IUSM-Pu due TREAT-AD Cen e
Miles one 2 Ta ge Enabling Package
LYN (LYN p o o-oncogene, S c amily y osine kinase)
Gene Symbol/Name
NCBI Gene ID
UniP o ID
Ensembl ID
LYN (LYN p o o-oncogene, S c amily y osine kinase)
4067
P07948
ENSG00000162711
Al e na i es
JTK8
Co esponding Au ho
Pa hum M. Wee awa na
Con ibu ing Au ho s
A i L. Beni ah, Timo hy I. Richa dson
Collabo a ing Au ho s
Supe ision
B en Clay on, Je Dage, Kun Huang, B uce Lamb, And ew
Meseca , Alan Palkowi z, Timo hy I. Richa dson
Da e App o ed by Admin Co e
Oc obe
15, 2025
Documen e sion
Ve sion 1.0
Documen e sion da e
No embe 6, 2025
Ci a ion
h ps://doi.o g/10.5281/zenodo.17545570
Sec ions o his wo k ha e been and will be submi ed o
pee e iew and p ospec i e publica ion
A ilia ions
Indiana Uni e si y School o Medicine
, Pu due Uni e si y
Funding
U54AG065181
1. ABSTRACT
The TaRge Enablemen o Accele a e The apy De elopmen o Alzheime ’s Disease (TREAT-AD) cen e s
we e es ablished o p o ide high-quali y esea ch ools and echnologies o alida e and ad ance he nex
gene a ion o d ug a ge s o Alzheime ’s disease (AD). Da a, me hods, and expe imen al esou ces a e
being openly dissemina ed h ough he AD Knowledge Po al o accele a e he disco e y and
cha ac e iza ion o AD- ele an he apeu ic a ge s. These esou ces a e compiled as Ta ge Enabling
Packages (TEPs) ha p o ide open access o alida ed eagen s, assays, and chemical ools. He e, he
IUSM-Pu due TREAT-AD Cen e p esen s a Miles one 2 (M2) TEP ha e alua es he expe imen al easibili y
o ini ia ing a d ug disco e y p og am a ge ing Lyn kinase by es ablishing and assessing ini ial in i o
assays and ool molecules.
Lyn is a S c- amily non ecep o y osine kinase ha se es as a c i ical egula o o immune signaling
h ough he phospho yla ion o bo h immuno ecep o y osine-based ac i a ion (ITAM) and inhibi o y
(ITIM) mo i s. Abe an Lyn ac i a ion has been implica ed in au oimmune diseases, hema ologic
malignancies, and neu odegene a ion, including AD, whe e Lyn exp ession and au ophospho yla ion a e
ele a ed in mic oglia su ounding amyloid plaques. To enable pha macological s udies o Lyn unc ion in
AD models, we pu sued a ligand-based op imiza ion s a egy s a ing om ima inib, based on he
obse a ion ha ima inib inhibi s Lyn kinase bu lacks he po ency and selec i i y equi ed o a sui able
p obe molecule. Gi en ha ima inib ac s as a ype II kinase inhibi o and he ad an ages o his binding
mode o kinase selec i i y, we easoned ha a ional modifica ions could enhance bo h po ency and
selec i i y wi hin he S c- amily o kinases and p o ide new chemo ype chemically dis inc om ima inib.
Th ough sys ema ic explo a ion o medium-leng h phenoxyme hyl and phenyl amide subs i u ions, we
iden ified a se ies o po en and selec i e ype II Lyn inhibi o s (e.g., TAD-0411662, TAD-0411836, TAD-
0411837), some exhibi ing single-digi nanomola Ki alues and >500- old selec i i y o e Hck, he closes
SFK homolog. Kinome-wide p ofiling confi med high specifici y owa d y osine kinases wi h minimal off-
a ge ac i i y, es ablishing hei sui abili y as in i o pha macological p obes, while also highligh ing
physicochemical p ope ies ha cu en ly limi hei use in in i o animal s udies ha equi ed b ain
pene a ion. These newly de eloped compounds ep esen bes in class Lyn-selec i e ype II inhibi o s
wi hin he SFKs and p o ide pha macological ools and associa ed da a o dissec ing Lyn-dependen
signaling pa hways in AD and o he diseases. Fu u e di ec ions o op imizing physicochemical p ope ies
o sufficien b ain exposu e in i o a e ou lined.
2. BACKGROUND
Lyn kinase, a membe o he S c amily (SFK) o non ecep o y osine kinases, plays a pi o al ole in
ansducing signals downs eam o immune ecep o s, p ima ily h ough he phospho yla ion o
immuno ecep o y osine-based ac i a ion mo i s (ITAMs) and immuno ecep o y osine-based inhibi o y
mo i s (ITIMs).1-3 Al hough he ITAM phospho yla ing unc ion o Lyn is conside ed edundan , as o he
SFK membe s, such as Fyn and Blk, a e capable o compensa ing o his ac i i y in i s absence, he ITIM
phospho yla ing unc ion o Lyn is unique.4,5 This dual unc ionali y enables Lyn o modula e cellula
ac i a ion h esholds and unc ion as a mas e egula o o immune and nonimmune signaling, pa icula ly
in hema opoie ic, ne ous sys em, epi helial, and endoc ine cells.6 S uc u ally, Lyn sha es he conse ed
domain a chi ec u e cha ac e is ic o SFKs, consis ing o an N- e minal SH4 domain ollowed by a unique
egion, SH3 and SH2 egula o y domains, and a ca aly ic SH1 domain.3 Se e al o he non ecep o y osine
kinase amilies, including Tec, FAK, Syk, JAK, and Abl, also exhibi modula a chi ec u es simila o hose o
SFKs.7 Among hese, he Abl amily is he mos closely ela ed o SFKs om bo h s uc u al and egula o y
pe spec i es. Like SFKs, Abl kinases main ain igh con ol o e hei enzyma ic ac i i y h ough
phospho yla ion-dependen ac i a ion and inhibi ion, as well as h ough he o ma ion o closed inac i e
con o ma ions media ed by in amolecula SH3 and SH2 domain in e ac ions. As a esul , SFKs o en
appea in he off- a ge kinase panels o mos ea ly Abl inhibi o de elopmen campaigns.8,9 Dys egula ion
o Lyn kinase, ei he h ough o e exp ession o cons i u i e ac i a ion, has been implica ed in a ious
pa hological condi ions, including au oimmune diseases, hema ological malignancies, solid umo s, and
neu odegene a i e diso de s such as AD.10-15
The exp ession o Lyn in mic oglial inc eases in esponse o amyloid be a (Aβ), a p ima y pa hological
hallma k o AD in humans and a pheno ype eplica ed in mu ine amyloidosis models o AD.16 Inc eased
Lyn exp ession has also been ound in human and mouse mic oglia compa ed o o he SFKs.17
Fu he mo e, pos -mo em AD pa ien b ains ha e also shown ele a ed Lyn le els in mic oglia.10
In e es ingly, he b oad-spec um S c kinase inhibi o PP1 (4-amino-5-(4-me hylphenyl)-7-( -
bu yl)py azolo[3,4-d]py imidine),18 which mainly a ge s Lyn, was shown o inhibi Aβ- igge ed
neu o oxin p oduc ion and imp o e neu onal su i al.10 Addi ionally, independen s udies ha e
documen ed inc eased Lyn ac i a ion in mouse mic oglia exposed o Aβ oligome s, akin o he mic oglia
om he pos -mo em AD pa ien b ains.19,20 In a de ailed s udy by Gwon e al., he ole o Lyn in AD was
explo ed, e ealing i s significan in ol emen in Aβ-induced neu o oxici y and au hype phospho yla ion
ia FcγRIIb2 phospho yla ion.21 The s udy obse ed a apid inc ease in Lyn ac i i y upon exposu e o
oligome ic Aβ1-42 in mouse neu onal cells. They also ound ha Lyn ac i a ion was inhibi ed in FcγRIIb2
knockou neu ons, sugges ing ha FcγRIIb2-Aβ1-42 in e ac ions media ed he ac i a ion o Lyn. An
analysis o AD pa ien b ain issue e ealed a h ee- old inc ease in Lyn's au ophospho yla ion in he
hippocampus compa ed o non-AD con ols. Fu he expe imen a ions confi med he di ec
phospho yla ion o he FcγRIIb2 ITIM a Ty 273 by Lyn upon exposu e o oligome ic Aβ1-42. In e es ingly,
he knockdown o Lyn exp ession significan ly supp essed Aβ1-42-induced cell dea h in neu oblas oma
and hippocampal cell lines, unde sco ing Lyn's ole in coun e ing Aβ1-42-induced neu o oxici y.
Addi ionally, phospho yla ed FcγRIIb2 by Lyn was ound o ec ui SHIP2 o FcγRIIb2, leading o au-
hype phospho yla ion. Las ly, a no el Lyn inhibi o (KICG2576) was shown o lessen Aβ-FcγRIIb2-media ed
neu onal cell dea h and escue Aβ-induced memo y impai men in mice. O e all, his s udy es ablishes
he ole o Lyn in AD in he con ex o Aβ- igge ed neu o oxici y and au hype phospho yla ion, which
a e he main pa hological ea u es o AD.
Despi e inc easing e idence suppo ing he ole o Lyn kinase in a ious disease condi ions, selec i e
inhibi o s a ge ing o Lyn emains an unme need. A p ima y hu dle a ises om he high deg ee o
s uc u al homology wi hin he SFKs, which include Fyn, Hck, Lck, Blk, c-S c, Fg , and Yes.1 These kinases
equen ly co-pa icipa e in immune ecep o signaling cascades, pa icula ly Fyn, Hck, and Lck, making i
essen ial o small-molecule inhibi o s a ge ing Lyn as a he apeu ic s a egy o be highly selec i e o Lyn
o e o he SFKs.22-24 This selec i i y is c i ical o a ibu ing pha macological pheno ypic ou comes o he
unc ion o Lyn. While some exis ing SFK inhibi o s ha a ge Lyn exhibi b oad kinome-wide selec i i y,
hey o en lack in a- amily specifici y and inhibi a b oad spec um o SFKs, limi ing hei u ili y as Lyn-
selec i e chemical p obes.
One a ional s a egy o achie e kinase selec i i y is o exploi inac i e con o ma ional s a es o he
ca aly ic domain.25 In con as o ype I inhibi o s ha bind he ATP-compe i i e ac i e (DFG-in/αC-in)
con o ma ion, ype II inhibi o s engage a DFG-ou inac i e con o ma ion, o en wi h αC-in posi ioning.26
This mode o binding mainly le e ages s uc u al diffe ences in allos e ic pocke s ha a e less conse ed
ac oss kinases, he eby offe ing enhanced selec i i y. In addi ion, kinases diffe in hei in insic
con o ma ional equilib ia and hei p opensi y o sample inac i e s a es, p o iding a unique oppo uni y
o design inhibi o s ha selec i ely s abilize hese less popula ed con o ma ions, as ecen ly demons a ed
by ex ensi e solu ion-phase NMR s uc u e elucida ion s udies.27 While ecen wo k has demons a ed he
easibili y o de eloping ype I1/2 inhibi o s ha p e e en ially bind he DFG-in/αC-ou inac i e
con o ma ion (second known inac i e con o ma ion) o achie e SRC-B sub amily selec i i y, which includes
Lyn, he e a e no epo s in he li e a u e explo ing he ype II inhibi o binding mode o de elop po en
Lyn inhibi o s wi h selec i i y wi hin he SFKs.28 P elimina y da a om ou g oup and o he s sugges ha
ce ain ype II inhibi o s, such as ima inib , masi inib and ba e inib, can po en ly inhibi Lyn kinase wi h
modes selec i i y o e o he SFK membe s.29,30 Gi en he cha ac e is ic biphasic h ee-s ep binding
mechanism o ype II inhibi o s31,32, which in ol es an ini ial con o ma ional selec ion ollowed by physical
binding and an induced fi s ep ha in ol es esidues dis al o he adi ional ATP binding si e, we
hypo hesized ha exis ing ype II inhibi o s could be a ionally modified o imp o e S c amily selec i i y
while e aining o enhancing Lyn po ency.
To his end, as a p oo o concep , we conduc ed a sys ema ic ligand-based op imiza ion o ima inib, a
known ype II kinase inhibi o , o de elop a se ies o no el ype II inhibi o s wi h enhanced Lyn selec i i y
wi hin he SFKs. This effo led o he iden ifica ion o se e al po en de i a i es ha exhibi significan
imp o emen s in in insic selec i i y ac oss he SFK amily, ep esen ing some o he mos selec i e
chemical p obes o Lyn epo ed o da e. He ein, we epo he design a ionale, syn hesis, and biological
e alua ion o hese compounds wi h he objec i e o ca alyzing and suppo ing u he in es iga ion in o
Lyn as a a ge o he ea men o AD.
3. RESULTS AND DISCUSSION
3.1 Inhibi o Design Ra ionale
Ima inib, also known comme cially as “Glee ec” o “Gli ec,” he ma ke ed mesyla e sal o ima inib, is a
po en and selec i e inhibi o o Abl (Bc -Abl) and was fi s epo ed by Zimme mann e al. in 1997.33 I
became he fi s FDA-app o ed kinase inhibi o o ea ing ch onic myelogenous leukemia (CML). The
medicinal chemis y effo ha led o he de elopmen o ima inib began wi h he iden ifica ion o lead
compound (1) om a sc eening campaign o p o ein kinase C (PKC) (se ine/ h eonine kinase) inhibi o s.
Compound 1 ea u es a phenylaminopy imidine scaffold subs i u ed a he 3ʹ-posi ion wi h a 3-py idyl
g oup (Figu e 1).34 Hi - o-lead op imiza ion o his scaffold led o compound 2, in which he 3-posi ion o
he phenyl ing is subs i u ed wi h a benzamide moie y, esul ing in a dual inhibi o o PKC-α and pla ele -
de i ed g ow h ac o ecep o (PDGF-R) wi h low mic omola inhibi o y ac i i y.35 In e es ingly,
in oduc ion o a so called “flag-me hyl g oup” a he 6-posi ion o he b idge phenyl ing o compound 2
comple ely diminished PKCα inhibi o y ac i i y and imp o ed PDGFR inhibi o y ac i i y o a submic omola
le el (IC50 = 0.1 µM), esul ing in he PDGFR-selec i e inhibi o 3.
Figu e 1. E olu ion o he clinically app o ed kinase inhibi o ima inib om a PKC hi (Compound 1). Ini ial SAR s udies
a ound 1 led o he iden ifica ion o he PKCα/PDGF-R dual inhibi o 2. In oduc ion o a “flag-me hyl” subs i u ion
on Compound 2 diminished PKCα ac i i y and enhanced PDGF-R po ency, yielding he PDGF-R selec i e inhibi o 3.
Subsequen SAR effo s explo ed wo majo s uc u al classes, ul ima ely leading o ima inib h ough inco po a ion
o a solubilizing N-me hylpipe azine moie y. (Highligh ed IC50 alues eflec ac i i y agains key kinase a ge s,
demons a ing inc easing selec i i y o PDGF-R and BCR-Abl, along wi h nanomola po ency agains Lyn kinase and
mode a e selec i i y o e Hck).
Subsequen ly, he op imiza ion o his compound as a po en and selec i e inhibi o o -Abl was epo ed,
culmina ing in he disco e y o ima inib.33 The SAR s udies explo ed 3-py idyl, 4-py idyl, and 3-indolyl
subs i u ions a he 3ʹ posi ion o he aminopy imidine ing, along wi h a ious subs i u ions a he 3ʹ
posi ion o he phenyl ing, classi ying he esul ing molecules in o wo s uc u al se ies (classes A and B),
in he p esence o absence o he “flag-me hyl g oup” a he 6-posi ion o he b idge phenyl ing (Figu e
1). Wi hin s uc u al class B, compounds bea ing a 3ʹ-py idyl g oup on he aminopy imidine ing and
a ious benzamide de i a i es a he 3-posi ion o he phenyl ing (as shown in Figu e 1) exhibi ed po en
-Abl inhibi ion wi h submic omola IC50 alues. Among hese, he ini ial lead compound (compound 3),
which showed submic omola PDGF-R inhibi o y ac i i y, also inhibi ed -Abl wi h an IC50 o 0.4 µM,
unc ioning as a dual inhibi o o PDGF-R and -Abl. Howe e , hese ea ly compounds suffe ed om poo
aqueous solubili y and low o al bioa ailabili y.33,34 To add ess hese limi a ions, a highly pola N-
me hylpipe azine moie y was in oduced a he 4-posi ion o he benzamide ing, connec ed ia a space
o he phenyl co e, esul ing in he chemical s uc u e o ima inib. This modifica ion no only imp o ed
aqueous solubili y and o al bioa ailabili y bu also enhanced po ency, educing he -Abl IC50 om 0.4 µM
o 38 nM and he PDGF-R IC50 om 0.1 µM o 50 nM, pa ing he way o he fi s clinically app o ed kinase
inhibi o . In addi ion, i displayed weak inhibi o y ac i i y agains he SFK membe c-S c, wi h an IC50 alue
o 15.7 µM. Ou in-house sc eening o ima inib agains he SFKs e ealed ha i po en ly inhibi s Lyn kinase
in i o, wi h a Ki alue o 119 nM and a 13- old selec i i y o e Hck, he closes ela i e o Lyn in he SFK
phylogene ic ee (Figu e 1).
Figu e 2. (A) Schema ic ep esen a ion o he h ee-s ep, biphasic ype II kinase inhibi o binding mechanism. Binding
p oceeds ia a con o ma ional selec ion (CS) s ep om he ac i e DFG-in o he inac i e DFG-ou con o ma ion,
ollowed by physical binding o o m he ini ial encoun e complex. This is subsequen ly ollowed by an induced fi
(IF) ansi ion, yielding he final inhibi o -bound complex. (B) Mul iple sequence alignmen o ep esen a i e S c
amily kinases (S c (P12931), Fyn (P06241), Hck (P08631), and Lyn (P12931)) om wo s uc u al g oups (SRC-A and
SRC-B), highligh ing key egions ele an o ype II inhibi o binding ha a e dis al o he ATP-binding pocke : he
linke egion (SH2-KD), p-loop, and ac i a ion loop. Residue diffe ences a e depic ed in colo .
Fo yea s, he ema kable selec i i y o ima inib o Abl o e c-S c emained unexplained in e ms o di ec
binding in e ac ions obse ed in X- ay c ys al s uc u es, as he key in e ac ing esidues a e iden ical, and
bo h enzymes adop he DFG-ou inac i e con o ma ion. I was only a e a decade ha he p ecise binding
mechanism o ima inib was elucida ed h ough ex ensi e NMR s udies and p e-s eady-s a e,
nonequilib ium s opped-flow kine ics expe imen s ha moni o ed in insic fluo escence quenching upon
inhibi o binding.7,27,32 The mechanism in ol es a h ee-s ep p ocess31,36 comp ising an ini ial
con o ma ional selec ion s ep, du ing which he kinase exis s in a p e-exis ing equilib ium be ween DFG-
in and DFG-ou con o ma ions (Figu e 2A). This is ollowed by selec i e binding o he inhibi o o he DFG-
ou s a e and subsequen ly by an induced-fi s ep cha ac e ized by a slow con o ma ional ea angemen
o he ac i a ion loop and changes in he p-loop. Acco ding o his mechanism, he expe imen ally
obse ed selec i i y a ises p ima ily om he slow ac i a ion loop ea angemen (induced fi s ep) and
he con o ma ional equilib ium be ween DFG-in and DFG-ou s a es (con o ma ional selec ion), which a e
go e ned by s uc u al ea u es in ol ing less conse ed esidues dis al o he adi ional ATP binding
pocke , a he han by di ec in e ac ions wi h esidues in he ATP binding pocke (Figu e 2B).
Gi en he empi ical e idence o mode a e selec i i y o ima inib o Lyn o e Hck obse ed in ou in-house
alida ion, and he unique h ee-s ep biphasic binding mechanism, which in ol es less conse ed esidues
bo h be ween and wi hin kinase amilies, loca ed dis al o he adi ional ATP binding pocke , we
hypo hesized ha sys ema ic modifica ions o he ima inib scaffold could yield a po en ype II inhibi o
wi h imp o ed selec i i y wi hin he SFKs. Ca e ul e alua ion o he o iginal ima inib SAR s udies desc ibed
abo e indica es ha i was limi ed o a ious subs i u ed benzamides and hei co esponding
he e ocycles a he 3-posi ion o he phenyl ing. As a esul , he ail egion, which in e ac s wi h he
allos e ic pocke and can influence ac i a ion loop dynamics, was es ic ed o ela i ely sho
subs i u ions. The inco po a ion o he N-me hylpipe azine moie y ha ga e ise o ima inib wi h an
ex ended ail egion, was p ima ily a s a egy o imp o e he solubili y and o al bioa ailabili y o he
o iginal se ies.34 In con as , we employed a ligand-based app oach ha modified he ail g oup o
selec i i y. We syn hesized a se ies o de i a i es bea ing ail g oups wi h medium-leng h, a egion o
chemical space ha was no explo ed in he o iginal ima inib SAR s udies, o in es iga e he impac o
hese modifica ions on Lyn inhibi o y ac i i y and selec i i y wi hin he SFKs.
Figu e 3. (A) O e iew o ail egion s uc u al modifica ions explo ed in his s udy. Ini ial modifica ions in ol ed he
in oduc ion o a phenoxyme hyl g oup wi h subs i u ions a he pa a (p), me a (m), and o ho (o) posi ions, yielding
compounds 4-28 and p obing chemical space no p e iously explo ed in he o iginal ima inib SAR. Compounds 29-
37 inco po a e a ious pa a subs i u ed phenyl g oups in he ail egion, e isi ing some epo ed analogs while also
in oducing new de i a i es. Compounds 38-40 ea u ing o ho-addi ions (Y = H, F, Cl) o assess i s impac on po ency
and selec i i y. (B) Gene al syn he ic ou e used o he p epa a ion o compounds 4-40.
As shown in Figu e 3A, we used phenoxyme hyl g oups bea ing diffe en subs i uen s a he pa a (p), me a
(m), and o ho (o) posi ions on he phenyl ing nex o he amide ca bonyl g oup o sys ema ically e alua e
he effec s o hese modifica ions on Lyn inhibi o y ac i i y and selec i i y wi hin he SFKs. The selec ion
o he phenoxyme hyl g oup as he ail moie y was based on wo c i e ia: (a) i posi ions he e minal
phenyl ing wo a oms dis al o he amide ca bonyl, in con as o he di ec ly a ached phenyl ing in he
o iginal ima inib SAR, he eby yielding a ail g oup o in e media e leng h, while p ese ing a molecula
a chi ec u e dis inc om he o iginal ima inib scaffold; and (b) he comme cial a ailabili y o a b oad
ange o subs i u ed phenoxyace ic acid de i a i es enabled he efficien syn hesis o a small, ocused
compound lib a y ia amide coupling wi h a common amine scaffold, affo ding final analogs in good o
excellen yields (Figu e 3B).
We also syn hesized a small se o de i a i es bea ing phenyl g oups wi h diffe en subs i uen s a he pa a
posi ion, di ec ly a ached o he amide ca bonyl g oup, simila o he o iginal ima inib SAR, o e isi some
o he epo ed chemical space and explo e new analogs o e alua ing hei inhibi o y ac i i y agains Lyn
kinase. Wi hin he o iginal SAR s udies, he “flag-me hyl g oup” a he 6-posi ion o he b idge phenyl ing
diminished PKC inhibi o y ac i i y while inc easing PDGF-R inhibi o y ac i i y, indica ing ha hese wo
enzymes a e sensi i e o his subs i u ion.33 Howe e , -Abl inhibi o y ac i i y was no affec ed by he
in oduc ion o he “flag-me hyl g oup,” as e idenced by equipo en inhibi ion o -Abl by bo h compounds
2 and 3 (IC50 = 0.4 µM). This p omp ed us o syn hesize compounds lacking he subs i u ion a his posi ion
as well as wo analogs bea ing Cl and F subs i uen s, espec i ely, o e alua e he impac o subs i u ion
a his posi ion on Lyn inhibi o y ac i i y and selec i i y. These compounds we e syn hesized simila ly o
he phenoxyme hyl de i a i es om comme cially a ailable s a ing ma e ials using an amide coupling
eac ion, as shown in Figu e 3B.
3.2 E alua ion o in i o Lyn Inhibi o y Ac i i y and Selec i i y
Ha ing syn hesized no el Lyn kinase inhibi o s, we nex de e mined hei inhibi o y ac i i y and selec i i y
wi hin he SFKs using he Ho Spo kinase assay, a adiome ic, fil a ion-based me hod widely ega ded as
he 'gold s anda d' o de ec ing kinase ac i i y due o i s high sensi i i y and eliabili y. This assay u ilizes
γ-32P-ATP as he phospha e dono , enabling he di ec measu emen o phospho yla ed subs a e
o ma ion.37 All compounds we e assayed agains ull-leng h Lyn kinase and Hck, wi h Hck selec ed as he
ini ial e e ence kinase o assess in insic selec i i y wi hin he SFK amily. Since Hck is he closes SFK
amily membe o Lyn, acco ding o phylogene ic analysis, we hypo hesized ha achie ing selec i e
Table 1. E alua ion o Lyn/Hck Inhibi ion by ail g oup phenoxyme hyl-subs i u ed analogs (4-28)
p-subs i u ion (R2, R3 = H)
Compound R1 Lyn Ki (nM) Hck Ki (μM) Lyn/Hck
4 (TAD-0411476)
F
330 ± 57
29.4 ± 4.4
88
5 (TAD-0411477)
Cl
275 ± 197
75
273
6 (TAD-0411478)
B
123 ± 21
47.3 ± 9.6
382
7 (TAD-0411659)
I
159 ± 12
8.6 ± 3.9
54
8 (TAD-0411669)
CH
3
377 ± 43
18.7 ± 5.3
50
9 (TAD-0411670)
CF3
115 ± 26
19.9 ± 1.2
174
10 (TAD-0411479)
OCH3
305 ± 88
34.5 ± 4.5
111
11 (TAD-0411671)
OCF3
149 ± 59
38.6
255
12 (TAD-0422249)
NH2
1.7 (μM)
15.0 ± 3.6
9
13 (TAD-0411658)
NO
2
60 (μM)
inac i e
NA
14 (TAD-0411657)
CN
658 ± 105
75
114
m-subs i u ion (R1, R3 = H)
Compound R2 Lyn Ki (nM) Hck Ki (μM) Lyn/Hck
15 (TAD-0411664)
F
152 ± 34
4.2 ± 1.0
28
16 (TAD-0411665)
Cl
254 ± 29
14.6 ± 1.4
57
17 (TAD-0411666)
B
377 ± 40
10.3 ± 3.3
27
18 (TAD-0411668)
CH
3
456 ± 37
13.5 ± 1.1
30
19 (TAD-0411737)
OCH3
6.8 ± 2.5 (μM)
34.9 ± 7.1
5
20 (TAD-0411836)
NO2
732 ± 2.5
14.2 ± 4.3
19
o-subs i u ion (R1, R2 = H)
Compound R3 Lyn Ki (nM) Hck Ki (μM) Lyn/Hck
21 (TAD-0411661)
F
92.5 ± 2.8
2.5
27
22 (TAD-0411662)
Cl
8.7 ± 3.9
0.57 ± 0.03
65
23 (TAD-0411663)
B
30.4 ± 9.2
1.0
33
24 (TAD-0411667)
CH3
34.6 ± 0.7
1.0
29
25 (TAD-0411832)
OCH3
47.6 ± 31.9
3.1 ± 0.3
65
26 (TAD-0411828)
NO2
47.7 ± 11.1
6.5 ± 0.7
136
o,p-subs i u ion (R2 = H)
Compound R1, R3 Lyn Ki (nM) Hck Ki (μM) Lyn/Hck
27 (TAD-0411834)
Cl, CH3
86 ± 23
43 ± 10
500
28 (TAD-0411734)
Cl, Cl
67 ± 8
3.9 ± 0.3
58
IC50 alues we e de e mined using he Ho Spo kinase assay (10 µM ATP) wi h ull-leng h p o ein and con e ed o Ki
alues using he Cheng-P usoff equa ion38 o acili a e di ec compa ison (𝐾𝐾𝑚𝑚
ATP alues: Lyn = 15 μM, Hck = 30 µM).
Values a e epo ed as he a e age o a leas wo independen expe imen s ± SEM. Inhibi o cons an s a e gi en in
nanomola (nM) o Lyn and mic omola (µM) o Hck.
inhibi ion o Lyn o e Hck would ansla e o imp o ed o e all selec i i y wi hin he SFKs. Table 1 and 2
summa izes he appa en Ki alues calcula ed om he IC50 alues, along wi h he Lyn o e Hck selec i i y
p ofiles o he compounds.
3.2.1 Effec o Tail G oup Phenoxyme hyl Subs i u ion on Lyn Inhibi o Ac i i y and
Selec i i y
The fi s -gene a ion o inhibi o s we syn hesized, bea ing pa a-subs i u ed phenoxy me hyl ail g oups
(compounds 4–14), displayed submic omola Lyn inhibi o y ac i i y, wi h some compounds showing
compa able po ency o ima inib, such as compound 6 (B subs i u ion) and 9 (CF3 subs i u ion). S ong
elec on-wi hd awing g oups (EWGs), bo h in e ms o esonance and induc i e effec s, such as ni o and
ni ile (compounds 13 and 14), as well as s ong elec on-dona ing g oups (EDGs) bea ing hyd ogen bond
dono s, such as amines (compound 12) a he pa a posi ion, end o nega i ely impac Lyn inhibi o y
ac i i y. In e es ingly, despi e his obse a ion, halogens, along wi h o he EWGs and EDGs lacking
hyd ogen bond dono s, we e gene ally well ole a ed a his posi ion ega dless o hei size, affo ding
compounds 4–11 wi h good Lyn inhibi o y ac i i y in he submic omola ange (< 400 nM) and imp o ed
Lyn o e Hck selec i i y compa ed o ima inib. No ably, some compounds achie ed excellen selec i i y o
o e 300- old, as obse ed o 6 (B subs i u ion). O e all, hese findings sugges ha subs i u ion a he
pa a posi ion wi h halogens, EWGs, o EDGs lacking hyd ogen bond dono s a e well ole a ed, wi h
minimal s e ic cons ain s, and se es as a key d i e o selec i i y, ei he by modula ing pola in e ac ions
wi hin he DFG-ou ex ended binding pocke o by diffe en ially influencing he ac i a ion loop
con o ma ional change associa ed wi h he induced-fi s ep o he binding mechanism be ween Lyn and
Hck.
Compounds 15–20 ep esen no el Lyn inhibi o s ea u ing me a-subs i u ed phenoxyme hyl ail g oups.
Simila o he fi s -gene a ion, he p esence o s ong EWGs, bo h by esonance and induc i e effec s, such
as ni o (compound 20), weakened Lyn inhibi o y ac i i y, while halogen subs i u ions (F, Cl, and B )
p o ided submic omola Lyn inhibi o y ac i i ies compa able o pa a-subs i u ed analogs, along wi h
mode a e imp o emen s in Lyn o e Hck selec i i y (25- o 60- old) compa ed o ima inib. In e es ingly,
unlike in he case o pa a-subs i u ion, me hoxy-subs i u ion a he me a-posi ion esul ed in educed Lyn
inhibi o y ac i i y. O e all, al hough halogen subs i u ions a he me a-posi ion yielded po en Lyn
inhibi o s, subs i u ion a he me a-posi ion was no a significan modula o o selec i i y, in con as o
he mo e p onounced selec i i y obse ed wi h pa a-subs i u ion.
Inhibi o s bea ing o ho-subs i u ed phenoxyme hyl ail g oups we e he mos success ul among all he
de i a i es, b eaking he submic omola ba ie and eaching low nanomola Lyn inhibi o y po ency. All
compounds, ega dless o he subs i u ion size o elec onic p ope ies, exhibi ed imp o ed Lyn inhibi o y
ac i i y and selec i i y, indica ing ha a wide ange o subs i u ions a e well ole a ed a his posi ion. In
pa icula , chlo ine subs i u ion a he o ho-posi ion (compound 22) yielded single-digi nanomola
po ency, wi h a Ki alue o 8.7 nM and 65- old selec i i y o Lyn o e Hck. Al hough he Lyn o e Hck
selec i i y was no as high as ha obse ed o he pa a-subs i u ed analogs, i was none heless imp o ed
and compa able o ha o he me a-subs i u ed se ies.
g oups, we iden ified a se ies o no el analogs ha exhibi ed low nanomola Lyn inhibi o y ac i i y wi h
significan ly imp o ed selec i i y o e closely ela ed amily membe s such as Hck and Fyn. Biochemical
cha ac e iza ion agains a b oade kinome panel consis ing o selec ed membe s om diffe en kinase
g oups e ealed ha he lead compounds main ained high specifici y o y osine kinases, while also
inhibi ing PDGF-R and c-Ki , simila o ima inib, as wo o he main off- a ge s. E alua ion o
physicochemical and ADME p ope ies highligh ed aqueous solubili y conce ns, along wi h high me abolic
clea ance in mouse mic osomes, pinpoin ing issues associa ed wi h he phenoxyme hyl moie y and i s
me abolism, ac o s ha equi e u he op imiza ion o in i o p obe de elopmen . Howe e , he
excellen MDCK pe meabili y obse ed o he lead compounds sugges s ha hey can s ill be used as in
i o chemical p obes o dissec ing Lyn-specific signaling pa hways in cellula models.
5. METHODS
5.1 Gene al P ocedu es
Unless o he wise no ed, all o he eagen s used in he syn hesis we e ob ained om comme cial sou ces
and used as ecei ed. All sol en s we e pu ified by passage h ough a sol en column composed o
ac i a ed alumina and s o ed unde an a gon o ni ogen a mosphe e. All o he syn heses we e ca ied
ou using flame-d ied glasswa e. No mal phase and e e se phase ch oma og aphy we e pe o med on a
Teledyne-ISCO Nex Gen 300 ins umen using p epacked silica gel o C18- unc ionalized silica gel columns
a ailable om Teledyne-ISCO, o on a Teledyne-ISCO Combiflash using p epacked silica gel columns
a ailable om Agela o Welch. 1H NMR and 13C NMR spec a we e eco ded in DMSO-d6 o CDCl3 as
sol en s on a B uke AVANCE III 500 spec ome e o 400 a 26 °C. Chemical shi s a e epo ed in pa s
pe million (ppm, δ) and e e enced o DMSO-d6 (2.50 ppm o 1H NMR and 39.52 ppm o 13C NMR) o
CDCl3 (7.26 ppm o 1H NMR and 77.2 ppm o 13C NMR). Coupling cons an s (J) a e epo ed in Hz, and
spin mul iplici ies a e desc ibed as s (single ), b (b oad single ), d (double ), ( iple ), q (qua e ), and m
(mul iple ). High- esolu ion mass spec a (HRMS) we e measu ed wi h an Agilen 6210 LC-TOF (ESI,APCI,
APPI) mass spec ome e . Pu i ies o he final compounds we e g ea e han 95%, as de e mined by
e e se-phase HPLC analysis on an Agilen 1260 analy ical HPLC sys em by ollowing me hod: g adien
able C; column Shimadzu Nexcol C18 (5 μm, 50 mm x 3.0 mm); mobile phase A, 1% o mic acid in H2O;
mobile phase B, 1% o mic acid in MeCN; flow a e, 0.5 mL/min; de ec ion wa eleng h, 214 nM; column
empe a u e, 40 °C.
Gene al P ocedu e A (Amide Coupling): To a s i ed solu ion o comme cially a ailable ca boxylic acids
(1.5 eq) in 6 mL DCM we e added CDI, (1.5 eq), and DIPEA (3 eq). The solu ion was allowed o s i a o
1 hou . To he solu ion was hen added 1 eq o he comme cially a ailable amine, 4-amino-2-[4-(3-py idyl)-
2-py imidinylamino] oluene. The solu ion was allowed o s i a o 24 hou s. The solu ion was hen
e apo a ed in acuo and he esul ing esidue was pu ified by flash ch oma og aphy (9:1 DCM:MeOH).
5.2 Syn hesis and Spec al Da a o Compounds 4-40
Compound 4 (TAD-0411476) N-{3-[4-(3-py idyl)-2-py imidinylamino]-4- olyl}(p-
fluo ophenoxy)ace amide
N
H
N
N
N N
H
O
O
F
Compound 4 was syn hesized acco ding o gene al p ocedu e A. (1H NMR: (500 MHz, DMSO) δ 10.04 (s,
1H), 9.29 – 9.23 (m, 1H), 8.96 (s, 1H), 8.69 (dd, J = 4.7, 1.7 Hz, 1H), 8.51 (d, J = 5.1 Hz, 1H), 8.46 (d , J = 8.1,
1.9 Hz, 1H), 7.95 (d, J = 2.2 Hz, 1H), 7.50 (ddd, J = 7.9, 4.7, 0.9 Hz, 1H), 7.43 (d, J = 5.2 Hz, 1H), 7.34 (dd, J =
8.2, 2.2 Hz, 1H), 7.22 – 7.12 (m, 3H), 7.06 – 7.00 (m, 2H), 4.68 (s, 2H), 2.21 (s, 3H). LRMS m/z (ESI+): 430.25
[M+H]+)
Compound 5 (TAD-0411477) N-{3-[4-(3-py idyl)-2-py imidinylamino]-4- olyl}(p-
chlo ophenoxy)ace amide
N
H
N
N
N N
H
O
O
Cl
Compound 5 was syn hesized acco ding o gene al p ocedu e A. (1H NMR: 400 MHz, DMSO δ 10.05 (s, 1H),
9.25 (d, J = 2.3 Hz, 1H), 8.94 (s, 1H), 8.68 (dd, J = 4.8, 1.7 Hz, 1H), 8.50 (d, J = 5.1 Hz, 1H), 8.45 (d , J = 8.1,
2.1 Hz, 1H), 7.94 (d, J = 2.2 Hz, 1H), 7.48 (dd, J = 8.0, 4.8 Hz, 1H), 7.43 (d, J = 5.1 Hz, 1H), 7.39 – 7.29 (m,
3H), 7.18 (d, J = 8.3 Hz, 1H), 7.05 – 7.00 (m, 2H), 4.70 (s, 2H), 2.20 (s, 3H). LRMS m/z (ESI+): 446.15 [M+H]+).
Compound 6 (TAD-0411478) N-{3-[4-(3-py idyl)-2-py imidinylamino]-4- olyl}(p-
b omophenoxy)ace amide
N
H
N
N
N N
H
O
O
B
Compound 6 was syn hesized acco ding o gene al p ocedu e A. (1H NMR: (500 MHz, DMSO) δ 10.07 (s,
1H), 9.26 (d, J = 2.3 Hz, 1H), 8.96 (s, 1H), 8.69 (dd, J = 4.8, 1.7 Hz, 1H), 8.51 (d, J = 5.1 Hz, 1H), 8.46 (d , J =
8.0, 2.0 Hz, 1H), 7.95 (d, J = 2.3 Hz, 1H), 7.51 – 7.45 (m, 3H), 7.43 (d, J = 5.2 Hz, 1H), 7.33 (dd, J = 8.2, 2.2
Hz, 1H), 7.18 (d, J = 8.3 Hz, 1H), 7.02 – 6.95 (m, 2H), 4.71 (s, 2H), 2.21 (s, 3H). LRMS m/z (ESI+): 490.30
[M+H]+)
Compound 7 (TAD-0411659) N-{3-[4-(3-py idyl)-2-py imidinylamino]-4- olyl}(p-
iodophenoxy)ace amide
N
H
N
N
N N
H
O
O
I
Compound 7 was syn hesized acco ding o gene al p ocedu e A. (1H NMR: 400 MHz, DMSO δ 10.06 (s,
1H), 9.26 (d, J = 2.3 Hz, 1H), 8.95 (s, 1H), 8.70 (dd, J = 4.8, 1.7 Hz, 1H), 8.52 (d, J = 5.1 Hz, 1H), 8.46 (d , J =
8.0, 2.0 Hz, 1H), 7.96 (d, J = 2.2 Hz, 1H), 7.67 – 7.60 (m, 2H), 7.48 (dd, J = 8.1, 4.7 Hz, 1H), 7.44 (d, J = 5.1
Hz, 1H), 7.33 (dd, J = 8.1, 2.2 Hz, 1H), 7.19 (d, J = 8.3 Hz, 1H), 6.90 – 6.83 (m, 2H), 4.71 (s, 2H), 2.22 (s,
3H). LRMS m/z (ESI+): 538.20 [M+H]+).
Compound 8 (TAD-0411669) N-{3-[4-(3-py idyl)-2-py imidinylamino]-4- olyl}(p- olyloxy)ace amide
N
H
N
N
N N
H
O
O
Compound 8 was syn hesized acco ding o gene al p ocedu e A. (1H NMR: (500 MHz, DMSO) δ 10.01 (s,
1H), 9.26 (d, J = 2.3 Hz, 1H), 8.95 (s, 1H), 8.69 (dd, J = 4.8, 1.7 Hz, 1H), 8.51 (d, J = 5.2 Hz, 1H), 8.45 (d , J =
8.0, 2.0 Hz, 1H), 7.96 (d, J = 2.2 Hz, 1H), 7.48 (dd, J = 8.0, 4.8 Hz, 1H), 7.43 (d, J = 5.1 Hz, 1H), 7.34 (dd, J =
8.2, 2.2 Hz, 1H), 7.18 (d, J = 8.3 Hz, 1H), 7.11 (d, J = 8.3 Hz, 2H), 6.92 – 6.86 (m, 2H), 4.64 (s, 2H), 2.23 (s,
3H), 2.21 (s, 3H). LRMS m/z (ESI+): 426.20 [M+H]+).
Compound 9 (TAD-0411670) N-{3-[4-(3-py idyl)-2-py imidinylamino]-4- olyl}[p-
( ifluo ome hyl)phenoxy]ace amide
N
H
N
N
N N
H
O
O
CF3
Compound 9 was syn hesized acco ding o gene al p ocedu e A. (1H NMR: (500 MHz, DMSO) δ 10.13 (s,
1H), 9.26 (dd, J = 2.4, 0.9 Hz, 1H), 8.96 (s, 1H), 8.68 (dd, J = 4.9, 1.7 Hz, 1H), 8.51 (d, J = 5.1 Hz, 1H), 8.46
(d , J = 8.0, 2.0 Hz, 1H), 7.96 (d, J = 2.2 Hz, 1H), 7.69 (d, J = 8.8 Hz, 2H), 7.47 (ddd, J = 8.1, 4.8, 0.9 Hz, 1H),
7.43 (d, J = 5.2 Hz, 1H), 7.33 (dd, J = 8.2, 2.2 Hz, 1H), 7.19 (dd, J = 8.7, 2.3 Hz, 3H), 4.83 (s, 2H), 2.21 (s, 3H).
LRMS m/z (ESI+): 480.35 [M+H]+)
Compound 10 (TAD-0411479) N-{3-[4-(3-py idyl)-2-py imidinylamino]-4- olyl}(p-
me hoxyphenoxy)ace amide
N
H
N
N
N N
H
O
O
OCH3
Compound 10 was syn hesized acco ding o gene al p ocedu e A. (1H NMR: (400 MHz, DMSO) δ 9.97 (s,
1H), 9.25 (d, J = 2.2 Hz, 1H), 8.94 (s, 1H), 8.68 (dd, J = 4.9, 1.7 Hz, 1H), 8.50 (d, J = 5.1 Hz, 1H), 8.45 (d , J =
8.0, 2.0 Hz, 1H), 7.95 (d, J = 2.2 Hz, 1H), 7.49 (dd, J = 8.0, 4.7 Hz, 1H), 7.42 (d, J = 5.1 Hz, 1H), 7.34 (dd, J =
8.3, 2.2 Hz, 1H), 7.17 (d, J = 8.3 Hz, 1H), 6.98 – 6.92 (m, 2H), 6.91 – 6.84 (m, 2H), 4.61 (s, 2H), 2.20 (s, 3H).
LRMS m/z (ESI+): 442.35 [M+H]+).
Compound 11 (TAD-0411671) N-{3-[4-(3-py idyl)-2-py imidinylamino]-4- olyl}(p-
ifluo ome hoxyphenoxy)ace amide
N
H
N
N
N N
H
O
O
OCF3
Compound 11 was syn hesized acco ding o gene al p ocedu e A. (1H NMR: (500 MHz, DMSO) δ 10.08 (s,
1H), 9.26 (dd, J = 2.4, 0.8 Hz, 1H), 8.96 (s, 1H), 8.69 (dd, J = 4.7, 1.7 Hz, 1H), 8.51 (d, J = 5.1 Hz, 1H), 8.46
(d , J = 8.0, 2.0 Hz, 1H), 7.96 (d, J = 2.2 Hz, 1H), 7.49 (ddd, J = 8.1, 4.9, 0.9 Hz, 1H), 7.43 (d, J = 5.2 Hz, 1H),
7.36 – 7.29 (m, 3H), 7.19 (d, J = 8.3 Hz, 1H), 7.13 – 7.06 (m, 2H), 4.75 (s, 2H), 2.21 (s, 3H). LRMS m/z (ESI+):
496.30 [M+H]+)
Compound 15 (TAD-0411664) N-{3-[4-(3-py idyl)-2-py imidinylamino]-4- olyl}(m-
fluo ophenoxy)ace amide
N
H
N
N
N N
H
O
O
F
Compound 15 was syn hesized acco ding o gene al p ocedu e A. (1H NMR: 400 MHz, DMSO δ 10.05 (s,
1H), 9.27 (d, J = 2.3 Hz, 1H), 8.96 (s, 1H), 8.69 (dd, J = 4.8, 1.7 Hz, 1H), 8.52 (d, J = 5.2 Hz, 1H), 8.46 (d , J =
8.0, 2.0 Hz, 1H), 7.96 (d, J = 2.2 Hz, 1H), 7.49 (dd, J = 8.0, 4.8 Hz, 1H), 7.44 (d, J = 5.1 Hz, 1H), 7.39 – 7.29
(m, 2H), 7.19 (d, J = 8.3 Hz, 1H), 6.95 – 6.74 (m, 3H), 4.74 (s, 2H), 2.22 (s, 3H). LRMS m/z (ESI+): 430.40
[M+H]+).
Compound 16 (TAD-0411665) N-{3-[4-(3-py idyl)-2-py imidinylamino]-4- olyl}(m-
chlo ophenoxy)ace amide
N
H
N
N
N N
H
O
O
Cl
Compound 16 was syn hesized acco ding o gene al p ocedu e A. (1H NMR: 400 MHz, DMSO δ 10.05 (s,
1H), 9.27 (d, J = 2.3 Hz, 1H), 8.96 (s, 1H), 8.70 (dd, J = 4.8, 1.6 Hz, 1H), 8.52 (d, J = 5.1 Hz, 1H), 8.47 (d , J =
8.0, 2.0 Hz, 1H), 7.96 (d, J = 2.2 Hz, 1H), 7.50 (dd, J = 8.0, 4.8 Hz, 1H), 7.44 (d, J = 5.1 Hz, 1H), 7.35 (ddd, J =
8.2, 5.2, 3.0 Hz, 2H), 7.20 (d, J = 8.3 Hz, 1H), 7.12 ( , J = 2.2 Hz, 1H), 7.02 (ddd, J = 17.9, 8.1, 2.2 Hz, 2H),
4.75 (s, 2H), 2.22 (s, 3H).LRMS m/z (ESI+): 446.30 [M+H]+).
Compound 17 (TAD-0411666) N-{3-[4-(3-py idyl)-2-py imidinylamino]-4- olyl}(m-
b omophenoxy)ace amide
N
H
N
N
N N
H
O
O
B
Compound 17 was syn hesized acco ding o gene al p ocedu e A. (1H NMR: (500 MHz, DMSO) δ 10.05 (s,
1H), 9.26 (d, J = 2.3 Hz, 1H), 8.95 (s, 1H), 8.69 (dd, J = 4.8, 1.6 Hz, 1H), 8.51 (d, J = 5.1 Hz, 1H), 8.46 (d , J =
8.1, 2.0 Hz, 1H), 7.96 (d, J = 2.2 Hz, 1H), 7.48 (dd, J = 8.1, 4.8 Hz, 1H), 7.43 (d, J = 5.2 Hz, 1H), 7.34 (dd, J =
8.1, 2.2 Hz, 1H), 7.30 – 7.22 (m, 2H), 7.22 – 7.15 (m, 2H), 7.06 – 7.00 (m, 1H), 4.74 (s, 2H), 2.21 (s, 3H).
LRMS m/z (ESI+): 490.35 [M+H]+).
Compound 18 (TAD-0411668) N-{3-[4-(3-py idyl)-2-py imidinylamino]-4- olyl}(m- olyloxy)ace amide
N
H
N
N
N N
H
O
O
Compound 18 was syn hesized acco ding o gene al p ocedu e A. (1H NMR: (500 MHz, DMSO) δ 10.01 (s,
1H), 9.26 (d, J = 2.2 Hz, 1H), 8.95 (s, 1H), 8.69 (dd, J = 4.8, 1.6 Hz, 1H), 8.51 (d, J = 5.1 Hz, 1H), 8.46 (d , J =
8.0, 2.0 Hz, 1H), 7.96 (d, J = 2.2 Hz, 1H), 7.49 (dd, J = 8.0, 4.8 Hz, 1H), 7.43 (d, J = 5.1 Hz, 1H), 7.34 (dd, J =
8.2, 2.2 Hz, 1H), 7.18 (d , J = 7.8, 3.7 Hz, 2H), 6.84 ( , J = 2.0 Hz, 1H), 6.79 (dd, J = 8.4, 2.4 Hz, 2H), 4.66 (s,
2H), 2.28 (s, 3H), 2.21 (s, 3H). LRMS m/z (ESI+): 426.20 [M+H]+).
Compound 19 TAD-0411737 N-{3-[4-(3-py idyl)-2-py imidinylamino]-4- olyl}(m-
me hoxyphenoxy)ace amide
N
H
N
N
N N
H
O
O
OCH3
Compound 19 was syn hesized acco ding o gene al p ocedu e A. (1H NMR: 400 MHz, DMSO δ 10.03 (s,
1H), 9.26 (d, J = 2.2 Hz, 1H), 8.96 (s, 1H), 8.69 (dd, J = 4.8, 1.6 Hz, 1H), 8.52 (d, J = 5.1 Hz, 1H), 8.46 (d , J =
8.0, 2.0 Hz, 1H), 7.96 (d, J = 2.1 Hz, 1H), 7.49 (dd, J = 8.0, 4.8 Hz, 1H), 7.44 (d, J = 5.2 Hz, 1H), 7.35 (dd, J =
8.3, 2.2 Hz, 1H), 7.21 ( d, J = 8.4, 3.9 Hz, 2H), 6.61 – 6.55 (m, 3H), 4.68 (s, 2H), 3.74 (s, 3H), 2.21 (s, 3H).
LRMS m/z (ESI+): 442.20 [M+H]+).
Compound 20 (TAD-0411836) (N-(4-me hyl-3-((4-(py idin-3-yl)py imidin-2-yl)amino)phenyl)-2-(3-
ni ophenoxy)ace amide)
N
H
N
N
N N
H
O
O
NO2
Compound 20 was syn hesized acco ding o gene al p ocedu e A. (1H NMR: (500 MHz, DMSO) δ 10.14 (s,
1H), 9.26 (d, J = 2.3 Hz, 1H), 8.97 (s, 1H), 8.68 (dd, J = 4.8, 1.7 Hz, 1H), 8.51 (d, J = 5.1 Hz, 1H), 8.46 (d , J =
8.0, 2.0 Hz, 1H), 7.95 (d, J = 2.2 Hz, 1H), 7.89 – 7.82 (m, 2H), 7.62 ( , J = 8.2 Hz, 1H), 7.52 – 7.47 (m, 2H),
7.44 (d, J = 5.1 Hz, 1H), 7.34 (dd, J = 8.2, 2.2 Hz, 1H), 7.20 (d, J = 8.3 Hz, 1H), 4.88 (s, 2H), 2.21 (s, 3H). LRMS
m/z (ESI+): 457.15 [M+H]+)
Compound 21 (TAD-0411661) N-{3-[4-(3-py idyl)-2-py imidinylamino]-4- olyl}(o-
fluo ophenoxy)ace amide
N
H
N
N
N N
H
O
O
F
Compound 21 was syn hesized acco ding o gene al p ocedu e A. (1H NMR: 400 MHz, DMSO δ 10.13 (s,
1H), 9.26 (d, J = 2.3 Hz, 1H), 8.96 (s, 1H), 8.70 (dd, J = 4.8, 1.7 Hz, 1H), 8.52 (d, J = 5.2 Hz, 1H), 8.47 (d , J =
8.1, 2.0 Hz, 1H), 7.95 (d, J = 2.2 Hz, 1H), 7.49 (dd, J = 8.0, 4.8 Hz, 1H), 7.44 (d, J = 5.1 Hz, 1H), 7.32 (dd, J =
8.2, 2.2 Hz, 1H), 7.29 – 7.22 (m, 1H), 7.19 (d, J = 8.3 Hz, 1H), 7.16 – 7.10 (m, 2H), 6.98 (ddd, J = 11.5, 6.4,
3.7 Hz, 1H), 4.81 (s, 2H), 2.22 (s, 3H). LRMS m/z (ESI+): 430.25 [M+H]+).
Compound 22 (TAD-0411662) N-{3-[4-(3-py idyl)-2-py imidinylamino]-4- olyl}(o-
chlo ophenoxy)ace amide
N
H
N
N
N N
H
O
O
Cl
Compound 22 was syn hesized acco ding o gene al p ocedu e A. (1H NMR: (500 MHz, DMSO) δ 10.11 (s,
1H), 9.25 (d, J = 2.3 Hz, 1H), 8.95 (s, 1H), 8.69 (dd, J = 4.8, 1.7 Hz, 1H), 8.51 (d, J = 5.1 Hz, 1H), 8.46 (d , J =
8.0, 2.0 Hz, 1H), 7.95 (d, J = 2.2 Hz, 1H), 7.50 – 7.41 (m, 3H), 7.33 – 7.26 (m, 2H), 7.19 (d, J = 8.3 Hz, 1H),
7.09 (dd, J = 8.3, 1.4 Hz, 1H), 6.99 ( d, J = 7.6, 1.4 Hz, 1H), 4.83 (s, 2H), 2.21 (s, 3H). LRMS m/z (ESI+): 446.25
[M+H]+).
Compound 23 (TAD-0411663) N-{3-[4-(3-py idyl)-2-py imidinylamino]-4- olyl}(o-
b omophenoxy)ace amide
N
H
N
N
N N
H
O
O
B
Compound 23 was syn hesized acco ding o gene al p ocedu e A. (1H NMR: 400 MHz, DMSO δ 10.05 (s,
1H), 9.26 (d, J = 2.3 Hz, 1H), 8.96 (s, 1H), 8.70 (dd, J = 4.7, 1.6 Hz, 1H), 8.52 (d, J = 5.1 Hz, 1H), 8.47 (d , J =
8.0, 2.0 Hz, 1H), 7.95 (d, J = 2.4 Hz, 1H), 7.62 (dd, J = 7.9, 1.6 Hz, 1H), 7.49 (dd, J = 8.0, 4.8 Hz, 1H), 7.44 (d,
J = 5.2 Hz, 1H), 7.38 – 7.31 (m, 2H), 7.20 (d, J = 8.3 Hz, 1H), 7.11 – 7.05 (m, 1H), 6.93 ( d, J = 7.6, 1.3 Hz,
1H), 4.83 (s, 2H), 2.22 (s, 3H).LRMS m/z (ESI+): 490.20 [M+H]+).
Compound 24 (TAD-0411667) N-{3-[4-(3-py idyl)-2-py imidinylamino]-4- olyl}(o- olyloxy)ace amide
N
H
N
N
N N
H
O
O
Compound 24 was syn hesized acco ding o gene al p ocedu e A. (1H NMR: (500 MHz, DMSO) δ 10.01 (s,
1H), 9.25 (d, J = 2.3 Hz, 1H), 8.95 (s, 1H), 8.69 (dd, J = 4.8, 1.7 Hz, 1H), 8.51 (d, J = 5.2 Hz, 1H), 8.46 (d , J =
8.1, 2.0 Hz, 1H), 7.95 (d, J = 2.2 Hz, 1H), 7.48 (dd, J = 8.0, 4.8 Hz, 1H), 7.43 (d, J = 5.2 Hz, 1H), 7.32 (dd, J =
8.2, 2.2 Hz, 1H), 7.21 – 7.10 (m, 3H), 6.87 ( , J = 7.8 Hz, 2H), 4.71 (s, 2H), 2.24 (s, 3H), 2.21 (s, 3H). LRMS
m/z (ESI+): 426.25 [M+H]+).
Compound 25 (TAD-0411832) N-{3-[4-(3-py idyl)-2-py imidinylamino]-4- olyl}(o-
me hoxyphenoxy)ace amide
N
H
N
N
N N
H
O
O
O
Compound 25 was syn hesized acco ding o gene al p ocedu e A. (1H NMR: (500 MHz, DMSO) δ 10.01 (s,
1H), 9.26 (d, J = 2.2 Hz, 1H), 8.96 (s, 1H), 8.69 (dd, J = 4.8, 1.7 Hz, 1H), 8.51 (d, J = 5.2 Hz, 1H), 8.46 (d , J =
8.0, 2.0 Hz, 1H), 7.96 (d, J = 2.4 Hz, 1H), 7.49 (dd, J = 8.0, 4.8 Hz, 1H), 7.43 (d, J = 5.2 Hz, 1H), 7.33 (dd, J =
8.1, 2.3 Hz, 1H), 7.19 (d, J = 8.3 Hz, 1H), 7.04 – 6.93 (m, 3H), 6.88 ( d, J = 7.6, 1.6 Hz, 1H), 4.68 (s, 2H), 3.80
(s, 3H), 2.21 (s, 3H). LRMS m/z (ESI+): 442.20 [M+H]+)
Compound 26 (TAD-0411828) N-{3-[4-(3-py idyl)-2-py imidinylamino]-4- olyl}(o-
ni ophenoxy)ace amide
N
H
N
N
N N
H
O
O
O2N
Compound 26 was syn hesized acco ding o gene al p ocedu e A. (1H NMR: (500 MHz, DMSO) δ 10.04 (s,
1H), 9.25 (dd, J = 2.3, 0.8 Hz, 1H), 8.96 (s, 1H), 8.69 (dd, J = 4.9, 1.7 Hz, 1H), 8.51 (d, J = 5.1 Hz, 1H), 8.46
(d , J = 8.1, 2.0 Hz, 1H), 7.97 – 7.90 (m, 2H), 7.66 (ddd, J = 8.9, 7.4, 1.7 Hz, 1H), 7.49 (ddd, J = 8.1, 4.8, 0.9
Hz, 1H), 7.44 (d, J = 5.1 Hz, 1H), 7.31 (ddd, J = 16.3, 8.4, 1.7 Hz, 2H), 7.20 (d, J = 8.3 Hz, 1H), 7.16 ( d, J =
7.7, 7.3, 1.1 Hz, 1H), 4.95 (s, 2H), 2.21 (s, 3H). LRMS m/z (ESI+): 457.55 [M+H]+)
Compound 27 (TAD-0411834) N-{3-[4-(3-py idyl)-2-py imidinylamino]-4- olyl}(4-chlo o-2-
olyloxy)ace amide
N
H
N
N
N N
H
O
O
Cl
Compound 27 was syn hesized acco ding o gene al p ocedu e A. (1H NMR: (500 MHz, DMSO) δ 10.04 (s,
1H), 9.26 (d, J = 2.3 Hz, 1H), 8.96 (s, 1H), 8.69 (dd, J = 4.8, 1.7 Hz, 1H), 8.51 (d, J = 5.1 Hz, 1H), 8.46 (d , J =
8.0, 2.0 Hz, 1H), 7.93 (d, J = 2.3 Hz, 1H), 7.48 (dd, J = 8.0, 4.8 Hz, 1H), 7.43 (d, J = 5.2 Hz, 1H), 7.31 (dd, J =
8.2, 2.2 Hz, 1H), 7.25 (d, J = 2.7 Hz, 1H), 7.22 – 7.17 (m, 2H), 6.90 (d, J = 8.7 Hz, 1H), 4.74 (s, 2H), 2.23 (s,
3H), 2.21 (s, 3H). LRMS m/z (ESI+): 460.15 [M+H]+)
Compound 28 (TAD-0411734) N-{3-[4-(3-py idyl)-2-py imidinylamino]-4- olyl}(2,4-
dichlo ophenoxy)ace amide
N
H
N
N
N N
H
O
O
Cl Cl
Compound 28 was syn hesized acco ding o gene al p ocedu e A. (1H NMR: 400 MHz, DMSO δ 10.12 (s,
1H), 9.26 (d, J = 2.3 Hz, 1H), 8.95 (s, 1H), 8.70 (dd, J = 4.8, 1.6 Hz, 1H), 8.52 (d, J = 5.1 Hz, 1H), 8.47 (d , J =
8.1, 2.0 Hz, 1H), 7.94 (d, J = 2.2 Hz, 1H), 7.61 (d, J = 2.6 Hz, 1H), 7.49 (dd, J = 8.0, 4.8 Hz, 1H), 7.44 (d, J =
5.2 Hz, 1H), 7.38 (dd, J = 8.9, 2.6 Hz, 1H), 7.30 (dd, J = 8.2, 2.2 Hz, 1H), 7.19 (d, J = 8.3 Hz, 1H), 7.13 (d, J =
9.0 Hz, 1H), 4.87 (s, 2H), 2.22 (s, 3H). LRMS m/z (ESI+): 480.20 [M+H]+).
Compound 29 (TAD-0411728) N-{3-[4-(3-py idyl)-2-py imidinylamino]-4- olyl}p-fluo obenzamide
N
H
N
N
N N
H
O
F
Compound 29 was syn hesized acco ding o gene al p ocedu e A. (1H NMR: 400 MHz, DMSO δ 10.23 (s,
1H), 9.29 (d, J = 2.2 Hz, 1H), 8.98 (s, 1H), 8.70 (dd, J = 4.8, 1.7 Hz, 1H), 8.53 (d, J = 5.1 Hz, 1H), 8.49 (d , J =
8.1, 2.0 Hz, 1H), 8.08 (d, J = 2.2 Hz, 1H), 8.07 – 8.02 (m, 2H), 7.53 (dd, J = 8.0, 4.8 Hz, 1H), 7.48 (dd, J = 8.2,
2.2 Hz, 1H), 7.44 (d, J = 5.1 Hz, 1H), 7.41 – 7.34 (m, 2H), 7.23 (d, J = 8.2 Hz, 1H), 2.24 (s, 3H). LRMS m/z
(ESI+): 400.20 [M+H]+).
Compound 30 (TAD-0411729) N-{3-[4-(3-py idyl)-2-py imidinylamino]-4- olyl}p-chlo obenzamide
N
H
N
N
N N
H
O
Cl
Compound 30 was syn hesized acco ding o gene al p ocedu e A. (1H NMR: 400 MHz, DMSO δ 10.28 (s,
1H), 9.29 (d, J = 2.2 Hz, 1H), 8.99 (s, 1H), 8.70 (d, J = 4.6 Hz, 1H), 8.53 (d, J = 5.2 Hz, 1H), 8.49 (d, J = 8.3 Hz,
1H), 8.09 (s, 1H), 8.00 (d, J = 8.5 Hz, 2H), 7.62 (d, J = 8.5 Hz, 2H), 7.53 (dd, J = 8.0, 4.8 Hz, 1H), 7.48 (d, J =
8.3 Hz, 1H), 7.45 (d, J = 5.1 Hz, 1H), 7.23 (d, J = 8.3 Hz, 1H), 2.24 (s, 3H).LRMS m/z (ESI+): 416.25 [M+H]+).
Compound 31 (TAD-0411730) N-{3-[4-(3-py idyl)-2-py imidinylamino]-4- olyl}p-b omobenzamide
N
H
N
N
N N
H
O
B
Compound 31 was syn hesized acco ding o gene al p ocedu e A. (1H NMR: 400 MHz, DMSO δ 10.28 (s,
1H), 9.29 (d, J = 2.3 Hz, 1H), 8.98 (s, 1H), 8.70 (dd, J = 4.8, 1.7 Hz, 1H), 8.53 (d, J = 5.1 Hz, 1H), 8.51 – 8.47
(m, 1H), 8.09 (d, J = 2.1 Hz, 1H), 7.92 (d, J = 8.5 Hz, 2H), 7.76 (d, J = 8.5 Hz, 2H), 7.53 (dd, J = 8.0, 4.8 Hz,
FUNDING SOURCES
This wo k was p ima ily suppo ed by NIA g an U54AG065181 (Palkowi z, Lamb, Richa dson) o he IUSM
Pu due TREAT-AD Cen e .
KEY WORDS
Lyn, S c, Alzheime ’s disease, Type II Kinase Inhibi o s
DATA AVAILABILITY
Da ase s will be made a ailable ia he AD Knowledge Po al.
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