Ci a ion: del Co e, X.; Maes o, A.;
López-F ancés, A.; Palacios, F.;
Vica io, J. Syn hesis o
Te asubs i u ed Phospho us Analogs
o Aspa ic Acid as An ip oli e a i e
Agen s. Molecules 2022,27, 8024.
h ps://doi.o g/10.3390/
molecules27228024
Academic Edi o : Jakub Adamek
Recei ed: 16 Sep embe 2022
Accep ed: 15 No embe 2022
Published: 18 No embe 2022
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
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Copy igh : © 2022 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
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A ibu ion (CC BY) license (h ps://
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molecules
A icle
Syn hesis o Te asubs i u ed Phospho us Analogs o Aspa ic
Acid as An ip oli e a i e Agen s
Xabie del Co e †, Ai o Maes o †, Ad ián López-F ancés , F ancisco Palacios and Ja ie Vica io *
Depa amen o de Química O gánica I, Cen o de In es igación y Es udios A anzados “Lucio Lasca ay”,
Facul ad de Fa macia, Uni e si y o he Basque Coun y, UPV/EHU Paseo de la Uni e sidad 7,
01006 Vi o ia-Gas eiz, Spain
*Co espondence: ja ie [email p o ec ed]; Tel.: +34-945013891
† These au ho s con ibu ed equally o his wo k.
Abs ac :
An e icien gene al me hod o he syn hesis o a wide amily o
α
-aminophosphona e
analogs o aspa ic acid bea ing e asubs i u ed ca bons is epo ed h ough an aza-Re o ma sky
eac ion o
α
-iminophosphona es, gene a ed om
α
-aminophosphona es, in an umpolung p ocess.
In addi ion, he
α
-aminophosphona e subs a es showed
in i o
cy o oxici y, inhibi ing he g ow h
o ca cinoma human umo cell lines A549 (ca cinomic human al eola basal epi helial cell) and
SKOV3 (human o a ian ca cinoma). In iew o he possibili ies in he di e si y o he subs i uen s
ha o e he syn he ic me hodology, an ex ensi e p o ile s uc u e–ac i i y is p esen ed, measu ing
IC50 alues up o 0.34 µM in he A549 and 9.8 µM in SKOV3 cell lines.
Keywo ds:
Re o ma sky eac ion; e asubs i u ed
α
-aminophosphona es; aspa ic acid; an ip oli e a i e
e ec
1. In oduc ion
The inc ease in li e expec ancy is one o he g ea es achie emen s o humankind,
which is also linked o a - eaching consequences wi h implica ions o nea ly all socioe-
conomic sec o s [
1
]. Acco ding o a s udy published in The Lance in 2018 [
2
], by 2040, i
is expec ed ha 59 coun ies will ha e an a e age li e expec ancy o mo e han 80 yea s.
In pa icula , he a e age li e expec ancy in Spain is p edic ed o be he highes in he
wo ld and will each 85.8 yea s. In he pas , he main oo s o mo ali y we e associa ed o
in ec ious and pa asi ic diseases bu , due o he phenomenon o popula ion ageing, ch onic
and degene a i e diseases ha e become he main conce n o all heal hca e sys ems wo ld-
wide. Acco dingly, cance s igu e among he leading causes o mo bidi y and mo ali y
wo ldwide and ha e become one o he wo ld’s la ges heal h p oblems [3].
The sys emic ea men o cance implies a combina ion o su ge y, chemo he apy
and adia ion. O he op ions include immuno he apy, a ge ed he apy, lase o ho monal
he apy [
4
]. Chemo he apeu ic agen s possess he abili y o a el h oughou he body,
and selec i ely des oy as -g owing malignan cells [
5
]. He e, in he i s on o ba le,
d ug disco e y plays a c ucial ole in his a ea h ough he syn hesis and cha ac e iza ion o
d ug candida es and he e alua ion o hei an icance p ope ies, p io o he subsequen
clinical ials. Despi e he s ong e o s made in he las decades o he de elopmen o
e icien chemo he apeu ic agen s, he con inuous sea ch o newe , sa e and mo e po en
cy o oxic d ugs is an essen ial ask in science, especially due o he known abili y o cance
cells o de elop esis ance o he known he apies [6,7].
Among he innume able amoun o po en ially chemo he apeu ic molecules, we
ocused, in his case, on o ganophospho us compounds. In pa icula , phosphonic acids
and hei es e s a e a amily o compounds, cha ac e ized by he p esence o a s able C-P
bond in hei s uc u e, ha show in e es ing and use ul biological p ope ies [
8
], including
an icance ac i i y, such as he case o cyclophosphamide [
9
] o zolend ona e [
10
,
11
].
Molecules 2022,27, 8024. h ps://doi.o g/10.3390/molecules27228024 h ps://www.mdpi.com/jou nal/molecules
Molecules 2022,27, 8024 2 o 22
Speci ically,
α
-aminophosphonic acids a e bioisos e s o
α
-amino acids, whe e he la
ca boxylic acid g oup has been eplaced by a e ahed al phosphonic acid g oup ha shows
he ully oxidized phospho us a om a he co e [
12
]. Due o his isos e ic subs i u ion,
α
-aminophosphonic acid sca old is able o mimic he e ahed al geome y and nega i e
cha ge de elopmen ound in he ansi ion s a e o pep ide clea age, hus inhibi ing
enzymes implied in p o eolysis p ocesses (Figu e 1) [
13
–
15
]. Conside ing his, i is easy o
an icipa e ha a numbe o
α
-aminophosphonic acid de i a i es show in e es ing biological
ac i i ies, such as he bicidal [
16
,
17
], an imic obial [
18
–
21
] o an ioxidan [
22
,
23
], and some
o hem ha e been epo ed as po en ial d ugs, in pa icula , o he ea men o in ec ious
diseases [
24
,
25
]. Rema kably, some
α
-aminophosphona e de i a i es ha e been desc ibed
as an icance [26–29] agen s.
Molecules2022,27,xFORPEERREVIEW2o 23
bondin hei s uc u e, ha showin e es inganduse ulbiologicalp ope ies[8],
includingan icance ac i i y,suchas hecaseo cyclophosphamide[9]o zolend ona e
[10,11].Speci ically,α‐aminophosphonicacidsa ebioisos e so α‐aminoacids,whe e he
la ca boxylicacidg ouphasbeen eplacedbya e ahed alphosphonicacidg oup ha
shows he ullyoxidizedphospho usa oma heco e[12].Due o hisisos e ic
subs i u ion,α‐aminophosphonicacidsca oldisable omimic he e ahed algeome y
andnega i echa gede elopmen oundin he ansi ions a eo pep ideclea age, hus
inhibi ingenzymesimpliedinp o eolysisp ocesses(Figu e1)[13–15].Conside ing his,
i iseasy oan icipa e ha anumbe o α‐aminophosphonicacidde i a i esshow
in e es ingbiologicalac i i ies,suchashe bicidal[16,17],an imic obial[18–21]o
an ioxidan [22,23],andsomeo hemha ebeen epo edaspo en iald ugs,inpa icula ,
o he ea men o in ec iousdiseases[24,25].Rema kably,someα‐aminophosphona e
de i a i esha ebeendesc ibedasan icance [26–29]agen s.
Figu e1.α‐Aminophosphonicacidg oupmimics he ansi ions a eo pep ideclea age.
Aspa icacidisoneo he20buildingblockα‐aminoacidso p o eins ha isknown
oha epha macologicalac i i ya someglu ama e ecep o s[30].Themos in e es ing
ea u eo hes uc u eo aspa icacidis hep esenceo asecondacidicsidechain ha
mayin e ac wi ho he aminoacids,enzymeso p o einsin hebody.In hecon ex o
his esea ch, ollowing hebiosisos e ismapp oach, wopossiblephospho a edanalogs
maybedesigned omaspa icacidsca oldI:β‐phospho yla edα‐aminoacidsIIo β‐
ca boxylicα‐aminophosphona esIII(Scheme1).
Scheme1.Twopossibleisos e icsubs i u ionso heca boxylicmoie ybyaphosphonicacidg oup
inaspa icacidsca old.
While hesyn hesiso phosphona eanalogso aspa icacidby heisos e ic
subs i u iono heβ‐ca boxylicg oupiswelldocumen ed[31–33], hep epa a iono he
pa en α‐aminophosphona eanalogsIIIby hesubs i u iono heα‐ca boxylicmoie yhas
ecei edlessa en ion,andmos o hesubs a esa e epo edassingleexampleso
gene alme hodsleading oα‐aminophosphona eso aspa icacidde i a i es[32,34,35].
Inpa icula , he ea ea ewexamples epo ed o hesyn hesiso e asubs i u edα‐
aminophosphona esde i ed omaspa icacidasconc e eexampleso hescopeo
eac ions ha implyC‐Co C‐Pbond o ma ion[36–39].I iswellknown ha he
de elopmen o eac ionsleading o he o ma iono e asubs i u edca bonsisa
challenging ask,due o helacko eac i i yo hesubs a es,de i ed om he
gene a iono ahighlyc owdeds uc u e.
Figu e 1. α-Aminophosphonic acid g oup mimics he ansi ion s a e o pep ide clea age.
Aspa ic acid is one o he 20 building block
α
-amino acids o p o eins ha is known
o ha e pha macological ac i i y a some glu ama e ecep o s [
30
]. The mos in e es ing
ea u e o he s uc u e o aspa ic acid is he p esence o a second acidic side chain ha
may in e ac wi h o he amino acids, enzymes o p o eins in he body. In he con ex o his
esea ch, ollowing he biosisos e ism app oach, wo possible phospho a ed analogs may be
designed om aspa ic acid sca old I:β-phospho yla ed α-aminoacids II o β-ca boxylic
α-aminophosphona es III (Scheme 1).
Molecules2022,27,xFORPEERREVIEW2o 23
bondin hei s uc u e, ha showin e es inganduse ulbiologicalp ope ies[8],
includingan icance ac i i y,suchas hecaseo cyclophosphamide[9]o zolend ona e
[10,11].Speci ically,α‐aminophosphonicacidsa ebioisos e so α‐aminoacids,whe e he
la ca boxylicacidg ouphasbeen eplacedbya e ahed alphosphonicacidg oup ha
shows he ullyoxidizedphospho usa oma heco e[12].Due o hisisos e ic
subs i u ion,α‐aminophosphonicacidsca oldisable omimic he e ahed algeome y
andnega i echa gede elopmen oundin he ansi ions a eo pep ideclea age, hus
inhibi ingenzymesimpliedinp o eolysisp ocesses(Figu e1)[13–15].Conside ing his,
i iseasy oan icipa e ha anumbe o α‐aminophosphonicacidde i a i esshow
in e es ingbiologicalac i i ies,suchashe bicidal[16,17],an imic obial[18–21]o
an ioxidan [22,23],andsomeo hemha ebeen epo edaspo en iald ugs,inpa icula ,
o he ea men o in ec iousdiseases[24,25].Rema kably,someα‐aminophosphona e
de i a i esha ebeendesc ibedasan icance [26–29]agen s.
Figu e1.α‐Aminophosphonicacidg oupmimics he ansi ions a eo pep ideclea age.
Aspa icacidisoneo he20buildingblockα‐aminoacidso p o eins ha isknown
oha epha macologicalac i i ya someglu ama e ecep o s[30].Themos in e es ing
ea u eo hes uc u eo aspa icacidis hep esenceo asecondacidicsidechain ha
mayin e ac wi ho he aminoacids,enzymeso p o einsin hebody.In hecon ex o
his esea ch, ollowing hebiosisos e ismapp oach, wopossiblephospho a edanalogs
maybedesigned omaspa icacidsca oldI:β‐phospho yla edα‐aminoacidsIIo β‐
ca boxylicα‐aminophosphona esIII(Scheme1).
Scheme1.Twopossibleisos e icsubs i u ionso heca boxylicmoie ybyaphosphonicacidg oup
inaspa icacidsca old.
While hesyn hesiso phosphona eanalogso aspa icacidby heisos e ic
subs i u iono heβ‐ca boxylicg oupiswelldocumen ed[31–33], hep epa a iono he
pa en α‐aminophosphona eanalogsIIIby hesubs i u iono heα‐ca boxylicmoie yhas
ecei edlessa en ion,andmos o hesubs a esa e epo edassingleexampleso
gene alme hodsleading oα‐aminophosphona eso aspa icacidde i a i es[32,34,35].
Inpa icula , he ea ea ewexamples epo ed o hesyn hesiso e asubs i u edα‐
aminophosphona esde i ed omaspa icacidasconc e eexampleso hescopeo
eac ions ha implyC‐Co C‐Pbond o ma ion[36–39].I iswellknown ha he
de elopmen o eac ionsleading o he o ma iono e asubs i u edca bonsisa
challenging ask,due o helacko eac i i yo hesubs a es,de i ed om he
gene a iono ahighlyc owdeds uc u e.
Scheme 1.
Two possible isos e ic subs i u ions o he ca boxylic moie y by a phosphonic acid g oup
in aspa ic acid sca old.
While he syn hesis o phosphona e analogs o aspa ic acid by he isos e ic subs i u-
ion o he
β
-ca boxylic g oup is well documen ed [
31
–
33
], he p epa a ion o he pa en
α
-aminophosphona e analogs
III
by he subs i u ion o he
α
-ca boxylic moie y has ecei ed
less a en ion, and mos o he subs a es a e epo ed as single examples o gene al me hods
leading o
α
-aminophosphona es o aspa ic acid de i a i es [
32
,
34
,
35
]. In pa icula , he e
a e a ew examples epo ed o he syn hesis o e asubs i u ed
α
-aminophosphona es
de i ed om aspa ic acid as conc e e examples o he scope o eac ions ha imply C-C o
C-P bond o ma ion [
36
–
39
]. I is well known ha he de elopmen o eac ions leading o
he o ma ion o e asubs i u ed ca bons is a challenging ask, due o he lack o eac i i y
o he subs a es, de i ed om he gene a ion o a highly c owded s uc u e.
In his con ex , he aza-Re o ma sky eac ion is a widely used me hod o he syn hesis
o
β
-amino acids [
40
–
43
], as well as o he syn hesis o biologically ac i e
molecules [44–46]
.
Du ing he las yea s, he use o dialkylzinc eagen s has eme ged as an al e na i e o Zn
dus [
47
–
50
]. In his ega d, e y ecen ly, we ha e epo ed an enan ioselec i e aza-
Molecules 2022,27, 8024 3 o 22
Re o ma sky eac ion o
α
-phospho yla ed ke imines ha leads o he o ma ion o e a-
subs i u ed
α
-aminophosphona e de i a i es [
51
]. In iew o he in e es ing p ope ies o
aspa ic acid de i a i es and he po en ial o he isos e ic subs i u ion o a ca boxyla e by a
phosphona e g oup, he co esponding phospho us analogs o aspa ic acid may be e y
in e es ing subs a es om a biological poin o iew. Acco ding o his, as pa o ou ongo-
ing esea ch in o he iden i ica ion o new chemo he apeu ic agen s [
52
–
54
], wi h a special
ocus on o ganophospho us de i a i es [
55
–
57
], we hough ha he p epa a ion o a wide
amily o phospho a ed analogs o aspa ic acid and he s udy o hei an icance p ope ies
would be an in e es ing con ibu ion o he ield o o ganic and medicinal chemis y. Fo
all he easons men ioned abo e, he ein, we epo a gene al me hod o he syn hesis
o e asubs i u ed phospho a ed analogs o aspa ic acid h ough an aza-Re o ma sky
eac ion o
α
-ke iminophosphona es and he e alua ion o hei
in i o
cy o oxic ac i i y
agains se e al cance cell lines.
2. Resul s and Discussion
2.1. Chemis y
Du ing he las yea s, we ha e been in ol ed in he syn hesis o
α
-aminophosphona e
de i a i es h ough he addi ion o ca bon nucleophiles o imines. The mos ema kable
ea u e o ou app oach is ha
α
-iminophosphona e elec ophiles a e gene a ed by he
oxida ion o he pa en
α
-aminophosphona es. Thus, he global eac ion can be conside ed
as an umpolung p ocess, whe e he nucleophilic cha ac e o
α
-aminophosphona e species
has been in e ed. In pa icula , ollowing his app oach, we ha e epo ed he enan iose-
lec i e syn hesis o indolyl phosphoglycines [
58
] by he addi ion o indole de i a i es o
α
-
phospho a ed aldimines and he nucleophilic addi ion o cyanide [
59
], o ganome allics [
60
]
o ni ome hane species [
61
] o
α
-phospho a ed ke imines, o he p epa a ion o di e se
e asubs i u ed
α
-aminophosphona e de i a i es. Mo e ecen ly, we ha e ex ended his
s a egy o he enan ioselec i e Re o ma sky eac ion, using
α
-ke iminophosphona es as
he elec ophile sou ce [51].
Following his las app oach, we es ed he aza-Re o ma sky eac ion o di e en
imines
1–4
wi h e hyl iodoace a e, unde d y ai a mosphe e, in he p esence o dime hylz-
inc, a o ding he co esponding
β
-aminoes e s
4–6
e y good yields when aldimines o
ac i a ed ke imines we e used (Scheme 2).
Molecules2022,27,xFORPEERREVIEW3o 23
In hiscon ex , heaza‐Re o ma sky eac ionisawidelyusedme hod o he
syn hesiso β‐aminoacids[40–43],aswellas o hesyn hesiso biologicallyac i e
molecules[44–46].Du ing helas yea s, heuseo dialkylzinc eagen shaseme gedas
anal e na i e oZndus [47–50].In his ega d, e y ecen ly,weha e epo edan
enan ioselec i eaza‐Re o ma sky eac iono α‐phospho yla edke imines ha leads o
he o ma iono e asubs i u edα‐aminophosphona ede i a i es[51].In iewo he
in e es ingp ope ieso aspa icacidde i a i esand hepo en ialo heisos e ic
subs i u iono aca boxyla ebyaphosphona eg oup, heco espondingphospho us
analogso aspa icacidmaybe e yin e es ingsubs a es omabiologicalpoin o iew.
Acco ding o his,aspa o ou ongoing esea chin o heiden i ica iono new
chemo he apeu icagen s[52–54],wi haspecial ocusono ganophospho usde i a i es
[55–57],we hough ha hep epa a iono awide amilyo phospho a edanalogso
aspa icacidand hes udyo hei an icance p ope ieswouldbeanin e es ing
con ibu ion o he ieldo o ganicandmedicinalchemis y.Fo all he easonsmen ioned
abo e,he ein,we epo agene alme hod o hesyn hesiso e asubs i u ed
phospho a edanalogso aspa icacid h oughanaza‐Re o ma sky eac iono α‐
ke iminophosphona esand hee alua iono hei in i ocy o oxicac i i yagains
se e alcance celllines.
2.Resul sandDiscussion
2.1.Chemis y
Du ing helas yea s,weha ebeenin ol edin hesyn hesiso α‐
aminophosphona ede i a i es h ough headdi iono ca bonnucleophiles oimines.
Themos ema kable ea u eo ou app oachis ha α‐iminophosphona eelec ophiles
a egene a edby heoxida iono hepa en α‐aminophosphona es.Thus, heglobal
eac ioncanbeconside edasanumpolungp ocess,whe e henucleophiliccha ac e o
α‐aminophosphona especieshasbeenin e ed.Inpa icula , ollowing hisapp oach,
weha e epo ed heenan ioselec i esyn hesiso indolylphosphoglycines[58]by he
addi iono indolede i a i es oα‐phospho a edaldiminesand henucleophilicaddi ion
o cyanide[59],o ganome allics[60]o ni ome hanespecies[61] oα‐phospho a ed
ke imines, o hep epa a iono di e se e asubs i u edα‐aminophosphona e
de i a i es.Mo e ecen ly,weha eex ended hiss a egy o heenan ioselec i e
Re o ma sky eac ion,usingα‐ke iminophosphona esas heelec ophilesou ce[51].
Following hislas app oach,we es ed heaza‐Re o ma sky eac iono di e en
imines1–4wi he hyliodoace a e,unde d yai a mosphe e,in hep esenceo
dime hylzinc,a o ding heco espondingβ‐aminoes e s4–6 e ygoodyieldswhen
aldimineso ac i a edke imineswe eused(Scheme2).
Scheme2.Aza‐Re o ma sky eac iono imines1–4.
Scheme 2. Aza-Re o ma sky eac ion o imines 1–4.
Ini ially, he simple N- osylimine
1
de i ed om benzaldehyde (R = H) was p o ed
o be an excellen subs a e o he eac ion, a o ding
β
-phenylalanine de i a i e
5
a
e y good yield (Scheme 2). Nex , we we e in igued whe he he eac ion could also be
applicable o
α
-iminoes e s and, o his eason, we ied he same eac ion condi ions
using N- osyl-p o ec ed
α
-iminoes e
2
(R = CO
2
E ) as a subs a e. In his case, he eac ion
Molecules 2022,27, 8024 4 o 22
also p oceeded e icien ly o a o d qua e na y aspa ic acid de i a i e
6
(Scheme 2). In a
simila way, using benzaldehyde-de i ed
α
-iminophosphona e
3a
(R = P(O)(OMe)
2
), he
e asubs i u ed aspa ic acid de i a i e
7a
was ob ained in an excellen yield (Scheme 2).
Howe e , he eac ion condi ions, including he equi ed dime hylzinc and he eac ion
ime, p esen ed high a ia ions depending on he s uc u e o he imine. On he o he hand,
we also es ed a non-ac i a ed ke imine
4
, de i ed om ace ophenone. Un o una ely, in
his case, he Re o ma sky p oduc
8
was no obse ed, and he s a ing ma e ials we e
eco e ed unal e ed.
The subs a es ob ained om he aza-Re o ma sky eac ion we e ully cha ac e ized
on he basis o hei
1
H,
31
P, and
13
C NMR, IR spec a and HRMS (see Supplemen a y
Ma e ials). The mos cha ac e is ic pa e n o hese compounds in he
1
H NMR spec-
um is he signals co esponding o he wo p o ons o he me hylene g oup nex o he
e asubs i u ed ca bon a
δ
~3.5 ppm, which, because o he p esence o a chi al cen e in
he s uc u e, ha e a dias e eo opic cha ac e and appea as wo independen signals. In
he pa icula case o phospho a ed aspa ic acid de i a i e
7a
, hose signals appea as
wo double double s a
δ
= 3.59 and 3.46 ppm, showing a ecip ocal geminal coupling o
2
J
HH
= 16.4 Hz and addi ional icinal couplings wi h he phospho us a om o
3
J
PH
= 22.7
and 10.7 Hz, espec i ely. Acco dingly, he dime hyl phosphona e moie y in
7a
is seen as
wo ep esen a i e in ense double s a
δH
= 3.46 ppm (
3
J
PH
= 10.7 Hz) and
δH
= 3.48 ppm
(
3
J
PH
= 10.5 Hz), ypical o he dias e eo opic me hoxy g oups a he phosphona e moie y.
Rema kably, he signal co esponding o he NH g oup o
6a
appea s as a hin double a
δH
= 6.17 ppm, ha slowly in e changes wi h D
2
O, showing a s ong coupling wi h he
phospho us a om o
3
J
PH
= 11.2 Hz, which may be a ibu able o a weak acidic cha ac e
o he sul onamide moie y.
Likewise, in he
13
C NMR spec um o phospho yla ed de i a i e
7a
, undoub edly,
he mos cha ac e is ic signal is he double co esponding o he chi al qua e na y ca bon
(DEPT) a
δC
= 62.1 ppm, which shows a e y s ong ipso coupling wi h he phospho us
a om o
1
J
PC
= 153.8 Hz. The me hylene g oup nex o he chi al ca bon appea s as a double
a
δC
= 54.0 ppm wi h a geminal coupling wi h he phospho us a om o
2JCP = 7.0 Hz
,
weake han expec ed, possibly due o an un a o able angle in e ms o he coupling, which
can be ela ed o a dis o ion a he opology a he sp
3
chi al ca bon, a ibu ed o he
high s e ic hind ance p esen a he qua e na y cen e . The p esence o he es e g oup is
e iden om he chemical shi a
δC
= 170.2 ppm, ypical o ca boxylic g oups, which
appea s as a double , coupled wi h he phospho us a om wi h a icinal coupling cons an o
3
J
PC
= 8.0 Hz. The ac ha he icinal C-P coupling is s onge han he geminal suppo s
he p oposed dis o ion o he bonding angles a he qua e na y ca bon, as expec ed om
he high s e ic c owding.
In cong ui y wi h he p oposed s uc u e, he He e onuclea Mul iple Bond Co ela ion
Spec oscopy (HMBC) spec um o
7a
p esen s clea co ela ions o bo h dias e eo opic
me hylene p o ons wi h he ca bonyl g oup, he chi al e asubs i u ed ca bon and he
qua e na y a oma ic ca bon o he phenyl subs i uen .
Nex , in iew o he e icien p o ocol achie ed o he aza-Re o ma sky eac ion, we o-
cused ou e o s on he ex ension o he eac ion o he use o se e al
α
-ke iminophosphona e
subs a es
3
. In his ega d, we conside ed he syn hesis o
α
-iminophosphona e subs a es
3
om a o mal oxida ion o e ia y aminophosphona es
9
. Then, he subsequen addi ion
o a nucleophile species would a o d e asubs i u ed aminophosphona es
11
. The e-
o e, his syn he ic app oach can be conside ed globally as a ou e o he gene a ion o
e asubs i u ed
α
-aminophosphona es by he subs i u ion o hyd ogen in a isubs i u ed
α
-
aminophosphona e by a nucleophilic eagen and he complemen a y p ocess (‘umpolung
eac ion’) o he ypical elec ophilic subs i u ion o isubs i u ed
α
-amino-phosphona es
9
leading o unc ionalized α-aminophosphona es 10 (Scheme 3).
Following his app oach,
α
-ke iminophosphona es
3
we e i s gene a ed by a o mal
oxida ion o
α
-aminophosphona es
9
, ollowing he p ocedu e de eloped by ou esea ch
g oup. Then, using dime hylphosphona e-subs i u ed imines and e hyl iodoace a e, 19 e a-
Molecules 2022,27, 8024 5 o 22
subs i u ed aspa ic acid analogs
7
, bea ing di e en alpha-a oma ic subs i uen s, we e
e icien ly syn hesized (Scheme 4).
Molecules2022,27,xFORPEERREVIEW5o 23
complemen a yp ocess(‘umpolung eac ion’) o he ypicalelec ophilicsubs i u iono
isubs i u edα‐amino‐phosphona es9leading o unc ionalizedα‐aminophosphona es
10(Scheme3).
Scheme3.Gene als a egies o hesyn hesiso qua e na yα‐aminophosphona es.
Following hisapp oach,α‐ke iminophosphona es3we e i s gene a edbya o mal
oxida iono α‐aminophosphona es9, ollowing hep ocedu ede elopedbyou esea ch
g oup.Then,usingdime hylphosphona e‐subs i u ediminesande hyliodoace a e,19
e asubs i u edaspa icacidanalogs7,bea ingdi e en alpha‐a oma icsubs i uen s,
we ee icien lysyn hesized(Scheme4).
Scheme4.Phospho usanalogso aspa icacid7,syn hesized h oughanaza‐Re o ma sky eac ion.
Inaddi ion o hemodel eac ion,usinganiminewi hasimplephenylsubs i uen
(Scheme4,7a), he eac ion ole a es hep esenceo pa aandme aalkylsubs i uen sa he
a oma ic ing(Scheme4,7b–c),aswellass ongelec ondona ingg oupsa hepa a
posi iono hea oma icimine(Scheme4,7d).Se e alhalogen‐subs i u eda oma ic
ke imineswe ealsosuccess ullyusedin he eac ion,includinga oma ic ingscon aining
b omine(Scheme4,7e),chlo ine(Scheme4,7 –i)o luo ine(Scheme4,7j–n)a omsa
di e seposi ionsandincludingape luo ophenylsubs i uen (Scheme4,7o).
Fu he mo e,anexcellen esul wasobse edusinga oma iciminessubs i u edwi h
elec on‐wi hd awingg oupssuchasp‐ i luo ome hylo p‐ni osubs i uen s(Scheme4,
Scheme 3. Gene al s a egies o he syn hesis o qua e na y α-aminophosphona es.
Molecules2022,27,xFORPEERREVIEW5o 23
complemen a yp ocess(‘umpolung eac ion’) o he ypicalelec ophilicsubs i u iono
isubs i u edα‐amino‐phosphona es9leading o unc ionalizedα‐aminophosphona es
10(Scheme3).
Scheme3.Gene als a egies o hesyn hesiso qua e na yα‐aminophosphona es.
Following hisapp oach,α‐ke iminophosphona es3we e i s gene a edbya o mal
oxida iono α‐aminophosphona es9, ollowing hep ocedu ede elopedbyou esea ch
g oup.Then,usingdime hylphosphona e‐subs i u ediminesande hyliodoace a e,19
e asubs i u edaspa icacidanalogs7,bea ingdi e en alpha‐a oma icsubs i uen s,
we ee icien lysyn hesized(Scheme4).
Scheme4.Phospho usanalogso aspa icacid7,syn hesized h oughanaza‐Re o ma sky eac ion.
Inaddi ion o hemodel eac ion,usinganiminewi hasimplephenylsubs i uen
(Scheme4,7a), he eac ion ole a es hep esenceo pa aandme aalkylsubs i uen sa he
a oma ic ing(Scheme4,7b–c),aswellass ongelec ondona ingg oupsa hepa a
posi iono hea oma icimine(Scheme4,7d).Se e alhalogen‐subs i u eda oma ic
ke imineswe ealsosuccess ullyusedin he eac ion,includinga oma ic ingscon aining
b omine(Scheme4,7e),chlo ine(Scheme4,7 –i)o luo ine(Scheme4,7j–n)a omsa
di e seposi ionsandincludingape luo ophenylsubs i uen (Scheme4,7o).
Fu he mo e,anexcellen esul wasobse edusinga oma iciminessubs i u edwi h
elec on‐wi hd awingg oupssuchasp‐ i luo ome hylo p‐ni osubs i uen s(Scheme4,
Scheme 4.
Phospho us analogs o aspa ic acid
7
, syn hesized h ough an aza-Re o ma sky eac ion.
In addi ion o he model eac ion, using an imine wi h a simple phenyl subs i uen
(Scheme 4,
7a
), he eac ion ole a es he p esence o pa a and me a alkyl subs i uen s a he
a oma ic ing (Scheme 4,
7b–c
), as well as s ong elec on dona ing g oups a he pa a posi ion
o he a oma ic imine (Scheme 4,
7d
). Se e al halogen-subs i u ed a oma ic ke imines we e
also success ully used in he eac ion, including a oma ic ings con aining b omine (Scheme 4,
7e
), chlo ine (Scheme 4,
7 –i
) o luo ine (Scheme 4,
7j–n
) a oms a di e se posi ions and
including a pe luo ophenyl subs i uen (Scheme 4,
7o
). Fu he mo e, an excellen esul
was obse ed using a oma ic imines subs i u ed wi h elec on-wi hd awing g oups such as
p- i luo ome hyl o p-ni o subs i uen s (Scheme 4,
7p–q
). The eac ion can e en be ex ended
o he use o ke imines holding he e oa oma ic o biphenyl subs i uen s (Scheme 4,7 –s).
Nex , he syn he ic p ocedu e was ex ended o he use o o he di e en alkyl
iodoace a es. The eac ion using me hyl iodoace a e and a p- luo ophenyl subs i u ed
α-ke iminophosphona e a o ds he co esponding e asubs i u ed α-aminophosphona e
12
an excellen yield (Scheme 5). Unde he same condi ions, he eac ion using benzyl
iodoace a e e icien ly yields he benzyl-p o ec ed analogs o aspa ic acid
13a–b
(Scheme 5).
Molecules 2022,27, 8024 6 o 22
Molecules2022,27,xFORPEERREVIEW6o 23
7p–q).The eac ioncane enbeex ended o heuseo ke iminesholdinghe e oa oma ic
o biphenylsubs i uen s(Scheme4,7 –s).
Nex , hesyn he icp ocedu ewasex ended o heuseo o he di e en alkyl
iodoace a es.The eac ionusingme hyliodoace a eandap‐ luo ophenylsubs i u edα‐
ke iminophosphona ea o ds heco esponding e asubs i u edα‐
aminophosphona e
12anexcellen yield(Scheme5).Unde hesamecondi ions, he eac ionusingbenzyl
iodoace a ee icien lyyields hebenzyl‐p o ec edanalogso aspa icacid13a–b(Scheme
5).
Scheme5.Ex ensiono heaza‐Re o ma sky eac ion oo he alkylhaloace a ede i a i es.
Ino de oob ain heca boxylicacidand/o phosphonicacidde i a i eso he
aspa a eanalogs, hehyd olysiso heob ainedsubs a eswasa emp ed.Howe e ,
unde acidico basic ea men ,compounds7a,12o 13bled o he o ma iono complex
mix u es.On hecon a y, he ea men o benzyles e 13bunde hyd ogenp essu ein
hep esenceo apalladiumca alys a o ded heco espondingca boxylicacid14an
almos quan i a i eyield(Scheme6).
Scheme6.Hyd ogenolysiso benzyles e 13b.
While ying o u he unde s and hena u eo he eac ion,somecon ol
expe imen swe epe o medusingnon‐con en ionalhaloace a ede i a i es.Fo
ins ance, heuseo α‐b anchediodoace a e(Scheme7a) esul edinacomple elosso he
eac i i y ha maybeexplaineddue o heelec onice ec o he luo ine,which educes
henucleophilici yo hein e media especies.Incon as , heuseo abulkie
phosphona eins eado hees e g oupinc eases hes e icdemando henucleophile
(Scheme7b).Finally,whenusinge hyl3‐iodop opiona e, he o ma iono heenola e
in e media edoesno occu ,and heco espondingo ganozinchalidespeciesmaybe
o med,whicha eno usualinnucleophilicaddi ionsdue o hei low eac i i y(Scheme
7c).
Scheme 5. Ex ension o he aza-Re o ma sky eac ion o o he alkyl haloace a e de i a i es.
In o de o ob ain he ca boxylic acid and/o phosphonic acid de i a i es o he
aspa a e analogs, he hyd olysis o he ob ained subs a es was a emp ed. Howe e ,
unde acidic o basic ea men , compounds
7a
,
12
o
13b
led o he o ma ion o complex
mix u es. On he con a y, he ea men o benzyles e
13b
unde hyd ogen p essu e in he
p esence o a palladium ca alys a o ded he co esponding ca boxylic acid
14
an almos
quan i a i e yield (Scheme 6).
Molecules2022,27,xFORPEERREVIEW6o 23
7p–q).The eac ioncane enbeex ended o heuseo ke iminesholdinghe e oa oma ic
o biphenylsubs i uen s(Scheme4,7 –s).
Nex , hesyn he icp ocedu ewasex ended o heuseo o he di e en alkyl
iodoace a es.The eac ionusingme hyliodoace a eandap‐ luo ophenylsubs i u edα‐
ke iminophosphona ea o ds heco esponding e asubs i u edα‐
aminophosphona e
12anexcellen yield(Scheme5).Unde hesamecondi ions, he eac ionusingbenzyl
iodoace a ee icien lyyields hebenzyl‐p o ec edanalogso aspa icacid13a–b(Scheme
5).
Scheme5.Ex ensiono heaza‐Re o ma sky eac ion oo he alkylhaloace a ede i a i es.
Ino de oob ain heca boxylicacidand/o phosphonicacidde i a i eso he
aspa a eanalogs, hehyd olysiso heob ainedsubs a eswasa emp ed.Howe e ,
unde acidico basic ea men ,compounds7a,12o 13bled o he o ma iono complex
mix u es.On hecon a y, he ea men o benzyles e 13bunde hyd ogenp essu ein
hep esenceo apalladiumca alys a o ded heco espondingca boxylicacid14an
almos quan i a i eyield(Scheme6).
Scheme6.Hyd ogenolysiso benzyles e 13b.
While ying o u he unde s and hena u eo he eac ion,somecon ol
expe imen swe epe o medusingnon‐con en ionalhaloace a ede i a i es.Fo
ins ance, heuseo α‐b anchediodoace a e(Scheme7a) esul edinacomple elosso he
eac i i y ha maybeexplaineddue o heelec onice ec o he luo ine,which educes
henucleophilici yo hein e media especies.Incon as , heuseo abulkie
phosphona eins eado hees e g oupinc eases hes e icdemando henucleophile
(Scheme7b).Finally,whenusinge hyl3‐iodop opiona e, he o ma iono heenola e
in e media edoesno occu ,and heco espondingo ganozinchalidespeciesmaybe
o med,whicha eno usualinnucleophilicaddi ionsdue o hei low eac i i y(Scheme
7c).
Scheme 6. Hyd ogenolysis o benzyles e 13b.
While ying o u he unde s and he na u e o he eac ion, some con ol expe imen s
we e pe o med using non-con en ional haloace a e de i a i es. Fo ins ance, he use o
α
-b anched iodoace a e (Scheme 7a) esul ed in a comple e loss o he eac i i y ha may
be explained due o he elec onic e ec o he luo ine, which educes he nucleophilici y
o he in e media e species. In con as , he use o a bulkie phosphona e ins ead o he
es e g oup inc eases he s e ic demand o he nucleophile (Scheme 7b). Finally, when
using e hyl 3-iodop opiona e, he o ma ion o he enola e in e media e does no occu ,
and he co esponding o ganozinc halide species may be o med, which a e no usual in
nucleophilic addi ions due o hei low eac i i y (Scheme 7c).
Molecules2022,27,xFORPEERREVIEW7o 23
Scheme7.Reac ioncondi ionsasshownin heScheme5.(a)Reac ionwi hα‐ luo oiodoace a e.(b)
Reac ion es wi hα‐iodophosphona e.(c)Remo e e o ma sky eaxc ion.
Basedon hesecon ol eac ions,aswellason he eac ionpa hwaysp oposed o
simila p ocesses[62,63],we heo izea en a i eca aly iccycleinwhichacombina iono
elec onicands e ice ec sand hes abili yo hezinc‐enola ein e media ea e
conside ed(Scheme8).
Scheme8.P oposed eac ionmechanism.
Ashasbeenadd essedabo e,aspa icacidde i a i esha ep o ed oha easso ed
pha macologicalac i i ies[30].Thus,in iewo hedesc ibedbene i so heisos e ic
subs i u iono aca boxyla ebyaphosphona eg oup[12],nex ,weexplo ed he
applica ionso hesyn hesizedphospho a edanalogso aspa icacidasan icance
agen s.
2.2.BiologicalResul s
In i ocy o oxici yo hephospho a edaspa icacidde i a i eswase alua edby
es ing hei an ip oli e a i eac i i iesagains se e alhumancance celllines.Cell
coun ingki (CCK‐8)assaywasused o hee alua iono g ow hinhibi ion.Mo eo e ,
non‐malignan MRC5lung ib oblas swe e es ed o s udyingselec i e oxici y[64]and
chemo he apeu icdoxo ubicinisusedasa e e ence alue.Inaddi ion, isubs i u ed
aminophosphona e9a(Iminep ecu so ,R1=H,R2=P(O)(OMe)2)[59]and e asubs i u ed
aminophosphona e18(R1=Me,R2=P(O)(OMe)2)[60]a eusedas empla esino de o
e alua e hee ec o hesubs i uen s(Table1).
Scheme 7.
Reac ion condi ions as shown in he Scheme 5. (
a
) Reac ion wi h
α
- luo oiodoace a e.
(b) Reac ion es wi h α-iodophosphona e. (c) Remo e e o ma sky eaxc ion.
Molecules 2022,27, 8024 7 o 22
Based on hese con ol eac ions, as well as on he eac ion pa hways p oposed
o simila p ocesses [
62
,
63
], we heo ize a en a i e ca aly ic cycle in which a combi-
na ion o elec onic and s e ic e ec s and he s abili y o he zinc-enola e in e media e a e
conside ed (Scheme 8).
Molecules2022,27,xFORPEERREVIEW7o 23
Scheme7.Reac ioncondi ionsasshownin heScheme5.(a)Reac ionwi hα‐ luo oiodoace a e.(b)
Reac ion es wi hα‐iodophosphona e.(c)Remo e e o ma sky eaxc ion.
Basedon hesecon ol eac ions,aswellason he eac ionpa hwaysp oposed o
simila p ocesses[62,63],we heo izea en a i eca aly iccycleinwhichacombina iono
elec onicands e ice ec sand hes abili yo hezinc‐enola ein e media ea e
conside ed(Scheme8).
Scheme8.P oposed eac ionmechanism.
Ashasbeenadd essedabo e,aspa icacidde i a i esha ep o ed oha easso ed
pha macologicalac i i ies[30].Thus,in iewo hedesc ibedbene i so heisos e ic
subs i u iono aca boxyla ebyaphosphona eg oup[12],nex ,weexplo ed he
applica ionso hesyn hesizedphospho a edanalogso aspa icacidasan icance
agen s.
2.2.BiologicalResul s
In i ocy o oxici yo hephospho a edaspa icacidde i a i eswase alua edby
es ing hei an ip oli e a i eac i i iesagains se e alhumancance celllines.Cell
coun ingki (CCK‐8)assaywasused o hee alua iono g ow hinhibi ion.Mo eo e ,
non‐malignan MRC5lung ib oblas swe e es ed o s udyingselec i e oxici y[64]and
chemo he apeu icdoxo ubicinisusedasa e e ence alue.Inaddi ion, isubs i u ed
aminophosphona e9a(Iminep ecu so ,R1=H,R2=P(O)(OMe)2)[59]and e asubs i u ed
aminophosphona e18(R1=Me,R2=P(O)(OMe)2)[60]a eusedas empla esino de o
e alua e hee ec o hesubs i uen s(Table1).
Scheme 8. P oposed eac ion mechanism.
As has been add essed abo e, aspa ic acid de i a i es ha e p o ed o ha e asso ed
pha macological ac i i ies [
30
]. Thus, in iew o he desc ibed bene i s o he isos e ic sub-
s i u ion o a ca boxyla e by a phosphona e g oup [
12
], nex , we explo ed he applica ions
o he syn hesized phospho a ed analogs o aspa ic acid as an icance agen s.
2.2. Biological Resul s
In i o
cy o oxici y o he phospho a ed aspa ic acid de i a i es was e alua ed
by es ing hei an ip oli e a i e ac i i ies agains se e al human cance cell lines. Cell
coun ing ki (CCK-8) assay was used o he e alua ion o g ow h inhibi ion. Mo eo e ,
non-malignan MRC5 lung ib oblas s we e es ed o s udying selec i e oxici y [
64
] and
chemo he apeu ic doxo ubicin is used as a e e ence alue. In addi ion, isubs i u ed
aminophosphona e
9a
(Imine p ecu so , R
1
= H, R
2
= P(O)(OMe)
2
) [
59
] and e asubs i u ed
aminophosphona e
18
(R
1
= Me, R
2
= P(O)(OMe)
2
) [
60
] a e used as empla es in o de o
e alua e he e ec o he subs i uen s (Table 1).
Table 1. An ip oli e a i e ac i i y o subs a es 5–7, 9 and 18 agains lung and o a ian cance cell lines.
Molecules2022,27,xFORPEERREVIEW8o 23
Table1.An ip oli e a i eac i i yo subs a es5–7,9and18agains lungando a iancance cell
lines.
En yComp.R1R2IC50(μM)
SKOV3A549MRC5
15CH2CO2E H>5018.68±2.16>50
26CH2CO2E CO2E >5014.17±0.41>50
37aCH2CO2E P(O)(OMe)2>502.66±0.26>50
49aHP(O)(OMe)2>5017.56±1.3>50
518CH3P(O)(OMe)2>50>50n.d.
6Doxo ubicin0.13±0.098<0.1>50
Inap elimina ys udy, hemos simpleβ‐alaninesubs a e5showedsome
cy o oxici yagains heA549cellline,wi hmodes IC50 alueso 18.68±2.16μMbu high
selec i i yi compa edwi h heMCR5cellline(Table1,En y1).Asligh lyimp o edIC50
alueo 14.17±0.41μMwasobse ed o aspa icacides e 6in hesamecelllineand,
ema kably, heisos e icsubs i u iono hees e g oupbyaphosphona eg oupin7a
esul edinasigni ican lybe e IC50 alueo 2.66±0.26μM,s illwi hahighselec i i y
owa dnon‐malignan cells(Table1,En ies2–3).
Thep esenceo hees e g oup,howe e ,p o ed obe e y ele an o he
an ip oli e a i eac i i yo compound7asince e asubs i u edα‐aminophosphona e18,
lackingsuchasubs i uen ,showednocy o oxici yagains heA549celllineand
isubs i u edα‐aminophosphona e9ashowedahighe IC50 alueo 17.56±1.3μM(Table
1,En ies4–5).Noneo he es edcompoundsshowedin i o oxici yagains heSKOV3
cellline.
This i s es o ou subs a esdemons a ed ha hebes esul sinac i i ya e
ob ainedwhenbo hα‐phosphona eandβ‐ca boxyla eg oupsa ep esen in hes uc u e,
o mingaphospho usanalogo aspa icacid.Fo his eason,nex ,wepe o medas udy
o hee ec o hesubs i u iona heα‐a oma ic ingo heaspa icacidanalogsin o he
cy o oxici yagains heSKOV3andA549celllines(Table2).
Table2.An ip oli e a i eac i i yo phospho a edanalogso aspa icacid7,12–14agains lung
ando a iancance celllines.
En yComp.R1R2IC50(μM)
SKOV3A549MRC5
17aPhE >502.66±0.26>50
27b4‐Me‐C6H4E >500.34±0.0429.62±2.98
37c3‐Me‐C6H4E >502.00±0.52>50
47d4‐Cl3CS‐C6H4E 6.43±0.644.41±0.292.47±0.30
57e4‐B ‐C6H4E >501.08±0.09>50
67 4‐Cl‐C6H4E 24.12±1.453.09±0.14>50
77g3‐Cl‐C6H4E >502.96±0.3438.05±1.61
87h3,4‐Cl2‐C6H3E 6.94±0.631.05±0.4211.29±1.16
97i3‐Cl‐4‐MeO‐C6H3E >501.44±0.15>50
En y Comp. R1R2IC50 (µM)
SKOV3 A549 MRC5
15CH2CO2E H >50 18.68 ±2.16 >50
26CH2CO2E CO2E >50 14.17 ±0.41 >50
37a CH2CO2E
P(O)(OMe)
2>50 2.66 ±0.26 >50
49a H
P(O)(OMe)
2>50 17.56 ±1.3 >50
518 CH3
P(O)(OMe)
2>50 >50 n.d.
6Doxo ubicin 0.13 ±0.098 <0.1 >50
In a p elimina y s udy, he mos simple
β
-alanine subs a e
5
showed some cy o oxici y
agains he A549 cell line, wi h modes IC
50
alues o 18.68
±
2.16
µ
M bu high selec i i y
i compa ed wi h he MCR5 cell line (Table 1, En y 1). A sligh ly imp o ed IC
50
alue o
Molecules 2022,27, 8024 8 o 22
14.17
±
0.41
µ
M was obse ed o aspa ic acid es e
6
in he same cell line and, ema kably,
he isos e ic subs i u ion o he es e g oup by a phosphona e g oup in
7a
esul ed in
a signi ican ly be e IC
50
alue o 2.66
±
0.26
µ
M, s ill wi h a high selec i i y owa d
non-malignan cells (Table 1, En ies 2–3).
The p esence o he es e g oup, howe e , p o ed o be e y ele an o he an ip o-
li e a i e ac i i y o compound 7a since e asubs i u ed α-aminophosphona e 18, lacking
such a subs i uen , showed no cy o oxici y agains he A549 cell line and isubs i u ed
α
-
aminophosphona e
9a
showed a highe IC
50
alue o 17.56
±
1.3
µ
M (Table 1,
En ies 4–5
).
None o he es ed compounds showed in i o oxici y agains he SKOV3 cell line.
This i s es o ou subs a es demons a ed ha he bes esul s in ac i i y a e
ob ained when bo h
α
-phosphona e and
β
-ca boxyla e g oups a e p esen in he s uc u e,
o ming a phospho us analog o aspa ic acid. Fo his eason, nex , we pe o med a s udy
o he e ec o he subs i u ion a he
α
-a oma ic ing o he aspa ic acid analogs in o he
cy o oxici y agains he SKOV3 and A549 cell lines (Table 2).
Table 2.
An ip oli e a i e ac i i y o phospho a ed analogs o aspa ic acid
7, 12–14
agains lung and
o a ian cance cell lines.
Molecules2022,27,xFORPEERREVIEW8o 23
Table1.An ip oli e a i eac i i yo subs a es5–7,9and18agains lungando a iancance cell
lines.
En yComp.R1R2IC50(μM)
SKOV3A549MRC5
15CH2CO2E H>5018.68±2.16>50
26CH2CO2E CO2E >5014.17±0.41>50
37aCH2CO2E P(O)(OMe)2>502.66±0.26>50
49aHP(O)(OMe)2>5017.56±1.3>50
518CH3P(O)(OMe)2>50>50n.d.
6Doxo ubicin0.13±0.098<0.1>50
Inap elimina ys udy, hemos simpleβ‐alaninesubs a e5showedsome
cy o oxici yagains heA549cellline,wi hmodes IC50 alueso 18.68±2.16μMbu high
selec i i yi compa edwi h heMCR5cellline(Table1,En y1).Asligh lyimp o edIC50
alueo 14.17±0.41μMwasobse ed o aspa icacides e 6in hesamecelllineand,
ema kably, heisos e icsubs i u iono hees e g oupbyaphosphona eg oupin7a
esul edinasigni ican lybe e IC50 alueo 2.66±0.26μM,s illwi hahighselec i i y
owa dnon‐malignan cells(Table1,En ies2–3).
Thep esenceo hees e g oup,howe e ,p o ed obe e y ele an o he
an ip oli e a i eac i i yo compound7asince e asubs i u edα‐aminophosphona e18,
lackingsuchasubs i uen ,showednocy o oxici yagains heA549celllineand
isubs i u edα‐aminophosphona e9ashowedahighe IC50 alueo 17.56±1.3μM(Table
1,En ies4–5).Noneo he es edcompoundsshowedin i o oxici yagains heSKOV3
cellline.
This i s es o ou subs a esdemons a ed ha hebes esul sinac i i ya e
ob ainedwhenbo hα‐phosphona eandβ‐ca boxyla eg oupsa ep esen in hes uc u e,
o mingaphospho usanalogo aspa icacid.Fo his eason,nex ,wepe o medas udy
o hee ec o hesubs i u iona heα‐a oma ic ingo heaspa icacidanalogsin o he
cy o oxici yagains heSKOV3andA549celllines(Table2).
Table2.An ip oli e a i eac i i yo phospho a edanalogso aspa icacid7,12–14agains lung
ando a iancance celllines.
En yComp.R1R2IC50(μM)
SKOV3A549MRC5
17aPhE >502.66±0.26>50
27b4‐Me‐C6H4E >500.34±0.0429.62±2.98
37c3‐Me‐C6H4E >502.00±0.52>50
47d4‐Cl3CS‐C6H4E 6.43±0.644.41±0.292.47±0.30
57e4‐B ‐C6H4E >501.08±0.09>50
67 4‐Cl‐C6H4E 24.12±1.453.09±0.14>50
77g3‐Cl‐C6H4E >502.96±0.3438.05±1.61
87h3,4‐Cl2‐C6H3E 6.94±0.631.05±0.4211.29±1.16
97i3‐Cl‐4‐MeO‐C6H3E >501.44±0.15>50
En y Comp. R1R2IC50 (µM)
SKOV3 A549 MRC5
17a Ph E >50 2.66 ±0.26 >50
27b 4-Me-C6H4E >50 0.34 ±0.04 29.62 ±2.98
37c 3-Me-C6H4E >50 2.00 ±0.52 >50
47d 4-Cl3CS-C6H4E 6.43 ±0.64 4.41 ±0.29 2.47 ±0.30
57e 4-B -C6H4E >50 1.08 ±0.09 >50
67 4-Cl-C6H4E 24.12 ±1.45 3.09 ±0.14 >50
77g 3-Cl-C6H4E >50 2.96 ±0.34 38.05 ±1.61
87h 3,4-Cl2-C6H3E 6.94 ±0.63 1.05 ±0.42 11.29 ±1.16
97i 3-Cl-4-MeO-C6H3E >50 1.44 ±0.15 >50
10 7j 4-F-C6H4E >50 7.15 ±0.24 >50
11 7k 3-F-C6H4E >50 0.59 ±0.09 >50
12 7l 2-F-C6H4E >50 0.90 ±0.12 >50
13 7m 2,4-F2-C6H3E >50 2.24 ±0.31 >50
14 7n 3,4-F2-C6H3E >50 5.70 ±0.70 >50
15 7o C6F5E 20.46 ±2.75 3.65 ±0.21 >50
16 7p 4-CF3-C6H4E 9.80 ±0.60 20. 30 ±1.14 >50
17 7q 4-NO2-C6H4E >50 0.67 ±0.06 >50
18 7 5-Cl-2- hienyl E 11.77 ±0.60 1.04 ±0.28 >50
19 7s 4-Ph-C6H4E 17.01 ±1.22 1.48 ±0.40 >50
20 12 3-F-C6H4Me >50 24.11 ±4.01 n.d.
21 13a 3-F-C6H4Bn >50 12.75 ±2.38 n.d.
22 13b Ph Bn >50 5.20 ±0.16 >50
23 14 Ph H >50 20.61 ±1.05 >50
24 Doxo ubicin 0.13 ±0.098 <0.1 >50
Molecules 2022,27, 8024 9 o 22
The in oduc ion o me hyl g oups in o a bioac i e s uc u e esul s in a mo e lipophilic
cha ac e , o en esul ing in an imp o ed abili y o molecules o c oss cell memb anes [
65
,
66
].
Indeed, pa a and me a olyl-subs i u ed aminophosphona es
7b
and
7c
p esen ed imp o ed
IC
50
alues o 0.34
±
0.04 and 2.00
±
0.52
µ
M in he A549 cell line i compa ed wi h he
model s uc u e
7a
(Table 2, En ies 2–3 s. En y 1). Al hough compound
7c
showed a high
selec i i y i compa ed wi h he non-malignan cells, de i a i e
7b
showed some oxici y
owa d he MCR5 cell line. Un o una ely, no ac i i y was obse ed agains he SKOV3 cell
line o bo h compounds (Table 2, En ies 2–3). In addi ion, p- ichlo ome hyl hiophenyl
de i a i e
7d
showed good IC
50
alues o 6.43
±
0.64 and 4.41
±
0.29
µ
M in he SKOV3
and A549 cell lines, espec i ely, bu i p esen ed a e y low selec i i y owa d he MRC5
cell line (Table 2, En y 4).
Nex , he e ec o he subs i u ion o he
α
-a oma ic ing wi h di e en halogen a oms
was explo ed. Fi s , p-b omo-subs i u ed de i a i e
7e
p o ed o be a e y good g ow h
inhibi o o he A549 cell line wi h an IC
50
alue o 1.08
±
0.09
µ
M, and a good selec i i y
i compa ed o he SKOV3 o MCR5 cell lines, which showed alues o e 50
µ
M (Table 2,
En y 5). p-Chlo ophenyl de i a i e
7
showed e y good oxici y agains he A549 cell line
and some ac i i y in he SKOV3 cell line wi h IC
50
alues o 3.09
±
0.14 and
24.12 ±1.45 µM
,
espec i ely, and a good selec i i y agains he heal hy cells (Table 2, En y 6). Howe e ,
he m-chlo ophenyl isome
7g
was ound o be less e ec i e han he pa a isome and
oxic agains he heal hy cells (Table 2, En y 7). Rema kably, he combina ion o bo h
subs i uen s in subs a e
7h
esul ed in a s ong cy o oxic e ec agains all he cell lines and
a o al lack o selec i i y (Table 2, En y 8). Likewise, he combina ion o a me a-chlo o and
apa a-me hoxy g oup in he a oma ic ing o
7i
esul ed in a e y selec i e ac i i y agains
he A549 cell line, wi h an IC50 alue o 1.44 ±0.15 µM (Table 2, En y 9).
Al hough, gene ally, he e ec o luo ine subs i uen s on he ac i i y o o ganic com-
pounds is a he di icul o p edic , i is well known ha he in oduc ion o luo ine a oms
in o bioac i e molecules e y o en leads o inc eased ac i i ies [
67
–
69
]. Wi h his in mind,
we explo ed he e ec o he in oduc ion o di e en luo ine-con aining phospho a ed
analogs o aspa ic acid
7j–p
. Fi s , he in oduc ion o a pa a- luo ine subs i uen in
7j
had
a nega i e e ec on he cy o oxici y i compa ed wi h he model phenyl-subs i u ed com-
pound
7a
(Table 2, En y 10 s. En y 1). On he con a y, he me a- and o ho-subs i u ed
isome s
7k
and
7l
, compa ed wi h he model subs a e
7a
, showed a e y good cy o-
oxic e ec and a high selec i i y agains he A549 cell line and imp o ed IC
50
alues
o
0.59 ±0.09 and 0.90 ±0.12 µM
we e obse ed, espec i ely (Table 2, En ies 11 and
12 s. En y 1). Those alues could no be imp o ed by he combina ion o wo luo-
ine subs i uen s a he a oma ic ing and, al hough good IC
50
alues o 2.24
±
0.31 and
5.70 ±0.70 µM
we e ob ained o di luo o-subs i u ed subs a es
7m–n
in he A549 cell
line, hose we e signi ican ly highe han he alues ob ained o mono-subs i u ed sub-
s a es
7k-l
(Table 2, En ies 13 and 14 s. En ies 11 and 12). Mo eo e , pe luo ophenyl
de i a i e
7o
was ound o be ac i e agains he A549 and SKOV3 cell lines wi h IC
50
alues
o
3.65 ±0.21
and 20.46
±
2.75
µ
M, espec i ely, wi h a good selec i i y owa d he heal hy
cells (Table 2, En y 15).
Su p isingly, he in oduc ion o a pa a- i luo ome hyl elec on-wi hd awing g oup
a he a oma ic ing had a posi i e e ec on he cy o oxici y o subs a e
7p
in he SKOV3
cell line, while i had a nega i e e ec o he A549 cell line. In his case, IC
50
alues
o
9.80 ±0.60
and 20.3
±
1.14
µ
M we e ob ained o each cell line (Table 2, En y 16 s.
En y 1) and no oxici y was obse ed in he MCR5 cells. Aspa ic acid analog
7q
, bea ing
o he elec on poo a oma ic subs i uen s, such as a p-ni ophenyl g oup, showed e y good
oxici y in he A549 cell line and a high selec i i y wi h an IC
50
alue o
0.67 ±0.06 µM
(Table 2, En y 17). Finally, he e oa oma ic o biphenyl
α
-subs i u ed subs a es
7
and
7s
p esen ed e y good IC
50
alues o 1.04
±
0.28 and 1.48
±
0.40
µ
M in he A549 cell line and
mode a e cy o oxici y agains he SKOV3 cell line, wi h IC
50
alues o 11.77
±
0.60 and
17.01
±
1.22
µ
M, espec i ely. Bo h compounds we e ound o be selec i e i compa ed
wi h he non-malignan cells (Table 2, En ies 18 and 19).
Molecules 2022,27, 8024 16 o 22
= 7.3 Hz, CH
3
), 54.4 (d,
2
J
PC
= 7.6 Hz, CH
3
), 38.2 (CH
2
), 21.6 (CH
3
), 14.2 (CH
3
).
31
P NMR
(120 MHz, CDCl
3
):
δ
21.7.
19
F NMR (282 MHz, CDCl
3
)
δ−
137.6,
−
138.0. FTIR (nea )
νmax
3262 (N-H), 1738 (C=O), 1338 (O=S=O), 1249 (P=O), 1163 (O=S=O). HRMS (ESI-TOF) m/z:
calcd o C20H25F2NO7PS [M + H]+492.1052, ound 492.1060.
E hyl 3-(dime hoxyphospho yl)-3-((4-me hylphenyl)sul onamido)-3-(pe luo ophenyl)
p opanoa e
(7o)
. The gene al p ocedu e was ollowed, s a ing o m imine
3o
(457 mg,
1 mmol) o a o d 501 mg (92%) o
7o
as a colo less oil.
1
H NMR (400 MHz, CDCl
3
)
δ
7.52
(d,
3
J
HH
= 8.3 Hz, 2H), 7.16 (d,
3
J
HH
= 8.3 Hz, 2H), 6.23 (d,
3
J
PH
= 10.5 Hz, 1H), 4.18–4.07
(m, 2H), 3.98 (m, 1H), 3.80 (d,
3
J
PH
= 10.9 Hz, 3H), 3.75 (d,
3
J
PH
= 10.9 Hz, 3H), 3.45 (m,
1H), 2.36 (s, 3H), 1.24 ( ,
3
J
HH
= 7.1 Hz, 3H).
13
C{
1
H} NMR (101 MHz, CDCl
3
)
δ
170.1 (d,
3
J
PC
= 8.4 Hz, C
qua
), 145.9 (m, C
qua
), 144.1 (C
qua
), 140.8 (m, C
qua
), 137.7 (d,
4
J
PC
= 1.9 Hz,
Cqua ), 137.5 (m, Cqua ), 129.2 (CH), 127.1 (CH), 111.2 (m, Cqua ), 61.0 (CH2), 60.0 (d, 1JPC =
153.3 Hz, C
qua
), 55.7 (d,
2
J
PC
= 7.2 Hz, CH
3
), 54.9 (d,
2
J
PC
= 7.8 Hz, CH
3
), 40.3 (d,
4
J
FC
=
5.3 Hz, CH
2
), 21.4 (CH
3
), 14.1 (CH
3
).
31
P NMR (120 MHz, CDCl
3
):
δ
21.7.
19
F NMR (282
MHz, CDCl
3
)
δ−
135.0,
−
154.3,
−
162.8. FTIR (nea )
νmax
3284 (N-H), 1741 (C=O), 1333
(O=S=O), 1254 (P=O), 1166 (O=S=O). HRMS (ESI-TOF) m/z: calcd o C
20
H
22
F
5
NO
7
PS [M +
H]+546.0769, ound 546.0781.
E hyl 3-(dime hoxyphospho yl)-3-((4-me hylphenyl)sul onamido)-3-(4-( i luo ome hyl)
phenyl)p opanoa e
(7p)
. The gene al p ocedu e was ollowed, s a ing o m imine
3p
(435 mg, 1 mmol) o a o d 455 mg (87%) o
7p
as a whi e solid. M.p. (CH
2
Cl
2
-hexanes).
114–115
◦
C.
1
H NMR (400 MHz, CDCl
3
)
δ
7.47 (
3
J
HH
= 8.8 Hz,
3
J
FH
= 2.3 Hz, 2H), 7.42 (d,
3
J
HH
= 8.4 Hz, 2H), 7.34 (d,
3
J
HH
= 8.4 Hz, 2H), 7.12 (d,
3
J
HH
= 8.2 Hz, 2H), 6.24 (d,
3
J
PH
=
10.1 Hz, 1H), 4.18 (qd,
3
J
HH
= 7.2 Hz,
3
J
FH
= 1.7 Hz, 2H), 3.68 (dd,
3
J
PH
= 23.8 Hz,
2
J
HH
=
16.2 Hz, 1H), 3.58 (d,
3
J
PH
= 10.8 Hz, 3H), 3.49 (d,
3
J
PH
= 10.8 Hz, 3H), 3.44 (dd,
3
J
PH
= 8.2
Hz,
2
J
HH
= 16.2 Hz, 1H), 2.39 (s, 3H), 1.28 ( ,
3
J
HH
= 7.1 Hz, 3H).
13
C{
1
H} NMR (75 MHz,
CDCl
3
)
δ
170.0 (d,
3
J
PC
= 6.8 Hz, C
qua
), 143.7 (C
qua
), 138.9 (C
qua
), 138.6 (d,
2
J
PC
= 6.6 Hz,
C
qua
), 130.4 (dq,
2
J
FC
= 32.8 Hz,
5
J
PC
= 3.0 Hz, C
qua
), 129.3 (CH), 128.8 (d,
3
J
PC
= 5.0 Hz,
CH), 127.5 (CH), 124.7 (m, CH), 123.9 (q,
1
J
FC
= 272.4 Hz, C
qua
), 62.0 (d,
1
J
PC
= 152.8 Hz,
C
qua
), 61.3 (CH
2
), 54.8 (d,
2
J
PC
= 7.4 Hz, CH
3
), 54.4 (d,
2
J
PC
= 7.6 Hz, CH
3
), 38.1 (CH
2
),
21.6 (CH
3
), 14.3 (CH
3
).
31
P NMR (120 MHz, CDCl
3
):
δ
21.9.
19
F NMR (282 MHz, CDCl
3
)
δ
−
63.4. FTIR (nea )
νmax
3261 (N-H), 1735 (C=O), 1327 (O=S=O), 1263 (P=O), 1163 (O=S=O).
HRMS (ESI-TOF) m/z: calcd o C21H26F3NO7PS [M + H]+524.1114, ound 524.1121.
E hyl 3-(dime hoxyphospho yl)-3-((4-me hylphenyl)sul onamido)-3-(4-ni ophenyl)p opanoa e
(7q)
. The gene al p ocedu e was ollowed, s a ing o m imine
3q
(412 mg, 1 mmol) o
a o d 425 mg (85%) o
7q
as a whi e solid. M.p. (CH
2
Cl
2
-hexanes). 129–130
◦
C.
1
H NMR
(400 MHz, CDCl
3
)
δ
7.96 (d,
3
J
HH
= 9.0 Hz, 2H), 7.56 (dd,
3
J
HH
= 9.0 Hz,
3
J
PH
= 2.3 Hz, 2H),
7.50 (d,
3
J
HH
= 8.2 Hz, 2H), 7.17 (d,
3
J
HH
= 8.2 Hz, 2H), 6.30 (d,
3
J
PH
= 10.3 Hz, 1H), 4.16
(m, 2H), 3.62 (dd,
2
J
HH
= 16.5 Hz,
3
J
PH
= 6.2 Hz, 1H), 3.58 (d,
3
J
PH
= 10.8 Hz, 3H), 3.55 (d,
3
J
PH
= 10.7 Hz, 3H), 3.48 (dd,
2
J
HH
= 16.5 Hz,
3
J
PH
= 8.2 Hz, 1H), 2.41 (s, 3H), 1.27 ( ,
3
J
HH
=
7.2 Hz, 3H).
13
C{
1
H} NMR (101 MHz, CDCl
3
)
δ
169.7 (d,
3
J
PC
= 7.6 Hz, C
qua
), 147.4 (d,
5
J
PC
= 3.4 Hz, C
qua
), 144.1 (C
qua
), 142.5 (d,
2
J
PC
= 6.6 Hz, C
qua
), 138.8 (d,
4
J
PC
= 1.4 Hz, C
qua
),
129.4 (CH), 129.3 (d,
3
J
PC
= 5.0 Hz, CH), 127.5 (CH), 122.8 (d,
4
J
PC
= 2.6 Hz, CH), 62.3 (d,
1
J
PC
= 152.2 Hz, C
qua
), 61.4 (CH
2
), 54.9 (d,
2
J
PC
= 7.3 Hz, CH
3
), 54.6 (d,
2
J
PC
= 7.5 Hz, CH
3
),
38.3 (CH
2
), 21.7 (CH
3
), 14.2 (CH
3
).
31
P NMR (120 MHz, CDCl
3
):
δ
21.2. FTIR (nea )
νmax
3272 (N-H), 1743 (C=O), 1349 (O=S=O), 1244 (P=O), 1163 (O=S=O). HRMS (ESI-TOF) m/z:
calcd o C20H26N2O9PS [M + H]+501.1091, ound 501.1098.
E hyl 3-(5-chlo o hiophen-2-yl)-3-(dime hoxyphospho yl)-3-((4-me hylphenyl)sul onamido)
p opanoa e
(7 )
. The gene al p ocedu e was ollowed, s a ing o m imine
3
(407 mg,
1 mmol) o a o d 378 mg (76%) o
7
as a pale b own solid. M.p. (CH
2
Cl
2
-hexanes).
93–94
◦
C.
1
H NMR (400 MHz, CDCl
3
)
δ
7.48 (d,
3
J
HH
= 8.3 Hz, 2H), 7.17 (d,
3
J
HH
= 8.3 Hz,
2H), 6.79 (dd,
3
J
HH
= 4.0 Hz,
4
J
PH
= 3.4 Hz, 1H), 6.61 (d,
3
J
HH
= 4.0 Hz, 1H), 6.23 (d,
3
J
PH
=
7.2 Hz, 1H), 4.20 (q,
3
J
HH
= 7.2 Hz, 2H), 3.68 (d,
3
J
PH
= 10.7 Hz, 3H), 3.66 (d,
3
J
PH
= 10.5 Hz,
3H), 3.61 (m, 1H), 3.21 (dd,
2
J
HH
= 15.5 Hz,
3
J
PH
= 7.1 Hz, 1H), 2.40 (s, 3H), 1.30 ( ,
3
J
HH
=
7.2 Hz, 3H).
13
C{
1
H} NMR (101 MHz, CDCl
3
)
δ
169.5 (d,
3
J
PC
= 5.6 Hz, C
qua
), 143.6 (C
qua
),
Molecules 2022,27, 8024 17 o 22
138.7 (d,
4
J
PC
= 1.4 Hz, C
qua
), 135.9 (d,
2
J
PC
= 7.7 Hz, C
qua
), 132.3 (d,
5
J
PC
= 3.6 Hz, C
qua
),
129.3 (CH), 128.6 (d,
3
J
PC
= 7.3 Hz, CH), 127.5 (CH), 125.3 (d,
4
J
PC
= 2.8 Hz, CH), 61.4 (CH
2
),
60.0 (d,
1
J
PC
= 161.5 Hz, C
qua
), 55.2 (d,
3
J
PC
= 7.3 Hz, CH
3
), 54.5 (d,
3
J
PC
= 7.6 Hz, CH
3
),
39.0 (CH
2
), 21.7 (CH
3
), 14.2 (CH
3
).
31
P NMR (120 MHz, CDCl
3
):
δ
20.3. FTIR (nea )
νmax
3270 (N-H), 1735 (C=O), 1341 (O=S=O), 1243 (P=O), 1163 (O=S=O). HRMS (ESI-TOF) m/z:
calcd o C18H24ClNO7PS2[M + H]+496.0415, ound 496.0444..
E hyl 3-([1,1’-biphenyl]-4-yl)-3-(dime hoxyphospho yl)-3-((4-me hylphenyl)sul onamido)
p opanoa e
(7s)
. The gene al p ocedu e was ollowed, s a ing o m imine
3s
(443 mg,
1 mmol) o a o d 478 mg (90%) o
7s
as a whi e solid. M.p. (CH
2
Cl
2
-hexanes). 112–113
◦
C.
1
H NMR (400 MHz, CDCl
3
)
δ
7.56–7.46 (m, 4H), 7.45–7.40 (m, 5H), 7.39–7.33 (m, 2H),
7.13 (d,
3
J
HH
= 8.3 Hz, 2H), 6.21 (d,
3
J
PH
= 10.5 Hz, 1H), 4.20 (q,
3
J
HH
= 7.2 Hz, 2H), 3.69
(dd,
3
J
PH
= 23.4 Hz,
2
J
HH
= 16.4 Hz, 1H), 3.56 (d,
3
J
PH
= 10.7 Hz, 3H), 3.49 (m, 1H), 3.46
(d,
3
J
PH
= 10.6 Hz, 3H), 2.38 (s, 3H), 1.29 ( ,
3
J
HH
= 7.2 Hz, 3H).
13
C{
1
H} NMR (101 MHz,
CDCl
3
)
δ
170.3 (d,
3
J
PC
= 7.3 Hz, C
qua
), 143.3 (C
qua
), 141.0 (d,
5
J
PC
= 3.1 Hz, C
qua
), 140.1
(d,
4
J
PC
= 1.4 Hz, C
qua
), 139.2 (C
qua
), 129.2 (CH), 129.0 (CH), 128.8 (d,
3
J
PC
= 5.1 Hz, CH),
128.3 (C
qua
), 127.8 (CH), 127.7 (CH), 127.1 (CH), 126.5 (d,
4
J
PC
= 2.8 Hz, CH), 62.1 (d,
1
J
PC
=
153.9 Hz, C
qua
), 61.1 (CH
2
), 54.7 (d,
2
J
PC
= 7.4 Hz, CH
3
), 54.2 (d,
2
J
PC
= 7.6 Hz, CH
3
), 38.2
(CH
2
), 21.7 (CH
3
), 14.3 (CH
3
).
31
P NMR (120 MHz, CDCl
3
):
δ
22.2. FTIR (nea )
νmax
3308
(N-H), 1729 (C=O), 1332 (O=S=O), 1241 (P=O), 1161 (O=S=O). HRMS (ESI-TOF) m/z: calcd
o C26H31NO7PS [M + H]+532.1553, ound 532.1555.
Me hyl 3-(dime hoxyphospho yl)-3-(3- luo ophenyl)-3-((4-me hylphenyl)sul onamido)
p opanoa e
(12)
. The gene al p ocedu e was ollowed, s a ing o m imine
3k
(417 mg,
1 mmol) o a o d 372 mg (81%) o
12
as a whi e solid. M.p. (CH
2
Cl
2
-hexanes). 115–116
◦
C.
1
H NMR (400 MHz, CDCl
3
)
δ
7.48 (d,
3
J
HH
= 8.3 Hz, 2H), 7.20–7.09 (m, 4H), 7.00 (m, 1H),
6.91 (m, 1H), 6.21 (b d,
3
J
HH
= 7.8 Hz, 1H), 3.73 (s, 3H), 3.64 (dd,
3
J
PH
= 24.4 Hz,
2
J
HH
=
16.2 Hz, 1H), 3.56–3.50 (m, 6H), 3.46 (dd,
2
J
HH
= 16.2 Hz,
3
J
PH
= 7.6 Hz, 1H), 2.39 (s, 3H).
13
C{
1
H} NMR (101 MHz, CDCl
3
)
δ
170.6 (d,
3
J
PC
= 6.8 Hz, C
qua
), 162.3 (dd,
1
J
FC
= 246.0 Hz,
4
J
PC
= 3.0 Hz, C
qua
), 143.7 (C
qua
), 139.0 (C
qua
), 137.1 (m, C
qua
), 129.5 (dd,
3
J
FC
= 8.1 Hz,
4
J
PC
2.8 Hz, CH), 129.3 (CH), 127.5 (CH), 123.7 (dd,
3
J
PC
= 5.1 Hz,
4
J
FC
= 3.0 Hz, CH), 116.1
(dd,
2
J
FC
= 24.1 Hz,
3
J
PC
= 4.8 Hz, CH), 115.5 (dd,
2
J
FC
= 21.1 Hz,
5
J
PC
= 2.9 Hz, CH), 62.0
(dd,
1
J
PC
= 153.7 Hz,
4
J
FC
= 1.4 Hz, C
qua
), 54.9 (d,
2
J
PC
= 7.7 Hz, CH
3
), 54.4 (d,
2
J
PC
= 7.6 Hz,
CH
3
), 52.3 (CH
3
), 38.2 (CH
2
), 21.6 (CH
3
).
31
P NMR (120 MHz, CDCl
3
):
δ
21.7.
19
F NMR
(282 MHz, CDCl
3
)
δ−
112.9. FTIR (nea )
νmax
3281 (N-H), 1729 (C=O), 1330 (O=S=O), 1243
(P=O), 1159 (O=S=O). HRMS (ESI-TOF) m/z: calcd o C
19
H
24
FNO
7
PS [M + H]
+
460.0990,
ound 460.1004.
Benzyl 3-(dime hoxyphospho yl)-3-(3- luo ophenyl)-3-((4-me hylphenyl)sul onamido)
p opanoa e
(13a)
. The gene al p ocedu e was ollowed, s a ing o m imine
3k
(417 mg,
1 mmol) o a o d 449 mg (84%) o
13a
as a whi e solid. M.p. (CH
2
Cl
2
-hexanes). 78–79
◦
C.
1
H NMR (400 MHz, CDCl
3
)
δ
7.47 (d,
3
J
HH
= 8.2 Hz, 2H), 7.41–7.31 (m, 5H), 7.17–7.08 (m,
4H), 7.03–6.98 (m, 1H), 6.94–6.87 (m, 1H), 6.24 (d,
3
J
PH
= 10.2 Hz, 1H), 5.19 (d,
2
J
HH
= 12.3
Hz. 1H), 5.13 (d,
2
J
HH
= 12.3 Hz, 1H), 3.73–3.63 (m, 1H), 3.57–3.34 (m, 7H), 2.38 (s, 3H).
13
C{
1
H} NMR (101 MHz, CDCl
3
)
δ
169.9 (d,
3
J
PC
= 6.9 Hz, C
qua
), 162.3 (dd,
1
J
FC
= 246.0 Hz,
4
J
PC
= 3.0 Hz, C
qua
), 143.6 (C
qua
), 139.0 (d,
4
J
PC
= 1.3 Hz, C
qua
), 137.1 (C
qua
), 135.6 (C
qua
),
129.4 (dd,
3
J
FC
= 8.2 Hz,
4
J
PC
= 2.5 Hz, CH), 129.3 (CH), 128.7 (CH), 128.7 (CH), 128.5 (CH),
127.5 (CH), 123.7 (dd,
3
J
PC
= 5.0,
4
J
FC
= 2.9 Hz, CH), 116.0 (dd,
2
J
FC
= 24.0 Hz,
3
J
PC
= 4.7 Hz,
CH), 115.4 (dd,
2
J
FC
= 21.1 Hz,
5
J
PC
= 3.1 Hz, CH), 67.0 (CH
2
), 61.9 (dd,
1
J
PC
= 153.6 Hz,
4
J
FC
= 1.6 Hz, C
qua
), 54.8 (d,
2
J
PC
= 7.5 Hz, CH
3
), 54.3 (d,
2
J
PC
= 7.7 Hz, CH
3
), 38.2 (CH
2
),
21.6 (CH
3
).
31
P NMR (120 MHz, CDCl
3
):
δ
21.6.
19
F NMR (282 MHz, CDCl
3
)
δ−
112.9.
FTIR (nea )
νmax
3281 (N-H), 1735 (C=O), 1331 (O=S=O), 1247 (P=O), 1154 (O=S=O). HRMS
(ESI-TOF) m/z: calcd o C25H28FNO7PS [M + H]+536.1303, ound 536.1322.
Benzyl 3-(dime hoxyphospho yl)-3-((4-me hylphenyl)sul onamido)-3-phenylp opanoa e
(13b)
.
The gene al p ocedu e was ollowed, s a ing o m imine
3a
(367 mg, 1 mmol) o a o d
476 mg (92%) o
13b
as a whi e solid. M.p. (CH
2
Cl
2
-hexanes). 84–85
◦
C.
1
H NMR (400 MHz,
CDCl
3
)
δ
7.46 (d, J= 8.3 Hz, 2H), 7.37–7.29 (m, 7H), 7.20–7.15 (m, 1H), 7.12–7.06 (m, 4H),
Molecules 2022,27, 8024 18 o 22
6.22 (d,
3
J
PH
= 11.1 Hz, 1H), 5.14 (d,
2
J
HH
= 12.2 Hz, 1H), 5.09 (d,
2
J
HH
= 12.2 Hz, 1H), 3.65
(dd, 3JPH = 22.3 Hz, 2JHH = 16.6 Hz, 1H), 3.49 (dd, 2JHH = 16.6 Hz, 3JPH = 7.5 Hz, 1H), 3.37
(d,
3
J
PH
= 10.7 Hz, 3H), 3.31 (d,
3
J
PH
= 10.7 Hz, 3H), 2.34 (s, 3H).
13
C{
1
H} NMR (101 MHz,
CDCl
3
)
δ
169.8 (d,
3
J
PC
= 8.1 Hz, C
qua
), 143.1 (C
qua
), 139.0 (d,
4
J
PC
= 1.4 Hz, C
qua
), 135.5
(C
qua
), 134.1 (d,
2
J
PC
= 7.3 Hz, C
qua
), 129.0 (CH), 128.4 (CH), 128.4 (CH), 128.2 (CH), 128.2
(CH), 128.0 (d,
3
J
PC
= 5.0 Hz, CH), 127.8 (d,
4
J
PC
= 2.6 Hz, CH), 127.4 (CH), 66.5 (CH
2
),
61.9 (d,
1
J
PC
= 154.1 Hz, C
qua
), 54.4 (d,
2
J
PC
= 7.4 Hz, CH
3
), 53.8 (d,
2
J
PC
= 7.7 Hz, CH
3
),
37.7 (CH
2
), 21.4 (CH
3
).
31
P NMR (120 MHz, CDCl
3
):
δ
22.0. FTIR (nea )
νmax
3322 (N-H),
1739 (C=O), 1337 (O=S=O), 1248 (P=O), 1163 (O=S=O). HRMS (ESI-TOF) m/z: calcd o
C25H29NO7PS [M + H]+518.1397, ound 518.1372.
P ocedu e o he Ob en ion o 3-(Dime hoxyphospho yl)-3-((4-me hylphenyl)sul onamido)-
3-phenylp opanoic acid 14
A mix u e o aminophosphona e
13b
(518 mg, 1 mmol) and Pd-C 10% (106 mg,
0.1 mmol) in MeOH (50 mL) we e s i ed o 12 h unde H
2
a mosphe e (75 psi). The
mix u e was hen il e ed on celi e and concen a ed unde educed p essu e o yield
p oduc
14
as a whi e solid (402 mg, 94%), a e c ys alliza ion in MeOH. M.p. (MeOH).
145–146
◦
C.
1
H NMR (400 MHz, CDCl
3
)
δ
9.91 (b s, 1H), 7.48 (d,
3
J
HH
= 8.2 Hz, 2H), 7.33
(d,
3
J
HH
= 7.6 Hz, 2H), 7.20 (m, 1H), 7.17–7.09 (m, 4H), 6.60 (d,
3
J
PH
= 10.6 Hz, 1H), 3.67
(dd,
3
J
PH
= 23.9 Hz,
2
J
HH
= 16.0 Hz, 1H), 3.52 (d,
3
J
PH
= 10.8 Hz, 3H), 3.49 (m, 1H), 3.47
(d,
3
J
PH
= 10.6 Hz, 3H), 2.38 (s, 3H).
13
C{
1
H} NMR (101 MHz, CDCl
3
)
δ
173.5 (d,
3
J
PC
= 7.7
Hz, C
qua
), 143.3 (C
qua
), 139.2 (d,
4
J
PC
= 1.4 Hz, C
qua
), 134.1 (d,
2
J
PC
= 7.6 Hz, C
qua
), 129.2
(CH), 128.4 (d,
5
J
PC
= 3.0 Hz, CH), 128.3 (d,
3
J
PC
= 5.1 Hz, CH), 128.0 (d,
4
J
PC
= 2.6 Hz, CH),
127.6 (CH), 62.1 (d,
1
J
PC
= 155.6 Hz, C
qua
), 55.1 (d,
2
J
PC
= 7.3 Hz, CH
3
), 54.5 (d,
2
J
PC
= 7.9
Hz, CH
3
), 38.0 (CH
2
), 21.6 (CH
3
).
31
P NMR (120 MHz, CDCl
3
):
δ
21.8. FTIR (nea )
νmax
3500–2500 (O-H s ), 3271 (N-H s ), 1714 (C=O s ), 1337 (O=S=O s as), 1235 (P=O s ), 1163
(O=S=O s sim) cm
–1
. HRMS (ESI-TOF) m/z: calcd o C
18
H
23
NO
7
PS [M + H]
+
428.0927,
ound 428.0901.
P ocedu e o he Ob en ion o Dime hyl (1-((4-me hylphenyl)sul onamido)-1-phenyle hyl)
phosphona e 18
A solu ion o
3a
(367 mg, 1 mmol) in d y CH
3
CN (3 mL) was s i ed a oom empe -
a u e unde N
2
a mosphe e. To his mix u e, Me
2
Zn (1.7 mL, 1.2 M in oluene, 2 mmol)
was added and he mix u e was s i ed o 2h a oom empe a u e. The eac ion was
quenched by a slow addi ion o a sa u a ed aqueous solu ion o NH
4
Cl (1 mL) and d ied
o e anhyd ous MgSO
4
. The solid was emo ed by il a ion and washed wi h AcOE , and
he il a e was concen a ed a educed p essu e o yield he c ude p oduc , which was
pu i ied by column ch oma og aphy in silica gel (AcOE /Hexanes) o gi e 326 mg (85%) o
18
as a whi e solid [
60
]. M.p. (E
2
O/pen ane). 163–164
◦
C. Li . 161–162 (E
2
O).
1
H NMR
(400 MHz, CDCl
3
)
δ
7.53 (d,
3
J
HH
= 8.3 Hz, 2H), 7.40 (m, 2H), 7.22–7.18 (m, 3H), 7.11 (d,
3
J
HH
= 8.2 Hz, 2H), 5.78 (d,
3
J
PH
= 8.1 Hz, 1H), 3.70 (d,
3
J
PH
= 10.4 Hz, 3H), 3.35 (d,
3
J
PH
=
10.4 Hz, 3H), 2.36 (s, 3H), 1.97 (d,
3
J
PH
= 16.8 Hz, 3H).
13
C {
1
H} NMR (75 MHz, CDCl
3
)
δ
142.3 (C
qua
), 141.8 (d,
4
J
PC
= 1.7 Hz, C
qua
), 133.4 (C
qua
), 128.6 (CH), 128.4 (d,
3
J
PC
= 6.0 Hz,
CH), 128.1 (d,
4
J
PC
= 2.2 Hz, CH), 128.0 (d,
5
J
PC
= 2.9 Hz, CH), 126.7 (CH), 61.1 (d,
1
J
PC
=
152.2 Hz, C
qua
), 54.5 (d,
2
J
PC
= 7.1 Hz, CH
3
), 53.9 (d,
2
J
PC
= 7.0 Hz, CH
3
), 21.3 (CH
3
), 20.4
(d,
2
J
PC
= 5.2 Hz CH
3
).
31
P NMR (120 MHz, CDCl
3
)
δ
26.1. FTIR (nea )
νmax
3315 (N-H),
1327 (O=S=O), 1242 (P=O), 1166 (O=S=O). HRMS (ESI-TOF) m/z: calcd. o C
17
H
22
NO
5
PS
[M + Na]+406.0848, ound 406.0856.
3.2. Biology
3.2.1. Ma e ials
Reagen s and sol en s we e used as pu chased wi hou u he pu i ica ion. All
s ock solu ions o he in es iga ed compounds we e p epa ed by dissol ing he powe ed
ma e ials in app op ia e amoun s o dime hylsul oxide (DMSO). The inal concen a ion
Molecules 2022,27, 8024 19 o 22
o DMSO ne e exceeded 5% ( / ) in he eac ions. The s ock solu ion was s o ed a 5
◦
C
un il i was used.
3.2.2. Cell Cul u e
Human epi helial lung ca cinoma cells (A549) (ATCC
®
CCL-185
™
, ATCC, Manassas,
VA, USA) we e g own in Kaighn’s Modi ica ion o Ham’s F-12 Medium (ATCC
®
30-2004
™
,
ATCC, Manassas, VA, USA) and lung ib oblas cells (MRC5) (ATCC
®
CCL-171
™
, ATCC,
Manassas, VA, USA) we e g own in Eagle’s Minimum Essen ial Medium (EMEM, ATCC
®
30-2003
™
, ATCC, Manassas, VA, USA). Epi helial o a y adenoca cinoma cells (SKOV3)
(ATCC
®
HTB-77
™
, ATCC, Manassas, VA, USA) we e g own in McCoy’s 5A medium
(ATCC
®
30-2007
™
, ATCC, Manassas, VA, USA). All o hem we e supplemen ed wi h 10%
o e al bo ine se um (FBS) (Sigma-Ald ich, Mad id, Spain) and wi h 1% o NORMOCIN
solu ion (The mo Fishe , Wal ham, MA, USA). Cells we e incuba ed a 37
◦
C and 5%
CO
2
a mosphe e, and we e spli e e y 3–4 days o main ain monolaye co e age. Fo he
cy o oxici y expe imen s, he A549 and SKOV3 cells we e seeded in 96-well pla es a a
densi y o 2.5–3
×
10
3
cells pe well and incuba ed o e nigh o achie e 70% o con luence
a he ime o exposi ion o he cy o oxic compound.
3.2.3. Cy o oxici y Assays
Cells we e exposed o di e en concen a ions o he cy o oxic compounds and we e
incuba ed o 48 h. Then, 10
µ
L o cell coun ing ki -8 was added in o each well o an
addi ional wo hou s’ incuba ion a 37
◦
C. The abso bance o each well was de e mined
by an Au oma ic Elisa Reade Sys em (The mo Scien i ic Mul iskan FC Au oma ic Elisa
Reade Sys em, The mo Scien i ic, Shanghai, China) a 450 nm wa eleng h.
4. Conclusions
In conclusion, we epo an e icien me hodology o he p epa a ion o phos-
phona e analogs o aspa ic acid, holding a a ie y o subs i uen s a hei
α
-a oma ic
ing.
α
-Ke iminophosphona es a e gene a ed by he oxida ion o hei pa en e ia y
α
-aminophosphona es and a subsequen aza-Re o ma sky eac ion wi h alkyl iodoace a e
de i a i es. Mo eo e , his me hodology has been success ully ex ended o aldimines
and ac i a ed ke imines, a o ding he Re o ma sky p oduc s in high yields. This s a egy
allows he possibili y o asso ed s uc u al di e si y in he esul an sca old depending
on he s a ing imine o alkyl iodoace a e. Mo eo e , he phospho a ed analogues o as-
pa ic acid
6
showed
in i o
cy o oxici y inhibi ing he g ow h o human umo cell lines
SKOV3 (human o a ian ca cinoma) and A549 (ca cinomic human al eola basal epi helial
cell), and a high selec i i y owa d he MRC5 non-malignan lung ib oblas s. The mos
ac i e subs a es we e p o ed o be e hyl es e de i a i es. Al hough p- olyl de i a i es
showed he bes esul agains A549, he in oduc ion o a i luo ome hylphenyl moie y
in he pa a posi ion exhibi ed he mos ema kable IC
50
alue agains he SKOV3 cell line.
Mo eo e , he majo i y o he compounds we e selec i e owa d he non-malignan cells.
The bes IC
50
alues ob ained a e 9.80
µ
M in he SKOV3 cell line o
α
-aminophosphona e
7p
, wi h a p- i luo ophenyl subs i uen , and 0.34
µ
M in he A549 cell line o subs a e
7k
,
holding a p-me hylphenyl moie y. Mos o he compounds p esen ed in his s udy show
low mic omola ac i i y and a high selec i i y owa d he non-malignan cells. I has also
been p o ed ha he absolu e con igu a ion o he e asubs i u ed s e eocen e does no
ha e any in luence on he biological ac i i y o he phospho a ed aspa ic acid de i a i es,
since bo h enan iome s o subs a e 7k showed simila IC50 alues.
Supplemen a y Ma e ials:
The ollowing suppo ing in o ma ion can be downloaded a : h ps://
www.mdpi.com/a icle/10.3390/molecules27228024/s1,
1
H,
13
C,
31
P and
19
F copies o compounds
5,6,7,12,13,14 and 18. 2D-NMR o compound 7a.
Au ho Con ibu ions:
Concep ualiza ion, X.d.C., A.M., F.P. and J.V.; me hodology: e alua ion
o he
in i o
an ip oli e a i e ac i i y, X.d.C.; me hodology: o ganic syn hesis, A.M. and A.L.-F.;
Molecules 2022,27, 8024 20 o 22
so wa e, X.d.C. and A.M.; alida ion, J.V.; o mal analysis, X.d.C. and A.M.; in es iga ion, X.d.C.,
A.M. and A.L.-F.; esou ces, F.P. and J.V.; da a cu a ion, X.d.C., A.M. and A.L.-F.; w i ing—o iginal
d a p epa a ion, J.V.; w i ing— e iew and edi ing, X.d.C., A.M., A.L.-F., F.P. and J.V.; isualiza ion,
F.P. and J.V.; supe ision, J.V.; p ojec adminis a ion, F.P. and J.V.; unding acquisi ion, F.P. and J.V.
All au ho s ha e ead and ag eed o he published e sion o he manusc ip .
Funding:
Financial suppo by Minis e io de Economía, Indus ia y Compe i idad (PID2021-122558OB-
I00) and Gobie no Vasco (GV-IT1701-22) is g a e ully acknowledged. X.d.C. and A.L.-F. hank he
Basque Coun y Go e nmen o a p edoc o al g an .
Ins i u ional Re iew Boa d S a emen : No applicable.
In o med Consen S a emen : No applicable.
Da a A ailabili y S a emen :
The da a p esen ed in his s udy a e a ailable in he supplemen a y
ma e ials ile o on eques om he co esponding au ho (
1
H,
13
C,
19
F and
31
P-NMR and HRMS
spec a and cy o oxici y essays).
Acknowledgmen s:
The au ho s hank o he echnical and human suppo p o ided by SGIke
(UPV/EHU/ERDF, EU).
Con lic s o In e es : The au ho s decla e no con lic o in e es .
Sample A ailabili y: Samples o he compounds a e no a ailable.
Re e ences
1.
Léga é, J. Popula ion Aging: Economic and Social Consequences. In In e na ional Encyclopedia o he Social & Beha io al Sciences,
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