Synthesis, hydrolyses and dermal delivery of N-alkyl-N-alkyloxycarbonylaminomethyl (NANAOCAM) derivatives of phenol, imide and thiol containing drugs

Article abstract of DOI:10.1016/j.bmcl.2006.03.061

Synthesis, characterization and hydrolysis in aqueous buffers of novel N-alkyl-N-alkyloxycarbonylaminomethyl (NANAOCAM) derivatives of substituted phenols, theophylline (Th) and 6-mercaptopurine (6MP) were carried out. The mechanism of hydrolysis was further investigated by synthesis, characterization and hydrolysis of N-aryl-N-alkyloxycarbonylaminomethyl (NArNAOCAM) derivatives of phenols. The hydrolysis follows pseudounimolecular first order kinetics and operates by way of an S_N1-type mechanism. Topical delivery of selected derivatives of acetaminophen (APAP), Th and 6MP was examined in in vitro diffusion cell experiments from IPM across hairless mice skins. The prodrug of APAP and 6MP increased permeation across the skin by about 2- and 4-fold, respectively, compared to the parent drug. NANAOCAM promoieties can act as novel prodrug derivatives of phenol, imide and thiol containing drugs for enhancing topical absorption.

Full text of DOI:10.1016/j.bmcl.2006.03.061

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3590–3594
Synthesis, hydrolyses and dermal delivery of N-alkyl-N-
alkyloxycarbonylaminomethyl (NANAOCAM) derivatives
of phenol, imide and thiol containing drugs
Susruta Majumdar and Kenneth B. Sloan*
Department of Medicinal Chemistry, University of Florida, Gainesville, FL 32610, USA
Received 22 February 2006; revised 21 March 2006; accepted 21 March 2006
Available online 17 April 2006
Abstract—Synthesis, characterization and hydrolysis in aqueous buffers of novel N-alkyl-N-alkyloxycarbonylaminomethyl
(NANAOCAM) derivatives of substituted phenols, theophylline (Th) and 6-mercaptopurine (6MP) were carried out. The mecha-
nism of hydrolysis was further investigated by synthesis, characterization and hydrolysis of N-aryl-N-alkyloxycarbonylaminomethyl
(NArNAOCAM) derivatives of phenols. The hydrolysis follows pseudounimolecular first order kinetics and operates by way of an
SN1-type mechanism. Topical delivery of selected derivatives of acetaminophen (APAP), Th and 6MP was examined in in vitro dif-
fusion cell experiments from IPM across hairless mice skins. The prodrug of APAP and 6MP increased permeation across the skin
by about 2- and 4-fold, respectively, compared to the parent drug. NANAOCAM promoieties can act as novel prodrug derivatives
of phenol, imide and thiol containing drugs for enhancing topical absorption.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Drugs containing polar functional groups like phenols,
thiols or imides pose problems of membrane perme-
ability and biphasic solubility which limit their dermal
delivery. The prodrug approach, which involves mask-
ing these polar functional groups as simple alkyl esters
or acyloxymethyl (ACOM, R0COOCH2–) and alkyl-
oxycarbonyloxymethyl (AOCOM, R0OCOOCH2–) es-
ters which then hydrolyse to the native drug either
enzymatically or chemically, has proven useful in
numerous cases.1 However, these derivatives may prove
to be too unstable or may not have the desired biphasic
properties to permeate the skin effectively. Replacing
the oxygen atom in OCH2 with nitrogen (N–R, R =
alkyl) in R0OCOOCH2– to give a NANAOCAM
(R0OCONRCH2–, R = alkyl) promoiety could give
medicinal chemists an additional handle and flexibility
to improve solubility (better balance between solubili-
ties in lipid and water) and stability (enzymatic vs
chemical) of prodrugs of polar molecules. Recent stud-
ies have shown that a drug or a prodrug needs to have
adequate lipid as well as water solubility to permeate
the skin effectively.1b,c Introduction of heteroatoms like
N into a promoiety has been shown to reduce crystal
lattice energy and increase biphasic solubility of drug
molecules. Only one example of the use of a NANAO-
CAM promoiety has been reported: 6MP.2 The use of
a close analogue of NANAOCAM, R0CONRCH2–,
has been reported for carboxylic acids.3 Here we ex-
tend the use of the NANAOCAM promoiety to phe-
nols and imide containing compounds like Th, and
investigate the mechanism of chemical hydrolysis. In
addition, we will present data from dermal penetration
studies of some NANAOCAM prodrugs of APAP
(phenol containing drug), Th (imide containing drug)
and 6MP (thiol containing drug).
The NANAOCAM derivatives of phenols, Th, 6MP
and dimethylaminobenzoic acid were synthesized by
alkylating the parent compound with NANAOCAM
4
chloride in the presence of triethylamine and CH2Cl2
(Table 1, compounds 1–10). The corresponding alkylat-
ing agent was synthesized as reported by Siver and
Sloan2 by reacting a molar equivalent of formaldehyde
with methyl amine to generate 1,3,5-trimethylhexahy-
drotriazene. Reaction of the 1,3,5-trimethylhexahydro-
triazene with the appropriate chloroformate gave the
corresponding NANAOCAM chlorides (Scheme 1).
Keywords: NANAOCAM; NArNAOCAM; APAP; Th; 6MP;
Prodrugs; Dermal delivery.
For the synthesis of NArNAOCAM derivatives of phe-
nols,4 we used a protocol developed by Moreira et al.3c
for the synthesis of alkylcarbonylaminomethyl chloride.
*
Corresponding author. Tel.: +1 352 846 1957; fax: +1 352 392
9455; e-mail: sloan@cop.ufl.edu
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.03.061

S. Majumdar, K. B. Sloan / Bioorg. Med. Chem. Lett. 16 (2006) 3590–3594
3591
Table 1. Hydrolysis of NANAOCAM derivatives of substituted phenols, Th, 6MP and dimethylaminobenzoic acid, and NArNAOCAM derivatives
of p-nitrophenol at pH 8.8 and 46 ꢁC, and the influence of pKa of leaving groups and substituents on rates of hydrolysis
O
R'
O
N
R
Y
Y = Phenol, Th, 6MP, dimethylaminobenzoic acid
R’= CH3; R = Alkyl or Aryl.
8
Compound
R
Y
logk (sꢀ1
)
t1/2 (min)
pKa
rꢀ
1
2
CH3
CH3
p-MeOCHN–C6H4O–
p-NC–C6H4O–
p-OHC–C6H4O–
o-OHC–C6H4O–
p-MeOC–C6H4O–
p-MeOOC–C6H4O–
p-O2N–C6H4O–
6-MP
ꢀ5.4937
ꢀ4.11
ꢀ3.81
ꢀ3.026
ꢀ4.21
ꢀ4.39
ꢀ3.28
ꢀ3.22
ꢀ4.56
ꢀ1.2953
ꢀ4.54
—
3600
149
75
9.5
0.19
0.99
0.94
7.95
7.66
6.79
8.05
8.47
7.14
7.5
3
CH3
CH3
4
12
5
CH3
CH3
189
290
22
0.82
0.74
1.25
6
7
CH3
CH3
8
19
9
CH3
CH3
Th
420
0.22
400
>24 h
519
8.6
5.03
10
11
12
13
p-Me2N–C6H4COO–
p-O2N–C6H4O–
p-O2N–C6H4O–
p-O2N–C6H4O–
p-MeO–C6H4–
p-EtOOC–C6H4–
C6H5
ꢀ4.65
Representative prodrug structures:
O
O
R'
O
N
N
S
R
N
R'
O
N
R
O
H
N
N
N
N
N
O
N
6MP
Th
R
N
O
O
NaOH
CH3
NH2
CH3
Cl
HN
O
N
X
O
RNH2
CH2=O
+
Py
N
N
TM SCl
R
R
R
N
C H3 O CO Cl
(CH2 O)n
O
O
X
X
R'
+
R'
O
N
R
Cl
O
O
Cl
O
N
N
CH2Cl2
R
R
CH3
CH3
Y
N
Cl
O
N
X
O
O
p-nitrophenol
T EA
CH2Cl2
TEA
O
R'
R'
Y-H
+
O
N
Y
X
O
N
R
Cl
R
Y = Phenol, Th, 6MP or
dimethylaminobenzoic acid
X = O CH3, CO O C2 H5, H.
Y = p-nitrophenol.
CH3
R, R' =
Com pounds11-13.
Compounds 1-10
Scheme 2.
Scheme 1.
fully characterized by UV,6 NMR6 and elemental
analysis.
We have expanded the synthesis to NANAOCAM chlo-
ride and NArNAOCAM chloride5 in this case. A substi-
tuted aryl amine was reacted with pyridine and
methylchloroformate to generate a N-arylcarbamic acid
methyl ester. The N-arylcarbamic acid methyl ester was
then reacted with excess trimethylsilylchloride and para-
formaldehyde to generate the desired alkylating agent,
NArNAOCAM chloride (Scheme 2). NArNAOCAM
chloride was then used to alkylate a phenol (Table 1,
compounds 11–13). All compounds synthesized were
To be therapeutically useful, a prodrug must hydrolyse
to the active drug molecule at an appropriate rate at
the target site. Kinetic hydrolysis studies on NANAO-
CAM prodrugs were thus carried out. Compound 8
reverted to 6MP with a half-life of 91 min in pH 7.1 buff-
er at 32 ꢁC and an SN1 mechanism of hydrolysis was
proposed initially by Siver and Sloan2 but not firmly

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S. Majumdar, K. B. Sloan / Bioorg. Med. Chem. Lett. 16 (2006) 3590–3594
-3
-3.5
-4
carbon and lead to the expulsion of the N-alkylcarbamic
acid alkyl ester as in path ‘b.’ This particular mechanism
however can be ruled out because of the lower pKa of
YH (ꢁ5.2–9.5) compared to the N-alkylcarbamic acid
alkyl ester (ꢁ14) making Yꢀ a better leaving group than
the N-alkylcarbamic acid alkyl ester anion.
7
-4.5
-5
3.5
5.5
7.5
9.5
pH
Figure 1. pH rate profile of 7.
A SN1 pathway is another possibility (path ‘c,’ Scheme 3).
The lone pair of electrons on nitrogen can donate its
electrons to stabilize an incipient carbocation with Yꢀ
leaving. This carbocation can subsequently react with
water to give hydroxymethyl-N-alkylcarbamic acid alkyl
ester which then falls apart to give N-alkylcarbamic acid
alkyl ester. However, none of the approaches provides
an insight into the mechanism of hydrolysis since both
SN1 and SN2 mediated hydrolyses are dependent on
the leaving group ability of the ionized parent molecule,
Yꢀ. However, the fact that hydrolyses were pH indepen-
dent strongly favours an SN1 mechanism. To more
clearly define the mechanism of hydrolysis, we next syn-
thesized prodrugs with an aryl group on the nitrogen in-
stead of a simple alkyl group. If the mechanism is SN2,
the rate of hydrolysis should be accelerated by an elec-
tron-withdrawing substituent on the N-aryl group where
a positive charge on CH2 in N–CH2–O is increased mak-
ing the CH2 a better target for nucleophilic attack.
While if the mechanism were SN1, the rate of hydrolysis
should be decelerated by an electron-withdrawing sub-
stituent on the N-aryl group where a positive charge
on CH2 in N–CH2–O is destabilized. Thus, NArNAO-
CAM (R0OCONRCH2–, R = aryl, R0 = CH3) promoi-
eties were used to alkylate p-nitrophenol (Table 1, 11–
13). The half-lives of hydrolyses clearly illustrate that
when an N-alkyl group (Table 1, t1/2 for 7 = 22 min)
was replaced by N-aryl (Table 1, t1/2 for 11 and
13 = 400–520 min, while t1/2 for 12 > 24 h), the rates of
hydrolyses were slower. An electron-donating substitu-
ent on the N-aryl ring like –OCH3 stabilizes the SN1
transition state more than an electron-withdrawing
group like –COOC2H5 or –H and the half-lives are in
the order of 12 > 13 > 11. Thus, hydrolysis of NAr-
NAOCAM derivative of phenols follows an SN1-type
of mechanism and by inference so do NANAOCAM
derivatives of phenols, Th, 6MP and carboxylic acids
like dimethylaminobenzoic acid. Evidence in the litera-
ture supports the conclusion that the mechanism of
hydrolysis is SN1. N-Alkylamidomethyl esters of carbox-
ylic acids also hydrolyse by an SN1 mechanism.3
established. Hydrolysis of NANAOCAM derivatives of
phenols at 32 ꢁC was much slower so rates of hydrolysis
were determined at 46 ꢁC and in pH 8.8 buffer for deriv-
atives 1–10 (Table 1).7 Thus, rates of hydrolysis were
determined where the leaving group Y was phenol, Th,
6MP or dimethylaminobenzoic acid in order to deter-
mine the effect of the pKa of YH on rate.
When logk (rate constant) was plotted against the pKa
of the leaving group (Yꢀ), a negative correlation was
found (slope = ꢀ0.93, r2 = 0.98; plot not shown). Rate
of hydrolysis of NANAOCAM-Y was dependent on
the nucleofugacity of the parent compound. The effect
of electron-withdrawing groups in the para position of
the phenol on rates of hydrolysis was also quantified
using the Hammett plots between rꢀ substituents9 ver-
sus logk. A plot of logk versus sigma (rꢀ) for 1–7 was
linear and indicated that rates of hydrolysis were depen-
dent on the electron-withdrawing effect imparted by the
substituent at the para position (slope = q = 0.96; plot
not shown). Rates of hydrolysis were also independent
of the pH of the buffer. When rates were studied in var-
ious buffers (pH 4.6, 7.1 and 8.8) at 46 ꢁC for 7 and logk
was plotted against buffer pH, a line of zero slope was
obtained (Fig. 1).
Several mechanisms for base catalysed hydrolysis of
NANAOCAM-Y can be proposed.
O
c
c
:
R
O
N
Y
b
R'
a
-
OH
Path ‘a’ shows an SN2-type of pathway where hydroxide
ion acts as a nucleophile and displaces Y to give a
hydroxymethyl-N-alkylcarbamic acid alkyl ester.
Similarly the hydroxide anion can attack the methylene
O H 2
-
+ O H
-
Y
+
Y H
O
O
O
O H 2
+
+
O
N
O
N
R
Y
O
N
O H 2
R
R
- C H 2 = O
O
H
+
O H 2
O
N
R
Scheme 3.

S. Majumdar, K. B. Sloan / Bioorg. Med. Chem. Lett. 16 (2006) 3590–3594
3593
Table 2. Water solubility (SAQ), solubility in IPM (SIPM), log partition coefficients between IPM and pH 4.0 buffer and flux from IPM through
hairless mice skins (JMIPM) of NANAOCAM prodrugs and parent drugs
Compound
SAQ (mM)
SIPM (mM)
logKIPM:4.0
JMIPM (lmole cm2 hꢀ1
)
% of parent drug obtained after diffusion
APAP
1
Th
100
1.91
14.13
0.34
2.69
0.02
1.35
ꢀ1.72
ꢀ0.45
ꢀ0.51
ꢀ0.04
ꢀ1.75
0.03
0.51
1.11
—
23
—
63
—
100
45.71
46
0.48
0.16
9
9.33
1.12
1.45
6MP
813
0.0038
0.015
NANAOCAM-carboxylic acid conjugates are chemical-
ly too unstable to serve as useful derivatives (compared
to phenols which are chemically stable) because the leav-
ing group has a pKa of ꢁ3–5 (compared to 6.8–9.5), e.g.,
10, t1/2 ꢁ 6 min at pH 7.4 and 37 ꢁC. The NArNAO-
CAM promoiety enhances the chemical stability of
phenolic derivatives compared to NANAOCAM pro-
moiety, and by analogy they should increase the stability
of carboxylic acid conjugates too. Thus, the implication
of mechanism of hydrolysis of NANAOCAM-Y conju-
gates in prodrug design is the design of a more stable
NArNAOCAM analogue of carboxylic acids which
can be conveniently formulated but would hydrolyze
independent of enzymes and be clinically useful. We
are currently investigating the application of NArNAO-
CAM prodrug technology to carboxylic acids like
naproxen.
O–CH2 of ROCOOCH2 with a substituted nitrogen
(N–R; R = alkyl) makes it possible to increase solubility
in a membrane and increases permeability across a
biological barrier such as skin.
In conclusion, NANAOCAM and NArNAOCAM pro-
moieties can act as soft alkyl derivatives of polar drugs.
The mechanism of chemical hydrolysis is believed to be
SN1 with phenol, Th or 6MP acting as the nucleofuge.
Proof-of-mechanism being SN1 comes from the in-
creased stability of NArNAOCAM derivatives com-
pared to that of NANAOCAM prodrugs. These
NArNAOCAM derivatives can potentially be useful in
designing stable carboxylic acid prodrugs whose phar-
maceutical formulation should be easier than that of
their NANAOCAM counterparts. The delivery of phe-
nol and thiol containing drugs like APAP and 6MP
can be enhanced by utilizing the NANAOCAM promo-
iety. The flux of APAP was increased by 2- and 4-fold
for 6MP from IPM across hairless mouse skin. Synthesis
of homologous series of NANAOCAM prodrugs of
APAP, Th and 6MP in order to optimize flux across
the skin is currently under progress. A more water-solu-
ble NANAOCAM derivative should further enhance
permeation of APAP and 6MP and also increase the
permeation of Th across the skin.
Although NANAOCAM-phenols hydrolyse by an SN1-
type of pathway, they are chemically too stable to revert
to the parent drug at a sufficient rate to be effective pro-
drugs based on chemical hydrolysis. To evaluate if these
derivatives hydrolyse on their passage through the skin
and increase permeation, selected prodrugs 1, 8 and 9
were analyzed in in vitro skin permeation studies (Table
2). The physicochemical characterization of these select-
ed NANAOCAM derivatives10 and the parent drugs
was also carried out (Table 2). In vitro skin permeation
studies through hairless mouse skin were carried out
using suspensions in IPM.10 The steady-state flux was
calculated from the slope of cumulative amounts of drug
permeated versus time. The in vitro diffusion cell exper-
iments from IPM through hairless mouse skin show
reversion of NANAOCAM prodrugs to the parent
drug. The permeation of 1 and 8 was enhanced com-
pared to the parent drug, while the permeation of 9
was lower than that of Th.11
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2006.03.061.
References and notes
1. (a) Bundgard, H. Design and Application of Prodrugs. In
A Textbook of Drug Design and Development; Krogsgaard-
Larsen, P., Bundgard, H., Eds.; Harwood: Reading, UK,
1991; pp 113–191; (b) Sloan, K. B.; Wasdo, S. Med. Res.
Rev. 2003, 23, 763; (c) Rautio, J.; Nevalainen, T.; Taipale,
H.; Vepsalainen, J.; Gynther, J.; Pederson, T.; Jarvinen, T.
J. J. Med. Chem. 2000, 43, 1489.
2. Siver, K. G.; Sloan, K. B. J. Pharm. Sci. 1990, 79, 66.
3. (a) Moreira, R.; Calheiros, T.; Cabrita, J.; Mendes, E.;
Pimentel, M.; Iley, J. Pharm. Res. 1996, 13, 70; (b) Iley, J.;
Moreira, R.; Calheiros, T.; Mendes, E. Pharm. Res. 1997,
14, 1634; (c) Moreira, R.; Mendes, E.; Calheiros, T.;
Bacelo, M. J.; Iley, J. Tetrahedron Lett. 1994, 35, 7107.
4. General procedure for alkylation reactions: the alkylation
of phenols with NANAOCAM-Cl and NArNAOCAM-Cl
was carried out by refluxing equimolar equivalents of
The increase in permeation of prodrugs 1 and 8 can be
attributed to the increase in biphasic solubility of the
prodrugs compared to the parent,1b,c,12 while lower per-
meation of 9 was because of the large decrease in water
solubility which could not be compensated by the in-
crease in lipid solubility of the prodrug compared to
Th. Thus, NANAOCAM derivatives of phenols, imides
and thiols represent a novel class of prodrugs which are
sufficiently chemically stable to allow formulation but
sufficiently enzymatically labile to revert to the parent
drug at a useful rate. Shorter chain NANAOCAM, pro-
drugs of phenols, have both higher lipid and water sol-
ubilities compared to ACOM and AOCOM phenolic
conjugates.11 Thus, replacing the oxygen atom in

3594
S. Majumdar, K. B. Sloan / Bioorg. Med. Chem. Lett. 16 (2006) 3590–3594
phenol, TEA in CH2Cl2 for 1 h followed by addition of the
to 2.9 mL of buffer in a cuvette such that the final
concentration was about 10ꢀ5 M. Half-lives were
calculated from the plot of log(A1 ꢀ At) or log
(ꢀ(A1 ꢀ At)) versus time.
alkylating agent to the reaction mixture. The contents
were stirred overnight and worked up by washing with
water. The CH2Cl2 solution was dried over Na2SO4 and
then concentrated to an oil which was purified by
crystallization or column chromatography.
8. Jencks, W. P.; Regenstein, J. In Handbook of Biochemistry
and Molecular Biology: Physical and Chemical Data,
3rd ed.; Fasman,G. D., Ed.; CRC: Cleveland,1976,
Vol. I, pp 305–351.
5. Majumdar, S.; Sloan, K. B. Synth. Commun. submitted for
publication.
6. 1H NMR (400 MHz; CDCl3; Me4Si) of 1: d 7.6 (s, 1H), d
7.39 (d, 2H), d 6.96–6.87 (2d, 2H), d 5.28–5.21 (2s, 2H), d
3.72–3.7 (2s, 3H), d 3.0–2.97 (2s, 3H), d 2.0 (s, 3H). UV of
1: kmax (pH 8.8 buffer)/nm 243.4 (e/L molꢀ1 cmꢀ1
0.95 · 104). UV: kmax (pH 7.1 buffer)/nm 240
(e/L molꢀ1 cmꢀ1 1.01 · 104, 0.09 · 104). 1H NMR
(400 MHz; CDCl3; Me4Si) of 12: d 8.2 (d, 2H), d
7.27–7.42 (m, 5H), d 7.05 (d, 2H), d 5.67 (s, 2H), d 3.75
(s, 3H). UV of 12: kmax (pH 8.8 buffer)/nm 305 nm
(e/L molꢀ1 cmꢀ1 1.19 · 104).
9. Exner, O. Correlation Analysis of Chemical Data; Plenum
Press: New York, 1988.
10. Experimental protocol for determination of solubili-
ties, partition coefficients and flux through hairless
mouse skin is reported in: Wasdo, S.; Sloan, K. B.
Pharm. Res. 2004, 21, 940, and references cited in
this paper.
11. Majumdar, S.; Sloan, K. B. 231st American Chemical
Society National Meeting and Exposition Abstracts, Atlan-
ta, GA, March 26–20, 2006.
7. The rates of hydrolysis in aqueous buffers were determined
by UV spectroscopy. An aliquot (100 lL) of a stock
solution of compound dissolved in acetonitrile was added
12. Roberts, W. J.; Sloan, K. B. J. Pharm. Sci. 1999, 88, 515.
13. Physicochemical analysis of this derivative was reanalyzed
and new values are reported in this paper.