Lead optimization of HMBA to develop potent HEXIM1 inducers

Article abstract of DOI:10.1016/j.bmcl.2014.01.025

The potency of a series of Hexamethylene bis-acetamide (HMBA) derivatives inducing Hexamethylene bis-acetamide inducible protein 1 (HEXIM1) was determined in LNCaP prostate cancer cells. Several compounds with unsymmetrical structures showed significantly improved activity. Distinct from HMBA, these analogs have increased hydrophobicity and can improve the short half-life of HMBA, which is one of the factors that have limited the application of HMBA in clinics. The unsymmetrical scaffolds of the new analogs provide the basis for further lead optimization of the compounds using combinatorial chemistry strategy.

Full text of DOI:10.1016/j.bmcl.2014.01.025

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1410–1413
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Lead optimization of HMBA to develop potent HEXIM1 inducers
a,c,
Bo Zhong a, Rati Lama a, Wannarasmi Ketchart b, Monica M. Montano b, , Bin Su
⇑
⇑
a Department of Chemistry, College of Sciences and Health Professions, Cleveland State University, 2121 Euclid Ave., Cleveland, OH 44115, USA
b Department of Pharmacology, Case Western Reserve University School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106, USA
c Center for Gene Regulation in Health and Disease, College of Sciences & Health Professions, Cleveland State University, 2121 Euclid Ave., Cleveland, OH 44115, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The potency of a series of Hexamethylene bis-acetamide (HMBA) derivatives inducing Hexamethylene
bis-acetamide inducible protein 1 (HEXIM1) was determined in LNCaP prostate cancer cells. Several com-
pounds with unsymmetrical structures showed significantly improved activity. Distinct from HMBA,
these analogs have increased hydrophobicity and can improve the short half-life of HMBA, which is
one of the factors that have limited the application of HMBA in clinics. The unsymmetrical scaffolds of
the new analogs provide the basis for further lead optimization of the compounds using combinatorial
chemistry strategy.
Received 4 November 2013
Revised 7 January 2014
Accepted 9 January 2014
Available online 17 January 2014
Keywords:
HMBA
HEXIM1
Published by Elsevier Ltd.
Derivatives
Drug development
Hexamethylene bis-acetamide (HMBA), a small molecule, was
investigated by the National Cancer Institute due to its potent
anti-cancer and cell differentiation activities.1,2 The molecule failed
at the Phase II clinical trial because of the dose-dependent toxic-
ity.1 HMBA achieves its biological activity via Hexamethylene
bis-acetamide inducible protein 1 (HEXIM1).3 HMBA significantly
induces HEXIM1 expression in various cell lines at millimolar con-
centrations.3 However, for the agent to reach the active concentra-
tion in patients, it has to be administered via infusion of a high
dosage,1,4 which causes significant toxicity.
HEXIM1 binds to 7SK RNA, a highly abundant non-coding RNA.
Together they act as potent inhibitors of positive transcription
elongation b (P-TEFb) and lead to inhibition of transcription.5–8
The regulation of the relative ratio of inactive to active P-TEFb in
cells by HEXIM1/7SK RNA plays a critical role in a wide range of
cellular gene expression such as estrogen and glucocorticoid recep-
tor regulated genes.7,9 HEXIM1 also has an important role in heart
development and remodeling.10,11 HEXIM1 expression is decreased
in a model of pathological hypertrophy in the adult heart12 and de-
creased HEXIM1 expression augmented angiotensinogen induced
pathological hypertrophy.13 Recent research suggests that HEXIM1
suppresses cancer metastasis and Human immunodeficiency virus
(HIV) replication.11,14,15 Another group reported on the inhibitory
role of HEXIM1 in prostate tumorigenesis.16 Thus, drug candidates
that can enhance the expression of HEXIM1 will have a great
application in the treatment of cancer, heart disease, and acquired
immunodeficiency syndrome (AIDS).
HMBA is the most potent and specific inducer for HEXIM1 thus
far. However, this occurs only in in vitro cell cultures at concentra-
tions ranging from one to five millimolars.2,3 It is difficult to reach
high concentrations of HMBA in blood circulation due to its toxic-
ity effects. There is an urgent need to optimize HMBA and improve
its ability to induce HEXIM1 expression.11,17 In the previous studies
numerous HMBA analogs have been developed to improve the
activity for inducing cell differentiation, but their ability to induce
HEXIM1 expression has never been evaluated.18 To obtain better
HEXIM1 inducers, we designed and synthesized HMBA symmetri-
cal and unsymmetrical derivatives. The structure of HMBA is rep-
resented as three different moieties, A, B and C, (Fig. 1), which
was used as the basis for our combinatorial chemistry approach
to generate the new analogs. Since the direct binding target of
HMBA is still unknown, the derivatives can be explored only via
the traditional medicinal chemistry strategy, that is, ligand based
modification. Moreover, HMBA is a water-soluble molecule, which
contributes greatly to its short half-life and poor tissue distribu-
tion. In the compound design, we introduced functional groups
that can increase the hydrophobicity of the molecule.
O
H
N
N
C
⇑
Corresponding authors. Tel.: +1 216 368 3378; fax: +1 216 368 1300 (M.M.M.);
H
A
B
O
tel.: +1 216 687 9219; fax: +1 216 687 9298 (B.S.).
E-mail addresses: monica.montano@case.edu (M.M. Montano), B.su@csuohio.
edu (B. Su).
Figure 1. HMBA analog design.
0960-894X/$ - see front matter Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2014.01.025

B. Zhong et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1410–1413
1411
R1
RCOCl or RSO2Cl
DMF, K2CO3
CH3I, NaH, DMF
R2
R2
R1
N
N
H2N
NH2
H
H
1a. R1=1,6-hexylene
2a1. R1=1,6-hexylene, R2=acetyl (HMBA)
2a2. R1=1,6-hexylene, R2=methanesulfonyl
2a3. R1=1,6-hexylene, R2=benzoyl
1b. R1=1,4-phenylene-bis(methylene)
1c. R1=1,4-cyclohexylene-bis(methylene)
1d. R1=1,4-cyclohexylene
2a4. R1=1,6-hexylene, R2=propionyl
1e. R1=1,5-pentylene
2a5. R1=1,6-hexylene, R2=butyryl
1f. R1=1,3-propylene
2a6. R1=1,6-hexylene, R2=2-(4-methoxyphenyl)acetyl
2b1. R1=1,4-phenylene-bis(methylene), R2=acetyl
2b2. R1=1,4-phenylene-bis(methylene), R2=methanesulfonyl
2c1. R1=1,4-cyclohexylene-bis(methylene), R2=acetyl
2c2. R1=1,4-cyclohexylene-bis(methylene), R2=methanesulfonyl
2d1. R1=1,4-cyclohexylene, R2=acetyl
2d2. R1=1,4-cyclohexylene, R2=methanesulfonyl
2e1. R1=1,5-pentylene, R2=acetyl
2e2. R1=1,5-pentylene, R2=methanesulfonyl
2f1. R1=1,3-propylene, R2=acetyl
2f2. R1=1,3-propylene, R2=methanesulfonyl
R1
3a1. R1=1,6-hexylene, R2=acetyl
R2
R2
N
N
3a2. R1=1,6-hexylene, R2=methanesulfonyl
3b1. R1=1,4-phenylene-bis(methylene), R2=acetyl
3b2. R1=1,4-phenylene-bis(methylene), R2=methanesulfonyl
3c1. R1=1,4-cyclohexylene-bis(methylene), R2=acetyl
3c2. R1=1,4-cyclohexylene-bis(methylene), R2=methanesulfonyl
3d1. R1=1,4-cyclohexylene, R2=acetyl
3d2. R1=1,4-cyclohexylene, R2=methanesulfonyl
3e1. R1=1,5-pentylene, R2=acetyl
3e2. R1=1,5-pentylene, R2=methanesulfonyl
3f1. R1=1,3-propylene, R2=acetyl
3f2. R1=1,3-propylene, R2=methanesulfonyl
Scheme 1. Synthesis of symmetrical HMBA analogs.
H
CH3COCl
TCA
H2N
Boc
N
Boc
R3
N
N
DMF, K2CO3
H
H
O
H
RCOCl
H
N
NH2
N
N
DMF, K2CO3
O
H
O
4a1. R3=2-(4-methoxyphenyl)acetyl
4a2. R3=2-(4-benzyloxyphenyl)acetyl
4a3. R3=4-methoxybenzoyl
4a4. R3=[1,1'-biphenyl]-4-carboxyl
4a5. R3=4-chloro-3-nitrobenzoyl
Scheme 2. Synthesis of unsymmetrical HMBA analogs.
The synthesis of the symmetrical and unsymmetrical deriva-
tives is described in Schemes 1 and 2, respectively. For the sym-
metrical analogs, moieties A and C were kept identical. Besides
the acetyl group of HMBA, methanesulfonyl group, propionyl
group or butyryl group were introduced. In addition, a bulky ben-
zoyl group was introduced. The active proton in the amide moiety
was replaced with methyl group to generate a series of analogs
with lower solubility. For the moiety B, various linkers such as
1,3-propylene, 1,5-pentylene, 1,4-cyclohexylene, 1,4-phenylene-
bis(methylene), 1,4-cyclohexylene-bis (methylene) were tested for
optimization. Since the starting materials 1,4-di(aminomethyl)cyclo-
hexane 1c and 1,4-diaminocyclohexane 1d are mixture of cis- and
trans-isomers, the corresponding products 2c1, 2c2, 3c1, 3c2, 2d1,
2d2, 3d1, 3d2 are also mixture of cis- and trans-isomers. The further
separation of cis- and trans-isomers was not carried out in this
preliminary study. For the unsymmetrical analogs, the A and B
moieties were kept the same as HMBA while the C moiety was
modified with aryl or alkyl amide. A total of 32 compounds were
synthesized, and these compounds were examined for their
potency to induce HEXIM1 in LNCaP prostate cancer cells. HMBA
(5 mM) was used as a positive control.
The biological activities of HMBA analogs were determined by
assessing HEXIM1 expression using western blot analyses. The
analogs were screened at 500 lM, a concentration that was 10
times lower than the concentration of HMBA used. Cell morphol-
ogy was examined after the treatment to exclude compounds that
caused cell toxicity. An increment of 2.5 fold in HEXIM1 expression
compared to the control was set as a cutoff to select candidates for
further analyses. In addition, solubility and structural characteris-
tics were considered when selecting the candidates. Among the
derivatives, 3b1, 3b2, 3c2, 2d1, 3d1, 2f2, 2a4 and 2a5 induced
HEXIM1 expression above 2.5 fold compared to the vehicle treat-
ment (Fig. 2). Compounds 2c1 and 4a3 also reached this cutoff.
However, cell morphology slightly changed after the treatment
with these two compounds, which excluded them from further
investigation. Compounds 3a2, 3d2, 3f2 did not reach the cutoff
for HEXIM1. The reason could be that precipitation of these com-
pounds during the treatment hindered entry to cells. Therefore,
these three compounds were screened at lower concentrations.
Compounds 4a1 and 4a4 have unsymmetrical novel structure,
and are selected as candidates for further optimization. These
two compounds were included for further analysis even if they

1412
B. Zhong et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1410–1413
5
4
3
2
1
0
500μM
Figure 2. Effect of HMBA analogs on HEXIM1 expression. LNCaP cells were treated with vehicle, HMBA (5 mM) and its analogs at 500 lM for 24 h. Level of HEXIM1 (upper
panel) and b-actin (lower panel) was analyzed by Western blot of cell extracts with HEXIM1 antibody and b-actin antibody respectively. The band intensities of HEXIM1 and
b-actin were quantified using ImageJ (NIH) and listed below the bands. The results are the representative of three independent experiments.
6
4
2
0
100μM
Figure 3. Lower concentration of HMBA analogs on HEXIM1 expression. LNCaP cells were treated with vehicle, HMBA (5 mM) and identified analogs at 100 lM for 24 h. Level
of HEXIM1 (upper panel) and b-actin (lower panel) was analyzed by Western blot of cell extracts with HEXIM1 antibody and b-actin antibody respectively. The band
intensities of HEXIM1 and b-actin were quantified using ImageJ (NIH) and listed below the bands. The results are the representative of three independent experiments.
did not reach the cutoff. Structurally, the aromatic and cyclohexyl-
ene moiety in compounds 3b1, 3b2, 2c1, 3c2, 2d1, and 3d1 re-
sulted in enhanced activity. Compounds 2a4 and 2a5 with
terminal propionyl and butyryl moieties exhibited better potency
as well, suggesting that the bigger alkyl group at the terminal moi-
ety allowed for enhanced activity. It is also possible that due to the
higher hydrophobicity of these analogs when compared to HMBA,
there is enhanced distribution of these compounds in the cells
leading to improved biological activity (Fig. 2).
Some compounds were further evaluated at a lower concentra-
tion of 100
lM (Fig. 3). The compounds did not precipitate in cell
culture, suggesting they were water soluble at 100
lM.

B. Zhong et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1410–1413
1413
40
30
20
10
0
Figure 4. Dose-dependent study of compound 4a1 and HMBA on HEXIM1 expression. Level of HEXIM1 (upper panel) and b-actin (lower panel) was analyzed by Western blot
of cell extracts with HEXIM1 antibody and b-actin, antibody respectively.
Compounds 3c2, 2d1, 3d1, 2f2, 3f2, and 4a4 were not able to in-
duce HEXIM1 expression, at 100 M. However, compounds 3a2
and 3d2 showed significant activity at 100 M that was not ob-
served at 500 M. The variation in potency could be due to the pre-
cipitation of the two compounds at 500 M, which hampers cell
penetration. Compounds 3b1 and 3b2 with the aromatic ring at
B moiety showed good activity at 100 M, and induced HEXIM1
expression by 3.8 and 2 fold, respectively. Compounds 2a4, 2a5,
and 4a1 exhibited reduced activity at lower concentrations, sug-
gesting dose dependent stimulation of HEXIM1 expression by
these compounds. Due to the unsymmetrical structural character-
istics of 4a1, a dose-dependent study was performed and the re-
sults are presented in Figure 4. The compound dose-dependently
induced HEXIM1 expression suggesting that the unsymmetrical
structure and increased hydrophobicity contribute to improved
biological activity. Considering the aromatic moiety of compounds
3b1 and 3b2 and the bulky terminal moiety of compounds 2a4 and
2a5, more potent analogs could be developed if several of these
structural units are combined in the new drug design.
In brief, our findings have provided unique molecular scaffolds
that significantly induced HEXIM1 expression in prostate cancer
cells, and have opened a new lead optimization direction of HMBA.
To the best of our knowledge, this is the first study focusing on the
lead optimization of HMBA to generate more potent HEXIM1
inducers. The results support the potential of unsymmetrical
HMBA derivatives as lead compounds. More potent HEXIM1 induc-
ers have potential application in cancer, AIDS, and heart disease
treatment. Further lead optimization of HMBA to generate more
potent HEXIM1 inducers is currently underway in our laboratory.
Department of Development (ODOD) to B.S. and NIH grant
CA092440 to M.M.M.
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References and notes
l
1. Andreeff, M.; Stone, R.; Michaeli, J.; Young, C. W.; Tong, W. P.; Sogoloff, H.;
Ervin, T.; Kufe, D.; Rifkind, R. A.; Marks, P. A. Blood 1992, 80, 2604.
2. Young, C. W.; Fanucchi, M. P.; Declan Walsh, T.; Baltzer, L.; Yaldaei, S.; Stevens,
Y. W.; Gordon, C.; Tong, W.; Rifkind, R. A.; Marks, P. A. Cancer Res. 1988, 48,
7304.
3. Turano, M.; Napolitano, G.; Dulac, C.; Majello, B.; Bensaude, O.; Lania, L. J. Cell.
Physiol. 2006, 206, 603.
4. Conley, B. A.; Egorin, M. J.; Sinibaldi, V.; Sewack, G.; Kloc, C.; Roberts, L.;
Zuhowski, E. G.; Forrest, A.; Van Echo, D. A. Cancer Chemother. Pharmacol. 1992,
31, 37.
5. He, N.; Pezda, A. C.; Zhou, Q. Mol. Cell. Biol. 2006, 26, 7068.
6. Michels, A. A.; Fraldi, A.; Li, Q.; Adamson, T. E.; Bonnet, F.; Nguyen, V. T.; Sedore,
S. C.; Price, J. P.; Price, D. H.; Lania, L.; Bensaude, O. EMBO J. 2004, 23, 2608.
7. Shimizu, N.; Ouchida, R.; Yoshikawa, N.; Hisada, T.; Watanabe, H.; Okamoto, K.;
Kusuhara, M.; Handa, H.; Morimoto, C.; Tanaka, H. Proc. Natl. Acad. Sci. U.S.A.
2005, 102, 8555.
8. Yik, J. H.; Chen, R.; Nishimura, R.; Jennings, J. L.; Link, A. J.; Zhou, Q. Mol. Cell
2003, 12, 971.
9. Ogba, N.; Chaplin, L. J.; Doughman, Y. Q.; Fujinaga, K.; Montano, M. M. Cancer
Res. 2008, 68, 7015.
10. Montano, M. M.; Doughman, Y. Q.; Deng, H.; Chaplin, L.; Yang, J.; Wang, N.;
Zhou, Q.; Ward, N. L.; Watanabe, M. Circ. Res. 2008, 102, 415.
11. Zhou, Q.; Yik, J. H. N.; Chen, R. 2006 US10/928,009, US10/928,009.
12. Espinoza-Derout, J.; Wagner, M.; Salciccioli, L.; Lazar, J. M.; Bhaduri, S.;
Mascareno, E.; Chaqour, B.; Siddiqui, M. A. Circ. Res. 2009, 104, 1347.
13. Mascareno, E.; Galatioto, J.; Rozenberg, I.; Salciccioli, L.; Kamran, H.; Lazar, J.
M.; Liu, F.; Pedrazzini, T.; Siddiqui, M. A. J. Biol. Chem. 2012, 287, 13084.
14. Ketchart, W.; Smith, K. M.; Krupka, T.; Wittmann, B. M.; Hu, Y.; Rayman, P. A.;
Doughman, Y. Q.; Albert, J. M.; Bai, X.; Finke, J. H.; Xu, Y.; Exner, A. A.; Montano,
M. M. Oncogene 2012, 32, 3829.
l
15. Ruefli, A. A.; Smyth, M. J.; Johnstone, R. W. Blood 2000, 95, 2378.
16. Mascareno, E. J.; Belashov, I.; Siddiqui, M. A.; Liu, F.; Dhar-Mascareno, M.
Prostate 2012, 72, 1035.
17. Richon, V. M.; Webb, Y.; Merger, R.; Sheppard, T.; Jursic, B.; Ngo, L.; Civoli, F.;
Breslow, R.; Rifkind, R. A.; Marks, P. A. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 5705.
18. Breslow, R.; Jursic, B.; Yan, Z. F.; Friedman, E.; Leng, L.; Ngo, L.; Rifkind, R. A.;
Marks, P. A. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 5542.
Acknowledgments
This work was supported by Center for Gene Regulation in
Health and Disease (GRHD) of Cleveland State University and Ohio