Preparation and biological evaluation of synthetic and polymer-encapsulated congeners of the antitumor agent pactamycin: Insight into functional group effects and biological activity

Article abstract of DOI:10.1016/j.bmc.2015.02.022

The synthesis and biological analysis of a number of novel congeners of the aminocyclopentitol pactamycin is described. Specific attention was paid to the preparation of derivatives at crucial synthetic branch points of the parent structure, and biological assays revealed a number of insights into the source of pactamycin's biological activity. Additionally, the encapsulation of pactamycin and select derivatives into the PRINT

Full text of DOI:10.1016/j.bmc.2015.02.022

Accepted Manuscript
Preparation and biological evaluation of synthetic and polymer-encapsulated
congeners of the antitumor agent pactamycin: insight into functional group ef-
fects and biological activity
Robert J. Sharpe, Justin T. Malinowski, Federico Sorana, J. Christopher Luft,
Charles J. Bowerman, Joseph M. DeSimone, Jeffrey S. Johnson
PII:
S0968-0896(15)00116-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.02.022
BMC 12085
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 December 2014
1 February 2015
11 February 2015
Please cite this article as: Sharpe, R.J., Malinowski, J.T., Sorana, F., Christopher Luft, J., Bowerman, C.J.,
DeSimone, J.M., Johnson, J.S., Preparation and biological evaluation of synthetic and polymer-encapsulated
congeners of the antitumor agent pactamycin: insight into functional group effects and biological activity,
Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.02.022
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Preparation and biological evaluation of synthetic and polymer-encapsulated congeners of the antitumor agent pactamycin:
insight into functional group effects and biological activity
Robert J. Sharpe,a Justin T. Malinowski,a Federico Sorana,a J. Christopher Luft, a,b Charles J. Bowerman,a
Joseph M. DeSimone,a-h and Jeffrey S. Johnsona
aDepartment of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290, United States
bDepartment of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
27599-3290, United States
cLineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290, United States
d Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695-8613, United States
e Carolina Center of Cancer Nanotechnology Excellence, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290, United
States
f Institute for Nanomedicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290, United States
g Institute for Advanced Materials, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290, United States
h Sloan-Kettering Institute for Cancer Research, Memorial Sloan-Kettering Cancer Center, New York, New York, 10065-9321, United States
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Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
Preparation and biological evaluation of synthetic and polymer-encapsulated
congeners of the antitumor agent pactamycin: insight into functional group effects and
biological activity
Robert J. Sharpe,a Justin T. Malinowski,a Federico Sorana,a J. Christopher Luft, a,b Charles J. Bowerman,a
Joseph M. DeSimone,a-h and Jeffrey S. Johnsona,*
aDepartment of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290, United States
bDepartment of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290,
United States
cLineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290, United States
d Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695-8613, United States
e Carolina Center of Cancer Nanotechnology Excellence, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290, United States
f Institute for Nanomedicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290, United States
g Institute for Advanced Materials, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290, United States
h Sloan-Kettering Institute for Cancer Research, Memorial Sloan-Kettering Cancer Center, New York, New York, 10065-9321, United States
ARTICLE INFO
ABSTRACT
Article history:
Received
The synthesis and biological analysis of a number of novel congeners of the aminocyclopentitol
pactamycin is described. Specific attention was paid to the preparation of derivatives at crucial
synthetic branch points of the parent structure, and biological assays revealed a number of
insights into the source of pactamycin’s biological activity. Additionally, the encapsulation of
pactamycin and select derivatives into the PRINT© nanoparticle technology was investigated as
a proof-of-concept, and evidence of bioactivity modulation through nanoparticle delivery is
demonstrated. This work has provided heretofore unrealized access to a large number of novel
compounds for further evaluation.
Received in revised form
Accepted
Available online
Keywords:
Pactamycin
Nanoparticles
2009 Elsevier Ltd. All rights reserved
.
Structure Activity Relationships
Structural Derivatization
———
Corresponding author. Tel.: +9198434936
*
Email address: jsj@unc.edu

scientists at the former Upjohn Chemical Co.,3 pactamycin
represents the most complex aminocyclitol antibiotic ever
discovered. Researchers at Upjohn showed it to be active against
Gram-positive and Gram-negative bacteria as well as against a
number of cancer cell lines in-vitro.4 More recent biological
studies have demonstrated pactamycin to have potent antiviral
(complete inhibition of polio-infected HeLa cells at 10-7 M) and
antiprotozoal qualities (P.f. K1: IC50 = 14.2 nM). 5 Unfortunately,
this promising biological profile is hindered by pactamycin’s
high cytotoxicity against human eukaryotic cell lines (MRC-5:
IC50 = 95 nM).5 X-ray crystallographic studies have shown that
the source of this activity stems from pactamycin’s ability to bind
to the 30S ribosomal subunit acting as an RNA dincucleotide
mimic.6 A complex array of H-bonding interactions within the
30S site enables pactamycin to act as a universal inhibitor of
translocation. Its impressive biology has attracted the attention of
a multidisciplinary field in hopes of transforming pactamycin
into a suitable therapeutic (Figure 1).
1. Introduction
Pharmaceutical development through organic synthesis
remains a critical feature of the drug discovery process.1 Upon
identification of an initial hit via high-throughput screening, a
significant amount of structural modification is often required
before a lead candidate can be advanced to clinical trials. Natural
molecules are often identified as initial hits in these screenings;
however, later modification of their complex structures toward
the preparation of useful drug molecules can be hindered by the
deficiency of a practical and flexible chemical synthesis.2 As a
result, the continued advancement of synthetic organic
methodology is critical for facile and flexible drug discovery and
development.
Pactamycin (1, Figure 1) is an example of a valuable natural
target that has yet to reach its full medicinal potential, at least in
part due to its structural complexity. Isolated in 1961 from a
fermentation broth of Streptomyces pactum var. pactum by
Figure 1. Structures of pactamycin (1) and natural, synthetic, and biosynthetic congeners.
synthetic studies have been reported by Isobe, Knapp, Looper,
Nishikawa, and our group in the past decade.17 In 2011,
Hanessian and coworkers described the first total synthesis of
In addition to 1, a number of naturally-occurring structural
congeners have been isolated from related Streptomyces bacteria,
displaying varied bioactivities. 7-Deoxypactamycin (2) and
jogyamycin (3) have shown increased antiprotozoal activity
relative to 1.7 A third natural analog, pactamycate (4), has also
pactamycin in 32 steps from L-threonine, enabling previously
unrealized access to synthetic congeners.
18
Since this initial
publication, Hanessian has demonstrated the efficacy of his route
to deliver pactamycin derivatives at the C1-dimethylurea and the
C3 aniline positions such as compounds 7 and 8.19
8
been reported. Alternatively, biosynthetic engineering studies
pioneered by Mahmud and coworkers have provided researchers
with the first series of unnatural structural analogs such as TM-
025F (5) and TM-026F (6), which display comparable activities
to pactamycin against plasmodium falciparum. 9 These data have
renewed promise for pactamycin analogs in drug development.
Moreover, encapsulation of natural cytotoxic agents into
nanoparticles (NPs) has also shown improved clinical benefits,
the most germane of these being reduction of undesired toxic side
effects and increased therapeutic delivery to the target of interest.
This approach has been successfully implemented in the case of
doxorubicin (Doxil©),10 paclitaxel (Abraxane©)11 and others.12
More recently, Bind Therapeutics13 and Cerulean14 have ongoing
clinical trials in NP formulations of cancer therapeutics
Our group began work on the total synthesis of 1 in 2009, and
this work culminated in 2013 with a 15-step, asymmetric
synthesis from commercially available 2,4-pentanedione (Figure
2). 20 Critical to our approach was to assemble the molecule in a
fashion such that key functional groups were installed both in
their native form and in a late-stage fashion; we surmised that
this approach would provide the greatest possible flexibility,
facilitating investigations of structure-activity relationships at all
critical branch points. To this end, we envisaged a synthon such
as 9 in which exploitation of appropriate functional handles at the
correct stage would install the requisite functionalities.
(docetaxel, irinotecan, and camptothecin).
DeSimone and
coworkers have demonstrated the use of the Particle Replication
in Non-Wetting Templates (PRINT®) technology to modulate
the activity of cytotoxic agents such as docetaxel, reducing
unwanted side-effects and increasing therapeutic activity in
vivo.15 To the best of our knowledge, however, the incorporation
of pactamycin or its congeners into NPs of any type with the goal
of bioactivity attenuation has not yet been explored.
While an efficient chemical synthesis of 1 might provide the
most flexibility in structural derivatization, the inherent
complexity of the molecule has rendered this a difficult
undertaking.
The heavily-compacted and heteroatom-rich
functionality in pactamycin presents a number of challenges
toward selective structural modification. Additionally, while the
unique functional groups present in the molecule (salicylate,
dimethylurea, aniline) offer novel branch points for structural
diversification, methods with which to install these moieties are
underexplored in the literature.16 To these aims, a number of

entry
1
H-Nu
product
% yielda
83
14a
2
3
14b
43
14c
14d
95
71
Figure 2. Synthon analysis of 1 showing key branch points for structural
derivatization.
NH2
4
5
86
59
14e
14f
2-fluorenyl aniline
6
7
14g
87
8
47
--b
-- c
-- d
14h
--
9
10
11
--
BnNH2
NaN3
--
a
Isolated yield;b TBS deprotection was observed as the sole product;c No
reaction was observed;d Conditions: NaN3 (1.1 equiv), Oxone (0.5 equiv),
CH3CN:H2O (9:1), rt; only TBS deprotection was observed in this reaction.
We first pursued the preparation of pactamycin congeners at the
C3-aniline position, inspired by a related epoxide-opening
strategy by Hanessian and co-workers (Table 1).18,19 We were
encouraged by the aniline flexibility demonstrated by Hanessian
in their earlier report and hoped that epoxide 11 would participate
in related transformations with functionally and electronically
diverse anilines.
Figure 3. Pactamycin: endgame strategy and synthesis completion
A summary of our disclosed synthesis endgame is described in
Figure 3, wherein ketone intermediate 10 (synthesized in ten
steps from 2,4-pentanedione in gram quantities) would serve as
our first point of derivatization.20 Nucleophilic methylation of 10
proceeded in good yield to provide carbinol 11 in 75% yield of a
single diastereomer at C5. Sc(OTf)3-promoted addition of m-
acetylaniline installed the substituted C3-aniline necessary for
elaboration to 1, upon which silyl deprotection afforded tetraol
12. Introduction of the remaining salicylate moiety to the C6-
hydroxymethylene of 12 was accomplished via reaction with the
previously reported acyl electrophile 13, which upon
hydrogenative removal of the Cbz protecting group delivered
pactamycin in 15 steps and 1.9% overall yield. With this strategy
established, we shifted our focus to examining the route’s
flexibility toward analog preparation.
Indeed, we were pleased to find that epoxide 11 reacted readily
in the presence of a number of substituted anilines in moderate to
excellent yields, providing anilines 14a-h. Notably, while the
installation of m-acetylaniline to 11 requisite for elaboration to
pactamycin necessitated superstoichiometric amounts of
Sc(OTf)3, all subsequent anilines examined in the reaction
proceeded to completion using 50 mol % Sc(OTf)3. We suspect
that this is an outgrowth of both low nucleophilicity and poor
solubility of the parent m-acetylaniline under the reaction
conditions. With the aniline tolerance established, we turned our
attention to alternative nitrogen nucleophiles. Unfortunately, all
non-aromatic nitrogen sourcfes (RNH2, R2NH, (–)N3) failed to
react with 11, giving either no reaction or starting material
decomposition. Addition products 14a-h were carried through the
endgame sequence described previously, delivering derivatives
15a-h (Figure 4). In the case of addition product 14g, the aryl
bromide was reduced during hydrogenolysis, delivering α-
naphthyl anilide 15g.
Herein, we delineate our efforts in the synthesis and biological
evaluation of novel pactamycin congeners. Additionally, we
report the first studies incorporating pactamycin and select
derivatives into polymeric NPs, fabricated using the PRINT®
technology, with the goal of enhancing activity and selectivity
while mitigating unwanted toxicities.
2. Results and discussion
2.1. C3-aniline
Table 1. Addition of substituted anilines and other nitrogen nucleophiles to
epoxide 11

synthesis of 1 utilized an early-stage N–H insertion reaction to
install the urea.20 Synthetic diversification from this early
intermediate would be a significant challenge. Consequently, we
envisaged a similar isocyanate formation/trapping strategy from
carbinol intermediate 11 via the acid-catalyzed elimination of
dimethylamine (Figure 6).
After some experimentation, treatment of 11 with NH4Cl in
H2O and MeOH resulted in complete elimination of
dimethylamine and convergence to a single product. However,
1H NMR analysis revealed that the intermediate isocyanate had
a Hydrogenation of the of bromide functionality was observed.
Figure 4. Synthesis of C3 aniline pactamycin derivatives
2.2. C1-dimethylurea
Hanessian’s approach toward preparation of pactamycin analogs
at the C1 dimethylurea position substituent relied on the trapping
of an in-situ generated isocyanate electrophile late in the
synthesis.18 This tactic proved effective in the preparation of a
series of functionalized ureas in good yields.19 By contrast, our
aConditions: (a) Oxone, CH3CN:H2O (9:1), rt; (b) NH4Cl, MeOH:H2O (8:1), 85 °C; (c) TBAF, THF, 0°C; (d) H2 (1 atm), Pd(OH)2/C (0.5 mass equiv.), MeOH,
rt; (e) Aniline (10 equiv.), Sc(OTf)3 (50 mol %), PhMe, 50 °C; (f) K2CO3, 13, DMA, rt.
Figure 5. Preparation of de-6-MSA pactamycate and pactamycate analogsa
this route established, we resolved to prepare a subset of varied
C3 pactamycate structures. Beginning with anilides 14b, 14c,
and 14e already in hand from the C3 derivitization studies, TBS
deprotection followed by oxazolidinone formation gave the
corresponding diols, which upon desilylation, acylation and
hydrogenolysis, provided pactamycate derivatives 22a-c.
2.3. C5-tertiary alcohol
We next began examining reactivity of the ketone in 10 with the
goal of diversification at C5 (Table 2). To this end, we were
n
pleased to find this ketone reacted readily with ethyl, hexyl, and
vinyl magnesium bromide to give the corresponding carbinols in
good yields. Unfortunately, when larger alkyl (entries 4-5, 8)
Figure 6. Failed isocyanate formation/trapping strategy from 11.
and aryl nucleophiles (entries 6-7) were examined, we observed
only un-productive side reactions21 or complete starting material
recovery. With this limitation established, we speculated that
hydride might also be a suitable nucleophile. In the event,
undergone intramolecular ring closure with the C2-carbamate to
furnish imidazolidinone 17 in 73% yield. Although this result
rendered our intermolecular isocyanate trapping strategy
unfeasible, we postulated that this reactivity might be exploited
toward analog preparation of the naturally-occurring congener
pactamycate 4 (Figure 5).
Table 2. Addition of nucleophiles to ketone 10a
Selective deprotection of the C7-TBS ether in 16 with oxone
furnished the corresponding secondary alcohol 18 in 84% yield,
which we postulated would serve as a more nucleophilic trapping
agent for the intermediate isocyanate. Indeed, upon treatment of
18 with NH4Cl in H2O:MeOH, the in-situ generated isocyanate
underwent trapping by the unprotected C7 hydroxyl to afford
oxazolidinone 19 in 73% yield. Removal of the TBDPS
protecting group with TBAF provided triol 20, which upon Cbz
deprotection, afforded De-6-MSA pactamycate 21, which has
been previously characterized.18b This result confirmed that the
intermediate isocyanate generated from 18 had indeed been
engaged by the C7 hydroxyl (and not the C2 carbamate). With
entry
1
R
product
% yieldb
75
Et
23a

2
3
nHexyl
73
43b
23b
treatment of 10 with NaBH4 in MeOH at -45 °C provided alcohol
23c in good yield with analogous stereofidelity to that observed
in the addition of carbon nucleophiles.
--
4
5
--
--
--c
--c
With addition products 23a-c in hand, we proceeded in the
synthesis to complete C5 analog preparation (Figure 7). We
were surprised to find, however, that subjection of these
intermediates to the optimized conditions for C3 m-acetylaniline
installation gave only significant amounts of recovered starting
material. Increasing the loading of Sc(OTf)3 or the reaction
time/temperature had seemingly no effect. It seems reasonable
that this addition is sluggish either due to poor coordination of
the Lewis acid or by an unfavorable substrate conformation for
addition relative to the parent C5-methyl compound (11). In
order to circumvent this issue, we turned to the strategy of
Hannesian and coworkers wherein the required C3 m-
acetylaniline was incorporated via an 2-(prop-1-en-2-yl)aniline
6
7
8
9
--
--
--d
--d
--e
88f
PhCH2
H
--
23c
a Isolated yields; bProduct could not be elaborated further; c No reaction was
f
observed;
Conditions: NaBH4 , MeOH, -45 °C.
d
e
Undesired side products isolated;
complex mixture;
aConditions: (a) m-propenylaniline (10 equiv), Sc(OTf)3 (50 mol %), C7H8, 50 °C ; (b) OsO4 (10 mol %), NMO (5 equiv), THF:Acetone:H2O (5:5:1), 0 °C to rt;
(c) NaIO4 (3.5 equiv), THF:H2O (1:1), rt; (d) TBAF, THF, 0 °C; (e) K2CO3, 13, DMA, rt; (f) H2 (1 atm), Pd(OH)2/C (0.5 mass equiv.), MeOH, rt.
Figure 7. Preparation of C5 pactamycin analogsa
surrogate.18 The acetophenone was later revealed via oxidation.
3. Results and Discussion
In our system, this strategy also proved effective, providing 2-
3.1. Biological Evaluation.
(prop-1-en-2-yl)aniline anilines 24a-c in good yields. Johnson-
Lemieux oxidation of the resulting alkenes provided the desired
m-acetylanilines 25a-c in good yields over the three-step
sequence. Completion of the remaining synthetic sequence
provided C5 pactamycin derivatives 26a-c.
Having prepared a library of novel compounds, we set out to
examine their varied biological profiles. Specifically,
compounds were tested against breast, ovarian, and lung
carcinoma cell lines. Additionally, the human embryonic cell
line for which pactamycin’s toxicity has been established (MRC-
5) was assayed for comparison.5 The results for all derivatives are
summarized in Table 4. As anticipated, pactamycin (entry 1)
displayed exceptional potency, showing nanomolar inhibition
against all three carcinoma cell lines. For comparison, the
penultimate intermediate in our synthesis of pactamycin (28)
bearing Cbz protection at the C2-aminomethine (entry 2) showed
a dramatic decrease in activity relative to 1. In order to better
understand the effect of chirality on the parent pactamycin
structure, ent-pactamycin (ent-(1)) (entry 3) was synthesized in
high enantiomeric purity via a slight modification of the
previously published route (see supporting information) and
2.4. C6,C7-hydroxyl
The final point of diversification centered on manipulation of
the salicylate-bearing C6 ester in 1 (Table 3). Gratifyingly,
efficient monoacylation of tetraol 12 was accomplished with a
variety of aliphatic and aromatic acyl electrophiles in good yields
(entries 1-5). The resulting monoesters were subjected to the
previously employed conditions for Cbz deprotection, providing
derivatives 27b-e. However, in the case of the differentiated
methoxyphenol 27a (entry 1), only decomposition was observed
upon hydrogenolysis.
Cognizant of the documented bioactivity difference across
multiple cell lines observed between 1 and its 7-deoxy congener
(2),7 reduction of the C7-hydroxyl to its corresponding methylene
was also probed. Unfortunately, all conditions explored (from a
number of different intermediates in our route) failed to deliver
the desired C7-methylene. As an alternative strategy, we
envisaged masking of the C7 hydroxyl via its ester might serve
the same purpose (i.e. removal of the H-bonding interaction at
C7).6 To this end, C6-C7 bis-acylated derivatives (entries 6-8)
were synthesized with varying degrees of steric encumbrance.
Cbz hydrogenolysis provided the diesters 27f-h.
Table 3. Functionalization of C6 (C7) alcohols.
entry
R2
product
yield 1a
yield 2a
R1

27ab
27bb
27cd
27dd
27ed
27fe
57
43
60
83
87
86
92
--c
62
73
76
61
38
53
8
27hf
76
72
1
2
3
4
5
6
7
H
H
Isolated yields; bX=OCH2CN; Conditions: K2CO3, DMA, rt;c Only
a
decomposition was observed; dX=Cl; Conditions: 2,4,6-collidine, CH2Cl2, -78
°C to rt; e X=OAc; Conditions: NEt3, DMAP (10 mol %), CH2Cl2, 0 °C to rt;
f
X=Cl; Conditions: NEt3, DMAP (10 mol %), CH2Cl2, 0 °C to rt. See
supporting information for details on electrophile preparation.
H
assayed in our study. As illustrated in Table 4, ent-(1) showed a
threefold order of magnitude decrease in bioactivity, illustrating
the impact of the natural enantiomer of 1 to effective cell-growth
inhibition.
Generally, all C3-aniline derivatives (entries 4-11) showed a
marginal to significant decrease in activity relative to 1 across all
cell lines, although 15b (entry 5) showed comparable activity
against A549 (EC50 = 141 nM) with a marginal decrease in
MRC5 activity. With regard to the pactamycate series of
analogs, De-6-MSA pactamycate 21 (entry 12) showed only
minor cell-growth inhibition. This was not an altogether
H
H
Ac
Piv
27gf
Table 4: Biological examination of pactamycin and synthetic analogsa
Entry
Structure
Code Number
A549
EC50
(lung
MDA-MB-231
EC50
(breast cancer) (ovarian cancer) (human lung
fibroblast)
SK-OV-3
EC50
MRC-5
EC50
cancer)
1
2
3
160 nM
11.8 µM
2.1 µM
124 nM
10.4 µM
1.2 µM
129 nM
12 µM
1.6 µM
53 nM
1
n.t.b
28
933 nM
ent-(1)
4
Ar =
15a
800 nM
659 nM
1.4 µM
380 nM
5
6
7
8
141 nM
1 µM
556 nM
n.t. b
434 nM
600 nM
4 µM
314nM
582 nM
682 nM
2326 nM
15b
15c
15d
15e
777 nM
884 nM
4 µM
3.3 µM
1.6 µM
9
324 nM
376 nM
145 nM
431 nM
860 nM
15f
10
2.21 µM
1.84 µM
2.44 µM
15g

11
12
760 nM
800 nM
n.t.b
436 nM
366 nM
15h
21
6 µM
3.8 µM
2933 nM
Entry
Structure
Code Number
A549
EC50
(lung
MDA-MB-231
EC50
(breast cancer) (ovarian cancer) (human lung
fibroblast)
SK-OV-3
EC50
MRC-5
EC50
cancer)
13
14
15
Ar =
n.t.b
n.t.b
n.t.b
n.t.b
n.t.b
n.t.b
n.t.b
n.t.b
n.t.b
n.t.b
n.t.b
n.t.b
22a
22b
22c
R = C2H5
Ar =
16
17
18
19
n.t.b
n.t.b
n.t.b
n.t.b
n.t.b
n.t. b
2063 nM
10.9 µM
6.5 nM
49 nM
26a
26b
26c
29
R = C6H14
Ar =
R = H
Ar =
32 nM
83 nM
50 nM
356 nM
7 nM
91 nM
R = H
Ar =
20
88 nM
203 nM
103 nM
129 nM
27b
R =

21
22
23
114 nM
118 nM
194 nM
79 nM
300 nM
352 nM
80 nM
75 nM
436 nM
105 nM
100 nM
366 nM
27c
27d
27e
Entry
Structure
Code Number
A549
EC50
(lung
MDA-MB-231
EC50
(breast cancer) (ovarian cancer) (human lung
fibroblast)
SK-OV-3
EC50
MRC-5
EC50
cancer)
24
25
26
R = Ac
137 nM
175 nM
588 nM
458 nM
1.93 µM
2.44 µM
123 nM
86 nM
132 nM
396 nM
778 nM
27f
27h
27g
593 nM
a Assays were carried out as triplicates; b Not toxic.
findings.19 Namely, no significant gain (or loss) of biological
activity was observed when the salicylate ester was altered
relative to the parent pactamycin structure. These results further
support the hypothesis that the C6 ester side chain has a limited
role in the key binding event of 1 in the 30S ribosome.19 The
three prepared C6,C7 bis-acylated derivatives (entries 24-26)
showed a linear decrease in activity with steric encumbrance of
the ester group. These results suggest that the C7 hydroxyl in 1
plays a larger role in the bioactivity of the structure than the C6
hydroxymethylene.
Upon collection of these initial data, derivatives 15f, 26c, 27f,
27c, and ent-(1) were identified as the most promising
compounds and were assayed via the NCI 60 human tumor cell
line screen. Upon initial one-dose screening, all five compounds
were found to have sufficient activity to merit the subsequent
five-dose assay. These derivatives were evaluated to determine
GI50 (50% growth inhibition) values. The results of these assays
are summarized in Table 5. Additionally, the previously
documented cell data for 1 is shown for comparison.
unexpected result, however, as biological assays of 21 conducted
by Hanessian and coworkers also showed little promising
activity.19Altering the C3 aniline position of the pactamycate
parent structure (entries 13-15) resulted in complete loss of
biological activity. These results, in combination with those of
the pactamycin C3 analogs, speak to the importance of the m-
acetyl functionality in 1 to its bioactivity.22
The results of compounds bearing diversity at C5 are shown in
entries 16-19. In combination with the C5 derivatives previously
described (vide supra), an additional derivative 29 (entry 19) was
prepared bearing alternate functionality at the C3 aniline for
comparison (see supporting information). Extending the length
of the carbon chain at C5 (entries 16-17) had significantly
deleterious effects to bioactivity as a complete loss of carcinoma
activity was observed, leaving only low inhibition of MRC-5.
However, removing alkyl functionality altogether at C5 (entries
18-19) had the opposite effect, as these C5 protio analogs
displayed the greatest activity across all cell lines of any
compound tested in our study (including pactamycin). We
speculate that these results are primarily a function of adjusting
the lipophilicity of the structure relative to 1.23
The results of our diversification of the C6 hydroxymethylene
(entries 22-25) are in agreement with Hanessian’s earlier
As expected based on our initial screen, ent-(1) showed multiple
orders of magnitude loss in activity across the entire assay. By

contrast, compound 26c bearing a secondary hydroxyl at C5
demonstrated exceptional activity, showing nM inhibition
throughout the screen and outperforming pactamycin in multiple
cell lines. Derivatives 27c (modified salicylate ester) and 27f
(C6, C7 diacetoxypactamycin) also demonstrated general nM
activity in the assay. The final derivative 15f bearing a fluorenyl
aniline at C3 showed a general decrease in biological activity
relative to 1 by factors of 10-100.
Scanning electron microscopy (SEM) analysis confirmed
uniform particle size and shape regardless of compound identity,
and drug loading of each sample was found to be ~10% as
determined by HPLC.26 NP-encapsulated compounds NP-1, NP-
15e, and NP-26c, were then examined in our assay, and the
results are given in Table 6 where the baseline toxicity values for
each compound are restated for comparison.
In vitro cytotoxicity analysis of the derivative NP
formulations showed bimodal effects on therapeutic activity. In
the A549 assay, nanoparticle delivery increased the cytotoxicity
of the therapeutic cargo. NP-1 demonstrated an EC50 threefold
more potent than pactamycin itself (52 nm to 160 nm,
respectively). NP-26c showed a near fivefold increase in potency
when compared to the unadultered small molecule (6.5 nM to 32
nM, respectively). Even compounds 15e, a less active drug in
comparison to 1, showing a nominal reduction in EC50 value for
the A549 cell line. Of significant interest was the increase in
selectivity observed for 26c, wherein the EC50 for A549
decreased while the EC50 for MDA-MB-231 and MRC5
increased.
3.2. Nanoparticle Fabrication-Biological Evaluation.
With these studies completed, we set out to examine the
efficacy of pactamycin and select analogs to activity modulation
via nanoparticle encapusulation.
Polymeric PRINT®
nanoparticles were fabricated by encapsulating compounds 1,
15e, and 26c, in poly(d,l-lactide) using previously described
methods.15b, 24 Compounds 15e and 26c were selected on the basis
of observing the effect of nanoformulation on derivatives both
more and less bioactive than 1. PRINT NPs containing 1 and
derivatives 15e and 26c all showed similar hydrodynamic radii
and PDI as determined by dynamic light scattering (DLS).25
Table 5. Summary GI50 values from NCI-60 cell line screeninga
GI50 (µM)
MOLT-4
NCI-H322M
HCT-15
SNB-19
M14
1b
(ent)-1
1.19
3.72
20.0
3.07
3.01
2.50
1.50
7.26
2.04
26c
0.046
0.016
0.16
27c
0.12
0.33
0.65
0.19
0.19
0.20
0.12
0.26
0.17
15f
27f
<0.10
0.12
0.78
1.07
1.46
1.40
0.88
0.73
0.61
1.37
0.73
0.33
0.48
10.2
0.57
0.68
0.53
0.61
0.34
7.31
0.03
<0.10
0.12
0.52
0.10
OVCAR-3
RXF 393
DU-145
MCF7
<0.10
<0.10
<0.01
<0.01
0.041
0.064
0.15
0.051
aData obtained from NCI-60 screening. See supporting information for comprehensive results. MOLT-4, leukemia cell line; NCI-H322M, nonsmall-cell lung
cancer cell line; HCT-15, colon cancer cell line; SNB-19, CNS tumor cell lines; M14, melanoma; OVCAR-3, ovarian cancer cell line; RXF 393, renal cancer cell
line; DU-145, prostate cancer cell line; MCF7, breast cancer cell line. bData can be accessed from the CAS: 23668-11-3 at the following website:
http://dtp.cancer.gov/dtpstandard/dwindex/index.jsp.
established a heretofore undocumented proof-of-concept for the
modulation of the pactamycin structure via the use of the
PRINT® nanoparticle delivery vehicle. A wider range of cell-
based assays and the further examination of pactamycin
Table 6. Cell-based assay comparison for compounds 1, 15e, 26c and NP
counterparts.a
A549
EC50
160 nM
MDA-MB-231
EC50
124 nM
MRC-5
EC50
53 nM
derivatives using the PRINT technology is planned and will be
reported in due course.
Compound
1
Acknowledgements
NP-1
52 nM
884 nM
693 nM
32 nM
117 nM
3.3 µM
5.5 µM
50 nM
52 nM
2.3 µM
1.8 µM
6.5 nM
18 nM
15e
NP-15e
26c
The project described was supported by Award No. R01
GM084927 from the National Institute of General Medical
Sciences and by the University Cancer Research Fund. R.J.S
acknowledges an NSF Graduate Research Fellowship. R.J.S. and
J.T.M. acknowledge an ACS Division of Organic Chemistry
graduate fellowship. F.S. acknowledges BI Research Italia S.a.s.
di BI IT S.r.l. and University of Camerino (Italy) for a doctoral
fellowship. Maribel Portillo is acknowledged for experimental
assistance and preparation of select derivatives.
NP-26c
6.5 nM
724 nM
a Assays were carried out as triplicates
4. Conclusions
Supplementary Data
In summary, we have demonstrated the efficacy of our
synthetic approach to efficient and modular preparation of a
number of varied analogs of the complex aminocyclitol
pactamycin. These results have provided additional insight into
the roles that each functional group plays in providing the
observed activity of the parent structure. Additionally, we have
Supplementary data associated with this article can be found in
the online version at URL

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