2-(nitrooxi)etil 2-amino-3-metilbutanoato

2-(Nitrooxy)ethyl 2-amino-3-methylbutanoate: A Comprehensive Reference

1. Identity and Chemical Characterization

Names and Classification

2-(Nitrooxy)ethyl 2-amino-3-methylbutanoate is the IUPAC systematic name for a synthetic organic molecule that combines two pharmacologically relevant moieties: an organic nitrate ester (–ONO₂) and the amino acid valine. The compound is catalogued in the National Library of Medicine MeSH system under unique identifier C555709, mapped to the headings Nitrates and Valine / analogs & derivatives, and is explicitly noted as "a nitric oxide donor."

The compound belongs to the class of nitric oxide amino acid esters, specifically the valine or norvaline derivative group. It is most commonly referred to as valine nitrooxy ethyl ester or, equivalently, valine ethylene glycol nitrate, along with any pharmaceutically acceptable salts thereof. In the peer-reviewed sports science literature it is abbreviated as 2-NO or ENO (ethyl nitrooxy). Other synonyms encountered in the scientific literature and patent filings include 2-nitrooxy ethyl 2-amino 3-methylbutanoate and 2-nitrooxy-ethyl-2-amino-3-methylbutanoate.

Chemical Structure

The compound has a structure that incorporates both amino acid and nitrate ester functionalities. Structurally, it is a valine ester in which the carboxylic acid group of L-valine (2-amino-3-methylbutanoic acid) is esterified with 2-nitrooxy ethanol (ethylene glycol mononitrate), yielding a molecule that carries both a primary amine (from the valine backbone) and a terminal organic nitrate group (–O–NO₂). The molecular formula is C₇H₁₄N₂O₅.

Physical Form and Stability

The compound easily dissociates in water into the valine derivative and associated ion. It is extremely stable in salt form and thus possesses a long shelf life. The preferred compounds of this class have a reported half-life of approximately 5 hours.

Natural Occurrence and Botanical Source

2-(Nitrooxy)ethyl 2-amino-3-methylbutanoate is not a naturally occurring substance. It is a synthetic molecule produced through organic chemistry, specifically the esterification of the amino acid valine with an ethylene glycol nitrate moiety. While this specific molecule is relatively new in the context of nutritional science, its components have roots in naturally occurring substances: the nitrooxy (nitrate) group relates to natural nitrates found in plants such as beetroot, which have been recognized for their cardiovascular properties. The valine component is a branched-chain amino acid (BCAA) that occurs naturally in virtually all protein-containing foods, including meat, dairy products, soy, and legumes.

2. Traditional and Historical Use

Because 2-(nitrooxy)ethyl 2-amino-3-methylbutanoate is a wholly synthetic compound, it has no documented history of traditional or indigenous use in any medicinal system. No records exist in Ayurvedic, traditional Chinese medicine, European folk medicine, or any other formal ethnobotanical tradition for this molecule, because it did not exist as a discrete chemical entity until modern synthetic organic chemistry made it available.

The two functional components of the molecule, however, do connect to longstanding traditions:

• Dietary nitrates (the nitrooxy moiety): The nitrooxy (nitrate) group relates to natural nitrates found in plants such as beetroot, which have been prized for cardiovascular benefits for centuries. Nitrate-rich vegetables were commonly used in folk remedies to support heart health, enhance stamina, and promote circulation, traditions that align with modern findings that dietary nitrates can improve blood flow and reduce blood pressure.
• L-Valine (the amino acid moiety): Valine, as part of the branched-chain amino acid (BCAA) family, has been incorporated into athletic nutrition practice since at least the mid-twentieth century and is found in traditional high-protein dietary preparations across many cultures. The amino acid component, resembling branched-chain amino acids (BCAAs), reflects practices widely utilized in herbal and nutritional remedies for muscle recovery, energy, and overall vitality.

The compound itself first appeared in the scientific literature in 2010 as a novel dietary supplement ingredient studied in the context of sport nutrition and nitric oxide enhancement.

3. Key Constituents and Mechanisms of Action

The Organic Nitrate Ester Moiety

The functional driver of the compound's principal pharmacological interest is its organic nitrate ester (–ONO₂) group. The important biological secondary messenger NO can be generated from exogenous nitrovasodilators and NO donors. Nitrate esters are nitrovasodilators and NO mimetics, believed to be biotransformed to NO in vivo.

Both the relaxing effect on vascular smooth muscle and the effect on platelets are considered to be due to a stimulation of soluble guanylate cyclase by nitric oxide derived from the organic nitrate ester molecule, through metabolization catalyzed by enzymes such as glutathione S-transferase, cytochrome P-450, and possibly esterases. The cyclic GMP produced by the guanylate cyclase acts via cGMP-dependent protein kinase.

Nitrovasodilators relax vascular smooth muscle by stimulating soluble guanylate cyclase and thus by increasing the formation of cyclic GMP (cGMP). This nucleotide is responsible for relaxation, most likely by decreasing cytosolic free Ca²⁺ by one or several mechanisms.

Postulated Pressure-Dependent Bioactivation

A notable hypothesis specific to 2-(nitrooxy)ethyl 2-amino-3-methylbutanoate is that its biological activation may depend on physical stress. It is now believed that the 2-NO molecule may be activated via increased blood pressure, secondary to physical stress. This would distinguish it mechanistically from simpler inorganic nitrate supplements and could explain why some effects are more evident during or after exercise than in sedentary conditions.

Nitric Oxide and Vascular Function

Nitric oxide (NO) functions as a signaling molecule, with a noted effect in promoting vasodilation by acting on vascular smooth muscle. Through this and other mechanisms, NO results in increased blood flow at rest and during exercise.

The Valine Amino Acid Component

Valine is an essential branched-chain amino acid (BCAA) that participates in protein synthesis and energy metabolism in skeletal muscle. The preferred compounds of this class are the valine or norvaline derivatives of the nitric oxide amino acid ester, known as valine nitrooxy ethyl ester (or valine ethylene glycol nitrate), which possess many of the required characteristics necessary to fulfill the role of boosting NO levels. Whether the valine moiety itself confers any physiologically meaningful anabolic or anti-catabolic effect in vivo after the ester bond is hydrolyzed has not been demonstrated in studies specific to this molecule.

Nitrate Tolerance — A Class Effect

A pharmacological consideration relevant to all organic nitrate esters is the development of tolerance with repeated use. Repeated administration of organic nitrates causes tolerance development characterized by a diminished relaxing effect and an attenuated rise in cGMP. Whether this tolerance phenomenon applies to 2-(nitrooxy)ethyl 2-amino-3-methylbutanoate specifically has not been examined in published clinical studies.

4. Scientific Evidence by Area of Use

The human clinical evidence base for 2-(nitrooxy)ethyl 2-amino-3-methylbutanoate is extremely limited, consisting of a small number of published studies, all from a single research group (Bloomer et al., University of Memphis), and all involving small samples of healthy men. There are no systematic reviews, meta-analyses, randomized controlled trials in patient populations, or independent replications of any finding. All conclusions must therefore be characterized as preliminary.

4.1 Circulating Nitrate/Nitrite (NOx) as a Biomarker of Nitric Oxide

Study type: Acute, randomized, crossover human study.
Key finding: 2-(Nitrooxy)ethyl 2-amino-3-methylbutanoate has a small effect on increasing circulating nitrate/nitrite and does not cause any change in hemodynamic variables within the 1-hour postingestion period in a sample of resistance-trained men.

This foundational observation — a reported approximately 6.7% increase in plasma NOx following oral ingestion — was reported by Bloomer and colleagues (2010, J Strength Cond Res) and is referenced across subsequent work by the same group. Oral intake of 2-nitrooxy ethyl 2-amino 3-methylbutanoate demonstrated an approximately 6.7% increase in circulating nitrate/nitrite; however, no measures of exercise performance were obtained.

Evidence strength: Weak. The effect size is small (6.7%), the observation window was only one hour, subjects were sedentary during measurement, and no exercise performance outcomes were assessed in this initial study.

4.2 Resistance Exercise Performance (Topical Gel Form)

Study type: Randomized, crossover, placebo-controlled human trial.
Population: Fourteen resistance-trained men (24 ± 1 years old) reported to the laboratory on 2 occasions to undergo exercise testing, which consisted of arm curl isometric force and muscular endurance (3 sets to fatigue using 80, 65, and 50% of 1 repetition maximum [1RM]: total of 9 sets).

Intervention: The gel (2-nitrooxy ethyl 2-amino 3-methylbutanoate; mixed in tea tree oil) or placebo (tea tree oil) was applied topically by the subjects for 7 days before each test day, with 7–10 days separating the randomly ordered conditions.

Key results: No statistically significant differences were noted between conditions for performance variables (p > 0.05). However, when using a load of 50% of 1RM, 6.2% more repetitions were performed when using the gel as compared with when using the placebo; 19.9% more repetitions were performed by 8 subjects noted to be "responders" to gel treatment.

Blood lactate and muscle pump significantly increased with exercise (p < 0.0001) but were not different between conditions (p > 0.05). Minimal change was noted in nitrate/nitrite, and the heart rate and perceived exertion were nearly identical between conditions (p > 0.05).

Conclusion per authors: 2-Nitrooxy ethyl 2-amino 3-methylbutanoate gel has a modest (6.2%), nonstatistically significant effect on exercise performance, in particular when men use a load equal to 50% 1RM in the biceps curl exercise.

Limitations noted by authors: Studies inclusive of a larger sample size are needed to extend these initial findings. The study used a topical (transdermal) delivery form, not an oral form, and was confined to a single upper-body exercise. The responder analysis (19.9% more repetitions in 8 of 14 subjects) is post-hoc and cannot be used to make general claims about efficacy.

Evidence strength: Weak. Null primary outcome; the exploratory 6.2% difference did not reach statistical significance. Single study with n = 14; single research group.

4.3 Blood Pressure and Plasma NOx Following Acute Exercise

Study type: Randomized, double-blind, crossover human trial.
Population and protocol: 15 healthy men (28.5 ± 0.8 yrs) reported to the lab on two different days separated by two weeks. They were randomly assigned in a double-blind, cross-over manner to receive either a 2-NO tablet or a placebo tablet before performing two, two-minute sets of sit-up exercise (exercise sets performed 20 minutes apart). Systolic (SBP) and diastolic (DBP) blood pressure and heart rate were measured before and for 20 minutes following each set of exercise.

Key finding: 2-NO can lower blood pressure in response to acute exercise in a sample of healthy, normotensive men, and the change in blood pressure may be associated with an increase in circulating NOx. The authors also proposed the hypothesis that the 2-NO molecule may be activated via increased blood pressure, secondary to physical stress.

Evidence strength: Preliminary. The study is small (n = 15), acute in design (single-dose), restricted to normotensive, healthy young men, and has not been replicated. Blood pressure changes in normotensive individuals during low-intensity exercise (sit-ups) require careful contextual interpretation.

5. Body Systems and Health Areas Associated with the Compound

Cardiovascular and Vascular System

The primary mechanistic interest in this compound relates to its capacity to donate nitric oxide and thereby promote vasodilation. Nitrovasodilators, as a class, induce vasodilation primarily through the donation of nitric oxide, which activates soluble guanylate cyclase in vascular smooth muscle cells, leading to increased cyclic guanosine monophosphate (cGMP) levels and subsequent relaxation of smooth muscle. In the context of 2-(nitrooxy)ethyl 2-amino-3-methylbutanoate specifically, it has been observed that the preferred compounds do not cause hypotension in normotensive or hypotensive individuals; therefore, upon administration, a hypertensive individual will experience the vasodilatory effect, which will result in a decrease in blood pressure. The decrease in blood pressure may be up to a normotensive blood pressure. Individuals with normal blood pressure will not experience the vasodilatory effect, and their blood pressure will remain stable.

Patent filings further describe the compound's potential application in the prophylaxis or treatment of conditions associated with impaired blood circulation, as well as for pulmonary hypertension and other respiratory conditions (U.S. Patents 8,946,160 and 8,883,844), though no clinical trial evidence in these patient populations has been published.

Skeletal Muscle and Athletic Performance

Nitric oxide dietary supplements are popular within the sport and bodybuilding community. The interest in 2-(nitrooxy)ethyl 2-amino-3-methylbutanoate in this context derives from the hypothesis that increasing circulating NOx will augment skeletal muscle blood flow and oxygen delivery during resistance exercise, translating into greater muscular endurance or strength. As described above, the available human evidence is limited and shows only a non-statistically significant trend toward improved repetition performance at lower loads.

Hemodynamic Variables

Across the available studies, no significant changes in resting heart rate were observed. 2-(Nitrooxy)ethyl 2-amino-3-methylbutanoate has a small effect on increasing circulating nitrate/nitrite and does not cause any change in hemodynamic variables within the 1-hour postingestion period in a sample of resistance-trained men. Blood pressure responses appear to be exercise-context-dependent rather than resting-state effects.

6. Dosage Forms and Reported Dosages

Oral (Tablet) Form

The compound was studied by Bloomer and colleagues in an oral tablet form, referenced as the route of administration in the foundational 2010 study that reported a ~6.7% increase in NOx. In the blood pressure and NOx study, subjects were randomly assigned to receive either a 2-NO tablet or a placebo tablet before exercise. The precise dose administered in milligrams in these human studies is not disclosed in the publicly available abstracts or summaries reviewed; the full-text manuscripts would be required to confirm the exact dose used.

Patent claims for the class of valine nitrooxy ethyl ester compounds specify: Therapeutically effective amounts include but are not limited to 0.5 to 30 mg of the compound.

Topical Gel Form

The gel formulation consisted of 2-nitrooxy ethyl 2-amino 3-methylbutanoate mixed in tea tree oil, applied topically by the subjects for 7 days before each test day. The specific concentration or mass applied per dose is not reported in available abstracts.

Other Formulations

Compounds described as valine nitrooxy ethyl ester (or valine ethylene glycol nitrate) are among the preferred forms noted in patent filings as well-suited to boosting NO levels. No inhalation or intravenous formulations have been studied for dietary supplement use.

7. Safety Considerations and Potential Interactions

Observed Safety Profile in Small Human Studies

In the human studies conducted to date — all short-term, acute or 7-day protocols in healthy young men — no significant adverse hemodynamic effects were reported. 2-(Nitrooxy)ethyl 2-amino-3-methylbutanoate does not cause any change in hemodynamic variables within the 1-hour postingestion period in a sample of resistance-trained men.

The patent literature claims that the compound does not produce hypotension in people with normal or low blood pressure: It has been observed that the preferred compounds do not cause hypotension in normotensive or hypotensive individuals. Therefore, upon administration, a hypertensive individual will experience the vasodilatory effect, which will result in a decrease in blood pressure. The decrease in blood pressure may be up to a normotensive blood pressure.

Nitrate Tolerance (Organic Nitrate Class Effect)

Since repeated administration of organic nitrates, particularly nitroglycerin, leads to tolerance, i.e., a decrease in the vasodilator effect, it was studied whether tolerance was a peripheral phenomenon occurring in the vascular smooth muscle, and was due to an impairment of GC activation. In isolated circular strips of bovine coronary arteries, 90-minute pretreatment with nitroglycerin greatly lowered the relaxing as well as the cGMP increasing response to nitroglycerin, indicating tolerance induction. Whether this tolerance phenomenon applies to 2-(nitrooxy)ethyl 2-amino-3-methylbutanoate at the doses used in dietary supplement contexts has not been specifically studied.

Interaction with Phosphodiesterase-5 (PDE5) Inhibitors

As a nitric oxide donor and organic nitrate ester, 2-(nitrooxy)ethyl 2-amino-3-methylbutanoate belongs to a class of compounds for which clinically established drug interactions exist. Nitrovasodilators induce vasodilation through the donation of nitric oxide, which activates soluble guanylate cyclase in vascular smooth muscle cells, leading to increased cGMP levels. This NO-mediated mechanism reduces preload and afterload, distinguishing nitrovasodilators from other vasodilator classes that act via different pathways, such as calcium channel blockade. PDE5 inhibitors (e.g., sildenafil, tadalafil) also elevate cGMP by preventing its breakdown; co-administration of any nitrate-based compound with PDE5 inhibitors is associated with potentially severe hypotension as a well-documented class interaction. No specific interaction data exist for this particular molecule.

Limitations of the Current Safety Database

The safety data for this compound are very limited. All published human studies are acute or short-term (7-day), conducted in small samples of healthy young men, and focused on efficacy rather than comprehensive safety assessment. No studies in women, older adults, individuals with cardiovascular disease, or other clinical populations have been published. No long-term safety data exist. Absent broader study, the compound's safety profile in vulnerable populations cannot be characterized from available evidence.

8. Overall Evidence Summary

2-(Nitrooxy)ethyl 2-amino-3-methylbutanoate is a synthetic organic nitrate ester of the amino acid valine, classified by the National Library of Medicine as a nitric oxide donor. This compound is structurally related to nitrooxy derivatives and branched-chain amino acids. Its proposed mechanism — releasing NO to activate soluble guanylate cyclase and relax vascular smooth muscle — is consistent with the well-established pharmacology of organic nitrate esters as a class.

However, the human evidence base is very limited. Direct clinical studies on 2-(nitrooxy)ethyl 2-amino-3-methylbutanoate are currently limited. The available published literature consists of fewer than three small crossover trials, all from a single research group, in healthy young men, with acute or short-term designs. The effects observed — a small (~6.7%) increase in circulating NOx after oral ingestion, a non-significant 6.2% trend toward more repetitions in one exercise study, and an apparent blood pressure reduction in the context of acute exercise — are preliminary findings that require independent replication in larger and more diverse populations before any conclusions can be drawn about efficacy. No regulatory body (FDA, EFSA, EMEA, or equivalent) has issued a safety or efficacy assessment for this specific compound as a dietary ingredient.

References

• U.S. National Library of Medicine MeSH Browser — 2-(nitrooxy)ethyl 2-amino-3-methylbutanoate (C555709)
• Bloomer RJ, Williams SA, Canale RE, Farney TM, Kabir MM. Acute effect of nitric oxide supplement on blood nitrate/nitrite and hemodynamic variables in resistance trained men. J Strength Cond Res. 2010 Oct;24(10):2587–92. (PubMed PMID: 20885188)
• Bloomer RJ, Alleman RJ Jr, Cantrell GS, Farney TM, Schilling BK. Effects of 2-nitrooxy ethyl 2-amino 3-methylbutanoate gel on resistance exercise performance and blood nitrate/nitrite in resistance trained men. J Strength Cond Res. 2012 Jun;26(6):1680–7. (PubMed PMID: 21921823)
• Bloomer RJ et al. Impact of 2-nitrooxy-ethyl-2-amino-3-methylbutanoate on blood pressure and plasma nitrate/nitrite following acute exercise in normotensive men. Medical Research Archives. 2016;3(6).
• U.S. Patent 8,946,160 — Nitric oxide amino acid esters for improving vascular circulation, and prophylaxis or treatment of a condition associated with impaired blood circulation.
• U.S. Patent 8,883,844 — Nitric oxide releasing amino acid ester for treatment of pulmonary hypertension and other respiratory conditions.
• Hanson J et al. Nitrate esters as nitric oxide donors: SS-nitrates. J Med Chem. 2001;44(9):1357–1365. (PubMed PMID: 11348172)
• Kukovetz WR, Holzmann S. Mechanism of vasodilation by nitrates: role of cyclic GMP. Cardiology. 1987;74 Suppl 1:12–19. (PubMed PMID: 2886220)
• Axelsson KL et al. Mechanisms of nitrate-induced vasodilatation and tolerance. Eur J Clin Pharmacol. 1990;38 Suppl 1:S7–12. (PubMed PMID: 1972361)
• Ahlner J, Andersson RGG, Torfgård K, Axelsson KL. Organic nitrate esters: clinical use and mechanisms of actions. Cardiovasc Drugs Ther. 1991;5(4):847–858.
• Evgenov OV et al. Stimulators and activators of soluble guanylate cyclase: review and potential therapeutic indications. Handb Exp Pharmacol. 2009;191:33–70. PMC3299283.
• Wylie LJ et al. Dietary inorganic nitrate as an ergogenic aid: an expert consensus derived via the modified Delphi technique. Sports Med. 2022. PMC9474378.