of lipopolysaccharide gene rfaC. Pharmacological disruption is accomplished through addition of polymyxin B nonapeptide (PMBN), an outer membrane disruptor. (f) Checkerboard assays show pharmacological disruption of LPS by
PMBN does not have an effect on the antimicrobial activity of CIP, yet potentiates both 99 and 102 (LEI- 800).
Fig 33: shows (a) A selection of 2D classes, box size in angstroms indicated. (b) Processing scheme (see Methods for description). (c) FSC curve for the final reconstruction as output by cryoSPARC. (d) Euler angle distribution as output by cryoSPARC. (e). Local resolution map to illustrate resolution distribution from <4 A next to the DNA and compound to >7 at the ends of the cleavage-reunion domain. (fH. Map-to-model curve. (g). Comparison of Gyr-Mu217-102 (LEI-800) map (grey surface) to the gepotidacin EM structure 6RKW (cartoon model). Note the significant change in the position of the CTDs and DNA.
Fig 34: shows (a) Overview of the model of the Gyr-Mu217-102 complex. GyrA (8-524) and
GyrB (402-790) are depicted as beige and coral cartoon representations. The modelled central part of
Mu217 DNA is shown in teal green. Two molecules of 102 (LEI-800) observed in a single gyrase heteroteramer are shown as golden van der Waals spheres. Uniform colour scheme is used throughout the manuscript. (b) A close-up of the 102 (LEI-800) binding pocket on the DNA-binding surface of GyrA.
GyrA is shown as molecular surface, DNA as cartoon representation and 102 (LEI-800) as van der Waals spheres. (c) Molecular interactions between GyrA and 102 (LEI-800). 102 (LEI-800) is shown as stick representation. Main GyrA residues important for 102 (LEI-800) binding are labelled. Two key hydrogen bonds between side-chain of Ser97 and N3 nitrogen of central pyrrolidine ring of 102 (LEI-800), and main chain nitrogen of Ser172 and sulfonic acid of 102 (LEI-800) are shown in gold; distances in Angstrom are indicated. (d) A comparison of 102 (LEI-800) (current study), ciprofloxacin (CIP) (PDB:2XCT) simocyclinone D8 (SD8) (PDB:4CKL) binding sites on GyrA DNA-binding surface. 102 (LEI-800) is shown as golden, SD8 as pink and CIP as grey stick representations. A quinoline ring of 102 (LEI-800) shares binding pocket with the aminocoumarin moiety of SD8, but unlike SD8, 102 (LEI-800) does not interfere with DNA binding. (e) Coulomb potential density map for 102 (LEI-800). (f) A 2D diagram of 102 (LEI-800) binding site generated by LigPlot. Key hydrogen bonds to Ser87 and Ser172 are shown in gold and distances in A are indicated. Spiked red arcs show non-bonded interactions with residues within 3.9 A distance.
DETAILED DESCRIPTION
[0099] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
P345823NL 23
[00100] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.
All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.
[00101] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.
Definitions
[00102] The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure.
[00103] The invention concerns amongst other things the treatment of a disease. The term “treatment”, and the therapies encompassed by this invention, include the following and combinations thereof: (1} hindering, e.g. delaying initiation and/or progression of, an event, state, disorder or condition, for example arresting, reducing or delaying the development of the event, state, disorder or condition, or a relapse thereof in case of maintenance treatment or secondary prophylaxis, or of at least one clinical or subclinical symptom thereof; (2) preventing or delaying the appearance of clinical symptoms of an event, state, disorder or condition developing in an animal {e.g. human) that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; and/or (3) relieving and/or curing an event, state, disorder or condition (e.g., causing regression of the event, state, disorder or condition or at least one of its clinical or subclinical symptoms, curing a patient or putting a patient into remission). The benefit to a patient to be treated may be either statistically significant or at least perceptible to the patient or to the physician. It will be understood that a medicament will not necessarily produce a clinical effect in each patient to whom it is administered; thus, in any individual patient or even in a particular patient population, a treatment may fail or be successful only in part, and the meanings of the terms “treatment” and “prophylaxis” and of cognate terms are to be understood accordingly. The compositions and methods described herein are of use for therapy and/or prophylaxis of the mentioned conditions.
[00104] The term “prophylaxis” includes reference to treatment therapies for the purpose of preserving health or inhibiting or delaying the initiation and/or progression of an event, state, disorder or condition, for example for the purpose of reducing the chance of an event, state, disorder or condition occurring.
The outcome of the prophylaxis may be, for example, preservation of health or delaying the initiation and/or progression of an event, state, disorder or condition. It will be recalled that, in any individual patient
P345823NL 24 or even in a particular patient population, a treatment may fail, and this paragraph is to be understood accordingly.
[00105] The term “antibiotic” refers to a compound that inhibits the growth of or destroys microorganisms, such as bacteria (e.g. Gram-positive bacteria, or Gram-negative bacteria). An “antibacterial” is an antibiotic that is active against bacteria. Compounds of the invention are antibacterial, in particular with activity against bacteria (such as Gram-negative bacteria).
[00106] The term “C:-C4 alkyl” used herein covers any saturated straight or branched chain alkyl moiety having from 1 to 4 (e.g. 1, 2, 3 or 4) carbon atoms. The term includes, e.g., methyl, ethyl, propyl (n-propyl or isopropyl) and butyl (n-butyl, sec-butyl or tert-butyl).
[00107] The term “alkylene chain” by itself or as part of another substituent means a divalent radical derived from a saturated straight chain alkyl with the general formula -CnHzs-, wherein n is an integer. A 1, 2 or 3 membered alkylene chain is intended to cover any saturated straight chain alkylene moiety having 1, 2 or 3 carbon atoms. The term “alkylene” is synonymous with the term “alkanediyl”.
[00108] The terms “halo” or "halogen" as used herein includes reference to F, Cl, Br or |, for example F,
Cl or Br. In a particular class of embodiments, halogen is F or Cl, of which F is more common.
[00109] The term “haloalkyl” refers to an alkyl group (alkyl being defined as above) where one or more hydrogen atoms are substituted by a corresponding number of halogens. For example, the term “C4-Ca haloalkyl” is intended to cover any saturated straight or branched chain alkyl moiety having from 1 to 4 (e.g. 1, 2, 3 or 4) carbon atoms where one or more hydrogen atoms are substituted by a corresponding number of halogen atoms. For example, “C1-C4 haloalkyl” covers, but is not limited to, trifluoromethyl (-
CFs), 2,2,2-trifluorcethyl, 4-chlorobutyl, 3-bromopropyl, and the like.
[00110] The term “5- or 6-membered heteroaryl group” may be any aromatic 5 or 6 membered ring system comprising from 1 to 4 heteroatoms independently selected from ©, S and N (in other words from 1 to 4 of the atoms forming the ring system are selected from O, S and N, the rest being carbon). 5- or 6- membered heteroaryl groups will be monocyclic. A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of 5- or 6-membered heteroaryl groups include pyrrolyl, e.g. 1-pyrrolyl, 2- pyrrolyl or 3-pyrrolyl, furanyl, e.g. 2-furanyl or 3-furanyl, thiophenyl, e.g. 2-thiophenyl or 3-thiophenyl, pyrazolyl, e.g. 3-pyrazolyl, imidazolyl, e.g. 2-imidazolyl or 4- imidazolyl, oxazolyl, e.g. 2-oxazolyl, 4-oxazolyl or 5-oxazolyl, isoxazolyl, e.g. 3-isoxazolyl, 4-isoxazolyl or 5-isoxazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, pyridinyl, e.g. 2-pyridyl, 3-pyridyl or 4-pyridyl, pyridazinyl pyrimidinyl, e.g. 2-pyrimidyl or 4-pyrimidyl, pyrazinyl, triazinyl and thiazolyl, e.g. 2-thiazolyl, 4- thiazolyl or 5-thiazolyl.
[00111] Each of the above terms (e.g., “alkyl”, “haloalkyl” and “heteroaryl”) are intended to cover the unsubstituted forms of the indicated radical, unless stated otherwise (e.g. by being explicitly stated as being substituted). The term “substituted” as used herein in reference to a moiety means that one or more, e.g. 1, 2 or 3, of the hydrogen atoms in said moiety are replaced independently of each other by the corresponding number of the described substituents.
[00112] It will, of course, be understood that substituents are only at positions where they are chemically possible, the person skilled in the art being able to decide (either experimentally or theoretically) without
P345823NL 25 inappropriate effort whether a particular substitution is possible. For example, amino or hydroxy groups with free hydrogen may be unstable if bound to carbon atoms with unsaturated (e.g. olefinic) bonds.
Additionally, it will of course be understood that the substituents described herein may themselves be substituted by any substituent, subject to the aforementioned restriction to appropriate substitutions as recognised by the skilled person.
[00113] Where steric issues determine placement of substituents on a group, the isomer having the lowest conformational energy may be preferred.
[00114] Where a compound, moiety, process or product is described as “optionally” having a feature, the disclosure includes such a compound, moiety, process or product having that feature and also such a compound, moiety, process or product not having that feature. Thus, when a moiety is described as “optionally substituted”, the disclosure comprises the unsubstituted moiety and the substituted moiety.
[00115] Where two or more moieties are described as being “independently” or “each independently” selected from a list of atoms or groups, this means that the moieties may be the same or different. The identity of each moiety is therefore independent of the identities of the one or more other moieties.
[00116] The term “pharmaceutically acceptable” as used herein includes reference to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings or animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. This term includes acceptability for both human and veterinary purposes.
[00117] The term “pharmaceutically acceptable salts” is meant to include salts of the active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent.
Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galacturonic acids and the like (see, for example, Berge et al., “Pharmaceutical Salts”, Journal of
Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.
P345823NL 26
[00118] The neutral forms of the compounds are preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents.
[00119] Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.
[00120] Certain compounds of the present invention possess asymmetric carbon atoms (optical centres) or double bonds; the racemates, diastereomers, tautomers, geometric isomers and individual isomers are encompassed within the scope of the present invention. The compounds of the present invention do not include those which are known in the art to be too unstable to synthesize and/or isolate.
[00121] The symbol 5 denotes a point of attachment of a moiety to the remainder of a compound.
[00122] The term “prodrug” as used herein represents compounds which are transformed in vivo to the parent compound or other active compound, for example, by hydrolysis in blood. An example of such a prodrug is a pharmaceutically acceptable ester of a carboxylic acid. A thorough discussion is provided in
T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the A.C.S. Symposium Series,
Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and
Pergamon Press, 1987; H Bundgaard, ed, Design of Prodrugs, Elsevier, 1985; and Judkins, et al.
Synthetic Communications, 26(23), 4351-4367 (1996); and The organic chemistry of drug design and drug action by Richard B Silverman in particular pages 497 to 546; each of which is incorporated herein by reference.
[00123] The term “pharmaceutical formulation” as used herein includes reference to a formulation comprising at least one active compound and optionally one or more additional pharmaceutically acceptable ingredients, for example a pharmaceutically acceptable carrier. Where a pharmaceutical formulation comprises two or more active compounds, or comprises at least one active compound and one or more additional pharmaceutically acceptable ingredients, the pharmaceutical formulation is also a pharmaceutical composition. Unless the context indicates otherwise, all references to a “formulation” herein are references to a pharmaceutical formulation.
[00124] The term “product” or “product of the invention” as used herein includes reference to any product containing a compound of the present invention. In particular, the term product relates to compositions and formulations containing a compound of the present invention, such as a pharmaceutical composition, for example.
[00125] The term “therapeutically effective amount” as used herein refers to an amount of a drug, or pharmaceutical agent that, within the scope of sound pharmacological judgment, is calculated to (or will} provide a desired therapeutic response in a mammal (animal or human). The therapeutic response may for example serve to cure, delay the progression of or prevent a disease, disorder or condition.
P345823NL 27
[00126] The present invention also includes all pharmaceutically acceptable isotopically-labelled compounds of formulae (I) to (XVI), wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number most commonly found in nature.
[00127] Examples of stable isotopes suitable for inclusion in the compounds of the invention include isotopes of hydrogen, such as 2H, carbon, such as 11C and 13C, nitrogen, such as 15N and oxygen, such as 70 and 150.
[00128] Isotopically-labelled compounds can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described using an appropriate isotopically- labelled reagent in place of the non-labelled reagent previously employed.
[00129] It may be that the compounds of the invention are occupied with deuterium (2H) instead of hydrogen at a certain position or positions at an isotopic abundance greater than deuterium’s natural isotopic abundance (e.g. greater than about 0.015%). The position or positions may be occupied with deuterium at an isotopic abundance of greater than 50%. The position or positions may be occupied with deuterium at an isotopic abundance of greater than 90%, e.g. greater than 95%. The position or positions may be occupied with deuterium at an isotopic abundance of greater than 99%, e.g. greater than 99.5%.
[00130] Isotopic abundance can be determined using conventional analytical methods known to a person skilled in the art, such as mass spectrometry and nuclear magnetic resonance spectroscopy.
[00131] The term “homolog” as used herein refers a polypeptide, including polypeptides from the same or different bacterial species, having greater than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity to a reference polypeptide, and/or having the same or substantially the same properties, and/or performing the same or substantially the same function as the reference polypeptide.
[00132] As used herein, the term "conservative modifications” refers to modifications that are physically, biologically, chemically or functionally similar to the corresponding reference amino acid (such as a corresponding amino acid in SEQ ID NO: 1)_, e.g., has a similar size, shape, electric charge, chemical properties, including the ability to form covalent or hydrogen bonds, or the like. Such conservative madifications include, but are not limited to, one or more amino acid substitutions, additions and deletions.
[00133] For example, conservative amino acid substitutions include those in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
[00134] Amino acid residues are usually divided into families based on common, similar side-chain properties, such as: 1. nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, methionine), 2. uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, proline, cysteine, tryptophan), 3. basic side chains (e.g., lysine, arginine, histidine, proline), 4. acidic side chains (e.g., aspartic acid, glutamic acid), 5. beta-branched side chains (e.g. , threonine, valine, isoleucine) and
P345823NL 28 6. aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). A conservative substitution may also involve the use of a non-natural amino acid.
[00135] Non-conservative substitutions, i.e. exchanging members of one family against members of another family, may lead to substantial changes, e.g., with respect to the charge, dipole moment, size, hydrophilicity, hydrophobicity or conformation of the binding member, which may lead to a significant drop in the binding activity, in particular if amino acids are affected that are essential for binding to the target molecule. A non- conservative substitution may also involve the use of a non-natural amino acid.
[00136] Conservative and non-conservative modifications can occur naturally (due to a DNA mutation) or may be introduced by a variety of standard techniques known in the art, such as combinatorial chemistry, site-directed DNA mutagenesis, PCR-mediated and/or cassette mutagenesis, peptide/protein chemical synthesis, chemical reaction specifically modifying reactive groups in the parental binding member.
[00137] The following abbreviations are used herein:
ACN acetonitrile FSC Fourier shell correlation
AMP ampicillin HEPES N-2-hydroxyethylpiperazine-N-2- ethane sulfonic acid
AMR antimicrobial resistance HPLC high-performance liquid chromatography aq. aqueous HRMS high-resolution mass spectrometry
ATCC American Type Culture ICso half maximal inhibitory concentration
Collection
ATP adenosine triphosphate LB lysogeny broth
BCP bacterial cytological profiling LC-UV liquid chromatography ultraviolet
CAMHB cation adjusted Mueller-Hinton LEI-800 compound 102 broth
CFU colony-forming unit LEI-801 compound 99
CHAPSO 3-([3- LPS lipopolysaccharide
Cholamidopropyl]dimethylam monio)-2-hydroxy-1- propanesulfonate
Cl confidence interval MDR multidrug resistant
CIP ciprofloxacin MSC minimum synergistic concentration, cryo-EM Cryogenic electron MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-
Microscopy diphenyltetrazolium bromide
CTF contrast transfer function NBTIs Novel Bacterial Topoisomerase
Inhibitors
DAPI 4'8-diamidino-2-phenylindole NMR nuclear magnetic resonance
DCM dichloromethane OM outer membrane
DIC differential interference PCA principal component analysis contrast
DIPEA N,N-Diisopropylethylamine PCR polymerase chain reaction
DMAP 4-(Dimethylamino)} pyridine PMBN polymyxin B nonapeptide
DMF dimethylformamide rcf relative centrifugal field
DMSO dimethyl sulfoxide RIF rifampicin
EDTA ethylenediaminetetraacetic rpm revolutions per minute acid
EER electron-event representation RT room temperature
Equiv equivalents SD8 simocyclinone D8
ESBL extended-spectrum beta SNP single nucleotide polymorphisms lactamase
ESI electrospray lonization TBAF tetrabutylammonium fluoride
FC fold change TCEP tris(2-carboxyethyhphosphine hydrochloride
FICI Fractional Inhibitory TET tetracycline
P345823NL 29
Concentration Index
FM4-64 (N-{3- TFA trifluoroacetic acid
Triethylammoniumpropyi}-4- {8-{4-{Diethylamino) Phenyh
Hexalrieny) Pyridinium
Dibromide}
EXAMPLES
Synthesis
General Synthesis
[00138] The skilled person will appreciate that adaptation of methods known in the art could be applied in the manufacture of the compounds of the present invention.
[00139] For example, the skilled person will be immediately familiar with standard textbooks such as "Comprehensive Organic Transformations - A Guide to Functional Group Transformations”, RC Larock,
Wiley-VCH (1999 or later editions); "March's Advanced Organic Chemistry - Reactions, Mechanisms and
Structure”, MB Smith, J. March, Wiley, (5th edition or later); “Advanced Organic Chemistry, Part B,
Reactions and Synthesis”, FA Carey, RJ Sundberg, Kluwer Academic/Plenum Publications, (2001 or later editions); "Organic Synthesis - The Disconnection Approach", S Warren (Wiley), (1982 or later editions); "Designing Organic Syntheses" S Warren (Wiley) (1883 or later editions); “Heterocyclic Chemistry”, J.
Joule (Wiley 2010 edition or later); ("Guidebook To Organic Synthesis" RK Mackie and DM Smith (Longman) (1982 or later editions), etc., and the references therein as a guide.
[00140] The skilled person is familiar with a range of strategies for synthesising organic and particularly heterocyclic molecules and these represent common general knowledge as set out in text books such as
Warren “Organic Synthesis: The Disconnection Approach”; Mackie and Smith “Guidebook to Organic
Chemistry”; and Clayden, Greeves, Warren and Wothers “Organic Chemistry”.
[00141] The skilled chemist will exercise their judgement and skill as to the most efficient sequence of reactions for synthesis of a given target compound and will employ protecting groups as necessary. This will depend inter alia on factors such as the nature of other functional groups present in a particular substrate. Clearly, the type of chemistry involved will influence the choice of reagent that is used in the said synthetic steps, the need, and type, of protecting groups that are employed, and the sequence for accomplishing the protection / deprotection steps. These and other reaction parameters will be evident to the skilled person by reference to standard textbooks and to the examples provided herein.
[00142] Sensitive functional groups may need to be protected and deprotected during synthesis of a compound of the invention. This may be achieved by conventional methods, for example as described in
P. G. M. Wuts, Greene's Protective Groups in Organic Synthesis, John Wiley & Sons, Inc., Michigan, 2014.
[60143] All chemicals (Sigma-Aldrich, Fluka, Acros, Merck, Combi-Blocks, Fluorochem, TCI) were used as received. All solvents used for reactions were of analytical grade. THF, Et:O, DMF, ACN and DCM were dried over activated 4 A molecular sieves, MeOH over 3 A molecular sieves. H20 used in synthesis procedures was of Milli-Q-grade quality. Column chromatography was performed on silica gel (Screening
Devices BV, 40-63 um, 60 A). The eluent EtOAc was of technical grade and distilled before use.
P345823NL 30
Triethylamine was distilled over KOH, and triethylamine and pyridine were stored over KOH pellets.
Starting materials were coevaporated with toluene (3x) before use in water-sensitive reactions.
[60144] Reactions were monitored by thin layer chromatography (TLC) analysis using Merck aluminium sheets (Silica gel 60, F254). Compounds were visualized by UV-absorption (254 nm) and spraying for general compounds: KMnOa (20 g/L) and K2CO: (10 g/L) in H20, or for amines: ninhydrin (0.75 g/L) and acetic acid (12.5 mL/L) in ethanol, followed by charring at 150°C. 'H and 13C NMR experiments were recorded on a Bruker AV-300 (300/75 MHz), Bruker AV-400 (400/101 MHz), Bruker DMX-400 (400/101
MHz), Bruker AV- 500 (500/126 MHz) and Bruker AV-600 (600/151 MHz). Chemical shifts are given in ppm (5) relative to tetramethylsilane, as internal standard. Multiplicity: s = singlet, bs = broad singlet, d = doublet, dd = doublet of doublet, t = triplet, q = quartet, quint = quintet, non = nonet m = multiplet.
Coupling constants (J) are given in Hz. LC-MS measurements were performed on a Thermo Finnigan
LCQ Advantage MAX ion-trap mass spectrometer (ESI+) coupled to a Surveyor HPLC system (Thermo
Finnigan) equipped with a standard C18 (Gemini, 4.6 mm D x 50 mm L, 5 pm particle size, Phenomenex) analytical column and buffers A: H20, B: ACN, C: 0.1% aq. TFA. High resolution mass spectra were recorded on a LTQ Orbitrap (Thermo Finnigan) mass spectrometer or a Synapt G2-Si high definition mass spectrometer (Waters) equipped with an electrospray ion source in positive mode (source voltage 3.5 kV, sheath gas flow 10 mL/min, capillary temperature 250°C) with resolution R = 60000 at m/z 400 (mass range m/z = 150-2000) and dioctylphtalate (m/z = 391.28428) as a lock mass. Preparative HPLC was performed on a Waters Acquity Ultra Performance LC with a C18 column (Gemini, 150 x 21.2 mm,
Phenomenex) using a ACN in H20 (+0.2% TFA) gradient. All final compounds were determined to be > 95% pure by LC-UV analysis.
General Synthetic Procedures
[00145] Certain compounds of the invention can be made according to or by methods analogous to the methods described in General Scheme | and/or General Procedures A-L.
General Scheme |, i) benzophenone Br Br
Ho Tpsc H I re ner K N ©
N 7 N iii) BrPhLi > — —Ê
Td imidazole YY TIPSO 6 ps AA
R® 9 DMAP RE Re RK R3
R DCM R° cis : trans
Boc20
Et3N
DCM
Br i) MsCl, Et;N, DCM Br TBAF Br
Boc ii NaN3, DMSO Roe ACN Roc
N - — Afni
TIPSO
Ns 2 RY HO RS R9 RE RS 10
PPhs Cl Z
H20 XN Br ; me Ky 9 Canals >
Br (Re)n 7 2 Boc . 2 09 H
R’ S-NH N ii) TFA, DCM R7 SLNH N ie EtsN x1” B -— > x17
HN DCM Xa ‚ 4 R? a OR re RF xR), XR, ©
P345823NL 31
[00146] wherein X!, X2, X3, R®, R7, Rê, R® and n are as defined above. As the skilled person will appreciate, other compounds of the invention may be made by appropriate variation of the reagents used in this general scheme.
[00147] General procedure A: Boc deprotection
[00148] The Boc protected compound (1 equiv.) was dissolved in DCM (0.1 M). TFA was added dropwise (17% v/v) at 0°C and the mixture was allowed to steer at room temperature for 4 h. The reaction was quenched with sat. aq. Na:COs, diluted with water and extracted with DCM (3x). The combined organic layers were dried with MgSOsa, filtered and concentrated in vacuo. Purification of the crude material by column chromatography (0% > 10% MeOH (10% aq. NHs) in DCM) afforded the pure product.
[00149] General procedure B: sulfonation with sulfonyl chloride
[00150] A solution of amine (1 equiv.) and triethylamine (2 equiv.) in DCM (0.1 M) was cooled to 0°C after which dropwise a solution of the corresponding sulfonyl chloride (1.5 equiv.) in DCM (0.1 M) was added.
The reaction mixture was allowed to warm to room temperature and stirred for 1 h before sat. aq. Na2CO:3 was added. The mixture was extracted with DCM (2x), and the combined organic layers were dried over
Mg2SO,, filtrated and concentrated in vacuo. The residue was purified by column chromatography (2% > 5% MeOH (10% aq. NHs) in DCM) to afford the product.
General procedure C: sulfonation with arylbromide
[00151] To a microwave reaction tube equipped with a magnetic stir bar was added potassium metabisulfite (2 equiv.), TBAB (1.2 equiv.), sodium formate (2.2 equiv.), palladium acetate (0.1 equiv.), triphenylphosphine (0.3 equiv.), 1,10-phenathroline (0.3 equiv.} and DMSO (0.25 M). The mixture was put under nitrogen flow for 10 min before the corresponding bromide (1 equiv.) was added. After that, the reaction vessel was immersed in a 70°C preheated heating block for 4 h. After cooling, DIPEA (1.5 equiv.) the amine (1.5 equiv.) and THF (0.5 M) were added to the reaction mixture. Subsequently, a solution of
N-chlorosuccinimide (2 equiv.) in THF (0.5 M) was added, the reaction was stirred at room temperature overnight. The mixture was then diluted with water, extracted with ethyl acetate. The organic layer was washed with brine and dried over Na2SO.. After filtration, the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (2% > 5% MeOH (10% aq. NHs) in
DCM).
General procedure D: Suzuki-Miyaura cross-coupling
[00152] The bromobenzyl compound (1 equiv.) was reacted with the corresponding boronic acid (1.2 equiv.), Pd(PPh3)4 (0.015 equiv.), K2CO:3 (4 equiv.) in 1,4-dioxane and water (1:3, 0.1 M} in a sealed microwave tube. The reaction mixture was degassed with under nitrogen flow for 15 min and then stirred overnight at 90°C. The reaction mixture was then filtered over a silica gel pad with EtOAc and concentrated in vacuo. The crude product was purified by column chromatography (1% > 10% MeOH (10% ag. NHs) in DCM) to give the pure product.
General procedure E: TIPS protection
[00153] The prolinol was co-evaporated in vacuo twice with toluene. The prolinol (1 equiv.), imidazole (1.5 equiv.) and DMAP (0.05 equiv.) were dissolved in dry DCM (0.5 M). TIPS-CI was added dropwise at 0°C after which the reaction mixture was allowed to warm to RT and was stirred for 16 h. The mixture was
P345823NL 32 then poured onto sat. aq. NH4Cl and extracted with DCM (4x). The combined organic layers were washed with brine (2x), dried with MgSO, filtered and concentrated in vacuo. Purification of the crude material by column chromatography (80% > 100% Et20 in pentane with 1% triethylamine) afforded the pure product.
General procedure F: pyrrolidine a-arylation
[00154] The amine (1 equiv.) and benzophenone (1.2 equiv.) were combined and co-evaporated in vacuo twice with toluene. These were then dissolved in dry Et20 (0.5 M) and transferred to a flame dried flask under argon atmosphere. n-Butyllithium (1 equiv.) was added dropwise at -78°C and the solution was steered for 10 min. Subsequently, the corresponding aryllithium solution (1.5 equiv, prepared according to general procedure G) was dropwise added at -78°C after which the mixture was removed from the cooling bath and left to stir for 2 h while reaching room temperature. The mixture was then quenched with
MeOH at -78°C and diluted with Et2O. This was then washed with water and brine. The aqueous layer was extracted with Et2O (3x) and the combined organic layers were dried with MgSO, filtered and concentrated in vacuo. Purification of the crude material by column chromatography (2% > 20% Et20 in pentane with 1% triethylamine and 10% toluene) afforded the separate stereoisomers as crude products.
General procedure G: preparation of aryllithium
[00155] 1,4-Dibromobenzene (1.5 equiv. to amine general procedure F) was co-evaporated in vacuo twice with toluene. This was then dissolved in dry Et2O (0.75 M) and transferred to a flame dried flask under argon atmosphere. n-butyllithium (1.5 equiv. to amine) was added dropwise at -78°C and the mixture was steered for 10 min. The mixture was then warmed to room temperature and steered for an additional 30 min before addition to the appropriate solution with imine intermediate.
General procedure H: Boc protection
[00156] The crude product of the pyrrolidine alkylation reaction (1 equiv.) was dissolved with di-tert-butyl dicarbonate (2.5 equiv.) and triethylamine (2 equiv.) in DCM (0.06 M) and the mixture was steered at room temperature. Upon completion, the reaction was diluted with DCM and washed with water and brine. The organic layer was dried with MgSOsu, filtered and concentrated in vacuo. Purification of the crude material by column chromatography (1% > 3% EtOAc in pentane) afforded the pure product.
General procedure |: TIPS deprotection
[00157] The TIPS protected starting material (1 equiv.) was dissolved in ACN (0.08 M). To this was added TBAF (5 equiv., 1 M in THF) and the mixture was steered at room temperature. The mixture was subsequently concentrated in vacuo on silica. Purification of the crude material by column chromatography (5% > 40% EtOAc in pentane) afforded the pure product.
General procedure J: mesylation
[00158] The primary alcohol (1 equiv.) and triethylamine (3 equiv.) were dissolved in dry DCM (0.05 M). A solution of MsCI (1.5 equiv.) in dry DCM (0.05 M) was added dropwise at 0°C and the mixture was steered at room temperature for 1.5 h. The mixture was quenched with the addition of water and extracted with DCM (3x). The combined organic layers were washed with brine, dried with MgSO,4, filtered and concentrated in vacuo. After characterization, the crude product was immediately used for further reactions.
General procedure K: azide substitution
P345823NL 33
[60159] The crude mesylated starting material (1 equiv.) and sodium azide (6 equiv.) were dissolved in dry DMF (0.1 M). The mixture was heated to 65°C and left to steer overnight. The reaction was diluted with Et2O and washed with water (3x) and brine. The organic layer was dried with MgSO, filtered and concentrated in vacuo. Purification of the crude material by column chromatography (15% > 20% EtOAc in pentane) afforded the pure product.
General procedure L: Staudinger reduction of azide
[00160] The azide compound (1 equiv.), (Ph)sP (2 equiv.) and water (2 equiv.) were dissolved in THF (0.1
M). The mixture was then refluxed at 60°C for 64 h. The reaction mixture was concentrated in vacuc and redissolved in Et20. This solution was thereafter extracted with 1 M aq. HCI (2x) and the aqueous layer washed with Et20 (2x). The pH of the aqueous layer was then adjusted to >12 with 2 M aq. NaOH and extracted with DCM (6x). The combined organic layers were dried with MgSO,, filtered and concentrated in vacuo. Purification of the crude material by column chromatography (5% > 50% MeCH (10% aq. NHs3) in EtOAc) afforded the pure product. intermediate Synthesis
N'-Tritylethane-1,2-diamine (104)
THN ~~
[00161] 2 Ethylenediamine (103) (267 mL, 4.00 mol) and K2CO: (66.3 g, 440 mmol) were suspended in DCM (700 mL) after which a solution of trityl chloride (112 g, 400 mmol) in DCM (700 mL) was added dropwise over 40 min. The reaction-mixture was stirred overnight at RT, filtered, concentrated under reduced pressure and co-evaporated with toluene to yield the product (123 g, quant.) which was used without further purification. ‘H NMR (400 MHz, CDCls) 86 7.48 (d, J = 7.6 Hz, 6H), 7.26 (t, J = 7.7 Hz, 6H), 7.17 (t, J = 7.3 Hz, 3H), 279, J=59 Hz 2H), 2.21 (1, J = 6.0 Hz, 2H), 1.51 (bs, 3H). 3C NMR (100 MHz, CDCls) 6 146.24, 128.76, 127.89, 126.34, 70.77, 46.60, 42.89.
N'-(4-Bromobenzyl)-N2-tritylethane-1,2-diamine (105)
THN ny
[00162] TO. 4-Bromobenzaldehyde (0.95 g, 5.1 mmol) was dissolved in methanal {10 mL), and M'-tritylethane-1,2-diamine (1.7 g, 5.6 mmol, 1.1 equiv.) was dissolved in THF (5 mL} and added thereto. The mixture was stirred at room temperature for 1 h, after which sodium borohydride (0.29 g, 7.6 mmol, 1.5 equiv.) was slowly added, followed by stirring overnight. Water (40 mL) was added to the mixture and the mixture was extracted with DCM (3x 30 mL). The organic layers were combined, dried with Na2SO0,, filtered and purified by column chromatography (2% > 5% MeOH {10% aq. NHs) in DCM) to yield title compound 105 {1.04 g, 2.04 mmol, 40%). tert-Butyl (4-bromobenzyl)(2-(tritylamino)ethyl)carbamate (106)
TrtHN Wy
[00163] Br Di-fert-butyl-dicarbonate (0.16 g, 0.73 mmol) was dissolved in DCM (1 mL) and slowly added to a mixture of 105 (0.52 g, 1.10 mmol} and triethylamine (0.30 mL, 2.16 mmol) in
DCM (5 mL). The reaction mixture was stirred at room temperature overnight, after which it was concentrated in vacuo. The residue was purified by column chromatography (5% > 10% EtOAc in pentane) to yield the title product 106 (0.33 g, 0.65 mmol, 59%).
P345823NL 34 'H NMR (400 MHz, CDCl3) 8 7.57 — 6.94 (m, 19H), 4.34 (5, 2H), 3.42 — 3.19 (m, 2H), 2.36 — 2.14 (m, 2H), 1.64 (bs, 1H), 1.54 — 1.32 (m, SH). 13C NMR (101 MHz, CDCls) § 156.21, 146.02, 137.50, 131.68, 128.63, 127.96, 127.35, 126.41, 121.28, 80.10, 70.86, 50.16, 47.43, 42.22, 28.51. tert-Butyl (4-(pyridin-3-yl)benzyl}{2-(tritylamino)ethyl)carbamate (107)
TRO
BN
[00164] | S The mixture of 106 (0.30 g, 0.52 mmol), 3-pyridinylboronic acid (86 mg, 0.70 mmol), Pd(PPhs)4 (30 mg, 26 umol), potassium carbonate (6.31 g, 2.2 mmol) in water (2 mL) and 1,4-dioxane (6 mL) was deoxygenated under nitrogen flow and sealed. The mixture was heated to 90°C and stirred overnight. The reaction mixture was then filtrated, concentrated and was purified by column chromatography (30% EtOAc in pentane) to yield 107 (0.23 g, 0.40 mmol, 76%). 'H NMR (400 MHz, CDCl) 8 8.82 (s, 1H), 8.58 (dd, J = 4.8, 1.7 Hz, 1H), 7.90 — 7.73 (m, 1H), 7.59 — 7.09 (m, 21H), 4.47 (s, 2H), 3.47 — 3.20 (m, 2H), 2.38 — 2.17 (m, 2H), 1.59 — 1.34 (m, SH).
BC NMR (101 MHz, CDCls) 8 156.11, 148.56, 148.35, 146.05, 138.61, 136.79, 136.37, 134.35, 128.65, 127.94, 127.36, 126.39, 123.67, 80.04, 70.87, 50.35, 47.50, 42.25, 28.54. tert-Butyl (2-aminoethyl){4-(pyridin-3-yl)benzyljcarbamate (108)
HN ~y
Boc
[00165] > To a solution of 107 (0.20 g, 0.35 mmol) and triethylsilane (0.40 mL, 2.5 mmol) in DCM (10 mL) on ice bath, was added TFA (0.15 mL). The reaction mixture was stirred at room temperature overnight, after which it was basified by adding sat. aq. Na2CO: (10 mL), extracted with
DCM (3x 25 mL). The combined organic layers were dried over Na2SO,, filtered and purified by column chromatography (5% MeOH (10% aq. NHaz) in DCM) to yield 108 (0.10 g, 0.32 mmol, 91%). ‘H NMR (400 MHz, CDCl3) § 8.84 (d, J= 2.4 Hz, 1H), 8.58 (dd, J=4.8, 1.7 Hz, 1H), 7.87 (dt, J= 7.9, 2.0
Hz, 1H), 7.55 (d, J = 8.1 Hz, 2H), 7.42 — 7.31 (m, 3H), 4.53 (s, 2H), 3.30 (d, J = 27.2 Hz, 2H), 2.97 — 2.68 (m, 2H), 1.47 (s, 9H). 3C NMR (101 MHz, CDCls) ò 156.20, 148.51, 148.27, 138.59, 136.80, 136.26, 134.28, 128.46, 127.33, 123.61, 80.10, 50.89, 50.06, 40.57, 28.49. tert-Butyl (2-(isoquinoline-5-sulfonamido)ethyl}{(4-(pyridin-3-yl)benzyljcarbamate (109) ohn,
Boc
[00166] NF Nig 109 (100 mg, 0.19 mmol, 83%) was synthesized from 108 (79 mg, 0.23 mmol) according to general procedure B.
H NMR (400 MHz, CDCI3) 8 9.36 — 9.30 (m, 1H), 8.81 (s, 1H), 8.63 — 8.55 (m, 2H), 8.43 (d, J=6.1 Hz, 1H), 8.38 (dd, J=7.4, 1.2 Hz, 1H), 8.16 (d, J=8.2 Hz, 1H), 7.86 (d, J= 8.0 Hz, 1H), 7.84 (t, J= 8.2 Hz, 1H), 7.46 (d, J = 8.6 Hz, 2H), 7.39 (dd, J= 8.0, 4.8 Hz, 1H), 7.22 (dd, J = 8.2, 3.2 Hz, 2H), 4.39 (s, 2H), 3.41 3.24 (m, 2H), 3.13 — 2.99 (m, 2H), 1.43 (s, 9H).
P345823NL 35 3C NMR (101 MHz, CDCls) ö 156.57, 153.18, 148.34, 147.93, 144.90, 137.92, 136.71, 136.11, 134.55, 134.35, 133.36, 133.00, 131.19, 129.03, 127.90, 127.24, 125.88, 123.70, 117.37, 51.37, 46.66, 42.19, 28.26.
N-{2-Aminoethyl)isoquinoline-5-sulfonamide (111)
ON ne,
CO
[00167] N# Isoquinoline-5-sulfonic acid (110) (10.0 g, 47.8 mmol) was dissolved in SOCl2 (60 mL) and DMF (1.2 mL). The mixture was refluxed at 60°C until TLC analysis showed the complete conversion of the starting material. The SOCI2 was evaporated in vacuo, and the reaction mixture was washed with DCM and then filtered. The crude sulfonyl chloride formed was immediately used in the following reaction.
[00168] Ethylene diamine (15.1 mL, 227 mmol) was added dropwise to a cooled (0°C) and stirred solution of the crude sulfonyl chloride (10.0 g, 37.7 mmol) in DCM (600 mL). The mixture was then stirred at room temperature for 2 h. The reaction mixture was diluted with sat. aq. Na2CO: (10 mL}, washed with brine (50 mL) and extracted with DCM (3x). The organic layers were combined, dried over MgSQ, filtered and concentrated. The residue was then co-evaporated with toluene to remove the remaining ethylene diamine giving 111 (10.3 g, 35.0 mmol, 93%) as a dark yellow solid that was used without further purification.
N-(2-({4-Bromobenzyl)amino)ethyl)isoquinoline-5-sulfonamide (112a}
Pry
[00169] NF 111 (0.10 g, 3.96 mmol) and 4-bromobenzaldehyde (0.36 g, 1.93 mmol) were dissolved in THF (20 mL) in the presence of activated 3 A molecular sieves. Then, sodium triacetoxyborohydride (0.84 g, 3.96 mmol} and glacial acetic acid (110 pL, 1.93 mmol) were added. The reaction mixture was stirred overnight, after which sat. aq. Na2COa (5 mL) was added to quench the reaction. The mixture was then diluted with brine (5 mL), extracted with Et20 (10 mL) and DCM (3x). The combined organic layers were dried over MgSO,, filtered and concentrated in vacuo. The crude product purified by column chromatography (1% > 10% MeOH (10% aq. NHs) in DCM) to give 112A (0.64 g, 1.52 mmol, 79%). ‘H NMR (400 MHz, CDCls) § 9.35 (d, J = 1.0 Hz, 1H), 8.65 (d, J = 6.1 Hz, 1H}, 8.45 — 8.40 (m, 2H), 8.20 (dt, J=8.2, 1.2 Hz, 1H), 7.69 (dd, J = 8.2, 7.4 Hz, 1H), 7.38 — 7.31 (m, 2H), 7.00 — 6.95 (m, 2H), 3.49 (s, 2H), 3.02 — 2.96 (m, 2H), 2.65 — 2.59 (m, 2H). 3C NMR (101 MHz, CDCls) 8 153.46, 145.24, 138.59, 134.28, 133.70, 133.44, 131.59, 131.30, 129.67, 129.10, 126.05, 121.01, 117.26, 52.53, 47.42, 42.54.
N-(2-(((6-Bromopyridin-3-yl)methyljamino)ethyljisoquinoline-5-sulifonamide (112b)
P345823NL 36 om
OO N“ Br
[00170] N# 111 (1.00 g, 3.98 mmol) and 6-bromonicotinaldehyde (0.36 g, 1.94 mmol) were dissolved in THF (20 mL) in the presence of activated 3 A molecular sieves. Then, sodium triacetoxyborohydride (0.84 g, 3.98 mmol) and glacial acetic acid (122 pL, 1.94 mmol) were added. The reaction mixture was stirred overnight. Sat. aq. Na2CO: (5 mL) was added to the reaction, which was then diluted with brine (10 mL) and extracted with Et2O (10 mL) and DCM (3x). The organic layers were combined, dried over MgSO,, filtered and concentrated in vacuo. The crude product was purified by column chromatography (1% > 10% MeOH (10% aq. NHa3) in DCM) to afford title compound 112b (0.73 g, 1.73 mmol, 89%). ‘H NMR (400 MHz, CDCl3) 6 9.39 (bs, 1H), 8.73 — 7.70 (m, 1H), 8.51 — 8.41 (m, 2H), 8.26 — 8.21 (m, 1H), 8.19-8.16 (m, 1H), 7.78 — 7.68 (m, 1H), 7.49 — 7.36 (m, 2H), 3.59 (s, 2H), 3.13 — 3.00 (m, 2H), 2.72 — 2.65 (m, 2H).
N-(2-((4-Bromo-3-chlorobenzyl)amino)ethyl)isoquinoline-5-sulfonamide (112c)
Phy
[00171] NF 111 (1.00 g, 3.98 mmol) and 3-chloro-4-bromoaldehyde (0.43 g, 1.94 mmol) were dissolved in THF (20 mL) in the presence of activated 3 A molecular sieves. Then, sodium triacetoxyborohydride (0.84 g, 3.98 mmol) and glacial acetic acid (122 pL, 1.94 mmol) were added. The reaction mixture was stirred overnight. Sat. aq. Na2CO: (5 mL) was added to quench the reaction, which was then diluted with brine (10 mL) and extracted with Et2O (10 mL) and DCM (3x). The organic layers were combined, dried over MgSO., filtered and concentrated in vacuo. The crude product was purified by column chromatography (1% > 10% MeOH (10% ag. NHa) in DCM) to afford title compound 112c (0.8 g, 1.76 mmol, 91%). ‘H NMR (400 MHz, CDCl3) ö 9.39 (bs, 1H), 8.77 — 8.67 (m, 1H), 8.51 — 8.41 (m, 2H), 8.26 — 8.22 (m, 1H), 7.78 —= 7.867 (m, 1H), 7.55 — 7.50 (m, 1H), 7.29 — 7.27 (m, 1H), 6.93 — 6.89 (m, 1H), 3.53 (s, 2H), 3.13 - 2.99 (m, 2H), 2.70 — 2.62 (m, 2H).
N-(2-((4-Bromo-3-fluorobenzyl)amino)ethyl)isoquinoline-5-sulfonamide (112d)
For
[00172] NF 111 (1.00 g, 3.98 mmol) and 3-fluoro-4-bromoaldehyde (0.39 g, 1.9 mmol) were dissolved in THF (20 mL) in the presence of activated 3 A molecular sieves. Then, sodium triacetoxyborohydride (0.84 g, 4.0 mmol) and glacial acetic acid (122 pL, 1.94 mmol) were added. The reaction mixture was stirred overnight. Sat. aq. Na2CO: (5 mL) was added to the reaction, which was then diluted with brine (10 mL) and extracted with Et2O (10 mL) and DCM (3x). The organic layers were combined, dried over MgSO,, filtered and concentrated in vacuo. The crude product was purified by column chromatography (1% > 10% MeOH (10% aq. NHs) in DCM) to afford title compound 112d (0.62 g, 1.4 mmol, 73%).
P345823NL 37 'H NMR (400 MHz, CDCls) 8 9.39 (bs, 1H}, 8.74 — 8.70 (m, 1H), 8.50 — 8.39 (m, 2H), 8.26 — 8.21 (m, 1H), 7.75-7.70 (m, 1H), 7.48 — 7.43 (m, 1H), 6.99 — 6.94 (m, 1H), 6.86 — 6.81 (m, 1H), 3.58 (s, 2H), 3.10 - 3.00 (m, 2H), 2.71 — 2.64 (m, 2H). tert-Butyl (4-bromobenzyl)(2-{isoquinoline-5-sulfonamido)ethyl}carbamate (113a) oi,
Boc cb >
[00173] NS 112a (2.26 g, 5.38 mmol) and NaHCO: (500 mg, 5.92 mmol) were suspended in THF (15 mL) and cooled to 0°C. Boc:0 (1.26 g, 5.92 mmol) was then carefully added to this mixture, followed by 6 h of stirring. The reaction was diluted with sat. aq. Na2CO: (5 mL), followed by dilution with brine (10 mL) and extraction with DCM (3x). The combined organic layers were dried over
MgSQ,, filtered and concentrated in vacuo. The crude product was purified by column chromatography (20% > 40% EtOAc in pentane) to give title compound 113a (2.42 g, 4.65 mmol, 86%).
H NMR (400 MHz, CDCl3) 8 9.37 (s, 1H), 8.66 (d, J = 6.1 Hz, 1H), 8.45 - 8.32 (m, 2H), 8.21 (d, J = 8.2
Hz, 1H), 7.69 (t, J = 7.8 Hz, 1H), 7.39 — 7.30 {m, 2H), 6.96 (d, J = 8.2 Hz, 2H), 4.27 (s, 2H), 3.39 - 3.20 (m, 2H), 3.11 — 2.89 (m, 2H), 1.43 (s, 9H). 3C NMR (101 MHz, CDCl3) ò 154.93, 153.37, 145.25, 136.89, 133.64, 133.29, 131.83, 131.32, 129.17, 128.94, 126.00, 121.41, 117.41, 81.25, 51.35, 46.77, 42.63, 28.43. tert-Butyl ((6-bromopyridin-3-yl})methyl)(2-(isoquinoline-5-sulfonamido}ethyl}carbamate (113b}
A mn
CO N“ Br
[00174] N 112b (0.73 g, 1.7 mmol) and NaHCO: (0.16 g, 1.9 mmol) were dissolved in THF (10 mL) and cooled to 0°C. Boc20 (0.4 g, 1.9 mmol) was carefully added and the mixture was stirred overnight. The reaction was quenched with sat. aq. Na2CO: (5 mL), washed with brine (10 mL) and extracted with DCM (3x). The organic layers were combined, dried over MgSO., filtered and concentrated in vacuo. This yielded crude product 113b (1.1 g, 1.73 mmol, 100%). which was directly used in the next step. tert-Butyl ((6-bromopyridin-3-yl)methyl)(2-(isoquinoline-5-sulfonamido)ethyljcarbamate (113c)
Hoey
Boc
[00175] N# 112C (0.8 g, 1.76 mmol) and NaHCO: (0.16 g, 1.94 mmol) were dissolved in THF (10 mL) and cooled to 0°C Boc20 (0.4 g, 1.9 mmol) was carefully added and the mixture was stirred overnight. The reaction was diluted with sat. aq. Na2CO: (5 mL), washed with brine (10 mL) and extracted with DCM (3x). The organic layers were combined, dried over MgSO,, filtered and concentrated in vacuo. This yielded crude product 113c (0.98 g, 1.76 mmol, 100%) which was directly used in the next step. tert-Butyl ((6-bromopyridin-3-yl)methyl}{(2-(isoquinoline-5-sulfonamido)ethyljcarbamate (113d)
P345823NL 38
Hy
Boc
[00176] NF 112d (0.60 g, 1.4 mmol) and NaHCO: (0.16 g, 1.9 mmol) were dissolved in THF (10 mL) and cooled to 0°C. Boc20 (0.4 g, 1.9 mmol) was carefully added and the mixture was stirred overnight. The reaction was diluted with sat. aq. Na2CO: (5 mL), washed with brine (10 mL) and extracted with DCM (3x). The organic layers were combined, dried over MgSO., filtered and concentrated in vacuo. This yielded crude product 113d (1.0 g, 1.4 mmol, 100%) which was directly used in the next step. 4-Bromo-N-(2-(isoquinoline-5-sulfonamido)ethyl)benzamide (114)
Py
[00177] N# To a mixture of 111 (0.25 g, 1.0 mmol), 4-bromobenzonic acid (0.26 g, 1.3 mmol) and HATU (0.38 g, 1.0 mmol) in DCM (30 mL) was added DIPEA (0.18 mL, 1.0 mmol), followed by stirring overnight. The reaction mixture was then washed with brine, and the organic layer was dried over Na2SO,, filtered and purified by column chromatography (3% MeOH (10% ag. NHs3) in
DCM) to yield title compound 114 (0.20 g, 0.43 mmol, 43%). ‘H NMR (400 MHz, CDClz) 8 9.25 (s, 1H), 8.56 (d, J= 6.2 Hz, 1H), 8.42 — 8.36 (m, 2H), 8.14 (d, J=8.2
Hz, 1H), 7.66 (t, J=7.8 Hz, 1H), 7.52 — 7.46 (m, 2H), 7.46 — 7.39 (m, 2H), 7.13 (t, J = 5.7 Hz, 1H), 3.76 — 3.83 (m, 2H), 3.54 — 3.48 (m, 2H), 3.23 — 3.18 (m, 2H). 3C NMR (101 MHz, CDCl3) 8 167.62, 153.25, 145.03, 134.15, 133.84, 133.37, 132.40, 131.81, 131.18, 129.10, 128.68, 126.51, 126.15, 117.42, 43.60, 40.12. 2-(Tritylamino)ethan-1-ol (116)
[00178] |" OH To a solution of trityl chloride (1.4 g, 5.0 mmol) and K2CO: (0.76 g, 5.5 mmol) in
DCM (17 mL) at 0 °C was added dropwise ethanolamine (115) (1.5 mL, 25 mmol). The reaction was allowed to warm to room temperature and stirred for 3 h before sat. ag. NaHCO: (15 mL) and H20 (15 mL) were added. The organic layer was collected and the aqueous layer extracted with DCM (3x 30 mL). The combined organic layers were dried over Na2SOyq, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (20% EtOAc in pentane) to yield 116 (1.5 g, 4.95 mmol, 99%). 'H NMR (400 MHz, CDClz) 8 7.65 — 7.52 (m, 6H), 7.42 — 7.31 (m, 6H), 7.27 (t, J=7.3 Hz, 3H), 3.71 (t, J = 5.3 Hz, 2H), 2.69 (bs, 1H), 2.41 (t, J = 5.3 Hz, 2H), 2.06 (bs, 1H).
BC NMR (101 MHz, CDCls) 8 145.88, 128.62, 127.85, 126.34, 70.55, 62.47, 45.60. 2-({4-Bromobenzyl}oxy)-N-tritylethan-1-amine (117) 0
[00179] Br A solution of 116 (1.5 g, 4.95 mmol) in DMF (4 mL) was cooled on ice, and carefully NaH (60% in oil, 0.25 g, 6.25 mmol) was added. 4-Bromo-benzylbromide (0.92 g, 3.3 mmol) was added. The reaction mixture was stirred at room temperature overnight, after which the reaction was
P345823NL 39 quenched with water (10 mL). The mixture was extracted with EtOAc (3x 10 mL). The combined organic layers were dried over Na2SOy, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (20% EtOAc in pentane) to yield title compound 117 (0.78 g, 1.65 mmol, 50%). "HNMR (400 MHz, CDCls) 8 7.51 — 7.41 (m, 8H), 7.29 — 7.22 (m, 6H), 7.20 — 7.12 (m, 5H), 4.37 (s, 2H), 3.58 (t, J= 5.3 Hz, 2H), 2.37 (t, J = 5.3 Hz, 2H). 13C NMR (101 MHz, CDCls) 8 146.17, 137.60, 131.56, 129.30, 128.78, 127.93, 126.38, 121.47, 72.10, 70.76, 70.60, 43.32. 2-((4-(Pyridin-3-yl)benzyl)oxy)-N-tritylethan-1-amine {118}
THAN
Os
[00180] NZ 118 (400 mg, 0.85 mmol, 51%) was synthesized from 117 (780 mg, 1.65 mmol) according to general procedure D.
H NMR (400 MHz, CDCl3) 8 8.84 (dd, J = 2.3, 0.9 Hz, 1H), 8.58 (dd, J = 4.8, 1.6 Hz, 1H), 7.86 (ddd, J = 7.9,2.4,1.6 Hz, 1H), 7.60 — 7.53 (m, 2H), 7.52 — 7.46 (m, 6H), 7.44 — 7.38 (m, 2H), 7.35 (ddd, J= 7.9, 4.8, 0.9 Hz, 1H), 7.30 — 7.23 (m, 6H), 7.20 — 7.15 (m, 3H), 4.50 (s, 2H), 3.65 (t, J= 5.3 Hz, 2H), 2.41 (t, J =5.3 Hz 2H). 3C NMR (101 MHz, CDCl3) ò 148.51, 148.32, 146.17, 138.63, 137.13, 136.45, 134.38, 128.77, 128.34, 127.88, 127.21, 128.33, 123.64, 72.42, 70.75, 70.84, 43.33. 2-((4-(Pyridin-3-yl)benzyl)oxy)ethan-1-amine (119)
HN
Os
[00181] NZ 119 (100 mg, 0.44 mmol, 54%) was synthesized from 118 (380 mg, 0.81 mmol) according to general procedure A. ‘H NMR (400 MHz, CDCl3) 8 8.84 (dd, J = 2.4, 0.9 Hz, 1H), 8.59 (dd, J = 4.8, 1.6 Hz, 1H), 7.87 (ddd, J = 7.9,2.4,1.6 Hz, 1H), 7.61 — 7.55 (m, 2H), 7.50 — 7.44 (m, 2H), 7.37 (ddd, J=7.9, 4.8, 0.9 Hz, 1H), 4.60 (s, 2H), 3.57 (t, J = 5.3 Hz, 2H), 2.93 (t, J = 5.3 Hz, 2H). 3C NMR (101 MHz, CDCl3) 5 148.56, 148.35, 138.44, 137.28, 136.42, 134.40, 128.50, 127.29, 123.66, 72.79, 72.66, 41.99.
[00182] N-{2-({4-Bromobenzyl)}{methyl)amino)ethyl)isoquinoline-5-sulfonamide (120)
Fe
CO | Br
[00183] Nus 112a (0.58 g, 1.4 mmol), formaldehyde (46 mg, 1.5 mmol) and sodium triacetoxyborohydride (0.59 g, 2.8 mmol) were suspended in THF (15 mL) and MeOH (2.5 mL).
The reaction mixture was stirred at room temperature overnight, after which it was diluted with sat. aq.
NaHCO: (10 mL) and extracted with DCM (3x). The organic layers were combined, dried over MgSO,, filtered and concentrated. The crude product was purified by column chromatography (1% > 10% MeOH (10% aq. NHs) in DCM) to give title compound 120 (0.38 g, 0.88 mmol, 64%).
P345823NL 40
[00184] {S}-2-{{{Triisoprapyisilyiioxyimethylipyrrolidine {122a} meso pM
[00185] Following general procedure E, L-prolinot {1214} (5.00 g, 49.4 mmol} was reacted with TIPS-CI (12.7 mL, 59.3 mmol), imidazole (8.73 g, 99.0 mmol) and DMAP, (0.302 g, 2.47 mmol} to afford the title compound as a colourless oil (2.89 g, 11.2 mmol, 23%) which was stored at -20°C io avoid degradation.
TH NMR (400 MHz, CDCl) 8 3.71 - 3.61 {m, 2H), 3.23 - 3.13 (my, 1H), 3.03 - 2.98 (m, 1H), 2.87 - 2.87 im, 1H), 2.05 (s, 1H), 1.82 = 1.70 (m, 2H}, 1.54 — 1.46 (m, 1H), 1.13 - 1.04 (m, 21H).
WG NMR (101 MHz, CDCls} 5 85.92, 80.19, 46.50, 27.35, 25.41, 17.90, 11.87.
[00186] {R}-2-{{{Triisopropyisilyloxyimethylipyrrolidine (122b) meso
[00187] Following general procedure E, R-prolingl (121k) (3.00 g, 29.7 mmol) was reacted with TIPS-CI (7.82 mL, 35.8 mmol}, imidazole {4.04 g, 59.3 mmol) and DMAP (0.181 q, 1.48 mmol) to afford the title compound as a colourless oil (2.40 g, 9.30 mmol, 31%) which was stored at -20°C {o avoid degradation.
TH NMR (300 MHz, CDCl) 6 3.78 — 3.56 (m, ZH), 3.26 — 3.09 (m, 1H), 3.08 - 2.93 (m, 1H), 2.93 - 2.78 {m, 1H), 2.02 (5, 1H), 1.88 - 1.81 (m, 3H), 1.60 ~ 1.41 (m, 18), 1.21 ~ 0.91 (m, 21H).
BC NMR {75 MHz, COC) © 88.00, 80.27, 48.58, 27.43, 25.48, 17.88, 11.84. {2R.58)-2-{4-Bromopheny-&-{{{triisopropyisiiyi)oxyimethylipyrrolidine {123a)
Br
A
[00183] TIPSO Following general procedure F, 1224 (1.57 g, 8.08 mmol} was reacted with n-BuLi {1.6 M in hexanes, 3.8 mL, 8.1 mmol}, benzophenone (1.33 g, 7.31 mmol} and bromobenzenelithium (8.13 mmol, prepared according to general procedure G) to afford the title compound as a crude mixture with benzhydrel (1.5 g crude, 35% purity, 1.6 mmol product, 25%). ‘H NMR (400 MHz, CDCls) 8 7.39 — 7.05 (m, 4H + benzhydrol-Ar), 3.88 — 3.83 {m, 2H}, 3.70 - 3.81 (m, 2H, 312, J=76 4.2 Hz 1H), 2.07 - 1.97 (m, 1H), 1.79 — 1.68 {m, 2H), 1.80 — 1.50 (m, 1H), 1.07 - 1.00 {m, 21H).
BC NMR (101 MHz, CDCi3) § 142.78, 131.38, 128.29 — 81 (+ benzhydrol-Ar), 75.78, 65.26, 62.11, 60.26, 34.28, 27.79, 18.02, 11.90. {25,85)-2-{4-Bromophenyii-&-{{{triisopropyisilyvlioxyimethyiipyrrolidine (123b}
Br
LO
[00189] nps Following general procedure F, 122a (1.57 g, 8.02 mmol} was reacted with n-Buli (1.6 M in hexanes, 3.8 mL, 8.1 mmol), benzophenone (1.33 g, 7.31 mmol} and bromobenzenelithium (8.13 mmol, prepared according to general procedures 3) to afford the titie compound as a crude midure with benzophenone (0.35 g crude, 80% purity, 0.48 mmol product, 89}.
P345823NL 41
TH NMR (400 MHz, CDCl) 5 7.41 - 7.13 {m, 4H + benzophenone-Ar}, 4.23 - 4.15 (m, 1H), 3.83 (dd, J = 57, 1.7 Hz, 2H}, 3.83 3.45 (m, 1H), 2.21 = 2.12 {m, 1H}, 2.03 ~ 1.82 {m, 1H), 1.86 ~ 1.53 (m, 2H}, 1.09 -1.04 (m, 21H).
BC NMR (101 MHz, CDOls)} 5 144.45, 131.38-127 48 (+ benzophenone-Ar), 120.37, 68.05, 60.42, 59.86, 25.09, 27.70, 18.13, 12.04.
[00130] i23,58}-2-{4-Bromophenyi}-5-{{{triisopropyisilyjoxy}methyBpyrrolidine {123c}
Br 0
[00191] need" Following general procedure F, 122b {2.39 4, 9.28 mmol) was reacted with n-BuLi {1.6 M in hexanes, 5.8 mL, 9.3 mmel), benzophenone (2.03 g, 11.1 mmol) and bromobenzaenelithium (13.98 mmol, prepared according to general procedure G) to afford the title compound as a crude mixture with benzhydrol (2.15 g crude, 50% purity, 2.81 mmol product, 28%).
TH NMR (300 MHz, COCK) 6 7.41~ 7.13 (m, 4H + benzhydrobAr, 4.08 — 3.87 (m, 11), 3.79 ~ 3.57 (m, 2H), 3.32 ~ 3.11 (my, 2H), 2.16 ~ 1.98 (my, 1H), 1.80 —~ 1.67 {m, 2H), 1.87 — 1.47 (m, 1H), 1.18 ~ 0.83 (im, 21H).
BC NMR (75 MHz, CDCI) 8 143.13, 130.44, 127.50-125.63 (+ benzhydrolAn, 126.48, 119.58, 65.14, 81.23, 59.48, 332.43, 28.85, 17.14, 11.05.
[00192] {ZR 5R}-2-{4-Bromophenyi}-S-{{{triisopropyisilylloxyimethyiipyrrolidine (123d)
Br 5 esd Following general procedure F, 122b (2.39 g, 9.28 mmol) was reacted with n-BuLi (1.6 M in hexanes, 5.8 mL, 9.3 mmol), benzophenone (2.03 g, 11.1 mmol) and bromobenzenelithium (13.9 mmol, prepared according to general procedure G) to afford the title compound as a crude mixture with benzophenone (0.430 g crude, 60% purity, 0.625 mmol product, 7%). ‘H NMR (300 MHz, CDClz} § 7.43 — 7.21 (m, 4H + benzophenone-Ar), 4.26 — 4.15 (m, 1H), 3.68 — 3.58 (m, 2H), 3.56 — 3.46 (m, 1H), 2.21 -= 2.13 (m, 1H), 2.03 — 1.93 (m, 1H), 1.74 — 1.53 (m, 2H), 1.08 — 1.04 (m, 21H). 3C NMR (75 MHz, CDCls) ò 143.60, 130.61-125.14 (+ benzophenone-Ar), 119.36, 65.12, 59.43, 58.92, 3416, 26.73, 17.15, 11.07. tert-Butyl (2R,55)-2-(4-bromophenyl)-5-({(triisopropylsilyljoxy)methyl)pyrrolidine-1-carboxylate (1243)
Br
[00133] TIPSO Following general procedure H, 123a (0.85 g crude, 35% purity, 0.91 mmol product) was reacted with Boc2O (494 mg, 2.26 mmol) and triethylamine (0.252 mL, 1.81 mmol) for 1.5 h to afford the title compound as a colourless oil (0.315 g, 0.615 mmol, 68%). 'H NMR (500 MHz, 333 K, CDClI3) 6 7.38 (d, J= 8.4 Hz, 2H), 7.16 (d, J= 8.4 Hz, 2H), 4.69 (bs, 1H), 4.07 -3.99 (m, 1H), 3.88 — 3.76 (m, 2H), 2.25 -2.15(m, 1H), 2.13 - 2.04 (m, 1H), 1.99 - 1.91 (m, 1H), 1.91 — 1.83 (m, 1H), 1.32 —1.23 (m, 9H), 1.10 - 1.05 (m, 21H).
P345823NL 42 8C NMR (126 MHz, 333 K, CDCl) 8 155.22, 144.18, 131.36, 127.76, 120.25, 79.79, 64.84, 62.84, 60.89, 34.60, 28.48, 27.21, 18.22, 12.35.
[00194] tert-Butyl (25,5S)-2-(4-bromophenyl)-5-(((triisopropylsilyl)oxy}methyl)pyrrolidine-1- carboxylate (124b)
Br ae CT
[00195] nps Following general procedure H, 123b (0.16 g crude, 60% purity, 0.23 mmol product) was reacted with Boc2O (36 mg, 0.16 mmol) and triethylamine (18 pL, 0.13 mmol) for 4.5 h to afford the title compound as a colourless oil (45 mg, 65 pmol, 28%).
Rotamer equilibrium (3:2).
Major rotamer 'H NMR (300 MHz, CDCl3) ò 7.41 (d, J = 8.5 Hz, 2H), 7.00 (d, J= 8.4 Hz, 2H), 4.79 (d, J = 8.5Hz, 1H), 4.18 (ddt, J= 7.7, 5.2, 2.6 Hz, 1H), 3.98 (dd, J = 9.8, 5.2 Hz, 1H), 3.80 (dd, J =9.8, 2.8 Hz, 1H), 2.66 — 2.50 (m, 1H), 2.10 — 1.90 (m, 2H), 1.60 (dd, J =12.5, 6.1 Hz, 1H), 1.15 (s, 8H), 1.11 - 1.03 (m, 21H).
Major rotamer 13C NMR (75 MHz, CDCls) ö 153.98, 145.26, 131.28, 127.12, 120.02, 79.33, 63.61, 61.95, 59.77, 33.46, 28.24, 25.18, 18.14, 12.10.
Minor rotamer *H NMR (300 MHz, CDCls) 8 7.41 (d, J=8.5Hz, 2H), 7.00 (d, J= 8.4 Hz, 2H), 4.91 (d, J = 8.5 Hz, 1H), 4.12 -4.01 (m, 1H), 3.91 (dd, J = 9.3, 3.4 Hz, 1H), 3.61 (dd, J= 9.4, 7.8 Hz, 1H), 2.50 - 2.31 {m, 1H), 2.00 (m, 2H), 1.60 (dd, J =12.5, 6.1 Hz, 1H), 1.44 (s, 9H), 1.13 — 1.03 (m, 21H).
Minor rotamer 13C NMR (75 MHz, CDCls) 8 153.98, 143.67, 131.55, 126.98, 120.02, 79.93, 63.61, 61.32, 59.74, 31.97, 28.64, 25.18, 18.14, 12.10. tert-Butyl (2S,5R)-2-(4-bromophenyl}-5-({(triisopropylsilyl}oxy)methyl)pyrrolidine-1-carboxylate {124c)
Br ge CV
[00196] need] Following general procedure H, 123c (2.15 g crude, 50% purity, 2.61 mmol product) was reacted with BoczO (1.42 g, 6.52 mmol) and triethylamine (0.727 mL, 5.22 mmol) for 1.5 h to afford the title compound as a colourless oil (0.904 g, 1.76 mmol, 67%). "HNMR (500 MHz, 333 K, CDCl3) 6 7.38 (d, J = 8.4 Hz, 2H), 7.16 (d, J = 8.4 Hz, 2H), 4.69 (bs, 1H), 4.06 —3.99 (m, 1H), 3.97 (dd, J = 9.4, 3.7 Hz, 1H), 3.86 (s, 1H), 2.28 —- 2.14 (m, 1H), 2.14 — 2.03 (m, 1H), 2.03 -1.91 (m, 1H), 1.91 — 1.82 (m, 1H), 1.27 (s, 9H), 1.13 — 1.03 (m, 21H). 3C NMR (126 MHz, 333 K, CDCls) 8 155.24, 144.32, 131.37, 127.77, 120.26, 79.80, 64.85, 62.85, 60.91, 34.55, 28.49, 27.20, 18.23, 12.36.
[00197] tert-Butyl (2R,5R)-2-(4-bromophenyl}-5-(((triisopropylsilyl)oxy)methyl}pyrrolidine-1- carboxylate (124d)
Br
[00198] mesg Following general procedure H, 123d (0.430 g crude, 60% purity, 0.625 mmol product) was reacted with Boc2O (0.341 g, 1.56 mmol} and triethylamine (0.174 mL, 1.25 mmol} for 4.5 h to afford the title compound as a colourless oil (0.133 g, 0.252 mmol, 44%).
P345823NL 43
Rotamer equilibrium (3:2).
Major rotamer 'H NMR (300 MHz, CDCI3) 6 7.41 (d, J=8.5Hz, 2H), 7.00 (d, J= 8.4 Hz, 2H), 4.79 (d, J = 8.5 Hz, 1H), 4.18 (ddt, J= 7.7, 5.2, 2.6 Hz, 1H), 3.98 (dd, J = 9.8, 5.2 Hz, 1H), 3.81 (dd, J = 9.8, 2.8 Hz, 1H), 2.66 — 2.48 (m, 1H), 2.13 — 1.87 (m, 2H), 1.60 (dd, J = 11.0, 6.2 Hz, 1H), 1.15 (s, 9H), 1.11 — 1.01 (m, 21H).
Major rotamer '*C NMR (75 MHz, CDCls) ò 153.94, 145.22, 131.25, 127.09, 120.00, 79.28, 63.59, 61.92, 59.74, 33.44, 28.21, 25.16, 18.12, 12.08.
Minor rotamer "H NMR (300 MHz, CDCls) 8 7.41 (d, J = 8.5 Hz, 2H), 7.00 (d, J= 8.4 Hz, 2H), 4.91 (d, J = 8.4 Hz, 1H), 4.12-4.02 (m, 1H), 3.91 (dd, J= 9.3, 3.4 Hz, 1H), 3.61 (dd, J= 9.4, 7.8 Hz, 1H), 2.48 - 2.35 (m, 1H), 2.13 -1.89 (m, 2H), 1.60 (dd, J = 11.4, 5.8 Hz, 1H), 1.44 (s, 9H), 1.12 — 1.04 (m, 21H).
Minor rotamer 13C NMR (75 MHz, CDCls) 8 153.80, 143.64, 131.52, 126.95, 120.27, 79.88, 63.59, 61.29, 59.71, 31.94, 28.61, 25.16, 18.12, 12.08. tert-Butyl (2R,5S)-2-(4-bromophenyl}-5-(hydroxymethyl)pyrrolidine-1-carboxylate (125a)
Br
[060189] HO Following general procedure |, 124a (0.494 g, 0.963 mmol) was reacted with TBAF (1 M in THF, 4.82 mL, 4.82 mmol) for 2.5 h to afford the title compound as a colourless oil (0.313 g, 0.877 mmol, 91%). 'H NMR (400 MHz, CDClIz) 8 7.44 (d, J= 8.4 Hz, 2H), 7.13 (d, J = 8.5 Hz, 2H), 4.79 (t, J = 7.0 Hz, 1H), 416 (p, J=6.3 Hz, 1H),3.78 (d, J =8.9 Hz, 2H), 2.34 - 2.15 (m, 1H), 2.11 — 1.91 (m, 1H), 1.90 - 1.73 (m, 1H), 1.63 (bs, 1H), 1.21 (s, SH).
BC NMR (101 MHz, CDCl) 6 143.41, 131.49, 127.42, 120.43, 80.97, 67.55, 62.96, 61.68, 34.36, 28.17, 27.19. tert-Butyl (2S,5S)-2-(4-bromophenyl)-5-(hydroxymethyl)pyrrolidine-1-carboxylate (125b)
Br gee I
[00200] wo Following general procedure |, 124b (48 mg, 94 pmol) was reacted with
TBAF (1 M in THF, 0.468 mL, 0.468 mmol) for 1.25 h to afford the title compound as a colourless oil (19 mg, 53 umol, 57%).
H NMR (300 MHz, CDCls} 5 7.43 (d, J = 8.4 Hz, 2H), 6.99 (d, J = 8.1 Hz, 2H), 4.83 (dd, J= 8.1, 2.4 Hz, 1H), 4.31 (tt, J = 7.3, 3.4 Hz, 1H), 3.85 - 3.64 (m, 2H), 2.45 -2.26 (m, 1H), 2.17 —- 1.98 (m, 1H), 1.75 - 1.57 (m, 2H), 1.16 (s, SH). 3C NMR (75 MHz, CDCls) 6 156.33, 144.10, 131.45, 127.04, 120.35, 80.68, 67.35, 62.46, 61.27, 33.51, 28.16, 26.18. tert-Butyl (2S,5R)})-2-(4-bromophenyl)-5-(hydroxymethyl}pyrrolidine-1-carboxylate (125c)
Br ge CT
[00201] vo] Following general procedure |, 124¢ (0.910 g, 1.78 mmol) was reacted with
TBAF (1 Min THF, 8.87 mL, 8.87 mmol) for 2.5 h to afford the title compound as a colourless oil (0.527 g, 1.478 mmol, 83%).
P345823NL 44 'H NMR (400 MHz, CDCls) 8 7.44 (d, J= 8.4 Hz, 2H), 7.13 (d, J = 8.2 Hz, 2H), 4.80 (t, J = 7.0 Hz, 2H), 422-412 (m, 1H), 3.78 (t, J = 5.4 Hz, 2H), 2.33 - 2.19 (m, 1H), 2.08 — 1.96 (m, 2H), 1.88 - 1.75 (m, 1H), 1.62 (bs, 1H), 1.21 (s, 9H).
BC NMR (101 MHz, CDCls) 6 131.53, 127.45, 120.48, 81.05, 67.73, 63.01, 61.75, 34.38, 28.21, 27.20.
[00202] tert-Butyl (2R,5R)-2-(4-bromophenyl}-5-(hydroxymethyl)pyrrolidine-1-carboxylate (125d)
Br
[00203] no’ Following general procedure |, 124d (0.133 g, 0.259 mmol) was reacted with TBAF (1 M in THF, 1.30 mL, 1.30 mmol) for 1.25 h to afford the title compound as a colourless oil (98 mg, quant). 'H NMR (300 MHz, CDCl) 8 7.43 (d, J= 8.4 Hz, 2H), 6.99 (d, J = 8.4 Hz, 2H), 4.83 (dd, J= 8.2, 2.4 Hz, 1H), 4.30 (tt, J=7.2, 3.2 Hz, 1H), 3.87 — 3.55 (m, 2H), 2.51 — 2.23 (m, 2H), 2.19 — 1.98 (m, 1H), 1.77 — 1.57 (m, 2H), 1.16 (s, SH).
BC NMR (75 MHz, CDCl) 6 156.26, 144.09, 131.43, 127.02, 120.32, 80.63, 67.16, 62.42, 61.22, 33.46, 28.14, 26.11. tert-Butyl (2R,55)-2-(4-bromophenyl}-5-({(methylsulfonyl}oxy)methyl}pyrrolidine-1-carboxylate (126a)
Br
[00204] MsO Following general procedure J, 125a (0.313 g, 0.877 mmol) was reacted with MsCI (0.102 mL, 1.32 mmol) and triethylamine (0.367 mL, 2.63 mmol) to afford the crude title compound (0.393 g, quant.). ‘H NMR (300 MHz, CDCl) 6 7.44 (d, J= 8.4 Hz, 2H), 7.11 (d, J = 8.4 Hz, 2H), 4.68 (bs, 1H), 4.49 (dd, J = 9.5,3.5Hz, 1H), 4.39 (bs, 1H), 4.23 (bs, 1H), 3.04 (s, 3H), 2.39 - 2.18 (m, 1H), 2.17 — 1.95 (m, 2H), 1.94 — 1.78 (m, 1H), 1.40 — 1.06 (m, 9H).
[00205] tert-Butyl (2S,5S)-2-(4-bromophenyl}-5-(((methylsulfonyl}oxy)methyl)pyrrolidine-1- carboxylate (126b)
Br ge CI wee’ Following general procedure J, 125b (19 mg, 53 pmol) was reacted with MsCl (6.2
HL, 80 umol) and triethylamine (22 HL, 0.16 mmol) to afford the crude title compound (24 mg, quant.).
Rotamer equilibrium (3:1).
Major rotamer 'H NMR (400 MHz, CDClI3) 8 7.43 (d, J=8.4 Hz, 2H), 6.97 (d, J=8.4 Hz, 2H), 4.85(d, J = 8.3 Hz, 1H), 4.49 — 4.40 (m, 1H), 4.40 — 4.32 (m, 2H), 3.05 (s, 3H), 2.55 - 2.34 (m, 1H), 2.21 - 2.05 (m, 1H), 1.96 (dd, J= 13.3, 7.2 Hz, 1H), 1.72 (dd, J = 12.6, 7.1 Hz, 1H), 1.17 (s, 9H).
Major rotamer *C NMR (101 MHz, CDCls) § 143.89, 131.48, 126.92, 120.46, 80.40, 69.28, 61.62, 56.86, 37.19, 32.98, 28.15, 25.15;
Minor rotamer 'H NMR (400 MHz, CDCls} 5 7.43 (d, J=8.4 Hz, 2H), 6.97 (d, J=8.4 Hz, 2H), 4.97 (d, J = 8.3 Hz, 1H), 4.49 — 4.41 (m, 1H), 4.30 — 4.24 (m, 1H), 4.19 (dd, J = 9.6, 7.3 Hz, 1H), 3.05 (s, 3H), 2.59 —
P345823NL 45 2.29 (m, 1H), 2.23 — 2.04 (m, 1H), 1.96 (dd, J= 13.3, 7.2 Hz, 1H), 1.72 (dd, J= 12.6, 7.1 Hz, 1H}, 1.47 (s,
SH).
Minor rotamer *C NMR (101 MHz, CDCI) ò 143.89, 131.71, 126.80, 120.46, 80.40, 68.74, 61.13, 56.78, 37.70, 31.92, 28.53, 25.72. tert-Butyl (2S,5R)-2-(4-bromophenyl}-5-(((methylsulfonyl)oxy)methyl)pyrrolidine-1-carboxylate (126¢)
Br gee CJ
[00206] ed Following general procedure J, 125c (0.526 g, 1.48 mmol) was reacted with MsCI {0.171 mL, 2.22 mmol) and triethylamine (0.618 mL, 4.43 mmol) to afford the crude title compound (0.691 g, quant.). "HNMR (300 MHz, CDCls) 8 7.43 (d, J= 8.5 Hz, 2H), 7.12 (d, J = 8.5 Hz, 2H), 4.68 (bs, 1H), 4.49 (dd, J = 9.5, 3.5 Hz, 1H), 4.39 (bs, 1H), 4.28 — 4.15 (m, 1H), 3.04 (s, 3H), 2.38 — 2.20 (m, 1H), 2.19 - 1.95 (m, 2H), 1.95 — 1.80 (m, 1H), 1.19 (s, SH).
BC NMR (75 MHz, CDCI) ö 154.85, 146.74, 131.45, 127.37, 120.45, 80.58, 69.62, 62.46, 57.62, 45.95, 37.39, 28.17, 27.17. tert-Butyl (2R,5R)-2-(4-bromophenyl)-5-({{methylsulfonyl}oxy)methyl)pyrrolidine-1-carboxylate (126d)
Br
[00207] meo” Following general procedure J, 125d (98 mg, 0.28 mmol) was reacted with
MsCI (32 HL, 0.41 mmol) and triethylamine (0.115 mL, 0.824 mmol) to afford the crude title compound (0.121 g, quant).
Rotamer equilibrium (3:1).
Major rotamer *H NMR (300 MHz, CDCl3) 8 7.43 (d, J = 8.4 Hz, 2H), 6.98 (d, J= 8.3 Hz, 2H), 4.85 (d, J= 8.3 Hz, 1H), 4.54 — 4.31 (m, 3H), 3.05 (s, 3H), 2.56 — 2.31 (m, 1H), 2.25 - 2.04 (m, 1H), 1.96 (dd, J = 13.3, 7.3 Hz, 1H), 1.72 (dd, J = 12.5, 7.0 Hz, 1H), 1.17 (s, SH).
Major rotamer 13C NMR (75 MHz, CDCIz) ò 153.94, 143.84, 131.38, 126.86, 120.36, 80.27, 69.18, 61.53, 56.78, 37.10, 32.89, 28.06, 25.06.
Minor rotamer *H NMR (300 MHz, CDCI3) 8 7.43 (d, J = 8.4 Hz, 2H), 6.98 (d, J= 8.3 Hz, 2H), 4.96 (d, J = 8.3 Hz, 1H), 4.84 — 4.30 (m, 1H), 4.27 (dd, J=7.6, 2.6 Hz, 1H), 4.20 (dd, J= 9.4, 7.3 Hz, 1H}, 3.06 (s, 3H), 2.61 -2.31 (m, 1H), 2.25 — 2.04 (m, 1H), 1.96 (dd, J = 13.3, 7.3 Hz, 1H), 1.72 (dd, J = 12.5, 7.0 Hz, 1H), 1.47 (s, 9H).
Minor rotamer '3C NMR (75 MHz, CDCI) 8 153.94, 143.84, 131.81, 126.75, 120.36, 80.90, 68.73, 61.04, 56.70, 37.60, 31.85, 28.43, 25.64. tert-Butyl (2S,5R)})-2-(azidomethyl)-5-(4-bromophenyl)pyrrolidine-1-carboxylate {1273}
P345823NL 48
Br
[00208] N= Following general procedure K, 126a (0.391 g, 0.905 mmol) was reacted with
NaN: (0.353 g, 5.43 mmol) for 65 h to afford the title compound as a colourless oil (0.239 g, 0.627 mmol, 69%). 'H NMR (500 MHz, 333 K, CDCl) 8 7.42 (d, J=8.4 Hz, 2H), 7.12 (d, J= 8.4 Hz, 2H), 4.73 (t, J= 7.2 Hz, 1H), 4.08 (h, J = 3.7 Hz, 1H), 3.70 (dd, J= 12.0, 3.9 Hz, 1H), 3.49 (dd, J = 12.0, 7.5 Hz, 1H), 2.31 - 2.20 (m, 1H), 2.10 = 1.97 (m, 1H), 1.93 — 1.80 (m, 2H), 1.29 (s, 9H). 13C NMR (126 MHz, 333 K, CDCls) 8 154.10, 142.40, 130.60, 126.58, 119.60, 79.54, 61.74, 57.63, 53.52, 33.15, 27.42, 27.23. tert-Butyl (2S,5S)-2-(azidomethyl}-5-(4-bromophenyl)pyrrolidine-1-carboxylate (127b}
Br ee OI
[00209] ‘ae Following general procedure K, 126b (24 mg, 55 ymol) was reacted with
NaNs (21 mg, 55 pmol) for 17 h to afford the title compound as a colourless oil (11 mg, 29 pmol, 53%).
Rotamer equilibrium (7:3).
Major rotamer 'H NMR (400 MHz, CDClI3) 8 7.43 (d, J=8.4 Hz, 2H), 6.98 (d, J=8.4 Hz, 2H), 4.86 (d, J = 7.7 Hz, 1H), 4.30 - 4.19 (m, 1H), 3.66 (dd, J=12.1, 6.5 Hz, 1H), 3.50 (dd, J = 12.1, 2.9 Hz, 1H), 2.57 - 235(m, 1H), 2.15-2.00 (m, 1H), 1.89 — 1.77 (m, 1H), 1.76 — 1.63 (m, 1H), 1.17 (s, 9H).
Major rotamer 13C NMR (101 MHz, CDCls) ö 153.02, 143.21, 130.46, 125.99, 119.39, 79.20, 60.78, 56.50, 51.49, 32.06, 27.20, 24.93.
Minor rotamer 'H NMR (400 MHz, CDCl3) 8 7.43 (d, J = 8.4 Hz, 2H), 6.98 (d, J= 8.4 Hz, 2H), 4.96 (d, J= 8.4 Hz, 1H), 4.14 (td, J= 8.1, 2.9 Hz, 1H), 3.57 (dd, J = 11.9, 2.7 Hz, 1H), 3.33 (dd, J= 11.9, 8.0 Hz, 1H), 264-229 (m, 1H), 2.15-1.96 (m, 1H), 1.83 — 1.79 (m, 1H), 1.78 — 1.64 (m, 1H), 1.47 (s, 9H).
Minor rotamer 13C NMR (101 MHz, CDClIz) 8 153.02, 141.83, 130.69, 125.87, 119.59, 79.71, 60.18, 56.45, 52.49, 30.94, 27.59, 25.55. tert-Butyl (2R,5S5)-2-(azidomethyl)-5-(4-bromophenyl)pyrrolidine-1-carboxylate (127c} cr
Boc
[00210] Ns Following general procedure K, 126c (0.691 g, 1.60 mmol) was reacted with
NaN: (0.621 g, 9.55 mmol) for 65 h to afford the title compound as a colourless oil (0.370 g, 0.970 mmol, 61%).
TH NMR (126 MHz, 333 K, CDCI:) 86 7.42 (d, J=8.5Hz, 2H), 7.12 (d, J=8.4 Hz, 2H), 4.72 (t, J = 7.1 Hz, 1H), 4.08 (h, J = 3.7 Hz, 1H), 3.70 (dd, J= 11.9, 3.8 Hz, 1H), 3.49 (dd, J= 11.7, 7.4 Hz, 1H), 2.31 - 2.18 (m, 1H), 2.11 = 1.96 (m, 1H), 1.93 — 1.78 (m, 2H), 1.29 (s, 9H).
BC NMR (126 MHz, 333 K, CDCls) 8 154.09, 142.41, 130.60, 126.58, 119.59, 79.53, 61.74, 57.62, 53.52, 33.16, 27.42, 27.22. tert-Butyl (2R,5R)-2-(azidomethyl}-5-(4-bromophenyl)pyrrolidine-1-carboxylate (127d)
P345823NL 47
Br
[00211] Ne Following general procedure K, 126d (0.121 g, 0.278 mmol) was reacted with
NaN: (0.109 g, 1.67 mmol) for 17 h to afford the title compound as a colourless oil (56 mg, 0.147 mmol, 53%).
Rotamer equilibrium (7:3).
Major rotamer 'H NMR (300 MHz, CDClI3) § 7.43 (d, J=8.5Hz, 2H), 6.98 (d, J= 84 Hz, 2H), 4.86 (d, J = 8.2 Hz, 1H), 4.33 - 4.21 (m, 1H), 3.66 (dd, J= 12.1, 6.5 Hz, 1H), 3.50 (dd, J= 12.1, 2.9 Hz, 1H), 2.56 — 2.32 (m, 1H), 2.17 — 2.00 (m, 1H), 1.88 —- 1.76 (m, 1H), 1.76 — 1.62 (m, 1H), 1.17 (s, 9H).
Major rotamer 13C NMR (75 MHz, CDCls) 8 152.97, 143.19, 130.42, 125.96, 119.35, 79.14, 60.75, 56.47, 51.45, 32.02, 27.16, 24.90.
Minor rotamer *H NMR (300 MHz, CDCls) 8 7.43 (d, J=8.5Hz, 2H), 6.98 (d, J=8.4 Hz, 2H), 4.96 (d, J = 8.3 Hz, 1H), 4.20 — 4.04 (m, 1H), 3.57 (dd, J = 12.0, 2.9 Hz, 1H), 3.33 (dd, J= 11.9, 7.9 Hz, 1H), 2.60 — 2.31 (m, 1H), 2.20 — 2.00 (m, 1H), 1.80 — 1.76 (m, 1H), 1.76 — 1.60 (m, 1H), 1.47 (s, 9H).
Minor rotamer 13C NMR (75 MHz, CDCl3) 5 152.97, 141.80, 130.64, 125.84, 119.55, 79.65, 60.14, 56.42, 52.46, 30.91, 27.55, 25.52. tert-Butyl (2S,5R)-2-(aminomethyl)-5-(4-bromophenyl)pyrrolidine-1-carboxylate (128a)
Br
[00212] H=N Following general procedure L, 127a (0.221 g, 0.597 mmol) was reacted with PPh; (0.304 g, 1.16 mmol) and water (21 pL, 1.16 mmol) to afford the tile compound as a colourless oil that crystalized upon storage (0.130 g, 0.366 mmol, 63%). ‘H NMR (500 MHz, 333K, CDCl3) 5 7.41 (d, J = 8.4 Hz, 2H), 7.11 (d, J=8.4 Hz, 2H), 4.73 (t, J= 7.3 Hz, 1H), 3.98 —-3.93 (m, 1H), 3.09 (dd, J=12.6, 5.6 Hz, 1H), 2.77 (dd, J= 12.6, 7.3 Hz, 1H), 2.30 - 2.18 (m, 1H), 2.08 — 1.96 (m, 1H), 1.88 — 1.73 (m, 2H), 1.49 (s, 2H), 1.28 (s, 9H). 3C NMR (126 MHz, 333K, CDCls) 5 154.64, 142.98, 130.56, 126.58, 119.41, 79.03, 61.73, 61.07, 45.74, 33.35, 27.44, 27.29. tert-Butyl (25,55)-2-(aminomethyl)-5-(4-bromophenyl)pyrrolidine-1-carboxylate (128b}
Br ge CI pan Following general procedure L, 127b (11 mg, 29 pmol} was reacted with PPh; (17 mg, 65 Hmol) and water (2 HL, 0.1 mmol) to afford the tile compound as a colourless oil that crystalized upon storage (8.7 mg, 24 umol, 83%).
Rotamer equilibrium (8:2).
Major rotamer "H NMR (400 MHz, CDCls) 8 7.43 (d, J=8.4 Hz, 2H), 6.98 (d, J= 8.5 Hz, 2H), 4.82 (d, J = 6.3Hz, 1H), 4.34 — 4.13 (m, 1H), 3.95 (bs, 2H), 3.14 (dd, J = 12.9, 5.9 Hz, 1H), 2.94 (dd, J = 12.9, 5.6 Hz, 1H), 2.42 — 2.27 (m, 1H), 2.20 — 2.03 (m, 1H), 1.87 — 1.74 (m, 1H), 1.74 — 1.62 (m, 1H), 1.15 (s, 9H).
Major rotamer 13C NMR (101 MHz, CDCls) 5 154.33, 142.97, 130.46, 126.04, 119.41, 79.59, 76.48, 61.06, 58.44, 44.14, 31.99, 27.16, 25.47.
P345823NL 48
Minor rotamer 'H NMR (400 MHz, CDCI3) ò 7.43 (d, J=8.4 Hz, 2H), 6.98 (d, J=8.5Hz, 2H), 4.94 (d, J = 8.4 Hz, 1H), 3.95 (bs, 3H), 3.05 (dd, J=12.5, 3.3 Hz, 1H), 2.69 (dd, J=12.6, 8.3 Hz, 1H), 2.42 - 2.27 (m, 1H), 2.20 — 2.03 (m, 1H), 1.87 —= 1.74 (m, 1H), 1.74 — 1.62 (m, 1H), 1.45 (s, SH).
Minor rotamer 13C NMR (101 MHz, CDCls) 8 154.33, 142.16, 130.61, 125.95, 119.41, 79.16, 76.48, 60.07, 59.71, 43.79, 31.01, 27.61, 24.87. tert-Butyl (2R,5S)-2-(aminomethyl)-5-(4-bromophenyl}pyrrolidine-1-carboxylate {128c} cr
Boc
[00213] H2N Following general procedure L, 127c (0.359 g, 0.942 mmol) was reacted with PPh3 (0.494 g, 1.83 mmol) and water (34 pL, 1.9 mmol) to afford the title compound as a colourless oil that crystalized upon storage (0.236 g, 0.066 mmol, 70%). "HNMR (500 MHz, CDCls) 8 7.41 (d, J=8.4 Hz, 2H), 7.11 (d, J = 8.4 Hz, 2H), 4.73 (t, J = 7.3 Hz, 1H), 4.00 - 3.88 (m, 1H), 3.09 (dd, J= 12.6, 5.7 Hz, 1H), 2.78 (dd, J= 12.6, 7.3 Hz, 1H), 2.30 — 2.20 (m, 1H), 2.06 — 1.96 (m, 1H), 1.87 — 1.75 (m, 2H), 1.28 (s, 9H).
BC NMR (126 MHz, CDCls) 8 154.65, 142.97, 130.55, 126.57, 119.41, 79.04, 61.74, 61.04, 45.73, 33.34, 27.44, 27.29. tert-Butyl (2R,5R)-2-(aminomethyl)-5-(4-bromophenyl)pyrrolidine-1-carboxylate (128d)
Br
[00214] HN Following general procedure L, 127d (56 mg, 0.147 mmol) was reacted with
PPh; (77 mg, 0.30 mmol) and water (5.3 pL, 0.23 mmol) to afford the title compound as a colourless oil that crystalized upon storage (34 mg, 95 umol, 65%).
Rotamer equilibrium (8:2).
Major rotamer 'H NMR (400 MHz, CDClI3) 8 7.43 (d, J = 8.4 Hz, 2H), 6.98 (d, J= 8.5 Hz, 2H), 4.82 (d, J = 6.3 Hz, 1H), 4.25 (bs, 3H), 3.14 (dd, J = 12.8, 5.5 Hz, 1H), 2.91 (dd, J= 12.8, 6.1 Hz, 1H), 2.45 - 2.23 (m, 1H), 2.19 — 2.03 (m, 1H), 1.92 = 1.75 (m, 1H), 1.75 — 1.59 (m, 1H), 1.15 (s, 9H).
Major rotamer 13C NMR (101 MHz, CDCls) ò 155.11, 144.02, 131.41, 127.01, 120.35, 80.41, 61.95, 59.46, 44.82, 32.91, 28.14, 26.21.
Minor rotamer 'H NMR (400 MHz, CDClI3) 8 7.43 (d, J = 8.4 Hz, 2H), 6.98 (d, J=8.5Hz, 2H), 4.94 (d, J = 8.4 Hz, 1H), 4.25 (bs, 3H), 3.98 (td, J = 8.2, 3.3 Hz, 1H), 3.06 (dd, J= 12.7, 3.4 Hz, 1H), 2.70 (dd, J = 12.6, 8.5 Hz, 1H), 2.45 - 2.23 (m, 1H), 2.19 - 2.03 {(m, 1H), 1.92 = 1.75 (m, 1H), 1.75 - 1.59 (m, 1H), 1.45 (s, SH).
Minor rotamer 13C NMR (101 MHz, CDClIz) & 153.86, 143.13, 131.58, 126.92, 120.40, 80.14, 61.03, 60.58, 44.85, 31.98, 28.58, 25.82. tert-Butyl (2R,5S)-2-(4-bromophenyl}-5-((isoquinocline-5-sulfonamido)methyl)pyrrolidine-1- carboxylate (129a)
P345823NL 49
Br of 5) 0
[00215] Nx Following general procedure B, 128a (0.113 g, 0.318 mmol) was reacted with quinocline-5-sulfonylchloride-HCI (87 mg, 0.38 mmol) and triethylamine (0.100 mL, 0.716 mmol) to afford the title compound as an off-white solid (0.166 g, 0.304 mmol, 95%). 'H NMR (400 MHz, CDCl3) 8 9.40 (s, 1H), 8.66 (d, J=6.2 Hz, 1H), 8.49 (d, J = 6.2 Hz, 1H), 8.45 (dd, J = 7.4,12Hz 1H), 8.25 (d, J= 8.2 Hz, 1H), 7.72 (t, J= 7.3 Hz, 1H), 7.56 (bs, 1H), 7.20 (d, J = 8.0 Hz, 2H), 6.75 (d, J=7.9 Hz, 2H), 4.62 (t, J = 7.5 Hz, 1H), 4.10 (t, J = 8.8 Hz, 1H), 3.29 — 2.95 (m, 2H), 2.23 - 2.12 (m, 1H), 2.07 — 1.90 (m, 1H), 1.73 — 1.56 (m, 2H), 1.15 (s, 9H).
BC NMR (101 MHz, CDCls) 8 156.78, 153.18, 145.19, 142.60, 134.41, 133.33, 133.14, 131.30, 131.22, 129.05, 126.92, 125.92, 120.29, 117.47, 81.15, 62.97, 58.07, 48.92, 34.35, 29.66, 27.95. tert-Butyl (2S,5S)-2-(4-bromophenyl)-5-{(isoquinoline-5-sulfonamido)methyl)pyrrolidine-1- carboxylate (129b)
Br o=S-ni nel 0
[00216] Nx Following general procedure B, 128b (8.7 mg, 24 pmol) was reacted with quinoline-5-sulfonylchloride: HCI (8 mg, 0.04 mmol) and triethylamine (10 pL, 73 pmol) to afford the title compound as an off-white solid (11 mg, 20 uymol, 84%).
IH NMR (400 MHz, CDCl3) 8 9.37 (s, 1H), 8.70 (d, J = 6.2 Hz, 1H), 8.45 (d, J = 6.1 Hz, 1H), 8.43 (dd, J = 7.4,1.2Hz, 1H), 8.21 (d, J=8.2 Hz, 1H), 7.79 — 7.64 (m, 1H), 7.40 (d, J = 8.5 Hz, 2H), 6.88 (d, J= 8.4
Hz, 2H), 4.72 (d, J=6.5 Hz, 1H}, 4.25 — 4.17 (m, 1H), 3.16 (dd, J = 12.4, 3.9 Hz, 1H), 3.06 (dd, J = 12.4, 7.5 Hz, 1H), 2.37 = 2.17 (m, 1H), 2.12 - 2.00 (m, 1H), 1.67 — 1.58 (m, 2H), 1.11 (s, 9H). 3C NMR (101 MHz, CDCls) 8 154.82, 152.35, 144.41, 142.58, 133.80, 132.49, 132.07, 130.49, 130.44, 128.22, 125.85, 124.96, 119.49, 116.54, 79.97, 61.23, 57.05, 47.66, 32.03, 27.08, 26.01. tert-Butyl (2S,5R)})-2-(4-bromophenyl}-5-((isoquinocline-5-sulfonamido)methyl)pyrrolidine-1- carboxylate (129c¢)
Br
Ozn ee CJ oo
[00217] Nx Following general procedure B, 128c (0.144 g, 0.630 mmol) was reacted with quinoline-5-sulfonylchloride HCI (0.172 g, 0.757 mmol) and triethylamine (0.207 mL, 1.48 mmol) to afford the title compound as an off-white solid (0.324 g, 0.593 mmol, 94%). 'H NMR (400 MHz, CDCl3) 8 9.40 (s, 1H), 8.65 (d, J=6.1 Hz, 1H), 8.49 (d, J=6.2 Hz, 1H), 8.45 (d, J = 7.4 Hz, 1H), 8.25 (d, J=8.2 Hz, 1H), 7.72 (t, J = 7.8 Hz, 1H), 7.61 (bs, 1H), 7.20 (d, J = 8.0 Hz, 2H), 6.75 (d, J=8.0Hz, 2H), 4.62 (t, J= 7.2 Hz, 1H), 4.19 — 3.98 (m, 1H), 3.38 — 2.91 (m, 2H), 2.29 — 2.10 (m, 1H), 2.10 — 1.89 (m, 1H), 1.76 — 1.53 (m, 2H), 1.14 (s, 9H).
P345823NL 50 3C NMR (101 MHz, CDCls) ö 156.69, 153.10, 145.03, 142.58, 134.30, 133.26, 133.05, 131.17, 131.09, 128.94, 126.85, 125.88, 120.15, 117.37, 80.96, 62.87, 57.99, 48.85, 34.25, 28.40, 27.85. tert-Butyl (2R,5 R)-2-(4-bromophenyl)-5-((isoquinoline-5-sulfonamido)methyl)pyrrolidine-1- carboxylate (129d)
Br ony a =
[00218] oe Following general procedure B, 128d (38 mg, 0.11 mmol} was reacted with quinoline-5-sulfonylchloride: HCI (37 mg, 0.16 mmol) and triethylamine (34 pL, 0.24 mmol) to afford the title compound as an off-white solid (57 mg, 0.10 mmol, 98%). 'H NMR (400 MHz, CDCl3) 8 9.36 (s, 1H), 8.67 (d, J=6.1 Hz, 1H), 8.45 (d, J = 5.8 Hz, 1H}, 8.43 (dd, J = 7.4,1.2Hz, 1H), 8.20 (d, J=8.2 Hz, 1H), 7.70 (t, J= 7.6 Hz, 1H), 7.40 (d, J= 8.4 Hz, 2H), 8.89 (d, J=8.4
Hz, 2H),4.72(d, J= 6.4 Hz, 1H), 4.27 — 4.16 (m, 1H), 3.16 (dd, J = 12.4, 4.4 Hz, 1H), 3.08 (dd, J = 12.4, 7.1 Hz, 1H), 2.36 — 2.19 (m, 1H), 2.09 — 2.00 (m, 1H), 1.78 — 1.52 (m, 2H), 1.10 (s, 9H). 130 NMR (101 MHz, CDCls) 8 154.65, 152.30, 144.27, 142.60, 133.79, 132.46, 132.03, 130.44, 130.38, 128.17, 125.83, 124.96, 119.43, 116.53, 79.84, 61.13, 57.00, 47.24, 31.92, 27.04, 25.75. tert-Butyl (2R,55)-2-(4-(6-fluoropyridin-3-yl)phenyl)-5-{(isoquinoline-5- sulfonamido)methyl)pyrrolidine-1-carboxylate (130a)
Zn F
UN fg”
DO
[00219] Nx Following general procedure D, 129a (84 mg, 0.15 mmol) was reacted with (6-flucropyridin-3-yl)boronic acid (26 mg, 0.18 mmol), K2CO: (85 mg, 0.62 mmol) and
Pd{(PPh3)4 (2.7 mg, 2.3 pmol) to afford the title compound as an off-white solid (78 mg, 0.14 mmol, 90%). 'H NMR (300 MHz, CDCls) 8 9.41 (s, 1H), 8.68 (d, J = 6.1 Hz, 1H), 8.52 (s, 1H), 8.48 (dd, J = 7.3, 1.3 Hz, 1H), 8.38 (d, J= 2.8 Hz, 1H), 8.25 (d, J=8.2 Hz, 1H), 7.94 (dd, J= 7.9, 2.7 Hz, 1H), 7.72 (t, J = 7.4 Hz, 1H), 7.24 (d, J = 7.9 Hz, 2H), 7.03 (dd, J= 8.4, 2.9 Hz, 1H), 6.98 (d, J= 9.4 Hz, 2H), 4.73 (t, J = 6.2 Hz, 1H), 4.20 — 4.11 (m, 1H), 3.28 — 3.05 (m, 2H), 2.30 — 2.18 (m, 1H), 2.09 — 1.96 (m, 3H), 1.79 — 1.71 (m, 1H), 1.70 — 1.61 (m, 1H), 1.17 (s, 9H).
BC NMR (75 MHz, CDCls) 6 162.19 (d, J = 239 Hz), 152.26, 144.72 (d, J = 15 Hz), 144.45, 142.76, 138.64 (d, J=7.9 Hz), 134.28, 133.66, 133.42 (d, J = 4.5 Hz), 132.36, 130.45, 128.25, 125.96, 125.13, 125.02, 116.68, 108.61 (d, J = 37.5 Hz), 80.33, 62.25, 57.16, 48.29, 33.54, 27.85, 27.12. tert-Butyl (2S,5S)-2-(4-(6-fluoropyridin-3-yl)phenyl}-5-({isoquinoline-5- sulfonamido)methyl)pyrrolidine-1-carboxylate (130b}
P345823NL 51 > F aN 0=8-xp Lr oo
[00220] Nx Following general procedure D, 129b (11 mg, 20 pmol} was reacted with (6-fluoropyridin-3-yl)boronic acid (3.5 mg, 25 pmol), K2COs (11 mg, 82 umol) and Pd(PPh3)4 (0.4 mg, 0.3 pmol) to afford the title compound as an off-white solid (5.9 mg, 10 pmol, 51%). 'H NMR (400 MHz, CDCl) 8 9.37 (s, 1H), 8.71 (d, J= 6.0 Hz, 1H), 8.47 (d, J= 6.2 Hz, 1H), 8.44 (dd, J = 73,12Hz 1H), 8.40 (d, J= 2.6 Hz, 1H), 8.22 (d, J=8.2 Hz, 1H), 7.95 (td, J= 8.1, 2.8 Hz, 1H), 7.75 — 7.67 (m, 1H), 7.46 (d, J=8.1 Hz, 2H), 7.11 (d, J= 8.3 Hz, 2H), 7.00 (dd, J= 8.5, 2.9 Hz, 1H), 4.82 d, J= 7.8 Hz, 1H), 4.31 — 4.22 (m, 1H), 3.26 — 3.05 (m, 2H), 2.39 — 2.25 (m, 1H), 2.18 — 2.06 (m, 1H), 1.79 — 1.66 (m, 2H), 1.11 (s, 9H). 3C NMR (101 MHz, CDCls) 8 161.37 (d, J = 412 Hz), 155.8, 152.36, 144.82 (d, J = 15 Hz), 144 43, 143.67, 138.69 (d, J = 8.3 Hz), 134.33, 133.84, 133.43 (d, J = 4.6 Hz), 132.51, 132.10, 130.48, 128.25, 126.00, 125.00, 124.98, 116.58, 108.62 (d, J = 37 Hz), 79.88, 61.47, 57.12, 47.79, 32.15, 27.09, 26.16. tert-Butyl (2S,5R)-2-(4-(6-fluoropyridin-3-yl)phenyl)-5-{(isoquinoline-5- sulfonamido)methyl}pyrrolidine-1-carboxylate (130c)
RT F
~_N 08x Nese +O
[00221] Nx Following general procedure D, 129c (0.10 g, 0..18 mmol) was reacted with (6-fluoropyridin-3-yl)boronic acid (31 mg, 0.22 mmol), K2CO: (0.101 g, 0.732 mmol) and
Pd(PPh3)4 (3.2 mg, 2.7 umo) to afford the title compound as an off-white solid (97 mg, 0.17 mmol, 94%). !H NMR (300 MHz, CDCls) 8 9.41 (s, 1H), 8.67 (d, J= 6.2 Hz, 1H), 8.50 (s, 2H), 8.48 (s, 1H), 8.38 (s, 1H), 8.25(,J=82Hz 1H), 7.95 (t, J=6.4 Hz, 1H), 7.73 (t, J=7.7 Hz, 1H), 7.25 (d, J = 7.8 Hz, 2H), 7.04 (s, 1H), 7.01 (s, 2H), 4.74 (s, 1H), 4.16 (s, 1H), 3.48 — 2.81 (m, 2H), 2.34 — 2.14 (m, 1H), 2.10 — 1.92 (m, 1H), 1.82-1.57 (m, 2H), 1.18 (s, 9H).
BC NMR (75 MHz, CDCl3) 8 163.09 (d, J = 239 Hz), 153.21, 145.61 (d, J = 15 Hz), 145.31, 143.72, 139.60 (d, J = 7.8 Hz), 135.16, 134.58, 134.37 (d, J = 5.1 Hz), 133.30, 131.36, 129.17, 126.87, 126.07, 126.00, 117.60, 109.54 (d, J = 38 Hz), 81.19, 63.17, 58.12, 49.15, 34.46, 28.73, 28.04. tert-Butyl (2R,5R)-2-(4-(6-fluoropyridin-3-yl)phenyl}-5-((isoquinoline-5- sulfonamido)methyl}pyrrolidine-1-carboxylate (130d)
P345823NL 52 23 F ~_N i” Sea | Nm
[00222] Nx Following general procedure D, 129d (57 mg, 0.10 mmol) was reacted with (6-flucropyridin-3-yl)boronic acid (18 mg, 0.13 mmol), KzCO: (58 mg, 0.42 mmol) and
Pd(PPhs)4 (1.8 mg, 1.6 pmol) to afford the title compound as an off-white solid (36 mg, 64 pmol, 61%). 'H NMR (300 MHz, CDCl) 8 9.37 (s, 1H), 8.70 (d, J= 6.1 Hz, 1H), 8.47 (d, J = 5.9 Hz, 1H), 8.44 (dd, J = 5 74,13Hz 1H), 8.40 (d, J= 2.6 Hz, 1H), 8.21 (d, J=8.3 Hz, 1H), 7.95 (td, J=7.9,26 Hz, 1H), 7.71 (t, J =8.0Hz, 1H), 746 (d, J=8.2Hz, 2H), 7.11 (d, J=8.2 Hz, 2H), 7.00 (dd, J= 8.6, 3.1 Hz, 1H), 4.82 d, J =7.1Hz, 1H), 4.35- 4.14 (m, 1H), 3.33 = 3.05 (m, 2H), 2.43 - 2.21 (m, 1H), 2.21 = 2.01 (m, 1H), 1.76 — 1.62 (m, 2H), 1.11 (s, 9H). 3C NMR (75 MHz, CDCl) 8 163.22 (d, J = 240 Hz), 155.87, 153.34, 145.80 (d, J = 15 Hz), 145.37, 144.70, 139.67 (d, J = 8.0 Hz), 135.31, 133.46, 133.05, 131.46, 129.23, 126.98, 125.99, 117.58, 109.59 (d, J=37 Hz), 80.81, 62.43, 58.13, 48.56, 33.10, 28.08, 27.04. tert-Butyl (2R,55)-2-([1,1'-biphenyl]-4-y1}-5-((isoquinoline-5-sulfonamido)methyl)pyrrolidine-1- carboxylate (130e) o=d-NH Ree J 0
[00223] NS Following general procedure D, 1294 (15 mg, 27 mol) was reacted with phenylboronic acid (4.0 mg, 33 pmol), K2CO: (15 mg, 0.11 mmol} and Pd(PPh3)4 (0.5 mg, 0.4 umol) to afford the title compound as an off-white solid (12 mg, 22 pmol, 80%). !H NMR (500 MHz, CDCl») 8 9.40 (d, J= 1.0 Hz, 1H), 8.69 (d, J = 6.1 Hz, 1H), 8.52 (d, J = 6.2 Hz, 1H), 8.47 (dd, J=7.3,1.2 Hz, 1H), 8.23 (dt, J=8.3, 1.1 Hz, 1H), 7.71 (dd, J= 8.2, 7.3 Hz, 1H), 7.55 (dd, J = 8.3,1.2Hz, 2H), 7.46 (t, J=7.7 Hz, 2H), 7.40 - 7.32 (m, 1H), 7.27 (d, J= 8.0 Hz, 2H), 6.88 (d, J= 7.7
Hz, 2H), 4.70 (s, 1H), 4.19 — 4.09 (m, 1H), 3.24 (s, 1H), 3.13 = 3.04 (m, 1H), 2.27 — 2.17 (m, 1H), 2.08 — 1.98 (m, 2H), 1.81 — 1.69 (m, 1H), 1.66 — 1.59 (m, 2H}, 1.15 (s, 9H).
BC NMR (126 MHz, CDCls) 5 152.31, 144.57, 141.60, 139.78, 138.85, 133.70, 132.37, 130.53, 128.30, 127.98, 126.44, 126.07, 125.00, 124.76, 116.76, 80.34, 62.44, 57.17, 48.60, 33.58, 28.03, 27.13. tert-Butyl (2R,5S5)-2-(4'-fluoro-[1,1'-biphenyl]-4-yl)-5-((isoquinoline-5- sulfonamido)methyl}pyrrolidine-1-carboxylate (130f)
P345823NL 53
F ee Ro J 0
[00224] Nx Following general procedure D, 1294 (15 mg, 27 pmol) was reacted with pyridin-3-ylboronic acid (4.6 mg, 33 umol), K2CO: (15 mg, 0.11 mmol) and Pd(PPh3)4 (0.5 mg, 0.4 Jmol) to afford the title compound as an off-white solid (12 mg, 21 Hmol, 75%). 'H NMR (500 MHz, CDCls) 8 9.40 (d, J= 1.0 Hz, 1H), 8.69 (d, J = 8.1 Hz, 1H), 8.51 (d, J = 6.0 Hz, 1H), 848(dd,J=7.3,1.3Hz, 1H), 8.23 (d, J= 8.2 Hz, 1H), 7.71 (t, J= 7.3 Hz, 1H), 7.52 — 7.44 (m, 2H), 7.22 (d, J=79 Hz, 2H), 7.14 (t, J= 8.7 Hz, 2H), 6.90 (d, J = 7.6 Hz, 2H), 4.71 (s, 1H), 4.18 — 4.10 (m, 1H), 3.22 (s, 1H), 3.13 = 3.05 (m, 1H), 2.27 — 2.17 (m, 1H), 2.07 — 1.98 (m, 1H), 1.79 — 1.70 (m, 1H), 1.82 (s, 1H), 1.18 (s, 9H).
BC NMR (126 MHz, CDCls) 6 181.61 (d, J = 247 Hz), 156.30, 152.30, 144.55, 141.65, 137.91, 135.91, 133.70, 132.39, 130.54, 128.30, 127.62 (d, J = 7.9 Hz), 125.94, 125.01, 124.84, 116.76, 114.83 (d, J = 21
Hz), 80.36, 62.39, 57.17, 48.52, 33.58, 27.99, 27.14. tert-Butyl (2S,5R)-2-({isoquinoline-5-sulfonamido)methyl}-5-(4-(pyridin-3-yl})phenyl)pyrrolidine-1- carboxylate {130g) € ~_N 0
[00225] Nx Following general procedure D, 129a (15 mg, 27 pmol) was reacted with pyridin-3-ylboronic acid (4.0 mg, 33 pmol), K2CO: (15 mg, 0.11 mmol) and Pd(PPhs)4 (0.5 mg, 0.4 umol) to afford the title compound as an off-white solid (11 mg, 21 umol, 76%). ‘H NMR (400 MHz, CDCl3) 8 9.41 (d, J = 0.8 Hz, 1H), 8.80 (d, J = 1.5 Hz, 1H), 8.69 (d, J = 6.1 Hz, 1H), 8.60 (dd, J = 4.8, 1.6 Hz, 1H), 8.51 (d, J = 6.1 Hz, 1H), 8.48 (dd, J=7.3, 1.2 Hz, 1H), 8.25 (d, J = 8.2 Hz, 1H), 7.84 (dt, J=7.9,1.8 Hz, 1H), 7.72 (t, J = 7.6 Hz, 1H), 7.39 (dd, J = 7.0, 4.8 Hz, 1H), 7.29 (s, 2H), 6.96 (d, J= 7.8 Hz, 2H), 4.73 (s, 1H), 4.23 -4.10 (m, 1H), 3.31 — 3.18 (m, 1H), 3.10 (t, J = 10.9 Hz, 1H), 230-219 (m, 1H), 2.10 — 2.00 (m, 2H), 1.79 — 1.69 (m, 1H), 1.66 — 1.58 (m, 1H), 1.16 (s, 9H).
Synthesis of Example Compounds
N-(2-((4-(Pyridin-3-yl)benzyl)amino)ethyl)isoquinoline-5-sulfonamide (1)
Og Ny
H
[00226] NZ N? 1 (12 mg, 29 pmol, 62%) was synthesized from 109 (20 mg, 38 umol) according to general procedure A. 'H NMR (400 MHz, CDCls) 5 9.33 (d, J= 1.1 Hz, 1H), 8.79 (dd, J = 2.4, 0.9 Hz, 1H), 8.63 (d, J = 6.1 Hz, 1H), 8.58 (dd, J = 4.8, 1.6 Hz, 1H), 8.50 — 8.42 (m, 2H), 8.17 (dt, J = 8.4, 1.1 Hz, 1H), 7.85 (ddd, J = 7.9,
P345823NL 54 24,16 Hz, 1H), 7.68 (dd, J= 8.2, 7.3 Hz, 1H), 7.45 (d, J= 8.6 Hz, 2H), 7.37 (ddd, J= 7.9, 4.8, 0.9 Hz, 1H), 7.22 (d, J = 8.6 Hz, 2H), 3.59 (5, 2H), 3.04 (t, J = 5.7 Hz, 2H), 2.69 (t, J = 6.1 Hz, 2H).
BC NMR (101 MHz, CDCls} ö 153.37, 148.40, 148.10, 145.14, 139.67, 136.58, 136.30, 134.48, 134.41, 133.55, 133.35, 131.31, 129.08, 128.69, 127.19, 126.01, 123.74, 117.33, 52.78, 47.60, 42.56.
HRMS [C23H22N402S +H]*: 419.15335 calculated, 419.15335 found.
N-{(2-((4-{Pyridin-3-yl)benzyl}Jaminoj}ethyl}naphthalene-1-sulfonamide (47)
OF Ny
H
[00227] N° 47 (50 mg, 39 pmol, 53% over two steps) was synthesized from 108 (24 mg, 73 pmol) and naphtalene 1-sulfonyl chloride (25 mg, 0.11 mmol) according to general procedure B, followed by general procedure A. 'H NMR (400 MHz, CDCls) § 8.81 (dd, J = 2.4, 0.9 Hz, 1H), 8.68 (dt, J = 8.6, 1.0 Hz, 1H), 8.59 (dd, J = 4.8,1.6 Hz, 1H), 8.28 (dd, J=7.3, 1.3 Hz, 1H), 8.06 (dt, J = 8.2, 1.1 Hz, 1H), 7.97 - 7.91 (m, 1H), 7.85 (ddd, J=7.9, 2.4, 1.6 Hz, 1H), 7.69 — 7.50 (m, 3H), 7.48 — 7.42 (m, 2H), 7.37 (ddd, J= 7.9, 4.8, 0.9 Hz, 1H), 7.23 — 7.16 (m, 2H}, 3.52 (s, 2H), 3.02 — 2.94 (m, 2H), 2.67 — 2.58 (m, 2H).
BC NMR (101 MHz, CDCls) ö 148.55, 148.30, 139.77, 136.70, 138.40, 134.53, 134.39, 134.36, 129.91, 129.27, 128.78, 128.50, 128.26, 127.24, 127.00, 124.48, 124.31, 123.72, 52.73, 47.37, 42.60.
HRMS [C24H23N202S+H]*: 418.15837 calculated, 418.15767 found.
N-(2-({(4-(Pyridin-3-yl)benzyl)amino)ethyl)quinazoline-6-sulfonamide (52) oH,
H
ND D cd bg
[00228] N N° 52 (12 mg, 29 pmol, 19% over two steps) was synthesized from 108 (50 mg, 0.15 mmol) and 5-bromoquinazoline (25 mg, 0.12 mmol) according to general procedure C followed by general procedure A.
H NMR (400 MHz, CDCl3) 8 9.51 (d, J = 0.8 Hz, 1H), 9.45 (s, 1H), 8.82 (dd, J = 2.3, 0.9 Hz, 1H), 8.60 (dd, J = 4.8, 1.6 Hz, 1H), 8.55 (dd, J = 2.0, 0.6 Hz, 1H), 8.27 (dd, J = 8.9, 2.0 Hz, 1H), 8.18 = 8.13 (m, 1H), 7.86 (ddd, J=7.9, 2.4, 1.6 Hz, 1H), 7.53 — 7.48 (m, 2H), 7.38 (ddd, J= 7.9, 4.8, 0.9 Hz, 1H), 7.35 — 7.30 (m, 2H), 3.73 (s, 2H), 3.17 — 3.06 {m, 2H), 2.83 — 2.72 (m, 2H).
BC NMR (101 MHz, CDCls) 5 161.48, 157.48, 151.46, 148.64, 148.30, 139.65, 139.50, 136.96, 136.27, 134.40, 130.89, 130.45, 128.83, 127.81, 127.40, 124.14, 123.75, 52.94, 47.60, 42.63.
HRMS [C22H21Ns02S+H]*: 420.14887 calculated, 420.14850 found.
N-(2-((4-(Pyridin-3-yl)benzyl)amino)ethyl)benzenesulfonamide (48)
P345823NL 55
OH
Os Ny
H
D
L
[00229] N 108 (24 mg, 73 pmol) and benzenesulfonyl chloride (19 mg, 0.11 mmol) were reacted according to general procedure B, followed by general procedure A to yield title compound 48 (8.8 mg, 24 pmol, 33% over two steps). 'H NMR (400 MHz, CDCl:) 8 8.83 (dd, J = 2.4, 0.9 Hz, 1H), 8.59 (dd, J = 4.8, 1.6 Hz, 1H), 7.91 — 7.83 (m, 3H), 7.61 7.46 (m, 5H), 7.41 — 7.31 (m, 3H), 3.72 (s, 2H), 3.08 — 3.02 (m, 2H), 2.78 — 2.71 (m, 2H).
BC NMR (101 MHz, CDCls) 8 148.55, 148.29, 139.89, 139.85, 136.81, 136.39, 134.41, 132.73, 129.22, 128.91, 127.35, 127.16, 123.73, 52.90, 47.58, 42.56.
HRMS [C29H2:N302S +H]*: 368.14272 calculated, 368.14201 found.
N-(2-({(4-(Pyridin-3-yl)benzyl)amino)ethyl)-1H-indazole-4-sulfonamide (49
OH
O2¢-N
H
=
CT
N N*
[00230] H 49 (8.0 mg, 20 umol, 13% over two steps) was synthesized from 108 (50 mg, 0.15 mmol) and 4-bromo-1H-indazole (25 mg, 0.12 mmol) according to general procedure C followed by general procedure A.
H NMR (400 MHz, MeOD) 6 8.91 (d, J = 2.3 Hz, 1H}, 8.63 (dd, J= 5.1, 1.5 Hz, 1H), 8.41 (d, J= 1.0 Hz, 1H), 8.30 (dt, J = 8.2, 1.9 Hz, 1H), 7.88 (d, J = 8.4 Hz, 1H), 7.85 - 7.80 (m, 3H), 7.72 (d, J = 7.1 Hz, 1H), 7.69-7.64 (m, 2H), 7.57 (dd, J = 8.5, 7.2 Hz, 1H), 4.34 (s, 2H), 3.23 — 3.17 (m, 4H).
HRMS [C2:H2:N502S+H]*: 408.14887 calculated, 408.14866 found
N-(2-((4-(Pyridin-3-yl}benzyl}amino}ethyl}-1H-indazole-6-sulfonamide (50)
OH
Os Ny
H
= lL
HN N
[00231] N= 50 (5.3 mg, 13 umol, 9% over two steps) was synthesized from 108 (50 mg, 0.15 mmol) and 6-bromo-1H-indazole (47 mg, 0.24 mmol) according to general procedure C followed by general procedure A. 'H NMR (400 MHz, MeOD) 0 8.80 (dd, J= 2.4, 0.9 Hz, 1H), 8.51 (dd, J= 4.9, 1.6 Hz, 1H), 8.37 (d, J= 5.0
Hz, 1H), 8.10 (ddd, J = 8.0, 2.3, 1.6 Hz, 1H), 7.61 (dd, J = 6.0, 2.3 Hz, 3H), 7.55 — 7.48 (m, 2H), 7.37 — 7.30 (m, 2H), 6.92 (d, J = 3.5 Hz, 1H), 3.67 (s, 2H), 3.07 (t, J= 6.3 Hz, 2H), 2.62 (t, J = 6.3 Hz, 2H).
HRMS [C21H21Ns02S+H]*: 408.14887 calculated, 408.14838 found.
N-Methyl-5-(N-(2-((4-(pyridin-3-yl)benzyl)amino)ethyl}sulfamoyl}picolinamide (51)
P345823NL 56 ohn,
H
N
“No
[00232] H 51 (4.0 mg, 9.0 umol, 6% over two steps) was synthesized from 108 (50 mg, 0.15 mmol) and 5-bromo-N-methylpicolinamide (52 mg, 0.24 mmol) according to general procedure C followed by general procedure A. 'H NMR (400 MHz, MeOD) 8 9.02 (dd, J = 2.3, 0.8 Hz, 1H), 8.80 (dd, J = 2.4, 0.9 Hz, 1H), 8.51 (dd, J = 4.9,1.6Hz, 1H), 8.36 (dd, J = 8.2, 2.3 Hz, 1H), 8.22 (dd, J = 8.2, 0.9 Hz, 1H), 8.10 (ddd, J= 8.0, 2.4, 1.6
Hz, 1H), 7.65 — 7.60 (m, 2H), 7.53 (ddd, J = 8.0, 4.9, 0.9 Hz, 1H), 7.44 — 7.38 (m, 2H), 7.24 — 7.09 (m, 2H), 3.77 (s, 2H), 3.09 (t, J = 6.4 Hz, 2H), 2.95 (s, 3H), 2.68 (t, J = 6.4 Hz, 2H).
HRMS [C2:Hz23NsO3S+H]*: 426.15944 calculated, 426.15879 found.
N-(2-((4-(Pyridin-3-yl)benzyl})amino)ethyl)quinazoline-7-sulfonamide (53
H ee
Vo
[00233] NZ N” 108 (50 mg, 0.15 mmol) and 8-bromoquinazoline (27 g, 0.13 umol) were reacted according to general procedure C, followed by general procedure A to yield title compound 53 (13 mg, 31 umol, 5% over two steps). 'H NMR (400 MHz, MeOD) 8 9.62 (d, J = 0.9 Hz, 1H), 9.36 (s, 1H), 8.78 (dd, J=2.4, 0.9 Hz, 1H), 8.54 — 8.48 (m, 2H), 8.31 (dd, J = 8.6, 0.7 Hz, 1H), 8.12 (dd, J = 8.6, 1.7 Hz, 1H), 8.08 (ddd, J= 8.0, 2.4, 1.6 Hz, 1H), 7.61 — 7.56 (m, 2H), 7.56 — 7.50 (m, 1H), 7.42 — 7.37 (m, 2H), 3.75 (s, 2H), 3.11 (t, J = 6.4 Hz, 2H), 2.70 (t, J = 6.4 Hz, 2H).
HRMS [Cz2H21NsO2S+H]*: 420.14887 calculated, 420.14857 found.
N-(2-((4-(Pyridin-3-yl)benzyl)amino)ethyl)-1H-pyrrolo[2,3-b]pyridine-4-sulfonamide (54)
Of Nn
H
NON? nN?
[00234] H 108 (50 mg, 0.15 mmol) and 4-bromo-7-azaindole (47 mg, 0.24 mmol) were reacted according to general procedure C, followed by general procedure A to yield title compound 54 (1.8 mg, 4.6 umol, 3% over two steps). 'H NMR (400 MHz, MeOD) 8 8.97 (s, 1H), 8.67 (d, J = 5.0 Hz, 1H), 8.41 (d, J= 5.0 Hz, 2H), 7.85 (d, J = 8.2 Hz, 2H), 7.76 (d, J=6.1 Hz, 1H), 7.71 — 7.862 (m, 3H), 7.55 (d, J = 5.0 Hz, 1H), 6.93 (d, J = 3.5 Hz, 1H), 4.34 (s, 2H), 3.25 — 3.16 (m, 4H).
HRMS [C21H21NsO2S+H]*: 408.14887 calculated, 408.14856 found. 3-Amino-N-(2-((4-(pyridin-3-yl)benzyl)amino)ethyl)isoquinoline-5-sulfonamide (55)
P345823NL 57
Os Ney
[00235] NF nN” 108 (50 mg, 0.15 mmol) and 5-bromoisoquinolin-3- amine (54 g, 0.24 mmol) were reacted according to general procedure C, followed by general procedure
A to yield title compound 55 (5.3 mg, 12 pmol, 8% over two steps). tH NMR (400 MHz, MeOD) 5 8.86 (d, J = 0.9 Hz, 1H), 8.79 (dd, J= 2.3, 0.8 Hz, 1H), 8.52 (dd, J=4.9,1.6
Hz, 1H), 8.20 (dd, J= 7.3, 1.3 Hz, 1H), 8.09 (dt, J = 8.0, 2.0 Hz, 1H), 8.03 (dt, J = 8.2, 1.2 Hz, 1H), 7.61 — 7.55 (m, 2H), 7.54 — 7.50 (m, 1H), 7.40 (d, J= 1.1 Hz, 1H), 7.32 = 7.05 (m, 5H), 3.59 (s, 2H), 3.02 (t, J = 6.2 Hz, 2H), 2.57 (t, J = 6.2 Hz, 2H).
BC NMR (101 MHz, MeOD) & 154.08, 148.69, 148.27, 140.76, 138.38, 137.37, 136.45, 135.54, 135.39, 135.23, 132.96, 130.43, 130.19, 128.17, 125.50, 124.89, 121.58, 87.10, 53.45, 48.94, 43.08.
HRMS [C23H24NsO2S+H]*: 434.16452 calculated, 434.18422 found.
N-(2-(([1,1'-Biphenyl]-4-ylmethyl)amino)ethyl)isoquinoline-5-sulfonamide (56)
Of Nn ®
H
0
[00236] Nu Phenylboronic acid (28 mg, 0.23 mmol) was subjected to general procedure D with 113a (0.10 g, 0.198 mmol) followed by general procedure A to yield the title compound 56 (60 mg, 33 Hmol, 17% over two steps).
IH NMR (400 MHz, CDCI3} 8 9.32 (d, J= 1.0 Hz, 1H), 8.66 (d, J = 8.1 Hz, 1H), 8.44 (ddd, J= 7.4, 5.8, 1.1
Hz, 2H), 8.15 (dt, J = 8.2, 1.1 Hz, 1H), 7.66 (dd, J = 8.2, 7.3 Hz, 1H), 7.59 — 7.53 (m, 2H), 7.50 — 7.40 (m, 4H), 7.39 — 7.31 (m, 1H), 7.19 = 7.14 (m, 2H), 3.57 (s, 2H), 3.07 — 2.94 (m, 2H), 2.71 — 2.62 (m, 2H). 3C NMR (101 MHz, CDCl3) 0 153.45, 145.31, 140.78, 140.23, 138.59, 134.36, 133.65, 133.42, 131.37, 129.12, 128.91, 128.44, 127.43, 127.28, 127.12, 126.01, 117.31, 52.86, 47.39, 42.52.
HRMS [C2H23N302S+H]*: 418.15837 calculated, 418.15782 found.
N-(2-((4-(Thiophen-3-yl}benzyl)amino)ethyl)isoquinoline-5-sulfonamide (57)
H
OTN
H ob Co
[00237] NFA = Thien-3-ylboronic acid (31 mg, 6.23 mmol) was subjected to general procedure D with 113a (0.10 g, 0.19 mmol) followed by general procedure A to provide 57 (39 mg, 90 umol, 47% over two steps). !H NMR (400 MHz, CDCl3) 8 9.33 (d, J= 1.0 Hz, 1H), 8.67 (d, J = 6.1 Hz, 1H), 8.45 — 8.41 (m, 2H), 8.16 (dt, J=8.2,1.1 Hz, 1H), 7.67 (dd, J= 8.2, 7.3 Hz, 1H), 7.51 — 7.45 (m, 2H), 7.43 (dd, J= 2.9, 1.4 Hz, 1H), 7.41 —7.35 (m, 2H), 7.16 — 7.09 (m, 2H), 3.54 (s, 2H), 3.02 — 2.94 (m, 2H), 2.70 — 2.62 (m, 2H). 13C NMR (101 MHz, CDCls) © 153.46, 145.34, 141.97, 138.42, 134.93, 134.31, 133.87, 133.45, 131.36, 129.13, 128.49, 126.59, 126.42, 126.36, 126.01, 120.35, 117.30, 52.89, 47.34, 42.50.
HRMS [C22H2N302S:+H]*: 424.11479 calculated, 424.11473 found.
P345823NL 58
N-(2-((4-(Furan-3-yl)benzyl)amino)ethyl)isoquinoline-5-sulfonamide (58)
OH
Og
SN ©
CO Wy
[00238] Furan-3-ylboronic acid (26 mg, 0.23 mmol) was subjected to general procedure D with 113a (0.10 g, 0.19 mmol) followed by general procedure A to provide 58 (6.0 mg, 15 ymol, 8% over two steps). !H NMR (400 MHz, CDCl:) 8 9.34 (d, J= 1.0 Hz, 1H), 8.68 (d, J = 6.1 Hz, 1H), 8.48 — 8.39 (m, 2H}, 8.18 (dt, J= 8.4, 1.1 Hz, 1H), 7.72 (dd, J= 1.5, 0.9 Hz, 1H), 7.68 (dd, J = 8.2, 7.4 Hz, 1H), 7.48 (t, J = 1.7 Hz, 1H), 7.41 — 7.36 (m, 2H), 7.17 — 7.09 (m, 2H), 6.69 (dd, J = 1.9, 0.9 Hz, 1H), 3.56 (s, 2H), 3.04 — 2.95 (m, 2H), 2.71 — 2.62 (m, 2H).
BC NMR (101 MHz, CDCls) 8 153.50, 145.41, 143.85, 138.59, 138.33, 134.32, 133.68, 133.46, 131.52, 131.38, 129.14, 128.49, 126.18, 126.05, 126.01, 117.28, 108.91, 52.90, 47.26, 42.50.
HRMS [C22H21N3O3S+H]*: 408.13764 calculated, 408.13725 found.
N-(2-(((4'-Fluoro-[1,1'-biphenyl]-4-yl)methyljamino)ethyl)isoquinoline-5-sulfonamide (59)
OH
5 On oO > D
Ns
[00239] F (4-Fluorophenylboronic acid (32 mg, 0.23 mmol) was subjected to general procedure D with 113a (0.10 g, 0.19 mmol) followed by general procedure A to provide 59 (41 mg, 99 umol, 52% over two steps). ‘H NMR (400 MHz, CDCls) 8 9.33 (d, J= 0.9 Hz, 1H), 8.67 (d, J = 6.1 Hz, 1H), 8.47 — 8.41 (m, 2H), 8.17 (dt, J=8.3,1.2 Hz, 1H), 7.67 (dd, J= 8.2, 7.3 Hz, 1H), 7.55 — 7.48 (m, 2H), 7.46 — 7.40 (m, 2H), 7.20 — 7.08 (m, 4H), 3.57 (s, 2H), 3.03 — 2.97 (m, 2H), 2.70 — 2.65 (m, 2H). 3C NMR (101 MHz, CDCl3} 8 162.57 (d, J = 243 Hz), 161.34, 153.46, 145.34, 139.27, 138.52, 136.91, 136.88, 134.31, 133.66, 133.44, 131.35, 129.12, 128.66 (d, J = 8.0 Hz), 128.52, 127.15, 126.01, 117.29, 115.76 (d, J = 23 Hz), 52.78, 47.35, 42.47.
HRMS [C24H22FN302S+H]*: 436.14895 calculated, 436.14842 found.
N-(2-({(4-(6-Fluoropyridin-3-yljbenzyl)amino)ethyl)isoquincline-5-sulfonamide (60)
OH
Og Ny
H
CO >
Ns = .
[00240] NF (6-Fluoropyridin-3-ylhboronic acid (32 mg, 0.23 mmol) was subjected to general procedure D with 113a (0.10 g, 0.19 mmol) followed by general procedure A to provide 60 (26 mg, 60 pmol, 32% over two steps). 'H NMR (400 MHz, CDCl:) 8 9.34 (d, J= 0.9 Hz, 1H), 8.67 (d, J = 6.1 Hz, 1H), 8.45 (dt, J= 7.1, 1.3 Hz, 2H), 8.39 (dt, J= 2.7, 0.9 Hz, 1H), 8.19 (dt, J=8.2, 1.1 Hz, 1H), 7.95 (ddd, J = 8.5, 7.6, 2.6 Hz, 1H), 7.69 (dd, J=8.2, 7.3 Hz, 1H), 7.46 — 7.41 (m, 2H), 7.26 — 7.21 (m, 2H), 7.01 (ddd, J = 8.5, 3.0, 0.7 Hz, 1H), 3.60 (s, 2H), 3.05-2.98 (m, 2H), 2.73 — 2.65 (m, 2H).
P345823NL 59 3C NMR (101 MHz, CDCls} 8 183.22 (d, J = 243 Hz), 153.47, 145.80 (d, J = 14.5 Hz) 145.34, 139.76 (d,
J=8.1Hz), 139.69, 135.69, 134.52 (d, J = 4.5 Hz), 134.30, 133.69, 133.48, 131.35, 129.14, 128.79, 127.21, 126.04, 117.27, 109.61 (d, J = 23 Hz), 52.76, 47.41, 42.51.
HRMS [C23H21FN402S+H]*: 437.14420 calculated, 437.14402 found.
N-{2-(((4'-Chloro-[1,1'-biphenyl]-4-yl)methyl)amino)ethyl)isoquinoline-5-sulfonamide {61} oN, ~
H oO
[00241] NA CI (4-Chlorophenyl)boronic acid (36 mg, 0.23 mmol) was subjected to general procedure D with 113a (0.10 g, 0.19 mmol) followed by general procedure A to provide 61 (80 mg, 0.14 mmol, 74% over two steps). 'H NMR (400 MHz, CDCl3) 8 9.32 (d, J= 1.0 Hz, 1H), 8.68 — 8.62 (m, 1H), 8.49 — 8.41 (m, 2H), 8.20 — 8.11 (m, 1H), 7.70 — 7.50 (m, 2H), 7.50 — 7.35 (m, 6H), 7.24 — 7.14 (m, 2H), 3.59 (d, J = 6.9 Hz, 2H), 3.05 —2.95(m, 2H), 2.73 — 2.60 (m, 2H). 130 NMR (101 MHz, CDCls) 8 153.42, 145.27, 139.17, 138.97, 138.73, 134.31, 133.85, 133.48, 133.41, 131.33, 129.10, 129.03, 128.60, 128.33, 127.09, 126.01, 117.31, 52.74, 47.40, 42.43.
HRMS [C24H22CIN3O2S+H]*: 452.11840 calculated, 452.11913 found.
N-(2-(((4'-Methyl-[1,1'-biphenyl]-4-yl)methyl)amino)ethyl}isoquinoline-5-sulfonamide (62)
H
ON ©
H oo
[00242] NF p-Tolylboronic acid (31 mg, 0.23 mmol} was subjected to general procedure D with 113a (0.10 g, 0.19 mmol) followed by general procedure A to provide 62 (46 mg, 0.11 mmol, 58% over two steps). 'H NMR (400 MHz, CDCl3) 8 9.32 (d, J= 1.1 Hz, 1H), 8.67 (d, J = 6.1 Hz, 1H), 8.47 — 8.39 (m, 2H), 8.16 (dt, J=8.2,1.1 Hz, 1H), 7.66 (dd, J = 8.2, 7.3 Hz, 1H), 7.49 — 7.42 (m, 4H), 7.28 — 7.21 (m, 2H), 7.18 — 7.12 (m, 2H), 3.55 (s, 2H), 3.02 — 2.96 (m, 2H), 2.69 — 2.63 (m, 2H), 2.40 (s, 3H). 3C NMR (101 MHz, CDCls) § 153.46, 145.35, 140.17, 138.29, 137.89, 137.22, 134.34, 133.66, 133.42, 131.37, 129.63, 129.13, 128.41, 127.08, 126.96, 126.00, 117.30, 52.87, 47.34, 42.51, 21.22.
HRMS [C2sH25N302S+H]*: 432.17402 calculated, 432.17384 found.
N-(2-({(4'-Cyano-[1,1'-biphenyl]-4-yI)methyl}amino)ethyl)isoquinoline-5-sulfonamide (63) oN, Cc
H
Cc
[00243] NF CN (4-Cyanophenyl)boronic acid (34 mg, 0.23 mmol) was subjected to general procedure D with 113a (0.10 g, 0.19 mmol) followed by general procedure A to provide 63 (17 mg, 40 pmol, 21% over two steps).
P345823NL 60 'H NMR (400 MHz, CDCls) 8 9.33 (d, J = 1.0 Hz, 1H), 8.65 (dd, J = 6.1, 1.2 Hz, 1H), 8.45 (ddd, J = 7 4, 2.2,1.1 Hz, 2H), 8.18 (dt, J = 8.2, 1.0 Hz, 1H), 7.74 — 7.62 (m, SH), 7.51 — 7.45 (m, 2H), 7.26 — 7.20 (m, 2H), 3.61 (s, 2H), 3.06 — 2.99 (m, 2H), 2.73 — 2.61 (m, 2H).
BC NMR (101 MHz, CDCls) 6 153.40, 145.22, 145.20, 140.23, 138.03, 134.29, 133.65, 133.44, 132.69, 131.30, 129.09, 128.70, 127.64, 127.34, 126.03, 119.03, 117.29, 110.87, 52.72, 47.49, 42.51.
HRMS [C2sH22N402S+H]*: 443.15362 calculated, 443.15333 found.
N-{2-(((4'-(Trifluoromethyl}-[1,1'-biphenyl]-4-yl)methyljamino)ethyljisoquinoline-5-sulfonamide {64} od Nn ©
H
CO
N. =~
[00244] CFs (4-(Trifluoromethylyphenyl)boronic acid (44 mg, 0.23 mmol) was subjected to general procedure D with 113a (0.10 g, 0.19 mmol) followed by general procedure A to provide 64 (64 mg, 0.13 mmol, 68% over two steps).
H NMR (400 MHz, CDCl3) 8 9.33 (d, J= 1.0 Hz, 1H), 8.67 (d, J = 6.1 Hz, 1H), 8.48 — 8.42 (m, 2H), 8.18 (dt, J=8.2,1.1 Hz, 1H), 7.72 — 7.83 (m, 5H), 7.52 — 7.47 (m, 2H), 7.24 — 7.18 (m, 2H), 3.59 (s, 2H), 3.04 — 2.96 (m, 2H), 2.72 — 2.64 (m, 2H). 130 NMR (101 MHz, CDCls) 8 153.44, 145.27, 144.30, 139.70, 138.68, 134.29, 133.66, 133.46, 131.33, 129.36 (q, J = 32 Hz), 129.12, 128.60, 127.40, 127.36, 126.03, 125.82 (q, J = 3.9 Hz), 124.40 (q, J = 273
Hz), 117.28, 52.77, 47.42, 42.51.
HRMS [C2sH22F3N2028+H]*: 486.14576 calculated, 486.14555 found.
N-(2-((4-(Pyridin-4-yl)}benzyl)amino)ethyl)isoquinoline-5-sulfonamide (65)
OFM,
H
[00245] N~F SN pyridin-4-ylboronic acid (28 mg, 0.23 mmol) was subjected to general procedure D with 113a (0.10 g, 0.19 mmol) followed by general procedure A to provide 65 (38 mg, 91 umol, 48% over two steps). 'H NMR (400 MHz, CDCls) 8 9.33 (d, J = 1.0 Hz, 1H), 8.66 — 8.60 (m, 3H), 8.48 — 8.42 (m, 2H}, 8.18 (dt, J =8.3, 1.2 Hz, 1H), 7.69 (dd, J= 8.2, 7.3 Hz, 1H), 7.55 — 7.50 (m, 2H), 7.49 — 7.45 (m, 2H), 7.26 — 7.21 (m, 2H), 3.61 (s, 2H), 3.08 — 2.99 (m, 2H}, 2.74 — 2.65 (m, 2H).
BC NMR (101 MHz, CDClIs) 5 153.41, 150.22, 148.04, 145.21, 140.77, 136.91, 134.43, 133.61, 133.42, 131.34, 129.11, 128.74, 127.12, 126.04, 121.61, 117.33, 52.80, 47.59, 42.56.
HRMS [C23H22N402S5+H]*: 419.15362 calculated, 419.15335 found.
N-(2-({(3'-Fluoro-[1,1'-biphenyl]-4-yl)methyl}amino)ethyl)isoquinoline-5-sulfonamide (66)
P345823NL 61
OH
Os ©
[00246] NA (3-Fluorophenyl)boronic acid (32 mg, 0.23 mmol) was subjected to general procedure D with 113a (0.10 g, 0.19 mmol) followed by general procedure A to provide 66 (29 mg, 70 pmol, 37% over two steps). 'H NMR (400 MHz, CDCl3) 8 9.34 (d, J = 1.0 Hz, 1H), 8.68 (d, J = 6.1 Hz, 1H), 8.47 — 8.39 (m, 2H), 8.18 (dt,J=8.1,1.1Hz, 1H), 7.68 (dd, J= 8.2, 7.3 Hz, 1H), 7.50 — 7.44 (m, 2H), 7.44 — 7.31 (m, 2H), 7.28 — 7.23 (m, 1H), 7.22 = 7.16 (m, 2H), 7.07 — 7.00 (m, 1H), 3.58 (s, 2H), 3.04 — 2.95 (m, 2H), 2.71 — 2.64 (m, 2H}.
BC NMR (101 MHz, CDCl) 8 164.53, 153.49, 145.40, 143.08 (d, J = 8.7 Hz), 139.24, 138.98, 134.30, 133.70, 133.49, 131.38, 130.40 (d, J = 8.7 Hz), 129.15, 128.56, 127.28, 126.02, 122.76 (d, J = 2.8 Hz), 117.29, 114.22 (d, J = 23 Hz), 114.00 (d, J = 23 Hz), 52.82, 47.36, 42.50.
HRMS [Cz4H22FN2O2S +H]*: 436.14895 calculated, 436.14864 found.
N-(2-(({3'-Methyl-[1,1'-biphenyl]-4-yl)methyl)amino)ethyl}isoquinoline-5-sulfonamide (67)
H
OTN, ©
H ch
[00247] Nus m-Tolylboronic acid (31 mg, 0.23 mmol) was subjected to general procedure D with 113a {0.10 g, 0.19 mmol) followed by general procedure A to provide 67 (59 mg, 0.14 mmol, 74% over two steps). ‘H NMR (400 MHz, CDCl3) § 9.31 (d, J = 1.0 Hz, 1H), 8.65 (d, J = 6.1 Hz, 1H}, 8.49 — 8.39 (m, 2H), 8.14 (dt, J=8.2, 1.1 Hz, 1H), 7.65 (dd, J = 8.2, 7.3 Hz, 1H), 7.50 — 7.41 (m, 2H), 7.39 — 7.29 (m, 3H), 7.19 — 7.10 (m, 3H), 3.56 (s, 2H), 3.03 — 2.97 (m, 2H), 2.70 — 2.63 (m, 2H), 2.42 (s, 3H). 3C NMR (101 MHz, CDCls) 8 153.40, 145.23, 140.72, 140.29, 138.46, 138.44, 134.36, 133.62, 133.38, 131.34, 129.09, 128.79, 128.38, 128.15, 127.88, 127.25, 126.00, 124.19, 117.32, 52.84, 47.41, 42.50, 21.64.
HRMS [C2sH25N302S+H]*: 432.17402 calculated, 432.17371 found.
N-(2-({(3'-Cyano-[1,1'-biphenyl]-4-yI)methyl}amino)ethyl)isoquinoline-5-sulfonamide (68)
OH
Og ©
CD ye
[00248] NA (3-Cyanophenyl}boronic acid (34 mg, 0.23 mmol) was subjected to general procedure D with 113a (0.10 g, 0.19 mmol) followed by general procedure A to provide 68 (21 mg, 50 pmol, 26% over two steps). !H NMR (400 MHz, CDCl3) 8 9.35 (d, J= 1.0 Hz, 1H), 8.69 (d, J = 6.1 Hz, 1H), 8.45 (ddd, J= 7.1, 4.9, 1.1
Hz, 2H), 8.20 (dt, J = 8.2, 1.1 Hz, 1H), 7.86 — 7.82 (m, 1H), 7.80 (ddd, J= 7.8, 1.9, 1.2 Hz, 1H), 7.70 (dd,
J=8.2,74Hz 1H), 7.64 (dt, J=7.7,1.4 Hz, 1H), 7.55 (d, J= 7.7, 1H), 7.50 — 7.44 (m, 2H), 7.25 - 7.20 (m, 2H), 3.81 (s, 2H), 3.05 — 2.96 (m, 2H), 2.74 — 2.64 (m, 2H).
P345823NL 62 3C NMR (101 MHz, CDCls) ö 153.51, 145.43, 142.08, 139.86, 137.96, 134.27, 133.73, 133.53, 131.52, 131.38, 130.86, 130.71, 129.79, 129.16, 128.79, 127.31, 126.04, 118.97, 117.27, 113.10, 52.76, 47.38, 42.48.
HRMS [C2sH22N402S+H]*: 443.15362 calculated, 443.15321 found.
N-2-(({(3'(Trifluoromethyl)-[1,1'-biphenyl]-4-yl)methyl}amino)ethyl}isoquinoline-5-sulfonamide (69)
OH
OSE NON © 0 ye
[00249] NF (3-(Trifluoromethylyphenyl)boronic acid (43 mg, 0.23 mmol) was subjected to general procedure D followed by general procedure A to provide 69 (37 mg, 80 pmol, 42% over two steps).
H NMR (400 MHz, CDCl3) 8 9.33 (d, J= 1.0 Hz, 1H), 8.68 (d, J = 6.1 Hz, 1H), 8.48 — 8.42 (m, 2H), 8.17 (dt, J=8.3,1.1 Hz, 1H),7.79(d, J=1.8 Hz, 1H), 7.74 (dt, J = 7.5, 1.7 Hz, 1H), 7.68 (dd, J = 8.2, 7.3 Hz, 1H), 7.62 — 7.53 (m, 2H), 7.51 — 7.45 (m, 2H}, 7.24 — 7.19 (m, 2H), 3.59 (s, 2H), 3.04 — 2.97 (m, 2H), 2.72 — 2.63 (m, 2H). 130 NMR (101 MHz, CDCls) 8 153.45, 145.31, 141.59, 139.52, 138.71, 134.31, 133.85, 133.45, 131.35, 131.21 (q, J = 32 Hz), 130.41, 129.39, 129.12, 128.63, 127.32, 126.02, 124.27 (q, J = 273 Hz), 124.06, 123.87, 117.27, 52.79, 47.41, 42.52.
HRMS [C25H22F3N202S+H]*: 486.14576 calculated, 486.14540 found.
N-(2-{((2'-Chloro-[1,1'-biphenyl]-4-yI)methyl)amino)ethyl)isoquinoline-5-sulfonamide (70)
H
OLN C) cl
H
Cc
[00250] NF (2-Chlorophenyl)boronic acid (36 mg, 0.23 mmol) was subjected to general procedure D with 113a (0.10 g, 0.19 mmol) followed by general procedure A to provide 70 (11 mg, 24 umol, 13% over two steps). 'H NMR (400 MHz, CDCl3) 8 9.35 (d, J= 1.0 Hz, 1H), 8.68 (d, J = 6.1 Hz, 1H), 8.49 — 8.42 (m, 2H), 8.19 (dt, J=8.3, 1.1 Hz, 1H), 7.69 (dd, J = 8.2, 7.4 Hz, 1H), 7.47 (dt, J= 7.1, 1.3 Hz, 1H), 7.39 — 7.23 (m, 6H), 7.23 -7.16 (m, 2H), 3.61 (s, 2H), 3.06 — 2.99 (m, 2H), 2.77 — 2.67 (m, 2H). 3C NMR (101 MHz, CDCls} § 153.47, 145.38, 140.16, 138.95, 138.42, 134.32, 133.68, 133.46, 132.53, 131.45, 131.37, 130.08, 129.68, 129.55, 129.14, 128.70, 127.66, 127.36, 127.00, 126.03, 117.29, 52.90, 47.40, 42.50.
HRMS [C24H22CIN302S+H]*: 452.11940 calculated, 452.11901 found.
N-(2-({(2'-Methyl-[1,1'-biphenyl]-4-yl)methyl)amino)ethyl)isoquinoline-5-sulfonamide (71)
P345823NL 63
H
Osi Ny ®
H
0
[00251] Nys o-Tolylboronic acid (31 mg, 0.23 mmol) was subjected to general procedure D with 113a (0.10 g, 0.19 mmol) followed by general procedure A to provide 71 (37 mg, 80 pmol, 42% over two steps). 'H NMR (400 MHz, CDCls) 8 9.35 (d, J= 1.1 Hz, 1H), 8.68 (d, J = 6.1 Hz, 1H), 8.48 — 8.43 (m, 2H), 8.19 (dt,J=8.2,1.1 Hz, 1H), 7.69 (dd, J = 8.2, 7.3 Hz, 1H), 7.30 — 7.12 (m, 8H), 3.58 (s, 2H), 3.03 — 2.98 (m, 2H), 2.72 — 2.67 (m, 2H), 2.26 (s, 3H).
BC NMR (101 MHz, CDCls) 8 153.47, 145.37, 141.54, 141.03, 137.97, 135.41, 134.31, 133.69, 133.47, 131.38, 130.47, 129.87, 129.42, 129.15, 127.74, 127.41, 126.02, 125.91, 117.30, 52.93, 47.38, 42.47, 20.60.
HRMS [CasH2sN3028+H]*: 432.17402 calculated, 432.17388 found.
N-(2-(((2'-Cyano-[1,1'-biphenyl]-4-yI)methyl)amino)ethyl)isoquinoline-5-sulfonamide (72) of Nn
DO °C
[00252] NA (2-Cyanophenylboronic acid (34 mg, 0.23 mmol) was subjected to general procedure D with 113a (0.10 g, 0.19 mmol) followed by general procedure A to provide 72 (44 mg, 0.10 mmol, 53% over two steps). "HNMR (400 MHz, CDCl3) 8 9.34 (d, J = 1.0 Hz, 1H), 8.67 (d, J = 6.1 Hz, 1H), 8.49 — 8.42 (m, 2H), 8.19 (dt, J=8.2,1.1 Hz, 1H), 7.76 (ddd, J= 7.8, 1.3, 0.6 Hz, 1H), 7.72 — 7.59 (m, 2H), 7.52 — 7.41 (m, 4H), 7.25 -7.19 {m, 2H), 3.59 (s, 2H), 3.06 — 2.97 (m, 2H), 2.72 — 2.63 (m, 2H). 3C NMR (101 MHz, CDCls) 8 153.44, 145.30, 145.17, 140.10, 137.14, 134.31, 133.84, 133.69, 133.44, 133.01, 131.34, 130.11, 129.12, 128.97, 128.30, 127.69, 126.05, 118.88, 117.31, 111.22, 52.75, 47 45, 42.49.
HRMS [C2sH22N402S+H]*: 443.15362 calculated, 443.15325 found.
N-(2-({(2'-Methoxy-[1,1'-biphenyl]-4-yl)methyl}Jamino)ethyl}isoquinoline-5-sulfonamide (73) of Nn >
CO °C
[00253] Nys (2-Methoxyphenyl)boronic acid (35 mg, 0.23 mmol) was subjected to general procedure D with 113a (0.10 g, 0.19 mmol) followed by general procedure A to provide 73 (28 mg, 80 pmol, 42% over two steps). !H NMR (400 MHz, CDCl3) 8 9.33 (d, J= 1.0 Hz, 1H), 8.68 (d, J = 6.1 Hz, 1H), 8.48 — 8.40 (m, 2H), 8.17 (dt, J=8.2, 1.1 Hz, 1H), 7.67 (dd, J = 8.2, 7.3 Hz, 1H), 7.48 — 7.41 (m, 2H), 7.36 = 7.27 (m, 2H), 7.17 — 7.12 (m, 2H), 7.05 — 6.96 (m, 2H), 3.81 (s, 3H), 3.56 (s, 2H), 3.04 — 2.97 (m, 2H), 2.71 — 2.65 (m, 2H).
P345823NL 64 3C NMR (101 MHz, CDCls) ö 156.52, 153.46, 145.39, 138.04, 137.62, 134.34, 133.66, 133.42, 131.38, 130.89, 130.27, 129.75, 129.13, 128.80, 127.68, 126.00, 120.96, 117.31, 111.29, 55.62, 52.92, 47.30, 42.46.
HRMS [C2sH25N303S+H]*: 448.16894 calculated, 448.16854 found.
N-{2-(((3',4'-Dichloro-[1,1'-biphenyl]-4-yl)methyljamino)ethyl}isoquinoline-5-sulfonamide (74)
H
ON ©
CO Op
[00254] NF CI (3,4-Dichlorophenyl)boronic acid (44 mg, 0.23 mmol) was subjected to general procedure D with 113a (0.10 g, 0.19 mmol) followed by general procedure A to provide 74 (13 mg, 27 pmol, 14% over two steps). 'H NMR (400 MHz, CDCls} 8 9.34 (d, J = 1.0 Hz, 1H), 8.68 (d, J = 6.1 Hz, 1H), 8.48 — 8.41 (m, 2H), 8.19 (dt, J=8.3, 1.1 Hz, 1H), 7.69 (dd, J = 8.2, 7.3 Hz, 1H), 7.64 (d, J = 2.1 Hz, 1H), 7.50 (d, J = 8.3 Hz, 1H), 7.46 — 7.41 (m, 2H), 7.39 (dd, J = 8.3, 2.2 Hz, 1H}, 7.22 — 7.17 (m, 2H), 3.58 (s, 2H), 3.04 — 2.92 (m, 2H), 2.74 — 2.63 (m, 2H). 130 NMR (101 MHz, CDCls) 8 153.50, 145.42, 140.85, 139.61, 137.82, 134.29, 133.71, 133.51, 131.58, 131.38, 130.85, 129.16, 128.95, 128.66, 127.15, 126.38, 126.03, 117.27, 52.79, 47.36, 42.50.
HRMS: found [C24H21Cl2N3O2S+H]* calculated for [C24H21Cl2N3O2S+H]* 486.08043 calculated, 486.07992 found.
N-(2-{((3',5'-Dichloro-[1,1'-biphenyl]-4-yl)methyl}amino)ethyl}isoquinoline-5-sulfonamide (75)
H
OG ©
NS
[00255] Cl (3,5-Dichlorophenylboronic acid (44 mg, 0.23 mmol) was subjected to general procedure D with 113a (0.10 g, 0.19 mmol) followed by general procedure A to provide 75 (5.0 mg, 10 uymol, 5% over two steps). 'H NMR (400 MHz, CDCl3) 5 9.35 (d, J= 1.0 Hz, 1H), 8.70 (d, J = 6.1 Hz, 1H), 8.48 — 8.41 (m, 2H), 8.20 (dt, J=8.2, 1.1 Hz, 1H), 7.70 (dd, J = 8.2, 7.3 Hz, 1H), 7.46 — 7.41 (m, 4H), 7.34 (t, J = 1.9 Hz, 1H), 7.23 — 7.18 (m, 2H), 3.59 (s, 2H), 3.02 — 2.96 (m, 2H), 2.71 — 2.65 (m, 2H).
HRMS [C24H2:Cl2N302S+H]*: 486.08043 calculated, 486.08022 found.
N-{2-({(4-(Naphthalen-2-yl)benzyl)amino)ethyl)isoquinoline-5-sulfonamide (76)
Of Nn ©
H
0
[00256] NLs Naphthalen-2-ylboronic acid (40 mg, 0.23 mmol} was subjected to general procedure D with 113a (0.10 g, 0.19 mmol} followed by general procedure A to provide 76 (80 mg, 0.17 mmol, 89% over two steps).
P345823NL 65 'H NMR (400 MHz, CDCls) 8 9.28 (d, J= 1.1 Hz, 1H), 8.65 (dd, J=6.2, 1.0 Hz, 1H), 8.46 (dd, J= 6.1, 1.0
Hz, 1H), 8.41 (dd, J=7.4,1.2 Hz, 1H), 8.09 (dq, J=8.2,1.4 Hz, 1H), 7.98 (d, J = 1.8 Hz, 1H), 7.91 — 7.81 (m, 3H}, 7.68 (dd, J=8.6, 1.9 Hz, 1H), 7.65 — 7.56 (m, 3H), 7.53 — 7.40 (m, 2H), 7.22 — 7.15 (m, 2H), 3.58 (s, 2H), 3.04 — 2.94 (m, 2H), 2.73 — 2.55 (m, 2H).
BC NMR (101 MHz, CDCls) 8 153.37, 145.18, 140.01, 138.45, 137.98, 134.35, 133.70, 133.59, 133.33, 132.66, 131.31, 129.06, 128.58, 128.54, 128.25, 127.71, 127.46, 126.43, 126.06, 125.98, 125.67, 125.44, 117.35, 52.79, 47.45, 42.44.
HRMS [C28H25N302S+H]*: 468.17402 calculated, 468.17387 found.
N-{2-({(4-(Benzo[d][1,3]dioxol-5-yl)benzyl)amino)ethyl})isoquinoline-5-sulfonamide {77)
H
Os Nn ©
H
»e
Ns 0
[00257] o—/ Benzo[d][1,3]dioxol-5-yIboronic acid (38 mg, 0.23 mmol) was subjected to general procedure D with 113a (0.10 g, 0.19 mmol) followed by general procedure A to provide 77 (24 mg, 50 pmol, 26% over two steps). ‘H NMR (400 MHz, CDCl) 8 9.34 (d, J= 1.0 Hz, 1H), 8.68 (d, J = 6.1 Hz, 1H), 8.47 — 8.40 (m, 2H), 8.18 (dt, J= 8.2, 1.1 Hz, 1H), 7.68 (dd, J = 8.2, 7.3 Hz, 1H), 7.45 — 7.38 (m, 2H), 7.18 = 7.11 (m, 2H), 7.06 — 7.00 (m, 2H), 6.93 — 6.85 (m, 1H), 6.01 (s, 2H), 3.56 (s, 2H), 3.05 — 2.96 (m, 2H), 2.74 — 2.62 (m, 2H). 13C NMR (101 MHz, CDCls) ö 153.49, 148.27, 147.24, 145.44, 140.06, 138.07, 135.18, 134.31, 133.69, 133.47, 131.38, 129.15, 128.48, 127.06, 126.01, 120.65, 117.29, 108.74, 107.68, 101.30, 52.80, 47.28, 42.44.
HRMS [C25H23N304S+H]*: 462.14820 calculated, 462.14761 found.
N-(2-((4-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)benzyl}amino)ethyl)isoquinoline-5-sulfonamide (78)
Of Ny ©
H
CO C
[00258] ol (2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)boronic acid (41 mg, 0.23 mmol) was subjected to general procedure D with 113a (0.10 g, 0.19 mmol,) followed by general procedure A to provide 78 (20 mg, 42 umol, 22% over two steps). 'H NMR (400 MHz, CDCls) 8 9.33 (d, J= 1.0 Hz, 1H), 8.68 (d, J = 6.1 Hz, 1H), 8.48 — 8.40 (m, 2H), 8.17 (dt,J=8.1,1.1Hz 1H), 7.67 (dd, J=8.2, 7.3 Hz, 1H), 7.44 — 7.38 (m, 2H), 7.16 — 7.10 (m, 2H), 7.10 — 7.03 (m, 2H), 6.93 (d, J = 8.3 Hz, 1H), 4.30 (s, 4H), 3.55 (s, 2H}, 3.02 — 2.96 (m, 2H), 2.70 — 2.62 (m, 2H).
BC NMR (101 MHz, CDCls) 8 153.48, 145.41, 143.82, 143.32, 139.69, 137.96, 134.37, 134.33, 133.68, 133.43, 131.38, 129.14, 128.46, 126.90, 126.00, 120.16, 117.71, 117.29, 115.85, 64.60, 64.57, 52.81, 47.28, 42.45.
HRMS [CzsH2sN:304S+H][*: 476.16385 calculated, 476.16340 found.
N-(2-(([2,3'-Bipyridin]-5-ylmethyl}amino}ethyl}isoquinoline-5-sulfonamide (79)
P345823NL 66
KTP
SON
[00259] NF nN? Pyridin-3-ylboronic acid (27 mg, 0.23 mmol) was subjected to general procedure D with 113b (0.10 g, 0.21 mmol) followed by general procedure A to provide 79 (10 mg, 24 pmol, 11% over two steps). 'H NMR (400 MHz, CDCl3) 8 9.33 (d, J = 1.0 Hz, 1H), 9.15 (dd, J = 2.4, 0.9 Hz, 1H), 8.68 — 8.61 (m, 2H), 8.49(dd,J=2.3,0.9 Hz, 1H), 8.47 — 8.43 (m, 2H), 8.29 (ddd, J= 8.0, 2.4, 1.7 Hz, 1H}, 8.18 (d, 8.3 Hz), 7.72 -7.55 (m, 3H), 7.41 (ddd, J = 8.0, 4.8, 0.9 Hz, 1H), 3.64 (s, 2H), 3.12 — 2.98 (m, 2H), 2.77 - 2.63 (m, 2H).
BC NMR (101 MHz, CDCls) 8 153.78, 153.46, 149.92, 149.79, 148.18, 145.28, 136.73, 134.65, 134.41, 134.39, 134.30, 133.68, 133.44, 131.32, 129.11, 126.06, 123.78, 120.40, 117.26, 50.33, 47.75, 42.60.
HRMS [C22H21Ns02S+H]*: 420.14887 calculated, 420.14839 found.
N-(2-(((6-Phenylpyridin-3-yl)methyl}amino)ethyl)isoquinoline-5-sulfonamide (80) of. 2s A
[00260] NA Phenylboronic acid (27 mg, 0.23 mmol) was subjected to general procedure D with 113b (0.10 g, 0.21 mmol) followed by general procedure A to provide 80 (24 mg, 57 umol, 27% over two steps). "HNMR (400 MHz, CDCl3) 8 9.32 (d, J = 1.0 Hz, 1H), 8.66 (d, J = 6.1 Hz, 1H), 8.47 — 8.41 (m, 1H), 8.16 (dt, J=8.2,1.1 Hz, 1H), 7.97 — 7.92 (m, 2H), 7.67 (dd, J = 8.2, 7.3 Hz, 1H), 7.61 (dd, J = 8.1, 0.9 Hz, 1H), 7.54 — 7.37 {m, 4H), 3.59 (s, 2H), 3.10 — 2.95 (m, 2H), 2.71 — 2.61 (m, 2H). 3C NMR (101 MHz, CDCl3) d 156.60, 153.47, 149.36, 145.30, 139.08, 136.57, 134.33, 133.71, 133.45, 133.37, 131.32, 129.10, 128.89, 126.94, 126.05, 120.42, 117.26, 50.35, 47.58, 42.58.
HRMS [C23H22N4O2S+H]*: 419.15362 calculated, 419.15334 found.
N-(2-(((6"-Fluoro-[2,3'-bipyridin]-5-yl)methyl)amino)ethyl)isoquinoline-5-sulfonamide (81)
KTP
[00261] NZ nN? F (6-Fluoropyridin-3-yl}boronic acid (30 mg, 0.23 mmol) was subjected to general procedure D with 113b (0.10 g, 0.21 mmol) followed by general procedure A to provide 81 (20 mg, 46 umol, 22% over two steps). "HNMR (400 MHz, CDCl3) 8 9.34 (d, J= 1.0 Hz, 1H), 8.77 (dt, J= 2.6, 0.8 Hz, 1H), 8.68 (d, J = 6.1 Hz, 1H), 8.50 — 8.39 (m, 4H), 8.20 (dt, J=8.2, 1.1 Hz, 1H), 7.70 (dd, J = 8.2, 7.3 Hz, 1H), 7.65 — 7.56 (m, 2H), 7.04 (ddd, J = 8.6, 3.0, 0.7 Hz, 1H), 3.64 (s, 2H), 3.08 — 3.00 (m, 2H), 2.73 — 2.67 (m, 2H). 13C NMR (101 MHz, CDCls) ö 164.06, (d, J = 243 Hz), 153.50, 152.83, 149.77, 146.20 (d, J = 14.5 Hz), 145.36, 139.88 (d, J = 8.1 Hz), 136.80, 134.27 (d, J = 4.5 Hz}, 133.76, 133.52, 132.93, 131.32, 129.13, 126.07, 120.13, 117.21, 109.70 (d, J = 23 Hz), 50.29, 47.63, 42.57.
P345823NL 67
HRMS [C22H20FNsO2S+H]*: 438.13945 calculated, 438.13902 found.
N-(2-({(6-(4-Fluorophenyl}pyridin-3-yl)methyl)amino)ethyl)isoquinoline-5-sulfonamide (82) oH
SNC
[00262] NZ F (4-Fluorophenyl)boronic acid (30 mg, 0.23 mmol) was subjected to general procedure D with 113b (0.10 g, 0.21 mmol) followed by general procedure A to provide 82 (25 mg, 57 pmol, 27% over two steps). 'H NMR (400 MHz, CDCls} 8 9.34 (d, J = 1.0 Hz, 1H), 8.68 (d, J = 6.1 Hz, 1H), 8.48 — 8.40 (m, 3H), 8.18 (dt, J=8.2, 1.1 Hz, 1H), 7.99 — 7.92 (m, 2H), 7.69 (dd, J = 8.2, 7.4 Hz, 1H), 7.59 (dd, J = 8.1, 0.9 Hz, 1H), 7.52 (dd, J =8.2, 2.3 Hz, 1H), 7.19 — 7.09 (m, 2H), 3.60 (s, 2H), 3.05 — 2.98 (m, 2H), 2.70 — 2.84 (m, 2H).
BC NMR (101 MHz, CDCls) 8 163.83 (d, J = 243 Hz), 155.65, 153.52, 149.38, 145.39, 136.61, 135.26, 134.25, 133.78, 133.51, 133.28, 131.33, 129.13, 128.75 (d, J = 8.0 Hz), 126.05, 120.08, 117.22, 115.81 (d, J=23 Hz), 50.33, 47.52, 42.56.
HRMS [C23H21FN402S+H]*: 437.14420 calculated, 437.14383 found.
N-(2-((3-Chloro-4-(pyridin-3-yl)benzyl)amino)ethyl)isoquinoline-5-sulfonamide (83) of Nn cl
[00263] NF nN” Pyridin-3-ylboronic acid (27 mg, 0.23 mmol) was subjected to general procedure D with 113c (0.10 g, 0.23 mmol) followed by general procedure A to provide 83 (11 mg, 24 pmol, 10% over two steps).
[00264] 'H NMR (400 MHz, CDCls) 6 9.35 (d, J= 1.0 Hz, 1H), 8.69 (d, J=6.1 Hz, 1H), 8.66 (dd, J= 2.2, 0.9 Hz, 1H), 8.63 (dd, J = 4.9, 1.7 Hz, 1H), 8.49 — 8.43 (m, 2H}, 8.21 (dt, J= 8.2, 1.1 Hz, 1H), 7.82 — 7.76 (m, 1H), 7.71 (dd, J = 8.2, 7.3 Hz, 1H), 7.38 (ddd, J=7.9, 4.9, 0.9 Hz, 1H), 7.31 (d, J = 1.7 Hz, 1H), 7.25 (dd, J=7.5,1.5Hz, 2H), 7.20 — 7.07 (m, 2H), 3.59 (s, 2H), 3.07 — 2.98 (m, 2H), 2.73 — 2.64 (m, 2H). 3C NMR (101 MHz, CDClIs) 8 153.51, 150.08, 148.90, 145.43, 141.53, 137.03, 135.83, 134.92, 134.29, 133.76, 133.54, 132.87, 131.41, 131.37, 129.54, 129.16, 126.72, 126.06, 123.06, 117.23, 52.33, 47 49, 42.56.
HRMS [C23Hz21CIN4O2S+H]*: 453.11466 calculated, 453.11422 found.
N-(2-({(2-Chloro-[1,1'-biphenyl]-4-yl)methyl)amino)ethyl)isoquinoline-5-sulfonamide (84)
ON Cl
DO TC
[00265] NA Phenylboronic acid (27 mg, 0.23 mmol) was subjected to general procedure D with 113¢ (0.10 g, 0.23 mmol) followed by general procedure A to provide 84 (23 mg, 51 mol, 22% over two steps).
P345823NL 68 'H NMR (400 MHz, CDCls) 8 9.41 — 9.29 (m, 1H), 8.70 (d, J = 6.1 Hz, 1H), 8.50 — 8.39 (m, 2H), 8.20 (d, J =8.2 Hz, 1H), 7.70 (t, J = 7.8 Hz, 1H), 7.48 — 7.35 (m, 5H), 7.32 = 7.18 (m, 2H), 7.07 (dd, J = 7.8, 1.7 Hz, 1H), 3.56 (s, 2H), 3.07 — 2.97 (m, 2H), 2.72 — 2.62 (m, 2H).
BC NMR (101 MHz, CDCls) 6 153.50, 145.43, 140.42, 139.43, 139.12, 134.28, 133.75, 133.51, 132.58, 131.53, 131.36, 129.54, 129.34, 129.14, 128.19, 127.76, 126.43, 126.04, 117.23, 52.33, 47.39, 42.53.
HRMS [C24H22CIN3O2S+H]*: 452.11940 calculated, 452.11890 found.
N-{2-({(3-Chloro-4-(6-fluoropyridin-3-yl}benzyl)amino)ethyl)isoquinoline-5-sulfonamide (85)
ON cl
[00266] NZ nN? F (6-Fluoropyridin-3-yl)boronic acid (30 mg, 0.23 mmol) was subjected to general procedure D with 113c {0.10 g, 0.23 mmol) followed by general procedure A to provide 85 (16 mg, 34 umol, 15% over two steps).
H NMR (400 MHz, CDCl3) 8 9.34 (d, J= 1.0 Hz, 1H), 8.68 (d, J = 6.1 Hz, 1H), 8.49 — 8.42 (m, 2H), 8.27 — 8.23 (m, 1H), 8.20 (dt, J = 8.3, 1.1 Hz, 1H), 7.89 (ddd, J= 8.5, 7.8, 2.5 Hz, 1H), 7.70 (dd, J= 8.2, 7.4
Hz, 1H), 7.33 — 7.08 (m, 3H), 7.00 (ddd, J = 8.4, 3.0, 0.7 Hz, 1H}, 3.57 (s, 2H), 3.06 — 2.97 (m, 2H), 2.71 — 2.63 (m, 2H).
BC NMR (101 MHz, CDCls) 8 163.11 (d, J = 243 Hz), 153.51, 147.92 (d, J = 14.5 Hz), 145.42, 142.32 (d,
J=8.1Hz), 141.78, 137.99, 134.88, 134.24, 133.77, 133.55, 132.91, 131.34, 129.57, 129.18, 128.35, 126.76, 126.05, 125.42, 117.20, 108.99 (d, J = 23 Hz), 52.28, 47.47, 42.55.
HRMS [C23H20CIFN4O2S+H]*: 471.10523 calculated, 471.10486 found.
N-(2-{((2-Chloro-4'-fluoro-[1,1'-biphenyl]-4-yl)methyl)amino)ethyl)isoquinoline-5-sulfonamide (86)
XIN ci
CO C
[00267] NZ F (4-Fluorophenyl)boronic acid (30 mg, 0.23 mmol) was subjected to general procedure D with 113c (0.10 g, 0.23 mmol) followed by general procedure A to provide 86 (20 mg, 43 pmol, 19% over two steps). 'H NMR (400 MHz, CDCls) 8 9.35 (d, J= 1.0 Hz, 1H), 8.71 (d, J = 6.1 Hz, 1H), 8.49 — 8.41 (m, 2H), 8.21 (dt, J=8.3, 1.2 Hz, 1H), 7.71 (dd, J = 8.2, 7.4 Hz, 1H), 7.43 — 7.36 (m, 2H), 7.25 — 7.16 (m, 2H), 7.16 — 7.09 (m, 2H), 7.07 (dd, J = 7.8, 1.8 Hz, 1H}, 3.56 (s, 2H), 3.05 — 2.96 (m, 2H), 2.72 — 2.61 (m, 2H). 3C NMR (101 MHz, CDCl) 5 163.73 (d, J = 243 Hz), 153.53, 145.50, 140.62, 138.59, 135.13, 134.25, 133.78, 133.57, 131.50, 131.37, 131.26 (d, J = 8.0 Hz), 129.40, 129.16, 126.49, 126.04, 117.21, 115.20 (d, J= 23 Hz), 52.32, 47.37, 42.52.
HRMS [C24H21CIFN302S+H]*: 470.10998 calculated, 470.1094 1 found.
N-(2-({3-Fluoro-4-(pyridin-3-yl)benzyljamino)ethyl)isoquinoline-5-sulfonamide (87)
P345823NL 69 of Nn F
[00268] NF nN” Pyridin-3-ylboronic acid (27 mg, 0.23 mmol) was subjected to general procedure D with 113d (0.10 g, 0.20 mmol) followed by general procedure A to provide 87 (10 mg, 23 pmol, 12% over two steps). !H NMR (400 MHz, CDCl3) 8 9.35 (d, J = 1.0 Hz, 1H), 8.77 (dq, J = 2.3, 1.0 Hz, 1H), 8.70 (d, J = 6.1 Hz, 1H), 8.61 (dd, J=4.8, 1.7 Hz, 1H), 8.50 — 8.42 (m, 2H), 8.21 (dt, J= 8.2, 1.1 Hz, 1H), 7.87 (ddt, J = 7.9, 2.3,1.7Hz, 1H), 7.71 (dd, J = 8.2, 7.3 Hz, 1H), 7.42 — 7.31 (m, 2H), 7.05 — 6.97 (m, 2H), 3.81 (s, 2H), 3.04 — 2.98 (m, 2H), 2.71 — 2.67 (m, 2H).
HRMS [C23H21FN402S+H]*: 437.14420 calculated, 437.14402 found.
N-(2-({(2-Fluoro-[1,1'-biphenyl]-4-yl)methyl)amino)ethyl)isoquinoline-5-sulfonamide (88) of Nn F 2s U 0 °C
[00269] NA Phenylboronic acid (27 mg, 0.23 mmol) was subjected to general procedure D with 113d (0.10 g, 0.20 mmol) followed by general procedure A to provide 88 (34 mg, 78 umol, 39% over two steps). ‘H NMR (400 MHz, CDCl3) 8 9.34 (d, J= 1.0 Hz, 1H), 8.68 (d, J = 6.1 Hz, 1H), 8.50 — 8.40 (m, 2H), 8.18 (dt, J=8.3, 1.1 Hz, 1H), 7.68 (dd, J = 8.2, 7.4 Hz, 1H), 7.52 (dt, J = 8.2, 1.5 Hz, 2H), 7.48 — 7.41 (m, 2H), 7.41 -7.28 (m, 2H), 7.01 — 6.92 (m, 2H), 3.57 (s, 2H), 3.08 — 2.95 (m, 2H), 2.74 — 2.62 (m, 2H). 3C NMR (101 MHz, CDCl3) 8 159.77 (d, J = 250 Hz), 153.48, 145.37, 141.23 (d, J = 7.2 Hz), 135.57, 134.30, 133.72, 133.48, 131.35, 130.82 (d, J= 3.9 Hz), 129.04 (d, J = 3.0 Hz), 129.13, 128.59, 127.93, 127.80, 126.03, 123.78 (d, J = 3.4 Hz), 117.25, 115.49 (d, J = 23 Hz), 52.42, 47.40, 42.55.
HRMS [C24H22FN302S +H]*: 436.14895 calculated, 436.14876 found.
N-(2-((3-Fluoro-4-(6-fluoropyridin-3-yl}benzyl)amino)ethyl)isoquinoline-5-sulfonamide (89) of Nn F
[00270] NZ N? F (6-Fluoropyridin-3-yl}boronic acid (30 mg, 0.23 mmol) was subjected to general procedure D with 113d (0.10 g, 0.20 mmol) followed by general procedure A to provide 89 (18 mg, 40 pmol, 20% over two steps). !H NMR (400 MHz, CDCl3) 8 9.35 (d, J= 0.9 Hz, 1H), 8.69 (d, J = 6.1 Hz, 1H), 8.45 (td, J= 7.3, 1.1 Hz, 2H), 8.36 (dq, J= 2.1, 1.0 Hz, 1H), 8.21 (dt, J = 8.3, 1.1 Hz, 1H), 7.97 (dddd, J = 8.5, 7.6, 2.6, 1.6 Hz, 1H), 7.70 (dd, J = 8.2, 7.4 Hz, 1H), 7.31 (t, J = 8.0 Hz, 1H), 7.07 — 6.97 (m, 3H), 3.60 (s, 2H), 3.04 — 2.98 (m, 2H), 2.71 — 2.64 (m, 2H). 13C NMR (101 MHz, CDCls) 8 163.14 (d, J = 243 Hz), 159.79 (d, J = 250 Hz), 153.52, 147.45 (dd, J = 15, 3.2 Hz), 145.43, 142.67, 141.65 (dd, J = 8.2, 3.7 Hz), 134.25, 133.76, 133.54, 131.36, 130.38, 129.44
P345823NL 70 (dd, J=4.4,1.4 Hz), 129.15, 126.05, 124.14, 123.31 (d, J = 14 Hz), 117.21, 115.69 (d, J = 23 Hz), 109.43 (d, J= 38 Hz), 52.37, 47.48, 42.57.
HRMS [C23H20F2N402S+H]*: 455.13478 calculated, 455.13426 found.
N-{2-(((2,4'-Difluoro-[1,1'-biphenyl]-4-yl)methyl)amino)ethyl)isoquinoline-5-sulfonamide (90)
KAP E
CO C
[00271] NF F (4-Fluorophenyl)boronic acid (30 mg, 0.23 mmol) was subjected to general procedure D with 113d (0.10 g, 0.20 mmol) followed by general procedure A to provide 90 (27 mg, 60 pmol, 30% over two steps). 'H NMR (400 MHz, CDCls} 8 9.34 (d, J = 0.9 Hz, 1H), 8.69 (d, J = 6.1 Hz, 1H), 8.48 — 8.41 (m, 2H), 8.20 (dt, J=8.3, 1.2 Hz, 1H), 7.70 (dd, J = 8.2, 7.4 Hz, 1H), 7.53 — 7.45 (m, 2H), 7.29 (t, J = 8.0 Hz, 1H), 7.16 —=7.10(m, 2H), 7.00 — 6.90 (m, 2H), 3.57 (s, 2H), 3.06 — 2.97 (m, 2H}, 2.71 — 2.63 (m, 2H).
BC NMR (101 MHz, CDCls) 8 162.35 (d, J = 288 Hz), 159.89 (d, J = 288 Hz), 153.51, 145.43, 141.34 (d,
J=17.1Hz), 134.26, 133.75, 133.53, 131.55 (d, J = 1.9 Hz), 131.38, 130.75 (d, J = 3.0 Hz), 130.67 (d, J = 2.9 Hz), 129.15, 126.92 (d, J = 13 Hz), 126.03, 123.84 (d, J = 3.3 Hz), 117.23, 115.67 (d, J = 1.8 Hz), 115.44 (d, J = 3.3 Hz), 52.40, 47.38, 42.54.
HRMS [CasH21F2N2O28+H]*: 454.13953 calculated, 454.13915 found.
N-(2-((4-(Pyridin-3-yl)benzyl)amino)ethyl}isoquinoline-5-carboxamide (91 og Mn
H
[00272] NF NZ To a solution of 108 (14 mg, 44 pmol), DIPEA (11.5 pL, 66 pmol) and the isoquinoline-5-carboxylic acid (8.3 mg, 48 mol.) in DCM (2 mL) was added HATU (17 mg, 44 ymol). The reaction mixture was stirred at room temperature overnight. after which the mixture was washed with water. The organic layer was dried over Na2SO4 and concentrated in vacuo, after which the residue was purified by column chromatography (5% MeOH (10% aq. NHs) in DCM). The product was then subjected to general procedure A to yield title compound 91 (13 mg, 33 Hmol, 81%).
H NMR (400 MHz, CDCls) 6 9.26 (d, J = 1.0 Hz, 1H), 8.80 (dd, J = 2.4, 0.9 Hz, 1H), 8.61 — 8.52 (m, 2H), 8.20 (dt, J= 6.0, 1.0 Hz, 1H), 8.04 (dt, J= 8.3, 1.1 Hz, 1H), 7.88 — 7.80 (m, 2H), 7.58 (dd, J = 8.2, 7.1 Hz, 1H), 7.54 — 7.49 (m, 2H), 7.46 — 7.40 (m, 2H), 7.36 (ddd, J=7.9, 4.8, 0.9 Hz, 1H), 6.81 (t, J = 5.3 Hz, 1H), 3.90 (s, 2H), 3.67 (gq, J = 5.6 Hz, 2H), 2.99 (d, J = 6.3 Hz, 2H). 3C NMR (101 MHz, CDCls) ö 168.31, 152.94, 148.61, 148.30, 144.32, 139.86, 136.93, 136.29, 134.34, 133.32, 133.17, 130.50, 129.37, 129.02, 128.82, 127.38, 126.34, 123.71, 118.36, 53.18, 48.10, 39.67.
HRMS [C24H22N4O +H]*: 383.18664 calculated, 383.18628 found.
N-Methyl-N-(2-((4-(pyridin-2-yl)benzyl)amino)ethyl)isoquinoline-5-sulfonamide (92/92)
P345823NL 71 oh
Sg N
[00273] NF 5 tert-Butyl (2-(isoquinoline-5-sulfonamido)ethyl}(4-(pyridin- 2-ylbenzylhcarbamate (109) (7.0 mg, 38 ymol), iodomethane (3.0 HL, 46 pmol) and NaOH (1.0 mg, 19 pmol) were dissolved in DMF (1 mL). The reaction mixture was stirred overnight at 80°C and then diluted with brine (3 mL) and extracted with DCM (3x). The crude product was subjected to general procedure A togive 92 (1.0 mg, 15% over 2 steps). 'H NMR (400 MHz, CDCl) 8 9.33 (d, J= 1.0 Hz, 1H), 8.85 (dd, J= 2.4, 0.9 Hz, 1H), 8.67 (d, J =6.2 Hz, 1H), 8.59 (dd, J= 4.8, 1.6 Hz, 1H), 8.51 (dt, J= 6.2, 0.9 Hz, 1H), 8.40 (dd, J= 7.4, 1.2 Hz, 1H), 8.20 (dt, J =8.2,1.1Hz, 1H), 7.88 (ddd, J= 7.9, 2.4, 1.6 Hz, 1H), 7.71 (dd, J= 8.2, 7.4 Hz, 1H), 7.57 — 7.53 (m, 2H), 7.37 (m, 3H), 3.82 (s, 2H), 3.34 (t, J = 6.1 Hz, 2H), 2.86 (m, 5H). '3C NMR (101 MHz, CDCl) 8 153.41, 148.60, 148.42, 145.35, 138.08, 136.93, 136.29, 134.40, 134.11, 133.80, 133.52, 131.42, 129.68, 128.91, 127.36, 126.01, 123.72, 117.78, 53.15, 49.64, 46.54, 34.88.
HRMS [C24H24N402S+H][*: 433.16927 calculated, 433.16907 found.
N-(2-(Isoquinoline-5-sulfonamido)ethyl)-4-(pyridin-3-yl}benzamide (93) of Nn sg N
[00274] NF Nig 93 (79 mg, 0.18 mmol, 79%) was synthesized from 114 (100 mg, 0.23 mmol) and pyridin-3-ylboronic acid (30 mg, 0.25 mmol) according to general procedure D.
H NMR (400 MHz, CDCls) § 9.23 (s, 1H), 8.70 (d, J= 2.2 Hz, 1H), 8.54 (dd, J = 4.8, 1.6 Hz, 1H), 8.50 — 8.41 (m, 2H), 8.39 (dd, J=7.4, 1.2 Hz, 1H), 8.08 (d, J=8.2 Hz, 1H), 7.77 (dt, J= 8.0, 2.0 Hz, 1H), 7.71 (1,
J=86Hz 3H), 7.60 (t, J= 7.8 Hz, 1H), 7.43 — 7.37 (m, 2H), 7.32 (dd, J = 8.0, 4.8 Hz, 1H), 3.64 —- 3.53 (m, 2H), 3.30 — 3.16 (m, 2H). 13C NMR (101 MHz, CDCls) 5 167.82, 153.19, 148.83, 147.83, 144.78, 140.50, 135.39, 134.63, 134.41, 133.60, 133.25, 133.16, 131.14, 129.02, 127.86, 126.99, 126.06, 123.91, 117.44, 87.10, 42.78, 40.35.
HRMS [C2aH20N4O3S+H]*: 433.13289 calculated, 433.13252 found.
N-(2-((4-(Pyridin-3-yl}benzyl}oxy)}ethyl}isoquinoline-5-sulfonamide (94) of Nn
[00275] NF nN” 94 (136 mg, 0.32 mmol, 76%) was synthesized from 119 (0.10 g, 0.44 mmol) and isoquinoline-5-sulfonyl chloride (106 mg, 0.40 pmol) according to general procedure B. 'H NMR (400 MHz, CDCl) 8 9.34 (d, J= 1.0 Hz, 1H), 8.80 (dd, J= 2.4, 0.9 Hz, 1H), 8.63 (d, J = 6.1 Hz, 1H), 8.60 (dd, J= 4.8, 1.6 Hz, 1H), 8.48 — 8.42 (m, 2H), 8.17 (dt, /=8.2, 1.1 Hz, 1H), 7.85 (ddd, J = 7.9, 2.4,16 Hz, 1H), 7.67 (dd, J=8.2, 7.3 Hz, 1H), 7.49 — 7.43 (m, 2H), 7.37 (ddd, J=7.9, 4.8, 0.9 Hz, 1H),
P345823NL 72 7.22 (d, J=82Hz, 2H), 6.30 (t, J= 5.9 Hz, 1H), 4.33 (s, 2H), 3.46 (t, J = 5.0 Hz, 2H), 3.27 — 3.17 (m, 2H).
BC NMR (101 MHz, CDCls} ö 153.36, 148.55, 148.17, 145.17, 137.45, 137.35, 136.22, 134.90, 134.47, 133.56, 133.13, 131.37, 129.11, 128.39, 127.22, 126.00, 123.74, 117.41, 72.79, 68.66, 43.15.
HRMS [C23H2iN303S +H]*: 420.13784 calculated, 420.13728 found.
N-{(2-(Methyl(4-{pyridin-3-yl)benzyl)amino)ethyl}isoquinoline-5-sulfonamide (95)
ON
[00276] NZ N” Pyridin-3-ylboronic acid (34 mg, 28 pmol) was subjected to general procedure D with 120 (0.10 g, 0.23 mmol) to provide 95 (45 mg, 0.10 mmol, 45%). 'H NMR (400 MHz, CDCl») 8 9.32 (d, J= 1.0 Hz, 1H), 8.84 (dd, J=2.4, 0.9 Hz, 1H), 8.65 (d, J = 6.1 Hz, 1H), 8.60 (dd, J= 4.8, 1.6 Hz, 1H), 8.48 — 8.41 (m, 2H), 8.18 (dt, J= 8.2, 1.1 Hz, 1H), 7.88 (ddd, J= 7.9, 24, 1.6 Hz, 1H), 7.68 (dd, J = 8.2, 7.3 Hz, 1H), 7.54 — 7.46 (m, 2H), 7.39 (ddd, J= 7.9, 4.9, 0.9 Hz, 1H), 7.29 — 7.23 (m, 2H), 3.38 (s, 2H), 3.06 — 2.97 (m, 2H), 2.49 — 2.38 (m, 2H), 1.91 (s, 3H).
BC NMR (101 MHz, CDCls) ö 153.42, 148.57, 148.27, 145.27, 138.08, 136.93, 136.27, 134.41, 134.21, 133.60, 133.42, 131.36, 129.61, 129.08, 127.20, 125.97, 123.72, 117.27, 61.72, 55.03, 41.24, 40.24.
HRMS [C24H24N402S+H]*: 433.16927 calculated, 433.16896 found.
N-({(2S,5R)-5-(4-(6-Fluoropyridin-3-yl)phenyl)pyrrolidin-2-yl)methyl)isoquinocline-5-sulfonamide (96)
FA F
~_N
Oo
Ve 0
[00277] Nx Following general procedure A, 1304 (53 mg, 94 pmol) was reacted with TFA to afford the title compound as a white solid (14 mg, 31 umol, 33%). "HNMR (400 MHz, CDCls) 8 9.37 (s, 1H), 8.71 (d, J= 8.1 Hz, 1H), 8.49 - 8.47 (m, 1H), 847 (d, J=1.2
Hz, 1H), 8.40 (d, J= 2.7 Hz, 1H), 8.22 (d, J= 8.2 Hz, 1H), 7.98 (ddd, J= 8.5, 7.6, 2.6 Hz, 1H), 7.75 - 7.68 (m, 1H), 7.42 (d, J = 8.3 Hz, 2H), 7.33 (d, J = 8.1 Hz, 2H), 7.02 (ddd, J = 8.5, 3.0, 0.7 Hz, 1H), 4.23 (dd, J =8.7,8.5 Hz, 1H), 3.50 — 3.42 (m, 1H), 3.04 (dd, J= 12.0, 4.2 Hz, 1H), 2.84 (dd, J = 12.0, 6.4 Hz, 1H}, 2.14 — 2.05 (m, 1H), 1.96 — 1.86 (m, 1H), 1.63 — 1.49 (m, 2H).
BC NMR (101 MHz, CDCl) 6 183.18 (d, J = 239 Hz), 153.56, 145.82 (d, J = 15 Hz), 145.40, 144.40, 139.76 (d, J = 8.0 Hz), 135.55, 134.65 (d, J = 4.8 Hz), 134.33, 133.70, 133.52, 131.38, 129.18, 127.18, 127.09, 126.08, 117.29, 109.61 (d, J = 37 Hz), 62.19, 56.05, 47.98, 34.48, 28.76.
HRMS [C2sH23FN4O2S + H]*: 463.15985 calculated, 463.15927 found. [a] 3 +47,9° (c = 1.00, CHC).
P345823NL 73
N-({{2R,5R)-5-(4-(6-Fluoropyridin-3-yl)phenyl}pyrrolidin-2-ylimethyl}isoquinoline-5-sulfonamide (97) “4 | F =~._N oz 9”
Nine “
[00278] Nx Following general procedure A, 130d (36 mg, 64 mmol) was reacted with TFA to afford the title compound as a white solid (22 mg, 48 pmol, 74%).
HNMR (400 MHz, CDClIs) ò 9.37 (d, J=1.1 Hz, 1H), 8.70 (d, J = 6.1 Hz, 1H), 8.51 — 8.47 (m, 1H), 8.46 (dd, J=4.7,1.2 Hz, 1H), 8.39 (dt, J= 2.6, 0.9 Hz, 1H), 8.22 (d, J= 8.2 Hz, 1H), 7.95 (ddd, J = 8.5, 7.6, 26Hz, 1H), 7.72 (dd, J=8.2, 7.4 Hz, 1H), 7.47 (d, J = 8.3 Hz, 2H), 7.28 (d, J = 6.4 Hz, 2H), 7.01 (ddd, J =8.5,3.1,0.7 Hz, 1H), 4.02 (dd, J = 8.4, 6.3 Hz, 1H), 3.60 — 3.49 (m, 1H), 3.01 (dd, J= 12.3, 4.4 Hz, 1H), 2.79 (dd, J=123,79 Hz, 1H), 2.23 - 2.13 (m, 1H), 2.08 — 1.96 (m, 1H), 1.75 - 1.63 (m, 1H), 1.54 — 1.41 (m, 1H). 3C NMR (101 MHz, CDCl3) ö 163.22 (d, J = 239 Hz), 153.50, 145.83 (d, J = 15 Hz), 145.39, 143.87, 139.74 (d, J = 8.0 Hz), 135.73, 134.52, 134.48, 133.69, 133.42, 131.39, 129.16, 127.33, 127.15, 126.086, 117.31, 109.62 (d, J = 38 Hz), 61.39, 57.24, 47.50, 34.76, 29.47.
HRMS [C25H23FN402S + H]*: 463.15985 calculated, 463.15925 found. {a} 5 +29.5° (c = 1.00, CHCl).
N-({(28,58)-5-(4-(6-Fluoropyridin-3-yl)phenyl)pyrrolidin-2-yl)methyl)isoquinoline-5-sulfonamide (98) ~_N
Do“
[00279] Nx Following general procedure A, 130b (5.8 mg, 10 pmol) was reacted with TFA to afford the title compound as a white solid (3.4 mg, 7.4 pmol, 71%). "HNMR (500 MHz, CDCls) 8 9.37 (s, 1H), 8.71 (d, J= 6.1 Hz, 1H), 8.47 (dd, J = 7.3, 1.2 Hz, 1H), 8.46 — 8.43 (m, 1H), 8.40 (dt, J=2.7, 0.8 Hz, 1H), 8.22 (d, J=8.2 Hz, 1H), 7.95 (ddd, J= 8.4, 7.6, 2.6 Hz, 1H), 7.72(dd, J=8.2,7.3 Hz, 1H), 7.48 (d, J = 8.4 Hz, 2H), 7.28 (d, J = 7.9 Hz, 2H), 7.01 (ddd, J = 8.5, 3.0, 0.7 Hz, 1H), 4.03 (dd, J = 8.4, 6.3 Hz, 1H), 3.58 — 3.52 (m, 1H), 3.01 (dd, J= 12.3, 4.4 Hz, 1H), 2.79 (dd,
J=12.3, 7.9 Hz, 1H), 2.23 — 2.16 (m, 1H), 2.06 — 1.99 (m, 1H), 1.70 (dg, J= 12.5, 8.6 Hz, 1H), 1.53 — 1.44 (m, 1H).
BC NMR (126 MHz, CDCls) 5 163.27 (d, J = 239 Hz), 153.53, 145.89 (d, J = 15 Hz), 145.48, 143.68, 139.75 (d, J = 8.0 Hz), 135.85, 134.52, 134.49, 133.71, 133.44, 131.43, 129.20, 127.39, 127.20, 126.086, 117.32, 109.64 (d, J = 38 Hz), 61.48, 57.30, 47.44, 34.72, 29.48.
P345823NL 74
HRMS [C2sH23FN402S + H]*: 463.15985 calculated, 463.15937 found. [a] & =-23.5° (c= 0.31, CHCl).
N+{({2R,55)}-5-{4-(6-Fluoropyridin-3-yl}phenyl}pyrrolidin-2-yl)methyl}isoguinoline-5-sulfonamide (99)
A
~_N ia =
[00280] C0) Following general procedure A, 130c (96 mg, 0.18 mmol) was reacted with TFA to afford the title compound as a white solid (54 mg, 0.12 mmol, 68%). 'H NMR {400 MHz, CDCl3) 8 9.37 (s, 1H), 8.69 (d, J = 6.1 Hz, 1H), 8.51 — 8.47 (m, 1H), 8.47 — 8.45 (m, 1H), 8.39 (dt, J = 2.7, 0.9 Hz, 1H), 8.21 (d, J = 8.2 Hz, 1H), 7.96 (ddd, J= 8.5, 7.8, 2.6 Hz, 1H), 7.71 (dd,
J=8.2,74Hz 1H), 7.41 (d, J= 8.3 Hz, 2H), 7.32 (d, J = 8.0 Hz, 2H), 7.02 (ddd, J = 8.5, 3.0, 0.7 Hz, 1H), 4.22 (dd, J= 8.6, 6.5 Hz, 1H}, 3.46 (ddt, J= 8.8, 6.5, 4.4 Hz, 1H), 3.04 (dd, J = 12.1, 4.3 Hz, 1H), 2.84 (dd, J= 12.0, 6.5 Hz, 1H), 2.14 — 2.05 (m, 1H), 1.97 — 1.84 (m, 1H), 1.63 — 1.46 (m, 2H).
BC NMR (101 MHz, CDCls) 8 163.13 (d, J = 239 Hz), 153.52, 145.76 (d, J = 15 Hz), 145.32, 144.46, 139.74 (d, J = 7.9 Hz), 135.45, 134.63 (d, J = 4.8 Hz), 134.33, 133.65, 133.47, 131.35, 129.14, 127.15, 127.03, 126.07, 117.29, 109.58 (d, J = 37 Hz), 62.13, 56.07, 48.03, 34.45, 28.74.
HRMS [CasH2aFN4O:S + H]*: 463.15985 calculated, 463.15918 found. [a] 5 -51.2° (c= 1.00, CHC).
N-{{(2S,5R)-5-{[1,1'-Biphenyl]-4-yl)pyrrolidin-2-yl)methyl)isoquinoline-5-sulfonamide (100) fap 0
[00281] Ny Following general procedure A, 130e (7.4 mg, 14 pmol) was reacted with TFA to afford the title compound as a white solid (8.4 mg, 9.9 pmol, 73%).
HNMR (850 MHz, CDCl3) 8 9.37 (s, 1H), 8.72 (d, J= 6.0 Hz, 1H), 8.47 (d, J= 6.1 Hz, 2H), 8.21 (d, J= 8.2 Hz, 1H), 7.71 (t, J = 7.8 Hz, 1H), 7.58 (d, J= 6.9 Hz, 2H), 7.48 (d, J = 8.1 Hz, 2H), 7.47 — 7.44 (m, 2H), 7.37 — 7.34 (m, 1H), 7.29 (d, J = 8.1 Hz, 2H), 4.22 (dd, J = 8.7, 6.5 Hz, 1H), 3.48 — 3.45 (m, 1H), 3.03 (dd, J= 12.1, 42 Hz, 1H), 2.84 (dd, J= 12.1, 6.4 Hz, 1H), 2.11 = 2.07 (m, 1H), 1.93 — 1.88 (m, 1H), 1.61 — 1.55 (m, 2H).
BC NMR (214 MHz, CDCl) 6 153.57, 145.47, 143.05, 140.96, 140.22, 134.31, 133.70, 133.50, 131.39, 129.18, 128.93, 127.37, 127.22, 127.19, 126.83, 126.05, 117.30, 62.36, 55.95, 47.90, 34.39, 28.78.
HRMS [C2osH2sN302S + H]*: 444.17402 calculated, 444.17390 found.
N-(((25,5R)-5-(4'-Fluoro-[1,1'-biphenyl]-4-yl}pyrrolidin-2-yl}methyl}isoquinoline-5-sulfonamide (101)
P345823NL 75 7) F 0 te” 0
[00282] Nx Following general procedure A, 130f (9.0 mg, 16 umol) was reacted with TFA to afford the title compound as a white solid (1.5 mg, 3.2 umol, 20%).
TH NMR (400 MHz, CDCl3) 8 9.37 (d, J=1.0 Hz, 1H), 8,71 (d, J = 6.1 Hz, 1H), 8.47 (m, 1H), 8.46 (m, 1H), 8.21 (d,J= 8.2 Hz, 1H), 7.71 (dd, J= 8.2, 7.3 Hz, 1H), 7.58 — 7.48 (m, 2H), 7.45 — 7.39 (m, 2H), 731-724 (m, 2H), 7.18 — 7.09 (m, 2H), 4.20 (dd, J= 8.8, 6.5 Hz, 1H), 3.51 — 3.39 (m, 1H), 3.03 (dd, J = 12.0, 4.2 Hz, 1H), 2.83 (dd, J = 11.9, 6.4 Hz, 1H), 2.13 - 2.03 (m, 1H), 1.97 — 1.82 (m, 1H), 1.63 — 1.51 (m, 2H). 13C NMR (101 MHz, CDCls) 8 162.55 (d, J = 246 Hz), 153.56, 145.42, 143.28, 139.17, 137.08 (d, J= 3.3
Hz), 134.33, 133.88, 133.51, 131.39, 129.18, 128.69 (d, J = 8.0 Hz), 127.05, 126.86, 126.08, 117.30, 115.77 (d, J = 21 Hz), 62.27, 55.93, 47.97, 34.44, 28.80.
HRMS [CzsH24FN302S + H]*: 462.16480 calculated, 462.16440 found.
N-(((28,5R)-5-(4-(Pyridin-3-yl}phenyl)pyrrolidin-2-yl)methyl)isoquinoline-5-sulfonamide (102). 7 ~_N
OO
[00283] Nx Following general procedure A, 130g (7.5 mg, 14 pmol) was reacted with TFA to afford the title compound as a white solid (4.2 mg, 9.3 umol, 68%).
[00284] 'H NMR (850 MHz, CDCl3) 8 9.38 (s, 1H), 8.84 (d, J= 2.4 Hz, 1H), 8.71 (d, J = 6.0 Hz, 1H), 8.60 (dd, J=4.8, 1.6 Hz, 1H), 8.49 — 8.47 (m, 1H), 8.47 (s, 1H), 8.22 (d, J= 8.2 Hz, 1H), 7.89 — 7.86 (m, 1H), 7.724, J=75Hz, 1H), 7.47 (d, J=8.2 Hz, 2H), 7.38 (ddd, J=7.8, 4.8, 0.9 Hz, 1H), 7.35 — 7.32 (m, 2H), 4.23 (dd, J=8.8,6.5Hz, 1H), 3.49 —3.44 (m, 1H), 3.04 (dd, J= 12.1, 4.2 Hz, 1H), 2.83 (dd, J=121,6.4
Hz, 1H), 2.13 — 2.08 (m, 1H}, 1.94 — 1.88 (m, 1H), 1.60 — 1.54 (m, 2H).
[00285] 13C NMR (214 MHz, CDCls) 8 153.58, 148.57, 148.38, 145.45, 144.32, 136.73, 136.44, 134.40, 134.32, 133.71, 133.54, 131.39, 129.19, 127.22, 127.13, 126.08, 123.74, 117.29, 62.24, 55.98, 47.99, 34.51, 28.78.
[00286] HRMS [C25H24N402S + H]*: 445.16927 calculated, 445.16913 found.
Antibacterial Activity
Materials and Methods
[00287] Reagents & materials. Buffers and salts were of ACS reagent grade or higher and were purchased commercially, from Carl Roth GmbH (Karlsruhe, Germany} and Sigma-Aldrich {Darmsiadt, {ermany}, biological materials and growth media were purchased from Sigma-Aldrich, Scharlab S.L. (Barcelona, Spain) and Fischer Scientific (Landsmeer, Netherlands). Antibiotics trimethoprim (Sigma-
P345823NL 76
Aldrich), ceftazidime (ceftazidime pentahydrate, Thermo Scientific, Landsmeer, Netherlands) and kanamycin (kanamycin monosulfate, MP biomedicals, lllkirch, France) were dissolved in DMSO stored in -20°C, apart from ciprofloxacin, which was used from a 3M aqueous solution containing 0.01% AcOH. All test compounds were used from 10 mM DMSO stock solutions made from the freeze dried powder and stored at -20°C.
[00288] Bacterial strains. Klebsiella pneumoniae ATCC 29665 (NCTC 11228), Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Acinetobacter baumannii ATCC BAA747 and
Staphylococcus aureus USA300 (ATCC BAA1717) belong to the American Type Culture Collection (ATCC). E. coli NCTC 13463 and 13846 belong to the National Collection of Type Cultures (UK Health
Security Agency). E. coli JW5503, JW3600, JW3602, JW3605 JW3594, JW3596 belong to the Keio
Collection52 of single-gene knockouts. E. coli strains 552059.1 and 552060.1 were isolated from urine£3 and acquired from the clinical Medical Microbiology department at the University Medical Center Utrecht.
E. coli mer-1 (EQAS 2016 412016126, mcr-1 positive, recovered during international antimicrobial resistance programs), W3110, BW25113, belong to the laboratory collection of N.I.M. Fluoroquinolone resistant E. coli strains 965, 991, 1022, 1075, 1104,1146, 1175, 1192, 1201, 1233 were isolated from blood cultures, positive ciprofloxacin resistance, during July-August 2021 and were acquired from the
Department of Medical Microbiology and Infection Prevention, Amsterdam University Medical Centre. The following reagents were obtained through BEI Resources, NIAID, NIH: E. coli, Strain MVAST0072, NR- 51488.
[00289] Antibacterial activity screen. From glycerol stocks, Staphylococcus aureus USA300, as the
Gram-positive representative strain, and Escherichia coli W3110, as the Gram-negative representative strain, were cultured on blood agar plates by overnight (18 + 2 h) aerobic incubation at 37°C. A single colony was transferred to tryptic soy broth (TSB) or lysogeny broth (LB). Cultures were grown to exponential phase (OD600 = 0.5) aerobically at 37°C. The bacterial suspensions were diluted 200-fold in cation adjusted Mueller-Hinton broth (CAMHB) and 99 uL were added in a library of test compounds (1
HL DMSO stock solution, per well in technical duplicates) in polypropylene 96-well microtiter plates to reach a volume of 100 uL and a final concentration of 100 pM for each test compound and a maximum of 1% DMSO. The plates were sealed with breathable membranes and incubated at 37°C overnight with constant shaking (600 rpm). Screening hits were selected from the wells where no visible bacterial growth was observed, as compared to the inoculum controls, containing 1% DMSO.
[00290] Minimum inhibitory concentration. MIC was determined by broth microdilution. Single colony cultures were grown to exponential phase (OD600 = 0.5) aerobically at 37°C. In case of strains from the
Keio collection®®, 50 pg/mL kanamycin was supplemented to the media tc ascertain a homogeneous population. The bacterial suspensions were diluted 100-fold in CAMHB and 50 uL was added to a 2-fold serial dilution series of test compounds (50 yL per well) in polypropylene 86-well microtiter plates to reach a volume of 100 pL. The plates were sealed with breathable membranes and incubated overnight at 37°C with constant shaking (600 rpm). The MIC was determined as the lowest concentration at which no visible bacterial growth was observed, as compared to the inoculum controls, from the median of a minimum of triplicates.
P345823NL 77
[00291] Mammalian cell culture. HepG2 and HEK293T cell lines (ATCC) were cultured at 37°C and 7%
CO: in DMEM (Sigma Aldrich, D6546) with GlutaMax, penicillin (100 pg/mL), streptomycin (100 pg/mL) and 10% Fetal Calf serum. Cells were passaged twice a week by first detaching using 0.05% trypsin in
PBS, and then diluting to appropriate confluence.
[00292] Cytotoxicity assay (MTT). Compound cytotoxicity was evaluated against HepG2 and HEK293T human cell lines using standard (3-(4,5-dimethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide (MTT) assay protocol®* with slight changes.
[00293] HepG2 and HEK293T cells were seeded at a density of 1.5 x 104 cells per well in a clear 96-well tissue culture treated plate in a final volume of 100 pL in Dulbecco's Modified Eagle Medium (DMEM), supplemented with Fetal Bovine Serum (1%), Glutamax and Pen/Strep.
[00234] Cells were incubated for 24 h at 37°C, 7% CO: to allow cells to attach to the plates. In addition to a single vehicle control, compounds (diluted from DMSO stock) were added into each well at eight concentrations ranging from 100 uM to 0.046 uM in three-fold dilutions (final DMSO concentration 0.5%).
Incubation was done for 24 h at 37°C, 7% CO:. After the incubation, MTT was added to each well at a final concentration of 0.40 mg/mL. The plates were then incubated for 2 h at 37°C, 7% CO:. Medium was carefully removed via suction, and purple formazan crystals were resuspended in 100 uL DMSO.
[00295] Absorbance was read at 570 nm using a Clariostar plate reader. The data was then analysed with GraphPad Prism software. ICs values were calculated using non-linear fitted curve with variable slope settings, with values adjusted for background (plotted ABSsamprie = (ABSsampLe — ABSBacksrouND) / (ABSvenicLe — ABSsackerounp)). Technical triplicates for each condition were used, along with biological duplicates.
[00296] Hemolytic activity. Whole defibrinated sheep blood (10631715, Fisher Scientific) was centrifuged (400 rcf) for 15 min at 4°C. The supernatant was discarded and the remaining blood cell suspension was mixed with phosphate buffered saline (PBS) and centrifuged (400 rcf) for 15 min at 4°C.
Washing cycles were repeated at least three times, until the supernatant was clear after centrifugation.
The packed blood cells were diluted 25-fold in PBS with 0.002% polysorbate 80 (p80). Test compounds were serially diluted 2-fold in U-bottom polypropylene 96-well microtiter plates in PBS with 0.002% p80 (75 pL). An equal volume (75 uL} of the blood cell suspension was added to all wells. Final concentrations of antibiotics ranged from 1.6 pM to 50 uM in triplicate. The well plates were incubated for 20 h at 37°C with continuous shaking (500 rpm). After incubation, plates were centrifuged (800 rcf) for 5 min, and 25 pL of supernatant was transferred to a clear UV-star flat-bottom polystyrene 96-well plate containing 100 pL ultrapure water, per well. Absorption was measured at 415 nm with a Spark® multimode microplate reader (Tecan, Switzerland). Data were corrected for the background response of 0.5% DMSO in the presence of cells with no antibiotic and normalized using the absorbance of 0.1%
Triton X-100 with blood cells, as 100% hemolytic activity.
[00297] Bacterial cytological profiling. Overnight E. coli BW25113 cultures were diluted 1:100 in fresh
LB medium in a 100 mL Erlenmeyer flask and incubated at 120 rpm and 37°C. When OD600 > 0.2, the cultures were diluted to OD600 = 0.1 in fresh LB medium with a final volume of 200 yL ina 2 mL
Eppendorf tube. The appropriate antibiotic or control treatment was applied and the samples were
P345823NL 78 incubated at 200 rpm and 30°C for 2 h. Subsequently, DAPI (2 ug/mL), FM4-64 (2 ng/mL) and SYTOX
Green (0.5 uM) were added to reach the desired concentration and the samples were incubated a further 10 min. The samples were then centrifuged at 3300 x g for 50 s and the pellets resuspended in half the original volume. From this suspension, 5 uL was spotted onto a 2.0% agarose pad for microscopy.
[00298] Microscopy was performed on a Zeiss Axio Observer Z1/7 inverted microscope. For differential interference contrast the transmitted light source was an Aquilla TL halogen lamp (3.0 V). For widefield fluorescence the Zeiss Colibri.2 LED lamps were configured as follows: DAPI (365 nm, 150 ms, 25.42%),
FM4-64 (590 nm, 1000 ms, 100%) and SYTOX Green (470 nm, 150 ms, 25.42%). Illuminated and reflected light were led through the following filters: DAPI (Zeiss 49: excitation = G 365, beam splitter = FT 395, emission = BP 445/50), FM4-84 (Zeiss 63: excitation = BP 572/25 (HE), beam splitter = FT 590 (HE), emission = BP 629/62 (HE)) and SytoxGreen (Zeiss 38: excitation = BP 470/40 (HE), beam splitter = FT 495 (HE), emission = BP 525/50 (HE)). Images were collected through a Plan-Apochromat 100x/1.40 objective in oil immersion (n = 1.518) with a Hamamatsu C9100-02 camera.
[00299] Specific information on the programs and parameters used during image analysis are displayed in Table 8 below. For the qualitative assessment of the bacterial morphology, the acquired images were first pre-processed in ImageJ57 (v 1.53m). Thereafter the brightness and contrast were adjusted to allow the clearest visual inspection of the particular phenotype. For the quantitative BCP, DIC images were first pre-processed and then a random forest classifier was trained with lllastik (v 1.3.3)58 to generate a segmented image. These binary masks were then combined together with the original DIC image and processed with the ImageJ plugin Microbe59 (v 5.13). MicrobeJ generated cell outlines and performed measurements on cell shape accordingly. The recorded cell outlines produced by MicrobeJ were exported and used as a guide to characterize the DNA shape within each bacterium using a custom- made ImageJ macro (Appendix Il). DAPI and SYTOX Green intensity were measured on raw images in a similar manner. Then by hand polygons were drawn that excluded bacteria to determine background intensity. After background correction the intensity was standardized to the DMSO control within the same biological replicate. For principal component analysis, the BCP assay was executed for three independently biological replicates. During each experiment, images were acquired until 24 or more bacteria in total had been observed. Following feature extraction for each replicate, principal component analysis was performed using GraphPad Prism {v 8.0.0) using multiple variable analysis with standardized data. PCs were selected based on the percent of total explained variance (minimum 80%).
[00300] Genomic studies Resistant mutant generation. Spontaneous resistant colonies were obtained by plating 100 pL of E. coli inoculum grown to an OD600 of 0.5 (~107 CFU) onto LB agar plates containing 5x the MIC of compound 60. The plates were incubated at 37°C and checked for growth after 24 and 48 h. Single colonies were picked after 48 h of incubation and their MIC was determined as described previously. Nine colonies with an increased MIC were selected for further analysis and stored as glycerol stocks.
[00301] Genomic studies Whole-genome sequencing. Nine spontaneous 60-resistant mutants of E. coli were selected for genome sequencing. DNA was extracted as described elsewhere. Briefly, E. coli cells were harvested from an overnight culture and re-suspended in lysis buffer (TE, SDS 10%,
P345823NL 79 proteinase K), incubated for 1 h at 37°C. Classical extraction with phenol-chloroform was performed and the aqueous layer was precipitated with absolute ethanol. The DNA pellet was washed with 70% ethanol, dried and solubilized in TE to perform RNA digestion with RNase 50 pg/mL (RNase A, Thermo Fischer).
Degraded RNA was removed by phenol/chloroform extraction followed by ethanol precipitation. DNA was re-suspended in nuclease-free water. Genome sequencing was performed using Illumina Novaseq 6000
PE150 at Novogene Co. Ltd. (Tianjin, China). Paired-end sequence reads were generated and mapped against the reference genome of E. coli W3110. The alignment to the reference genome was performed using the Burrow-Wheeler Alignment tool (BWA v0.7.8+!). SNP/InDel calling, annotation and statistics was performed using SAMtools*? (v0.1.19) and ANNOVAR® (v2015MAR22). The structural variant calling annotation and statistics was performed with BreakDancer v1.4.4 and ANNOVAR*3 (v2015MAR22). The wild type E. coli W3110 was also sequenced and compared to the reference genome to confirm that the mutations found in the 60-resistant colonies were unique and related to the antibiotic resistance.
Sequencing data is available at NCBI through the BioProject accession number PRJNA855320.
[00302] Construction of gyrA recombinant mutants in E. coli W3110. Mutants of the DNA gyrase subunit A encoding gene (gyrA) were constructed following the protocol for gene editing via the CRISPR-
Cas9 system. ** Strains and plasmids used are listed in Table 1, and primers used can be found in Table 2.
Table 1
Plasmid Characteristics Source or reference
E. coli W3110 Wild type (F-A1ph~1 IN{rnD, mi) Jensen, 199352
E. coli DH5a fhuA2 A (argF-lacZ)U169 phoA ginv44 $80 Meselson &
A(lacZ)M15 gyrA96 recAl relA1 endA1 thi-1 hsdR17 Yuan, 196852 pCas repA101(Ts), kan’, Pcas-cas9, Paras-Red, lacl9, Pie- Jiang et al, 201554 sgRNA-pMB1; temperature-sensitive replication vector pTargetF pMB1, aadA, sgRNA-fts Jiang et al, 20155 psgRNA-gyrA-M5 Plasmid used to insert the S97L mutation in gyrA psgRNA-gyrA-S83L Plasmid used to insert the S83L mutation in gyrA psgRNA-gyrA-D87N Plasmid used to insert the D97N mutation in gyrA
Table 2
Primers Sequence (5'to 3' gyrA_P 05 TTCTCTAGAGTCGACCTGCAGGAAGTCCGGCCCCGGGATGTST (SEQ ID NO: 27) gyrA_P TCGAGTAGGGATAACAGGGTAATATCTAGAGGTTTACCGGCGATTTTTCGGCATTCAT 08 (SEQ ID NO: 28) gyrA_P 09 ATCAGCCCTTCAATGCTGATG (SEQ ID NO: 29) gyrA_P
TCCGTAATTGGCAAGACAAAC (SEQ ID NO: 30) gyrA_P | CTCAGTCCTAGGTATAATACTAGTAGCATATAACGCAGCGAGAAGTTTTAGAGCTAGAAAT 11 AGCAAGTTAAAATAAG (SEQ ID NO: 31) gyrA_P 12 CTGCAGGTCGACTCTAGAGAATTCAAAAAAAGCACCGACTC (SEQ ID NO: 32) gyrA_P
TTTACTACGTTACATGCTGGTAGACGGTCAGGGTAACTTCG (SEQ ID NO: 33) gyrA_P
CCAGCATGTAACGTAGTAAAAATGGCTGCGCCATGCGGACGATC SEQ ID NO: 34
P345823NL 80 gyrA_P | CTCAGTCCTAGGTATAATACTAGTCATAGACCGCCGAGTCACCAGTTTTAGAGCTAGAAAT 17 AGCAAGTTAAAATAAG (SEQ ID NO: 35 gyrA_P 18 GGAGATCTGGCTGTTTATGACACGATCGTCCGCATGGCGCAG SEQ ID NO: 36 gyrA_P GTCATAAACAGCCAGATCTCCATGGGGATGGTATTTACCGATTACGTCACCAACGAC 19 SEQ ID NO: 37 gyrA_P | CTCAGTCCTAGGTATAATACTAGCTATGACACGATCGTCCGCAGTTTTAGAGCTAGAAATA 20 GCAAGTTAAAATAAG (SEQ ID NO: 38) gyrA_P 21 CTATAACACAATAGTAAGGATGGCGCAGCCATTCTCGCTGCG (SEQ ID NO: 39) gyrA_P | GCCATCCTTACTATTGTGTTATAGACCGCCGAGTCACCATGGGGATGG (SEQ ID NO: 22 40)
[00303] The following mutations were created: S97L in E. coli W3110. Two common mutations in the gyrA gene that give resistance to fluoroquinolone antibiotics were created in E. coli W3110, namely S83L and D87N. To create the gyrA [S97L] mutants, the construct psgRNA-gyrA-M5 was assembled as follows: The 20 nt spacer sequence was introduced by PCR on pTargetF using primers gyrA_P11 and gyrA_P12, the homology-directed repair arms were amplified from E. coli W3110 genomic DNA by PCR using primer pairs gyrA_P05 and gyrA_P013, and gyrA_P08 and gyrA_P14. The three PCR fragments generated were cloned into the Spel and Bglll digested pTargetF via Gibson assembly. To avoid off- target effects of CRISPR, besides desired point mutation for gyrA[S97L], four other silent point mutations were also included in HDR template. Consequently, the amino acid sequence of gyrA will remain the same in the generated mutant except for the desired mutation. Similarly, constructs psgRNA-gyrA-S83L and psgRNA-gyrA-D87N were created to introduce mutations S83L and D87N in gyrA, respectively. In psgRNA-gyrA-S83L, spacer sequence was introduced using primers gyrA_P12 and gyrA_P17, HDR template was amplified using primer pairs gyrA_P05 and gyrA_P18, and gyrA_P08 and gyrA_P19. In psgRNA-gyrA-D87N, spacer sequence was introduced using primers gyrA_P12 and gyrA_P20, HDR template was amplified using primer pairs gyrA_P05 and gyrA_P21, and gyrA_P08 and gyrA_P22. The verified constructs for gyrA engineering were transformed into E. coli W3110 carrying pCas9, and plated in LB containing kanamycin (50 pg/mL) for the selection of the pCas9 plasmid and spectinomycin (100 pg/mL) for the selection of psgRNA-gyrA-X. The plates were incubated at 30°C. Plasmids were cured by growing the strains with 0.5 mM IPTG with no antibiotics to lose first the psgRNA-gyrA plasmids. After losing the spectinomycin resistance, the strain was grown in LB with no antibiotics at 42°C to lose the pCas plasmid. Colony PCR was performed on plasmid-free colonies using primers gyrA_P9 and gyrA_P10, and PCR products were sequenced to confirm the desired mutation.
[00304] DNA gyrase supercoiling assay. The DNA gyrase inhibition assay was executed as instructed by the manufacturer (G1001 from Inspiralis Limited). A master mix was made including 0.5 ug relaxed plasmid (pBR322) in 35 mM Tris-HCI (pH 7.5), 24 mM KCI, 4 mM MgClz, 2 mM DTT, 1.8 mM spermidine, 1 mM ATP, 6.5% (w/v) glycerol, and 0.1 mg/mL albumin per reaction. Of this stock solution 27 uL was aliquoted for each reaction in 1.5 mL Eppendorf tubes and supplemented with 0.6 pL of the appropriate compound dilution or corresponding solvent with a final DMSO concentration of 2%. The reactions were started with 3 HL of enzyme (1 U) in dilution buffer (50 mM Tris-HCI (pH 7.5), 100 mM KCI, 2 mM DTT, 1 mM EDTA, and 50% (w/v) glycerol) for a final volume of 30 uL. Reactions were run at 37°C for 30 min and stopped with the addition of 30 uL Stop Dye (40% (w/v) sucrose, 100 mM Tris-HCI pH 8, 10 mM
P345823NL 81
EDTA, 0.5 mg/mL bromophenol Blue) and 30 pL of a chloroform:iscamyl alcohol solution (24:1 v/v). After brief vortexing and centrifugation at 2300 x g for 1 min, 20 pL of the aqueous phase was loaded onto a 1% (w/v) agarose gel in TAE (Tris-acetate 0.04 mM, EDTA 0.002 mM) gel. The gel was run at 85 V for 2 h followed by staining (15 min) in 1 pg/mL ethidium bromide and destaining (5-10 min) in water. DNA was visualized (602/50, UV Trans, auto optimal exposure) with a ChemiDoc MP (Bio-Rad Laboratories, Inc) and the percentage of supercoiled DNA relative to the total amount material per lane was determined with
ImageLab 6.1 software (Bio-Rad Laboratories, Inc). ICso curves were generated in GraphPad Prism (v 9.0.0) using the nonlinear regression curve fit variable slope with four parameters and least squares regression.
[00305] Checkerboard assay. Dilution series of both the test compound and polymyxin B nonapeptide (PMBN) were prepared in CAMHB media. To evaluate synergy, 25 uL of test compound solutions were added to wells containing 25 uL of PMBN solution. This was replicated in three columns for each combination so as to obtain triplicates. 50 pL of bacterial stock (0.5 x 105 CFU/mL) was added and the plates were sealed with breathable membranes. After incubation for 20 hours at 37 °C while shaking at 600 rpm, the breathable seals were removed and the plates shaken using a bench top shaker to ensure homogeneous bacterial suspensions. The plates were then transferred to a Tecan Spark plate reader and following another brief shaking (20 seconds) the OD600 was measured. The resulting OD800 values were transformed to a 2D gradient to visualize the growth/no-growth results
[00306] Synergy assay: Fractional Inhibitory Concentration Index (FICI). The FICI was calculated using Equation 1 where synergy is indicated by FICI £ 0.5.3
PIC = oben Mon (1)
MIC, Miles
Equation 1. Calculation of FICI. MSCant = MIC of antibiotic in combination with synergist; MICan = MIC of antibiotic alone; MSCsy = MIC of synergist in combination with antibiotic; MICsyn = MIC of synergist alone.
In cases where the MIC of the antibiotic or synergist was found to exceed the highest concentration tested, the highest concentration in the dilution series was used in determing the FICI and the result reported as < the calculated value.
[00307] Cryogenic electron microscopy sample preparation. Plasmids for the expression of E. coli
GyrA and GyrB (pET28-GyrATS and pET28-GyrBTS) were previously described?? and obtained as a gift of Prof. Valerie Lamour (IGBMC, University of Strasbourg). GyrA and GyrB were purified as described, 217 bp DNA (a strong gyrase binding site of phage Mu) was used as a substrate. E. coli GyrA and GyrB subunits were mixed in equimolar proportions to reconstitute the full DNA gyrase enzyme. 217 bp DNA was added to the complex in a 1:1 ratio to a final concentration of gyrase-DNA complex ~15 HM. The reconstituted complex was buffer exchanged using dialysis at 4°C overnight to cryo-EM buffer (25 mM
Na-HEPES pH 8, 30 mM potassium acetate, 2.5 mM magnesium acetate, 0.5 mM TCEP). After buffer exchange, the sample was concentrated to ~30 uM. Sample was supplemented with 100 uM 102 compound, 1 mM ADPNP and incubated for 30 min at 37°C. 8 mM CHAPSO was added, and sample was centrifuged (60 min at 21,000 x g) to remove potential aggregates.
P345823NL 82
[00308] Cryo-electron microscopy data collection and analysis. Aliquots of 4 pl of reconstituted complexes (~12 mg/ml) were applied to glow-discharged (Leica, 60 s/8 mA) Quantifoil haley carbon grids (R2/1, 300 copper mesh). After 30 s of incubation with 95% chamber humidity at 10°C, the grids were blotted for 6 s and plunge-frozen in liquid ethane using a Vitrobot mark IV (FEI).
[00309] Cryo-EM data were collected at the Polish national cryo-EM facility SOLARIS with a Titan
Glacios microscope (Thermo Fisher Scientific) operated at 200 kV, and images (movie frames) were collected at the calibrated physical pixel size of 0.95 A per pixel with a defocus range of -3 to -0.9 um.
The images were recorded in counting mode on a Falcon IV electron direct detector (Thermo) in EER format. A dose rate and exposure time was set to generate a total dose of ~40 electrons/A2. Statistics for cryo-EM data collection are listed in Table 3.
Table 3 800 _ Data collection and processing
Microscope ThermoFisher Glacios _
Magnification 45,000x
Voltage (kV 300 )
Electron dose (e:/Â2 39.91
Detector Falcon 4
Defocus range (-um 3-0.9 à Pixel size (A) 0.95 î
Symmetry imposed C1
Micrographs (no.) 10,362 ;
Initial particle images (no.) 1,575,192
Final particle images (no.) 132,680
Global map resolution (A) 4.23
FSC threshold 0.143
Refinement
Model resolution (A) 4.17
FSC threshold 0.143
Map sharpening B factor (A?) 129.4
Model composition
Non-hydrogen atoms 15,278
Protein residues 1784
Nucleotides 52 à
Ligands 2 à
Mean B factors (A?)
Protein 60.74 à
Nucleotide 75.47
Ligands 19.30 ;
R.m.s. deviations
Bond lengths (A) 0.004 |l
Bond angles (° 0.890
Validation
MolProbity score 2.67
Clashscore 5.61 à ‚ Ramachandran plot Favored (%) 87.76
Allowed (%) 11.00
Disallowed (%) 4128
P345823NL 83
[00310] All processing was done in cryoSPARC 3.3.1.% 10,362 movies were dose weighted and motion and CTF corrected in patch mode. Particles were picked with cryoSPARC template picker and 2x2 binned particles (1,575,192) were subjected to several rounds of 2D classification. Cleaned particles representing holoenzyme with secondary structure elements visible (183,664) underwent two rounds of 3D classification with 2 classes (CS3 ab initio, 134,472 particles retained) and was refined to 4.1 A resolution using non-uniform refinement procedure.*® Particles were re-extracted as unbinned using updated coordinates. Further non-uniform refinement of this particle set with local CTF correction’ resulted in a final map of estimated 4.23 A global resolution and 3.7 A local resolution around the drug binding site. Multiple attempts to improve the FSC and resolution by global and focussed classification in both cryoSPARC and Relion did not bring improvement nor highlighted a particular source of heterogeneity in the dataset; it was therefore concluded that in this case the resolution was limited by the quality of the original sample. The 4.23 A map displayed more visible side-chains and best local resolution for 102 binding pocket and therefore was selected for final refinement.
[00311] Model building and refinement. The closest available structure of E. coli gyrase (PDB:6RKV22) was used as a starting point for model building. BRKV model was rigid-body fitted in ChimeraX*® and manually adjusted in Coot®. Real-space refinement was performed in phenix.refine (using
Ramachandran restrains and secondary structure restraints). The model and restraints for 102 were obtained using Grade server (hitp://grade.globalphasing.org); 102 was initially manually fitted into the density and refined in Phenix. The model was refined against the 4.23 A map described above.
MolProbity5t was used to validate the structures. Statistics for the final model is reported in Table 3.
Antibacterial activity
[00312] To discover the antibiotic activity against Gram-negative bacteria, compounds of the invention were assayed against two different strains of Gram-negative bacteria; E. coli and K. pneumoniae. Data is presented in Table 4.
Table 4 oo Mcp IVe
Compound . , 2K = Compound oo , = K es pneumonie BCH pneumonke 1 6.25 12.5 72 25 50 47 25 >50 73 25 50 48-55 >50 >50 74 25 >50 56 6.25 12.5 75-85 >50 >50 57 6.25 12.5 86 50 >50 58 25 25 87 25 50 59 3.1 6.25 88 12.5 25 60 6.25 6.25 89 6.25 12.5 61 6.25 >50 90 12.5 12.5 62 >50 >50 91-94 >50 >50 63 6.25 12.5 95 >50 50 64 >50 >50 96 3.1 12.5 65 12.5 25 97 12.5 25 66 12.5 25 98 >50 >50 67 >50 >50 99 >50 >50 68 12.5 25 100 1.6 6.25
P345823NL 84 69 25 >50 101 1.6 3.1 70 25 50 102 31 6.25 71 12.5 50
[00313] Many of the compounds of the invention showed good to excellent control over one or both of the bacterial strains tested (e.g. Compounds 1, 56, 57, 59, 60, 61, 63, 89, 96, 100, 101 and 102).
Cytotoxicity
[00314] Compounds 56, 57, 59, 60, 61, 63, 89 and 96-102 were tested for cytotoxicity using human kidney (HEK293T) and liver (HepG2) cell lines by means of an MTT assay measuring metabolic activity as an indicator of cell viability, proliferation, and compound cytotoxicity. Data is presented in Table 5.
Table 5 ‘Compound Ce compound CoM [00315] C
Compound WEKzesT Hepoz SOMPOUNd HEKzeaT mMepoz ompo 56 10.4 16.7 96 22 >50 57 6.6 8.2 97 39 >50 unds 59 26 1.7 98 49 >50 60 60 >50 >50 99 >50 >50 61 1.0 28 100 8.1 37 and 83 14.7 38 101 3.2 13 102 89 8.9 6.8 102 >50 >50 were found to be non-cytotoxic (ICse > 50 pM). Compound 102 was the most potent compound with a MIC of 3.1 HM, with no cytotoxicity or hemolytic activity observed (Table 5, Figure 26). Also of note, compound 102 was found to be bactericidal against E. coli, when evaluated at higher concentrations (Figure 27). 102 showed selectivity towards the Gram-negative strains E. coli and K. pneumoniae among a selection of pathogenic species (Table 6).
Table 6. Antimicrobial spectrum of isoquincline sulfonamides. stan 6 402 9g cp
K. pneumoniae ATCC 29665 25 12.5 12.5 0.012
P. aeruginosa ATCC 27853 >50 >50 >50 0.377
A. baumannii ATCC BAA747 >50 25 25 0.755
E. coli 0.024
W3110 6.25 0.8 1.6 0.024
BW25113 6.25 1.8 3.1 0.024
ATCC 25922 12.5 3.1 6.25 0.024
S. aureus
ATCC 29213 >50 >50 >50 0.755
ATCC BAA1717 >50 >50 >50 >0.755
[06316] Furthermore, this activity was maintained when 102 was tested on several clinical isolates of E. coli (Table 7), including multidrug resistant (MDR), mcr-1 positive, and extended-spectrum beta lactamase (ESBL) producing strains.
Table 7. MIC (uM) of selection of compounds against clinical isolates of E. coli and potentiation by addition of 4 uM PMBN.
P345823NL 85 . . Resistance 60 + 101 + 102 + CIP +
E. coli strain rofile 60 PMBN 101 PMBN 102 PMBN CIP PMBN
MIC MSC FC MIC MSC EC MIC MSC FC MIC MSC mer-1 MCR 50 125 a 125 31 a 25 6.25 id >32 232
NCTC13463 ESBL 125 18 8 31 04 8 31 04 8 1 1
NCTC13846 MDR (mer-1) >50 25 >2 125 31 4 50 125 4 232 >32
MVASTO072# MDR 50 3.1 #8 125 08 #8 25 15 {8 232 >32 552059.1% 50 31 {#8 125 08 16 25 0.78 NN 1 1 652060. 1# 50 31 16 125 08 18 25 15 {8 1 1 1075 MDR >50 25 22 50 625 8 >50 12.5 >4 0.13 0.13
PMBN: Polymyxin B nonapeptide, CIP: ciprofloxacin, MSC: minimum synergistic concentration,
FC: fold change in MIC after addition of 4 uM PMBN, MCR: mobile colistin resistance, MDR: multidrug resistant, ESBL: extended spectrum beta lactamase, # urinary tract infection isolates.
[00317] In view of this excellent profile, compound 102 was selected for further profiling. Closely related inactive compound 99 was chosen as a negative control compound.
Bacterial cytological profiling
[00318] To gain more information about the MoA of compound 102, an imaging-based approach was employed termed bacterial cytological profiling (BCP), developed by Pogliano and co-workers.2! This method exploits the fact that bacteria often undergo drastic morphological changes as a result of antibiotic treatment and that antibiotics with differing MoAs result in visually distinct phenotypes. BCP allows for the quantification of these different phenotypes and, by constructing a reference map of antibiotics, the phenotypic similarity of cells treated with 102 was compared to cells treated with a number of clinically-used reference antibiotics. To this end, the morphological effects of 102 on E. coli were compared to a known cell wall synthesis inhibitor (ampicillin (AMP)), an RNA synthesis inhibitor (rifampicin (RIF}), a protein synthesis inhibitor (tetracycline (TET)), a DNA synthesis inhibitor (CIP) along with negative control (DMSO treatment) as well as a structurally similar inactive control (compound 99).
[00319] To visualize these changes with fluorescence microscopy, the treated bacteria were stained with a membrane permeable DNA dye (DAPI), a lipophilic membrane dye (FM4-64) and a membrane impermeable DNA dye to signify loss of membrane integrity (SYTOX Green) (Figure 28). Clear morphological changes were seen in response to all active compounds. Notably, 102 induced elongation of the bacteria and condensation of the DNA, which resembled to some extent the effects observed in bacteria treated with CIP (Figure 28). To obtain a more quantitative image analysis, 24 features that describe cell dimensions and fluorophore intensities of individual bacteria, were extracted (see Table 8, below).
Table 8. Parameters used during image analysis
Software Analysis Parameters/Command
ImageJ?! (v 1.53m) Qualitative BCP — DAPI run("Subtract Background...", channel "rolling=15"); run("Mean...", "radius=1");
Qualitative BCP — FM4-64 run("Subtract Background...", channel "rolling=5"}, run("Enhance Contrast...” "saturated=0.3"); run{("Mean...", "radius=1");
P345823NL 86
Qualitative BCP — DIC . channel No preprocessing
Quantitative BCP — DIC run("Gaussian...", "radius=1.5"); channel
Quantitative BCP — DAPI Custom made script (Figure S1) shape descriptors
Quantitative BCP — DAPI Custom made script (Figure S2) and SYTOX Green intensity llastik? (v 1.3.3) DIC image segmentation All features selected
Classifier trained for label “cell” or “background”
Microbed® (v 5.131) Quantitative BCP — DIC Image used for bacteria detection: DIC shapes channel
Segmentation based on: binary mask generated by lllastik.
Bacteria detection = fit shape (rod shaped)
Attributes: Area (1.5 — max); Length (1.5 — max); Width (0.5 — max); Circularity (0 — max)
Enabled options: Exclude on Edges;
Shape descriptors; Segmentation (default settings); Intensity; Shape;
Profile (medial, default settings)
[00320] Principle component analysis of these 24 features revealed that the inactive control 99 clustered with DMSO, while the reference antibiotics clustered separately from one another (Figure 29a,c). 102 formed a cluster by itself, which was interesting considering its apparent similarity to CIP. This led to the suggestion that 102 may have a unique MoA related to the inhibition of DNA synthesis.
Genomic studies
[00321] To identify the cellular target of compound 102, it was aimed to obtain drug-resistant mutants and subsequently performing comparative genomics studies to identify SNPs that correlate to the resistance phenotype. To this end, E. coli (107 CFU) were plated on solid agar containing compound 68 (5x MIC) (Figure 30a) and nine viable colonies were isolated and screened for resistance (Figure 30b). All colonies had at least an 8-fold increase in MIC for compound 60 and cross-resistance was also subsequently found with compound 162 and compound 101, while no cross-resistance was found with common antibiotics (Table 9).
Table 9. MIC of known antibiotics and compounds against selected 80-resistant (60-r) selected isolates of 1% W3110.
Antibiotic ModeofAcion ~~ 0 1 6.25 >100 100 >100 60 6.25 >100 100 >100 101 1 6 >50 6.25 3.1 102 3.1 >50 12.5 12.5
Imipenem PBP - cell wall 0.375 (0.1) (0.75 (0.2) 0.375 (0.1) 10.375 (0.1)
Meropenem PBP - cell wall 0.05 (0.02) (0.09 (0.04) 0.05 (0.02) 0.05 (0.02)
Ceftazidime PBP - cell wall 0.75 (0.4) 0.375(0.2) 0.75(0.4) 0.75 (0.4)
Colistin Outer membrane 0.19 (0.5) 0.375 (1) 0.375 (1) 0.18 (0.5)
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Trimethoprim Folic acid synthesis [0.375 (0.16) [0.375 (0.16) [0.18 (0.08) 0.375 (0.16)
Neomycin Fr synthesis 5 (1) 075(05) 156) 0.75 (0.5)
Chloramphenicol (805) synthesis 2 4 (ay 12.4 (4) 12.4 (4) 12.4 (4)
Ciprofloxacin DNA replication as) (0.007) (0.007%) (0.007)
[00322] Of interest, one of the colonies showing resistance to 60 exhibited an enhanced sensitivity towards 102 and 101.
[00323] Whole-genome sequencing of the strains showing resistance to compound 60 revealed that each had mutations in a gene encoding bacterial DNA gyrase (Figure 31), a heterotetrameric protein complex comprising two GyrA and two GyrB subunits. Eight of the colonies were found to contain mutations in
GyrA, primarily in the N-terminal winged-helix (A84V, S97L) or the tower domains (S172A, A179V, N338S,
P324L) (Figure 30c¢), and one in GyrB (A456E). Mapping these mutation sites on a recently published cryo-EM structural model of DNA gyrase?? revealed their spatial proximity and suggested a potential binding pocket (Figure 30d). To validate that these mutations in gyrA were indeed responsible for the resistance towards 60, a CRISPR/Cas9 system was used to introduce the highly resistant gyrA S97L mutation, found in mutants 60-r1 and 60-r2 (Figure 30b) in an E. coli wild type background. It was confirmed that bacteria containing the S97L mutation were resistant against compounds 60, 101 and 102 (Table 10).
Table 10. CRISPR-gyrA editing. Target validation by introduction of point mutations with CRISPR/Cas9 on an E. coli W3110 background. MIC values are in agreement with sequenced spontaneous mutants.
WT WT 6.25 16 3.1 0.024
S97L 102-resistant >50 >50 >50 0.024
S83L FQ-resistant 1 25 3.1 6.25 >1.5
D87N FQ-resistant 2 50 3.1 6.25 >1.5
[00324] This combined with the whole-genome sequencing results strongly suggests that DNA gyrase is the primary target of the isoquinoline sulfonamides.
DNA gyrase supercoiling assay
[00325] DNA gyrase is a well-validated antibiotic target, which is responsible for introducing negative supercoils in DNA (Figure 32a), a process that is required for DNA synthesis and proliferation of bacteria.
DNA gyrase is a target of the fluoroquinolone antibiotics including CIP. To test whether 102 was able to directly interact with DNA gyrase, 102 was incubated in a concentration-dependent manner with recombinant E. coli DNA gyrase and measured its supercoiling activity in a gel-based assay. The activity of 102 was compared to inactive control 99 (Figure 32b). CIP was taken along as a positive control and produced an ICs value of 925 nM, which is in line with previously reported values.?* Remarkably, 102 inhibited DNA gyrase activity with an I1Cs0 of 35 nM, thereby making it >25-fold more potent than CIP.
Mirroring the phenotypic screening results, control compound 99 was 13-fold less potent than 102,
P345823NL 88 thereby confirming that the cis-2R, SR is the distomer. Taken together, these biochemical studies demonstrate that the compounds of the invention are potent inhibitors of DNA gyrase.
Cellular penetration
[00326] Biochemical data showed that 102 is a nanomolar range inhibitor of DNA gyrase, but its antimicrobial activity is in the low micromolar range (Figure 32e). It was hypothesized that this discrepancy could be due to active efflux by TolC, a major bacterial transporter or poor cellular uptake caused by the Gram-negative OM.2* To distinguish between these possibilities, it was first checked whether 102 and 99 showed enhanced potency in the Afo/C efflux pump mutant. No increase in potency was observed, suggesting that the isoquinoline sulfonamides are not substrates of the TolC efflux pump.
Next, 102 and 99 were tested on E. coli strains containing loss-of-function mutations to outer membrane lipopolysaccharide (LPS) assembly genes (ArfaC, ArfaY, Arfal, ArfaP and ArfaD) (Table 11).
Table 11. MIC (UM) on E. coli strains of the Keio collection. © BW25113 parent 0024 >50 31
JW5503 ATolC 0.024 >50 3.1
JW3600 ArfaY 0.024 >50 1.6
JW3602 Arfal 0.024 >50 1.6
JW3605 ArfaP 0.048 12.5 0.4
JW3594 ArfaD >0.048 31 0.4
JW3596 ArfaC 0.024 3.1 0.4
[00327] While the LPS biosynthesis mutations did not lead to the increase in activity of CIP, the antibacterial effect of 102 was strongly potentiated in all mutants. Of note, the activity of 99 was also potentiated in the ArfaC, ArfaP and ArfaD strains. A similar increase in sensitivity was observed when E. coli was co-treated with 102 or 99 in presence of polymyxin B nonapeptide (PMBN) (Figure 32e}, a well- characterized25 disrupting agent of the Gram-negative OM.2 The synergistic effect of PMBN and compounds 99 and 102 was further explored with a checkerboard assay. Results are shown in Table 12, below.
Table 12. Result table from checkerboard assays of antibiotics and PMBN as synergist on E. coli W3110.
Ciprofloxacin 0.025 0.05 1.5-2 99 6.25 200 £0.28 102 0.39 3.13 <0.25
MSC: minimum synergistic concentration, MIC minimum inhibitory concentration, FICI:
Fractional Inhibitory Concentration Index, PMBN: Polymyxin B nonapeptide.
[00328] Clear synergy was seen for 102 and 99, while none was observed for CIP (Figure 32f). The potentiating effect of PMBN on 102 was maintained across the panel of clinical isolates tested (Table 7).
Thus, both genetic and pharmacological studies indicate that the Gram-negative OM limits the cellular penetration of the isoquinoline sulfonamides.
Cryo-EM studies
P345823NL 89
[00329] To validate presumed binding pocket of 102 on the GyrA subunit, cryogenic electron microscopy (cryo-EM) was used to determine a medium-resoclution structure of E. coli gyrase holocomplex (AzBz2) bound to the substrate 217-bp dsDNA, nucleotide analogue ADPNP and 102. A single dataset collected on 200 kV Glacios microscope allowed us to visualise enzyme-DNA complex in the wrapped state (see
Figure 33) and reconstruct a model for the cleavage-reunion domain of the enzyme (residues 8-524 of
GyrA and 405-804 of GyrB) similarly to Lamour and coworkers, at the resolution of 4.2 A (Figure 34a).
CTDs of GyrA with wrapped DNA and ATPase domains of GyrB were also observed but not modelled.
While overall conformation of the complex was comparable to the available E. coli gyrase cryo-EM structure in complex with gepotidacin (PDB: SRKW and 6RKYV), there was a striking difference in the position of CTDs which were shifted upwards to form a narrow angle with the GyrB ATPase domains (Figure 33). The DNA substrate was seemingly intact, suggesting 102 unlike fluoroquinolones, does not stabilize cleaved DNA complex. Guided by the induced mutations (Figure 33b), identified 102 was easily identified as the distinct density, clearly separated from both DNA and protein (Figure 34a,e). One molecule of 102 was bound to each GyrA subunit, occupying a horseshoe-like hydrophobic pocket on the
DNA-binding surface (Figure 34b) just underneath the DNA but far away (>20 A) from the catalytic residues. The curved shape of 102 perfectly corresponded to the shape of the pocket, while the opposite would be the case for a distomer 99. The quinoline ring was lodged between His45 and Leu98 of GyrA while the pyridine ring is positioned right on top of the GyrA lle112 and Phe96. Remarkably, two hydrogen bonds anchoring 102 in place were made by the side chain of GyrA Ser87 (to the N3 of the central pyrrolidine ring) and the main chain of GyrA Ser172 (to the sulfonic acid moiety) (Figure 34c,f). Therefore, not only the main mutation (S97A) selected upon exposure to 102 would clearly prevent the interaction, but also the initial SAR observation that nitrogen-containing linker is of crucial importance is now fully substantiated. Similarly, side-chain interaction with Ser172 explains how S172A mutation can slightly potentiate compound 102 activity.
[00330] The compounds of the invention exhibit potent, Gram-negative-specific antibacterial activity with little mammalian cell toxicity. Cell morphology and mutation selection clearly pointed to DNA gyrase as the target for compounds of the invention after which a cryo-EM study revealed that the compounds bind to a previously unexploited site on the GyrA subunit. Somewhat surprisingly, compounds of the invention show no affinity for binding to DNA, as is common in other DNA gyrase inhibitors.
When directly compared to the clinically used fluoroquinolone CIP, compounds of the invention were found to exhibit a number of key differences: 1) Morphological studies with bacterial cytological profiling indicated that E. coli cells respond differently to CIP treatment than to compounds of the invention; 2) No significant cross-resistance was found between CIP and compounds of the invention in strains harboring resistance-inducing point mutations, further highlighted by the susceptibility of CIP resistant clinical isolates to compounds of the invention; and 3) Structural insights derived from cryo-EM studies reveal no overlap in the binding site of compounds of the invention in the DNA gyrase compared with that of CIP.
Based on this evidence (but without wishing to be bound by theory), it is thought that compounds of the invention disrupt DNA gyrase activity via a novel mechanism that is distinct from that of the fluoroquinolones. While CIP was found to generally exhibit lower MIC values than compounds of the
P345823NL 90 invention against susceptible strains, it is interesting to note that compounds of the invention are much more potent inhibitors of DNA gyrase supercoiling activity, an effect most likely ascribed to the Gram- negative OM.
Indeed, synergy assays with PMBN indicate that once past the OM, the antibacterial activity of compounds of the invention is significantly enhanced.
P345823NL 91
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GL <Gequencelala seguenceliNuubsc="Ss>
GE <IiNSsDseqd> 2 <INSDSeq length>856</INSDSeq length> id <INSDSeq moliype>AA/INIDIeq molLype»>
Gs <INSDSeq division>PAT</INSDSeg division» ëë <INSDSeq feature-table> £7 <INSDFeature> oa CINEDFeature keyvsource</INSDFeature key» 5D “INSDFeabture Location». .856</INSDFeature location»
Fi} <INSDFesture gualis>
Tl <INSDOualifier> 2 <INSDQualifler named>mol type“ /INSDQuali fier names
Ty <INSDQualifier valuevprotein</INSDQualifisn wvalus>
TA </INSDQuali fier!» iE CINSDQualifiler id="glve
TE CINZDQuallfier namerorganism</IN3DQualifier name> 7 <“INSDQualifier valus>Corynebacterium diphtheriae/INSDQualijier value»
Te </INSDQualifier>
IE </INSDFaature quals> 23 </INSDFeatune> </INZDSey feature-table> <INEDSeq sequence MSDDLLGGDGYDRVHPIDLNEEME TSY IDYAMSVIVGRALPEVRDGLKPVHRRILY
AMYDSGYRPDRSYVKSARPVSDTMGQFHPHGDSAIYD TLVRLAQDWNMRY PMVDGQGNFGSRGNDGPAAMRYTE
CRLTPLAMEMVRDIRENTVDFSPNYDGKTQEPDVLPSRVPNLIMNGSNGIAVGMATNI PPHNLRELGEAIFWLL
DNPEADEASALEACMKYVKGPDFPTAGQIVGSQGINDAYTTGRGSIRMRGVTSIEEEGNRQIIVITELPYQVNP
DNMISNIAEQVRDGKLAGISKIEDESSDRVGMRIVVTLKRDAVPRVVLNNLYKHSQLQTNFGANMLSIVDGVPR
TLRLDOQMLRHYVTHQIEVIVRRTQYRLDEAEKRAHILRGLVKALDMLDEVIALIRRSPTVDIARTGLMELLTVD
EIQADAILAMQLRRLAALERQKIIDELAEIELEIADYKDILARPERQRAIVRDELAEIVDKYGDDRRTQIIAAT
GDVTEEDLIARENVVVTITSTGYAKRTKVDAYKSQRRGGKGVRGAELKQDDVVRHFFVSSTHDWILFFTNFGRV
YRLKAYELPEASRTARGQHVANLLEFQPEERIAQVIQIQSYEDAPYLVLATAQGRVKKSRLSDYESNRSGGLIA
INLNEGDKLIGAALCDNDDDLLLVSEEGQSIRFNANDDQLRPMGRATAGVKGMRFKGDDQLLAMTVVKPDAFLL
VATSGGYGKRTSLDEY SPQGRGGQGVLTFKYTPKRGKLIAAVVVDEDDEILAITSAGGVIRTVVNQIRPSSRAT
MGVRLVNLEDGVELLAIDRNVEGE GEEAAEAVATGVVDGPAERGKQTEVDLGVDSADEEA</ INSDS ey saqu ance 33 </INSDSeg>
Sá </SegusncsDara>
SL <Sequencebala seguantaliliicghar=n4ns
HE CINSDSag» ad <INSDSeq length>833</INSD3eq Length> 28 <INSDSeq moltype>AA“/INSDSeq moltype> 23 <INSDSeq division>PAT</INSDSeg division:
Sa <INSLSeq feature-itabled
Gi <INSDFeabture> el <IMaDFesature key>source“/INSDFeature key> 43 <INSDFeature location>l..833</INSDFeature locations ed <INSDFeature quals> IJ
EN CINBDOualifierns 3E <INSDQualifier name>mol type“ /INSDQualifier name» 37 <INSDQualifier valuerprotein</INSDQualifier value» 38 </INSDQuali jier
Ga <INSDQualifier 1d=Vgdn>
LO <INSDQualifier namevorganism“/INSDQualifier name> iel <INSDOualifier valuerEnterococcus faecalis</INSDgualifien value»
LOE </INSDOualilfier>
Lis </INSDPeature guals> 104 </INSDFeaturer 195% </IN3DSeq featura-table>
CINSDIag sequence>MSEEIRENIQDVNLTSEMKE SFIDYAMSVIVARALPDVRDGLKPVHRRILYGMNEL
GVTPDKPHKKSARIVGDVMGKYHPHGDIAIYESMVRMAQPFSYRAMLVDGHGNFGSVDGDGAAAMRY TEARMSK
IALEMLRDINKNTVDFQGNYDDSEQEPVVLPARFPNLLVNGTTGIAVGMATNIPPHNLSEVIDATSLLMDNPDV
TTNELMEVLPGPDFPTGGLVMGKSGIRRAYETGKGSITVRAKVELTEMPNGKERILVTELPYMVNKAKLIERIS
ELHRDKRIEGITDLRDESSREGMRIVIDVRRDVSASVVLNNLYKMTALQTSFGFNMLAIEKGVPKILSLKRILE
NYVEHQKEVITRRTIFDKNKAEARAHILEGLRIALDHIDEIIAIIRGSQSDDEAKATLIERFEFSDRQAQAILD
MRLRRLTGLERDKIENEYQELLKFIADLEDILARPERVIEIIKTELNDVRTKFGDARRTELLVGEVLSLEDEDL
IEEEEVVITLTNNGY IKRMANSEFRAQRRGGRGVQGMGVHDDDFVKNLVSCSTHDTLLFFTNTGKVYRAKGYE I
PEYGRTAKGIPVINLLGIDSTEKIQAIISVEGKAEAGKYLFFTTLKGTVKRTAVTAFSNIRSNGLIAISLKEDD
ELVNVVTTNGNQKMIIGTHAGYSVTFDENTVRDMGRTASGVRGIRLRENDYVVGAAILDENKEVLVITENGYGK
RTKASEYPVKGRGGKGIKTANITEKNGPLAGLTTVNGDEDILLITNKGVIIRFNVDSVSQTGRATLGVRLMRME
DGAKVVTMAVVEPEEVEEEIVEVVETTENETTSETEE</INSDSedq sequenced an </INSDSea> ine </SeguenceDatar ins <“SequenceData semmenceIDNuber=sSs>
LAD <INSDSeq>
LAE <INSDSeq lengih>823</INSDSeq length»
LL <INSDSeq moliype>AA</INSDSeq moltype»
LLS CINZDSeq divizion»PAT</INSDS=q division»
Lie <“INSDSeq feature-itable> iiD <INSDFeature»> iia <INSDFesture keyrsource“/INSDFealure key»
LAY <INSDFeature location>l..823</INIDFeaturs location
LAE <INSDFeature guals> ie <INSDQualifier>
Lal <INMEDQualifier namermol type</INsDoualifier name>
Lal CINZDOQuallifier valuesprotein“/INSDgualifier value»
LEE </INSDOualilfier> 123 <INSDQualifier id="g5"> ize <INSDQualifier name>organism</INSDQualifisr names ijk <INSDQualiiler value>Enterococcus faecium“/INSDQGualifler value» ize </INSDQuali fier!»
La </INSDFearture qualss
Law </INSDFaaturel u
LADE </INSDSeq feature-fablex <INBSDSeq sequence >MSEEIRENTHDVNLTSEMKDSFIDYAMSVIVARALPDVRDGLKPVHRRILYGMNEL
GVTPDKAHKKSARIVGDVMGKYHPHGDSAIYESMVRMAQPFSYRYMLVDGHGNFGSVDGDGAAAMRY TEARMSK
IATEMLRDINKNTVDFQSNYDDTEKEPVVLPARFPNLLVNGTTGIAVGMATNIPPHNLSEVVAAIDLLMDNPDV
TTNELMEVLPGPDFPTGGLVMGKSGIRRAYETGKGSITVRAKVEITEMPNGKERIIVTELPYMVNKAKLIERIS
ELHRDKRIEGITDLRDESSREGMRIVIDIRRDASASVILNNLYKLTSLONSFGFNMLAIEKGVPKVLSLKQILE
NYIEHQREVITRRTVFEKEKAEARAHILEGLRIALDHIDEIIAIIRGSKSDDEAKATMIERFDLSDRQSQAILD
MRLRRLTGLEREKIENEYQELLKLIEDLTDILARPERVTEIIKNELADLNQRFGDKRRTELLVGEVLSLEDEDL
IEEEEIVITLTNNGY IKRLANNEFRAQRRGGRGVQGMGIHNDDFVKNLVSCSTHDTLLFFTNNGKVYRAKGYEI
PEYGRTAKGIPIINLLGIDSSESIQATIIAVTGEAEEGHYLFFTTRQGTVKRTSVTAFANIRSNGLIAIGLKEDD
ELVNVLLTDGQSNIIIGTHNGYSVTFKEEAVRDMGRTASGVRGIRLREEDYVVGAALMKEEQEVLVITEKGYGK
RTKVSEYPVKGRGGKGIKTANITEKNGPLAGLTTVSGDEDIMLITDKGVIIRFNVSTVSQTGRSTLGVRLMKME
ADTKVVTMAAVEPEAAEQTVEEQGSEE</INSDSeq sequence»
LSL </INSDSear
LS <j Gaemquencebata>
LEG <Sequencebata seguanteliMushao="EY> ize <IiNSsDseqd> 13D <INSDSeq length»835</INilSeq length>
LIB <INSDSeq moliype>AA/INIDIeq molLype»> 1a <INSDSeq division>PAT</INSDSeg division»
LEB <INSDSeq feature-table>
Lau <INSDFeature>
TaD CINEDFeature keyvsource</INSDFeature key»
Lad “INSDFeabture locabtion>l..835</IN3DFeature location» iid <INSDFeature gualis> 14 <INSDOualifier> idd <INSDQualifler named>mol type“ /INSDQuali fier names ij <INSDQualifier valuerprotein</INSDQuali fien value» i4ë </INSDQuali fier!»
TAY CINSDQualifler id='g8N2>
TAG CINZDQuallfier namerorganism</IN3DQualifier name> iin <INSDQualifier value>Erysipelothrix rhusiopathiae</INSDRualifisr valus> 185 </INSDQualifier> 151 </INSDFaature quals> ian </INSDFeaLure>
HE </INZDSey feature-table>
CINEDSeq sequence MEDNTQSYDKIKQRNISEEMKKSFVSYAMSVIVSRALPDVRDGLKPVHRRILYAMN
DLGMTSDKPYKKSARIVGEVIGKYHPHGDTAVYDSMVRMAQEFSYRYMLIDGHGNFGS IDGDGAAAMRY TEARM
SKISMELIRDINKNTVDFIDNYDGEEREPVVLPSRFPNVLVNGGTGIAVGMATNIPPHNLGEVIDATIALIDNP
DITIKELMEDY IFGPDFPTGALLLGRSGIKSAFETGRGSVVMRAKVDIEEMKNGKPRIIISEIPYQVNKATLVE
KIATLVRDKEIDGITDLRDE SNREGIRIVVELRREVQAEVVLNQLYRLTALQSSFGVNMLALVNGRPELLNLLQ
VLSHYRDHQIEIVTRRTQFELKKAEDRAHILQGIMIALDHIDEVISIIRSSKDDPEAITRLNEAFDLTEIQSKA
VLDMQLRRLTGLQRDKVENEFNELTILIVDLKD ILANHDRLLTITIKDELIEIKTKFGDDRRSE IVEADIDMLDE
DLIPVEDIVVTMTMNGYIKRTTVDSFNTONRGGKGVRGISTYDEDTVDQFIAMSTHDYLLLFTNLGKVYRIRGE
NVPSSSRTSKGIPVVNLLNLTEGETVKTLVKVAKDDE SKYAFFVTKQGIVKRVEVQEFESIROQNGKIAITLRED
DELVGVRMTNGDNE II IGGSNGKAVRFDENEVRSMGRTASGVIGFNVDEGEVVGIATDREGQY ILAVTEKGYGK
RTD IAEYRRTKRGAKGVKTVNITEKNGNLVSLRAVNGDEEALIISNEGTVIRTEISNIGIYGRSTIGVRLINVG
ETDSVSQVAILQPTVEEPDEEQTTDQVEPVNEKEIEIAE</ INSDS aq sequence 155 <STHEDS eq
L3G </SeguenceDatas
LE <Sequencebata zegvencaiDNumber=njn)»
LEE <INSDSeq>
Lis “INSDSeqg length>842</INSDSeq lengths
LED <INSDSeq moltype>dAA/INSDSeg moltype> ial <INSDSeq divislon»PAT</INIDSeg division ief <INSDSeq Ieatureriabier 153 “INSDFeaturex
Lhd <INSDFeature key>source“/INSDFealure key> 18% <IMaDFeature location>l..842</INSDFeature location»
LO <INSDFeature qualsh
LET <CINSDQualifiers
LEE <INSDQualifier name>mol_ type /INSDQualiiier named
LES <INSDQualifier value»protein</INSDOQualijier value» ijn </INSDQualifier>
IE “INSDoualifier id="g3rx>
LHR <INSDGualifier namerorganism</INSDQualifisr name> 1 <INSDQualifier valuerListeria monocytogenes</INsLgualifier value 174 <SINSDOualifier>
LEE </INSDFeaturs duals»
Lia </INSDEeaturer
ATE </INSD8eq featura-tabler
LINSDEeq sequence >MAETPNQRITE INLNKEMRTSFLDYAMSVIVARALPDVRDGLKPVHRRILYAMNDL
GITSDKAYKKSARIVGEVIGKYHPHGDTAVYFTMVRMAQDF SYRNMLVDGHGNF GSVDGDMAAAMRY TEARMSK
ISMELLRDINKDTIDYADNYDGSEREPVILPARFPNLLVNGSSGIAVGMATNIPTHHLGEVIDGVLALSHDPDI
TIRDLMEYIPGPDFPTAGMIMGRSGIRRAYESGRGSITVRGRVDIEEKKNGKETIVITEIPYQVNKARLVERIA
ELAREKKIDGITSLNDESDRSGMRIVIEVRRDISASVIVNNLFKMTALQTTFGINMLALVDNHPKVLNLKE ILY
HYLEHQKVVIRRRTEFELRKAEARAHILEGLRIALDNIDAIIKLIRGSKTSDVAKEGLMTQFNLSDKQAQATILD
MRLQRLTGLEREKIEEEYONLVALINDLKAILADDERILEIIREELEEIKVKYADKRRTEILAGDLVSLEDEDL
IPEEEVAITLTKRGY IKRLPLSTYRSQRRGGRGIQGMSTHEDDFVEHLVATSTHDTLLFFTNTGKVYRSKGYEV
PEYGRTAKGIPIINLLGIESQEQVNAVINLSEFTDDSYLFFTTKHGVVKRTTLSQFAKIRQSGLRAVELRENDE
LISVOMTDGSKNMIIATKHGQSIYFPEENIRVMGRTAAGVRGIRLREDDEVIGMEVLEDDEKVLVVTEKGY GKQ
TPASQYPLRNRGGMGVKTVTITEKNGNLVAMKTVTGEEDLMLMTVSGVLIRFEIDTVSQTGRSAMGVKLIRLDE
DEKVATVAKVPKEEDEVELEEEIDETLITQVPDESFEDAPGSDIEE</[INSDSeqg sequence
Ln </INSDSedg>
TED </SeguenceData>
LE <Sequencalata semuencernNumLer=N8N> in: <INSDSeq»
Las <INSDSeq lengith>822</IN3DSeq length»
LSA <INSDSeq moltype>AA</INSDSeqg moltypes
TRL <INSDSeq division»PAT</INEDIag division>
Lee “INSDSeq fealure-table> 1a <INSDFeature»
IEE <INSDFeature key>source</INSDFeature key»
RG <INSDFesture location>l..B822</INSDieaturs location» 130 <INSD¥eature guals>
RR <INSDoualifier>
Lan <INSDQualifier namermol type</INSDQualifier name>
Las <INSDOQualifier valuesprotein</INSDLQualifier valued hd <SINSDOualifier>
Lah LINSDGualiifler i0='g58%> ia <INSDQualifier name>organism</INSDQualifier names 137 <INSDQualifier value>Streptococcus pneumoniae serotype
A</INSDOualifier valuer iss </INSDQvalifier: u
Tan </INZDFeature guals> 200 </INSDFeature>
SO </INSDSeqg faature-tabled <INSDSedq sequence >MQDKNLVNVNLTKEMKASFIDYAMSVIVARALPDVRDGLKPVHRRILYGMNELGVT
PDKPHKKSARITGDVMGKYHPHGDSSIYEAMVRMAQWWSYRYMLVDGHGNFGSMDGDSAAAQRYTEARMSKIAL
EMLRDINKNTVDFVDNYDANEREPLVLPARFPNLLVNGATGIAVGMATNIPPHNLGETIDAVKLVMDNPEVTTK
DLMEVLPGPDFPTGALVMGKSGIHKAYETGKGSIVLRSRTE IETTKTGRERIVVTEFPYMVNKTKVHEHIVRLV
QEKRIEGITAVRDESNREGVRFVIEVKRDASANVILNNLFKMTOMQTNFGFNMLAIQNGIPKILSLRQILDAYI
EHQKEVVVRRTRFDKEKAEARAHILEGLLIALDHIDEVIRIIRASETDAEAQAELMSKFKLSERQSQAILDMRL
RRLTGLERDKIQSEYDDLLALIADLADILAKPERVSQIIKDELDEVKRKFSDKRRTELMIGOVLSLEDEDLIEE
SDVLITLSNRGYIKRLDODEFTAQKRGGRGVQGTGVKDDDFVRELVSTSTHDHLLFFTNKGRVYRLKGYEIPEY
GRTAKGLPVVNLLKLDEDESIQTVINVESDRSDDAYLFFTTRHGIVKRTSVKEFANIRONGLKALNLKDEDELI
NVLLAEGDMDIIIGTKFGYAVRFNQSAVRGMSRIATGVKGVNLREGDTVVGASLITDQDEVLIITEKGYGKRTV
ATEYPTKGRGGKGMQTAKITEKNGLLAGLMTVQGDEDILMI ITDTGVMIRTNLANISQTGRATMGVKVMRLDQDA
QIVTFTTVAVAEKEEVGTENETEGEA</INSDSec sequence» 203 </INSDSec:> 204 <SBequencelata>
Sal <Hegquencelbata seguansellMonhar=nat >
Li LINSDSeq»
Zij <INSDSeq iength>828/INSDSeq Llengih>
Zan <INSDSeq moltype>AA</INSDSeq moliype> 203 CINBDSeq divisionsPAT</INSDSeg division»
ZLD <INSISeq fealure-tabie»> 2 <INSDFesture> aE <INSDFeature keyrsource</INIiDiFezaturs key>
SL CINZDFeature location>l..828<«</IN3D¥Feature location»
Lh <“INSDPearure duals» 215 <INSDQualifier>
Zia “INSDgvelifier named>mol type</INSDQualifier name>
SAT <INSDQualifier valuerprotein</INSDQualifisr value»
Zie </INSDQvalifier: zie <INSDQualifiler id="gqgSdN>
SED CINMEDOualifier namerorganism</INSDQualifier name
Pa CINZDQualifier valuerStreptococcus pyogenes serotype M3 </INSDQvalifier valus> 282 </INSDQualifier> zal </INSDFeature guals> 22d x“/INSDFeacturex
Zal </INSDSeg feature-tabled> <IiNsDSeq sequence >MODRNLIDVNLTSEMKTSFIDYAMSVIVARALPDVRDGLKPVHRRILYGMNELGVT
PDKPHKKSARITGDVMGKYHPHGD SSIYEAMVRMAQWWSYRHMLVDGHGNFGSMDGDGAAAQRYTEARMSKIAL
ELLRDINKNTVNFQDNYDGSEREPVVLPARFPNLLVNGATGIAVGMATNIPPHNLAES IDAVKMVMEHPDCTTR
ELMEVIPGPDFPTGALVMGRSGIHRAYDTGKGSIVLRSRTEIETTQTGRERIVVTEFPYGVNKTKVHEHIVRLA
QEKRLEGITAVRDESSREGVRFVIEIRREASATVILNNLFKLTSLQTNFSFNMLAIENGVPKILSLRQIIDNYI
SHQKEVIIRRTRFDKDKAEARAHILEGLLIALDHLDEVIAIIRNSETDVIAQTELMSRFDLSERQSQAILDMRL
RRLTGLERDKIQSEYDDLLALIADLSDILAKPERIITIIKEEMDE IKRKYANPRRTELMVGEVLSLEDEDLIEE
EDVLITLSNKGY IKRLAQDEFRAQKRGGRGVQGTGVNNDDFVRELVSTSTHDTLLFFTNFGRVYRLKAYEIPEY
GRTAKGLPIVNLLKLEDGETIQTIINARKEETAGKSFFFTTKOGIVKRTEVSEFNNIRONGLRALKLKEGDQOLI
NVLLTSGQDDIIIGTHSGYSVRFNEASIRNMGRSATGVRGVKLREDDRVVGASRIQDNQEVLVITENGFGKRTS
ATDYPTKGRGGKGIKTANITPKNGQLAGLVTVDGTED IMVITNKGVIIRTNVANISQTGRATLGVKIMKLDADA
KIVTFTLVQPEDSSIAEINTDRENSISKNKDN<./INSDSeg sequence» 227 </INSDSeg> u 22% </SeguenceData> 2328 <SequencaData zouencalDiiumbar="10" 230 <INSDSeq>
ZS <INSDSeq length>887</INSDSeq length»
ESE “INSDSeqg moliypevAA</INSDSeq moltype>
TEE <INSDSeq diviglon»PAT</INSDSeqg division
ZG <INSDSeq feature-table> zjn <INSDFearurer 228 <IN3DFeature key>source“/INSDFeature key> aid <INSDFeature locatlonrl..887</INSDFeaturs locabion> 238 <INSDFeature quals> ax <INSDQualifiers
SAD <INSDQualifier name>mol type“ /INSDQualifier name» pa <INSDQualifier value>protein</IN3DQualifier value» dz </INSDOuaiifiers
Fal <INSDOualifier ld=vgids> zj <INSDQualifler namedorganism</INSDQualifisr name> 235 <INSDgualifier value»>Staphylococcus aureus“ /INSDQGualifier value» ad </INSDOualifier:>
Pa “/INSDPeature guals>
An </INBDFeaturne» vA </INSDSeg feature-tabler <INSDSeq seduenc:>MAELPQSRINERNITSEMRESFLDYAMSVIVARALPDVRDGLKPVHRRILYGLNEQ
GMTPDKSYKKSARIVGDVMGKYHPHGDSSIYEAMVRMAQDFSYRYPLVDGQGNFGSMDGDGAAAMRY TEARMTK
ITLELLRDINKDTIDFIDNYDGNEREPSVLPARFPNLLANGASGIAVGMATNIPPHNLTELINGVLSLSKNPDI
SIAEIMEDIEGPDFPTAGLILGKSGIRRAYETGRGSIQMRSRAVIEERGGGRORIVVTEIPFQVNKARMIEKIA
ELVRDKKIDGITDLRDETSLRTGVRVVIDVRKDANASVILNNLYKOTPLOTSFGVNMIALVNGRPKLINLKEAL
VHYLEHQKTVVRRRTQYNLRKAKDRAHILEGLRIALDHIDEIISTIRESDTDKVAME SLQOORFKLSEKQAQAIL
DMRLRRLTGLERDKIEAEYNELLNYISELEAILADEEVLLQLVRDELTEIRDRFGDDRRTE IQLGGFEDLEDED
LIPEEQIVITLSHNNY IKRLPVSTYRAQNRGGRGVQGMNTLEEDFVSQLVTLSTHDHVLFFTNKGRVYKLKGYE
VPELSRQSKGIPVVNAIELENDEVISTMIAVKDLESEDNFLVFATKRGVVKRSALSNF SRINRNGKIAISFRED
DELIAVRLTSGQEDILIGTSHASLIRFPESTLRPLGRTATGVKGITLREGDEVVGLDVAHANSVDEVLVVTENG
YGKRTPVNDYRLSNRGGKGIKTATITERNGNVVCITTVTGEEDLMIVTNAGVIIRLDVADISQNGRAAQGVRLI
RLGDDQFVSTVAKVKEDAEDETNEDEQSTSTVSEDGTEQQREAVVNDETPGNAIHTEVIDSEENDEDGRIEVRQ
DFMDRVEEDIQQSSDEE“/INSDSeg sequencer
El </INSDSedg>
ZEE </SeguenceData> 252 <Sequencalata sopeancalliumbe r=» 254 CINIDSeq> zijl <INSDSeq lengith>808</IN3DSeq length» 2% <INSDSeq moltype>AA</INSDSeqg moltypes
Zj <INSDSeq division>PATA/INSDSeg division»
EE “INSDSeq fealure-table> in <INSDFeature»
ZE <INSDFeature key>source</INSDFeature key»
Zei <INSDFesture location»l..808</INSDFeature location» 252 <INSDFealure gualz>
Da <INSDOualifisr> 294 <INSDQualifier namermol type</INSDQualifier name>
SEN CINMEDOualifier valuesprotein</INSDLQualifier valued
ZEE <SINSDOualifier>
ZET <INSDQualifler in=\gizr>
ZEE <INSDQualifier name>organism</INSDQualifier names
FEE <INSDQualifier value>Clostridioides difficile“ /INSDOualifier valuer
A70 </INSDQvalifier: u
ZL CAINSDFeature guals>
ZE </INSDFeature>
SE </INSDSeqg faature-tabled <iNSDSeq sequence >MEENNKILPIEIAEEMKKSY IDYSMSVIAGRALPDVRDGLKPVHRRILYSMSELNL
TPDKPYRKSARIVGYVLGKYHPHGDTAVYYAMVRMAQDFSTRALLVDGHGNFGSVDGDSPAAMRYTEAKMSKLS
LELLRDIEKETVDFKPNFDESLKEPSVLPARYPNLLVNGSNGIAVGMATSIPPHNLAEVIDATVYLIDNPECSV
DDLIKFVQGPDFPTAAIIMGKESIAEAYRTGRGKVKVRSRAFIEELPKGKQQIIVTEIPYQVNKAKLVERIAEL
VKEKRIEGISDLRDESNRNGMRIVIELKRDANANIVLNNLYKHSQMEDTFSIIMLALVDGQPRVLNLKOILYHY
IKHQEDVVTRRTKFELNKAEARAHILEGLKIALDNIDAVISLIRASKTGQOEAKIGLIEKFKLTEIQAQAILDMR
LQRLTGLERDKIEAEYEDLIKKINRLKE ILADERLLLNVIKNEITIIKENYSDERRTE IRHAEGE IDMRDL ISD
EEIAITLTHFGYIKRLPSDTYKSQKRGGRGISALTTREEDFVRHLVTTTTHSRLLFFTNKGRVFKLNAYEIPEG
KRQAKGTAIVNLLQLSADEKIATLIPIDGNDENEYLLLATKKGIVKKTKREEFKNINKSGLIAIGLRDDDELIG
VELTDGKQEVLLVTKEGMS IRFDENDIRYMGRTAMGVKGITLSKEDFVVSMNLCSKGTDVLVVSKNGFGKRTNI
EEYRSQIRAGKGIKTYNISEKTGTIVGADMVNEDDEIMIINSDGVLIRIRVNEISLFGRVTSGVKLMKTNDEVN
VVSIAKINIEEE</INSDSeqg sequence» 2E </INSDSeg> 2 <SBequencelata> aE <HGegquencelbata seguanselilMonhar=nigy >
Li LINSDSeq» 270 <INSDSeq iength>838/INSDSeq lengih>
EEG <INSDSeq moltype>AA</INSDSeq moliype>
ZE: CINBDSeq divisionsPAT</INSDSeg division» 282 <INSISeq feature-table> 283 <INSDFesture>
ZG <INSDFeature keyrsource</INIiDiFezaturs key>
SEL CINZDFeature location>l..838«</IN3D¥Feature location»
LEE <“INSDPFeacure gqualis>
GET <INSDOQualifier>
ER “INSDgvelifier named>mol type</INSDQualifier name> 253 <INSDuvalifler valuerprotein</INSDQualifisr value» 280 </INSDQualifisan»
Du <INSDQuelifier id="gl3ns
ZE <INSDOQualifier namerorganism“/INSDQualifier name
Sul CINZDQualifier value Mycobacterium tuberculosis</IN3DQuallfier value» ad </INSDOualiiier>
E35 </INSDFeature guals> 236 </INSDFealure»
ER </INSDSeg feature-tabled>
LINEDSeq sequence >MITDTTLPPDDSLDRIEPVDIEQEMQORSY IDYAMSVIVGRALPEVRDGLKPVHRRVL
YAMFDSGFRPDRSHAKSARSVAETMGNYHPHGDASIYDSLVRMAQPWSLRYPLVDGQGNFGSPGNDPPAAMRYT
EARLTPLAMEMLRE IDEETVDFIPNYDGRVQEPTVLPSRFPNLLANGSGGIAVGMATNIPPHNLRELADAVEWA
LENHDADEEETLAAVMGRVKGPDFPTAGLIVGSQGTADAYKTGRGS IRMRGVVEVEED SRGRTSLVITELPYQV
NHDNFITSIAEQVRDGKLAGISNIEDQSSDRVGLRIVIEIKRDAVAKVVINNLYKHTQLQTSFGANMLAIVDGV
PRTLRLDQLIRYYVDHQLDVIVRRTTYRLRKANERAHILRGLVKALDALDEVIALIRASETVDIARAGLIELLD
IDEIQAQAILDMQLRRLAALERQRIIDDLAKIEAEIADLED ILAKPERQRGIVRDELAEIVDRHGDDRRTRIIA
ADGDVSDEDLIAREDVVVTITETGYAKRTKTDLYRSQKRGGKGVQGAGLKQDDIVAHFFVCSTHDLILFFTTQG
RVYRAKAYDLPEASRTARGQHVANLLAFQPEERIAQVIQIRGYTDAPYLVLATRNGLVKKSKLTDFD SNRSGGI
VAVNLRDNDELVGAVLCSAGDDLLLVSANGQSIRFSATDEALRPMGRATSGVQGMRFNIDDRLLSLNVVREGTY
LLVATSGGYAKRTAIEEYPVQGRGGKGVLTVMYDRRRGRLVGALIVDDDSELYAVTSGGGVIRTAARQVRKAGR
QTKGVRLMNLGEGDTLLAIARNAEESGDDNAVDANGADQTGN: /INSISeg seguence> 239 </INSDSeg> u 200 </SeguenceData>
ZL <SequenceData saguencaliunbaer="313"%
ZOE <iNSDSed> 303 <INSDSeq lengih>1273</INSDSeqg lengths
Sid “INSDSeqg moliypevAA</INSDSeq moltype>
RIE <INSDSeq diviglon»PAT</INSDSeqg division
Se <INSDSeq feature-table>
ST CINEDEeatures 208 <IN3DFeature key>source“/INSDFeature key>
S08 <INSDFeature locatlonrl..1273</INSDFeature location»
FAQ <INSDFeature quals>
IHL <INSDQualifiers win <INSDQualifier name>mol type“ /INSDQualifier name»
SLS <INSDQualifier value>protein</IN3DQualifier value»
Lid </INSDOQualijier>
ZLD <INSDOualifier id="giár>
Sid <INSDQualifler namedorganism</INSDQualifisr name>
SAE <INSDgualiifler value Mycobacterium leprae“/INSDGualifier value»
TLE </INSDOualifier:>
Sl “/INSDPeature cuals>
SED </INSDFealure»>
SEL «/INSDSeg featurse-tabier> <INSDSegq seduence>MIDITLPPGDGSIQRVEPVDIQQOEMQORSYIDYAMSVIVGRALPEVRDGLKPVHRRV
LYAMLDSGFRPDRSHAKSARSVAE TMGNYHPHGDASIYDTLVRMAQPWSLRYPLVDGOGNFGSPGNDPPAAMRY
CVSGNSLVRLLFGKSIRIGDIVTGAQFNSDNPIDLKVLDRHGNPVVADYLFHSGEHSTYTVRTTEGYEITGTSN
HPLLCLVNVGGIPTLLWKLIGEIRSGDYVVLQRIPPVEFGPADWYSTMEALLFGAFISGGFVFQDHAGFNSLDR
DYFTMVVNAYDTVVGGLRCISSRITVSGSTLLELDVYNLIEFKKTRLSGLCGQRSADKLVPDWLWHSPSTVKRA
FLQALFEGEGFSSILSRNIIEISYSTLSERLAADVOOMLLEFGVVSERYCHTVNEYKVVIANRAQVEMFFTOVG
FGVTKQAKLIRDVVSMSPCVGMDINCVPGLATFIRKHCDNRWVEEDSFNQHNVDCVOHWHHHSAEIVGHIADPD
IRAIVTDLTDGRFYYARVASVTDTGIQPVFSLHVDTEDHSFLTNGFISHNTEARLTPLAMEMLREIDEETVDFI
SNYDGRVQEPMVLPSRFPNLLANGSGGIAVGMATNIPPHNLYELADAVFWCLENHDADEETMLVAVMERVKGPD
FPTAGLIVGSQGIADAYKTGRGSIRIRGVVEVEEDSRGRTSLVITELPYQVNHDNFITSIAEQVRTGRLAGISN
VEDQGSDRVGVRIVIE IKRDAVAKVVLNNLYKHTQLQTSFGANMLSIVDGVPRTLRLDOMICYYVEHQLDVIVR
RTTYRLRKANERAHILRGLVKALDALDEVITLIRASQTVDIARVGVVELLDIDDIQAQAILDMQLRRLAALERQ
RIIDDLAKIEVEIADLGDILAKPERRRGIIRNELTEIAEKYGDDRRTRIIAVDGDVNDEDLIAREEVVVTITET
GYAKRTKTDLYRSQKRGGKGVQGAGLKQDDIVRHFFVCSTHDWILFFTTQGRVYRAKAYELPEASRTARGQHVA
NLLAFQPEERIAQVIQIRSYEDAPYLVLATRAGLVKKSKLTDFDSNRSGGIVAINLRDNDELVGAVLCAADGDL
LLVSANGQSIRFSATDEALRPMGRATSGVQGMRFNADDRLLSLNVVREDTYLLVATSGGYAKRTSIEEYPMQGR
GGKGVLTVMYDRRRGSLVGAIVVDEDSELYAITSGGGVIRTTARQVRQAGROTKGVRLMNLGEGDTLLAIARNA
EESADGVSVKVMISRSRVLSFFGSDSNTSPDRT</ INEDS eg sequence 223 </INSDSeg>
IEA <SBequencelata>
SEL <HGegquencelbata seguanselilMonhar=nl4Y >
SAE LINSDSeq»
Su <INSDSeq iength>8398/INSDSeq lengih>
IER <INSDSeq moltype>AA</INSDSeq moliype>
DE CINBDSeq division»PAT</IN3D3eq division»
TIO <INSISeq feature-table>
FIL <INSDFesture> 33 <INSDFeature keyrsource</INIiDiFezaturs key>
SSS CINZDFeature location>l..839«</IN3D¥Feature location»
Sd <“INSDPFeacure gqualis> 345 <INSDQualifier> sid “INSDgvelifier named>mol type</INSDQualifier name>
SI <INSDQualifier valuerprotein</INSDQualifisr value» 238 </INSDQualifisan»
II <INSDQuelifier id='gl&ns
BAD <INSDOQualifier namerorganism“/INSDQualifier name
SAL CINZDQualifiler value Mycobacterium avium subsp. avium</INEDQualifier value»
RE </INSDOQualijier>
S42 </INSDFeature guals> 2d x“/INSDFeacturex
TA </INSDSeg feature-tabled>
CINEDSeq sequence >MTDTTLPPGGDAADRVEPVD IQQEMORSY IDYAMSVIVGRALPEVRDGLKPVHRRV
LYAMYDSGFRPDRSHAKSARSVAETMGNYHPHGDASIYDTLVRMAQPWSLRY PLVDGQGNFGSPGND PPAAMRY
TEARLTPLAMEMLRQIDEETVDFIPNYDGRVQEPTVLPSRFPNLLANGSGGIAVGMATNIPPHNLGELAEAVEFW
ALDNYEADEEATLAAVMERVKGPDFPTSGLIVGTQGIADAYKTGRGSIRMRGVVEVEEDSRGRTSLVITELPYQ
VNHDNFITSIAEQVRDGKLAGISNIEDQSSDRVGLRIVIELKRDAVAKVVLNNLYKHTQLQTSFGANMLAIVDG
VPRTLRLDQLIRHYVDHQLDVIVRRTTYRLRKANERAHILRGLVKALDALDEVIALIRASETVDIARQGLIELL
DIDEIQAQAILDMQLRRLAALERQRIIDDLAKIEAETIADLED ILAKPERQRGIVRDELAEIVEKHGDARRTRIV
AADGDVSDEDLIAREDVVVTITETGYAKRTKTDLYRSQKRGGKGVQGAGLKQDD IVRHFFVCSTHDWILFFTTQ
GRVYRAKAYELPEASRTARGQHVANLLAFQPEERIAQVIQIRSYEDAPYLVLATRNGLVKKTKLTDFDSNRSGG
IVAINLRDNDELVGAVLCSAEDDLLLVSANGQS IRFSATDEALRPMGRATSGVQGMREFNADDY LLSLNVVREGT
YLLVATSGGYAKRTAIEEYPVQGRGGKGVLTVMYDRRRGRLVGALIVDEDSELYAITSGGGVIRTAAGQVRKAG
RQTKGVRLMNLGEGDTLLAIARNAEEAADEAVEESDGAAGSDG/INSDSeq sequence 287 </INSDSeg> zae </SeguenceData> iss <SequenceData zouencalDiiumbar="18">
THO <INSDSeq>
SL <INSDSeq length>839</INSDSeq length»
ILE CINZDSeq moltype»AAS/INSDSey moltype>
FEE <INSDSeq diviglon»PAT</INSDSeqg division
Sh <INSDSeq feature-table> 355 LIMIDFeaturas 258 <IN3DFeature key>source“/INSDFeature key>
TR <INSDFeature locatlonrl..839</INSDFeaturs locabion> 358 <INSDFeature quals>
IEG <INSDQualifiers
HED <INSDQualifier name>mol type“ /INSDQualifier name»
Sel <INSDQualifier value>protein</IN3DQualifier value»
JEE </INSDOQualijier> 25: <INSDOualifier id="g1jn>
SAG <INSDQualifler namedorganism</INSDQualifisr name>
Tat <INSDQualifier value»Mycobacteroides abscessus</INSDQualifier value»
IEE </INSDOualifier:>
IT “/INSDPeature guals>
GEE </INSDFeature»
Zen </INSDSeg feature-tabler
CINBDSeq sequence>MIDTTLPPGGDDAVDRIEPVDIQQEMORSYTIDYAMSVIVGRALPEVRDGLKPVHRR
VLYAMYDSGFRPDRSHAKSARSVAETMGNYHPHGDASIYDTLVRMAQPWSLRYPLVDGQGNFGSPGNDPAAAMR
YTEARLTPLAMEMLRE IDEE TVDFIPNYDGRVMEPTVLPSRFPNLLANGSGGIAVGMATNMPPHNLRELAEAVY
WALDNHEADEE TTLKAVCEKITGPDFPTSGLIVGTQGIHDAYTTGRGSIRMRGVAE IEEDSKGRTSLVITELPY
QVNHDNF ITSIAEQVRDGKIAGISNIEDQSSDRVGLRIVVVLKRDAVAKVVLNNLYKHTQLQTSFGANMLS IVD
GVPRTLRLDQLIRLYVNHQLDVIIRRTRYRLRKANERAHILRGLVKALDALDEVIALIRASQTVDIARTGLIEL
LDVDEIQAQAILDMQLRRLAALERQKIIDDLAKIEAE IADLEDILAKPERQRAIVKDELAE ITEKYGDDRRTRI
ISADGDVADEDLIAREDVVVTSTETGYAKRTKTDLYRSQKRGGKGVOGAGLKODDIVKHFFVCSTHDWILFFTT
KGRVYRAKAYDLPEAARTARGQHVANLLAFQPEERIAQVIQIKSYEDAPYLVLATKNGLVKKSKLTEFDSNRSG
GLVAVNLRDGDELVGAVLCSAEDDLLLVSAHGQSIRFSATDEALRPMGRATSGVQGMRFNGEDDLLSLNVVREG
TYLLVATSGGYSKRTAIEEYPVQGRGGKGVLTVQYDPRRGSLVGALVVDEESELYAITSGGGVIRTIAKOVRKA
GRQTKGVRLMNLGEGDTLLAIAHNADEGDADPDEDAAGTTAGE<./INSDSeq sequencer
SF </INSUE aa»
STZ </SeqenceData>
ST “SequenceData seguencellsumben="i8r> 274 <INSDSeq>
SES <INSDSeq leng:ih>863</INSDSeq length»
Tie <INSDSeq moliype>AA</INSDSeq moltype>
I <IMEDZeq division>PAT«/INSDSeg division
IED <INSDSeq festure-table>
Sin <INSDFeature»
Soi <INSDFeature key>source</INSDFeature key»
SEL <INSDFeature focation>l..863</IN3DF¥eature location
IEE <INSDFeature guals> 282 SINSDOualifie>
Jed <INSDgualifier namexmol type“/INSDgualifier name>
TEE <INSDQualifier valuerprotein</INSDQualifier value»
IRE </INSDOualifier:>
BEY CINSUQualifler in=NgSij>
HARE <INSDQualifier name>organism</INiDQualifier named
SED <INSDQualifier value>Campylobacter jejuni subsp. jejuni serotype 0:2 </INSDQualifier value» 230 </INSDQuali jier
TSL </INSDFeature guals> 382 </INSDFeature>
Iw </INSDSeg feature-tabled> <INSDSeq sequsnce>MENIFSKDSDIELVDIENSIKSSYLDYSMSVIIGRALPDARDGLKPVHRRILYAMQ
NDEAKSRTDFVKSARIVGAVIGRYHPHGDTAVYDALVRMAQDFSMRYPSITGQGNFGSIDGDSAAAMRYTEAKM
SKLSHELLKDIDKDTVDFVPNYDGSESEPDVLPSRVPNLLLNGSSGIAVGMATNIPPHSLNELIDGLLYLLDNK
DASLEE IM9OFIKGPDFPTGGIIYGKKGIIEAYRTGRGRVKVRAKTHIEKKTNKDVIVIDELPYQTNKARLIEQI
AELVKERQIEGISEVRDESNKEGIRVVIELKREAMSE IVLNNLFKSTTME STFGVIMLAIHNKEPKIFSLLELL
NLFLTHRKTVIIRRTIFELQKARARAHILEGLKIALDNIDEVIALIKNSSDNNTARDSLVAKFGLSELQANAIL
DMKLGRLTGLEREKIENELAELMKEIARLEEILKSETLLENLIRDELKEIRSKFDVPRITQIEDDYDDIDIEDL
IPNENMVVTITHRGY IKRVPSKQYEKQKRGGKGKLAVTTYDDDF IE SFFTANTHDTLMEFVTDRGQLYWLKVYKI
PEGSRTAKGKAVVNLINLQAEEKIMAIIPTTDFDESKSLCFFTKNGIVKRTNLSEYQNIRSVGVRAINLDENDE
LVTAIIVQRDEDEIFATGGEENLENQE IENLDDENLENEE SVSTQGKMLFAVTKKGMC IKFPLAKVREIGRVSR
GVTAIKFKEKNDELVGAVVIENDEQEILSISAKGIGKRTNAGEYRLQSRGGKGVICMKLTEKTKDLISVVIVDE
TMDLMALTSSGKMIRVDMQSIRKAGRNTSGVIVVNVENDEVVSIAKCPKEENDEDELSDENFGLDLQ&/INSDS
Sz sequence 355 </INSDSec:>
ERE <SBequencelata> hE <HGegquencelbata seguanselilMonhar=niTYs
SEG LINSDSeq»
SD <INSDSeq iength>8984/INSDSeq Llengih> 40340 <INSDSeq moltype>AA</INSDSeq moliype>
SDL CINBDSeq divisionsPAT</INSDSeg division» 402 <INSISeq feature-table> 305 <INSDFesture> 404 <INSDFeature keyvsource</INSDFeature key> 4050 CINZDFeature location>l..894</IN3D¥Feature location» 405 <“INSDPFeacure gqualis> 407 <INSDQualifier> 409 “INSDgvelifier named>mol type</INSDQualifier name> 438 <INSDQualifier valuerprotein</INSDQualifisr value» 410 </INSDQualifisan»
ALE <INSDQuelifier id=vglevs
ALE <INSDOQualifier namerorganism“/INSDQualifier name
Als CINZDQualifier value>Vibrio cholerae“/INSDQualifier value» 4734 </INSDOualilfier> 415 </INSDPeature guals> 414 </INSDFeaturer d17 </IN3DSeq featura-table> <iNSDseq sequence >MSDLARE ITPVNIEDELRSSYLDYAMSVIVGRALPDVRDGLKPVHRRVLFAMNVLG
NDWNKAYKKSARVVGDVIGKYHPHGDSAVYDTIVRMAQPFSLRYMLVDGQGNFGSIDGDSAAAMRY TEVRMSKI
AHELLADLDKETVDYVPNYDGTEQIPAVLPTKIPNLLINGSSGIAVGMATNIPPHNLGEVIDGCLAY INNEAIT
IDELMDY IPGPDFPTAALISGRKGIIDAYKTGRGKVYMRSKAEIEADKNGKETIIVSE IPYQVNKARLIEKIAE
LVKEKKVEGISALRDE SDKDGMRIVIECKRDAVGEVVLNNLYANTQLQTTFGINMVALDNGQPKLFNLKEMLKC
FVDHRREVVTRRTIFELRKARERAHILEGLALALANIDEIIELIRHAATPAEAKEGLIARGWDLGNVSAMLERA
GTDAARPEWLEPQYGIRDGKYFLTEQQAQAILDLRLHKLTGLEHEKILDEYKQLLEEIAALMHILASTERLMEV
IREELEAIRAGFSDARRTEITAATHDIDMEELIAREDVVVTLSHEGYVKYQLLSDYEAQRRGGKGKSATKMKEE
DYIERLLVANTHDNILLFSTRGKTYRMKVYQLPLASRTARGKPIVNLLPLEENERITAILPVTEFSEDKFIFMA
TGDGTVKKTSLDQFANVRANGLIALNLRDEDSLIGVDITDGESEIMLFSKFGKVVRFKESEETAVVDENGOPVL
DENGQPE IKFKGVRPMGRTAAGVRGMKLAEGDQVVSLIVPKAEGDVLTVTENGY GKRTSLSEYPTKGRGTQGVV
SIKVSERNGSVVGAVQVAEGDEFMMITNAGTLVRTRVAEVSQVGRNTQGVTLIRTSEGESVVGLQRIDEIEESE
LPEGEEELVENDAPVAQDDDGEQE</ INSDSaqg zequence> 419 </INSDSeg> u 430 </SeguenceData>
JEL <SequenceData saguencaliunbaer="318">
ZZE <iNSDSed>
A423 <INSDSeq length>880</INSDSeq length»
En “INSDSeqg moliypevAA</INSDSeq moltype> 4x5 <INSDSeq diviglon»PAT</INSDSeqg division dd <INSDSeq feature-table> dij <INSDFearurer 32E <INSDF Feature key>source“/INSDFeature key> 32 <INSDFeature location>1..880</INSDFeature locabion> 430 <INSDFeature quals>
ASL <INSDQualiifier> 4% <INSDQualifier name>mol type“ /INSDQualifier name» 443 <INSDQualifier value>protein</IN3DQualifier value» dze </INSDOuali fier» jah <INSDOualifier id="qggZ0r>
A456 <INSDQualifler namedorganism</INSDQualifisr name> 43% <INSDQualifier value>Haemophilus influenzae</INSDQualifier value> 435 <fINSDUualifler»> 43% “/INSDPeature guals> dd </INBDFeaturne» ddl </INSDSeg feature-tabler <INSDSeq sequence>MIDSIQSSITPVNIEEELKSSYLDYAMSVIVGRALPDVRDGLKPVHRRVLFSMDRE
GNTANKKYVKSARVVGDVIGKYHPHGDSAVYDTIVRMAQPFSLRYMLVDGQGNFGSIDGDAPAAMRYTEVRMOK
ITQALLTDLDKETVNFSPNYDGELMIPDVLPTRIPALLANGSSGIAVGMATNIPPHNLNEVLNGCLAYIDKNEI
TIDELMQHIPGPDFPTAALINGRKGIEEAYRTGRGKVYVRARATVETNEKGREQIIVSELPYQVNKAKLVEKIA
ELIREKKIEGISNITDLSNKEGIRIEIDIKRDAVGEVVLNHLYSLTQMOVTFGINMVALDHGOPRLFNLKEIIE
AFVLHRREVVTRRSIFELRKARERTHILEGLAVARSNIDEMIAIIRNSKNREEAATSISSRSWTLHSDIINLLD
ASARPDELEENLGIQGEQYYLSPAQVNAILELRLHRLTGIAFEEVIKEYEELLVKIADLLHILSSAERLMEVIR
EELEEVKAQFGDDRLTEITAASGDIDLEDLIAQEDVVVTLSHEGYVKYQPLTDYEAQRRGGKGKSATKMKEEDF
IEKLLVANTHDTILCFSSRGRLYWLKVYQLPQASRGARGRPIVNILPLQENERITAILPVSAYEEDKFVVMATA
GGIVKKIALTEFSRPRSNGIIALNLRDEDELIGVDITDGSNE IMLFSSQGRVVRFAENAVRAMGRLATGVRGIK
LALTNDISDDESAVEIEDISDDNAEASLDLNIDKVVSLVVPKGEGAILTATONGYGKRTQLSEYPTKSRNTKGV
ISIKVSERNGKVVAATQVEETDQIMLITDAGTLVRTRVSEVSIVGRNTQGVRLIRTADDEHVVSLERVCDADED
DSLEESSSEE</INSDS aq sequences dd <SINEDE E> ddd </SeguenceData> 4490 <Sequencalata sopeancalliumber="18n> ddd JINSDSeq> 337 <INSDSeq lengith>877</IN3DSeq length» 448 <INSDSeq moltype>AA</INSDSeqg moltypes
Aan <INSDSeq division>PATA/INSDSeg division» 450 “INSDSeq fealure-table> 40% <INSDFeaturer
HI <INSDFeature key>source</INSDFeature key» 457 <INSDFesture locatiocn>l..877</INSDieaturs location» 454 <INSDFealure gualz>
A55 <INSDQuelifier>
Ana <INSDQualifier namermol type</INSDQualifier name>
ANY CINMEDOualifier valuesprotein</INSDLQualifier valued 45% <SINSDOualifier> 45% LINSDGualiifler aa=swgdly>
ZED <INSDQualifier name>organism</INSDQualifier names 451 <INSDQualifier value>Klebsiella pneumoniae subsp. pneumoniae</INIDQuallfier values» 382 </INSDQvalifier: 4873 </INZDFeature guals> 484 </INSDFeature> den x/INSDSeg faature-tabled <“iNSDSeq sequence>MSDLAREITPVNIEEELKNSYLDYAMSVIVGRALPDVRDGLKPVHRRVLYAMNVLG
NDWNKAYKKSARVVGDVIGKYHPHGDSAVYDTIVRMAQPFSLRYMLVDGQGNFGSIDGDSAAAMRY TEIRLAKI
AHELMADLEKE TVDFVDNYDGTERIPDVMPTKIPNLLVNGASGIAVGMATNIPPHNLTEVINGCLAYVDDEDIS
IEGLMAHIPGPDFPTAAIINGRRGIEEAYRTGRGKVY IRARAEVEVDAKSGRETIIVHEIPYQVNKARLIEKIA
ELVKEKRVEGISALRDESDKDGMRIVIEVKRDAVGEVVLNNLYSQTQLQVSFGINMVALHHGQPKIMNLKDIIA
AFVRHRREVVTRRTIFELRKARDRAHILEALAVALANIDPIIELIRRAPTPAEAKTALVAQAWDLGNVAAMLER
AGDDAARPEWLEPEFGVRDGKYYLTEQQAQATILDLRLOKLTGLEHEKLLDEYKELLEQIAELLHILGSADRLIME
VIREELELIRDQFGDERRTE ITANSADINIEDLINQEDVVVTLSHQGYVKYQPLTDYEAQRRGGKGKSAARIKE
EDFIDRLLVANTHDTILCFSSRGRLYWMKVYQLPEASRGARGRPIVNLLPLEADERITAILPVREYEEGVNVEFM
ATASGTVKKTALTEFSRPRSAGIIAVNLNEGDELIGVDLTSGQODEVMLFSAAGKVVRFKEDAVRAMGRTATGVR
GIKLAENDSVVSLIIPRGEGAILTVTONGYGKRTAAAEYPTKSRATQGVISIKVTERNGSVVGAVQVDDCDQIM
MITDAGTLVRTRVSEVSIVGRNTQGVILIRTAEDENVVGLQRVAEPVDDEELDAIDGSAAEGDDDIVPEADIDD
DIAEDEE/INSDSeqd sequence 487 </INSDSeq> doe <j Gaemquencebata> ien <“SequsrnceData segquanceliNuubsc=NgON> 470 <IiNSsDseqd> 47 <INSDSeq length»878</INIlSeq length> 472 <INSDSeq moliype>AA/INIDIeq molLype»> 373 <INSDSeq division>PAT</INSDSeg division»
A774 <INSDSeq feature-table>
ATL <INSDFesture>
Vie CINEDFeature keyvsource</INSDFeature key» 477 “INSDFeabture locabtion>l..878</IN3DFeature location» 473 <INSDFeature gualis> 373 <INSDOualifier> 48a <INSDQualifler named>mol type</INSDRualifizp names
JEL <INSDQualifier valuerprotein</INSDQuali fien value»
A480 </INSDQualifiern>
ARI <INSDguelifier id=Vg2i"n>
Add v“INSDQualifier namerorganism</IN3DQualifier name> duh <INSDQualifier valuse>Enterobacter cloacae</INSDQualifier value
AE </INSDQualifier> - 487 </INSDFaature quals> 4948 </INSDPeatune> jas </INZDSey feature-table>
CINEDSeq sequence MSDLAREITPVNIEEELKSSYLDYAMSVIVGRALPDVRDGLKPVHRRVLYAMNVLG
NDWNKAYKKSARVVGDVIGKYHPHGDIAVYDTIVRMAQPFSLRYMLVDGOGNFGSVDGDSAAAMRYTEIRMSKI
AHELMADLEKE TVDEVDNYDGTERIPDVMPTKIPNLLVNGSSGIAVGMATNIPPHNITEVINGCLAYIDDEDIS
IEGLMEHIPGPDFPTAAIINGRRGIEEAYRTGRGKIYIRARAEVEADAKTGRETIIVHEIPYQVNKARLIEKIA
ELVKEKRVEGISALRDESDKDGMRIVIEIKRDAVGEVVLNNLYSQTQLQVSFGINMVALHHGQPKIMNLKE ILS
AFVRHRREVVTRRTIFELRKARDRAHILEALAVALANIDPIIELIRRAPTPAEAKAAL ISRPWDLGNVAAMLER
AGDDAARPEWLEPEFGVRDGQYYLTEQQAQATILDLRLOKLTGLEHEKLLDEYKELLEQIAELLHILGSAERLME
VIREELELVRDQFGDARRTE ITANSSDINIEDLINREDVVVTLSHQGYVKYQPLTDYEAQRRGGKGKSAARIKE
EDFIDRLLVANTHDTILCFSSRGRLYWMKVYQLPEASRGARGRPIVNLLPLEANERITAILPVREYEEGVNVEFM
ATASGTVKKTALTEFSRPRSAGIIAVNLNEGDELIGVDLTSGSDEVMLFSAAGKVVRFKENAVRAMGRTATGVR
GIKLAGEDSVVSLIIPRGEGAILTVTONGYGKRTAEGEYPTKSRGTQGVISIKVTERNGSVVGAVQVDDADQIM
MITDAGTLVRTRVSEISVVGRNTQGVILIRTAEDENVVGLQRVAEPVDDEELDSIDGSVAEGDDDIAPEAD TDD
DVADDADE</ I NSLS aq seqguanca> 39: </INSDSeg> u aan </Segquaencebata>
As <SSequencebaha segsenceinNvumbsc=N2iN»
Gud CINSDSag» 43h <INSDSeq length>»+878</INSDSeg Length> dee <INSDSeq moltype>AA“/INSDSeq moltype> 457 <INSDSeq division»PAT</INSDSeqg division: 338 <INSLSeq Ieature-labier 388 <“INSDFearure»
SDO <IMaDFesature key>source“/INSDFeature key>
SOL CINSDFesature locations>l..878B</IN3DFeature location
LOE <INSDFeature quals> IJ
L435 CINBDOualifierns
Hid <INSDQualifier name>mol type“ /INSDQualifier name» 305 <INSDQualifier value>protein</INSDQualifisr value»
SEE </INSDQuali jier
Ba <INSDQualifier id="g23Ns>
SOR <INSDQualifier namevorganism“/INSDQualifier name>
So <INSDOualifier valuerSalmonella typhimurium“/INSDguali fier value»
SEN </INSDOualilfier>
DIE </INSDPeature guals>
S12 </INSDFeature> iz </IN3DSeq featura-table> <iNSDseq sequence >MSDLAREITPVNIEEELKSSYLDYAMSVIVGRALPDVRDGLKPVHRRVLYAMNVLG
NDWNKAYKKSARVVGDVIGKYHPHGDSAVYDTIVRMAQPFSLRYMLVDGQGNFGSIDGDSAAAMRYTEIRLAKI
AHELMADLEKE TVDEVDNYDGTEKIPDVMPTKIPNLLVNGSSGIAVGMATNIPPHNLTEVINGCLAYIDNEDIS
IEGLMEHIPGPDFPTAAIINGRRGIEEAYRTGRGKVY IRARAEVEADAKTGRETIIVHEIPYQVNKARLIEKIA
ELVKDKRVEGISALRDESDKDGMRIVIEVKRDAVGEVVLNNLYSQTQLQOQVSFGINMVALHHGQPKIMNLKDIIS
AFVRHRREVVTRRTIFELRKARDRAHILEALAIALANIDPIIELIRRAPTPAEAKAALISRPWDLGNVAAMLER
AGDDAARPEWLEPEFGVRDGOYYLTEQQAQAILDLRLOKLTGLEHEKLLDEYKELLEQIAELLHILGSADRLME
VIREEMELIRDQFGDERRTE ITANSADINIEDLISQEDVVVTLSHQGYVKYQPLTDYEAQRRGGKGKSAARIKE
EDFIDRLLVANTHDTILCFSSRGRLYWMKVYQLPEASRGARGRPIVNLLPLEANERITAILPVREYEEGVNVEFM
ATASGTVKKTALTEFSRPRSAGIIAVNLNDGDELIGVDLTSGSDEVMLFSAAGKVVRFKEDAVRAMGRTATGVR
GIKLAGDDKVVSLIIPRGEGAILTVTONGYGKRTAADEYPTKSRATQGVISIKVTERNGSVVGAVQVDDCDQIM
MITDAGTLVRTRVSEISVVGRNTQGVILIRTAEDENVVGLQRVAEPVDDEELDAIDGSVAEGDEDIAPEAE SDD
DVADDADE /INSDSeg seguence> 515 </INSDSeg> u
Sia </SeguenceData>
SA <SequenceData saguencalMiunbaer="23% 2%
SAR <INSDSeq>
LL <INSDSeq length>875</INSDSeq length»
Lal CINZDSeq moltype»AAS/INSDSey moltype>
SEL <INSDSeq diviglon»PAT</INSDSeqg division:
LY <INSDSeq feature-table>
SE CINEDEeatures
Did <INSDF Feature key>source“/INSDFeature key>
BE <INSDFeature locatlonrl..875</INSDFeaturs locabion>
Lae <INSDFeature quals>
Sa <INSDQualifiers a <INSDQualifier name>mol type“ /INSDQualifier name»
La <INSDQualifier value>protein</IN3DQualifier value»
SEO </INSDOQualijier>
S53 CINSDOualifisr id="qggZ4r>
S22 <INSDQualifler namedorganism</INSDQualifisr name>
BI <INSDQualifier value»>Shigella dysenteriae</INSDQualifier value>
Sá <fINSDUualifler»>
LL “/INSDPeature guals>
NRE </INSDFealure»>
DST «/INSDSeg featurse-tabier> <INSDSeq seduencs>MSDLAREITPVNIEEELKSSYLDYAMSVIVGRALPDVRDGLKPVHRRVLYAMNVLG
NDWNKAYKKSARVVGDVIGKYHPHGDSAVYDTIVRMAQPFSLRYMLVDGQGNFGSIDGDSAAAMRYTEIRLAKI
AHELMADLEKE TVDEVDNYDGTEKIPDVMPTKIPNLLVNGSSGIAVGMATNIPPHNLTEVINGCLVYIDDEDIS
IEGLMEHIPGPDFPTAAIINGRRGIEEAYRTGRGKVY IRARAEMEVDAKTGRETIIVHEIPYQVNKARLIEKIA
ELVKEKRVEGISALRDESDKDGMRIVIEVKRDAVGEVVLNNLYSQTQLOQVSFGINMVALHHGQPKIMNLKDIIA
AFVRHRREVVTRRTIFELRKARDRAHILEALAVALANIDPIIELIRHAPTPAEAKTALVANPWQLGNVAAMLER
AGDDAARPEWLEPEFGVRDGLYYLTEQQAQATILDLRLOKLTGLEHEKLLDEYKELLDQIAELLRILGSADRLIME
VIREELELVREQFGDKRRTE ITANSADINLEDLITQEDVVVTLSHQGYVKYQPLSEYEAQRRGGKGKSAARIKE
EDFIDRLLVANTHDHILCFSSRGRVYSMKVYQLPEATRGARGRPIVNLLPLEQDERITAILPVTEFEEGVKVEM
ATANGTVKKTVLTEFNRLRTAGKVAIKLVEGDELIGVDLTSGEDEVMLFSAEGKVVRFKESSVRAMGCNTTGVR
GIRLGEGDKVVSLIVPRGDGAILTATONGYGKRTAVAEYPTKSRATKGVISIKVTERNGLVVGAVQVDDCDQIM
MITDAGTLVRTRVSEISIVGRNTQGVILIRTAEDENVVGLQRVAEPVDEEDLDTIDGSAAEGDDE IAPEVDVDD
EPEEE</ INEDS zg sequence
LET <SINEDE E> na </SeguenceData>
NE <Sequencalata sopeancalliumber="280%>
Ha CINIDSeq> 5a <INSDSeq lengith>871</IN3DSeq length»
SAA <INSDSeq moltype>AA</INSDSeqg moltypes
SAL <INSDSeq division»PAT</INEDIag division>
Lan “INSDSeq fealure-table> oF LINSDFeature>
DAE <INSDFeature key>source</INSDFeature key»
S545 <INSDFesture locatiocn>l..871</INSDieaturs location»
S59 <INSD¥eature guals>
BHA <INSDoualifier>
SED <INSDQualifier namermol type</INSDQualifier name>
SE CINMEDOualifier valuesprotein</INSDLQualifier valued
Dd <SINSDOualifier>
LEE LINSDGualiifler in=\gg5r> 55e <INSDQualifier name>organism</INSDQualifier names 557 <INSDQualifier value>Legionella pneumophila subsp. pneumophila</iNiDOuali filer values 35E </INSDQvalifier: 554 </INZDFeature guals>
RES </INSDEFeature>
LOL </INSDSeqg faature-tabled <INSDSedq sequence >MSKGYIQIMVYLAREVLPVNIEDELRQSYLDYAMSVIVGRALPDVRDGLKPVHRRV
LFAMSELGNDWNKPYKKSARVVGDVIGKYHPHGDTAVYDTIVRMAQPFSMRYLLIDGOGNFGSVDGDAPAAMRY
TEVRMSKVAHALLADLDKETVDFSPNYDETEFAPVVLPSRIPNLLVNGSSGIAVGMATNIPPHNLTEVINACIA
LVDEPDTSLEDLMEIIPGPDFPTAAIINGRAGIIEGYRTGKGRVVIRARTEIETDESSGRQSIIIQELPYQVNK
ARLIERIAELVRDKKVEGISGLRDESDKQGMRVVIELKRNE VADVVLNNLFAHTOMONVFGINMVALVDGQPRT
LNLKQILEYFIKHRREVVTRRTIFELKKARSRAHLLEGLGIALANIDEMIALIKQSPTPQDAKSALLSKIWQPG
LVKAMLEKAGSNASRPDDLTEEYGLHENGYKLSEAQAQAILELRLHRLTALEQDKIINEFEELLNLIKELLDIL
ASPERLMQVIRDELIEIKSQFGDERRTE ITASQEDLTIEDLITEEDVVVTLSHQGYVKYQPITAYQAQRRGGKG
KSATHVKDEDFVERLVIASTHDTLLCFSNHGKLYWLKAYQLPQASRASRGRPIINILPLAEGEEINAMLPVREY
KDGSYVFMATKKGTVKKVPLNAFSRPRSNGIIAVDLEEDDSLVGVDITDGTRDIMLFTDAGKVIRFDENKVRPM
GRTARGVRGIRVEKDQAVKSLVVVDPNGGTILTATENGYGKRTHIDEYRVSGRGGQGVISIQVTERNGKVVRSL
QVTDNDEAML ITDKGTLVRFKVNELSVIGRNTQGVRLINVSSGETVVGMQKIVDLGEELEEAEDSSLNAEDNSD
E</INSDSeyg sequence 563 </INSDSear
LA <j Gaemquencebata>
LED <Gequencelala segquanceliNuubsc=tgdN>
LEE <IiNSsDseqd>
S57 <INSDSeq length>890</INSDSeq length>
Ha <INSDSeq moliype>AA/INIDIeq molLype»>
BAS <INSDSeq division>PAT</INSDSeg division»
SEO <INSDSeq feature-table>
LHL <INSDFeature>
LEE CINEDFeature keyvsource</INSDFeature key»
LS “INSDFeabture Location». .890</INSDFeature location»
Ld <INSDFesture gualis> 375 <INSDOualifier>
Sá <INSDQualifler named>mol type“ /INSDQuali fier names
BEE <INSDQualifier valuevprotein</INSDQualifisn wvalus>
BER </INSDQuali fier!»
SEAT <INSDguelifier id="g28">
SEO CINZDQuallfier namerorganism</IN3DQualifier name>
Sul <INSDQualifier value>Yersinia pestis subsp. pestis bv.
Medievalis/INSDQualifier value»
BER </INSDQualifier> u
DE </INSDFaature quals>
Bd </INSDFeature> 385 </INZDSey feature-table>
CINEDSeq sequence MSDLAREITPVNIEEELKSSYLDYAMSVIVGRALPDVRDGLKPVHRRVLEFAMNVLG
NDWNKPYKKSARVVGDVIGKYHPHGDSAVYDTIVRMAQPFSLRYMLVDGQGNFGSVDGDSAAAMRY TEIRMSKI
AHELLADLEKDTVDFVPNYDGTEQIPAVMPTRIPNLLVNGSSGIAVGMATNIPPHNLSEVIDGCLAY IEDENIS
IEGLMEY IPGPDFPTAAIINGRRGIEEAYRTGRGKVY IRARAEVEADAKTGRETIIVHEIPYQVNKARLIEKIA
ELVKEKRVEGISALRDESDKDGMRIVIE IKRDAVGEVVLNNLYSLTQLQVTFGINMVALSQGQPKLLNLKDILV
AFVRHRREVVTRRTIFELRKARDRAHILEALAIALANIDPIIELIRRASTPAEAKAGLIASPWELGNVASMLER
AGDDAARPEWLEAEFGIRDGKYYLTEQQAQAILDLRLOKLTGLEHEKLLDEYKELLTLIGELIFILENPDRLME
VIREELVAIKEQYNDLRRTE ITANTSDINIEDLINQEDVVVTLSHQGYVKYQPLSDYEAQRRGGKGKSAARIKE
EDFIDRLLVANTHDTILCFSSRGRLYWMKVYQLPEASRGARGRPIVNLLPLEPNERITAILPVREYEEGCHVFM
ATASGTVKKTALTEFSRPRSAGIIAVNLNEGDELIGVDLTDGSNEVMLFSALGKVVRFPEGQVRSMGRTATGVR
GINLNGDDRVISLIIPRGDGEILTVTENGYGKRTAVAEYPTKSRATQGVISIKVSERNGKVVGAVQVAPTDQIM
MITDAGTLVRTRVSEVSVVGRNTQGVTLIRTAEDEHVVGLQRVAEPEEDDDILDGESSEGEEGSEENAALNAPS
DDVADEDTDAEDDTDVEDDN< / I NSDS eq sequencer 387 </INSDSeg> IJ
Ges </SegusncsDara>
LE LSequencebalta seqguantailiioghar="a8Y
LOG CINSDSag»
Lu “INSDSeq length>923</INSD3eq Length>
Se <INSDSeq moltype>AA“/INSDSeq moltype> 532 <INSDSeq division>PAT</INSDSeg division: 534 <INSLSeq feature-itabled
BE <INSDFeabure>
Re <IMaDFesature key>source“/INSDFeature key>
Sa <INSDFearure location>l..923</IN3DFeatiure location»
La <INSDFeature quals> IJ
Is <INSDQOualifier»
RESIN <INSDQualifier name>mol type“ /INSDQualifier name»
SD: <INSDQualifier valuerprotein</INSDQualifier value»
ADD </INSDQuali jier ai <INSDQualifier Ld=TgRYRL
Si <INSDQualifier namevorganism“/INSDQualifier name>
SON <INSDOualifier value>Pseudomonas aeruginosa</INSDGualiflier value» £08 </INSDOualilfier>
LOT </INSDPeature guals>
SDE </INSDFeaturer
AD </IN3DSeq featura-table> <iNSDseq sequence >MGELARKE ILPVNIEDELKQSYLDYAMSVIVGRALPDARDGLKPVHRRVLYAMSELG
NDWNKPYKKSARVVGDVIGKYHPHGDTAVYDTIVRMAQPFSLRYMLVDGQGNFGSVDGDNAAAMRY TEVRMAKL
AHELLADLEKE TVDWVPNYDGTEQIPAVMPTKIPNLLVNGSSGIAVGMATNIPPHNLGEVIDGCLALMDNPDLT
VDELMQYIPGPDFPTAGIINGRAGIIEAYRTGRGRIYIRARAVVEEMEKGGGREQIIITELPYQLNKARLIEKI
AELVKEKKIEGISELRDESDKDGMRVVIELRRGEVGEVVLNNLYAQTQLQSVFGINVVALVDGQPRTLNLKDML
EVFVRHRREVVTRRTVYELRKARERGHILEGQAVALSNIDPVIELIKSSPTPAEAKERLIATAWESSAVEAMVE
RAGADACRPEDLDPQYGLRDGKYYLSPEQAQAILELRLHRLTGLEHEKLLSEYQEILNLIGELIRILTNPARLM
EVIREELEAVKAEFGDARRTEIVASQVDLTIADLITEEDRVVTISHGGYAKSQPLAAYQAQRRGGKGKSATGMK
DEDYIEHLLVANSHATLLLFSSKGKVYWLRTFE IPEASRTARGRPLVNLLPLDEGERITAMLQIDLEALQQNGG
ADDDLDEAEGAVLEGEVVEAAEVEEVEGETAELVAEPTGAY IFMATAFGTVKKTPLVQFSRPRSSGLIALKLEE
GDTLIAAAITDGAKEVMLFSSAGKVIRFAESVVRIMGRNARGVRGMRLGKGQQL ISMLIPESGAQILTASERGF
GKRTPLSKFPRRGRGGQGVIAMVTNERNGALIAAVQVQEGEE IMLISDQGTLVRTRVDEVSLSGRNTQGVTLIK
LASDEVLVGLERVQEPSGGDDEDLPEGEEAAESLGESAESESEPAAEAEGNEE </ INSDS eg sequenced
Sid </INSDSeq> ”
S12 </SeguenceData>
ALS <SequenceData ssqvueanoselDNumber=Ngen)»>
Sid <iNSDSed>
SLE <INSDSeq length>904</INSDSeq length»
SLE “INSDSeqg moliypevAA</INSDSeq moltype> 517 <INSDSeq diviglon»PAT</INSDSeqg division:
SLE <INSDSeq feature-table>
L173 LIMIDFeaturas
AED <INSDF Feature key>source“/INSDFeature key>
BEL <INSDFeature locatlonrl..904</INSDFeaturs locabion>
Gld <INSDFeature quals>
E03 <INSDQualifiers dad <INSDQualifier name>mol type“ /INSDQualifier name»
Sen <INSDQualifier value>protein</IN3DQualifier value»
SEE </INSDOuaiifiers
SET <INSDOualifier id="qgga81>
HIE <INSDQualifler namedorganism</INSDQualifisr name> a2 <INSDQualifier valuerAcinetobacter baumannii</INSD2ualifier valus>
S30 </INSDOualifier:>
SSL “/INSDPeature cuals>
ESE </INSDFeature»
EE «/INSDSeg featurse-tabier> <INSDSeq sequenca>MSVSEIRPIATEDELKHSYLDYAMSVIVSRALPDVRDGLKPVHRRVLYAMHELGND
YNKAYKKSARVVGDVIGKYHPHGDLAVYETIVRMAQDFSLRYLLVDGQGNFGSIDGDSAAAMRY TEVRMTKLAH
ELLADLEKDTVDWEDNYDGSERIPEVLPTRVPNLLINGAAGIAVGMATNMAPHNMTEVVNACLAYADNPNISIE
GLMEYITGPDFPTGGIIYGKSGIVDAYRTGKGRLHIRGKYHFEEDEKTGRTTIVFTEIPYQVNKARVIERIAEL
VKEKKLEGISELRDESDKEGMRIAIDLKRGENAEVVVNNLFLNTQLENSFSINMVCLDNGQPKLMNLKDIIAAF
IRHRQEVVTRRTMFELRKARERGHILEGLTVALANIDEIIETIKTSANPAEARERLLAGEWAGGGVVALLEKAG
AISVRPDEIEGEDPNRPFGLSDSIYRLSPTQVGAILELRLHRLTGLEQDKLHAEYTEILGQIAELTAILNDFNL
ILMGVIREELAQVLQQYGDARRTEIVESRVDFCREDLIPEEQVVLTVSQTGYAKTQPLSDYQAQRRGGRGKSATS
MKDDDFIQHLIVASNHATVLCFTNVGKVYRLKVFEVPQASRGAKGRPIVNLLPLDATETVTAILPLTEFPENHY
VFMATASGTVKRVELEQFANIRSNGLRAIELNEEDTLIGVAITDGNQQIMLFSNEGKAIRFAETDVRAMGRTAK
GVRGMRVSFASSTLSEEDADVENDDSDDNDDSADSSLVSRIVSLVVVPETGEVLCASANGYGKRTPVNDFPTKK
RGGKGVIAIKTSERNGELVGAVSIDETKELLLISDGGTLVRTRAAE VAMTGRNAQGVRLIRLSEEETLVGVVSI
EAVEDEEELLESEVDTTETDSEEAVSNNEDTSEE</ INSDS ag zecduence> £50 <SINEDE E>
ELE </SeguenceData> £37 <Sequencalata sopeancalliumler="2T8s
HR CINIDSeq>
Aid <INSDSeq Length>43</INSDSeq length» 240 <IMEDZeq moliype>DNA</INSDSeqg moltyper
GAL <INSDSeq division»PAT</INEDIag division>
GA “INSDSeq fealure-table> cis LINSDFeature:» dd <INSDFeature key>source</INSDFeature key»
S495 <INSDFeature location>l..43</INIDFeaturs location»
Add <INSDFealure gualz> ada <INSDOualifisr>
S48 <INSDQualifier namermol type</INSDQualifier name>
S40 <INSDOualifier valuerother DNA /INSDQualifier value»
GLO <SINSDOualifier>
S50 LINSDGualiifler in=\gg8%> she <INSDQualifier nemssorganism“/TINSDOQualifier name
S53 <INSDQualifier value>synthetic construct /INSDouallfier valuer
A54 </INSDQvalifier:
SE </INSDFeature quals>
SEG </INSDEFeature>
SLY <INSD¥eature»
LIE <“INSDFeslture key>misce feature /INSDFesature key» £53 <INSDFeature location>l..43</IN3DFeature location»
S60 <INSDFesture duels» aad CINZDQuallifier id=sgs5nx dal CINADGualifler name»note/INSDOualifier name>»
AED <INSDQualifier value»>gyrA POS“ /INSDGualifier values
S&á <fINSDUualifler»>
GEL x“/INSDFeature gquals>
EEE </INSDFealure»>
LET </INSDSeg feature-tabler
Ha <INSDSeg zequence>ttctctagagtcgacctgcaggaagteccggeccegggatgtgt/INSDSeq sag usnea»
SR </INSDSeqg>
SEG <SBequencelata>
GFL <HGegquencelbata seguanselilMonhao="2®Y >
SS LINSDZeqg>
SE <INSDSeq length>58</INSDSeg length> £74 <INSDSeq molityperDNAC/TNSDSeg moliype>
SEH <INSDSeq division>PAT/THSDSeq divisions
A74 <INSDSeg feabture-tablel
Gij <INSDFeature> sis <INSDFearure key>sources/INSDFeature key>
Dijk JIiNSDFeature lccation>l..58/INSDFeature Location bei <“INSDPFeacure ouals:»>
SRL <INSDQualifier»>
SER “INSDgvelifier named>mol type</INSDQualifier name>
AR <INSDQualifilier valuerother DNA</INSDGueliiier value»
BE </INSDQvalifier:
AEH <INSDQuelifier id="g3ons
ARE CINMEDOualifier namerorganism</INSDQualifier name on “INSDoualifier value>synthetic construct“/INSDgualifiern value
LEE </INSDOualiiier>
LED </INSDFeature guals> 430 </INSDFealure» -
OSL <INSDFeabture>
Suz <INEDFeature key misc feature</INSDhFsature key> [ORE <INSDFeature Iscation>»l..58</INSDFealure location
Ghd CINEDFeature gualse
Ha <INSboualifier LON gv >
Ze <INSDQualifier name>note</INSDQualifier name>
SY <INSDQualifier value>gyrA POB</INSDQualifier valuse>
A38 </INSDQualiËier>
Hug </INSDFeature guals»
TOO </INSDPeature>
GOL </INSDSeg feature-teblex>
OL
<INSDSeq sequencertegagtagggataacagggtaatatctagaggtttaccggcgattttteggcatte at /INSISeg sequenced 702 </INEDS egy u
Und </SecguenceData>
Tan <SequenceData saguencalMiunbaer="28%%
Ga <iNSDSed>
TO <INSDSeq liengith>2l1</INSDSeg lengths
Ee: x“INSDSeq moliyperDNA</INSDSeg moltypex
Tin <“INSDSeg division>PAT/INSDSecg division»
Jin <INSDSeq feature-itableX
Ti CINEDEeatures
Tie <INSDFeature keyssource“/INSDFealure hay» zij <INSDFeature locationsl1..21</INSDFeature Location»
TA <INSDFeature quals> u
TLE <INSDQualifiers
TLE CINEDQUalifiler name>mol type“ /INSDgualifier named
VLT <“INSDOQualifier valuse>other DNA</TINSDOualifier value>
ALE </INSDOQualijiier>
TIN <INSDOualifier id=vglis>
Tan <INSDuualifier namedorganism</INSDOualifisr name>
TR <INSDQualifier valuersynthetic construct</INSDQualifier valued ey </INSDOualifier:>
Tas </INSDFeature guals>
Tad </INSDFeature»>
ED <INSDFeature:>
TES <INSDFeature keyrmisc feature</INSDFeature key»
Ta <INSUFeature location>l..21</INSDFeature location»
Tan <INGDFeature quals> u
Tan <INSDQuelifier id='eggjnn
TRG <INSDOualifier namernote</INSDOualifisr name>
SL <INSDQualifier value>gyrA POS</iNSDQualifier value»
TSE </INSDOualilfier>
TEE </INSDPeature guals>
Tod </INSDFeature> u
TIN </INSDEeg Zearture-tabier
ERS <INSDSeq sequence>atcageccttcaatgetgatg</INsSDdeq sequence»
FR </INSDSeg>
TIE <SBequencelata>
PRE <Sequencebata segoantallomhar=m309 >
A LINSDSeq>
JAL <INSDSeg length>21</INSDSeq length>
Tau <INSDSeq molityperDNAC/TNSDSeg moliype>
Fan <INSDSeq division>PAT/THSDSeq divisions
Tad <INBDZeq feabture-tablel
TAN <INSDFeature
FAG <“INSDFearure keyrsource</INSDieature key
Ta STNEDFeabture locationsl..21</INSDFeaeture Locations
Fan <“INSDPFeacure gqualis>
Tas <INSDQualifier»>
THO <INSDgualifier name>mol type</INSDOualifier name>
TH CINBDQualifiler valuerother DNAC/IN3DGualifler value»
TER </INSDQualifisan»
FE <INSDQuelifier id="glvr
TE CINMEDOualifier namerorganism</INSDQualifier name
TLL “INSDoualifier value>synthetic construct“/INSDgualifiern value
ThE </INSDOQualijiier>
EN) </INSDFeature guals> 758 </INSDFeature> u
Tha <INSDFeabture>
Tal <INSDFsature key>misc feature“ /INSDFeature key>
TOL CINSDFesature locationr>l..21</INSDFeature location
TEE CINEDFeature gualse
JEL <INSboualifier LEARY > dea <INSDQualifier name>note</INSDQualifisr name>
TED <INSDQualifier value>gyrA PlO</INSDQualifier valuse>
TAH </INSDQuali fier»
Ta </INSDFaature cualss»
TAR </INSDPeature>
Tou </INSDSeq feature-tabled>
EO “INSDSeq sequancertecegtaattggecaagacaaac</INIDSeqg sequencer
TE <SINBDE eg
TIE </SeguenceData>
EE <Sequencelata sepience liane n="31%>
Tg JINSDSeq>
FE <INSDSeg lengbh>T7</ INSDSeq length
FERS <INEDZaqg moltype>DNA</INSD3eg moltvpe:r>
TE <INSDSeq divisicon>PATA/INSDSeag division» is “INSDSeq feature-tabled>
Tin LINSDFeatures»
TED <INSDFeature kevyrsource</INSDFesture kev:
TE <INSDFesture Location>l..T7</INSDFeature location»
RZ <INSDFeature duals»
Tal <INSDQualifier>
TRG <INSDQualifier namermol type</INSDQualifier name>
TRE <INSDOualifier valuerother DNA4/INSDQualilfier value»
TEE <SINSDOualifier> a LINSDGualiifler ia=wgl3dy>
TER <INSDQualifier nemssorganism“/TINSDOQualifier name
TES <INSDQualifier value>synthetic construct /INSDouallfier valuer 790 </INSDQvalifier:
Te </INSDFeature quals>
Tan </INSDEFeature>
Tal <INSD¥eature»
Fd <INSDFeabure key>misce feature /INSDFesature key»
Tak <INSDFeature location>l..77</IN3DFeature location»
TES <INSDFesture duels»
TE CINZDQuallifier id=vgdsn»
TOR CINADGualifler name>note</IN3Doualifier name>» ad <INSDQualifier value»>gyrA Pll</INSDGualifier values
BOD <fINSDUualifler»>
SDL “{INSDFeature quals>
GOE </INSDFealure»>
S03 </INSDSeg feature-tabler <INSDSeq seduencsrseteagtecctaggtataatactagtagcatataacgcagcgagaagttttagagcta gaaatagcaagttaaaataag:/INSDSeg sequence s05 </INSDSeg> u
SDE </SemtenceDala>
BOF <HGegquencelbata seguanselilMonhao=n3Z8Y 5s
HOE <INZDIeq>
HOD <INSDSeq length>4l</INSDSeg length> 214 <INSDSeq molityperDNAC/TNSDSeg moliype>
Si <INSDSeq division>PAT/THSDSeq divisions
Sia <INBDZeq feabture-tablel
SLS <INSDFeature>
RR <INSDFearure key>source</INIDFeature key»
FLL JIiNSDFeature location>l..4l</INSDFeature Location
GLE <“INSDPFeacure gqualis>
SLT <INSDQualifier»> 19 <INSDQualifier name>mol type</INsSDQualifier nama» zij <INSDouelijler valuerother DNA</INSDGueliiier value»
S20 </INSDQualifisan»
SZ <INSDQualifiler id="g38N>
BEE <INSDOQualifier namerorganism“/INSDQualifier name
SES “INSDoualifier value>synthetic construct“/INSDgualifiern value
Hd </INSDOQualijier> 2ED </INSDFeature guals>
Dd </INSDFealure» u
Sa <INSDFeabture>
B28 <INEDFeature key misc feature</INSDhFsature key>
Bow <INSDFeature Iscation>l..41</INSDFealure location
SSU CINEDFeature guals>
Ea <INSboualifier LO=NgBor>
Sie <INSDQualifier name>note</INSDQualifier name>
Sl <INSDQualifier value>gyrA Pl2</INSDQualifier value> 8324 <ATNaSDQuali fis»
RA </INSDFeature guals»
STE </INSDFeature>
BRE </INSDSeg feature-teblex>
BE IJ
“INSDSeq sequsnceretgcaggtcgactctagagaattcaaaaaaagcaccgacte/INSDLSeq seque nce>
REE </INSDSegr £40 </SecuenceDatas
Sa <SequenceData saguencalMiunbaer="33"% 842 <iNSDSed>
SAS <INSDSeq lengith>4l</INSDSeg lengths
Sad x“INSDSeq moliyperDNA</INSDSeg moltypex
H4a5 <INSDSeq division»PATL/INIDSeg division»
EEE <INSDSeq feature-itableX
S47 CINEDFeature> £48 <INSDFeature keyssource“/INSDFealure hay» 244 <INSDFeature locationsl..41</INSDFeature Location»
Se CINSDFeature gualis>
BEL <INSDQualifiers
Fh SINEDOualifiler name>mol type</IN3DQualifier name»
HELE <“INSDOQualifier value>other DNA</INSDOuallfier value> and </INSDOQualijier> 255 CINSDOualifisr id="qgZ&r> 256 <INSDuualifier namedorganism</INSDOualifisr name>
SR <INSDQualifier valuersynthetic construct</INSDQualifier valued
BAS <fINSDUualifler»>
Sil </INSDFealture cauels»>
GEO </INSDFealure»>
SEL <INSDFearture:»
HE2 <INSDFeature keyrmisc feature</INSDFeature key» £532 <INSDFeature locations>l..4l1</INSDFeature Locations
Sad <INGDFeature quals> u
SEE <INSDQualifier id="gSin>
BoE <INSDOualifier namernote</INSDOualifisr name> aa SINEDOualifiler valua>gyrA P13</INSDQualifier values
HEE </INSDOualilfier>
ZLD </INSDPeature vuals> 270 </INSDFeaturer -
DEL </INSDEeg Zearture-tabier es <INSDSeq sequence>tttactacgttacatgctggtagacggtcagggtaactteg<//INSDSeq seque near
GTL </INSDE ag»
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ETH “SequenceData seguencellNumben="Z4®> ijd <INSDSeq>
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Sad <INSDQualifier namerorganism“/INSDQualifier name>
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BEE </INSDFeaturer - 328 “INSDFeaturex
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Wi <INSDQualifier id="qg3S">
SHE <INSDgualifier namedorganism</INSDQualifier name> 359 <INBDQualifiler valuersynthetic construct</INSDoualifier value»
SEG </INSDQuali fier!» wsl </INSDFearture qualss
GEDE </INSDFeacturer
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Ged <INSDFeature key>misc feature</INSDFeature key> 365 <INSDFesture locaticon»l1..42</INSDFeature Location»
Bad <INSDFesture gualz> u
Sa CINSDQualifier id="gBan>
SHB <INSDQualifier namernote</INZDOualifier name>
Eee CIMNEDOualifier valuergyrA Pl8“/INSD{ualifier value:
STD </INSDOualifiers> u u
WIL </INSDFeature duels»
GTZ </INZDFeature> 373 </INSD8eq featura-tabler <INSDSeq sequence>ggagatctggetgtttatgacacgategtecgecatggegeag</INSDSeq seu sneer
GTL </INSDSeg>
SEG <j Gaemquencebata>
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IE <INSDSed> 373 <INSDSeq iength>57</INSDSeq length>
ZED <INSDSeq moliype>DNA/INSDSeg moltyper
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SEE <INSDieq festure-table>
GES <INSDFeature>
Gna “INSDFearture keyrsourced/INIDFeaturs Key»
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LEE <INSDFeature quels» u
BET <INSDOualifier>
S88 <INSDQualifier name>mol type</INSDQualifiar DATE >
G89 <INSDQualiiler valuerother DNA /INSDQualiiler value»
Sag </INSDQuali fier!» u u
Ge <INSDguelifier id="gs0">
GE “INSDOualifier namevorganism /INSDQualifier named
Gos <INSDQualifier valuse>synthetic construct“ /INSDGualifier valuer
BEd </INSDQualifier> 355 <SINSDFaature guals> 358 </INSDFealurex> -
Sh <INSDFeature>
Gl <“INSDFearure key>misce feature«/IN3UFsature key>
Gis JIiNSDFeature lccation>l..5S7/INSDFeature Location
Li <“INSDPFeacure ouals:»>
LOO <INSDQualifier id="qSS5> 1502 <INSDQualifier name>note</INSDQualifier name>
Lijns CINSDQualifier valuergyrA P19</INSDQgualijien valued 100g </INSDQuali fier 100% </INSDFeature quals>
Goa </INSDEFeature> u
ADT </INSDSeg faature-table> <INSDSeq sequence>gtecataaacagccagatctccatggggatggtatttaccgattacgtcaccaacga ce“ /INSDSeg sequenced 1009 </INSDSeg> u
REARS </SeguenceDatas
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LGR <INSDSeq diviglon»>PAT</INSDSeg divisions 1Gia <INSDSeq feature-itablel
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LOE <CINSDQualifiers
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LOS! CINEDEeatures naa <INSDFeature key>misc feature“/INSDFealure key> ins <INSDFeature locationsl..76</INSDFeature Location»
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TONE <INSDguelifier id="g5ë">
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Less </INSDOualiiier> 1038 </INSDFeature guals>
Laas </INSDFealure» -
LOS </INSDSeg feature-tabled>
LOE
<INSDSeq sequencevctcagtcctaggtataatactagctatgacacgatcgtcecgcagttttagagctag aaatagcaagttaaaataag/INSDSeq sequenced
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Lhd </SeguenceData>
Ian <Sequencalata sopeancallilumber="388 ide “INSDSeg» 13a <INSDSey lenghh>42</IN3DSeq length 134s <INEDSeq moltype>DNA</INSDSeg moltype:>
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LOD <INSDQualifier>
LONE <INSDQualifier namermol type</INSDQualifier name> an <INSDOualifier valuerother DNA</INSDQualifier value»
LORE <SINSDOualifier>
REN LINSDGualiifler in=\gg3r>
LOE <INSDQualifier nemssorganism“/TINSDOQualifier name 10a <INSDQualifier value>synthetic construct /INSDouallfier valuer 1082 </INSDQualifisan» 1083 </INSDFearure guals> hed </INSDEFeature>
Leal <INSD¥eature»
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Lean <INSDFeature location>l..42</IN3DFeature location» eae <INSDFesture duels»
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LT CINADGualifler name>note</IN3Doualifier name>»
LEL <INSDQualifier value>gyrA P21</INSDgualifier value»
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LOTS </INSDFeature uals
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LOTS </INSDSeg feature-tabler
LOTS u <IN3D3eq zeguence>ctataacacaatagtaaggatggegcagccattetecgetgeg“/INSDSeq sequ ancer
OTT </INSDSeg>
KE! </SemtenceDala>
LOS “Sequsrcebala seconde liMNgachev="gds>
GEG LINSDSeq>
Lewd <INSDSeq length>48</INSDSeg length> 1OE2 <INSDSeq moltype>DNA</INSDSeg moiiyper ijE) <INSDSeq division»PAT</INSDSeg divisions
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Long <INSDFearure key>source</INIDFeature key»
LOST JIiNSDFeature lccation>l..48/INSDFeature Location hea <“INSDPearure duals»
LGES <INSDQualifier»> 1030 “INSDgvelifier named>mol type</INSDQualifier name>
TOBA CINBDQualifiler valuerother DNAC/IN3DGualifler value» 1082 </INSDQualifisan»
RRA <INSDQuelifier id=vgidns
Load CINMEDOualifier namerorganism</INSDQualifier name
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LGDB7 </INSDFeature guals> ij38 </INSDFealure» - 1a <INSDFeabture>
LAO <INSDFsature key>misc feature“ /INSDFeature key>
TRON <INSDFeature Iscation>l..48</INSDFealure location
LOE CINEDFeature gualse
LDS <INSDQualifler in='g58%>
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Linn <INSDQualifier value>gyrA P22</INSDQualifier value> iine </INSDQualiËier> aay </INSDFeature guals»
Lildë </INSDPeature>
LA </INSDSeg feature-teblex> “INSDSeq sequsencergccatccttactattgtgttatagaccgccgagtcaccatggggatgg: /INSDSe oO seguence> 111 </TNSDSeg> 112 <fSeguenceDalar
LLL: </ST26SeguencelLiscing>

Claims (41)

ConclusiesConclusions 1. Verbinding met formule I: ROR Î G8 ON re. . wt R Ry iad er N7 4 Ss XE Rg, a Sg EN RE AE ah DS X (Rohn {th waarbij: een enkele van de X', X2, en X3 gelijk is aan N, terwijl de resterende twee van de X1, X2 en X3 gelijk zijn aan CRY; R'is geselecteerd uit H, halo, C4-C4 alkyl, en C1-C4 haloalkyl; R? is geselecteerd uit H, halo, Cs-Cs alkyl, en C4-C4 haloalkyl; of Rt en R? samen een 1-, 2-, of 3-ledige alkyleenketen, optioneel gesubstitueerd, vormen met, indien chemisch mogelijk, één, twee, of drie groepen die onafhankelijk van elkaar geselecteerd zijn uit halo, C4-C4 alkyl, en C+-C4 haloalkyl, CN; alle R32 en R% onafhankelijk van elkaar geselecteerd zijn uit halo, C:-C4 alkyl, en C1-C4 haloalkyl, CN, OR? en NRR, waarbij R? geselecteerd is uit H, C+-C4 alkyl, en C1-Ca haloalkyl, en alle R? onafhankelijk van elkaar geselecteerd zijn uit H en C+4-C4 alkyl, R* is geselecteerd uit H, C4-C4 alkyl, en C+4-C4 haloalkyl; R5 is geselecteerd uit fenyl en een 5- of 6-ledige heteroarylgroep, optioneel gesubstitueerd met 1 of 2 R% groepen; alle R® onafhankelijk van elkaar geselecteerd zijn uit halo, C1-Cs alkyl, en C1-C4 haloalkyl, CN, -O-C+-C4 alkyl, en -O-C+-C4 haloalkyl; alle R7 onafhankelijk van elkaar geselecteerd zijn uit H, halo, C+-C4 alkyl, en C:- Ca haloalkyl, CN, -0-C+-C4 alkyl, en -0-C+-Ca4 haloalkyl; n gelijk is aan 0, 1, 2, of 3; en p gelijk is aan 0, 1, 2, of 3;1. Compound of formula I: ROR Î G8 ON re. . wt R Ry iad er N7 4 Ss XE Rg, a Sg EN RE AE ah DS X (Rohn {th where: one of X', X2, and X3 is N, while the remaining two of X1, X2, and X3 are CRY; R' is selected from H, halo, C4-C4 alkyl, and C1-C4 haloalkyl; R? is selected from H, halo, Cs-C8 alkyl, and C4-C4 haloalkyl; or Rt and R? together form a 1-, 2-, or 3-membered alkylene chain, optionally substituted, with, if chemically possible, one, two, or three groups independently selected from halo, C4-C4 alkyl, and C+-C4 haloalkyl, CN; each of R32 and R% are independently selected from halo, C1-C4 alkyl, and C1-C4 haloalkyl, CN, OR? and NRR, where R? is selected from H, C+-C4 alkyl, and C1-C8 haloalkyl, and each R? are independently selected from H and C+4-C4 alkyl, R* is selected from H, C4-C4 alkyl, and C+4-C4 haloalkyl; R5 is selected from phenyl and a 5- or 6-membered heteroaryl group, optionally substituted with 1 or 2 R% groups; each R® are independently selected from halo, C1-C8 alkyl, and C1-C4 haloalkyl, CN, -O-C+-C4 alkyl, and -O-C+-C4 haloalkyl; each R7 are independently selected from H, halo, C+-C4 alkyl, and C:- C8 haloalkyl, CN, -O-C+-C4 alkyl, and -O-C+-C4 haloalkyl; n equals 0, 1, 2, or 3; and p equals 0, 1, 2, or 3; of een farmaceutisch aanvaardbaar zout, een farmaceutisch aanvaardbare stereo-isomeer, een farmaceutisch aanvaardbaar solvaat, of een farmaceutisch aanvaardbaar progeneesmiddel daarvan.or a pharmaceutically acceptable salt, a pharmaceutically acceptable stereoisomer, a pharmaceutically acceptable solvate, or a pharmaceutically acceptable prodrug thereof. 2. Verbinding volgens conclusie 1, waarbij Rt en R? samen een 2- of een 3-ledige alkyleenketen vormen, optioneel gesubstitueerd met één, twee, of drie groepen die onafhankelijk van elkaar geselecteerd zijn uit halo, C4-C4 alkyl, en C4-C4 haloalkyl, CN.A compound according to claim 1, wherein Rt and R? together form a 2- or 3-membered alkylene chain, optionally substituted with one, two, or three groups independently selected from halo, C4-C4 alkyl, and C4-C4 haloalkyl, CN. 3. Verbinding volgens conclusie 1, waarbij R* gelijk is aan H, en R2 gelijk is aan H.3. A compound according to claim 1, wherein R* is H, and R2 is H. 4. Verbinding volgens conclusie 1, waarbij de verbinding een verbinding is met formule IIA of met formule IIB: Ee ee HL SN, (FRY R7 ee ar J es RE re x Fg” “ey RE he No “ny, RS Fog en {Rhy {HAY Lig ~F Os t hs i Pi oo IR a Ne ey Re BS ESN KE eh se WEN ee A (18, waarbij: alle R3 en R® onafhankelijk van elkaar geselecteerd zijn uit H, halo, C1-C4 alkyl, en C1-C4 haloalkyl, en CN; en m gelijk is aan 0, 1, of 2.4. The compound of claim 1, wherein the compound is a compound of formula IIA or of formula IIB: Ee ee HL SN, (FRY R7 ee ar J es RE re x Fg” “ey RE he No “ny, RS Fog en {Rhy {HAY Lig ~F Os t hs i Pi oo IR a Ne ey Re BS ESN KE eh se WEN ee A (18, wherein: each of R3 and R® are independently selected from H, halo, C1-C4 alkyl, and C1-C4 haloalkyl, and CN; and m is 0, 1, or 2. 5. Verbinding volgens conclusie 4, waarbij alle R3 en R® gelijk zijn aan H.5. A compound according to claim 4, wherein all of R3 and R® are H. 6. Verbinding volgens conclusie 4 of conclusie 5, waarbij m gelijk is aan 1.6. A compound according to claim 4 or claim 5, wherein m is 1. 7. Verbinding volgens een der voorgaande conclusies, waarbij R® is:7. A compound according to any preceding claim, wherein R® is: 4 rab Ae Xa waarbij: X* gelijk is aan N, CH, of CR"; X5 gelijk is aan N, CH, of CR"; R'is geselecteerd uit halo, C4-C4 alkyl, en C4-C4 haloalkyl, en CN; R'is geselecteerd uit halo, C1-Ca alkyl, en C4-C4 haloalkyl, en CN; qgelijk is aan 0, 1, of 2; en ’ het verbindingspunt aangeeft met de rest van de verbinding, op voorwaarde dat, wanneer X* gelijk is aan N, X5 gelijk is aan CH of aan CR", en dat, wanneer X5 gelijk is aan N, X* gelijk is aan CH of aan CR.4 rab Ae Xa where: X* is N, CH, or CR"; X5 is N, CH, or CR"; R' is selected from halo, C4-C4 alkyl, and C4-C4 haloalkyl, and CN; R' is selected from halo, C1-C8 alkyl, and C4-C4 haloalkyl, and CN; q is 0, 1, or 2; and ’ denotes the point of attachment to the remainder of the compound, provided that when X* is N, X5 is CH or CR" and when X5 is N, X* is CH or CR. 8. Verbinding volgens conclusie 7, waarbij X* gelijk is aan N.8. A compound according to claim 7, wherein X* is N. 9. Verbinding volgens conclusie 7 of conclusie 8, waarbij X5 gelijk is aan CH of aan CR", optioneel waarbij X5 gelijk is aan CH.9. A compound according to claim 7 or claim 8, wherein X5 is CH or CR", optionally wherein X5 is CH. 10. Verbinding volgens conclusie 7 of conclusie 9, waarbij X* gelijk is aan CH of aan CR? optioneel waarbij X* gelijk is aan CH.10. A compound according to claim 7 or claim 9, wherein X* is CH or CR? optionally wherein X* is CH. 11. Verbinding volgens conclusie 7 of conclusie 10, waarbij X5 gelijk is aan N.11. A compound according to claim 7 or claim 10, wherein X5 is N. 12. Verbinding volgens een der conclusies 7 en 9 tot en met 11, waarbij R'is geselecteerd uit halo, CHs, CF3, en CN.A compound according to any one of claims 7 and 9 to 11, wherein R' is selected from halo, CH3, CF3, and CN. 13. Verbinding volgens een der conclusies 7 tot en met 10 en 12, waarbij R™ geselecteerd is uit halo, CHs, CF3, en CN.A compound according to any one of claims 7 to 10 and 12, wherein R™ is selected from halo, CH3, CF3, and CN. 14. Verbinding volgens een der conclusies 7 tot en met 13, waarbij q gelijk is aan O.14. A compound according to any one of claims 7 to 13, wherein q is O. 15. Verbinding volgens een der conclusie 7 tot en met 13, waarbij q gelijk is aan 1, en waarbij R3 geselecteerd is uit halo, CHs, CFs, en CN.A compound according to any one of claims 7 to 13, wherein q is 1, and wherein R3 is selected from halo, CH3, CF3, and CN. 16. Verbinding volgens een der voorgaande conclusies, waarbij R® niet gesubstitueerd is of gesubstitueerd is met een enkele R3 groep, optioneel waarbij R5 niet gesubstitueerd is.A compound according to any preceding claim, wherein R® is unsubstituted or is substituted with a single R3 group, optionally wherein R5 is unsubstituted. 17. Verbinding volgens een der conclusies 1 tot en met 6 of 14, waarbij R® niet A SN, ë hil “gy be 7 gesubstitueerd “ís, en waarbij * het verbindingspunt aangeeft met de rest van de verbinding.A compound according to any one of claims 1 to 6 or 14, wherein R™ is not substituted by A, β, γ, and wherein * indicates the point of attachment to the remainder of the compound. 18. Verbinding volgens een der voorgaande conclusies, waarbij p gelijk is aan O of aan 1, optioneel waarbij p gelijk is aan 0.18. A compound according to any preceding claim, wherein p is 0 or 1, optionally wherein p is 0. 19. Verbinding volgens een der conclusies 1 tot en met 15, waarbij p gelijk is aan 1, en waarbij R32 gelijk is aan halo.A compound according to any one of claims 1 to 15, wherein p is 1, and wherein R32 is halo. 20. Verbinding volgens een der voorgaande conclusies, waarbij R+ geselecteerd is uit H, C+-C4 alkyl, en optioneel geselecteerd is uit H en CHs.A compound according to any preceding claim, wherein R+ is selected from H, C+-C4 alkyl, and is optionally selected from H and CH3. 21.Verbinding volgens een der voorgaande conclusies, waarbij R* gelijk is aan H.21. A compound according to any preceding claim, wherein R* is H. 22. Verbinding volgens conclusie 1, waarbij de verbinding een verbinding is met formule (V) £4 : (RE Cull LN. pe A ff hp ET Noy KY ey EE H I | (Ry, os de eh ey } Ne 1 pp i XE + > XE {Wh waarbij: q gelijk is aan 0, 1, of 2; X* gelijk is aan N, CH, of CR"; X5 gelijk is aan N, CH, of CR": R19 is geselecteerd uit halo, C4-C4 alkyl, en C4-C4 haloalkyl, en CN; en R' is geselecteerd uit halo, C1-C4 alkyl, en C1-C4 haloalkyl, en CN,22. A compound according to claim 1, wherein the compound is a compound of formula (V) → 4 : (RE Cull LN. pe A ff hp ET Noy KY ey EE H I | (Ry, os de eh ey } Ne 1 pp i XE + > XE {Wh where: q is 0, 1, or 2; X* is N, CH, or CR"; X5 is N, CH, or CR": R19 is selected from halo, C4-C4 alkyl, and C4-C4 haloalkyl, and CN; and R' is selected from halo, C1-C4 alkyl, and C1-C4 haloalkyl, and CN, op voorwaarde dat, wanneer X* gelijk is aan N, X5 gelijk is aan CH of aan CR", en dat, wanneer X5 gelijk is aan N, X* gelijk is aan CH of aan CR, of een farmaceutisch aanvaardbaar zout, een farmaceutisch aanvaardbare stereo-isomeer, een farmaceutisch aanvaardbaar solvaat, of een farmaceutisch aanvaardbaar progeneesmiddel daarvan.provided that when X* is N, X5 is CH or CR", and that when X5 is N, X* is CH or CR, or a pharmaceutically acceptable salt, a pharmaceutically acceptable stereoisomer, a pharmaceutically acceptable solvate, or a pharmaceutically acceptable prodrug thereof. 23. Verbinding volgens conclusie 20, waarbij R°, R2, R32 R3 R+, R'" R' X4 X5 p en q zijn zoals gedefinieerd in welke dan ook der conclusies 2, 3, 8 tot en met 16, of 18 tot en met 21.A compound according to claim 20, wherein R°, R2, R32, R3, R+, R'" R' X4, X5, p and q are as defined in any one of claims 2, 3, 8 to 16, or 18 to 21. 24. Verbinding geselecteerd uit: eee baas ess ne Hod JL OT B JJ | HO iP Sy pa I a YY Noa Soy PEN Fs H, i, pj BS, ms RH, el Rb) Troe’24. Connection selected from: eee baas ess ne Hod JL OT B JJ | HO iP Sy pa I a YY Noa Soy PEN Fs H, i, pj BS, ms RH, el Rb) Troe’ i. A 8 Le ~ ng A Ne, A EN LI 6 fd Seg gee Sop RF N oh hou OM OM Q be Ta “Ny i LUNE SES «oy Bll Romper oo, Sy SEER Rey SNR eh TN AS ER NL bot Le Rha as + 3 + CB bE a pn oF Ong eest ì HOE ob B JL we ie Sa En, ge eK 5 py oT 5 in, § = ay FF we {J LJ Ke (a F ‘bY CL, Re € a Se oF 5 FT Rin 2 : OF gd ‚PO 5 3 etn R Aa Cadi i vr Osj As & A nd oy ny Se, PN a han ; Roy, iy ‚3 dS Fg dn 3 ef PT EN o Ee 9 PT Ee Ad ve Be DPA Rh A STL "y * en A hs Na > and San . of een farmaceutisch aanvaardbaar zout, een farmaceutisch aanvaardbare stereo-isomeer, een farmaceutisch aanvaardbaar solvaat, of een farmaceutisch aanvaardbaar progeneesmiddel daarvan.i. A 8 Le ~ ng A Ne, A EN LI 6 fd Seg gee Sop RF N oh hou OM OM Q be Ta “Ny i LUNE SES «oy Bll Romper oo, Sy SEER Rey SNR eh TN AS ER NL bot Le Rha as + 3 + CB bE a pn oF Ong est ì HOE ob B JL we ie Sa En, ge eK 5 py oT 5 in, § = ay FF we {J LJ Ke (a F 'bY CL, Re € a Se oF 5 FT Rin 2 : OF gd 'PO 5 3 etn R Aa Cadi i vr Osj As & A nd oy ny Se, PN a han ; Roy, iy '3 dS Fg dn 3 ef PT EN o Ee 9 PT Ee Ad ve Be DPA Rh A STL "y * and A hs Na > and San . or a pharmaceutically acceptable salt, a pharmaceutically acceptable stereoisomer, a pharmaceutically acceptable solvate, or a pharmaceutically acceptable prodrug thereof. 25. Verbinding die in staat is om te binden met een hoefijzervormige hydrofobe pocket van de DNA bindingsoppervlak GyrA subeenheid van DNA-gyrase.25. Compound capable of binding with a horseshoe-shaped hydrophobic pocket of the DNA binding surface GyrA subunit of DNA gyrase. 26. Verbinding volgens conclusie 25, waarbij het DNA-gyrase een DNA-gyrase is van een gramnegatieve of van een grampositieve bacterie, optioneel een DNA- gyrase van een gramnegatieve bacterie.26. A compound according to claim 25, wherein the DNA gyrase is a DNA gyrase from a gram-negative or a gram-positive bacterium, optionally a DNA gyrase from a gram-negative bacterium. 27.Verbinding volgens conclusie 25 of conclusie 26, waarbij de verbinding bindt met ten minste één aminozuur in een positie die geselecteerd is uit de groep die bestaat uit 92, 97, 98, 169, 172, 266, 267, 268, en 269 van SEQID NO: 1, of met een overeenstemmend aminozuurresidu in een homoloog daarvan, of een conservatieve modificatie daarvan, optioneel waarbij de verbinding bindt met ten minste één aminozuurresidu dat geselecteerd is uit de groep die bestaat uit M92, S97, L98, N169, $172, Y266, Q267, V268, en N269 van SEQID NO: 1, of een overeenstemmend aminozuurresidu in een homoloog daarvan, of een conservatieve modificatie daarvan.27. A compound according to claim 25 or claim 26, wherein the compound binds to at least one amino acid in a position selected from the group consisting of 92, 97, 98, 169, 172, 266, 267, 268, and 269 of SEQID NO: 1, or to a corresponding amino acid residue in a homologue thereof, or a conservative modification thereof, optionally wherein the compound binds to at least one amino acid residue selected from the group consisting of M92, S97, L98, N169, $172, Y266, Q267, V268, and N269 of SEQID NO: 1, or to a corresponding amino acid residue in a homologue thereof, or a conservative modification thereof. 28. Verbinding volgens conclusie 27, waarbij de verbinding bindt met aminozuur S97 en/of S172 van SEQID NO: 1, of met een overeenstemmend aminozuurresidu in een homoloog daarvan, of een conservatieve modificatie daarvan.28. The compound of claim 27, wherein the compound binds to amino acid S97 and/or S172 of SEQID NO: 1, or to a corresponding amino acid residue in a homologue thereof, or a conservative modification thereof. 29. Verbinding volgens conclusie 27 of 28, waarbij het homoloog in overeenstemming is met welke dan ook van SEQID NO: 2,3, 4,5, 6, 7,8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, of 26.29. The compound of claim 27 or 28, wherein the homologue is any of SEQID NO: 2,3, 4,5, 6, 7,8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26. 30. Verbinding volgens een der conclusies 25 tot en met 29, waarbij de verbinding in het bezit is van een structuur zoals gedefinieerd in welke dan ook van de conclusies 1 tot en met 24.30. A compound according to any one of claims 25 to 29, wherein the compound has a structure as defined in any one of claims 1 to 24. 31. Verbinding volgens een der voorgaande conclusies, waarbij de verbinding in het bezit is van een ICso die kleiner is dan ongeveer 10 uM in een DNA-gyrase supercoiling assay, optioneel waarbij de verbinding in het bezit is van een ICso die kleiner is dan ongeveer 500 nM.A compound according to any preceding claim, wherein the compound has an IC50 of less than about 10 µM in a DNA gyrase supercoiling assay, optionally wherein the compound has an IC50 of less than about 500 nM. 32. Formulering, omvattende een verbinding volgens een der voorgaande conclusies, en optioneel een farmaceutisch aanvaardbare drager.32. A formulation comprising a compound according to any preceding claim and optionally a pharmaceutically acceptable carrier. 33. Formulering volgens conclusie 32, waarbij de formulering een parenterale formulering of een orale formulering is.33. A formulation according to claim 32, wherein the formulation is a parenteral formulation or an oral formulation. 34. Formulering volgens conclusie 32 of 33, waarbij de formulering een parenterale formulering is, optioneel waarbij de formulering een formulering is voor een intraveneuze injectie.34. A formulation according to claim 32 or 33, wherein the formulation is a parenteral formulation, optionally wherein the formulation is a formulation for intravenous injection. 35. Verbinding volgens een der conclusies 1 tot en met 31, of formulering volgens een der conclusie 32 tot en met 34, voor gebruik als geneesmiddel.35. A compound according to any one of claims 1 to 31, or a formulation according to any one of claims 32 to 34, for use as a medicament. 36. Verbinding volgens een der conclusies 1 tot en met 31, of formulering volgens een der conclusies 32 tot en met 34, voor gebruik bij de behandeling van een bacteriële infectie.36. A compound according to any one of claims 1 to 31, or a formulation according to any one of claims 32 to 34, for use in the treatment of a bacterial infection. 37. Verbinding of formulering volgens conclusie 35, waarbij de bacteriële infectie een gramnegatieve bacteriële infectie is, optioneel waarbij de bacteriële infectie een infectie is die veroorzaakt wordt door bacteriën die geselecteerd zijn uit Campylobacter jejuni, Vibrio cholerae, Escherichia coli, Haemophilus influenzae, Klebsiella pneumoniae, Enterobacter cloacae, Salmonelle enterica, Shigella dysenteriae, Legionella pneumophila, Yersinia pestis, Pseudomonas aeruginosa, of Acinetobacter baumannii.37. The compound or formulation of claim 35, wherein the bacterial infection is a gram-negative bacterial infection, optionally wherein the bacterial infection is an infection caused by bacteria selected from Campylobacter jejuni, Vibrio cholerae, Escherichia coli, Haemophilus influenzae, Klebsiella pneumoniae, Enterobacter cloacae, Salmonelle enterica, Shigella dysenteriae, Legionella pneumophila, Yersinia pestis, Pseudomonas aeruginosa, or Acinetobacter baumannii. 38. Verbinding of formulering volgens conclusie 36 of 38, waarbij de bacteriële infectie een infectie is die veroorzaakt is door bacteriën die geselecteerd zijn uit38. A compound or formulation according to claim 36 or 38, wherein the bacterial infection is an infection caused by bacteria selected from E. Coli, K. pneumoniae, Klebsiella, Acinetobacter, P. Aeruginosa, Salmonella, Helicobaceter, en optioneel een infectie is die veroorzaakt wordt door E. Coli ofE. Coli, K. pneumoniae, Klebsiella, Acinetobacter, P. Aeruginosa, Salmonella, Helicobaceter, and optionally an infection caused by E. Coli or K. pneumoniae.K. pneumoniae. 39. Verbinding of formulering volgens conclusie 36, waarbij de bacteriële infectie een grampositieve bacteriële infectie is, optioneel waarbij de grampositieve bacteriële infectie een infectie is die veroorzaakt wordt door Bacillus anthracis, Corynebacterium diphteriae, Enterococcus faecalis, Enterococcus faecium, Erysipelothrix rhusiopathiae, Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Cloistridioides difficile, Mycobacterium tuberculosis, Mycobacterium leprae, mycobacterium avium, of Mycobacterium abscessus.39. The compound or formulation of claim 36, wherein the bacterial infection is a gram-positive bacterial infection, optionally wherein the gram-positive bacterial infection is an infection caused by Bacillus anthracis, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Erysipelothrix rhusiopathiae, Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Cloistridioides difficile, Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium avium, or Mycobacterium abscessus. 40. Werkwijze voor het behandelen van een bacteriéle infectie in een patiént, omvattende het aan de patiënt toedienen van een werkzame hoeveelheid van een verbinding volgens een der conclusies 1 tot en met 31, of van een formulering volgens een der conclusie 32 tot en met 34.40. A method of treating a bacterial infection in a patient comprising administering to the patient an effective amount of a compound according to any one of claims 1 to 31, or a formulation according to any one of claims 32 to 34. 41. Gebruik van een verbinding volgens een der conclusies 1 tot en met 31, of van een formulering volgens een der conclusies 31 tot en met 34, als een DNA- gyrase inhibitor, optioneel waarbij het gebruik in vitro is.41. Use of a compound according to any one of claims 1 to 31, or of a formulation according to any one of claims 31 to 34, as a DNA gyrase inhibitor, optionally wherein the use is in vitro.
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