Design, synthesis and structure-activity relationship studies of a novel focused library of 2,3,4-substituted oxazolidines with antiproliferative activity against cancer cell lines

Article abstract of DOI:10.1016/j.ejmech.2017.06.022

In the present work we describe the synthesis and antiproliferative evaluation of a focused library of 30 novel oxazolidines designed by modification of N-substituent, by ring variation, by alkyl variation or by extension of the structure. It was noted that carbamate and N,O-aminal groups were essential for activity. In general, replacement of the phenyl ring with pyridinyl was not tolerated. However, the introduction of a second phenyl ring with an appropriate spacer at the 3- or 4-position of the first phenyl ring generally enhanced the cytotoxic profile. Among all the prepared compounds, 24 was the most potent compound found in this class, being active on four of five cancer cell lines and it was 5-fold and 10-fold more potent than the lead compounds against HL60 and JURKAT cells, respectively. In addition, it showed relevant activity against MCF-7 and HCT-116 cells, which were resistant to lead. Moreover, 24 showed little antiproliferative activity against VERO, indicating low toxicity to normal cells. Thus, this compound has the potential to be developed as an anticancer agent.

Full text of DOI:10.1016/j.ejmech.2017.06.022

Accepted Manuscript
Design, synthesis and structure-activity relationship studies of a novel focused library
of 2,3,4-substituted oxazolidines with antiproliferative activity against cancer cell lines
Saulo F. Andrade, Bárbara G. Oliveira, Larissa C. Pereira, Jonas P. Ramos, Angélica
R. Joaquim, Martin Steppe, Elaine M. Souza-Fagundes, Ricardo J. Alves
PII:
S0223-5234(17)30468-3
10.1016/j.ejmech.2017.06.022
EJMECH 9517
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 October 2016
Revised Date: 31 May 2017
Accepted Date: 12 June 2017
Please cite this article as: S.F. Andrade, Bá.G. Oliveira, L.C. Pereira, J.P. Ramos, Angé.R. Joaquim, M.
Steppe, E.M. Souza-Fagundes, R.J. Alves, Design, synthesis and structure-activity relationship studies
of a novel focused library of 2,3,4-substituted oxazolidines with antiproliferative activity against cancer
cell lines, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.06.022.
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ACCEPTED MANUSCRIPT
Design, synthesis and structure-activity relationship studies of a novel
focused library of 2,3,4-substituted oxazolidines with antiproliferative
activity against cancer cell lines
Saulo F. Andradea,b, Bárbara G. Oliveiraa, Larissa C. Pereiraa, Jonas P. Ramosc,
Angélica R. Joaquimb, Martin Steppeb, Elaine M. Souza-Fagundesc and Ricardo J.
Alvesa,*
aDepartamento de Produtos Farmacêuticos, Faculdade de Farmácia, Universidade
Federal de Minas Gerais (UFMG), Av. Antônio Carlos, 6627, Belo Horizonte, MG
31.270-901, Brazil
bPrograma de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal do Rio
Grande do Sul (UFRGS), Av. Ipiranga, 2752, Porto Alegre, RS 90610-000, Brazil
cDepartamento de Fisiologia e Biofísica, Instituto de Ciências Biológicas, Universidade
Federal de Minas Gerais (UFMG), Brazil
*Corresponding author
Tel: + 55 31 3409 6955
Fax: + 55 31 3409 6935
ricardodylan@farmacia.ufmg.br
ABSTRACT
In the present work we describe the synthesis and antiproliferative evaluation of a
focused library of 30 novel oxazolidines designed by modification of N-substituent, by
ring variation, by alkyl variation or by extension of the structure. It was noted that
carbamate and N,O-aminal groups were essential for activity. In general, replacement of
the phenyl ring with pyridinyl was not tolerated. However, the introduction of a second
phenyl ring with an appropriate spacer at the 3- or 4-position of the first phenyl ring
generally enhanced the cytotoxic profile. Among all the prepared compounds, 24 was
the most potent compound found in this class, being active on four of five cancer cell
lines and it was 5-fold and 10-fold more potent than the lead compounds against HL60
and JURKAT cells, respectively. In addition, it showed relevant activity against MCF-7
and HCT-116 cells, which were resistant to lead. Moreover, 24 showed little
antiproliferative activity against VERO, indicating low toxicity to normal cells. Thus,
this compound has the potential to be developed as an anticancer agent.
Keywords: Oxazolidines, Chiral, Anti-cancer, Pro-apoptotic, Library

ACCEPTED MANUSCRIPT
Graphical Abstract
Oxazolidine: essential for the activity
Methyl groups: important for the activity
O
SAR
Carbamate: important
for the activity
O
O
O
N
O
Extension of
the structure
O
O
N
O
Replacement with pyridinyl
decrease the activity
O
N
Second ring and
appropriate spacer:
important for the activity
H
2- X = NO2
3- X = COOCH3
X
IC50 (µM)
IC50 (µM)
Compound 3
HL60 - 32 ± 11; JURKAT - 48 ± 4;
MCF-7 - >50; HCT116 - >50
HL60 - 7 ± 1; JURKAT - 5 ± 3
MCF-7 - 31 ± 6 ; HCT116 - 17 ± 1
Highlights
► Focused library of 30 novel chiral oxazolidines was prepared.
► Antiproliferative activity of compounds was assessed on cancer cell lines.
► Some relevant structural requirements for cytotoxic activity were found.
► Introduction of a second ring with appropriate spacer enhanced the activity.
► Compound 24 was more potent than lead on four of five cancer cell lines.

ACCEPTED MANUSCRIPT
1. Introduction
Chemotherapy plays a central role in the treatment of cancer. It assists in curing the
disease and/or in improving the quality of the patient’s life [1]. In recent decades,
several new drugs have been launched to treat different types of cancer that did not
respond to chemotherapy. However, many currently available drugs show severe side
effects and dose-limiting toxicity. In addition, resistance of tumor cells to multiple
structurally unrelated cytotoxic drugs (multidrug resistance) is frequently observed
[1,2]. As successful cancer treatment remains a challenging goal, several groups
continue make efforts to discover novel, selective and less toxic chemotherapeutic
agents [3-5].
During an ongoing program to identify new anticancer compounds we reported the
antiproliferative activity of compound 1 against leukemia cell lines, with low toxicity
against human peripheral blood mononuclear cell - PBMC (Fig. 1) [6]. In order to shed
some light on the mechanism responsible for the cytotoxicity and antiproliferative
properties, investigations were carried out. It was observed that compound 1 induced
exposure of phosphatidylserine and DNA fragmentation on HL60 cells, which could be
blocked by pretreatment of cells with Z-VAD-FMK. In addition, compound 1 induced
caspase-3 activation and an additional analysis revealed an increased percentage of
apoptotic HL60 cells after treatment with 1 [6]. These results indicated that 1 has great
pro-apoptotic potential. This is an important finding because apoptosis is one of the
most important pathways used in targeting the discovery of new anticancer drugs [7].
O
O
O
N
O
X
1- X = 3-NO2
2- X = 4-NO2
3- X = 4-COOCH3
Fig. 1. Structure of cytotoxic oxazolidine 1-3.
Encouraged by the promising activity of compound 1, we performed an initial study on
its structure-activity relationship. In this study, we prepared 25 compounds to evaluate
the importance of ring substituent and stereochemistry of oxazolidine on anticancer
activity [8]. It was observed that introduction of hydrophobic and electron withdrawing
COOCH3 or NO2 groups were important for the activity of this class of compound. The
unsubstituted compounds (X = H) were inactive against cancer cell lines. The presence
of hydrophilic and electron withdrawing COOH, or hydrophobic and electron donor
OMe, resulted in poor activity. Regarding the substituent position, a substituent at the 3-
or 4-position was important for the activity. All ortho-substituted compounds were
inactive. The S isomers were generally more active than their enantiomers; in some
cases, S isomers were 10 times more potent. Finally, no significant difference in activity
was observed between S isomers bearing COOCH3 and NO2 at the 3- or 4-position.
However, para-substituted compounds appeared to be more selective than were meta-
substituted against cancer cell lines. Based on these results, it appeared to us that
compounds 2 and 3 provided good leads for further chemical modification (Fig. 1).

ACCEPTED MANUSCRIPT
In a second study, we described the synthesis and evaluation of a series of oxazolidines
designed by isosteric replacement or rigidification of the oxymethylene spacer of
compounds 2-3 [9]. We found that alkene derivatives (E or Z) were more active than
were lead compounds against LNCaP cells, but they retained their activity against
MDA-MB-231 cells and were slightly more potent against HL60 and JURKAT cells.
Considering the difficulty of obtaining the alkenes, and the modest improvement, it
appeared reasonable to continue using compounds 2-3 as lead compounds for further
chemical modification.
In a continued effort to enhance the cytotoxic profile of this new class, we decided to
investigate the importance of other functionalities for antiproliferative activity. The
purpose of this study was to prepare a new series of oxazolidines designed by
modification of N-substituent, by ring variation, by alkyl variation, or by extension of
the structure, and to evaluate this new series against cancer cells in order to establish the
relevant structural requirements for cytotoxic activity in order to obtain more potent and
selective compounds (Fig. 2).
O
O
O
N
Y = Naphthyl, pyridinyl or
substituted pyridinyl
O
Y
11-16
Ring variation
O
O
O
R
R
O
O
Z
Modification of
N-substituent
O
N
N
O
Alkyl variation
N
O
O
O
O
17-18
4-10
2- X = NO2
3- X = COOCH3
O2N
R = H or -CH2CH2CH2CH2-
X
O2N
Z = BnO, alkyl, Ph,
phenylethyl or phenylethenyl
Extension of the structure
O
W = Appropriate spacer or Ph
A = Alkyl, Ph or aryl
O
N
O
O
A
W
19-31
Fig. 2. Design of new series of oxazolidines.
2. Results and discussion
2.1. Chemistry
Initially, key compounds 1-3, 32 and 35 were prepared as previously reported (Fig. 3)
[8]. In brief, commercially available -serine was protected with tert-butoxycarbonyl
D
and the carboxylic acid was converted to the methyl ester by treatment with methyl
iodide and potassium carbonate to give 33 in 78% overall yield [10,11]. Acetonide
formation of 33 was carried out using 2,2-dimethoxypropane (DMP) and BF3.OEt2 to
afford acetonide 34 in 77% yield [12]. Next, the methyl ester of acetonide 34 was
reduced to alcohol 35 using NaBH4 in 85% yield [13]. Lastly, reaction of alcohol 35
with appropriate substituted phenol in Mitsunobu conditions provided 1-3 or 32 in 63-
73% yield [14].

ACCEPTED MANUSCRIPT
O
O
O
O
O
O
O
N
NH2
O
N
O
NH
O
OH
a, b
O
O
N
OH
HO
c
d
H3CO
O
H3CO
e
HO
X
1- X = m-NO2
2- X = p-NO2
3- X = p-COOCH3
32- X = m-COOCH3
O
O
35
D-serine
33
34
Fig. 3. Preparation of key compounds 1-3, 32 and 35. Reagents and conditions: a)
Boc2O, NaOH, t-BuOH/H2O (1:1); b) CH3I, K2CO3, DMF; c) DMP, BF3.OEt2, acetone;
d) NaBH4, THF/MeOH (7:3), 0 ºC → reflux; e) Appropriate substituted phenol, PPh3,
DIAD, toluene, 80 ºC.
In order to prepare the N-substituent variation series, an initial attempt to selectively
remove Boc in presence of N,O-aminal was carried out (Fig. 4). We did not find in the
literature a protocol for this selective removal; however, Murray and coworkers report
on the Boc deprotection in presence of acetal using ZnBr2 [15]. Despite several attempts
to employ their method, including variation of solvent (THF or CH2Cl2), time (5–24 h)
and temperature (25–65 ºC), we just recovered starting material. Bu4NF and
HCl/organic solvent systems were also investigated [16,17]. After five days, only
starting material was recovered using Bu4NF (10 eq, THF, reflux). Selective N,O-
aminal removal was observed with another method. Treatment of compound 2 with a
saturated solution of HCl in THF gave 37 in 45% yield (Fig. 4). The reaction was
monitored for 9 h. After 3 h, no evolution was observed and large amount of starting
material was detected, even after 9 h. Thus, it was necessary to purify the product by
silica gel column chromatography. Considering that compound 37 is a key intermediate
for the synthesis of the N-alkyl variation series, we decided to find a better method to
accomplish this transformation. After trying different conditions, we found that
treatment of compound 2 with TsOH (5 eq) in MeOH/water (95:5) under reflux gave 37
in 52% yield in good purity without using column chromatography [18].
O
O
H
N
O
O
O
O
N
NH
O
O
d or e
OH
a, b or c
O
x
36
2
O2N
O2N
37
O2N
Fig. 4. Selective removal of Boc and N,O-aminal attempts. Reagents and conditions: a)
ZnBr2 (5 eq), CH2Cl2 or THF, 5 or 24 h, 25 or 65 ºC; b) Bu4NF (10 eq), THF, reflux, 5
d; c) HCl, THF or (CH3)2CO, r. t., 9 h; d) HCl, THF, r. t.; e) TsOH, MeOH/H2O (95:5),
reflux.
Given the difficulty of removing Boc, this protecting group was replaced by Cbz which
can be smoothly removed by hydrogenolysis (Fig. 5). In this case we used a precursor
bearing a COOMe group instead of NO2 because of the well-known susceptibility of
this latter group to reduction with H2. Thus, compound 40 was prepared from D-serine
in a very similar manner to that previously described in Fig. 3 for Boc derivatives
[13,14]. Unfortunately, catalytic hydrogenation or catalytic-transfer hydrogenation of

ACCEPTED MANUSCRIPT
compound 40 did not furnish the desired product 41 [19]. Instead, we observed only the
formation of the product 42 resulting from simultaneous Cbz and N,O-aminal
deprotection (Fig. 5).
We did not find any previous report of hydrogenolysis of N,O-aminal in this type of
compound. We concluded that compounds of this class bearing N-unsubstituted N,O-
aminal are probably unstable and cannot be isolated. We were thus forced to abandon
our initial synthetic strategy.
O
O
O
O
N
O
O
NH2
N
O
O
N
O
H3CO
O
HO
OH
3 steps
b
a
HO
O
O
39
40
38
H3COOC
D
-serine
c,d
NH2
X
H
N
OH
O
O
O
H3COOC
42
41
H3COOC
Fig. 5. Preparation of compound 40 and Cbz-deprotection attempt. Reagents and
conditions: a) NaBH4, THF/MeOH (7:3), 0 ºC → reflux; b) Nipagin, DIAD, PPh3,
Toluene, 80 ºC; c) H2, Pd-C (10%), THF or MeOH, r. t., 2 h; d) HCOONH4 (4 eq), Pd-C
(10%), Isopropanol, 80 ºC, 30 min.
A novel strategy was adopted, as shown in Fig. 6. Initially, N,O-aminal and Boc were
simultaneously removed by treatment of compound 2 with TFA in CH2Cl2 to give
amine 43 in quantitative yield [20]. The commercially available carboxylic acids 44a-f
were activated with NHS/EDC to afford activated esters 45a-f in 80-100% yield [21].
The esters 45a-f were reacted with amine 43 to give amides 46a-f in 40-56% yield [21].
An initial attempt to introduce N,O-aminal was carried out using DMP and BF3.Et2O as
previously described (Fig. 3). However, in this case this method did not lead to
acetonide formation. Nitrogen is more nucleophilic in carbamate than in amide, due to
carbonyl cross-resonance with oxygen. Thus, more drastic conditions were employed to
accomplish acetonide formation. Amides 46a-f were reacted with DMP using catalytic
TsOH under reflux to give the N-substituted series 5-10 in 36-75% yield [22].
Compound 4 (Z = Benzyloxy) was prepared in a similar manner as described for 40
(Fig. 5).

ACCEPTED MANUSCRIPT
O
O
NH3
O
Z
O
OH
O
O
N
NH
a
O
d
.
c
OH
Z
O
N
CF3COO
O
O2N
O
43
46 a-f
O2N
O2N
2
5-10
O
O
O2N
O
N
b
Z
OH
Z
O
44 a-f
O
45 a-f
44a, 45a,
46a, 5
44c, 45c,
46c, 7
44b, 45b,
46b, 6
Z =
H3C
44d, 45d,
46d, 8
44e, 45e, 44f, 45f,
46f, 10
46e, 9
Fig. 6. Preparation of N-substituent variation series. Reagents and conditions: a)
CF3COOH, CH2Cl2; b) EDC, NHS, CH2Cl2; c) NaHCO3, THF/H2O (1:1); d) DMP,
TsOH, Toluene/CHCl3 (6:4), 80 ºC.
The alkyl variation series was prepared from N,O-aminal deprotected compound 37
(Fig. 7). Treatment of 37 with paraformaldehyde in presence of catalytic amount of
TsOH (5 mol%) in toluene at 85 ºC gave 17 in 36% yield (Fig. 7) [23]. This modest
yield could be explained by the formation of a little more polar byproduct, which was
identified as compound 47 (Fig. 8). The proposed mechanism for the ring formation is
shown in Fig. 8 and was rationalized as follows. Partial cleavage of paraformaldehyde
would give intermediate I that could react with 37 to give the byproduct 47 in 25%
yield. To the best of our knowledge, this is the first report of the formation of a
tetrahydro-1,3,5-dioxazepine ring by this method. Derivative 18 was prepared from 37
using cyclopentanone and TsOH in toluene at 80 ºC in 40% yield with 48% of starting
material recovered [24]. We tried to improve the yield by increasing temperature or
reaction time, but in both cases we observed the formation of a polar product resulting
from Boc and N,O-aminal cleavage. Thus, poor yields were obtained in these cases.
O
O
R
R
O
O
O
O
N
NH
O
OH
a
17 - R = H
18 - R = -CH2CH2CH2CH2-
O2N
37
O2N
17-18
Fig. 7. Preparation of alkyl variation series. Reagents and conditions: a)
Paraformaldehyde or cyclopentanone, TsOH, toluene, 80 or 85 ºC.

ACCEPTED MANUSCRIPT
O
Heat
H
n
Heat
H
n
1.
2.
HO
O
HO
O
OH
HO
O
Solvent
H
H
Toluene
I
p-Formaldehyde
Formaldehyde
+H+
-H2O
HO
O
H2O
O
OH
OH
HO
O
OH
I
Boc
Boc
Boc
H2O
O
NH
Boc
+H+
NH
-H+
-H2O
NH
+
OH
O
OH
O
O
O
37
O
O
O
O
O
N
-H+
NH
O
O
47
O2N
Fig. 8. Proposed mechanism for the tetrahydro-1,3,5-dioxazepine ring formation.
In order to prepare the extension of the structure series, methyl esters 3 and 32 were
hydrolyzed under alkaline conditions to give acids 48 and 49 in 83 and 94% yield,
respectively (Fig. 9) [8,25]. Acid 48 was activated with NHS/EDC to afford activated
ester 50 in quantitative yield. The ester 50 was coupled with the appropriate amine to
give amides 19-22 in 60-90% yield (Fig. 9) [21]. Treatment of acids 48 or 49 with EDC,
DMAP and the appropriate aniline gave the amides 23 or 28 in 48 and 77% yield,
respectively (Fig. 9) [26].
The retro-amide and -sulfonamide series were prepared from nitro derivatives 1-2 (Fig.
9). Initially, the nitrocompounds were reduced with H2 in presence of Pd-C to give
anilines 51 and 52 that were used immediately without further purification. Anilines
were coupled with p-toluic acid, mesyl or tosyl chloride to afford the retro-amides 24
and 29 in 57-60% yield (2 steps) or retro-sulfonamides 25, 26 and 30 in 40-70% yield (2
steps) [26,27].
Finally, the ring variation series 11-16 and biphenyl derivatives 27 and 31 were
synthesized from the previously described alcohol 35 (Fig. 10). This compound was
treated with appropriate naphthol, hydroxypyridine or phenylphenol under Mitsunobu
conditions to give these derivatives in 31-68% yield [14].

ACCEPTED MANUSCRIPT
O
O
O
O
N
O
O
O
O
O
N
N
O
O
O
a
O
O
b ou c
O
A
48 - para
49 - meta
19-23
28
3 - para
32 - meta
HO
H3CO
H
H
N
H
N
N
N
m-
p-
p-
p-
p-
p-
A =
N
N
22
N
H
19
20
21
23
28
O
O
O
O
O
O
O
N
N
N
O
O
O
O
d
O
A
e or f
O2N
N
H
H2N
24-26
29-30
2- para
1- meta
51- para
52- meta
O
O O
S
O O
S
A =
p-
m-
m-
p-
p-
S
O O
O
24
25
26
30
29
Fig. 9. Preparation of amides (19-22; 23 and 28), retro-amides 24-26 and -sulfonamides
29-30: a) NaOH, (CH3)2CO/H2O (3:1); b) I- EDC, NHS, CH2Cl2; II- Appropriate
amine, CH2Cl2 (compounds 19-22); c) I- EDC, DMAP, CH2Cl2; II- Appropriate aniline
(compounds 23 and 28); d) H2, Pd-C, THF; e) Amides: p-toluic acid, EDC, DMAP,
CH2Cl2/THF; f) Sulfonamides: appropriate sulfonyl chloride, CH2Cl2/THF/Pyridine.
N
Cl
11
12
13
O
O
O
O
N
O
N
a
O
O
Y =
N
N
HO
O2N
N
Y
14
15
16
11-16
35
31
27
Fig. 10. Preparation of ring variation series. Reagents and conditions: a) Appropriate
naphthol, hydroxypyridine or phenylphenol, PPh3, DIAD, toluene or THF, 80 ºC.
2.2. Biological evaluation
2.2.1. Antiproliferative activity
The antiproliferative activity of prepared compounds was assessed on five human
cancer cell lines, namely, HL60 promyelocytic leukemia, JURKAT T-cell leukemia,
MDA-MB-231 and MCF-7 breast carcinoma cells and HCT-116 colon carcinoma cells
[28]. Cytotoxic effects on normal cells were evaluated using VERO African green

ACCEPTED MANUSCRIPT
monkey kidney cells. The results are summarized in Table 1 and expressed as the
concentration of drug inhibiting cell growth by 50% (IC50).
In general, compounds 4 (p-NO2) and 40 (p-COOCH3) bearing a Cbz group were as
active against these five cell lines as were leads 2 (p-NO2) and 3 (p-COOCH3) bearing
Boc. The isosteric replacement of carbamate oxygen with methylene resulted in poor
activity. Amides 5-10 were inactive in almost all cases. Amides with neopentyl (5), t-
butyl (8) and methyl (9) aliphatic substituents were inactive against all lines. These
results indicated that a carbamate group is essential in this case and replacement by an
amide is not tolerated. However, compound 6, which is methylene isoster of 4 (p-NO2,
Cbz) retained activity against HL60 cells although it was less active against MDA-MB-
231. Concerning HL60 cells, comparing the activity of 6 with 7 or 10, one observes that
rigidification or carbon chain contraction resulted in poor activity.
The ring variation series showed variable results. Replacement of the phenyl ring with
piridinyl did not enhance the activity. Compounds 13 and 14 bearing a 2-pyridinyl
moiety, substituted at position 5 with an electron withdrawing group, were less active
than lead 2, indicating that the insertion of nitrogen at the 2-position is unfavorable.
Compounds 15 and 16 that possessed 3-pyridinyl or 4-pyridinyl were inactive against
all cancer cell lines. This shows that bioisosterism is not observed with these rings.
Concerning naphthyl analogues 11-12, introduction of a new phenyl ring at the 2- and
3-positions was not well tolerated, since compound 11 had little activity. However,
introduction of a new ring at the 3- and 4-position was favorable. Compound 12 was
more active than leads 2 and 3 against leukemia, MCF-7 and HCT-116 cells and
retained the activity against MDA-MB-231 cell line. This result suggests that bulky
groups in this region could be accommodated in a hydrophobic pocket of a bioreceptor.
Interestingly, 12 possesses a naphthyl ring that is electron-rich, and we have reported
previously that electron withdrawing groups enhance activity. Hence, bulky electron
withdrawing groups could increase the biological activity of this class. This is why we
planned the extension of the structure series.
Compound 37, which did not possess N,O-aminal, was inactive against all lines, thus
indicating that the oxazolidine ring is essential for the activity of this class. As expected,
compound 43 resulting from N,O-aminal and Boc removal was also inactive.
Compound 17 without N,O-aminal methyl groups was two-fold less potent than lead 2
against HL60 cell line and it was inactive on MDA-MB-231 cells, indicating that
methyl groups are important for the activity. In addition, compound 18 bearing a spiro
ring was more active than 2 on leukemia cells suggesting that bulky groups in this
region enhance the activity of this class. In addition, 18 was less cytotoxic against
VERO cells (IC50 > 100 µM) indicating low toxicity towards normal cells. The
unexpected product 47 that possessed a tetrahydro-1,3,5-dioxazepine ring was less
potent than 17 against HL60 cells. Thus, we concluded that ring expansion was not
favorable.
Turning to the extension of the structure series, in most cases these compounds retained
or showed enhanced activity as compared to lead 3. In this series, the ester group was
replaced by amide or sulfonamide in order to get more stable compounds. Amides 19-
21 bearing propyl, isopropyl or diethyl substituents retained their activity on leukemia
lines, except in one case. Compound 19 (n-propyl) was two-fold more potent than lead
against JURKAT cells. Nevertheless, the methylpiperazinamide derivative 22 was
inactive against all cell lines. Analysis of the results obtained with compounds 19–22
suggested that, in general, no enhancement in activity was observed by increasing alkyl
chain length. On the other hand, amide 23, bearing a phenyl ring substituent, was more

ACCEPTED MANUSCRIPT
active than 3 against leukemia cells. This result is in accordance with that previously
observed with naphthyl derivative 12.
Table 1
Antiproliferative activity of novel oxazolidines series against cancer cells.
Compd IC50 (µM)a
HL60
28 ± 2
32 ± 11
24 ± 7
>50
36 ± 5
>50
>50
>50
>50
>50
16 ± 1
35 ± 15
>50
>50
>50
57 ± 1
15 ± 3
34 ± 16
46 ± 14
26 ± 4
>50
12 ± 1
7 ± 1
14 ± 0
>50
JURKAT MDA-MB-231 MCF-7 HCT-116 VERO
>50
48 ± 4
>50
>50
>50
>50
>50
>50
>50
>50
28 ± 1
>50
>50
>50
37 ± 2
25 ± 6
48 ± 14
>50
>50
>50
>50
>50
>50
55 ± 1
42 ± 1
>50
>50
>50
>50
>50
>50
>50
47 ± 11
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
35 ± 7
>50
>50
>50
>50
NDb
>50
>50
>50
>50
>50
>50
31 ± 6
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
25 ± 5
>50
>50
>50
>50
NDb
>50
>50
>50
>50
>50
>50
17 ± 1
50 ± 7
>100
>100
>100
>100
>100
>100
>100
>100
>100
44 ± 3
>100
>100
>100
>100
NDb
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
37
40
43
47
Cisc
>50
>50
42 ± 2
20 ± 11
39 ± 2
44 ± 8
>50
20 ± 4
5 ± 3
14 ± 7
>50
>100
82 ± 38
73 ± 1
>100
NDb
>100
>100
46 ± 4
NDb
59 ± 17 63 ± 5
NDb
>50
NDb
>50
>50
>50
NDb
18 ± 3
19 ± 1
21 ± 2
>50
>50
30 ± 6
>50
21 ± 0
>50
>50
>50
49 ± 2
>50
>50
>50
>50
>50
>100
>50
>50
31 ± 8
>50
>50
>50
>50
>50
>50
>50
>50
>100
>100
>100
NDb
>100
>100
>100
NDb
22 ± 2
>50
NDb
40 ± 3
NDb
>50
>50
>50
>50
89 ± 4
3 ± 1
NDb
92 ± 3
NDb
79 ± 18
31 ± 6
26 ± 1
a Each data represents mean ± SD of at least three independent experiments.
b Not determined.
c Cisplatin was used as a positive control.
The retro-amide and -sulfonamide series showed good results. Retroamide 24 was the
most potent compound found in this class to date. This compound was active on four
out of five cancer cell lines and it was about 5 and 10-fold more active than lead 3
against HL60 and JURKAT cell lines, respectively. In addition, it had relevant activity

ACCEPTED MANUSCRIPT
against MCF-7 and HCT-116 cells which were resistant to leads. Moreover, 24 showed
little antiproliferative activity against VERO indicating low toxicity to normal cells.
Curiously, it possesses an electron donor nitrogen bonded to the first phenyl ring and it
has been noted before that introduction of an electron donor group results in poor
activity. Thus, this good profile could be attributed to the low electron density in the
second ring due to the presence of carbonyl. This hypothesis is apparently confirmed by
comparing the activity of 24 with 23, since 24 was more active against all cell lines.
Isosteric replacement of amide with sulfonamide resulted in decreased activity. Retro-
sulfonamide 25 was less potent than 24 against all lines and 25 had antiproliferative
activity on normal cells. Mesyl retro-sulfonamide 26 and p-biphenyl compound 27 were
inactive against all cancer cells. These findings indicate that, in general, introduction of
an electron donor group results in poor activity and that the second phenyl ring, as well
as the amide or sulfonamide spacer between the phenyl rings, are important for the
activity of compounds 24 and 25. Comparing the activity of retro-amide 24 and its
meta-derivative 29, it was noted that in this case para-substitution was favorable.
Compound 24 was more active than 29 against all cancer cells, being at least ten-fold
more potent on JURKAT cells. However, this same effect was not observed in the case
of retro-sulfonamides. Compounds 25 and 30 showed comparable activity.
2.2.2. DNA fragmentation induction
We also investigated the cytotoxicity of compounds by evaluating DNA fragmentation
as indicative of pro-apoptotic potential. The assay was based on the principle that
apoptotic cells, among other typical features, are characterized by DNA fragmentation
and, consequently, loss of nuclear DNA content. The use of a fluorochrome such as
propidium iodide, which is capable of binding and labeling DNA, makes it possible to
obtain a rapid and precise evaluation of cellular DNA content by flow cytometric
analysis and subsequent identification of hypodiploid cells [29].
Some of the most potent compounds (2, 3, 4, 12, 18, 23, 24, 25 and 30) were incubated
at 50 µM with HL60 or JURKAT cells. After 24 h, DNA fragmentation was evaluated.
As demonstrated in Fig. 11, significant increases in DNA fragmentation were detected
after treatment with all compounds at 50 µM in HL60 cells. However, only compounds
designed by extension of the structure, 24, 25 and 30 (retro-amide and -sulfonamide
series) induced relevant DNA fragmentation on JURKAT cells. This could explain the
good results obtained with this strategy. These three compounds showed good activity
against these two cell lines. Thus, apparently the cytotoxicity of this class is related to
pro-apoptotic potential. In addition, compounds 2, 3, 4, 12, 18 and 23 were, in general,
less active than 24, 25 and 30 and induced less DNA fragmentation on JURKAT cells.

ACCEPTED MANUSCRIPT
Fig. 11. Effect of compounds 2, 3, 4, 12, 18, 23, 24, 25 and 30 on the DNA content of
HL60 and JURKAT cells. DNA content was assessed by staining with propidium iodide
and flow cytometric analysis. Representative data with means ± SD from two
independent experiments performed in duplicate.
3. Conclusions
In summary, we have described herein the synthesis and cytotoxic evaluation of four
series of analogues of chiral oxazolidines 2 and 3. Some important structure-activity
relationship findings were revealed. The presence of a carbamate group and the
oxazolidine ring were essential for the activity. The introduction of a new ring at the 3-
and 4-position was favorable. Removal of N,O-aminal methyl groups decreased the
activity, while good results were obtained with the extension of the structure strategy. In
general, these compounds retained or enhanced their activity in comparison with lead 3.
In most cases, amides bearing an aliphatic substituent (19–21) retained the activity on
leukemia lines. The introduction of a second phenyl ring with an appropriate spacer at
the 3- or 4-position of the first phenyl ring generally enhanced the cytotoxic profile.
Among all prepared compounds, 24 was the most potent, being active on four out of
five cancer cell lines and it was about five and ten-fold more potent than lead against
HL60 and JURKAT cells. In addition, it showed relevant activity against MCF-7 and
HCT-116 cells which were resistant to lead, and did not show cytotoxicity towards
normal cells. Thus, compound 24 can be considered for further development as a
potential chemotherapeutic agent.
4. Experimental section
4.1. General
1
Melting points were determined on Microquímica MQAPF 301 apparatus. The H
and 13C NMR spectra were obtained on a Bruker Avance DPX-200 or DRX-400
spectrometer. The proton and carbon chemical shifts (δ) are given with respect to TMS.
Optical rotations were measured on an ADP220 Bellinghan + Stanley Ltd polarimeter.
IR spectra were recorded on a Spectrum One, Perkin-Elmer ATR system. Reactants
were obtained from commercial suppliers and used without further purification. Column
chromatography was performed on silica gel 60 0.063-0.200 mm/70-230 mesh Merck.
Flash column chromatography was performed on silica gel 60 0.040-0.063 mm/230-400

ACCEPTED MANUSCRIPT
mesh Merck. THF and toluene were dried over Na/benzophenone and distilled prior to
use.
4.2. Synthesis of (R)-2-[[(1,1-dimethylethoxy)carbonyl]amino]-3-(4-nitrophenoxy)-1-
propanol 37
To a stirred solution of 2 (0.045 g, 0.128 mmol) in a mixture of MeOH/H2O 95:5 (3
mL) was added TsOH monohydrate (0.120 g, 0.64 mmol) at 60 ºC. After 30 min, the
mixture was concentrated under reduced pressure and then EtOAc (40 mL) was added.
The organic layer was washed with 0.5 M aqueous HCl (40 mL), 1M aqueous NaOH
(40 mL) and water (40 mL), dried over Na2SO4, filtered and concentrated to give 37 as
21
yellow oil (0.020 g, 52% yield): [α]D +26 (c 1.0, CH2Cl2); IR v 3399, 3083, 2977,
1
2931, 2881, 1684, 1607, 1592, 1510, 1496, 1459, 1339 cm-1; H NMR (200 MHz,
CDCl3) δ (ppm): 8.18 (d, 2H, Ar, J = 9.1 Hz), 6.97 (d, 2H, Ar, J = 9.1 Hz), 5.12 (d, 1H,
13
NH, J = 7.2 Hz), 4.20-3.74 (m, 5H, CHN, CH2O, CH2OAr), 1.44 (s, 9H, CH3); C
NMR (50 MHz, CDCl3) δ (ppm): 163.36 (Ar), 155.76 (C=O), 141.77, 125.93, 114.52
(Ar), 80.29 (C), 67.30 (CH2OAr), 61.80 (CH2O), 28.31 (CH3). HRMS (m/z): [MNa+]
calcd for C14H20N2NaO6, 335.1214; found, 335.1211.
4.3. Synthesis of benzyl (S)-4-hydroxymethyl-2,2-dimethyl-3-oxazolidinecarboxylate 39
To a stirred solution of 38 (3.40 g, 0.0116 mol) in anhydrous THF (20 mL) was
added NaBH4 (4.38 g, 0.116 mol) at 0 ºC. After 25 min, MeOH (8 mL) was added
dropwise. After gas evolution ceased, the mixture was stirred under reflux. After 40
min, the suspension was concentrated under reduced pressure and water (50 mL) and
EtOAc (100 mL) was carefully added to the resulting mixture at 0 ºC. The organic layer
was washed with water (50 mL), dried over Na2SO4, filtered and concentrated to give
21
25
39 as colorless oil (3.04 g, 99% yield): [α]D +20 (c 1, CHCl3), lit: [α]D +20.2 (c 1,
CHCl3) [13]; IR v 3443, 3034, 2984, 2940, 2881, 1681, 1587, 1534, 1498, 1406, 1348
1
cm-1; H NMR (200 MHz, CDCl3) δ (ppm) major rotamer: 7.32 (bs, 5H, Ar), 5.12 (bs,
2H, CH2Ph), 3.98-3.56 (m, 6H, CHN, CH2O, CH2OH, OH), 1.51, 1.45 (s, 6H, CH3); 13C
NMR (50 MHz, CDCl3) δ (ppm) major rotamer: 154.19 (C=O), 128.53, 127.97, 126.88
(Ar), 94.29 (C), 67.60, 65.29, 63.87 (CH2O, CH2OH, CH2Ph), 59.66 (CHN), 27.04,
24.58 (CH3). HRMS (m/z): [MNa+] calcd for C14H19NNaO4, 288.1206; found,
288.1202.
4.4. General procedure 1 - Mitsunobu reactions
To a stirred solution of 39 (0.100 g, 0.433 mmol), appropriate substituted phenol
(0.649 mmol) and PPh3 (0.182 g, 0.693 mmol) in anhydrous toluene (5 mL) was added
DIAD (0.14 mL, 0.693 mmol) at 80 ºC. After 3 h, EtOAc (40 mL) was added to the
resulting solution. The organic layer was washed with 0.5 M aqueous NaOH (40 mL)
and water (2 X 40 mL), dried over Na2SO4, filtered and concentrated. The residue was
purified by flash silica gel column chromatography eluting with Hexanes/EtOAc (9:1)
or (95:5).
4.4.1.
Benzyl
(S)-2,2-dimethyl-4-[[4-(methoxycarbonyl)phenoxy]methyl]-3-
oxazolidinecarboxylate (40)
White solid. 70% yield: mp 83-85 ºC; [α]D21 +48 (c 0.79, CH2Cl2); IR v 3063, 2990,
1
2955, 2904, 1713, 1697, 1602, 1507, 1437, 1411 cm-1; H NMR (200 MHz, CDCl3) δ

ACCEPTED MANUSCRIPT
(ppm) major rotamer: 7.84 (d, 2H, Ar, J = 8.4 Hz), 7.35 (bs, 5H, Ar), 6.80 (d, 2H, Ar, J
= 8.4 Hz), 5.15 (bs, 2H, CH2Ph), 4.31-3.91 (m, 5H, CHN, CH2O, CH2OAr), 3.86 (s, 3H,
13
OCH3), 1.65, 1.55 (s, 6H, CH3); C NMR (50 MHz, CDCl3) δ (ppm) major rotamer:
166.61 (COOCH3), 161.78 (Ar), 151.99 (C=O), 135.98, 131.52, 128.62, 128.27, 128.02,
122.83, 113.96 (Ar), 94.44 (C), 67.02, 66.37, 65.45 (CH2O, CH2OAr, CH2Ph), 55.22
(CHN), 51.77 (OCH3), 26.57, 22.91 (CH3). HRMS (m/z): [MNa+] calcd for
C22H25NNaO6, 422.1574; found, 422.1572.
4.5. Synthesis of (R)-1-hydroxy-3-(4-nitrophenoxy)-2-propylammonium trifluoroacetate
43
To a stirred solution of 2 (0.916 g, 2.60 mmol) in CH2Cl2 (17 mL) was added TFA
(6.55 mL). After 1 h, the mixture was concentrated under nitrogen to give 43 as yellow
oil (0.848 g, 100% yield): [α]D21 -7 (c 3.18, MeOH), IR v 3721-2141, 1779, 1668, 1611,
1593, 1513, 1498, 1343 cm-1; 1H NMR (200 MHz, CD3OD) δ (ppm): 8.22 (d, 2H, Ar, J
= 9.3 Hz), 7.16 (d, 2H, Ar, J = 9.3 Hz), 4.43-3.65 (m, 5H, CHN, CH2O, CH2OAr); 13C
NMR (50 MHz, CD3OD) δ (ppm): 164.35, 143.46, 126.82, 115.97 (Ar), 67.20
(CH2OAr), 59.85 (CH2O), 53.50 (CHN). HRMS (m/z): [MH+] calcd for C9H13N2O4,
213.0870; found 213.0872.
4.6. General procedure 2 - synthesis of NHS activated esters 45a-f
To a stirred solution of appropriate acid 44a-f (1.96 mmol) in CH2Cl2 (3 mL) were
added NHS (0.361 g, 3.14 mmol) and EDC (0.601 g, 3.14 mmol). After 3 h, the mixture
was diluted with CH2Cl2 (40 mL). The organic layer was washed with brine (2 X 40
mL), saturated aqueous NaHCO3 (40 mL) and brine (2 X 40 mL), dried over Na2SO4,
filtered and concentrated to give NHS activated esters (80-100% yield) that were used
immediately without further purification.
4.7.
Synthesis
of
benzyl
(S)-2,2-dimethyl-4-[(4-nitrophenoxy)methyl]-3-
oxazolidinecarboxilate 4
Synthesis of 4 was carried out as described in general procedure 1. Yellow oil. 76%
21
yield: [α]D +56 (c 2, CH2Cl2); IR v 3083, 2984, 2938, 2883, 1703, 1608, 1591, 1512,
1497, 1463, 1405, 1338 cm-1; 1H NMR (200 MHz, CDCl3) δ (ppm) major rotamer: 7.98
(d, 2H, Ar, J = 9.0 Hz), 7.37 (bs, 5H, Ar), 6.81 (d, 2H, Ar, J = 9.0 Hz), 5.17-5.14 (m,
13
2H, CH2Ar), 4.34-3.88 (m, 5H, CHN, CH2O, CH2OAr), 1.65, 1.56 (s, 6H, CH3); C
NMR (50 MHz, CDCl3) δ (ppm) major rotamer: 162.97 (Ar), 153.02 (C=O), 141.62,
135.83, 128.73, 128.47, 128.04, 125.83, 114.36 (Ar), 94.56 (C), 67.25, 66.90, 65.39
(CH2O, CH2OAr, CH2Ar), 54.97 (CHN), 26.59, 22.87 (CH3). HRMS (m/z): [MNa+]
calcd for C20H22N2NaO6, 409.1370; found, 409.1371.
4.8. General procedure 3 - synthesis of compounds 5-10
To a stirred solution of 43 (0.20 g, 0.61 mmol) in THF (2 mL) was added a solution
of NaHCO3 (0.086 g, 1.02 mmol) in water (2 mL). After 10 min, appropriate NHS
activated ester 45a-f (0.51 mmol) was added. After 2 h, the mixture was concentrated
under reduced pressure and then EtOAc (40 mL) was added. The organic layer was
washed with 0.5 M aqueous HCl (20 mL), 1 M aqueous NaOH (20 mL) and water (2 X

ACCEPTED MANUSCRIPT
20 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by silica
gel column chromatography eluting with Hexanes/EtOAc to give 46a-f.
To a stirred solution of appropriate substituted propanol 46a-f (0.15 mmol) in a
mixture of toluene (0.36 mL) and chloroform (0.27 mL) was added PTSA (0.001 g,
0.008 mmol) and DMP (0.074 mL, 0.6 mmol) at 80 ºC. After 2 h, the mixture was
cooled to room temperature and EtOAc (20 mL) was added. The organic layer was
washed with saturated aqueous NaHCO3 (20 mL) and water (20 mL), dried over
Na2SO4, filtered and concentrated. The residue was purified by silica gel column
chromatography eluting with appropriate mixture as indicated in each case.
4.8.1.
(S)-3-[(3,3-Dimethylbutanoyl)-2,2-dimethyl-4-[(4-
nitrophenoxy)methyl]oxazolidine (5)
Eluent hexanes:EtOAc (85:15). Colorless oil. 53% yield: [α]D21 +88 (c 0.2, CH2Cl2);
IR v 3083, 2953, 2871, 1646, 1608, 1593, 1514, 1497, 1464, 1403, 1387, 1363, 1341
1
cm-1; H NMR (400 MHz, CDCl3) δ (ppm): 8.22 (d, 2H, Ar, J = 9.2 Hz), 6.96 (d, 2H,
Ar, J = 9.2 Hz), 4.30-4.01 (m, 5H, CHN, CH2O, CH2OAr), 2.39 (d, 1H, CH2, J = 12.7
Hz), 2.14 (dd, 1H, CH2, J = 12.7, 3.6 Hz), 1.68, 1.59 (s, 6H, CH3), 1.08 (s, 9H,
C(CH3)3); 13C NMR (100 MHz, CDCl3) δ (ppm): 169.05 (C=O), 162.86, 142.16,
126.03, 114.51 (Ar), 95.71 (C), 67.85, 65.29 (CH2O, CH2OAr), 56.97 (CHN), 47.55
(CH2), 31.88 (C(CH3)3), 29.90 (C(CH3)3), 27.17, 23.04 (CH3). HRMS (m/z): [MNa+]
calcd for C18H26N2NaO5, 373.1734; found, 373.1735.
4.8.2.
(S)-2,2-Dimethyl-4-[(4-nitrophenoxy)methyl]-3-[3-
(phenyl)propanoyl]oxazolidine (6)
21
Eluent hexanes:EtOAc (75:25). Yellow oil. 68% yield: [α]D +75 (c 0.94, CH2Cl2);
IR v 3085, 2982, 2935, 2881, 1643, 1608, 1591, 1510, 1496, 1407, 1338 cm-1; 1H NMR
(200 MHz, CDCl3) δ (ppm): 8.17 (d, 2H, Ar, J = 9.1 Hz), 7.26-7.24 (m, 5H, Ar), 6.83
(d, 2H, Ar, J = 9.1 Hz), 4.07-3.65 (m, 5H, CHN, CH2O, CH2OAr), 3.16-3.01 (m, 2H,
CH2Ar), 2.98-2.59 (m, 2H, CH2), 1.66, 1.57 (CH3); 13C NMR (50 MHz, CDCl3) δ
(ppm): 169.22 (C=O), 162.65, 141.87, 140.83, 128.54, 126.33, 125.79, 114.38 (Ar),
95.56 (C), 67.43, 65.32 (CH2O, CH2OAr), 56.21 (CHN), 37.46 (CH2), 31.53 (CH2Ar),
26.88, 22.78 (CH3). HRMS (m/z): [MNa+] calcd for C21H24N2NaO5, 407.1577; found,
407.1578.
4.8.3.
(S)-2,2-Dimethyl-3-((E)-3-phenylpropenoyl)-4-[(4-
nitrophenoxy)methyl]oxazolidine (7)
Eluent hexanes:EtOAc (7:3). Yellow oil. 75% yield: [α]D21 +176 (c 0.9, CH2Cl2); IR
v 3059, 2983, 2936, 2881, 1650, 1607, 1591, 1510, 1496, 1463, 1450, 1397, 1339 cm-1;
1H NMR (200 MHz, CDCl3) δ (ppm): 8.12 (d, 2H, Ar, J = 9.0 Hz), 7.74 (d, 1H, CHalk, J
= 15.2 Hz), 7.49-7.34 (m, 5H, Ar), 6.90 (d, 2H, Ar, J = 9.0 Hz), 6.79 (d, 1H, CHalk, J
13
=15.2 Hz), 4.71-4.09 (m, 5H, CHN, CH2O, CH2OAr), 1.74, 1.65 (s, 6H, CH3); C
NMR (50 MHz, CDCl3) δ (ppm): 162.88, 162.74 (C=O, Ar), 143.26, 141.87 (CHalk,
Ar), 134.62, 129.96, 128.80, 127.81, 125.86 (Ar), 118.58 (CHalk), 114.39 (Ar), 95.91
(C), 68.36, 65.44 (CH2O, CH2OAr), 55.88 (CHN), 26.97, 22.92 (CH3). HRMS (m/z):
[MNa+] calcd for C21H22N2NaO5, 405.1421; found, 405.1422.
4.8.4.
(S)-3-[(2,2-Dimethylpropanoyl)-2,2-dimethyl-4-[(4-
nitrophenoxy)methyl]oxazolidine (8)
Eluent hexanes:EtOAc (7:3). Yellow oil. 36% yield: [α]D21 +50 (c 0.3, CH2Cl2); IR v
1
3083, 2959, 2881, 1647, 1607, 1592, 1509, 1497, 1462, 1380, 1368, 1339 cm-1; H

ACCEPTED MANUSCRIPT
NMR (200 MHz, CDCl3) δ (ppm): 8.18 (d, 2H, Ar, J = 9.2 Hz), 7.02 (d, 2H, Ar, J = 9.2
Hz), 4.39-4.02 (m, 3H, CHN, CH2OAr), 3.68 (dd, 1H, CH2O, J = 9.6, 3.4 Hz), 3.50 (dd,
13
1H, CH2O, J = 9.6, 5.3 Hz), 1.32 (s, 6H, CH3), 1.19 (s, 9H, C(CH3)3); C NMR (50
MHz, CDCl3) δ (ppm): 178.49 (C=O), 163.45, 141.69, 125.96, 114.56 (Ar), 100.31 (C),
66.22, 58.53 (CH2O, CH2OAr), 48.70 (CHN), 38.77 (C(CH3)3), 27.47 (C(CH3)3), 24.19
(CH3).
4.8.5. (S)-3-(Acetyl)-2,2-dimethyl-4-[(4-nitrophenoxy)methyl]oxazolidine (9)
21
Eluent hexanes:EtOAc (1:1). Yellow solid. 49% yield: mp 99-102 ºC; [α]D +67 (c
0.66, CH2Cl2); IR v 3116, 2984, 2931, 1652, 1610, 1590, 1522, 1494, 1464, 1405, 1346
1
cm-1; H NMR (200 MHz, CDCl3) δ (ppm): 8.16 (d, 2H, Ar, J = 7.3 Hz), 6.94 (d, 2H,
Ar, J = 7.3 Hz), 4.55-3.89 (m, 5H, CHN, CH2O, CH2OAr), 2.18 (s, 3H, CH3CO), 1.62,
1.55 (s, 6H, CH3); 13C NMR (50 MHz, CDCl3) δ (ppm): 167.38 (C=O), 162.79, 141.96,
125.90, 114.40 (Ar), 95.50 (C), 67.84, 65.43 (CH2O, CH2OAr), 57.16 (CHN), 26.88
(CH3CO), 23.82, 22.79 (CH3). HRMS (m/z): [MNa+] calcd for C14H18N2NaO5,
317.1108; found, 317.1111.
4.8.6. (S)-3-(Benzoyl)-2,2-dimethyl-4-[(4-nitrophenoxy)methyl]oxazolidine (10)
21
Eluent hexanes:EtOAc (7:3). Yellow solid. 51% yield: mp 132-135 ºC; [α]D +100
(c 0.4, CH2Cl2); IR v 3083, 2983, 2936, 2885, 1635, 1608, 1591, 1509, 1494, 1462,
1
1446, 1398, 1338 cm-1; H NMR (400 MHz, CDCl3) δ (ppm): 8.17 (d, 2H, Ar, J = 9.2
Hz), 7.78 (d, 2H, Ar, J = 7.6 Hz), 7.51 (t, 1H, Ar, J = 7.6 Hz), 7.43 (t, 2H, Ar, J = 7.6
Hz), 7.01 (d, 2H, Ar, J = 9.2 Hz), 4.53 (bs, 1H, CHN), 4.35 (dd, 1H, CH2OAr, J = 9.4,
4.4 Hz), 4.27 (dd, 1H, CH2OAr, J = 9.4, 6.4 Hz), 4.05 (dd, 1H, CH2O, J = 11.0, 3.8 Hz),
3.90 (dd, 1H, CH2O, J = 11.0, 4.6 Hz), 2.15 (s, 6H, CH3); 13C NMR (100 MHz, CDCl3)
δ (ppm): 167.84 (C=O), 163.28, 141.91, 133.76, 131.97, 128.69, 127.02, 125.96, 114.60
(Ar), 66.97, 61.55 (CH2O, CH2OAr), 50.43 (CHN), 30.89 (CH3). HRMS (m/z): [MNa+]
calcd for C19H20N2NaO5, 379.1264; found, 379.1265.
4.9. Synthesis of compounds 11-14
Synthesis of 11-14 was carried out as described in general procedure 1. In this case,
compound 39 was replaced by compound 35.
4.9.1.
1,1-Dimethylethyl
(S)-2,2-dimethyl-4-[[(1-naphthyl)oxy]methyl]-3-
oxazolidinecarboxylate (11)
27
Yellow oil. 31% yield: [α]D +65 (c 1.11, CH2Cl2); IR v 3054, 2978, 2935, 2878,
1690, 1596, 1580, 1508, 1459, 1384, 1375, 1364 cm-1; H NMR (200 MHz, CDCl3) δ
1
(ppm): 8.27 (bs, 1H, Ar), 7.80 (bs, 1H, Ar), 7.48-7.37 (m, 4H, Ar), 6.94 (bs, 1H, Ar),
4.34-4.11 (m, 5H, CHN, CH2O, CH2OAr), 1.69, 1.64, 1.52 (s, 15H, CH3, C(CH3)3); 13C
NMR (50 MHz, CDCl3) δ (ppm) major rotamer: 154.09 (Ar), 152.36 (C=O), 134.43,
127.42, 126.42, 125.88, 125.47, 121.96, 120.34, 104.88 (Ar), 93.62 (C), 80.26
(C(CH3)3), 66.40, 65.45 (CH2O, CH2OAr), 55.72 (CHN), 28.39 (C(CH3)3), 26.81, 23.04
(CH3). HRMS (m/z): [MNa+] calcd for C21H27NNaO4, 380.1832; found, 380.1834.
4.9.2.
1,1-Dimethylethyl
(S)-2,2-dimethyl-4-[[(2-naphthyl)oxy]methyl]-3-
oxazolidinecarboxylate (12)
21
White solid. 52% yield: mp 62-66 ºC; [α]D +76 (c 0.69, CHCl3); IR v 3058, 2980,
2934, 2879, 1708, 1692, 1628, 1600, 1512, 1458, 1376, 1365 cm-1; 1H NMR (200 MHz,
CDCl3) δ (ppm): 7.78-7.16 (m, 7H, Ar), 4.38-4.06 (m, 5H, CHN, CH2O, CH2OAr),

ACCEPTED MANUSCRIPT
13
1.70, 1.64, 1.54 (s, 15H, CH3, C(CH3)3); C NMR (50 MHz, CDCl3) δ (ppm) major
rotamer: 156.38 (Ar), 152.30 (C=O), 134.46, 129.28, 129.02, 127.58, 126.63, 126.42,
123.74, 118.61, 106.83 (Ar), 94.10 (C), 80.48 (C(CH3)3), 66.69, 65.40 (CH2O,
CH2OAr), 56.12 (CHN), 28.49 (C(CH3)3), 26.84, 23.08 (CH3). HRMS (m/z): [MNa+]
calcd for C21H27NNaO4, 380.1832; found, 380.1839.
4.9.3.
1,1-Dimethylethyl
(R)-4-[[(5-chloro-2-pyridyl)oxy]methyl]-2,2-dimethyl-3-
oxazolidinecarboxylate (13)
25
Solvent: anhydrous THF (5 mL). Colorless crystal. 59% yield: mp 61-65 ºC; [α]D
+49 (c 3.34, CH2Cl2); IR v 3063, 2978, 2936, 2879, 1694, 1590, 1565, 1458, 1385,
1
1375, 1364 cm-1; H NMR (200 MHz, CDCl3) δ (ppm): 8.04 (d, 1H, Ar, J = 2.6 Hz),
7.48 (bd, 1H, Ar, J = 8.7 Hz), 6.66 (d, 1H, Ar, J = 8.7 Hz), 4.44-3.96 (m, 5H, CHN,
CH2O, CH2OAr), 1.58, 1.54, 1.44 (s, 15H, CH3, C(CH3)3); 13C NMR (50 MHz, CDCl3)
δ (ppm) major rotamer: 161.71 (Ar), 151.64 (C=O), 145.02, 138.52, 124.19, 111.87
(Ar), 93.95 (C), 80.42 (C(CH3)3), 65.21, 64.86 (CH2O, CH2OAr), 56.06 (CHN), 28.36
(C(CH3)3), 27.43, 23.10 (CH3). HRMS (m/z): [MNa+] calcd for C16H23ClN2NaO4,
365.1239; found, 365.1243.
4.9.4.
1,1-Dimethylethyl
(R)-2,2-dimethyl-4-[[(5-nitro-2-pyridyl)oxy]methyl]-3-
oxazolidinecarboxilate (14)
Solvent: anhydrous THF (5 mL). Yellow oil. 43% yield: [α]D25 +60 (c 2.41, CH2Cl2);
IR v 3083, 2979, 2936, 2883, 1692, 1603, 1579, 1518, 1478, 1459, 1386, 1376, 1365,
1344 cm-1; 1H NMR (200 MHz, CDCl3) δ (ppm): 9.01 (d, 1H, Ar, J = 2.6 Hz), 8.32 (bd,
1H, Ar, J = 9.0 Hz), 6.80 (d, 1H, Ar, J = 9.0 Hz), 4.59-3.99 (m, 5H, CHN, CH2O,
CH2OAr), 1.57, 1.54, 1.44 (s, 15H, CH3, C(CH3)3); 13C NMR (50 MHz, CDCl3) δ (ppm)
major rotamer: 166.66 (Ar), 152.16 (C=O), 144.63, 139.55, 134.02, 111.20 (Ar), 93.59
(C), 80.60 (C(CH3)3), 65.95, 65.21 (CH2O, CH2OAr), 55.84 (CHN), 28.33 (C(CH3)3),
27.43, 24.21 (CH3). HRMS (m/z): [MNa+] calcd for C16H23N3NaO6, 376.1479; found,
376.1477.
4.10. General procedure 4 - synthesis of compounds 15-16
To a stirred solution of 35 (0.300 g, 1.30 mmol), appropriate hydroxypyridine (0.247
g, 2.60 mmol) and PPh3 (0.681 g, 2.60 mmol) in anhydrous THF (15 mL) was added
DIAD 95% (0.54 mL, 2.60 mmol) at 80 ºC. After 3 h, the mixture was concentrated
under reduced pressure and EtOAc (100 mL) was added. The organic layer was
extracted with cold 0.1 M aqueous HCl (2 X 40 mL). 1 M aqueous NaOH (60 mL) was
added to the aqueous layer and the resulting mixture was extracted with EtOAc (2 X
100 mL). The organic layer was dried over Na2SO4, filtered and concentrated. The
residue was purified by flash silica gel column chromatography eluting with
Hexanes/EtOAc (7:3).
4.10.1.
1,1-Dimethylethyl
(S)-2,2-dimethyl-4-[[(3-pyridyl)oxy]methyl]-3-
oxazolidinecarboxylate (15)
25
Yellow oil. 53% yield: [α]D +58 (c 3.46, CH2Cl2); IR v 3058, 2979, 2936, 2880,
1689, 1575, 1477, 1427, 1385, 1376, 1365 cm-1; H NMR (200 MHz, CDCl3) δ (ppm):
1
8.29-8.18 (m, 2H, Ar), 7.25-7.13 (m, 2H, Ar), 4.23-3.87 (m, 5H, CHN, CH2O,
CH2OAr), 1.58, 1.53, 1.46 (s, 15H, CH3, C(CH3)3); 13C NMR (50 MHz, CDCl3) δ (ppm)
major rotamer: 154.57 (Ar), 152.21 (C=O), 142.07, 138.32, 123.71, 120.69 (Ar), 93.49
(C), 80.58 (C(CH3)3), 66.17, 65.00 (CH2O, CH2OAr), 55.79 (CHN), 28.29 (C(CH3)3),

ACCEPTED MANUSCRIPT
27.41, 24.12 (CH3). HRMS (m/z): [MH+] calcd for C16H25N2O4, 309.1809; found,
309.1818.
4.10.2.
1,1-Dimethylethyl
(S)-2,2-dimethyl-4-[[(4-pyridyl)oxy]methyl]-3-
oxazolidinecarboxylate (16)
25
Yellow oil. 63% yield: [α]D +58 (c 4.24, CH2Cl2); IR v 2979, 2936, 2882, 1688,
1591, 1571, 1501, 1464, 1385, 1376, 1365 cm-1; H NMR (200 MHz, CDCl3) δ (ppm):
1
8.36-8.33 (m, 2H, Ar), 6.83-6.80 (m, 2H, Ar), 4.21-3.85 (m, 5H, CHN, CH2O,
CH2OAr), 1.55, 1.49, 1.43 (s, 15H, CH3, C(CH3)3); 13C NMR (50 MHz, CDCl3) δ (ppm)
major rotamer: 164.37 (Ar), 152.20 (C=O), 150.95, 110.21 (Ar), 93.51 (C), 80.63
(C(CH3)3), 65.63, 64.92 (CH2O, CH2OAr), 55.50 (CHN), 28.24 (C(CH3)3), 27.38, 24.04
(CH3).
4.11.
Synthesis
of
1,1-dimethylethyl
(S)-4-[(4-nitrophenoxy)methyl]-3-
oxazolidinecarboxylate 17
To a stirred suspension of 37 (0.080 g, 0.256 mmol) and TsOH monohydrate (0.003
g, 0.0158 mmol) in anhydrous toluene (2 mL) was added p-formaldehyde (0.029 g,
0.962 mmol) at 85 ºC. After 6 h, EtOAc (20 mL) was added. The organic layer was
washed with saturated aqueous NaHCO3 (20 mL), water (20 mL), dried over Na2SO4,
filtered and concentrated. The residue was purified by silica gel column
21
chromatography (hexanes/EtOAc 85:15) to give 17 as a yellow oil (36% yield): [α]D
+61 (c 0.46, CHCl3); IR v 3083, 2976, 2874, 1697, 1608, 1592, 1513, 1497, 1477, 1460,
1391, 1367, 1339 cm-1;1H NMR (200 MHz, CDCl3) δ (ppm): 8.20 (d, 2H, Ar, J = 9.2
Hz), 7.01 (d, 2H, Ar, J = 9.2 Hz); 4.87-4.83 (m, 2H, NCH2O), 4.26-4.03 (m, 5H, CHN,
13
CH2O, CH2OAr), 1.50 (s, 9H, C(CH3)3); C NMR (50 MHz, CDCl3) δ (ppm): 163.30
(Ar), 152.69 (C=O), 141.73, 125.89, 114.51 (Ar), 81.05 (C), 79.25 (NCH2O), 69.76,
67.13 (CH2O, CH2OAr), 53.72 (CHN), 28.34 (CH3). HRMS (m/z): [MNa+] calcd for
C15H20N2NaO6, 347.1214; found, 347.1213.
4.11.1. 1,1-Dimethylethyl (S)-6-[(4-nitrophenoxy)methyl]tetrahydro-1,3,5-dioxazepine-
5-carboxylate (47)
21
Yellow oil. 25% yield: [α]D +70 (c 0.4, CHCl3); IR v 3083, 2977, 2933, 2886,
1696, 1608, 1592, 1512, 1498, 1463, 1392, 1367, 1339 cm-1; H NMR (200 MHz,
1
CDCl3) δ (ppm): 8.21 (d, 2H, Ar, J = 9.0 Hz), 6.99 (d, 2H, Ar, J = 9.0 Hz), 5.61-5.45
(m, 1H, OCH2O), 4.95-4.62 (m, 4H, OCH2O, NCH2O, CHN), 4.30-4.10 (m, 4H, CH2O,
13
CH2OAr), 1.50 (s, 9H, C(CH3)3); C NMR (50 MHz, CDCl3) δ (ppm): 163.36 (Ar),
154.62 (C=O), 141.74, 125.89, 114.50 (Ar), 97.07 (OCH2O), 81.48 (C), 74.01
(NCH2O), 69.31, 66.60 (CH2O, CH2OAr), 54.17 (CHN), 28.24 (CH3). HRMS (m/z):
[MNa+] calcd for C16H22N2NaO7, 377.1319; found, 377.1314.
4.12.
Synthesis
of
1,1-dimethylethyl
3-[(4-nitrophenoxy)methyl]-1-oxa-4-
azaspiro[4.4]nonane-4-carboxylate 18
To a stirred suspension of 37 (0.050 g, 0.160 mmol), TsOH monohydrate (0.003 g,
0.016 mmol) in anhydrous toluene (2 mL) was added cyclopentanone (0.21 mL, 2.4
mmol) at 80 ºC. After 4 h, the mixture was concentrated under reduced pressure and the
residue was directly purified by silica gel column chromatography (hexanes/EtOAc 9:1)
21
to give 18 as yellow oil (0.024 g, 40% yield; 48% of recovered starting material): [α]D
+48 (c 0.42, CHCl3); IR v 3085, 2970, 2874, 1688, 1608, 1592, 1513, 1498, 1464, 1387,

ACCEPTED MANUSCRIPT
1
1366, 1333 cm-1; H NMR (200 MHz, CDCl3) δ (ppm): 8.20 (d, 2H, Ar, J = 9.0 Hz),
7.06-7.02 (m, 2H, Ar), 4.29-3.85 (m, 5H, CHN, CH2O, CH2OAr), 2.27-1.70 (m, 8H,
CH2), 1.51 (s, 9H, C(CH3)3); 13C NMR (50 MHz, CDCl3) δ (ppm) major rotamer:
163.50 (Ar), 152.25 (C=O), 141.65, 125.90, 114.57 (Ar), 103.22 (C), 80.97 (C(CH3)3),
67.22, 65.24 (CH2O, CH2OAr), 55.32 (CHN), 37.14, 35.24 (CH2C), 28.49 (CH3), 24.77,
24.15 (CH2). HRMS (m/z): [MNa+] calcd for C19H26N2NaO6, 401.1683; found,
401.1682.
4.13.
Synthesis
of
1,1-dimethylethyl
(S)-2,2-dimethyl-4-[[[[(4-succinimid-1-
yl)oxy]carbonyl]phenyloxy]methyl]-3-oxazolidinecarboxylate 50
To a stirred solution of 48 (0.220 g, 0.627 mmol) in CH2Cl2 (5 mL) was added NHS
(0.115 g, 1.00 mmol) and EDC (0.192 g, 1.00 mmol) at room temperature. After 3 h,
EtOAc (20 mL) was added. The organic layer was washed with brine (2 X 10 mL),
dried over Na2SO4, filtered and concentrated to give 50 as colorless semi-solid (0.280 g,
100% yield) that was used immediately without further purification.
4.14. General procedure 5 - synthesis of compounds 19-22
To a stirred solution of 50 (0.080 g, 0.179 mmol) in CH2Cl2 (1 mL) was added the
appropriate amine (1.79 mmol). After 4 h, the mixture was concentrated under reduced
pressure and then EtOAc (20 mL) was added. The organic layer was washed with
saturated aqueous NaHCO3 (10 mL), water (10 mL), dried over Na2SO4, filtered and
concentrated. The residue was purified by silica gel column chromatography eluting
with appropriate mixture as indicated in each case.
4.14.1.
1,1-Dimethylethyl
(S)-2,2-dimethyl-4-[[[4-
(propylamino)carbonyl]phenoxy]methyl]-3-oxazolidinecarboxylate (19)
22
Eluent hexanes:EtOAc (65:35). Colorless semi-solid. 60% yield: [α]D +49 (c 0.70,
CH2Cl2); IR v 3328, 2969, 2934, 2876, 1690, 1634, 1606, 1575, 1504, 1465, 1385,
1376, 1365 cm-1; 1H NMR (200 MHz, CDCl3) δ (ppm): 7.71-7.68 (m, 2H, Ar), 6.91 (d,
2H, Ar, J = 8.0 Hz), 6.36 (bs, 1H, NH), 4.22-3.70 (m, 5H, CHN, CH2O, CH2OAr), 3.34
(q, 2H, CH2NH, J = 6.5 Hz), 1.59-1.46 (m, 17H, C(CH3)2, C(CH3)3, CH2CH3), 0.92 (t,
13
3H, CH2CH3, J = 7.3 Hz); C NMR (50 MHz, CDCl3) δ (ppm) major rotamer: 167.02
(C=O amide), 160.83 (Ar), 152.24 (C=O), 128.60, 127.45, 114.20 (Ar), 93.57 (C),
80.56 (C(CH3)3), 66.11, 65.10 (CH2O, CH2OAr), 55.85 (CHN), 41.62 (CH2NH), 28.35
(C(CH3)3), 27.45, 24.17 (CH3), 22.85 (CH2CH3), 11.34 (CH2CH3). HRMS (m/z):
[MNa+] calcd for C21H32N2NaO5, 415.2203; found, 415.2203.
4.14.2.
1,1-Dimethylethyl
(S)-2,2-dimethyl-4-[[[4-(1-
methylethylamino)carbonyl]phenoxy]methyl]-3-oxazolidinecarboxylate (20)
22
Eluent hexanes:EtOAc (65:35). Colorless solid. 75% yield: mp 43-44 ºC; [α]D +57
(c 1.13, CH2Cl2); IR v 3317, 2974, 2935, 2877, 1690, 1630, 1606, 1573, 1503, 1458,
1
1385, 1365 cm-1; H NMR (200 MHz, CDCl3) δ (ppm): 7.71 (d, 2H, Ar, J = 7.9 Hz),
6.96 (d, 2H, Ar, J = 7.9 Hz), 5.98 (d, 1H, NH, J = 7.4 Hz), 4.32-3.84 (m, 6H, CHN,
CH2O, CH2OAr, CH(CH3)2), 1.63, 1.58, 1.50 (s, 15H, C(CH3)2, C(CH3)3), 1.25 (d, 6H,
13
CH(CH3)2, J = 6.6 Hz); C NMR (50 MHz, CDCl3) δ (ppm) major rotamer: 166.05
(C=O amide), 160.70 (Ar), 152.22 (C=O), 128.54, 127.32, 114.10 (Ar), 93.47 (C),
80.55 (C(CH3)3), 66.00, 65.20 (CH2O, CH2OAr), 55.82 (CHN), 41.66 (CH(CH3)2),
28.31 (C(CH3)3), 27.43, 24.14 (CH3), 22.73 (CH(CH3)2). HRMS (m/z): [MNa+] calcd
for C21H32N2NaO5, 415.2203; found, 415.2201.

ACCEPTED MANUSCRIPT
4.14.3. 1,1-Dimethylethyl (S)-4-[[[4-(diethylamino)carbonyl]phenoxy]methyl]-2,2-
dimethyl-3-oxazolidinecarboxylate (21)
22
Eluent hexanes:EtOAc (7:3). Colorless oil. 63% yield: [α]D +45 (c 0.80, CH2Cl2);
IR v 2974, 2935, 2877, 1693, 1626, 1607, 1576, 1513, 1457, 1424, 1377, 1364 cm-1; 1H
NMR (200 MHz, CDCl3) δ (ppm): 7.33 (d, 2H, Ar, J = 8.0 Hz), 6.95 (d, 2H, Ar, J = 8.0
Hz), 4.26-3.88 (m, 5H, CHN, CH2O, CH2OAr), 3.41 (bs, 4H, CH2CH3), 1.63, 1.57, 1.50
13
(s, 15H, C(CH3)2, C(CH3)3), 1.18 (bs, 6H, CH2CH3); C NMR (50 MHz, CDCl3) δ
(ppm) major rotamer: 171.07 (C=O amide), 159.02 (Ar), 152.21 (C=O), 129.58, 128.05,
114.17 (Ar), 93.44 (C), 80.46 (C(CH3)3), 65.86, 65.03 (CH2O, CH2OAr), 55.82 (CHN),
40.98 (CH2CH3), 28.29 (C(CH3)3), 27.41, 22.93 (CH3), 13.65 (CH2CH3). HRMS (m/z):
[MNa+] calcd for C22H34N2NaO5, 429.2360; found, 429.2368.
4.14.4.
1,1-Dimethylethyl
(S)-2,2-dimethyl-4-[[[4-(4-methylpiperazin-1-
yl)carbonyl]phenoxy]methyl]-3-oxazolidinecarboxylate (22)
It was not necessary to purify by silica gel column chromatography. Yellow semi-
solid. 90% yield: [α]D22 +91 (c 0.42, CH2Cl2); IR v 2976, 2936, 2879, 2792, 1692, 1630,
1
1606, 1576, 1512, 1456, 1425, 1385, 1365 cm-1; H NMR (200 MHz, CDCl3) δ (ppm):
7.37 (d, 2H, Ar, J = 8.6 Hz), 6.97-6.90 (m, 2H, Ar), 4.27-3.63 (m, 9H, CHN, CH2O,
CH2OAr, CH2NCO), 2.42 (bs, 4H, CH2N), 2.32 (s, 3H, CH3N), 1.63, 1.57, 1.50 (s, 15H,
13
C(CH3)2, C(CH3)3); C NMR (50 MHz, CDCl3) δ (ppm) major rotamer: 170.04 (C=O
amide), 159.47 (Ar), 152.12 (C=O), 128.92, 128.09, 114.14 (Ar), 93.38 (C), 80.39
(C(CH3)3), 65.89, 65.11 (CH2O, CH2OAr), 55.42 (CHN), 54.82 (CH2NCO, CH2N),
45.83 (CH3N), 28.23 (C(CH3)3), 27.34, 24.09 (CH3). HRMS (m/z): [MH+] calcd for
C23H36N3O5, 434.2649; found, 434.2646.
4.15. General procedure 6 - synthesis of compounds 23 and 28
To a stirred solution of 48 or 49 (0.043 g, 0.123 mmol) in CH2Cl2 (2 mL) was added
EDC (0.038 g, 0.196 mmol) and DMAP (0.002 g, 0.0123 mmol). After 5 min, aniline or
p-toluidine (0.49 mmol) was added. After 4 h, EtOAc (20 mL) was added. The organic
layer was washed with 0.5 M aqueous HCl (10 mL), saturated aqueous NaHCO3 (10
mL), water (2 X 10 mL), dried over Na2SO4, filtered and concentrated. The residue was
purified by silica gel column chromatography eluting with appropriate mixture as
indicated in each case.
4.15.1. 1,1-Dimethylethyl (S)-4-[[[4-(phenylamino)carbonyl]phenoxy]methyl]-2,2-
dimethyl-3-oxazolidinecarboxylate (23)
22
Eluent hexanes/EtOAc (85:15). White solid. 48% yield: mp 150-151 ºC; [α]D +44
(c 0.50, CH2Cl2); IR v 3415, 3011, 2972, 2932, 2874, 1692, 1662, 1602, 1595, 1509,
1494, 1470, 1435, 1393, 1365 cm-1; 1H NMR (200 MHz, CDCl3) δ (ppm): 8.23 (bs, 1H,
NH), 7.81-7.72 (m, 2H, Ar), 7.65 (d, 2H, Ar, J = 7.5 Hz), 7.31 (t, 2H, Ar, J = 7.5 Hz),
7.09 (t, 1H, Ar, J = 7.5 Hz), 6.93 (d, 2H, Ar, J = 8.6 Hz), 4.24-3.81 (m, 5H, CHN,
13
CH2O, CH2OAr), 1.61, 1.56, 1.50 (s, 15H, C(CH3)2, C(CH3)3); C NMR (50 MHz,
CDCl3) δ (ppm) major rotamer: 165.51 (C=O amide), 161.16 (Ar), 152.33 (C=O),
138.21, 128.93, 127.52, 124.17, 120.23, 114.39 (Ar), 93.69 (C), 80.70 (C(CH3)3), 66.06,
65.16 (CH2O, CH2OAr), 55.68 (CHN), 28.40 (C(CH3)3), 27.50, 24.20 (CH3). HRMS
(m/z): [MNa+] calcd for C24H30N2NaO5, 449.2047; found, 449.2049.

ACCEPTED MANUSCRIPT
4.15.2.
1,1-Dimethylethyl
(S)-2,2-dimethyl-4-[[[3-(4-
methylphenylamino)carbonyl]phenoxy]methyl]-3-oxazolidinecarboxylate (28)
23
Eluent hexanes/EtOAc (8:2). Yellow semi-solid. 77% yield: [α]D +13 (c 1.39,
CHCl3); IR v 3315, 2978, 2934, 2879, 1689, 1653, 1595, 1516, 1485, 1455, 1388, 1377,
1
1365 cm-1; H NMR (200 MHz, CDCl3) δ (ppm): 8.30 (s, 1H, NH), 7.77 (bs, 1H, Ar),
7.62-7.58 (m, 2H, Ar), 7.52-7.35 (m, 2H, Ar), 7.14 (d, 2H, Ar, J = 8.0 Hz), 7.09-7.07
(m, 1H, Ar), 4.33-3.90 (m, 5H, CHN, CH2O, CH2OAr), 2.33 (s, 3H, CH3Ar), 1.63-1.47
13
(m, 15H, C(CH3)2, C(CH3)3); C NMR (100 MHz, CDCl3) δ (ppm) major rotamer:
164.90 (C=O amide), 158.16 (Ar), 152.58 (C=O), 135.92, 135.59, 134.00, 129.97,
129.33, 120.79, 120.50, 119.50, 111.05 (Ar), 93.65 (C), 80.91 (C(CH3)3), 66.21, 65.50
(CH2O, CH2OAr), 55.04 (CHN), 28.51 (C(CH3)3), 27.68, 24.12 (CH3), 20.90 (CH3Ar).
HRMS (m/z): [MNa+] calcd for C25H32NaN2O5, 463.2203; found, 463.2213.
4.16. General procedure 7 - synthesis of compounds 51-52
To a solution of 2 or 1 (0.200 g, 0.568 mmol) in THF (4 mL) was added Pd-C 10%
w/w (0.020 g). The suspension was stirred under hydrogen atmosphere at room
temperature. After 2.5 h, the mixture was diluted with CH2Cl2 (15 mL) and filtered. The
resulting amine solution 51 or 52 was used immediately without further purification.
4.17. General procedure 8 - synthesis of compounds 24 and 29
To a stirred suspension of p-toluic acid (0.116 g, 0.852 mmol) in CH2Cl2 (5 mL) was
added EDC (0.261 g, 1.36 mmol) and DMAP (0.010 g, 0.0852 mmol). After
dissolution, a solution of amine 51 or 52 was added (0.568 mmol, general procedure 7).
After 3 h, EtOAc (60 mL) was added. The organic layer was washed with 0.5 M
aqueous HCl (30 mL), saturated aqueous NaHCO3 (30 mL), water (2 X 30 mL), dried
over Na2SO4, filtered and concentrated. The residue was purified by silica gel column
chromatography (hexanes/EtOAc 8:2).
4.17.1.
1,1-Dimethylethyl
(S)-2,2-dimethyl-4-[[4-(4-
methylbenzoylamino)phenoxy]methyl]-3-oxazolidinecarboxylate (24)
White solid. 57% yield, (2 steps): mp 100-101 ºC; [α]D22 +45 (c 1.42, CH2Cl2); IR v
3308, 2976, 2874, 1694, 1645, 1613, 1601, 1573, 1510, 1467, 1386, 1376, 1365 cm-1;
1H NMR (200 MHz, CDCl3) δ (ppm): 8.10 (s, 1H, NH), 7.74 (d, 2H, Ar, J = 6.9 Hz),
7.52 (bs, 2H, Ar), 7.21 (d, 2H, Ar, J = 6.9 Hz), 6.89 (d, 2H, Ar, J = 8.4 Hz), 4.12-3.78
(m, 5H, CHN, CH2O, CH2OAr), 2.39 (s, 3H, CH3Ar), 1.62, 1.58, 1.50 (s, 15H, C(CH3)2,
13
C(CH3)3); C NMR (50 MHz, CDCl3) δ (ppm) major rotamer: 165.67 (C=O amide),
155.24 (Ar), 151.60 (C=O), 141.94, 132.00, 131.60, 129.18, 127.00, 122.10, 114.80
(Ar), 94.00 (C), 80.22 (C(CH3)3), 66.30, 65.16 (CH2O, CH2OAr), 55.68 (CHN), 28.41
(C(CH3)3), 26.76, 24.26 (CH3), 21.38 (CH3Ar). HRMS (m/z): [MNa+] calcd for
C25H32N2NaO5, 463.2203; found, 463.2207.
4.17.2.
1,1-Dimethylethyl
(S)-2,2-dimethyl-4-[[3-(4-
methylbenzoylamino)phenoxy]methyl]-3-oxazolidinecarboxylate (29)
23
Yellow solid. 60% yield (2 steps): mp 115-117 ºC; [α]D +50 (c 1.53, CHCl3); IR v
3314, 2979, 2935, 2878, 1690, 1656, 1599, 1491, 1387, 1377, 1365 cm-1; 1H NMR (200
MHz, CDCl3) δ (ppm): 8.13 (s, 1H, NH), 7.75 (d, 2H, Ar, J = 8.2 Hz), 7.45-7.16 (m,
5H, Ar), 6.74-6.70 (m, 1H, Ar), 4.23-3.81 (m, 5H, CHN, CH2O, CH2OAr), 2.39 (s, 3H,
13
CH3Ar), 1.62, 1.58, 1.50 (s, 15H, C(CH3)2, C(CH3)3); C NMR (50 MHz, CDCl3) δ

ACCEPTED MANUSCRIPT
(ppm) major rotamer: 165.72 (C=O amide), 159.07 (Ar), 151.92 (C=O), 142.20, 139.34,
132.07, 129.64, 129.28, 127.03, 112.82, 110.71, 106.78 (Ar), 93.58 (C), 80.43
(C(CH3)3), 66.16, 65.34 (CH2O, CH2OAr), 56.03 (CHN), 28.42 (C(CH3)3), 26.76, 24.36
(CH3), 21.35 (CH3Ar). HRMS (m/z): [MNa+] calcd for C25H32N2NaO5, 463.2203;
found, 463.2202.
4.18. General procedure 9 - synthesis of compounds 25, 26 and 30
To a stirred solution of amine 51 or 52 (0.568 mmol, general procedure 7) was
added pyridine (1.5 mL) and mesyl or tosyl chloride (1.70 mmol). After 3 h, EtOAc (60
mL) was added. The organic layer was washed with 1 M aqueous HCl (60 mL), water
(2 X 60 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by
silica gel column chromatography eluting with appropriate mixture as indicated in each
case.
4.18.1.
1,1-Dimethylethyl
(S)-2,2-dimethyl-4-[[4-(4-
toluenesulfonylamino)phenoxy]methyl]-3-oxazolidinecarboxylate (25)
23
Eluent hexanes:EtOAc (7:3). White solid. 70% yield (2 steps): mp 101-102 ºC; [α]D
+55 (c 0.47, CHCl3); IR v 3248, 2979, 2936, 2879, 1688, 1598, 1508, 1465, 1389, 1365
1
cm-1; H NMR (200 MHz, CDCl3) δ (ppm): 7.59 (bs, 2H, Ar), 7.18 (d, 2H, Ar, J = 7.6
Hz), 7.05 (s, 1H, NH), 6.93 (bs, 2H, Ar), 6.74 (d, 2H, Ar, J = 8.4 Hz), 4.19-3.70 (m, 5H,
CHN, CH2O, CH2OAr), 2.35 (s, 3H, CH3Ar), 1.57, 1.53, 1.47 (s, 15H, C(CH3)2,
13
C(CH3)3); C NMR (100 MHz, CDCl3 + drops of DMSO) δ (ppm) major rotamer:
156.38 (Ar), 152.23 (C=O), 143.17, 136.33, 129.75, 129.34, 127.14, 124.71, 114.92
(Ar), 93.47 (C), 80.53 (C(CH3)3), 66.15, 65.08 (CH2O, CH2OAr), 55.95 (CHN), 28.31
(C(CH3)3), 27.43, 24.18 (CH3), 21.40 (CH3Ar). HRMS (m/z): [MNa+] calcd for
C24H32N2NaO6S, 499.1873; found, 499.1883.
4.18.2.
1,1-Dimethylethyl
(S)-2,2-dimethyl-4-[[4-
(methylsulfonylamino)phenoxy]methyl]-3-oxazolidinecarboxylate (26)
Eluent hexanes:EtOAc (7:3). Yellow solid. 57% yield (2 steps): mp 108-110 ºC;
[α]D23 +55 (c 0.47, CHCl3); IR v 3246, 2978, 2935, 2880, 1687, 1610, 1508, 1466, 1388,
1
1366 cm-1; H NMR (200 MHz, CDCl3) δ (ppm): 7.15 (m, 3H, Ar, NH), 6.87 (d, 2H,
Ar, J = 8.8 Hz), 4.21-3.74 (m, 5H, CHN, CH2O, CH2OAr), 2.90 (s, 3H, CH3SO2), 1.58,
13
1.54, 1.47 (s, 15H, C(CH3)2, C(CH3)3); C NMR (50 MHz, CDCl3) δ (ppm) major
rotamer: 156.74 (Ar), 151.53 (C=O), 129.43, 124.46, 115.34 (Ar), 93.52 (C), 80.65
(C(CH3)3), 66.20, 65.09 (CH2O, CH2OAr), 55.86 (CHN), 38.58 (CH3SO2), 28.32
(C(CH3)3), 27.43, 24.17 (CH3). HRMS (m/z): [MNa+] calcd for C18H28N2NaO6S,
423.1560; found, 423.1562.
4.18.3.
1,1-Dimethylethyl
(S)-2,2-dimethyl-4-[[3-(4-
toluenesulfonylamino)phenoxy]methyl]-3-oxazolidinecarboxylate (30)
23
Eluent hexanes:EtOAc (75:25). White semi-solid. 40% yield (2 steps): [α]D +48 (c
0.79, CHCl3); IR v 3244, 2978, 2935, 2880, 1693, 1600, 1495, 1466, 1385, 1366 cm-1;
1H NMR (400 MHz, CDCl3) δ (ppm): 7.67 (d, 2H, Ar, J = 7.8 Hz), 7.22 (d, 2H, Ar, J =
7.8 Hz), 7.12-7.08 (m, 1H, Ar), 6.77-6.61 (m, 4H, Ar, NH), 4.21-3.74 (m, 5H, CHN,
CH2O, CH2OAr), 2.38 (s, 3H, CH3Ar), 1.66-1.47 (m, 15H, C(CH3)2, C(CH3)3); 13C
NMR (100 MHz, CDCl3) δ (ppm) major rotamer: 159.19 (Ar), 152.37 (C=O), 143.81,
137.74, 136.11, 130.06, 129.62, 127.30, 113.58, 111.42, 107.76 (Ar), 93.57 (C), 80.69
(C(CH3)3), 66.07, 65.18 (CH2O, CH2OAr), 55.90 (CHN), 28.43 (C(CH3)3), 27.56, 24.26

ACCEPTED MANUSCRIPT
(CH3), 21.53 (CH3Ar). HRMS (m/z): [MNa+] calcd for C24H32N2NaO6S, 499.1873;
found, 499.1876.
4.19. Synthesis of compounds 27 and 31
Synthesis of 27 and 31 was carried out as described in general procedure 1. In this
case, compound 39 was replaced by compound 35. The residue was purified by flash
silica gel column chromatography eluting with Hexanes/EtOAc (975:25) or (95:5).
4.19.1.
1,1-Dimethylethyl
(S)-4-[[(4-biphenyloxy]methyl]-2,2-dimethyl-3-
oxazolidinecarboxylate (27)
White solid. 68% yield: mp 86-87 ºC; [α]D22 +57 (c 0.98, CH2Cl2); IR v 2979, 2935,
2879, 1693, 1608, 1584, 1519, 1487, 1464, 1384, 1376, 1364 cm-1; 1H NMR (200 MHz,
CDCl3) δ (ppm): 7.57 (bs, 4H, Ar), 7.44 (bs, 2H, Ar), 7.36 (bs, 1H, Ar), 7.07 (bs, 2H,
Ar), 4.33-3.93 (m, 5H, CHN, CH2O, CH2OAr), 1.80, 1.68, 1.55 (s, 15H, C(CH3)2,
13
C(CH3)3); C NMR (50 MHz, CDCl3) δ (ppm) major rotamer: 157.99 (Ar), 151.66
(C=O), 140.70, 133.90, 128.66, 128.11, 126.68, 114.86 (Ar), 93.50 (C), 80.45
(C(CH3)3), 66.78, 65.32 (CH2O, CH2OAr), 56.02 (CHN), 28.47 (C(CH3)3), 27.52, 24.30
(CH3). HRMS (m/z): [MNa+] calcd for C23H29NNaO4, 406.1989; found, 406.1987.
4.19.2.
1,1-Dimethylethyl
(S)-4-[[(3-biphenyloxy]methyl]-2,2-dimethyl-3-
oxazolidinecarboxylate (31)
22
Colorless oil. 51% yield: [α]D +55 (c 1.54, CH2Cl2); IR v 2978, 2934, 2878, 1689,
1597, 1571, 1478, 1422, 1385, 1376, 1364 cm-1; H NMR (200 MHz, CDCl3) δ (ppm):
1
7.62 (bs, 2H, Ar), 7.46-7.38 (m, 4H, Ar), 7.24 (bs, 2H, Ar), 7.00 (bs, 1H, Ar), 4.35-3.96
(m, 5H, CHN, CH2O, CH2OAr), 1.68, 1.63, 1.54 (s, 15H, C(CH3)2, C(CH3)3); 13C NMR
(50 MHz, CDCl3) δ (ppm) major rotamer: 158.86 (Ar), 151.66 (C=O), 142.65, 140.88,
129.70, 128.64, 127.14, 119.75, 113.61, 113.34 (Ar), 93.50 (C), 80.43 (C(CH3)3), 66.78,
65.32 (CH2O, CH2OAr), 56.06 (CHN), 28.42 (C(CH3)3), 27.50, 24.26 (CH3). HRMS
(m/z): [MNa+] calcd for C23H29NNaO4, 406.1989; found, 406.1990.
4.20. Cell lines
HL60 cells (human promyelocytic leukemia) and JURKAT (T cell leukemia) were
cultivated in the logarithmic phase of growth in RPMI 1640 (Sigma-Aldrich, St. Louis,
MO) supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin (GIBCO
BRL, Grand Island, NY), enriched with 2 mM of L-glutamine and 10% of fetal bovine
serum. MCF-7 and MDA-MB-231 (human breast cancer), HCT-116 (colon carcinoma)
and Vero cells (kidney epithelial cells of the African Green Monkey) were cultivated in
D-MEM (Sigma-Aldrich, St. Louis, MO), enriched with 2 mM of L-glutamine and 5%
of fetal bovine serum.
4.21. Analysis of cell viability
HL60 and JURKAT were cultured in 96 wells plate at densities of 50,000 and
100,000 cells/well, respectively. MCF-7, MDA-MB-231, HCT-116 and Vero cells were
seeded at density of 10,000 cells/well. The plates were pre-incubated in a 5% CO2/95%
air-humidified atmosphere at 37 ºC for 24 h to allow adaptation of cells prior to the
addition of the test compounds. All substances were dissolved in dimethyl sulfoxide
(DMSO), prior to dilution. The 50% inhibitory concentration (IC50) was determinated

ACCEPTED MANUSCRIPT
over a range of concentrations (10 nM-100 µM). All cell cultures were incubated in a
5% CO2/95% air-humidified atmosphere at 37 ºC for 48 hours. Cell viability was
estimated measuring the rate of mitochondrial reduction of yellow tetrazolium salt MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma-Aldrich, St.
Louis, MO) to insoluble purple formazan crystals [28]. After incubation with the test
compounds, MTT solution (20 µL; 5 mg/mL) was added to each well and incubated for
4 hours. At the end of this incubation, the supernatant was removed and 200 µL of 0.04
M HCl in isopropyl alcohol were added to dissolve the formazan crystal. The optical
densities (OD) were measured with a spectrophotometer at 590 nm. Results were
normalized with DMSO control (0.05%) and expressed as percentage of cell viability
inhibition. Interactions of compounds and media were estimated on the basis of the
variations between drug-containing medium and drug-free medium to control for false-
positive or false-negative results. The 50% inhibitory concentration (IC50) values were
obtained graphically from dose-effect curves using Prism 5.0 (GraphPad Software Inc.).
4.22. DNA fragmentation assay
Cell cycle status and quantification of DNA fragmentation (hypodiploid DNA-
content) were performed by propidium iodide staining [29]. HL60 and JURKAT cells
were treated with compounds 2, 3, 4, 12, 18, 23, 24, 25 and 30 at the final concentration
of 50 µM and incubated in a 5% CO2/95% air-humidified atmosphere at 37 ºC for 24
hours. DMSO (0.5%) was used as negative control. Afterwards, the cells were
centrifuged, resuspended in a hypotonic fluorochrome solution - HFS (50 µg/mL PI in
0.1% sodium citrate plus 0.1% Triton X-100) and incubated at 4 ºC for 4 h. The PI
fluorescence of 20,000 individual nuclei was measured using a FACScalibur flow
cytometer (Becton Dickinson Immunocytometry Systems, San Jose, Calif.). Data were
analyzed using FlowJo software (TreeStar Inc, CA). All results were expressed as the
mean ± SD of two independent experiments carried out in triplicate. These data were
analyzed using Student's t-test for paired comparisons, using Prism 5.0 (GraphPad
Software Inc.). Statistical significance was determined as P < 0.05.
Acknowledgements
We are thankful to FAPEMIG, FAPERGS, CNPq and CAPES for financial support
and fellowships.
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