1
Fi s syn hesis o me ged hyb ids phospho yla ed azi ino[2,1-b]
benzo[e][1,3]oxazine de i a i es as an icance agen s.
Vic o Ca amiñana, Ana M. Ochoa de Re ana, Jesús M. de los San os*, F ancisco Palacios*
Depa men o O ganic Chemis y I, Facul y o Pha macy and Lasca ay Resea ch Cen e , Uni e si y o he
Basque Coun y (UPV/EHU), Paseo de la Uni e sidad 7, 01006 Vi o ia, Spain.
E-mail add esses: jesus.delossan[email p o ec ed]us / ancisco.pa[email p o ec ed]s
Keywo ds: an ip oli e a i e e ec , azi idines, 2H-azi ines, me ged hyb id compounds,
phospho us subs i u ed azi ino[2,1-b]benzo[e][1,3]oxazines.
Abs ac :
This wo k desc ibes a s aigh o wa d dias e eoselec i e syn he ic access o azi ino[2,1-
b]benzo[e][1,3]oxazines con aining phospho us subs i uen s such as phosphona e o phosphine
oxide, by means o nucleophilic addi ion o unc ionalized phenols o he C–N double bond o
2H-azi ine de i a i es. In addi ion, he cy o oxic e ec on cell lines de i ed om human lung
adenoca cinoma (A549) and human emb yonic kidney (HEK293) was also sc eened. Some
azi ino[2,1-b]benzo[e][1,3]oxazines 4 and 6 exhibi ed e y good ac i i y agains he A549 cell
line in i o. Fu he mo e, selec i i y owa ds cance cell (A549) o e (HEK293), and non-
malignan cells (MCR-5) has been de ec ed.
1. In oduc ion
In he inc easing ield o i e e sible inhibi o d ug disco e y, he e is a new e i al o he
in e es in co alen binding d ugs owing o he FDA app o al o sa e and e icien co alen
he apeu ics and a be e unde s anding o he p o i s o he co alen binding mechanism [1,2].
These ac s a e making i e e sible inhibi o s a obus comeback in cance he apeu ics [3,4].
Co alen inhibi o s own many ad an ages o e e e sible inhibi o s, including less equen
d ug dosing due o he p olonged in e al o ac ion, imp o ed biochemical e iciency, he
po en ial o p e en de e mined d ug esis ance mechanisms, and a lowe e ec i e dose may
esul in high po en ial o g ea e he apeu ic index [5]. In ecen imes, wi h he g ow h o
a ge ed co alen inhibi o s, many new d ugs con ain elec ophilic moie ies ac ing as
“wa heads”. Se e al molecules wi h a di e si y o elec ophilic wa heads, including ke one,
This is he accep ed manusc ip o he a icle ha appea ed in inal o m in Eu opean Jou nal o Medicinal Chemis y 185 :
(2020) // A i cle ID 111771, which has been published in inal o m a h ps://doi.o g/10.1016/j.ejmech.2019.111771. © 2019 Else ie
Masson SAS unde CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/)
2
es e , ni ile, α,β-unsa u a ed ca bonyl, epoxide, o azi idine unc ionali ies ha e been
ecognized as co alen inhibi o s [6]. The e o e, ob aining a sca old wi h an app op ia e
pla o m o inse ion o an elec ophilic moie y a he igh ajec o y ela i e o he
nucleophilic pa ne is being used as a u he challenge.
The azi idine h ee-membe ed ing is a s uc u al componen ound in a wide ange o
biologically ac i e agen s, na u al p oduc s, and ela ed compounds. Azi idine-based inhibi o s
displayed high selec i i y o pa asi e p o ozoon cys eine p o ease [7], while azi idine-2,3-
dica boxyla e was used as ypanocidal agen s a ge ing he majo cys eine p o ease o
T ypanosoma b ucei [8]. In addi ion, a bio inyla ed dibenzylazi idine-2,3-dica boxyla e was
designed by Leippe e al. [9] as an i e e sible cys eine p o ease inhibi o o a ge he mala ia
pa asi e Plasmodium alcipa um. The an i umo and an ibio ic p ope ies o some o hese
compounds, including azinomycin B [10,11], mi omycin C [12,13], madu opep in [14], and
FR-900482 [15], a close cousin o mi omycin C since hey sha e a simila s uc u al analogy,
a e widely known. The he apeu ic applica ions o some o he s a e mo e a ied [16]. Fo
ins ance, icellomycin [17] displays high in i o ac i i y agains G am-posi i e bac e ias, as
well as, mul id ug esis an s ains o S aphylococcus au eus; howe e , azicemicin [18,19]
shows inhibi o y ac i i ies owa d G am-nega i e bac e ias and mycobac e ias (Figu e 1).
O
O
O
OH
N
O
N
H
OOH
O
N
OH
AcO
Azinomycin B
NH
O
Mi omycin C
H2N
O
O
N
OCONH2
H2N
H
N
ON
NH
H2N
HN
OH
O
Ficellomycin
NO
OH
OHC
OH
NH
OCONH2
FR-900482
HO
O
OH
O
OH O
O
OH H
OH
Azicemicin A
N
Figu e 1. Rep esen a i e examples o co alen d ugs wi h azi idine wa heads. Elec ophiles a e
highligh ed.
3
Mi omycins a e a class o pha maceu icals wi h e y po en an ibac e ial and an i-cance
ac i i y. Fo ins ance, mi omycin C (Figu e 1) has been in ex ensi e clinical use o mo e han
20 yea s o i s ac i i y agains bladde umo s, s omach, b eas , esophagus, colo ec al, and non-
small-cell lung cance s [20]. This class o chemo he apy agen s a e he classical bio educ i e
d ugs in eg a ing a quinone moie y o educ i e ac i a ion in i o and subsequen elec ophilic
si es o DNA alkyla ion and co alen c oss-linking. The azi idine ing p esen in hese
chemo he apeu ics seems o cause DNA monoalkyla ion ia acid-ac i a ion o p oduce a
p o ona ed azi idine ha ing opens elie ing he s ain ela ed o he h ee-membe ed ing
[21,22]. The e o e, azi idines a e po en alkyla ing agen s which may ac as co alen d ugs
h ough hei capabili y o ac as DNA c oss-linking agen s ia nucleophilic ing opening o he
h ee-membe ed he e ocycle [23].
To o e come he d as ic side e ec s ela ed o a single d ug, he de elopmen o hyb id
molecules in oducing wo o mo e po en ially pha macopho es o concu en ly modula e
se e al a ge s o mul i ac o ial diseases, ha e been ecognized as a popula app oach o
mul id ug he apy [24]. Whe eas azi idine compounds a e aluable as eac i e building blocks
on hei own [25,26,27], se e al azi idine-con aining p oduc s ha e p o ed o possess
biological ac i i y, essen ially owing o he p esence o he azi idine moie y. Mo eo e , om a
biological iewpoin , o ganophospho us compounds a e ascina ing compounds as i is known
ha phospho us subs i uen s may modi y he eac i i y o he e ocycles and con ol impo an
biological unc ions [28], including new an ileishmanial [29] and an ip oli e a i e agen s
[30,31]. S a egies o inco po a ion o o ganophospho us unc ionali ies in adequa e syn hons
ha e been ex ensi ely applied in he syn hesis o phospho yla ed azahe e ocycles [32,33],
aminophosphona es [34], o phosphina es [35]. Likewise, he benzoxazine amewo k is p esen
in many ac i e compounds o biological ele ance, such as an i-mala ials [36], an i-
mycobac e ials [37], an i-in lamma o y [38], as well as po en inhibi o s o me hionyl- RNA
syn he ase (MRS) [39] o DNA-PK enzyme wi h an ip oli e a i e ac i i y [40].
Conside ing hese ac s, he e we wish o accoun he syn hesis o new [1,3]benzoxazine-
azi idine me ged hyb ids wi h phospho us con aining g oups such as phosphine oxide o
phosphona e, h ough nucleophilic addi ion o unc ionalized salicylaldehydes o 2H-azi ines.
These new hyb id molecules, which may e ain he unc ional p ope ies o he pa en
molecules, a e ob ained by o e lapping o [1,3]benzoxazine and azi idine amewo ks, di ec ly
connec ed o he phospho us subs i uen (Figu e 2). Besides, hese new hyb id molecules we e
es ed o an ip oli e a i e ac i i y agains di e en human cance cell lines. Owing o he
4
inhe en in e es o hese new hyb ids, bo h in syn he ic and medicinal chemis y, his en ail an
in e es ing challenge. As a as we know, his is he i s example o he p epa a ion o
azi ino[2,1-b]benzo[e][1,3]oxazines and hei s udy as an ip oli e a i e agen s.
O
NPR
2
O
O
NHN
PR
2
O
2H-benzo[
e][1,3]oxazine
amewo k
Azi idine moie y
In oducing o ganophospho us
unc ionali ies
Figu e 2. Phospho yla ed azi ino[2,1-b]benzo[e][1,3]oxazines as new me ged hyb id
molecules.
2. Resul s and discussion
2.1. Chemis y
A i s , we an icipa ed ha nucleophilic addi ion o unc ionalized salicylaldehydes 1 o
phospho us subs i u ed 2H-azi ines 2, would p o ide a use ul app oach o he syn hesis o
unc ionalized azi ino-benzoxazine de i a i es 4 wi h phospho us subs i uen s a C–1 (Scheme
1). The e o e, he eac ion o 2H-azi ines 2 wi h salicylaldehyde de i a i es 1 was assessed.
Thus, as ou lined in Scheme 1, in an ini ial expe imen he nucleophilic addi ion o
salicylaldehyde 1a (R1 = H) o 2H-azi ine-phospha e 2a (R2 = Me, R = OiP ) was eadily
achie ed using E 3N in dichlo ome hane a oom empe a u e. Two equi alen s o he
salicyladehyde componen we e used in o de o ensu e ull con e sion. Unde hese eac ions
condi ions, he azi ino-benzoxazine de i a i e 4a was appa en ly ob ained as a mix u e o wo
isome s in a a io o 71:29 (Table 1, en y 1), as e idenced by he p esence o wo se s o signals
in 1H and 31P NMR spec a.
We ha e ecen ly epo ed a dias e eoselec i e syn he ic me hodology o he p epa a ion o
phospho us subs i u ed cyanoazi idines [41]. These compounds ha e been ob ained as a
mix u e o wo o ame s a ibu ed o he di e en geome ies a ound he phospho us a om.
Assuming we could ob ain a mix u e o o ame s a ound he P–C bond o azi ino-benzoxazine
de i a i e 4a, as be o e, we achie e a iable- empe a u e 1H NMR and 31P NMR s udies o 4a
5
(R = OiP , R1 = H, R2 = Me). These s udies suppo ed he hypo hesis ha he doubling o signals
N
R
2
PR
2
OCH
2
Cl
2
, 4 Å M.S.
0 ºC o
2a: R = O
i
P , R
2
= Me
2b: R = OE , R
2
= Me
2c: R = Ph, R
2
= Me
2d: R = Ph, R
2
= E
4
OH
O+E
3
N
O
N
OH
R
2
PR
2
O
R
1
R
1
R
1
O
N
R
2
PR
2
OH
O
CH
2
Cl
2
, 4 Å M.S.
0 ºC o
E
3
N
3
7
1a: R
1
= H
1b: R
1
= 3-OMe
1c: R
1
= 5-Me
1d: R
1
= 5-F
Scheme 1. Syn hesis o azi ino[2,1-b]benzo[e][1,3]oxazine phosphona es and phosphine
oxides.
in 1H, 31P, and 13C NMR spec a was due o he p esence o wo o ame s o compound 4a,
since bo h o ame s in e change by o a ion a ound he P–C bond (see supplemen a y da a).
Va iable- empe a u e 31P NMR s udy o 4a in DMSO-d6 showed a mix u e o wo isome s in
a a io o 81:19 a 18 ºC. Howe e , his mix u e end o equilib a e a highe empe a u es since
a 80 ºC he a io o o ame s changed o 62:38. Simila esul s has been obse ed o a iable-
empe a u e 1H NMR s udy (see supplemen a y da a). A sligh di e ence in o ame s a io can
be de ec ed by changing he sol en om CDCl3 o DMSO-d6 (see Table 1, en y 1). The ac
ha wo o ame s we e obse ed may be connec ed o he di e en geome ies a ound he
phospho us a om, as p e iously obse ed in a benzo ul ene-phosphine oxide [42], o mo e
ecen ly in a DFT s udy o he mos s able o ame o an azi idine-2-ca boxyla e [43].
The absence o coupling cons an obse ed in 13C NMR spec um be ween he di(iso-p opyl)
phosphona e subs i uen a C–1 and he me hyl g oup a C–1a o azi ino-benzoxazine 4a,
appea s o indica e a syn- ela ionship be ween bo h g oups [44]. Conclusi ely, he X- ay
di ac ion analysis no only es ablished he syn- ela ionship be ween he me hyl g oup and he
phospho us a om, bu also he an i- ela ionship be ween hose g oups and he hyd oxyl g oup
a C–7 (see supplemen a y da a).
6
Table 1
Azi ino[2,1-b]benzo[e][1,3]oxazine phosphona es and phosphine oxides 4 ob ained.
O
N
OH
R
2
PR
2
O
R
1
En y Compound
R R1 R2 Yield(%)[a]
Ro ame s
a io[b]
1 4a O
i
P H Me 81 71:29 (81:19)
[
c]
2 4b OE H Me 76 70:30
3 4c Ph H Me 55 76:24
4 4d Ph H E 54 81:19
5 4e Ph 3-OMe Me 80 76:24
6 4 Ph 3-OMe E 51 74:26
7 4g Ph 5-Me Me 84 75:25
8 4h Ph 5-Me E 75 74:26
9 4i Ph 5-F Me 96 92:8
[a]
Yield o isola ed pu i ied compounds 4.
[b]
Ro ame s a io calcula ed by
1
H NMR in CDCl3 solu ions.
[c]
Ro ame s a io calcula ed by 1H NMR in DMSO-d6 solu ions.
S a ing om 2H-azi ine-phosphona e 2a and salicylaldehyde 1a, azi ino-benzoxazine
de i a i e 4a (R = OiP , R1 = H, R2 = Me) was isola ed in 81% yield (Scheme 1, Table 1, en y
1), while he addi ion o salicylaldehyde 1a o unc ionalized 2H-azi ine-phosphona e 2b
p o ide 76% o azi ino-benzoxazine de i a i e 4b (R = OE , R1 = H, R2 = Me) (Scheme 1,
Table 1, en y 2).
A easonable mechanism o he cons uc ion o he e ocycles 4 can be explained h ough
ini ial nucleophilic addi ion o unc ionalized phenol 1 a he ca bon-ni ogen double bond o
2H-azi ine 2 o gi e in e media e 3. This nucleophilic addi ion is expec ed o a ise in a
dias e eoselec i e way h ough he less hinde ed ace. Hence, he nea ness o phenol g oup o
unc ionalized salicylaldehyde 1 o he C–N double bond o 2H-azi ine 2 on he o he side o
he phosphona e g oup appea s o be mo e a o able, due o he high exocyclic dihed al angle
o he sp3 hyb idized sa u a ed ca bon and he p esence o he la ge phospho us g oup. We ha e
p e iously obse ed his ema k in he nucleophilic addi ion o G igna d eagen s [44,45],
hyd ides [46], alcohols [45], o cyanide anion [41] o 2H-azi ines. A e he nucleophilic
addi ion, he cons uc ion o icyclic azi ino-benzoxazine amewo k in de i a i e 4 akes
7
place h ough a dias e eoselec i e in amolecula nucleophilic a ack o he azi idine moie y o
he ca bonyl g oup o in e media e 3, in a simila way o he p e iously epo ed o he eac ion
o 2H-azi ine wi h enola es de i ed om β-ke o es e s[47].
This p ocess was ex ended o he nucleophilic addi ion o salicylaldehyde 1a o 2H-azi ine-
phosphine oxides 2c,d (R = Ph) unde he same eac ion condi ions (Scheme 1). Azi ino-
benzoxazines 4c (R = Ph, R1 = H, R2 = Me, Table 1, en y 3) and 4d (R = Ph, R1 = H, R2 = E ,
Table 1, en y 4) we e ob ained in mode a e chemical yields and as a mix u e o wo o ame s.
Fu he in es iga ion o he p ocess e ealed ha his s a egy is also sui able o o he
salicylaldehyde de i a i es 1. As illus a ed in Table 1, he me hod is ole an o a a ie y o
unc ionalized salicylaldehydes 1 wi h a ying subs i u ions. Fo ins ance, 2H-azi ine-
phosphine oxides 2c,d (R = Ph) eac ed wi h o- anillin 1b (R1 = OMe), 5-
me hylsalicylaldehyde 1c (R1 = Me), o 5- luo osalizylaldehyde 1d (R1 = F) in he p esence o
E 3N, p oducing he co esponding azidinyl-benzoxazines 4e–i (see Table 1, en ies 5–9).
In o de o de ine he scope and limi a ions o he nucleophilic addi ion o unc ionalized
phenols o 2H-azi ines 2 and inc ease he di e si y o subs i uen s in ou subs a es, his
dias e eoselec i e app oach was ex ended o include he eac i i y o 2-hyd oxyace ophenone
5 owa d phospho us subs i u ed 2H-azi ines 2. Fo his pu pose, he nucleophilic addi ion o 5
o 2H-azi ine phosphona e 2a and phosphine oxides 2c,d we e pe o med using E 3N as he
base in CH2Cl2 o yield de i a i es 6a–c in mode a e yields (Table 2). These new azi ino-
benzoxazines 6 we e ob ained, as be o e, as a mix u e o wo o ame s in a a io a ound 85:15.
A simila con igu a ion o ha ound in he case o salicylaldehydes was obse ed o azi ino-
benzoxazine 6a and was unambiguously p o ed by X- ay di ac ion analysis (see
supplemen a y da a).
This me hodology desc ibes a s aigh o wa d dias e eoselec i e ou e o unc ionalized
azi ino[2,1-b]benzo[e][1,3]oxazine phosphona e and phosphine oxides 4 and 6 by means
nucleophilic addi ion o unc ionalized phenol, such as salicylaldehyde de i a i es 1 and 2-
hyd oxyace ophenone 5, o phospho us subs i u ed 2H-azi ines 2. To ou knowledge, his
app oach ep esen s he i s example o an azi ino[2,1-b]benzo[e][1,3]oxazine.
8
Table 2
Azi ino[2,1-b]benzo[e][1,3]oxazine phosphona e and phosphine oxides 6 ob ained.
N
R
2
PR
2
OCH
2
Cl
2
, 4 Å M.S.
0 ºC o
2a: R = O
i
P , R
2
= Me
2c: R = Ph, R
2
= Me
2d: R = Ph, R
2
= E
6a-c
OH
O+E
3
N
O
N
R
2
PR
2
O
5
Me MeHO
En y Compound R R2 Yield(%)[a]
Ro ame s
a io[b]
1 6a O
i
P Me 35 85:15 (
100:0)[c]
2 6b Ph Me 57 85:15
3 6c Ph E 62 87:13
[a]
Yield o isola ed pu i ied compounds 6.
[b]
Ro ame s a io calcula ed by
1
H NMR in
CDCl
3
solu ions. [c] Ro ame s a io calcula ed by 1H NMR in DMSO-d
6
solu ions.
2.2. Biological esul s
In i o cy o oxici y o ou newly p epa ed phospho yla ed azi ino[2,1-
b]benzo[e][1,3]oxazines 4 and 6 was assessed by es ing hei an ip oli e a i e ac i i ies agains
wo di e en human cance cell lines: A549 (ca cinomic human al eola basal epi helial cells)
and HEK-293 (human emb yonic kidney cells). Cell coun ing ki (CCK-8) assay was applied
o e alua e g ow h inhibi ion. Table 3 displays cell p oli e a ion inhibi o y ac i i ies as IC50
alues o all syn hesized compounds and chemo he apeu ic doxo ubicin (DOX).
As epo ed in able 3, es ed compounds exhibi ed a wide spec um o an ip oli e a i e
ac i i y agains he cance cell lines es ed in cul u e. In gene al, compounds 4 and 6 p esen ed
a g ea e selec i e cy o oxici y in he human lung adenoca cinoma cell line (A549) han in he
human emb yonic kidney cell line (HEK-293). Likewise, heal hy lung cells, such as MRC-5
non-malignan lung ib oblas s we e es ed o s udying selec i e cy o oxici y [48], and as
ou lined in able 3, none o he syn hesized compounds 4 and 6 exhibi ed any oxici y owa d
MRC-5 cells.
9
Table 3
An ip oli e a i e ac i i y o azi ino[2,1-b]benzo[e][1,3]oxazines 4 and 6.
O
N
R
2
PR
2
O
R
3
HO
R
1
En y Comp. R R1 R2 R3
Cy o oxici y IC50 (M)[a]
lung
A549
kidney
HEK-293 MRC-5
1 DOX – – – 0.48 ± 0.017[49] 5.68 ± 1.56 [50] >50 [51]
2 4a O
i
P H Me H 3.0
0.29 >50 >50
3 4b OE H Me H 15.8
2.44 n. .
[b]
>50
4 4c Ph H Me H 2.7
0.37 >50 >50
5 4d Ph H E H 10.4
0.68 >50 >50
6 4e Ph 3-OMe Me H 1.3
0.22 >50 >50
7 4 Ph 3-OMe E H 20.7
3.75 >50 >50
8 4g Ph 5-Me Me H 3.2
0.80 >50 >50
9 4h Ph 5-Me E H 12.9
0.90 >50 >50
10 4i Ph 5-F Me H 19.6
0.24 >50 >50
11 6a O
i
P H Me Me 10.3
1.67 >50 >50
12 6b Ph H Me Me 21.1
2.44 >50 >50
13 6c Ph H E Me 3.2
0.27 >50 >50
[a]
The cy o oxici y IC50 alues lis ed a e he concen a ions co esponding o 50% g ow h inhibi ion.
[b]
No epo ed.
Rega ding he new azi ino-benzoxazines 4 agains A549 cell line in i o, diisop opyl 4a
and die hyl azi ino-benzoxazine phosphona e 4b showed dissimila IC50 alues be ween 3.0
0.29 and 15.8 2.44 M wi h he mos e ec i e compound being diisop opyl azi ino-
benzoxazine 4a (R = OiP , R1 = R3 =H, R2 = Me) (Table 3, compa e en ies 1 and 2). As
p e iously epo ed [52], he p esence o a bulky g oup a he phosphona e es e moie y gi e
ise o an inc eased ac i i y. Azi ino-benzoxazine phosphine oxides 4c–i (Table 3, en ies 3–
10) showed IC50 alues be ween 1.3 0.22 and 20.7 3.75 M wi h he mos e ec i e
de i a i e being azi ino-benzoxazine phosphine oxide 4e (R = Ph, R1 = 3-OMe, R2 = Me, R3
=H) wi h an IC50 alue o 1.3 0.22 M. Conce ning he e ec o he subs i u ion a C–1a, in
16
107.4 Hz, CH)majo , 26.9 (CH2)majo , 26.8 (CH2)mino , 9.5 (CH3)majo , 9.2 (CH3)mino , ppm; 31P
NMR (120 MHz, CDCl3) δ 24.9majo , 23.7mino ppm; ESI-HRMS (CI) m/z calcd. o C24H25NO4P
([M+H]+) 422.1521 , ound 422.1508.
((1S*,1aS*,7S*)-7-Hyd oxy-1a,5-dime hyl-1,1a-dihyd o-7H-
azi ino[2,1-b]benzo[e][1,3]oxazin-1-yl)diphenylphosphine oxide
(4g). (1.65 g, 84%) as a yellow solid om 2H-azi ine 2c (1.27 g, 5 mmol,
1 eq) and 2-hyd oxy-5-me hylbenzaldehyde 1c (1.36 g, 10 mmol, 2 eq)
as desc ibed in he gene al p ocedu e. The c ude p oduc was pu i ied
by c ys alliza ion om hexane/CH2Cl2 (50:50) o a o d he i le compound 4g. mp 168–170
ºC; IR (nea ) max 3242, 3056, 2867, 1496, 1438, 1435, 1177, 1069, 724 cm–1; 1H NMR (400
MHz, CDCl3) δ 7.83–6.69 (m, 13H, A H), 5.75 (s, 1H, CHOH)majo , 5.48 (s, 1H, CHOH)mino ,
5.23 (bs, 1H, OH), 2.98 (d, 2JPH = 21.8 Hz, 1H, CH-P)majo , 2.64 (d, 2JPH = 22.3 Hz, 1H, CH-
P)mino , 2.25 (s, 3H, CH3)majo , 2.23 (s, 3H, CH3)mino , 1.88 (s, 3H, CH3)mino , 1.83 (s, 3H, CH3)majo
ppm; 13C {1H} NMR (100 MHz, CDCl3)
146.4mino , 146.3majo , 132.9, 132.5, 132.2, 132.2,
132.1, 132.0, 131.9, 131.9, 131.8, 131.4, 131.4, 131.3, 131.2, 130.4, 129.3, 129.0, 128.9, 128.8,
128.6, 128.5, 127.7, 120.2majo , 118.8mino , 117.1mino , 116.1majo (CA ), 80.8 (d, 3JPC = 4.6 Hz,
CH)mino , 76.7 (d, 3JPC = 3.6 Hz, CH)majo , 74.2 (d, 2JPC = 4.4 Hz, Cqua )majo , 74.0 (d, 2JPC = 5.4
Hz, Cqua )mino , 35.8 (d, 1JPC = 106.5 Hz, CH)mino , 32.8 (d, 1JPC = 107.6 Hz, CH)majo , 20.9
(CH3)majo , 20.8 (CH3)mino , 20.6 (CH3)mino , 20.1 (CH3)majo ppm; 31P NMR (120 MHz, CDCl3)
δ 25.7majo , 24.3mino ppm; ESI-HRMS (CI) m/z calcd. o C23H23NO3P ([M+H]+) 392.1416,
ound 392.1403.
((1S*,1aS*,7S*)-1a-E hyl-7-hyd oxy-5-dime hyl-1,1a-dihyd o-7H-
azi ino[2,1-b]benzo[e][1,3]oxazin-1-yl)diphenylphosphine oxide
(4h). (1.57 g, 75%) as a pale yellow solid om 2H-azi ine 2d (1.34 g, 5
mmol, 1 eq) and 2-hyd oxy-5-me hylbenzaldehyde 1c (1.36 g, 10 mmol,
2 eq) as desc ibed in he gene al p ocedu e. The c ude p oduc was
pu i ied by c ys alliza ion om hexane/E 2O (50:50) o a o d he i le compound 4h. mp 122–
124 ºC; IR (nea ) max 3243, 3060, 2977, 1619, 1590, 1495, 1438, 1175, 1119, 730, 701 cm–1;
1H NMR (400 MHz, CDCl3) δ 7.83–6.71 (m, 13H, A H), 5.73 (d, 3JHH = 3.9 Hz, 1H,
CHOH)majo , 5.48 (bs, 1H, CHOH)mino , 5.21 (d, 3JHH = 3.9 Hz, 1H, OH)majo , 3.89 (d, 3JHH = 2.4
Hz, 1H, OH)mino , 3.03 (d, 2JPH = 21.8 Hz, 1H, CH-P)majo , 2.69 (d, 2JPH = 22.7 Hz, 1H, CH-
P)mino , 2.25 (s, 3H, CH3)majo , 2.23 (s, 3H, CH3)mino , 2.25–2.06 (m, 2H, CH2), 1.11 ( , 3JHH =
7.4 Hz, 3H, CH3)mino , 1.03 ( , 3JHH = 7.4 Hz, 3H, CH3)majo ppm; 13C {1H} NMR (75 MHz,
DMSO)
146.4mino , 146.1majo , 134.4, 133.3, 133.0, 131.9, 131.8, 131.6, 131.5, 131.4, 131.3,
130.9, 130.8, 130.6, 130.5, 130.1, 129.1, 128.9, 128.8, 128.7, 128.6, 128.6, 128.3, 128.1, 127.1,
17
120.7, 120.5, 119.9, 116.3, 115.9, (CA ), 79.2 (d, 3JPC = 5.0 Hz, CH)mino , 77.3 (d, 2JPC = 5.2 Hz,
Cqua )mino , 76.8 (d, 2JPC = 4.5 Hz, Cqua )majo , 76.2 (d, 3JPC = 3.5 Hz, CH)majo , 35.6 (d, 1JPC =
106.0 Hz, CH)mino , 33.0 (d, 1JPC = 108.4 Hz, CH)majo , 26.3 (CH2), 20.4 (CH3)majo , 20.2
(CH3)mino , 9.3 (CH3)mino , 9.2 (CH3)majo ppm; 31P NMR (120 MHz, CDCl3) δ 25.1majo , 23.9mino
ppm; ESI-HRMS (CI) m/z calcd. o C24H25NO3P ([M+H]+) 406.1572, ound 406.1561.
((1S*,1aS*,7S*)-5-Fluo o-7-hyd oxy-1a-me hy-1,1a-dihyd o-7H-
azi ino[2,1-b]benzo[e][1,3]oxazin-1-yl) diphenylphosphine oxide
(4i). (1.90 g, 96%) as a whi e solid om 2H-azi ine 2c (1.27 g, 5 mmol,
1 eq) and 5- luo osalicylaldehyde 1d (0.84 g, 10 mmol, 1.2 eq) as
desc ibed in he gene al p ocedu e. The c ude p oduc was pu i ied by
c ys alliza ion om hexane/CH2Cl2 (50:50) o a o d he i le compound 4i. mp 169–171 ºC; IR
(nea ) max 3417, 2930, 2857, 1483, 1432, 1182, 1141, 732, 691 cm–1; 1H NMR (400 MHz,
CDCl3) δ 7.87-6.74 (m, 13H, A H), 5.92 (d, 3JHH = 4.3 Hz, 1H, CHOH)majo , 5.69 (d, 3JHH = 4.0
Hz, 1H, OH)majo , 5.47 (bs, 1H, CHOH)mino , 4.77 (bs, 1H, OH)mino , 2.91 (d, 2JPH = 21.7 Hz,
1H, CH-P)majo , 2.60 (d, 2JPH = 22.0 Hz, 1H, CH-P)mino , 1.85 (s, 3H, CH3)mino , 1.83 (s, 3H,
CH3)majo ppm; 13C {1H} NMR (75 MHz, DMSO)
159.1, 155.1, 144.6mino , 144.4majo , 134.3,
133.0, 133.0, 131.9, 131.7, 131.5, 131.4, 131.0, 130.9, 130.6, 130.5, 128.8, 128.8, 128.7, 128.3,
128.2, 122.0, 119.9, 117.9, 116.6, 116.3, 113.3, 113.0, (CA ), 79.3 (d, 3JPC = 4.7 Hz, CH)mino ,
75.7 (d, 3JPC = 3.0 Hz, CH)majo , 74.0 (d, 2JPC = 5.3 Hz, Cqua )mino , 73.6 (d, 2JPC = 4.3 Hz,
Cqua )majo , 35.2 (d, 1JPC = 105.5 Hz, CH)mino , 32.2 (d, 1JPC = 108.0 Hz, CH)majo , 20.1 (CH3)mino ,
19.7 (CH3)majo ppm; 31P NMR (120 MHz, CDCl3) δ 25.9majo , 24.5mino ppm; 19F {1H} NMR
(282 MHz, CDCl3) δ –119.8 ppm; ESI-HRMS (CI) m/z calcd. o C22H20FNO3P ([M+H]+)
396.1165, ound 396.1153.
4.1.3.2. Gene al p ocedu e and spec al da a o he eac ion o unc ionalized 2H-azi ines 2
wi h 2-hyd oxyace ophenone 5. To a 0 ºC solu ion o 2H-azi ine (5 mmol, 1 eq) 2 in CH2Cl2
(25 mL) and 4 Å M.S., was added d opwise 2-hyd oxyace ophenone 5 (1.20 mL, 10 mmol, 2
eq) and E 3N (2.09 mL, 15 mmol, 3 eq) unde a ni ogen a mosphe e. The eac ion mix u e was
allowed o each oom empe a u e and s i ed a he same empe a u e un il TLC showed he
disappea ance o 2H-azi ine 2 (72–120 h). 4 Å M.S. was il e ed h ough a sin e ed glass
acuum il a ion unnel wi h celi e and washed wi h CH2Cl2. The eac ion mix u e was washed
wi h wa e (315 mL) and ex ac ed wi h CH2Cl2 (15 mL). The o ganic laye s we e d ied o e
anhyd ous MgSO4, il e ed and concen a ed o d yness in acuum. The c ude p oduc was
pu i ied by c ys alliza ion o lash-column ch oma og aphy o a o d he co esponding azi ino-
benzoxazine de i a i es 6.
18
Diisop opyl ((1S*,1aS*,7S*)-7-hyd oxy-1a,7-dime hyl-1,1a-dihy-
d o-7H-azi ino[2,1-b]benzo[e][1,3]oxazin-1-yl)phosphona e (6a).
(0.63 g, 35%) as a whi e solid om 2H-azi ine 2a (1.27 g, 5 mmol, 1
eq) as desc ibed in he gene al p ocedu e a e s i ing a oom
empe a u e o 120 h. The c ude p oduc was pu i ied by lash-column ch oma og aphy (SiO2,
AcOE /hexane 50:50) o a o d he i le compound 6a. mp 122–124 ºC; IR (nea ) max 3331,
2981, 2934, 1612, 1587, 1374, 1280, 1228, 1011, 986 cm–1; 1H NMR (400 MHz, CDCl3) δ
7.48–7.46 (m, 1H, A H), 7.23–7.21 (m, 1H, A H), 7.06–6.99 (m, 1H, A H), 6.83–6.81 (m, 1H,
A H), 4.74–4.68 (m, 2H, CH), 3.27 (d, 3JPH = 5.7 Hz, 1H, OH)majo , 2.48 (bs, 1H, OH)mino , 2.38
(d, 2JPH = 12.3 Hz, 1H, CH-P)majo , 2.14 (d, 2JPH = 12.5 Hz, 1H, CH-P)mino , 1.90 (s, 3H,
CH3)mino , 1.84 (s, 3H, CH3)majo , 1.83 (s, 3H, CH3)mino , 1.63 (s, 3H, CH3)majo , 1.34–1.28 (m,
12H, CH3) ppm; 13C {1H} NMR (100 MHz, CDCl3)
148.1mino , 147.5majo , 130.4, 129.5, 126.6,
126.3, 126.2, 125.8, 123.2, 123.0, 117.4, 116.5 (CA ), 83.5 (d, 3JPC = 7.0 Hz, Cqua )mino , 83.5 (d,
3JPC = 4.9 Hz, Cqua )majo , 73.7 (d, 2JPC = 4.4 Hz, Cqua ), 71.4 (d, 2JPC = 6.4 Hz, OCH), 71.1 (d,
2JPC = 6.7 Hz, OCH), 32.0 (CH3), 31.4 (d, 1JPC = 211.5 Hz, CH), 24.2 (d, 3JPC = 3.4 Hz, CH3),
24.1 (d, 3JPC = 4.4 Hz, CH3), 24.1 (d, 3JPC = 3.0 Hz, CH3), 23.9 (d, 3JPC = 5.5 Hz, CH3), 20.3
(CH3)mino , 20.0 (CH3)majo ppm; 31P NMR (120 MHz, CDCl3) δ 19.6majo , 19.1mino ppm; ESI-
HRMS (CI) m/z calcd. o C17H27NO5P ([M+H]+) 356.1627, ound 356.1628.
((1S*,1aS*,7S*)-7-Hyd oxy-1a,7-dime hyl-1,1a-dihyd o-7H-
azi ino[2,1-b]benzo[e][1,3]oxazin-1-yl)diphenylphosphine oxide (6b).
(1.11 g, 57%) as a whi e solid om 2H-azi ine 2c (1.27 g, 5 mmol, 1 eq)
as desc ibed in he gene al p ocedu e a e s i ing a oom empe a u e o
96 h. The c ude p oduc was pu i ied by c ys alliza ion om E 2O and washed se e al imes
wi h pen ane o a o d he i le compound 6b. mp 163–165 ºC; IR (nea ) max 3250, 2977, 2930,
1613, 1594, 1435, 1185, 1157, 729 cm–1; 1H NMR (400 MHz, CDCl3) δ 7.76–6.82 (m, 14H,
A H), 2.79 (d, 2JPH = 21.5 Hz, 1H, CH-P)majo , 2.56 (bs, 1H, OH), 2.46 (d, 2JPH = 23.0 Hz, 1H,
CH-P)mino , 2.00 (s, 3H, CH3)mino , 1.90 (s, 3H, CH3)majo , 1.61 (s, 3H, CH3)majo , 1.20 (s, 3H,
CH3)mino ppm; 13C {1H} NMR (100 MHz, CDCl3 + CD3OD)
148.1mino , 147.4majo , 132.8,
132.4, 132.4, 132.3, 132.1, 131.5, 131.4, 131.2, 131.1, 131.0, 130.7, 130.3, 129.4, 129.2, 129.0,
128.9, 128.8, 128.7, 128.7, 128.6, 126.6, 126.3, 125.5, 123.3, 123.0, 122.4, 117.3, 116.4 (CA ),
82.7 (d, 3JPC = 3.5 Hz, Cqua ), 74.0 (d, 2JPC = 4.7 Hz, Cqua )majo , 73.6 (d, 2JPC = 5.7 Hz, Cqua )mino ,
35.7 (d, 1JPC = 107.1 Hz, CH)mino , 33.3 (d, 1JPC = 108.8 Hz, CH)majo , 32.2 (CH3), 20.5
(CH3)mino , 20.1 (CH3)majo ppm; 31P NMR (160 MHz, CDCl3) δ 25.2majo , 24.2mino ppm; ESI-
HRMS (CI) m/z calcd. o C23H23NO3P ([M+H]+) 392.1416, ound 392.1403.
19
((1S*,1aS*,7S*)-1a-E hyl-7-hyd oxy-7-me hyl-1,1a-dihyd o-7H-
azi ino[2,1-b]benzo[e][1,3]oxazin-1-yl)diphenylphosphine oxide (6c).
(1.25 g, 62%) as a yellow solid om 2H-azi ine 2d (1.34 g, 5 mmol, 1 eq)
as desc ibed in he gene al p ocedu e a e s i ing a oom empe a u e o
72 h. The c ude p oduc was pu i ied by c ys alliza ion om E 2O and
washed se e al imes wi h pen ane o a o d he i le compound 6c. mp 147–179 ºC; IR (nea )
max 3253, 2974, 2924, 1619, 1587, 1442, 1163, 910, 729, 698 cm–1; 1H NMR (400 MHz,
CDCl3) δ 7.79–6.85 (m, 14H, A H), 2.87 (d, 2JPH = 21.7 Hz, 1H, CH-P)majo , 2.74 (bs, 1H, OH),
2.53 (d, 2JPH = 23.1 Hz, 1H, CH-P)mino , 2.32–2.12 (m, 2H, CH2), 1.64 (s, 3H, CH3), 1.22–1.12
(m, 2H, CH3) ppm; 13C {1H} NMR (100 MHz, CDCl3)
148.4mino , 147.7majo , 133.2, 132.5,
132.2, 132.2, 132.1, 132.1, 132.1, 131.8, 131.7, 131.5, 131.3, 131.2, 129.6, 128.9, 128.7, 128.6,
128.5, 126.4, 124.9, 123.2, 116.8 (CA ), 82.9 (d, 3JPC = 3.4 Hz, Cqua ), 78.2 (d, 2JPC = 4.7 Hz,
Cqua ), 34.7 (d, 1JPC = 108.2 Hz, CH), 32.9 (CH3), 26.9 (CH2), 9.2 (CH3) ppm; 31P NMR (120
MHz, CDCl3) δ 25.0majo , 23.9mino ppm; ESI-HRMS (CI) m/z calcd. o C24H25NO3P ([M+H]+)
406.1572, ound 406.1558.
4.2. Biology
4.2.1. 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 DMSO. The inal concen a ion o DMSO ne e exceeded 10% ( / )
in eac ions. The s ock solu ion was s o ed a 5 ºC un il i was used.
4.2.2. Cy o oxici y assays.
Cells we e cul u ed acco ding o he supplie ´s ins uc ions. Cells we e seeded in 96-well pla es
a a densi y o 2–4 x 103 cells pe well and incuba ed o e nigh in 0.1 mL o media supplied
wi h 10% Fe al Bo ine Se um (Lonza) in 5% CO2 incuba o a 37 ºC. On day 2, d ugs we e
added and samples we e incuba ed o 48 hou s. A e ea men , 10 μL o cell coun ing ki -8
was added in o each well o addi ional 2 hou s incuba ion a 37 ºC. The abso bance o each
well was de e mined by an Au oma ic Elisa Reade Sys em a 450 nm wa eleng h.
20
Acknowledgmen s
The au ho s hank Minis e io de Ciencia, Inno ación y Uni e sidades (MCIU); Agencia
Es a al de In es igación (AEI) y Fondo Eu opeo de Desa ollo Regional (FEDER; RTI2018-
101818-B-I00, UE), and Gobie no Vasco (GV, IT 992-16) o suppo ing his wo k. We also
hank o echnical and human suppo p o ided by SGIke (UPV/EHU/ ERDF, EU).
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