N-Alkyl-N-alkyloxycarbonylaminomethyl (NANAOCAM) prodrugs of carboxylic acid containing drugs

Article abstract of DOI:10.1016/j.bmcl.2006.11.074

Synthesis and hydrolysis in aqueous buffers of novel N-alkyl-N-alkyloxycarbonylaminomethyl (NANAOCAM) and N-aryl-N-alkyloxycarbonylaminomethyl (NArNAOCAM) derivatives of carboxylic acid containing drugs were carried out. The hydrolysis follows a S_N1 type mechanism and is dependent on the nucleofugacity of the leaving group. Topical delivery of the NANAOCAM derivative of naproxen from IPM across hairless mice skin was examined in in vitro diffusion cell experiments. The prodrug was 4.5-fold less lipid soluble, 2.4-fold less water soluble and 3.6-fold less permeable than the parent drug.

Full text of DOI:10.1016/j.bmcl.2006.11.074

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1447–1450
N-Alkyl-N-alkyloxycarbonylaminomethyl (NANAOCAM)
prodrugs of carboxylic acid containing drugs
Susruta Majumdar and Kenneth B. Sloan*
Department of Medicinal Chemistry, University of Florida, PO Box 100485, Gainesville, FL 32610, USA
Received 27 September 2006; revised 17 November 2006; accepted 29 November 2006
Available online 1 December 2006
Abstract—Synthesis and hydrolysis in aqueous buffers of novel N-alkyl-N-alkyloxycarbonylaminomethyl (NANAOCAM) and
N-aryl-N-alkyloxycarbonylaminomethyl (NArNAOCAM) derivatives of carboxylic acid containing drugs were carried out. The
hydrolysis follows a SN1 type mechanism and is dependent on the nucleofugacity of the leaving group. Topical delivery of the
NANAOCAM derivative of naproxen from IPM across hairless mice skin was examined in in vitro diffusion cell experiments.
The prodrug was 4.5-fold less lipid soluble, 2.4-fold less water soluble and 3.6-fold less permeable than the parent drug.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Carboxylic acid containing drugs are ionized at physio-
logical pH and find it difficult to breach biological mem-
brane barriers. The prodrug approach which transiently
masks polar functional groups has proven useful in a
number of cases.1,2 Most carboxylic acid containing
drugs are derivatized as alkyl esters, acyloxymethyl
(ACOM) or alkyloxycarbonyloxymethyl (AOCOM) es-
ters. These carboxylic acid esters are hydrolysed in vivo
by ubiquitous esterases to yield the parent drug.3 Since
these types of prodrugs rely on enzymes to generate
the active principle, they are prone to biological variabil-
ity in tissues and cells and may not generate sufficient
amounts of the active principle at the target site to be
therapeutically useful.3 Replacing the oxygen atom in
AOCOM by nitrogen gives the N-alkyl-N-alkyloxycarb-
and may improve permeation through the skin as previ-
ously observed.
In the present paper, we report the synthesis of
NANAOCAM derivatives of carboxylic acid containing
drugs, investigate its mechanism of hydrolysis in aque-
ous buffers and probe the effect of the NANAOCAM
promoiety on penetration of a model carboxylic acid
containing drug, naproxen, across hairless mouse skin
in vitro from IPM.
Synthesis and hydrolysis studies. NANAOCAM deriva-
tives of five different carboxylic acids were made by
alkylating the parent drug with N-alkyl-N-alkyloxycarb-
onylaminomethyl chloride (NANAOCAM-Cl)6 (Table
1). The alkylating agent was synthesized in two steps.
Methylamine was reacted with formaldehyde in the
presence of an equivalent of NaOH to generate 1, 3,
5-trimethylhexahydrotriazine. Reaction of 1, 3, 5-trim-
ethylhexahydrotriazine with methylchloroformate over-
night in dichloromethane gave NANAOCAM-Cl in
quantitative amounts.4,7
onylaminomethyl
(NANAOCAM)
promoiety.
NANAOCAM conjugates of phenols, imides and thiols
hydrolysed by a SN1 type of pathway in aqueous buf-
fers4 and exhibited improved biphasic solubility and flux
from isopropyl myristate (IPM) across hairless mouse
skins.4,5 Extension of the application of the NANAO-
CAM promoiety to carboxylic acid containing drugs,
which exhibit lower pKa values (ꢀ3–5) compared to phe-
nols and imides (ꢀ8.6–10), should give prodrugs that
hydrolyse chemically rather than relying on enzymes,
N-Aryl-N-alkyloxycarbonylaminomethyl (NArNAO-
CAM) derivatives of naproxen were also made to
illustrate the mechanism of hydrolysis. An aromatic
amine was reacted with equimolar amounts of pyridine
and methyl chloroformate to yield N-arylcarbamic acid
methylester. Chloromethylation of N-arylcarbamic
acid methylester with paraformaldehyde and trimethyl-
silyl chloride (TMSCl) gave the corresponding
Keywords: Prodrugs; NANAOCAM; NArNAOCAM; Chemically
labile; Topical delivery; Dermal delivery; Permeation; Soft alkylation.
*
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.2006.11.074

1448
S. Majumdar, K. B. Sloan / Bioorg. Med. Chem. Lett. 17 (2007) 1447–1450
Table 1. Hydrolysis of NANAOCAM derivatives of carboxylic acid containing agents and NArNAOCAM derivatives of naproxen at pH 7.1 and
39 ꢁC and the influence of pKa of leaving groups on rates of hydrolysis
O
R'
O
N
R
Y
Y = -OOC-Drug
R’= CH3; R = Alkyl or Aryl.
Compound
R
Y
logk (sꢁ1
)
t1/2 (min)
pKa
1
2
3
4
5
6
7
8
CH3
CH3
Naproxen
Indomethacin
Ibuprofen
ꢁ2.24
ꢁ2.5
2
4.2
4.5
4.41
5.2
6.0
4.2
4.2
4.2
3.65
4.11
6
CH3
CH3
ꢁ2.55
ꢁ2.72
ꢁ3.55
ꢁ4.02
ꢁ3.63
ꢁ4.13
Dimethylaminobenzoic acid
Nalidixic acid
Naproxen
CH3
C6H5
40
119.62
49.83
156.46
p-MeO–C6H4–
p-EtOOC–C6H4–
Naproxen
Naproxen
NArNAOCAM-Cl. Alkylation of naproxen in the fol-
lowing step with NArNAOCAM-Cl gave the desired
NArNAOCAM prodrugs of naproxen. All compounds
synthesized were fully characterized by UV,8 NMR8
and elemental analysis.8
If the mechanism is SN1, the rate of hydrolysis should be
decreased by the N-aryl group where a positive charge
on CH2 in N–CH2–O is destabilized. Furthermore, as
in the case of phenols, the order of rates of hydrolyses
should be –COOEt > –H > –OMe which would be con-
sistent with a SN1 type of hydrolysis (Scheme 1). An
electron-donating substituent on the N-aryl ring like –
OCH3 stabilizes the SN1 transition state more than an
The hydrolysis of NANAOCAM and NArNAOCAM
conjugates was investigated in pH 7.1 buffer at 39 ꢁC.9
All the NANAOCAM-carboxylic acid prodrugs hydro-
lysed in aqueous buffers to release the parent molecule
exhibiting pseudo-unimolecular first order kinetics.
The lower pKa of carboxylic acids makes the rates of
hydrolysis faster than phenols, imides and thiols. A plot
of logk versus pKa for 1–5 was linear and indicated that
rates of hydrolysis were dependent on the nucleofugacity
of the leaving group (slope = q = 0.65, r2 = 0.92, plot
not shown). Previously the mechanism of hydrolysis of
NANAOCAM derivatives of phenols, imides and thiols
was established to be SN1. To investigate if a SN1 type of
hydrolysis also holds for NANAOCAM derivatives of
carboxylic acids, NArNAOCAM derivatives of a model
carboxylic acid drug, naproxen, were synthesized (6–8)
and their rates of hydrolysis measured (Table 1).
OH2
-
-
DRUG-COOH + OH
DRUG-COO
+
O
O
O
OH2
+
+
OH2
O
N
R
OOC-DRUG
O
N
O
N
R
R
-CH2=O
O
H
+
OH2
O
N
R
Scheme 1.
Representative prodrug structures:
O
R
N
O
O
O
O
N
O
O
CH3
O
O
MeO
N
O
1, R = Me, Naproxen prodrug
6-8, R = Aryl, Naproxen prodrug
2, R = Me, Indomethacin prodrug
Cl
O
O
N
O
O
O
O
O
O
N
O
3, R = Me, Ibuprofen prodrug
N
N
5, R = Me, Nalidixic acid prodrug

S. Majumdar, K. B. Sloan / Bioorg. Med. Chem. Lett. 17 (2007) 1447–1450
1449
electron-withdrawing group like –COOEt. Indeed, the
substituted N-aryl derivatives increased the half-lives
of hydrolyses from 2 min to between 50 and 150 min
in the order –COOEt > –H > –OCH3. N-Alkylamidom-
ethyl esters of carboxylic acids also hydrolyse by a SN1
mechanism2 so there is some precedence for this result.
occurs on the carbonyl functional group of the ester fol-
lowed by elimination of carboxylate ion to form N-hy-
droxymethyl-N-arylcarbamic
(Scheme 2).10
acid
methyl
ester
All three NArNAOCAM naproxen derivatives repre-
sent potentially useful prodrugs which are reasonably
stable but should be sufficiently labile to release the drug
at an appropriate rate to express its pharmacological
activity. Moreover, since 8 would eventually release p-
aminobenzoic acid, a component of sunscreens, it may
be the most attractive candidate.
The kinetics of decomposition of N-(40-ethyloxycarbon-
ylphenyl)-N-methyloxycarbonylaminomethyl ester of
naproxen, 8, was also studied in aqueous buffers at
39 ꢁC over a wide pH range (Fig. 1). The rates of hydro-
lysis were independent of the pH of buffers from pH 4.0
to 8.25 so the mechanism of hydrolyses along this pH
range is believed to be SN1. At pH 9.2 a sharp increase
in rates of hydrolysis was observed. This is probably due
to a change in the mechanism of hydrolysis from a SN1
to an addition-elimination where nucleophilic addition
The physicochemical characterizations of NANAO-
CAM prodrugs of naproxen and dimethylaminobenzoic
acid and the parent drugs were also carried out (Table
2).11 Solubility in IPM (SIPM) and partition coefficient
between IPM and pH 4.0 buffer (KIPM:4.0) were deter-
mined. Solubility in pH 4.0 buffer (Se4.0) was estimated
from the product of SIPM and KIPM:4.0 since direct mea-
surement of S4.0 was not possible owing to the instability
of 1 and 4 in buffer. 1 was 4.5-fold less lipid-soluble and
2.5-fold less water-soluble than naproxen, while 4 was
11.7-fold more lipid soluble and 5.5-fold more water sol-
uble than dimethylaminobenzoic acid. Since naproxen is
therapeutically more interesting, we investigated the
delivery of 1 across hairless mice skin in vitro from
IPM. However, the prodrug, being less water and lipid
soluble than the parent drug, performed poorly in com-
parison to naproxen since it is necessary to improve
both the water and lipid solubility of the prodrug com-
pared to the parent drug12 to improve topical delivery.
-3
-3.2
-3.4
-3.6
-3.8
-4
-4.2
-4.4
-4.6
-4.8
-5
3.5
5.5
7.5
9.5
Here we have reported NANAOCAM and NArNAO-
CAM promoieties as novel prodrugs of carboxylic acid
containing drugs which hydrolyse by a SN1 type of path-
way. NANAOCAM-carboxylic acids are chemically too
unstable to be therapeutically useful, however NAr-
NAOCAM derivatives, being more stable, may be more
useful for pharmaceutical formulation. NArNAOCAM-
naproxen conjugates are thus prodrugs of a carboxylic
acid which unlike ACOM or AOCOM esters are not
dependent on enzymatic hydrolysis and have sufficient
chemical stability to be formulated. The NArNAOCAM
promoiety can also act as spacers or linkers for conjuga-
tion of water solubilizing groups like amines and pep-
tides to a carboxylic acid functional group of a drug
molecule and thereby increase biphasic solubility and
flux through skin. Attempts to make NArNAOCAM-
naproxen derivatives with enhanced water solubility
are currently under investigation.
pH
Figure 1. pH rate profile of 8.
O
O
O
O
H
R
N
O
O
N
O
HO
N
O
-CH2=O
-
OH
COOEt
COOEt
COOEt
8
+
RCOOH
Scheme 2.
Table 2. Estimated solubility in pH 4.0 buffer (Se4.0), solubility in IPM (SIPM), log partition coefficients between IPM and pH 4.0 buffer (logKIPM:4.0),
flux from IPM through hairless mice skins (JMIPM), residual skin concentration of total drug species (Crs) and permeability coefficients from IPM
through hairless mouse skins (PMIPM) of NANAOCAM prodrugs and parent drugs
a
Compound
Se4.0
SIPM
logKIPM:4.0
JMIPM
(lmol cmꢁ2 hꢁ1
Crs
PMIPM
(cm hꢁ1
% of parent drug
obtained after diffusion
(mM)
(mM)
)
(lmol)
)
Naproxen
1
Dimethylaminobenzoic acid
0.27
0.11
0.38
2.09
23.49
5.16
4.21
1.95
1.67
1.05
1.35
0.36
0.1
2.71
4.21
0.015
0.019
—
100
4
46.83
a Calculated from JMIPM/SIPM
.

1450
S. Majumdar, K. B. Sloan / Bioorg. Med. Chem. Lett. 17 (2007) 1447–1450
3H), 3.85 (q, 1H), 3.7–3.65 (2s, 3H), 2.89–2.85 (2s, 3H),
1.57 (d, 3H). UV: kmax (pH 7.1 buffer) 271.5 and 245 nm
(e) 0.52 · 104, 0.77 · 104 L molꢁ1
Elemental analysis
References and notes
.
1. Bundgard, H. In Design and Application of Prodrugs. In a
Textbook of Drug Design and Development; Krogsgaard-
Larsen, P., Bundgard, H., Eds.; Harwood: Reading, UK,
1991; p 113.
2. (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,
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Bacelo, M. J.; Iley, J. Tetrahedron Lett. 1994, 35, 7107.
3. Beamount, K.; Webster, R.; Gardner, I.; Dack, K. Curr.
Drug Metab. 2003, 4, 461.
4. Majumdar, S.; Sloan, K. B. Bioorg. Med. Chem. Lett.
2006, 16, 3590.
5. Majumdar, S.; Sloan, K.B. 231st American Chemical
Society National Meeting & Exposition Abstracts, Atlan-
ta, GA, March 26–30, 2006.
6. General procedure for alkylation reactions: The alkylation
of carboxylic acids with NANAOCAM-Cl and NArNAO-
CAM-Cl was carried out by reacting equimolar equiva-
lents of carboxylic acid and TEA in CH2Cl2 for 1 h
followed by addition of the 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.
(Found: C, 65.05; H, 6.45; N, 4.2 Calcd for C18H21NO5:
C, 65.24; H, 6.39; N, 4.23%), yield = 97%, mp = 100–
101 ꢁC, Rf (0.49, ethyl acetate/hexane, 1:4). 1H
NMR(400 MHz; CDCl3; Me4Si) of 6: d 7.69 (t, 3H),
7.36–7.39 (dd, 1H), 7.13–7.17 (m, 5H), 6.9 (d, 2H), 5.5–
5.68 (dd, 2H), 3.93 (s, 3H), 3.88 (q, 1H), 3.65 (s, 3H), 1.57
(d, 3H). UV: kmax (pH 7.1 buffer) 272.8 and 245 nm (e
0.35 · 104, 0.83 · 104 L molꢁ1), yield = 60%, Rf (0.17,
ethyl acetate/hexane, 1:5).
9. 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
to 2.9 ml of buffer in a cuvette such that the final
concentration was about 10ꢁ5 M. Half-lives were calcu-
lated from the plot of log(A1 ꢁ At) or log(ꢁ(A1 ꢁ At))
versus time.
10. Bundgaard, H.; Rasmussen, G. J. Pharm. Res. 1991, 8,
1238.
11. Experimental protocol for determination of solubilities,
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.
12. (a) Sloan, K. B.; Koch, S. A. M.; Siver, K. G. Int. J.
Pharm. 1984, 21, 251; (b) Sloan, K. B. Adv. Drug Del. Rev.
1989, 3, 67; (c) Sloan, K. B. In Topical and Ocular Drug
Delivery; Sloan, K. B., Ed.; Merkel Dekker: New York,
1992; p 17; (d) Roberts, W. J.; Sloan, K. B. J. Pharm. Sci.
1999, 88, 515; (e) Sloan, K. B.; Wasdo, S. Med. Res. Rev.
2003, 23, 763.
7. Majumdar, S.; Sloan, K. B. Synth. Commun. 2006, 36,
3537.
8. 1H NMR(400 MHz; CDCl3; Me4Si) of 1: d 7.69 (t, 3H),
7.4 (d, 1H), 7.11–7.15 (m, 2H), 5.41–5.29 (dd, 2H), 3.91 (s,