Synthesis and Evaluation of 2-Alkylthio-4-(N-substituted sulfonamide)pyrimidine Hydroxamic Acids as Anti-myeloma Agents

Article abstract of DOI:10.1111/cbdd.12678

A series of pyrimidine hydroxamic acids with a sulfide substituent at the second position and a sulfonamide substituent at the fourth position have been synthesized and evaluated for their activity against human myeloma cell line RPMI 8226. Several compounds exhibited significant anti-cancer potency. It was found that representative compound 6a selectively killed cancerous but not normal cells. Moreover, compound 6a was effective in causing apoptosis in RPMI 8226 cells and exhibited promising HDAC-inhibitory activities.

Full text of DOI:10.1111/cbdd.12678

Chem Biol Drug Des 2016; 87: 472–477
Research Letter
Synthesis and Evaluation of 2-Alkylthio-4-(N-
substituted sulfonamide)pyrimidine Hydroxamic Acids
as Anti-myeloma Agents
Jinbao Xiang1, Crystal Leung2, Zhuoqi Zhang1,
Pyrimidine moiety, as a structural component of several
key biomolecules, has attracted great attention from
organic and medicinal chemists (4–7). Recently, we have
designed and synthesized a series of novel pyrimidine-
Cassie Hu2, Chao Geng1, Lili Liu3, Lang Yi1,
Zhiwei Li1,*, James Berenson2 and Xu Bai1,*
1The Center for Combinatorial Chemistry and Drug
Discovery, The School of Pharmaceutical Sciences, The
College of Chemistry, Jilin University, 1266 Fujin Road,
Changchun, Jilin 130021, China
based compounds (8–16). To explore our compounds as
potential anti-myeloma agents, we have screened our
pyrimidine-based library and identified 2-alkylthio-4-(N-
substituted sulfonamide)pyrimidine hydroxamic acids with
anti-myeloma activity in vitro. Moreover, the presence of
N-substituted sulfonamido substituent in the pyrimidine
ring is essential to achieve activity. These results are differ-
ent from the previously reported 2-aminopyrimidine
hydroxamic acids as anti-cancer HDAC inhibitors (such as
JNJ-16241199, Figure 1) (17–20). To the best of our
knowledge, there is no report of 2-alkylthiopyrimidine
hydroxamic acids as anti-cancer agents. Herein, the
synthesis and evaluation of the novel 2-alkylthio-4-(N-sub-
stituted sulfonamide)pyrimidine hydroxamic acids as anti-
myeloma agents are presented.
2Institute for Myeloma & Bone Cancer Research, 9201
West Sunset Blvd, Suite 300, West Hollywood, CA 90069,
USA
3Changchun Discovery Sciences, Ltd., 750 Jinbi Street,
Changchun, Jilin 130117, China
*Corresponding authors: Xu Bai, xbai@jlu.edu.cn; Zhiwei
Li, zhiweili@jlu.edu.cn
A series of pyrimidine hydroxamic acids with a sulfide
substituent at the second position and a sulfonamide
substituent at the fourth position have been
synthesized and evaluated for their activity against
human myeloma cell line RPMI 8226. Several com-
pounds exhibited significant anti-cancer potency. It
was found that representative compound 6a selectively
killed cancerous but not normal cells. Moreover, com-
pound 6a was effective in causing apoptosis in RPMI
8226 cells and exhibited promising HDAC-inhibitory
activities.
R1 substituted pyrimidine hydroxamic acids 6a–i were syn-
thesized by the method shown in Scheme 1. The compound
1 was reacted with N-benzyl-4-methoxybenzenesulfonamide
in the presence of K2CO3 as a base to afford ester 2. Hydrol-
ysis of 2 afforded acid 3. Intermediate 3 was converted to
hydroxamic acid 4 in a two-step process involving formation
of an activated acyl chloride followed by addition of hydroxy-
lamine (21). Oxidation of 4 with m-CPBA gave the sulfoxide
intermediate 5. Final pyrimidine hydroxamic acids 6a–i were
obtained by reacting sulfoxide 5 with various nucleophiles
(R1H).
Key words: anti-cancer activity, apoptosis, histone acetyla-
tion, multiple myeloma, pyrimidine hydroxamic acid, sulfide
substituent
Received 12 July 2015, revised 11 October 2015 and
accepted for publication 22 October 2015
R2 substituted pyrimidine hydroxamic acids 6j–s were pre-
pared by the method shown in Scheme 2. Compound 7
was reacted with thiourea in the presence of NaOEt as a
base to afford pyrimidine 8 (22). Condensation of 8 with 1-
iodopentane formed 9. Chlorination of 9 using phosphoryl
chloride yielded the desired 10. Coupling of 10 with nucle-
ophile (R2H) gave intermediate 11. Intermediate 11l (R2=H)
could be obtained from starting material 1. Reduction of 1
with Zn-NH4Cl furnished 12. Oxidation of 12 with m-CPBA
gave the sulfone 13. Condensation of 13 with pentane-1-
thiol formed intermediate 11l. Hydrolysis of 11 afforded acid
14. Intermediate 14 was converted to hydroxamic acid 6
(6k–p and 6r) in a two-step process involving formation of
Cancer is a group of different diseases characterized by
uncontrolled growth and spread of abnormal cells (1). Mul-
tiple myeloma accounts for approximately 1% of all can-
cers and 10% of hematologic malignancies (2). Despite
the fact that the significant advances have achieved in the
last two decade with the discovery of novel agents such
as proteasome inhibitors (bortezomib) and immunomodula-
tory drugs (thalidomide and lenalidomide), the disease
remains incurable (3). The continued commitment to the
arduous task of discovering non-traditional, efficient, and
safe anti-myeloma agents remains critically important.
472
ª 2015 John Wiley & Sons A/S. doi: 10.1111/cbdd.12678

Novel Anti-Myeloma Agents
Figure 1: Hydroxamic acid derivatives as anti-cancer HDAC inhibitors.
Scheme 1: Synthesis of compounds 6a–i. Reagents and conditions: (a) N-benzyl-4-methoxybenzenesulfonamide, K2CO3, CH3CN, reflux,
64%; (b) NaOH aq, 1,4-dioxane, 50 °C, 98%; (c) (i) (COCl)2, DMF, CH2Cl2, rt, (ii) NH2OH∙HCl, Et3N, DMF, CH2Cl2, 0 °C to rt, 60% for two
steps; (d) m-CPBA, CH2Cl2, rt, 65%; (e) thiol or pentan-1-amine, Et3N, CH3CN, rt, 32–60%; (f) pentan-1-ol, NaH, rt, 28%.
an activated acyl chloride followed by addition of hydroxy-
lamine. Alternatively, amidation of 14 with commercially
available NH2OTHP and removal of the tetrahydropyranyl
(THP) protecting moiety in acidic conditions led to the final
pyrimidine hydroxamic acid 6 (6j, 6q and 6s) (20).
Anti-proliferative activities of the pyrimidine hydroxamic
acids were evaluated using the human myeloma cell line
RPMI 8226 as an experimental model. As shown in
Table 1, our data demonstrated that among 20 com-
pounds tested, nine compounds (6a, 6e–i, 6m, 6o and
6s) exhibited significant anti-proliferative activity with IC50
below 10 lM. To our knowledge, this is the first example
of 2-alkylthiopyrimidine hydroxamic acids as anti-cancer
agents. It was observed that compound 6a is more potent
than both nitrogen and oxygen analog (entry 1 versus
entries 2 and 3), indicating that the thiol linker at the C-2
position is essential to enhance growth inhibitory effects.
Moreover, compounds 6a (entry 1) and 6e–i (entries 5–9)
showed better growth inhibition compared with 6d (entry
4), suggesting that the length of R1-group had effect on
activities. It was noteworthy that the presence of a N-sub-
stituted sulfonamido substituent (R2) in the pyrimidine ring
The a,b-unsaturated hydroxamic acid 6t was prepared by
the method shown in Scheme 3. Initially, reduction of 10
with LiAlH4 gave the alcohol 15 in only 14% yield. Treat-
ment of 10 with DIBAL-H formed the alcohol 15 in good
yield. Oxidation of 15 with MnO2 gave the aldehyde 16.
Coupling of 16 with N-benzyl-4-methoxybenzenesulfona-
mide followed by Wittig olefination yielded the ester 18
(23). Hydrolysis of 18 afforded acid 19. Intermediate 19
was converted to hydroxamic acid 6t in a two-step pro-
cess involving formation of an activated acyl chloride
followed by addition of hydroxylamine.
Chem Biol Drug Des 2016; 87: 472–477
473

Xiang et al.
Scheme 2: Synthesis of compounds 6j–s. Reagents and conditions: (a) thiourea, EtONa, EtOH, reflux, 98%; (b) CH3(CH2)4I, NaI,
tetrabutyl ammonium bromide, CH3Cl3, H2O, 50 °C, 53%; (c) POCl3, reflux, 89%; (d) nucleophile (R2H), K2CO3, CH3CN, reflux, 34–83%;
(e) Zn powder, NH4Cl aq, toluene, 80 °C, 56%; (f) m-CPBA, CH2Cl2, rt, 76%; (g) CH3(CH2)4SH, Et3N, CH3CN, rt, 89%; (h) NaOH aq, 1,4-
dioxane, 50 °C, 72–99%; (i) (i) (COCl)2, DMF, CH2Cl2, rt, (ii) NH2OH∙HCl, Et3N, THF, H2O, 0 °C to rt, 7–93% for two steps; (j) (i) NH2OTHP
[O-(tetrahydro-2H-pyran-2-yl)hydroxylamine], EDC∙HCl, Et3N, HOBt, CH2Cl2, THF, rt, (ii) CF3CO2H, CH2Cl2, MeOH, rt, 28–35% for two
steps.
appears to be essential to achieve activity (entry 1 versus
entries 10–12). These results are different from the previ-
ously reported 2-aminopyrimidine hydroxamic acids as
anti-cancer HDAC inhibitors where the R2-group is hydro-
gen atom (such as JNJ-16241199, Figure 1) (17–20). Fur-
ther studies were focused on the effects of the
sulfonamide moiety at the C-4 position. Compound 6m
with a p-fluoro group (entry 13) showed slightly more
active than compound 6a. Removal of p-methoxy of com-
pound 6a led to decreased inhibitory activity (6n, entry
14). Among all the tested compounds, compound 6o with
an m-methoxy group was found to be the most active
with the IC50 value of 5.6 lM (entry 15). Replacement of
the phenyl ring by a pyridyl group led to decreased inhibi-
tory activity (6p versus 6a, entry 16). Removal (6q versus
6a, entry 17) or insertion (6r versus 6a, entry 18) of a
methylene spacer between the amino and phenyl ring
reduced the activity; however, the insertion of a longer
chain, as in 6s (entry 19), enhanced the activity. It was
unexpected that the belinostat and panobinostat (6) ana-
log 6t is not substantially toxic to RPMI 8226 cells (entry
20). In consistent to the data published by others (24), the
positive control compound SAHA has an IC50 values of
1.0 lM.
In addition, the anti-cancer activity of representative com-
pound 6a against MM1S was further tested, and the pre-
liminary result indicated that the IC50 value was 7.5 lM. It
was interesting to note that representative compound 6a
did not present any detectable cytotoxicity against normal
peripheral blood mononuclear cell (PBMC) for the tested
concentrations from 0.078 to 20 lM (Figure 2), suggesting
that compound 6a is a potent anti-myeloma agent that
selectively kill cancerous but not normal cells.
To elucidate the possible molecular mechanism of com-
pounds 6’s anti-myeloma activity, we further analyzed the
apoptotic cell death induced by representative compound
6a. As shown in Figure 3, compound 6a induced RPMI
8226 cell apoptosis in drug dose-dependent way, confirm-
ing that apoptosis contributes to the anti-myeloma effect
of compound 6a.
474
Chem Biol Drug Des 2016; 87: 472–477

Novel Anti-Myeloma Agents
Scheme 3: Synthesis of compound 6t. Reagents and conditions: (a) DIBAL-H, THF, 50 °C, 76%; (b) MnO2, CHCl3, reflux, 70%;
(c) N-benzyl-4-methoxybenzenesulfonamide, K2CO3, CH3CN, rt, 53%; (d) ethyl 2-(diethoxyphosphoryl)acetate, NaH, THF, reflux, 66%;
(e) NaOH aq, 1,4-dioxane, 50 °C, 98%; (f) (i) (COCl)2, DMF, CH2Cl2, rt, (ii) NH2OH∙HCl, Et3N, THF, H2O, 0 °C to rt, 16% for two steps.
Table 1: Anti-proliferative activities of compounds
6 in RPMI
8226 cells. RPMI 8226 cells were cultured in the presence of
compounds 6 at a serial concentration for 48 h. IC50 were deter-
mined by MTS assaya
Entry
Compd.
IC50 (lM)
Entry
Compd.
IC50 (lM)
1
2
3
4
5
6
7
8
6a
6b
6c
6d
6e
6f
8.6 Æ 0.4
>10
>10
12
13
14
15
16
17
18
19
20
21
6l
>10
6m
6n
6o
6p
6q
6r
7.0 Æ 0.1
>10
>10
5.6 Æ 0.6
>10
>10
7.8 Æ 0.4
9.1 Æ 0.3
9.8 Æ 0.2
9.3 Æ 0.5
8.8 Æ 0.4
>10
6g
6h
6i
>10
6s
6t
6.8 Æ 0.1
Figure 2: Effects of Compound 6a on human PBMC
proliferation. PBMCs from healthy donor were cultured in the
presence of compound 6a at the indicated concentration for
24 h. Cell viability was measured by MTS assay.
9
10
11
>10
6j
6k
SAHA
1.0 Æ 0.1
>10
aData were obtained from four independent experiments.
Compounds 6 contain hydoxamic functional group, the
active motif of one type of HDAC inhibitors. To explore
possible HDAC-inhibitory activities of compounds 6, we
treated RPMI 8226 cells with representative compound
6a. Histone acetylation was determined by Western blot
analysis using antibody against acetylated histone H4.
As shown in Figure 4, compound 6a has the potent
close to SAHA. Histone acetylation signal induced by
10 lM of compound 6a is stronger than that induced by
5 lM of SAHA but weaker than that induced by 10 lM of
potential lead compound for further development in anti-
myeloma therapy. Moreover, it was noteworthy that cin-
namoyl HDAC inhibitors with a bulky substituent at the
orthoposition of the phenyl ring have been reported
recently (Pharmacyclics’ compound, Figure 1) (25), indi-
cating that compound 6a appears to be capable of
binding to HDACs.
In summary, the synthesis and evaluation of anti-cancer
activity of a series of pyrimidine hydroxamic acids with
various sulfide substituents at the second position and
SAHA, suggesting that compound 6a could be
a
Chem Biol Drug Des 2016; 87: 472–477
475

Xiang et al.
Figure 3: Compound 6a induced apoptosis in RPMI 8226 cells. RPMI 8226 cells were cultured in the presence of compound 6a at the
indicated concentration for 24 h. Apoptosis was determined by flowcytometry following Annexin V-FITC staining.
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